U.S. patent number 5,863,502 [Application Number 08/786,956] was granted by the patent office on 1999-01-26 for parallel reaction cassette and associated devices.
This patent grant is currently assigned to Sarnoff Corporation. Invention is credited to Zygmunt Marian Andrevski, William Ronald Roach, Peter David Southgate, Peter John Zanzucchi.
United States Patent |
5,863,502 |
Southgate , et al. |
January 26, 1999 |
Parallel reaction cassette and associated devices
Abstract
The invention provides a parallel reaction device for conducting
reactions therein comprising one or more reaction flow-ways, each
such reaction flow-way comprising one or more chambers connected
serially by fluid exchange channels, additional fluid exchange
channels connecting such reaction channels in parallel, valve means
for initiating and impeding the flow of fluids through such fluid
exchange channels, and means for moving the flow of fluids into and
out of such chambers.
Inventors: |
Southgate; Peter David
(Monmouth Junction, NJ), Andrevski; Zygmunt Marian
(Princeton, NJ), Roach; William Ronald (Rocky Hill, NJ),
Zanzucchi; Peter John (Lawrenceville, NJ) |
Assignee: |
Sarnoff Corporation (Princeton,
NJ)
|
Family
ID: |
21746101 |
Appl.
No.: |
08/786,956 |
Filed: |
January 23, 1997 |
Current U.S.
Class: |
422/417; 436/165;
436/180; 422/430 |
Current CPC
Class: |
B01J
19/0093 (20130101); B01L 3/50273 (20130101); F15C
5/00 (20130101); B01L 3/5025 (20130101); B01L
3/505 (20130101); B01L 3/502738 (20130101); B01J
19/0046 (20130101); B01J 2219/00828 (20130101); B01L
2400/0406 (20130101); B01L 2400/0655 (20130101); B01L
2300/0867 (20130101); B01L 2400/0481 (20130101); C40B
40/06 (20130101); B01J 2219/00783 (20130101); B01L
7/52 (20130101); B01J 2219/00585 (20130101); B01L
2200/10 (20130101); B01J 2219/00389 (20130101); B01J
2219/0059 (20130101); B01L 2300/1822 (20130101); B01J
2219/00353 (20130101); B01L 2300/123 (20130101); B01L
2300/185 (20130101); B01J 2219/00891 (20130101); C40B
60/14 (20130101); B01J 2219/00466 (20130101); B01J
2219/00722 (20130101); B01J 2219/00952 (20130101); B01J
2219/00833 (20130101); B01J 2219/005 (20130101); B01L
2300/0816 (20130101); G01N 2035/00247 (20130101); B01J
2219/00873 (20130101); B01J 2219/00822 (20130101); Y10T
436/2575 (20150115); B01J 2219/00831 (20130101) |
Current International
Class: |
B01L
3/00 (20060101); B01J 19/00 (20060101); F15C
5/00 (20060101); G01N 35/00 (20060101); G01N
021/03 () |
Field of
Search: |
;422/50,58,60,61,62,68.1,82.05,100,102,104 ;436/63,165,180 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Copy of International Search Report dated May 22, 1997, from
corresponding international application PCT/US97/00298..
|
Primary Examiner: Pyon; Harold Y.
Attorney, Agent or Firm: Burke; William J.
Government Interests
This invention was made with U.S. Government support under Contract
No. 70NANB5H1037. The U.S. Government has certain rights in this
invention.
Claims
What is claimed:
1. A device for conducting parallel reactions, comprising:
(a) a cassette formed of a body having an upper surface and a lower
surface and including an upper film or a lower film attached to the
upper or lower surface, respectively, wherein the upper or lower
film is formed of a flexible material;
(b) two or more reaction flow-ways in the cassette, wherein each
reaction flow-way comprises two or more fluid chambers which
comprise a first supply chamber and a first reaction chamber having
an upper wall and a lower wall, and wherein the fluid chambers are
serially connected by first fluid exchange channels;
(c) a valve for controlling the flow of fluid through a first fluid
exchange channel;
(d) a pump for moving fluids into or out of the fluid chambers;
and
(e) a first inlet port on the cassette connected to a first supply
chamber in each reaction flow-way by a second fluid exchange
channel.
2. The device of claim 1, wherein the upper and lower walls of each
first reaction chamber are formed of a portion of said upper film
which is attached to said upper surface, and a portion of said
lower film, which is attached to said lower surface and further
comprising at least one compression device for bringing the upper
and lower walls of the first reaction chambers together to minimize
the volume of the first reaction chambers.
3. The device of claim 1, further comprising:
(j) one or more waste chambers; and
(k) an exhaust port for evacuating one or more of the first
reaction chambers or the waste chambers.
4. The device of claim 1, further comprising:
(o) a permanent magnet that can be positioned adjacent to one or
more of the fluid chambers.
5. The device of claim 1, further comprising a valve which
comprises a hole extending through the body, further comprising a
fluid exchange channel proximate to but not intersecting the hole,
and a film having an embossed portion sealed to the body such that
the hole and the fluid exchange channel are covered, wherein
pressure applied to the film closes the valve.
6. The device of claim 1, further comprising:
(g) one or more second supply chambers, wherein two or more fourth
fluid exchange channels connect the second supply chamber to two or
more reaction flow-ways, which fourth fluid exchange channels
include two or more said valves so that fluid from the second
supply chamber can be directed to any one of the connected reaction
flow-ways to the exclusion of the other connected reaction
flow-ways; and
(h) one or more second inlet ports on the cassette each connected
to one of the second supply chambers by a separate third fluid
exchange channel.
7. The device of claim 6, further comprising:
(i) a fluid chamber interposed between the second supply chamber
and the connected reaction flow-way and connected to a fourth fluid
exchange channel.
8. The device of claim 1, wherein the body comprises recesses in
its upper or lower surface which, together with an associated upper
or lower film, form the first and second fluid exchange channels,
and a plurality of fluid chambers.
9. The device of claim 8, wherein a fluid chamber is formed in the
upper or lower surface and at least one first or second fluid
exchange channel is formed on an opposing upper or lower surface
located above or below that fluid chamber.
10. The device of claim 8, further comprising:
(f) at least one hole situated in the body so as to connect a first
or second fluid exchange channel formed at the upper or lower
surface of the body with a first or second fluid exchange channel
formed at the other surface.
11. The device of claim 1, further comprising:
(l) a heater for heating one or more of the fluid chambers;
(m) a cooler for cooling one or more of the fluid chambers; and
(n) a temperature monitor for monitoring the temperature of one or
more of the fluid chambers.
12. The device of claim 11, wherein the heater and the cooler
comprise a thermoelectric heat pump attached to a heat sink having
a heater element.
13. The device of claim 9, wherein the heater or the cooler can
change the temperature of a fluid chamber at a rate of at least
about 5.degree. C. per second.
14. The device of claim 1, further comprising
(p) a detection chamber or channel having a transparent wall.
15. The device of claim 14, further comprising:
(q) a light source adapted to direct light to the transparent wall
of a chamber or channel to illuminate the chamber or channel.
16. The device of claim 15, further comprising:
(r) a light detection device positioned to detect:
(1) the light reflected from an illuminated chamber or channel
having a transparent wall,
(2) the light transmitted through an illuminated chamber or channel
having a transparent wall, or
(3) the light emissions emanating from an excited molecule in a
chamber or channel having a transparent wall.
17. A device for conducting assays in parallel using fluids that
are confined to a disposable cassette comprising:
the disposable assay cassette, which comprises:
(i) at least two reaction flow-ways, including a first reaction
flow-way designed to receive and assay an experimental sample and a
second reaction flow-way designed to receive and assay a negative
control,
(ii) for each reaction flow-way, at least one supply chamber
connected thereto and containing fluids needed in the assay and at
least one reaction chamber,
(iii) a negative control supply chamber connected with the second
reaction flow-way and containing the negative control, and
(iv) a test sample supply chamber connected with the first reaction
flow-way designed to receive a test sample through an inlet
connected with the test sample supply chamber, valves for
controlling the flow of fluids in the cassette, and an instrument
comprising a temperature control unit for controlling in parallel
the temperature in a reaction chamber in each reaction flow-way,
valve actuators for opening and closing the valves in the cassette,
and one or more pumps for pushing fluid out of the various supply
chambers and reaction chambers of the cassette.
18. The device of claim 17, wherein the cassette further comprises
(v) a third reaction flow-way designed to receive and assay a test
sample and a positive control, (vi) connecting routes between the
test sample supply chamber and both the first and third reaction
flow-ways, wherein these connecting routes are controlled by valves
that allow selective flow between the test sample supply chamber
and either the first or third reaction flow-way, and (vii) a first
positive control supply chamber connecting with the third reaction
flow-way containing the positive control.
19. The device of claim 17, wherein the cassette further comprises
(1) a fourth reaction flow-way designed to receive and assay a
positive control, and (2) a second positive control supply chamber
connecting with the fourth reaction flow-way containing the
positive control.
20. The device of claim 17, wherein the cassette further comprises
(v) a third reaction flow-way designed to receive and assay a test
sample and a positive control, (vi) connecting routes between the
test sample supply chamber and both the first and third reaction
flow-ways, wherein these connecting routes are controlled by valves
that allow selective flow between the test sample supply chamber
and either the first or third reaction flow-way, (vii) a fourth
reaction flow-way designed to receive and assay a positive control,
(viii) a second positive control supply chamber connecting with the
fourth reaction flow-way containing the positive control, and (ix)
a first positive control supply chamber connecting with the third
reaction flow-way containing the positive control.
21. A method of conducting assays using a device for parallel
reactions, which method comprises:
(a) providing the device of claim 17 for conducting assays in
parallel, wherein reagents and control materials are pre-loaded
into the supply chambers;
(b) inserting a test sample into the test sample supply chamber;
and
(c) reacting (1) the test sample and (2) the negative control
sample in separate parallel flow-ways.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
This non-provisional U.S. national application, filed under 35
U.S.C. 111 (a) claims, under 35 U.S.C. 119(e)(1), the benefit of
the filing date of provisional U.S. application Ser. No.
60/010,513, filed under 35 U.S.C. 111 (b) on Jan. 24, 1996.
The present invention relates to a disposable parallel reaction
device for conducting reactions, which device can include a
component containing all necessary supply and reaction chambers and
connecting fluid exchange channels. The parallel reaction device is
particularly adapted for conducting polymerase chain reaction
("PCR") assays, and other scientific, forensic and diagnostic
assays. Synthetic reactions, including combinatorial chemistry, can
also be conducted in the device.
The PCR assay has provided a powerful method of assaying for the
presence of either defined segments of nucleic acids or nucleic
acid segments that are highly homologous to such defined segments.
The method can be used to assay body fluids for the presence of
nucleic acid specific for particular pathogens, such as the
mycobacterium causing Lyme disease, the HIV virus or other
pathogenic microbes. The microbe diagnostic assay functions by
adding, to a sample that may contain a target segment of nucleic
acid from the microbe's genome, at least one pair of "primers"
(i.e., relatively short nucleic acid segments or nucleic acid
analogs) that specifically bind to (i.e., "hybridize" with) the
target segment of nucleic acid. The first primer of a pair binds to
a first strand of the two-stranded target nucleic acid segment and,
when hybridized, can prime the enzymatic reproduction of a copy of
the second strand of the target nucleic acid segment in a direction
arbitrarily designated as the downstream direction. The second
primer of a pair binds to the second strand of the target nucleic
acid segment at a position downstream from the first primer
hybridization site and can prime the enzymatic reproduction of a
copy of the first strand of the target nucleic acid segment in the
upstream direction. (In the case where the sample is made up of
single-stranded target nucleic acids, the second primer will
hybridize with the theoretical second strand determined with the
Watson-Crick base-pairing rules.) To the sample are added the
monomer building blocks of nucleic acid and an enzyme that
specifically catalyzes nucleic acid reproduction from a single
strand of nucleic acid to which the short primer is bound. The
enzyme is preferably highly resistant to destruction by elevated
temperatures. The sample is heated to a DNA melting temperature to
separate the two strands of the sample nucleic acid and then cooled
to a replication temperature. The replication temperature allows
the primers to specifically bind to the separated strands and
allows the reproductive enzyme to operate. After this cycle, the
reaction mix contains two sets of the two stranded nucleic acid
segment for each target nucleic acid segment that was originally
present. Heating and replication temperature cycles are repeated
until sufficient amounts of the nucleic acid segment are created
through this exponential reproduction method. For instance, after
20 cycles the segment has been amplified as much as 2.sup.20 -fold,
or roughly 1,000,000-fold.
There are at least four critical problems associated with
automating the PCR reaction. First, the degree of amplification
achieved by the assay creates a large risk of contamination from
foreign DNA from handling. Thus far, this risk has been dealt with
in commercial, manual procedures by conducting the reactions in
"clean" facilities that are extremely expensive to construct and
maintain. For automation, this risk implies that all the reagents
needed and the reaction chamber for the amplification should be
contained in a disposable platform in which the sample can be
inserted in a controlled, one-time operation. This risk also
implies that sample preparation steps should be minimized and, to
the extent possible, conducted within a disposable platform.
Second, the high temperatures needed to "melt" the nucleic acid so
that the two strands separate imply that the reaction chamber must
be well-sealed against vapor loss, even while allowing the
insertion and removal of various reagent fluids. This goal is
particularly hard to achieve on a suitable, disposable
platform.
Third, the reactions should be conducted in relatively small
volumes, generally volumes of no more than about 100 .mu.l, to
conserve expensive reagents and minimize the amount of sample,
which could be a precious sample fluid or tissue that must be
conserved to allow for other types of testing or is available only
in a small amount.
Fourth, to provide assurance that a positive or negative result is
meaningful, it is preferable to perform multiple, parallel
reactions (for example, on positive and negative controls, in
addition to the sample) using the same reagents for each
reaction.
Recently, there have been a number of publications on the mechanics
of operating micro-scale reactors. These reactors are often
described as constructed on silicon-based materials using the
etching techniques developed by the semiconductor industry. This
literature, however, does not present an effective solution to the
problem of how to operate a disposable, high temperature
microreactor. The present invention provides an economical, high
temperature microreactor with effective valves suitable for use in
conducting multiple, parallel PCR assays, each using the same
reagents to assure meaningful results. The microreactor is also
suitably adapted for conducting automated assays even when high
temperature and considerably high vapor pressure are not a
particular concern.
SUMMARY OF THE INVENTION
In one embodiment, the present invention provides a device for
conducting parallel reactions, comprising:
(a) a cassette formed of a body having an upper surface, a lower
surface, and an edge, and including an upper film or a lower film
attached to the upper or lower surface, respectively, wherein the
upper or lower film is formed of a flexible material;
(b) two or more reaction flow-ways in the cassette, wherein each
reaction flow-way comprises two or more fluid chambers which
comprise a first supply chamber and a first reaction chamber having
an upper wall and a lower wall, and wherein the fluid chambers are
serially connected by first fluid exchange channels;
(c) a valve for controlling the flow of fluid through a first fluid
exchange channel;
(d) a pump for moving fluids into or out of the fluid chambers;
and
(e) a first inlet port on the cassette connected to a first supply
chamber in each reaction flow-way by a second fluid exchange
channel.
The first supply chamber is preferably a supply chamber having a
releasable seal blocking the outlet into the first fluid exchange
channel connecting the first supply chamber to its reaction
flow-ways; more preferably, the first supply chamber is an
internal-outlet supply chamber. The pump preferably comprises a
foot-pad pump with foot-pads designed to push on the first supply
chamber to open the sealed outlet and pump fluid into the connected
first fluid exchange channel. Preferably, the first supply chamber
is collapsible upon evacuation and fillable from a vacuum-collapsed
state to a defined volume.
In one aspect of the invention, the second fluid exchange channel
is releasably sealed so as to block the flow of fluids through the
second fluid exchange channel. Preferably, the second fluid
exchange channel is heat-sealed; more preferable, the second fluid
exchange channel is sealed at multiple locations to prevent fluid
communication between the first supply chambers.
In another aspect, the valve used in the context of the present
invention is a plunger-type valve that is controlled by a pressure
control means for:
(i) applying a positive pressure to the plunger-type valve such
that the plunger-type valve presses against the upper or lower film
so as to impede the flow of fluid in a first fluid exchange
channel, and
(ii) releasing the positive pressure to the plunger-type valve such
that the plunger-type valve releases from the flexible film so as
to permit the flow of fluid in the first fluid exchange channel.
Preferably, the plunger of the plunger-type valve is affixed to an
instrument from which the cassette is detachable.
The cassette can be formed of a body that comprises recesses in its
upper or lower surface which, together with an associated upper or
lower film, form the first and second fluid exchange channels, and
a plurality of fluid chambers. In the invention, it is preferred
that a fluid chamber is formed in the upper or lower surface and at
least one first or second fluid exchange channel is formed on an
upper or lower surface located above or below that fluid chamber.
The cassette of the present invention further comprises:
(f) at least one hole situated in the body so as to connect a first
or second fluid exchange channel formed at the upper or lower
surface of the body with a first or second fluid exchange channel
formed at the other surface.
Preferably, the portion of upper or lower film covering a said
fluid chamber made up of a recess in the body is embossed to mirror
the shape of the bottom of the fluid chamber such that when the
chambers is evacuated the film portion will invert to match the
shape of the bottom of the chamber. Preferably, one of the pumps is
a foot-pad pump having a foot pad that fits against the surface of
the inverted embossed film portion of said fluid chamber.
Preferably, the cassette further comprises:
(g) one or more second supply chambers, wherein two or more fourth
fluid exchange channels connect the second supply chamber to two or
more reaction flow-ways, which fourth fluid exchange channels
include two or more said valves so that fluid from the second
supply chamber can be directed to any one of the connected reaction
flow-ways to the exclusion of the other connected reaction
flow-ways; and
(h) one or more second inlet ports on the cassette each connected
to one of the second supply chambers by a separate third fluid
exchange channel.
Preferably, the device further comprises
(i) a metering chamber interposed between the second supply chamber
and the connected reaction flow-way. The combination of elements
(f), (g), (h), and optionally (i) forms a sample insertion device.
Preferably, the cassette has more than one such sample insertion
device and sufficient reaction flow-ways such that different
experimental samples can be reacted in parallel.
Preferably, the upper and lower walls of each first reaction
chamber are formed of an embossed portion of a said upper film and
an embossed portion of a said lower film, wherein the embossing
allows upper and lower walls of the first reaction chambers to be
brought together to minimize the volume of the first reaction
chambers. Preferably, at least one pump comprises a foot-pad pump
with upper and lower foot-pads designed to push together the upper
and lower walls of a first reaction chamber. Alternatively or in
addition, at least one of the pumps comprises gas pressure conduits
for applying a positive pressure to the flexible upper or lower
walls of a first reaction chamber so as to cause the flexible upper
or lower wall to press inward thereby decreasing the volume within
the first reaction chamber and impelling the flow of fluids
therefrom.
In a preferred embodiment, the cassette further comprises
(j) one or more waste chambers; and
(k) an exhaust port for evacuating one or more of the first
reaction chambers or the waste chambers.
Each embodiment of the invention can further comprise
(l) a heater for heating one or more of the fluid chambers;
(m) a cooler for cooling one or more of the fluid chambers; and
(n) a temperature monitor for monitoring the temperature of one or
more of the fluid chambers. Preferably, a foot-pad for pumping
fluid out of the fluid chamber is associated with a heater and
cooler for the fluid chamber; more preferably, the heaters and the
coolers comprise a thermoelectric heat pump attached to a heat sink
having a heater element. Preferably, the heaters and the coolers
can change the temperature of a fluid chamber at a rate of at least
about 5.degree. C. per second.
Additionally, each embodiment of the invention can further
comprise
(o) a permanent magnet that can be positioned adjacent to one or
more of the fluid chambers, or removed therefrom, wherein further
the invention comprises means for moving the magnet adjacent to or
away from the cassette.
Each embodiment of the invention can also comprise
(p) a detection chamber or channel having a transparent wall.
Further, each such embodiment can include
(q) a light source capable of directing light to the transparent
wall of a chamber or channel; and also
(r) a light detection device capable of detecting:
(1) the light reflected from an illuminated chamber or channel
having a transparent wall;
(2) the light transmitted through an illuminated chamber or channel
having a transparent wall; or
(3) the light emissions emanating from an excited molecule in a
chamber or channel having a transparent wall.
In a preferred embodiment, the invention includes at least one
valve that comprises:
(1) a shut-off means comprising a valve ball or pinch foot, and
(2) switching means for positioning the valve ball or pinch foot so
that the valve ball or pinch foot: (i) presses against the flexible
film to cut off flow through a first or second fluid exchange
channel, or (ii) releases away from the flexible film to allow flow
through the first or second fluid exchange channel. The switching
means preferably comprises spring loaded levers. Preferably, at
least one valve comprises:
(1) a spacer,
(2) a spacer spring means for normally pressing the spacer against
the flexible film so as to cut off the flow of fluids through a
first fluid exchange channel, and
(3) an electromagnet effective when activated to sufficiently
release the pressure against the flexible film to allow the flow of
fluids through the first or second fluid exchange channel.
In a preferred embodiment, the invention provides a device for
conducting assays in parallel using fluids that are confined to a
disposable cassette comprising the disposable assay cassette, which
comprises (i) at least two reaction flow-ways, including a first
reaction flow-way designed to receive and assay an experimental
sample and a second reaction flow-way designed to receive and assay
a negative control, (ii) for each reaction flow-way, at least one
supply chamber connected thereto and containing fluids needed in
the assay and at least one reaction chamber, (iii) a negative
control supply chamber connected with the second reaction flow-way
containing the negative control, and (iv) a test sample supply
chamber connected with the first reaction flow-way designed to
receive a test sample through an inlet connected with the test
sample supply chamber, valves for controlling the flow of fluids in
the cassette, and an instrument comprising a temperature control
unit for controlling in parallel the temperature in a reaction
chamber in each reaction flow-way, valve actuators for opening and
closing the valves in the cassette, and one or more pumps for
pushing fluid out of the various supply chambers and reaction
chambers of the cassette. Preferably, the cassette further
comprises (v) a third reaction flow-way designed to receive and
assay a test sample and a positive control, (vi) connecting routes
between the test sample supply chamber and both the first and third
reaction flow-ways, wherein these connecting routes are controlled
by valves that allow selective flow between the test sample supply
chamber and either the first or third reaction flow-way, and (vii)
a first positive control supply chamber connecting with the third
reaction flow-way containing the positive control. Also preferably,
the cassette further comprises (viii) a fourth reaction flow-way
designed to receive and assay a positive control, and (ix) a second
positive control supply chamber connecting with the fourth reaction
flow-way containing the positive control. As well, the cassette
preferably comprises (v) a third reaction flow-way designed to
receive and assay a test sample and a positive control, (vi)
connecting routes between the test sample supply chamber and both
the first and third reaction flow-ways, wherein these connecting
routes are controlled by valves that allow selective flow between
the test sample supply chamber and either the first or third
reaction flow-way, and (viii) a first positive control supply
chamber connecting with the third reaction flow-way containing the
positive control. Preferably, the pumps comprise one or more
foot-pad pumps. Further, the temperature control unit preferably
comprises a thermoelectric heat pump; and the thermoelectric heat
pump preferably is attached to a heat sink having a heater element.
Preferably, the valves of this embodiment comprise plunger-type
valves.
The invention further provides a method of conducting assays,
including chemical diagnostic assays, antibody-based assays and
nucleic acid amplification-based assays, using one of the
aforementioned devices, which method comprises
(a) providing the device for conducting assays in parallel, wherein
reagents and control materials are pre-loaded into the supply
chambers;
(b) inserting a test sample into the test sample supply chamber;
and
(c) reacting in parallel in separate reaction flow-ways (1) the
test sample and (2) the negative control.
Preferably, the reagents or control materials include binding
domains derived from antibodies; alternatively, the reagents or
control materials include fluids containing primers, nucleotide
triphosphates, and ions and buffers suitable for supporting a
nucleic acid amplification reaction. Preferably, the reacting
comprises reacting in separate reaction flow-ways (1) test sample
and (2) negative control with a suspension of nucleic acid-binding
beads, wherein the suspension of nucleic acid-binding beads is
provided by a separate supply chamber for each reaction flow-way;
and replacing the fluid suspending the nucleic acid-binding beads
with a fluid containing primers, nucleotide triphosphates, and ions
and buffers suitable for supporting a nucleic acid amplification
reaction. Preferably, the nucleic acid binding beads are
paramagnetic beads and the replacing step comprises (1)
magnetically locking the nucleic acid-binding beads in place while
pushing the suspending fluid into a waste chamber, (2) resuspending
the nucleic acid-binding beads in a wash fluid, wherein wash fluid
is introduced from a separate supply chamber for each reaction
flow-way, (3) magnetically locking the nucleic acid-binding beads
in place while pushing the suspending fluid into a waste chamber,
and (4) resuspending the nucleic acid-binding beads in the fluid
containing primers, nucleotide triphosphates, and ions and buffers
suitable for supporting a nucleic acid amplification reaction.
In a preferred embodiment, the invention relates to a method of
conducting nucleic acid amplification reactions using the
aforementioned device, which method comprises
(a) providing the device for conducting assays in parallel, wherein
reagents and control materials are pre-loaded into the supply
chambers, wherein the reagents or control materials include
primers, nucleotide triphosphates, and ions and buffers suitable
for supporting a nucleic acid amplification reaction;
(b) inserting a test sample into the test sample supply chamber;
and
(c) reacting in parallel in separate reaction flow-ways (1) the
test sample, (2) a negative control and (3) a mixture of the test
sample and a positive control. The present invention further
preferably relates to a method of conducting nucleic acid
amplification reactions using the aforementioned device, which
method comprises:
(a) providing the device for conducting assays in parallel, wherein
reagents and control materials are pre-loaded into the supply
chambers, wherein the reagents or control materials include
primers, nucleotide triphosphates, and ions and buffers suitable
for supporting a nucleic acid amplification reaction;
(b) inserting a test sample into the test sample supply chamber;
and
(c) reacting in parallel in separate reaction flow-ways (1) the
test sample, (2) a negative control, (3) a mixture of the test
sample and a positive control and (4) a positive control.
The invention still further provides a device comprising a cassette
suitable for conducting reactions therein, which cassette comprises
a body having one or more recesses and one or more embossed films
covering the recesses. Preferably, the cassette includes a hole
extends through the body, further comprising a fluid exchange
channel in communication with a valve, which valve is in
communication with the hole, and a film having an embossed portion
sealed to the body such that the hole and the fluid exchange
channel are covered. The device further comprises preferably a
pneumatically driven plunger for pressing the embossed film portion
at or about the valve, and pressure control means for (i) applying
a positive pressure to the pneumatically driven plunger such that
the plunger presses against the flexible film so as to close the
valve, and (ii) releasing the positive pressure to the
pneumatically driven plunger such that the plunger releases from
the flexible film so as to open the valve.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A, 1B and 1C show a top, side and bottom view of a cassette
of the invention.
FIG. 2A shows a side view of a Bursapak supply chamber.
FIG. 2B illustrates a method for sealing closed a fluid exchange
channel.
FIG. 2C illustrates how pressure can be used to open a Bursapak
supply chamber.
FIGS. 2D and 2E illustrate a foot-pad that can be used to
pressurize the fluid in the Bursapak supply chamber.
FIG. 3 schematically diagrams a parallel reaction device of the
invention.
FIG. 4A illustrates a cassette of the invention.
FIGS. 4B-4E show the cassette of FIG. 4A with various subsets of
the features thereof illustrated and numbered.
FIG. 5A and 5B show a plunger-type valve mechanism for regulating
fluid flow through a cassette.
FIG. 6 shows in perspective view the part of a plunger-type valve
located in the body of a cassette.
FIG. 7 shows the parts of a plunger-type valve located outside the
cassette (i.e., in the instrument).
FIGS. 8A, 8B and 8C show various configurations of valve mechanisms
for regulating fluid flow through a cassette.
FIGS. 9A and 9B show a magnetic spring valve mechanism for
regulating fluid flow through a cassette.
FIG. 10 shows a support device for rapidly heating and cooling a
reaction chamber and providing a foot-pad for a foot-pad pump.
FIGS. 11A and 11B show the operation of a foot-pad pump on a
reaction chamber.
FIG. 12 shows a schematic of accessory support devices for rapidly
heating or cooling a reaction chamber.
FIG. 13 shows another mechanism for rapidly heating or cooling a
reaction chamber.
FIG. 14 shows yet another mechanism for rapidly heating or cooling
a reaction chamber.
FIGS. 15A and 15B show two side views of a detection channel.
FIG. 16 shows an example of a magnet useful for locking
paramagnetic beads at a certain location in a cassette.
FIGS. 17A and B show a device for mounting a septum to the
cassette.
DEFINITIONS
The following terms used in this disclosure shall have the meanings
set forth below:
annealing temperature
PCR protocols and other nucleic acid amplification protocols often
use an "annealing temperature" less than the replication
temperature to accelerate the rate at which the primers bind to
(i.e., hybridize with) the sample nucleic acid; this annealing
temperature is typically between about 45.degree. C. and about
72.degree. C., often about 55.degree. C. Generally, the annealing
temperature will be about 5.degree. C. below the lowest T.sub.m for
the interaction between (a) one of the primers used in reaction and
(b) the target nucleic acid segment.
Bursapak chamber
a chamber formed in a solid support and having a film formed of a
flexible material that is sealed to the support at the edges of the
chamber and has an outlet channel that is blocked by a portion of
the film which is sealed over the outlet channel, wherein the seal
over the outlet is broken or removed by pressurizing the fluid
contents of the chamber at a pressure that does not affect the seal
at the edges of the chamber; preferably, the film is on one face of
the cassette body and the outlet is oriented toward the other.
cassette
a disposable device for conducting reactions therein having a
cassette body, one or more upper membranes or one or more lower
membranes which individually or in combination define one or more
supply chambers, one or more reaction chambers and fluid exchange
channels connecting the supply chambers to reaction chambers.
cassette body
a solid portion having sufficient depth and sturdiness to allow
cavities formed therein to provide the depth for fluid chambers and
fluid exchange channels.
collapsible upon evacuation
some of the chambers described below will preferably be filled by
first applying a vacuum to evacuate the chamber contents and then
filling the evacuated chamber with fluid--preferably, these
chambers are "collapsible" in that they have at least one flexible
film that collapses to minimize chamber volume.
connection (between fluid chambers, inlets or detection
channels)
two fluid chambers, inlets or detection channels are "connected" or
have a "route of connection" therebetween if there is one or more
fluid exchange channels joining the two such that fluid can move
from one to the other.
concentric Bursapak supply chamber
an internal outlet Bursapak supply chamber wherein the outlet
channel is located substantially in the center of the supply
chamber; "substantially in the center" means that the distance
between the center of the supply chamber and the geometric center
of the supply chamber is no more than about 20% of the length of
the supply chamber cross-section defined by the line joining the
center of the outlet and the geometric center of the supply
center.
DNA strand separation temperature
the temperature used in a nucleic acid amplification protocol to
separate the complementary strands of nucleic acid that may be
present in a sample; this temperature is typically between about
92.degree. C. and about 97.degree. C., preferably about 94.degree.
C.
elevated pressure
a pressure more than ambient atmospheric pressure.
fillable from a vacuum-collapsed state to a defined volume these
are chambers that unfold from the collapsed state to a first
volume;
preferably, the inserted fluid volume is within about 10% of the
first volume, more preferably within about 3% of the first volume.
The first volume is the maximum volume of fluid that can be
inserted into the chamber without affecting the integrity of the
chamber.
fluid chamber
the term "fluid chamber" encompasses reaction, supply, waste
metering and sample storage chambers, and other fluid containing
chambers. In those embodiments where contents of the chambers can
be pumped out using a foot-pad having a shape that conforms to a
covering film that is inverted to match the shape of the bottom of
the chamber, the chamber can be closed by maintaining the foot-pad
pressed against the inverted covering film.
fluid-tight
a space or chamber is fluid-tight if it retains an aqueous fluid in
the space at a temperature of 99.degree. C. for one hour; a seal
between two materials is fluid-tight if the seal is substantially
no more permeable to water than the most water-permeable such
material.
foot-pad
a plunger having a shape designed to conform to the inverted shape
of the covering film of a supply chamber; when the plunger presses
against the flexible film it pressurizes the fluid in the supply
chamber and, if an exit is available, pushes the fluid out of the
supply chamber.
foot-pad pump
a mechanical, electromechanical or pneumatic device that uses a one
or more, preferably two or more, foot-pads to press on one or more
fluid chambers such as supply chambers or reaction chambers to
pressurize the contents and push the contents out through an
unobstructed connected fluid exchange channel.
integral
parts or elements of a valve are integral to a body layer or to a
cassette if They cannot be facilely and reversibly detached from
that body layer or cassette.
internal outlet Bursapak supply chamber
a Bursapak supply chamber wherein the outlet channel is located
away from the edges of the supply chamber such that
fluid-containing space is interposed between the sealed outlet
channel and the edges chamber.
negative control
a material designed to be comparable to a sample to be assayed but
lacking the substance to be assayed for, such that a positive
result upon assaying a negative control would indicate a problem
with the assay protocol or assay reagents.
nucleic acid melting temperature or T.sub.m
the transition temperature for two-stranded duplex of nucleic acid
at which the equilibria shifts from favoring the base-paired duplex
to favoring the separation of the two strands.
positive control
a material designed to generate, in the absence of a problem with
the assay chemistry such as the presence of an interfering
substance, a positive assay result.
reaction flow-away
a series of two or more serially connected fluid chambers through
which fluids can move.
reduced pressure
a pressure less than ambient atmospheric pressure.
replication temperature
the temperature used in a nucleic acid amplification protocol to
allow the nucleic acid reproductive enzyme to reproduce the
complementary strand of a nucleic acid to which a primer is bound
(i.e., hybridized); this temperature is typically between about
69.degree. C. and about 78.degree. C., preferably about 72.degree.
C., when using a heat stable polymerase such as Taq polymerase.
serially connected
two or more fluid chambers are serially connected if there are
fluid exchange channels by which fluid from a first of the serially
connected chambers can pass to a second of the serially connected
chambers, and from there to a third of the serially connected
chambers, and so on until the fluid passes to the last of the
serially connected chambers.
substantially uniform temperature in the reaction chamber
where the temperature in a reaction chamber varies by no more than
about .+-.0.3.degree. C.
target nucleic acid segment
a segment of nucleic acid that is sought to be identified or
measured in a sample, such as a sequence intended, if present, to
be amplified in a nucleic acid amplification reaction such as a PCR
reaction, strand displacement assay or ligase chain reaction; the
target segment is typically part of a much larger nucleic acid
molecule found in the sample.
thermoelectric heat pump
a device for heating and cooling fluid chambers that is made up of
one or more thermoelectric blocks.
DETAILED DESCRIPTION
The cassette of the present invention includes at least one
reaction chamber and at least one supply chamber in combination
with interconnecting fluid exchange channels. The cassette
comprises a body into which the aforementioned chambers and
channels are formed such that when covered by a film and sealed, as
described below, the formed body with film can hold fluids. The
shape of the body can be any shape, although preferably it is a
flat square, rectangular or circular structure of length and width
or diameter substantially greater than its depth, such as, for
example, 3 cm.times.3 cm.times.3 mm, inter alia, and the length and
width or diameter can be further described with respect to a top or
bottom surface, and the depth can be further described with respect
to an edge. The chambers and channels prior to covering by the film
can be open to any surface of the body, preferably is open to the
top or bottom, more preferably is open to the top and bottom,
although each chamber or channel preferably is open to one side
only.
The present invention is described herein with respect to
particular embodiments; however, these embodiments should not be
construed as in any way limiting the scope of the present
invention, which includes all modifications encompassed within the
spirit and scope of the invention as described hereinbelow.
Exemplary Cassette
FIGS. 1A, 1B and 1C show a top view, cross-sectional view and
bottom view of a portion of one embodiment of a cassette 100
according to the invention. The cassette 100 has a body 105 in
which are defined inlet 130, first fluid exchange channel 141,
supply chamber 150, second fluid exchange channel 142, reaction
chamber 160, third fluid exchange channel 143 and waste chamber
170. The body 105 has first upper film 110A, second upper film
110B, third upper film 110C and lower film 120. In FIG. 1A, first
seal portion 111A (shaded area), second seal portion 111B (shaded
area) and third seal portion 111C (shaded area) show where first
upper film 110A, second upper film 110B and third upper film 110C,
respectively, are sealed against body 105. In FIG. 1C, shading 121
shows where lower film 120 is sealed against body 105. Inlet 130
has a septum 131. First, second and third upper films 110A-C are
collectively referred to as "upper films 110." Septum 131 can be,
for instance a bilayer material formed of an outer layer of silicon
or neoprene rubber and an inner layer of chemically inert material
such as tetrafluoroethylene homopolymer (e.g., Teflon, E. I. duPont
de Nemours and Co., Wilmington, Del.) facing the body 105. Second
upper film 110B and lower film 120 are embossed or shaped at
positions 161 and 162 to help form reaction chamber 160, as will be
described in greater detail below with reference to FIGS. 11A and
11B. First upper film 110A is embossed or shaped at the location of
supply chamber 150 so that first upper film protrudes above the
upper surface of body 105, creating a greater volume for supply
chamber 150 and facilitating the mechanism by which supply chamber
150 is emptied, as described further in the text below with
reference to FIGS. 2A and 2B. Third upper film 110A is embossed or
shaped at the location of waste chamber 170, which embossing
facilitates the mechanism by which the waste chamber is filled. A
valve 180 is formed in third fluid exchange channel 143. The outlet
151 of supply chamber 150 is sealed by a portion of first upper
film 110A. Supply chamber 150 is a Bursapak supply chamber, which
type of supply chamber is a particularly useful type of supply
chamber for use in the cassette of the invention. Because many of
the cassettes described below make use of this preferred type of
supply chamber, Bursapak supply chambers are described in more
detail in the following section.
Bursapak Supply Chambers
FIG. 2A shows a side view of a Bursapak supply chamber 150 having
supply cavity 155, which can contain a fluid. The Bursapak supply
chamber 150 has an inlet first fluid exchange channel 141, which is
preferably sealed, for instance by heat sealing at sealing location
141A, after the Bursapak supply chamber 150 has been filled with
fluid, and an outlet second fluid exchange channel 142 which is
initially sealed with a fourth seal portion 111D of first upper
film 110A. FIG. 2B shows the use of die 1300 to heat seal first
fluid exchange channel 141, at sealing location 141A. FIG. 2C
illustrated how pressure--indicated by the arrows--applied to the
fluid in Bursapak supply chamber 700 is effective to pull the seal
portion 111 away from the outlet second fluid exchange channel 142.
FIG. 2D illustrates a foot-pad 210 that can be used to apply
pressure to the fluid in Bursapak supply chamber 150 and pump it
through outlet second fluid exchange channel 142. Foot-pads can be
fabricated of any suitably sturdy material including, without
limitation, aluminum, plastics, rubber, alumina, copper, sintered
beryllia, and the like. Upper films 110 and lower films 120 are
preferably constructed of a flexible film such as a polyethylene,
polyvinylidene fluoride or polyethylene/polyethylene terephthalate
bi-layer film. Suitable films are available from Kapak Corporation,
Minneapolis, Minn. or E. I. duPont de Nemours and Co., Wilmington,
Del. Polyethylene/polyethylene-terephthalate bi-layer film such as
3M No. 5 or 3M No. 48 (3M Corp., Minn.) or Dupont M30 (DuPont de
Nemours, Wilmington, Del.) are particularly preferred. The
polyethylene layer is preferably positioned against body 105. FIG.
2E shows the foot-pad used to pump fluid out of Bursapak supply
chamber 150.
The first upper film 110A is embossed or shaped, for instance by
applying suitably shaped, heated dies to the first upper film 110A,
so that it can protrude away from the body 105 when the supply
chamber 150 is filled and will rest, without substantial
stretching, against the bottom of supply chamber 150 when the
supply chamber 150 is evacuated.
It is believed that the application of force through a foot-pad
results in the application of greater force per unit length at the
edges of the fourth seal portion 111D than at the edges of first
seal portion 111A, resulting in selective peeling of fourth seal
portion 111D. Whatever the mechanism, however, in operation
Bursapak chambers operate as illustrated in FIGS. 2A-2C. To assure
proper functioning, in some embodiments it may be necessary to seal
fourth portion 111D relatively more weakly, for instance using a
weaker adhesive or a lower temperature sealing die.
Materials, Dimensions for Cassette Components
Body 105 is preferably formed of a molded plastic, such as high
density polyethylene, but other materials that are suitably
resistant to the chemistries sought to be conducted on the parallel
reaction device, such as glass and silicon-based materials, can be
used. Where body 105 is plastic, it is preferably formed by a
molding process that is used to form cavities and channels that
will be sealed with upper and lower films 110 and 120 to form fluid
chambers and fluid exchange channels. Such cavities and channels
are formed in glass and silicon materials by chemical etching or
laser ablation. Upper and lower films 110 and 120 typically have a
thickness of from about 0.3 mils to about 5 mils, preferably from
about 1 mil to about 3 mils. For fluid chambers having a diameter
of about 1 cm or more, the film thickness is more preferably about
2 mils. Reaction chamber 130A typically has a thickness, between
upper and lower films 110 and 120, of from about 0.1 mm to about 3
mm preferably of from about 0.5 to about 1.0 mm and an area,
defined by the inner surface of upper or lower films 110 or 120, of
preferably from about 0.05 cm.sup.2 to about 2 cm.sup.2, more
preferably from about 0.1 cm.sup.2 to about 1 cm.sup.2, yet more
preferably about 0.5 cm.sup.2. The dimensions of reaction chamber
are preferably sized small enough to permit rapid thermal cycling
(on the order of about 10 seconds).
Fluid exchange channels typically have a diameter between about 200
and about 500 .mu.m. Supply chambers 150 typically have a volume
between about 5 and about 500 .mu.l, preferably from about 10 to
about 200 .mu.l, more preferably from about 30 to about 160 .mu.l.
Metering chambers preferably have a volume between about 5 and
about 50 .mu.l. Preferably, the total volume of each reaction
chamber 160 is between about 5 .mu.l and about 200 .mu.l, more
preferably, between about 10 .mu.l and about 100 .mu.l. Preferably,
each reaction chamber has a thickness (i.e., distance between upper
film 110 and lower film 120) of about 1 mm or less.
Upper and lower films 110 and 120 preferably are resistant to
temperatures as high as about 120.degree. C. and are between about
1 and about 6 mils in thickness, more preferably, between about 2
and about 4. The thinness of the membranes facilitates rapid heat
exchange between the reaction chamber and an adjacent heating or
cooling device.
Schematic of Parallel Reaction Device
FIG. 3 illustrates schematically a parallel reaction device 301
according to the invention having five reaction flow-ways, each
such flow-way, respectively, used for analyzing (A) a sample 300,
(B) a positive control 310, (C) a negative control 320, (D) a
positive control 330 combined with sample 300, and (E) a sample
300. Each of these samples and controls is introduced into one of
first through fifth lysing chambers 340A-E (collectively, lysing
chambers 340). Lysing reagents and washing buffer can be
distributed from first supply chamber 350 and second supply chamber
360, respectively, to all five lysing chambers 340. Waste can be
emptied from lysing chambers 340 into a single waste chamber 370.
The remaining contents of each of lysing chambers 340 can then be
transferred to one of first through fifth reaction chambers 380A-E,
respectively (collectively, reaction chambers 380). Amplification
reagents are added to each of reaction chambers 380 from a third
supply chamber 390. Waste can be emptied from reaction chambers 380
into waste chamber 370. The remaining contents of each of reaction
chambers 380A-E can then be transferred into one of first through
fifth storage chambers 399A-E, respectively. Each valve which
regulates the flow of fluids into and out of the various chambers
is separately diagrammed in FIG. 3 as an encircled letter "v."
It should be noted that some of the arrows in FIG. 3, which arrows
represent fluid channels, apparently pass through a fluid chamber.
These channels actually pass above or below the fluid chamber, as
is described further in the text below. As is described further
below, lysing chambers 340 and reaction chambers 380 preferably
have flexible upper film 110 and lower film 120 that can be
manipulated with a foot-pad pump or a gas pressure flow control
means. If both upper and lower walls of a fluid chamber are formed
with films 110 and 120, then channels passing through the region of
the device occupied by the lysis chambers 340 or reaction chambers
380 must pass adjacent to such chambers rather than above or below
the chambers.
Detailed Cassette--Structure
Another cassette 200 is illustrated in FIG. 4A. The illustrated
cassette 200 has planar dimensions of 31/4 inches by 55/16 inches,
although other sizes are contemplated, including for instance in
circumstances where the sizes of the fluid chambers and other
components of the cassette differ from those illustrated. Because
of the complexity of FIG. 4A, FIGS. 4B-4E show the body 205 of the
cassette together with illustrations of various subsets of the
components of body 205. In these illustrations the solid lines
connecting inlets, valves or fluid chambers represent fluid
exchange channels. Those fluid exchange channels represented by
dark lines are formed in the upper surface of body 205, while those
represented by lighter lines are formed in the lower surface of
body 205. At the top of FIG. 4B are illustrated the symbols used to
represent an inlet 230 or a supply chambers 250 of various sizes
(sizes recited for illustrative purposes only).
In FIG. 4B are illustrated: alpha first supply chamber 251A, beta
first supply chamber 251B, and so on through delta first supply
chamber 251D, which are connected to first inlet 231 by alpha
second fluid exchange channel 242A; alpha second supply chamber
252A, beta second supply chamber 252B, and so on through theta
second supply chamber 253H, which are connected to second inlet 232
by beta second fluid exchange channel 242B; alpha third supply
chamber 253A, beta third supply chamber 253B, and so on through
theta third supply chamber 253H, of which alpha, gamma, epsilon and
eta third supply chambers 253A, C, E and G are connected to third
inlet 233 by gamma second fluid exchange channel 243B and beta,
delta, zeta and theta third supply chambers 253B, 253D, 253F and
253H are connected to beta fourth inlet 234B, delta fourth inlet
234D, zeta fourth inlet 234F and theta fourth inlet 234H,
respectively; and alpha fourth supply chamber 254A, beta fourth
supply chamber 254B, and so on through theta fourth supply chamber
254H, of which alpha, gamma, epsilon and eta fourth supply chambers
254A, 254C, 254E and 254G are connected to fifth inlet 235 by delta
second fluid exchange channel 242D and beta, delta, zeta and theta
fourth supply chambers 254B, 254D, 254F and 254H are connected to
sixth inlet 236 by epsilon second fluid exchange channel 242E. The
connecting fluid exchange channels 215 between second fluid
exchange channels 242 and supply chambers 250 are represented by
solid circles.
Alpha first reaction chamber 261A can receive fluid from any of
seven supply chambers 250, which supply chambers 250 are alpha
first supply chamber 251A, alpha second supply chamber 252A, beta
second supply chamber 252B, alpha third supply chamber 253A, beta
third supply chamber 253B, alpha fourth supply chamber 254A and
beta fourth supply chamber 254B. Beta first reaction chamber 261B,
gamma first reaction chamber 261C and delta first reaction chamber
261D each can receive fluid, in a manner parallel to the
arrangement for alpha first reaction chamber 261A, from seven
supply chambers 250 as illustrated. Alpha first reaction chamber
261A, beta first reaction chamber 261B, gamma first reaction
chamber 261C and delta first reaction chamber 261D connect to alpha
second reaction chamber 262A, beta second reaction chamber 262B,
gamma second reaction chamber 262C and delta second reaction
chamber 262D, respectively, via alpha first fluid exchange channel
241A, beta first fluid exchange channel 241B, gamma first fluid
exchange channel 241C and delta first fluid exchange channel 241D,
respectively. Alpha second reaction chamber 262A, beta second
reaction chamber 262B, gamma second reaction chamber 262C and delta
second reaction chamber 262D connect to first waste chamber 271
under the control of alpha first valve 281A, beta first valve 281B,
gamma first valve 281C and delta first valve 281D,
respectively.
In FIG. 4C are illustrated alpha seventh inlet 237A and beta
seventh inlet 237B, which are connected to alpha fifth supply
chamber 255A and beta fifth supply chamber 255B, respectively.
Alpha fifth supply chamber 255A and beta fifth supply chamber 255B
are connected to alpha second reaction chamber 262A and beta second
reaction chamber 262B.
Exhaust port 275 allows the first reaction chambers 261, second
reaction chambers 262, first waste chamber 271, second waste
chamber 272, metering chamber 290 and detection channels 295 to be
evacuated prior to use. This evacuation is possible because all of
the first reaction chambers 261, second reaction chambers 262,
first waste chamber 271, second waste chamber 272, metering chamber
290 and detection channels 295 communicate when the appropriate
valves 280 are open. Alpha sealing position 276A and beta sealing
position 276B can be heat sealed when the evacuation process is
complete to lock the first reaction chambers 261, second reaction
chambers 262, first waste chamber 271, second waste chamber 272,
metering chamber 290 and detection channels 295 in the evacuated
state prior to operating the cassette.
In FIG. 4D, sixth supply chamber 256 is filled using alpha eighth
inlet 238A and is connected to metering chamber 290 under the
control of alpha second valve 282A. Seventh supply chamber 257 is
filled using beta eighth inlet 238B and is connected to metering
chamber 290 under the control of beta second valve 282B. From
metering chamber 290 fluid can be directed to either gamma second
reaction chamber 262C or delta second reaction chamber 262D under
the control of gamma second valve 282C and delta second valve 282D,
respectively.
In FIG. 4E, fluid from alpha second reaction chamber 262A can be
directed to alpha detection channel 295A under the control of alpha
third valve 283A. Corresponding connections from beta second
reaction chamber 262B through delta second reaction chamber 262D to
beta detection channel 295B through delta detection channel 295D,
respectively, are controlled by beta third valve 283B through delta
third valve 283D, respectively. Alpha eighth supply chamber 258A,
beta eighth supply chamber 258B, and so on, are respectively
connected to alpha detection channel 295A, beta detection channel
295B, and so on. Alpha eighth supply chamber 258A, beta eighth
supply chamber 258B, and so on are filled through ninth inlet
239.
Detailed Cassette--Operational Features
This discussion of operational features of the cassette structure
200 shown in FIGS. 4A-4E assumes that the supply chambers of that
structure are Bursapak supply chambers. The first supply chambers
251 can be used to store fluid having suspended paramagnetic beads
used in preparing nucleic acid from biological samples, which
paramagnetic beads are described in greater detail below. A
foot-pad pump operates propel in parallel the fluid and suspended
beads from the first supply chambers 251 to the connected first
reactions chambers 261. To assure that the beads are suspended the
foot-pad pump operating on the first supply chambers 251 and
foot-pad pump operating on the first reaction chambers 261 can
alternately be operated to move the fluid back and forth between
the first supply chambers 251 and first reaction chambers 261,
thereby agitating the fluid and resuspending the beads.
The second supply chambers 252 can contain a buffer solution, such
as a buffer solution used to wash the paramagnetic beads. The
associated foot-pad pump has four foot-pads designed to interact
with either (1) alpha second supply chamber 252A, gamma second
supply chamber 252C, epsilon second supply chamber 252E and eta
second supply chamber 252G or (2) beta second supply chamber 252B,
delta second supply chamber 252D, zeta second supply chamber 252F
and theta second supply chamber 252H. Alternatively, the pump has
two sets of four pads designed to interact with second supply
chambers 252.
The third supply chambers 253 alternate in size between supply
chambers 253 having volumes of 100 .mu.l and supply chambers 253
having volumes of 30 .mu.l. The 100 .mu.l supply chambers 253 can
be used to store cell lysis solutions while the 30 .mu.l supply
chambers 253 can be used to store solutions of primers.
Alpha, gamma, epsilon and eta fourth supply chambers 254A, 254C,
254E and 254G can be used to store a solution containing the
appropriate nucleotide triphosphates for a nucleic acid
amplification assay. Beta, delta, zeta and theta fourth supply
chambers 254B, 254D, 254F and 254H can be used to store solutions
containing the polymerase enzyme for the nucleic acid amplification
assay.
A desirable feature for a cassette such as that illustrated in
FIGS. 4A-4E is the ability to incorporate a positive control in one
or more, but not all, of the reaction flow-ways 265 (not identified
in Figures, first reaction flow-way 265A includes alpha first and
second reaction chambers 261A and 262A, second reaction flow-way
265B includes beta first and second reaction chambers 261B and
262B, and so on) . Thus, a material that should generate a positive
assay result can be inserted into sample that otherwise may or may
not produce a positive signal (i.e., experimental samples) or in
samples that should not produce a positive signal (i.e., negative
controls). In this way, the source of any substances that interfere
with the assay can be determined. Any failure of the reaction
flow-ways containing a positive control to generate a positive
signal or an appropriately strong positive signal would indicate
that a standard solution used in the assay contains a substance or
has a property that interferes with the assay. Fluids expected to
generate negative signals can also be incorporated into the
cassette.
Controls, e.g., fluids that have a predetermined amount of a
component to be tested for or that are known to lack the component,
can be inserted into alpha and beta second reaction chambers 262A
and 262B from alpha and beta fifth supply chambers 255A and 255B.
Note that this particular embodiment does not include a facile way
to introduce both a positive control and a test sample into a
reaction flow-way; however, modifications of the cassette 200 of
FIGS. 2A-2E that would allow such a means of introduction are
easily envisioned.
Not all Bursapak supply chambers 250 must be utilized. A Bursapak
supply chamber is avoided simply by not pumping its contents into
the connected reaction chambers.
It is desirable to contain all waste fluids in the cassette 200.
Thus, the illustrated cassette 200 has a first waste chamber 271
and a second waste chamber 272 (collectively waste chambers 270) of
sufficient volume to accommodate all the fluids introduced into the
cassette. Waste chambers 270 are prepared in an evacuated state
such that the films forming the outer wall of the waste chambers
270 (see film 110C of FIG. 1) rest against the inner surfaces of
the waste chambers 270. As fluid is pumped into the waste chambers
270, the film will flex outwardly to provide room for the inserted
fluid. It is desirable to confine the fluids to the cassette for
instance to isolate biohazards or, in the case of nucleic acid
amplification assays, to minimize the opportunity for aerosols to
spread nucleic acid through the lab creating the potential for
cross-contamination of other assays.
Supply chambers 250 are also evacuated in like manner prior to
filling. Most supply chambers 250 will, in a preferred embodiment,
be prefilled prior to shipment to the laboratory where the assay
will be conducted. Of course, the test sample will be inserted at
the lab site. Fluid insertion is best described with reference to
FIG. 1B. A needle can be inserted into septum 131 and used to
evacuate supply chamber 150, causing film 110A to collapse onto the
floor of supply chamber 150. Then, fluid can be inserted through
the septum into supply chamber 150. The first fluid exchange
channel is then blocked, for instance by heat sealing or by
crimping.
Focusing on delta reaction flow-way 265D, note that experimental
sample from sixth supply chamber 256 is first relayed to delta
second reaction chamber 262D while flow to delta first reaction
chamber 261D is blocked by operating a foot-pad pump minimize the
volume of delta first reaction chamber 261D. Typically, the first
reaction conducted on the experimental sample will occur in delta
first reaction chamber 261D. To move the experimental sample from
delta second reaction chamber 262D to delta first reaction chamber
261D, delta second valve 282D is closed, the foot-pad pump acting
on delta first reaction chamber 261D is released, and the foot-pad
pump acting on delta second reaction chamber 262D is operated to
pump the experimental sample into delta first reaction chamber
261D.
Foot-pad pumps that operate to drain a supply chamber 150 can
remain engaged with the supply chamber 150 to prevent back-flow
into the supply chamber 150.
Valves
FIGS. 5A, 5B, 6 and 7 illustrate yet another embodiment of the
invention that utilizes plunger-type valves to control the flow of
fluids in the cassette 100 or cassette 200. The operation of such a
plunger-type valve in a cassette 100 or 200 is illustrated above
with reference to FIGS. 5A and 5B. Plunger 810 has a plunger rod
811 and a piston 812. In the position illustrated in FIG. 5A,
plunger rod 811 is withdrawn away from such that third film 110C,
which is embossed to protrude away from the seat 181 of valve 180,
does not interfere with fluid flow from alpha third fluid exchange
channel 143A, into valve 180, and out through beta third fluid
exchange channel 143B. In FIG. 5B, plunger rod 811 presses film
110C against valve seat 181, blocking fluid flow. FIG. 6 shows a
three-dimensional view of valve 180, including valve seat 181 and
valve trough 182.
The plunger 810 can be constructed of numerous durable materials
including without limitation a plastic such as polycarbonate or
metal such as stainless steel or aluminum or the like. The diameter
of plunger rod 811 is typically from about 20 to about 100 .mu.m,
preferably about 60 .mu.m, while piston 812 typically has a
diameter from about 100 to about 300 .mu.m, preferably about 200
.mu.m. Preferably, the ratio of the cross-sectional area of the
piston 812 to that of the plunger rod 811 is at least about
10-fold, thereby providing a corresponding mechanical
advantage.
A pneumatic mechanism for operating plunger 810 is illustrated in
FIG. 7. Instrument 900 (not shown) has a pneumatic device 800
formed of first portion 800A and second portion 800B which can be
joined together, for instance, by bolts, rivets, adhesives or
snap-fitting pieces. Interposed between the first and second
portions 800A and 800B is flexible gasket 820, which can be formed
of a suitable film such as poly (2-chloro-1,3-butadiene) (e.g.,
Neoprene, DuPont de Neumours, Wilmington, Del.) or silicon rubber.
Flexible gasket 820 can be held in place by the clamping action of
first and second portions 800A and 800B, which adherent force can
be supplemented using heat sealing or adhesive. Pneumatic cavity
830 is formed in both first and second portions 800A and 800B and
has a cavity inlet 831. Fluid, preferably a gas, is inserted
through cavity inlet 831 to pressurize the part of pneumatic cavity
830 located above the gasket 820 and cause the gasket 820 to press
against plunger 810, causing plunger 810 to press against valve
seat 181. In the absence of such fluid pressure in pneumatic cavity
830, pump induced pressure in third fluid exchange channel 143A is
sufficient to displace (a) third upper cover into displacement
cavity 840 and (b) plunger 810 from the valve seat 181, thereby
allowing flow. Pneumatic device 800 can be formed of numerous
durable materials including without limitation a plastic such as
polycarbonate or metal such as brass or aluminum or the like.
As an alternate to the above method of plunger actuation, other
methods may be used which do not employ the piston. These include
motor driven cam or screw, and external hydraulic or pneumatic
cylinders.
In another valve embodiment of the invention shown in FIG. 8A with
reference to another cassette 600 (not shown). Valve ball 620 is
used to press lower film 120 flush against the lower surface of
first body layer 601 so as to block fluid flow through hole 632.
Valve ball 620 can be fabricated of any suitably material such as
nylon, high density polyethylene, polycarbonate and the like. Lower
film 120 is sealed to portions 601A and 601C of first body layer
601, but typically is not sealed to portion 601B. The sealing
between lower film 120 and portions 601A and 601C can be done
using, for instance, adhesives or by clamping the membrane between
body layer 601 and second body layer 602. First body layer 601,
second body layer 602 and third body layer 603 can be joined
together using, for instance, by bolts, rivets, adhesives or
snap-fitting pieces. Pressure can be applied to valve ball 620 to
press it against or release it from lower film 120 in a number of
ways. Note that the valve is designed so that valve ball 620 will
automatically center itself to properly seat itself against first
layer 601. FIG. 8A shows a spring loaded lever 640 that allows a
push motion to open the valve, where force is applied as indicated
by arrow "B". A push rod 643 (not illustrated) can be used to so
engage spring loaded lever 640. FIG. 8B illustrates another
embodiment that uses pull rod 641 to open the valve. The function
of both spring loaded level 640 and pull rod 641 depend on the
spring 642 formed from third body layer 603. Both types of rods can
be activated by a cam 650 that is driven by a shaft 652 (not
illustrated). In operation, liquid flow is, for instance, in the
direction indicated by arrow "A" and proceeds by first conduit 631
and second conduit 632. When valve ball 620 is seated against first
body layer 601, the valve is closed and flow is stopped. As the
valve ball 620 is withdrawn, lower film 120 deforms in response to
fluid pressure, into cavity 633 to form third conduit 633A (not
shown) linking second conduit 632 with fourth conduit 634. Fourth
conduit 634 connects with fifth conduit 635. FIG. 8C illustrates
the use of a cam 650 to activate a pull rod 641 that is spring
loaded with pull rod spring 651. All of the various pull rods 641
and pull rod springs 651 can be contained in a single base plate
604, such as that shown in FIG. 8C, which can be attached to the
instrument 900. The valve of FIG. 8C also differs in employing a
pinch foot 621 instead of a valve ball 620 and in seating the pinch
foot 621 against portion 601B instead of against the opening of
second conduit 632. In the illustrated embodiments of FIGS. 8A--8C,
the valves are normally in the closed position. The positioning of
the valves can be programmed and activated by controller 960 (not
shown). To further ensure that fluid flow is blocked prior to
attaching the cassette 600 to the base plate, temporary membranes
or seals can be employed to maintain the various fluids in their
chambers. These membranes could be broken by applying a light
pressure. Alternatively, the fluids could be frozen prior during
storage to attaching the parallel reaction device to the base
plate.
Alternatively, second and third body layer 602 and 603,
respectively, can be designed to be separable from first body layer
601, which contains fluid exchange channels and fluid chambers. In
this embodiment, prior to joining these separable parts, the valve
locations are not strongly closed to fluid flow, although the lower
film 120 can rest securely enough against portion 601B to prevent
inadvertent fluid flow. Where the valve includes a valve ball 620,
a ball retention film 615 is usefully sealed to the upper side of
second body layer 602 to assure that the value ball 620 does not
fall out of the device. The advantage of separating these pieces is
that the portions of the parallel reaction device containing
mechanical elements can be re-used while the fluid-handling portion
can be disposed of.
FIG. 9B shows a closed electromagnetic valve 380 for use in
controlling the flow of fluids in a cassette 300. Located in a
portion 700 of instrument 900, the electromagnetic valve 380 has a
spacer 730 that is pressed against a flexible upper film 110 by
first spacer spring 731 and second spacer spring 732. The first and
second spacer springs 731 and 732 or the spacer 730 are
sufficiently magnetic or magnetically permeable that they can be
drawn away from upper film 110 by activating electromagnetic coils
740. In FIG. 9A, The electromagnetic valve 380 is shown in the open
position with spacer 730 electromagnetically drawn away from valve
seat 381.
Auxiliary Blocks
FIG. 10 illustrates a part of instrument 900, reaction cell
servicing device 400, having upper auxiliary block 400A for moving
fluids into or out of a reaction chamber 160. Preferably, there
will be a corresponding, upwardly oriented lower auxiliary block
400B located underneath reaction chamber 160. Upper auxiliary block
400A is honeycombed with upper conduit 430A. Upper conduit 430A has
an upper inlet 431A and an upper outlet 432A. First upper portion
401A of upper auxiliary block 400A is fabricated of any suitably
sturdy material, but is preferably constructed of the same material
as third upper portion 403A. Second upper portion 402A is
preferably fabricated of a heat-insulating material, such as,
without limitation, nylon, polycarbonate and the like. Third and
fourth upper portions 403A and 404A are preferably fabricated of a
heat-conductive material, such as, without limitation, aluminum,
copper, sintered beryllia, and the like. Upper portions 401A-404A
can be joined using, for instance, bolts, rivets, adhesives or
snap-fitting pieces. Upper electrical heaters 440A are positioned
adjacent to the reaction chamber 160.
The upper and lower heaters 440A and 440B are generally thin layers
of conductive material that is separated from the heat-conductive
upper and lower sections 402A and 402B of upper and lower auxiliary
blocks 400A and 400B by a thin electrical insulation layer. Such an
insulation layer is formed, for example, by direct deposition onto
the substrate. For example, silicon nitride can be deposited from
the gas phase or aluminum oxide can be deposited using a liquid
carrier. The conducting layer forming upper and lower heaters 440A
and 440B are, for example, deposited by vacuum evaporation (e.g.,
for a nichrome conducting layer) or by deposition from the vapor
(e.g., for an indium tin oxide conducting layer). Alternately,
pre-formed heater sheets are cemented to the substrate, for
instance using an epoxy cement or the adhesive recommended by the
vendors. Appropriate heaters can be obtained from Elmwood Sensors
Inc. (Pawtucket, R.I.) or from Omega Engineering Inc. (Stamford,
Conn.). Typically, individual heater elements have planar
dimensions appropriate, alone or in combination with electrically
coupled heater elements, to match the size of the reaction chamber
to be heated.
Fourth upper portion 404A constitutes an upper foot-pad 404A' for a
foot-pad pump that operates to pump fluid out of a reaction chamber
160. In this context, where a foot-pad is associated with a heating
and cooling device, it is preferably fabricated of a material with
high thermal conductivity such as aluminum, copper, sintered
beryllia, and the like. The operation of the foot-pad pump 460,
which includes lower foot-pad 404B', is illustrated in FIGS. 11A
and 11B. When the upper and lower foot-pads 404A' and 404B' are
withdrawn away from reaction chamber 160, the reaction chamber 160
can be filled with fluid (see FIG. 11A). When the upper and lower
foot-pads 404A' and 404B' are brought towards each other (see FIG.
11B), fluid in reaction chamber 150 is pushed out either through
second fluid exchange channel 142 or third fluid exchange channel
143. The embossing at location 161 (for the upper film 110B) or at
location 162 (for lower film 120), allows the two films to be
pushed together without substantial stretching. The embossing of
upper film 110B and lower film 120 is done, for instance, by
applying suitably shaped, heated dies.
Preferably, instrument 900 has a device for pumping and controlling
reaction cell temperature, such as reaction cell servicing device
400, for each reaction chamber 160 in the cassette 100 or 200.
FIG. 12 shows a schematic of the accessory support devices for the
upper auxiliary block 400A of FIG. 11. Water is propelled through
upper and lower conduits 430A and 430B, respectively, from pump and
water cooler console 950. Pump and water cooler console 950 further
includes fluid valves operating under the control of controller
960. Electrical current is supplied to upper and lower heaters 440A
and 440B, respectively, by power supply 970, which is controlled by
controller 960. Controller 960 receives input from upper and lower
thermal sensors 450A and 450B, respectively.
In FIG. 13, upper auxiliary block 500A includes a set of paired
first and second upper thermoelectric blocks 511A and 512A,
respectively, while lower auxiliary block 500B has a set of paired
first and second lower thermoelectric blocks 511B and 512B,
respectively. First upper and first lower thermoelectric blocks
511A and 511B, respectively, are made of p-type semiconductor
material, while second upper and second lower thermoelectric blocks
512A and 512B, respectively, are made of n-type semiconductor
material. The thermoelectric blocks 511 and 512 are electrically
connected in series by upper and lower connectors 513A and 513B as
illustrated to form thermoelectric heat pumps. Such thermoelectric
heat pumps are available for instance from Tellurex Corp., Traverse
City, Mich. and Marlow Industries, Dallas, Tex. Upper and lower gas
inlet/outlets 510A and 510B are connected to upper and lower
manifolds 520A and 520B, respectively, formed by the space between
the upper and lower thermoelectric blocks 501A and 501B. Upper and
lower manifolds 520A and 520B (which are made up of the space
between thermoelectric blocks) are connected, respectively, to an
upper plurality of passageways 521A or a lower plurality of
passageways 521B. The outer portions of upper and lower auxiliary
blocks 500A and 500B are upper and lower heat sinks 504A and 504B,
respectively, which are preferably constructed of a heat-conductive
material such as, without limitation, aluminum, copper, sintered
beryllia, and the like. First upper air-tight collar 506A, second
upper air-tight collar 507A, first lower air-tight collar 506B and
second lower air-tight collar 507B help form upper and lower
manifolds 520A and 520B. Upper and lower thermal sensors 570A and
570B are connectable to a controller or a monitoring device by
upper and lower leads 571A and 571B, respectively.
It will be recognized that upper end-plate 502A viewed from
underneath or lower end-plate 502B viewed from above would have a
series of holes which are the outlets of upper and lower
passageways 521A and 521B. Another attribute of the auxiliary
blocks 500A and 500B is that the thermoelectric blocks typically
are arrayed in three dimensions rather than two.
Heating is achieved by applying voltage of the proper polarity to
upper first and second leads 508A and 509A and to lower first and
second leads 508B and 509B. Cooling is achieved by reversing the
polarity of the voltage. An important variable in the operation of
these heating and cooling devices is temperature uniformity. To
increase temperature uniformity, upper and lower first end-plates
502A and 502B are preferably constructed of a material of high
thermal conductivity, such as sintered beryllia. Other suitable
materials include, without limitation, ceramics containing metallic
aluminum. Preferably, the thermoconductivity of end-plates 502A and
502B is at least about 0.2 watt.multidot.cm.sup.-1
.multidot.K.sup.-1, more preferably at least about 2
watt.multidot.cm.sup.-1 .multidot.K.sup.-1. The upper and lower
temperature sensors 570A and 570B can be, without limitation,
thermocouples or resistive sensors. The upper and lower sensors
570A and 570B can, for example, be deposited on upper and lower
first end-plates 502A and 502B as thin films or they can be in the
form of thin wires embedded into holes in upper and lower first
end-plates 502A and 502B.
Upper and lower auxiliary blocks 500A and 500B provide an alternate
method of applying pressure to second upper film 110B and lower
film 120 to push fluid out of reaction chamber 160. When gas
pressure is applied through upper gas inlet/outlet 510A and
corresponding lower gas inlet/outlet 510B (not shown) of lower
auxiliary block 500B, the gas exiting upper and lower pressurized
fluid channels 521A and 521B (not shown) forces upper and lower
films 110 and 120 together, thereby forcing fluid from reaction
chamber 160.
Upper or lower auxiliary block 500A or 500B can contain a plurality
of upper or lower pressurized fluid channels 421A or 421B,
respectively, which are used to operate a gas pressure flow control
means. The fluid within these channels typically is a gas such as
oxygen or nitrogen. Gas of higher than atmospheric pressure can be
applied to the upper or lower pressurized fluid channels 421A or
421B from, for instance, a pressurized gas canister or a pump
applied to upper or lower gas inlet/outlet 410A or 410B. A vacuum,
usually a partial vacuum, can be applied to the upper or lower
pressurized fluid channels 421A or 421B using, for instance, a
vacuum pump. Numerous mechanisms for controlling the pressure of
the pressurized fluid channels will be recognized by those of
ordinary skill in the engineering arts.
FIG. 14 illustrate another upper auxiliary block 1500A and lower
auxiliary block 1500B that use thermoelectric heat pumps but use a
foot-pad pump instead of a gas-pressure mediated pumping device.
Upper and lower foot-pads 1505A and 1505B are used to pump fluid
out of reaction chamber 160. Thermoelectric blocks 1513 are used to
heat or cool as described above. Upper and lower heat sink thermal
sensors 1592A and 1592B are located in upper heat sink 1504A and
lower heat sink 1504B, respectively. Upper heat sink heater 1590A
and lower heat sink heater 1590B (connected to electrical power via
upper leads 1591A and lower leads 1591B, respectively) are used to
transfer heat to the thermoelectric blocks 1513, thereby allowing
thermoelectric blocks 1513 to operate at a higher temperature
range. Upper and lower sensors 1570A and 1570B are used to monitor
the temperature of the adjacent reaction chamber 160.
The speed with which the temperature of the reaction chamber 160 is
increased or decreased is important for optimizing some nucleic
acid amplification assays. During the temperature cycling important
for some nucleic acid amplification assays, it is important to
operate at a relatively lower temperature where the nucleic acid
sample is enzymatically reproduced and at a higher temperature
where the nucleic acid sample is melted to separate the two strands
of the nucleic acid. During the period when the assay apparatus
cycles between the two preselected temperatures believed to be
appropriate for a given nucleic acid amplification, various
unwanted chemistries can be expected to occur. For instance, as the
temperature increases from the lower temperature, the replication
enzyme can be expected to continue to function, although not
necessarily with the appropriate accuracy of replication achieved
at the prescribed lower temperature. At the higher temperature set
point, this unwanted enzymic activity is inhibited by the high
temperature. Thus, it is important to rapidly change the reaction
temperature between the two operating temperature plateaus.
One mechanism by which the temperature can rapidly be changed in
the reaction chamber is illustrated in FIG. 10. Assume that the
reaction chamber 160 is operating at lower plateau temperature "G".
Under these conditions, cooling water does not flow through upper
conduit 430A or corresponding lower conduit 430B (not shown). The
temperature is maintained by intermittently operating upper and
lower heaters 440A and 440B when the temperature in reaction
chamber 160 lowers beneath a temperature of G minus X (where X is a
temperature differential). At a pre-programmed time, the
temperature is raised to higher plateau temperature "H" by
activating upper and lower heaters 440A and 440B until a
temperature is reached that will lead to a temperature
stabilization at temperature H. Water flow through upper and lower
conduits 430A and 430B can be activated to minimize temperature
overshoots if needed. Temperature H is maintained by intermittently
operating upper and lower heaters 440A and 440B when the
temperature of the reaction chamber 160 lowers beneath a
temperature of H minus Y (where Y is a temperature differential).
To cycle back to temperature G, the controller activates the pump
451 (not illustrated) of console 450 to cause cooling water to flow
through upper and lower conduits 430A and 430B.
The performance of such a heater device and cooling device can be
simulated using a heat transfer simulation computer program using a
finite element approximation of the heat flow equation. The
simulation is conducted with the following assumptions: the
thickness of the reaction chamber 160 is 0.5 mm, the upper and
lower films were 0.1 mm thick and the insulation between the heater
and the auxiliary block was 0.025 mm thick. Such a simulation has
determined that a jump from 25.degree. C. to 75.degree. C. can be
achieved within 3.2 seconds, where, after 3.2 seconds, the
temperature in the reaction chamber is substantially uniform. The
reciprocal cooling step can be achieved within about 3 seconds,
resulting in a substantially uniform temperature in the reaction
chamber. Preferably, after this cooling step the variation in
temperature in the reaction chamber is no more than about
0.1.degree. C.
Using the heating and cooling devices of the present invention,
including the device described in the immediately preceding
paragraph, reaction chamber 160 temperatures between about
-20.degree. C. and about 100.degree. C. can be maintained.
In one preferred embodiment, when the parallel reaction device
includes more than one reaction flow-way, each such reaction
flow-way will include at least one reaction chamber 160 which will
have at least one heating and cooling device made up of
thermoelectric blocks 501 (such as the heating and cooling device
described in the paragraph immediately above) capable of being
aligned with a side of the reaction chamber. More preferably, each
such reaction chamber 160 will have a heating and cooling device on
each of two opposing sides. In another preferred embodiment, the
cross-sectional area of upper or lower first end-plate 502A or 502B
substantially matches the largest cross-sectional area of the
reaction chamber 160 to which it is intended to transfer heat.
The principles of temperature cycling for a reaction chamber 160
heated and cooled with upper and lower auxiliary blocks 500A and
500B or upper and lower blocks 1 500A and 1500B are the same as
those outlined above for the upper and lower auxiliary blocks 400A
and 400B of FIG. 10.
In another embodiment, the reaction chamber 160 is heated and
cooled by passing a heated or cooled fluid, preferably a gas,
either directly over one or more surfaces of the reaction chamber
160 or through a heat exchange apparatus that can be positioned
adjacent to one or more surfaces of the reaction chamber 160. The
apparatus illustrated in FIG. 10 can be modified to operate
pursuant to this embodiment by (a) removing (or not using) the
upper and lower heaters 440A and 440B and (b) adding a heater for
heating the fluid. The parallel reaction device preferably has two
fluid management systems, one for a hotter fluid and another for a
cooler fluid, together with the valving required to inject the
hotter or cooler fluid into the tubing leading to the reaction
chamber 160 as appropriate for maintaining a given temperature in
the reaction chamber. Particularly where the heating and cooling
fluid is a gas, the temperature of the gas soon after it has passed
by the reaction chamber 160 will provide a useful indication of the
temperature of the reaction chamber 160.
Where the auxiliary blocks act as foot-pads or for other footpads,
mechanical or electromechanical methods of drawing the foot-pads
towards or away from the fluid chamber on which it acts are well
known and include solenoids, pneumatically activated plungers,
screw mechanisms and the like.
Auxiliary blocks and other features useful in conjunction with this
invention are described in U.S. patent application Ser. No.
<11772A>, filed Oct. 31, 1996, titled "Assay System," Docket
No. DSRC 11772A, which is incorporated herein in its entirety by
reference.
Miscellaneous Pumps
Pumping action can also be achieved using, for instance,
peristaltic pumps, mechanisms whereby a roller pushes down on the
flexible film of a fluid chamber to reduce the volume of the
chamber, plungers that press on the flexible film of a fluid
chamber to reduce its volume, and other pumping schemes known to
the art. Such mechanisms include micro-electromechanical devices
such as reported by Shoji et al., "Fabrication of a Pump for
Integrated Chemical Analyzing Systems," Electronics and
Communications in Japan, Part 2, 70: 52-59, 1989 or Esashi et al.,
"Normally closed microvalve and pump fabricated on a Silicon
Wafer," Sensors and Actuators, 20: 163-169, 1989 or piezo-electric
pumps such as described in Moroney et al., "Ultrasonically Induced
Microtransport," Proc. MEMS, 91: 277-282, 1991.
Detection Devices
In a preferred embodiment, at least one reaction chamber 160 has a
transparent retaining wall that is generally formed of upper film
110 or lower film 120 (or two retaining walls are transparent).
Reaction chamber 160 can be a chamber where a reaction occurs, such
as one of lysing reaction chambers 340 or reaction chambers 380
(see FIG. 3), it can be a supply chamber containing samples,
controls or reagents, such as supply chambers 350, 360 and 390, or
it can be a storage chamber, such as one of storage chambers
399A-E. The parallel reaction device in this embodiment preferably
includes a light source capable of directing light to the
transparent upper or lower film 110 or 120 and a detection device
for detecting (a) the light reflected from an illuminated reaction
chamber 160, (b) the light transmitted through an illuminated
chamber 160, or (c) the light emissions emanating from an excited
molecule in a chamber 160. A membrane is "transparent" if it is 80%
transparent at a wavelength useful for detecting biological
molecules.
The detection device can incorporate optical fibers, optical
lenses, optical filters or other optical elements. Alternatively,
where detection uses fluorescence, detection and quantitation can
be done by photographing the detection channel 295 under
appropriate excitation light. With fiber optics or other suitable
optical devices, the size of the detection system that is adjacent
to the parallel reaction device is minimized. This size
minimization facilitates incorporating the detection system
together with a temperature control device (described more fully
below) into the parallel reaction device. A particularly preferred
light source is a solid state laser. The size of these light
sources also facilitates incorporating a number of auxiliary
components about the parallel reaction device. When a nucleic acid
amplification is conducted in an parallel reaction device that
incorporates current technology solid state lasers, the method used
to detect amplified nucleic acid uses a dye that absorbs light at a
wavelength higher than about 600 nm to indicate the presence of
amplified nucleic acid, as described below. Examples of such dyes
include Cy5.TM., one of a series of proprietary cyanine class dyes.
Cy5.TM., and the related dyes, are products of Biological Systems,
Inc. (Pittsburgh, Pa.). This particular dye is relatively
small,absorbs at about 650 nm and emits a fluorescent signal at
about 667 nm. Other, larger suitable dyes include structures
derived from seaweed such as allophycocyanin and
allophycocyanin-conjugated reagents (Sigma Chemical Co., St. Louis,
Mo.). These dyes absorb in the 630-750 nm range. The relatively
long wavelengths of the excitation light described above avoid much
of the background fluorescence associated with biological
materials, plastics or other possible components of the cassette
100. A preferred solid state laser source is a Laser Max, Inc.
(Rochester, N.Y.) Model LAS 200-635.5, which emits a light with a
wavelength at a maximum of 4. Other calorimetric detection methods,
for instance those utilizing biotin-avidin binding to associate
horse radish peroxidase with a hybridized pair of polynucleotide
sequences, can be used.
Signals from the detection device typically will be input into a
controller 960, where they can be used to determine the presence,
or absence, of material assayed for and the magnitude of the signal
indicating the presence of the material. From these data, the
amount of assay material can be calculated and the quality of the
assay as indicated by the controls can be quantitated. This
information is then stored for the assay report listing.
In a preferred embodiment, the cassette has one or more detection
channels 295. One such detection channel 295 is illustrated in
FIGS. 15A and 15B. It is made up of a number of fibers 297, which
together preferably transmit at least about 50% of light of a
wavelength useful in the detection procedure, confined to the
detection channel 295. The fibers 297 can be bound in place for
instance by cementing or crimping. The fibers 297 can be fabricated
of glass or suitably transparent plastics. The fibers 297 are
preferably between about 5 .mu.m and about 50 .mu.m in diameter,
more preferably about 20 .mu.m. The detection channel typically has
a width and depth of no more than about 3,000 .mu.m, preferably
between about 200 .mu.m and about 1,000 .mu.m. Microchannels
between the fibers 297 allow liquid to flow through the detection
channel 295. A detection-mediating molecule is bound to the fibers
297. Preferably the detection-mediating molecule is an
oligonucleotide that hybridizes with the nucleic acid to be
amplified in a nucleic acid amplification reaction and the nucleic
acid amplification reaction utilizes primers having a detectable
moiety. The detection-mediating molecules are bound to the fibers
297 by known methods. Preferably, discrete bands on the fibers such
as first band 296A, second band 296B and third band 296C have
separate detection-mediating molecules, which could be, for
instance, designed to detect two separate species to be amplified
in a nucleic acid amplification reaction and to provide a control
for non-specific hybridizations. To manufacture the banding pattern
of bound molecules, oligonucleotide synthesis procedures that
utilize photo-cleavable protecting groups and masks to protect
certain bands 296 from photocleavage can be used. Such synthesis
procedures are described in U.S. Pat. No. 5,424,186 (Fodor et al.).
The instrument 900 is preferably designed to provide heat control
at the detection channels 295 for conducting hybridization
reactions. In a preferred embodiment, the sides 298 of the
detection channel 295 are coated with a reflective coating so that
light incident from above will reflect and twice pass through the
detection channel 295. Such a reflective coating is provided by
metalizing, for instance using a sputtering or evaporation
process.
Alternatively, the detection channels 295 contain membranes 299
(not shown), such as a nylon membrane, to which a hybridization
probe has been bound. If two or more hybridization probes are used,
they are each bound to a specific region of the membranes 299 using
"dot blot" procedures such as are described in Bugawan et al., "A
Method for Typing Polymorphism at the HLA-A Locus Using PCR
Amplification and Immobilized Oligonucleotide Probes" Tissue
Antigens 44: 137-147, 1994 and Kawasaki et al., "Genetic Analysis
Using Polymerase Chain Reaction-Amplified DNA and Immobilized
Oligonucleotide Probes: Reverse Dot-Blot Typing", Methods in
Enzymology 218: 369-381, 1993. As described above, the
amplification product hybridized with the bound probe or probes has
attached-via the amplification primers--a detectable moiety.
Note that for cavities in the cassette 200 intended for use in
detection, such as detection channels 295, in a preferred
embodiment of the invention the upper film 110 over the cavity is
replaced with a cover 110' selected for its optical properties,
such as, without limitation, a cover 110' made of optical quartz.
Because pumping is effected elsewhere in the cassette, the cover
110' does not have to be flexible like an upper film 110.
While in a preferred embodiment detection is done in situ in the
cassette, in other embodiments the products of chemical reactions
effected in the cassette are removed and detection methods or
chemistries are done elsewhere, including in a different
cassette.
Paramagnetic Beads and High Field Gradient Magnet
Paramagnetic beads useful for facilitating chemical processes
conducted in a cassette 100 are available from several sources
including Bang Laboratories (Carmel, Ind.) for beads lacking
conjugated biomolecules, Dynal (Lake Success, N.Y.) for beads
conjugated to various antibodies (for instance, antibodies that
bind to the CD2 cell-surface receptor) and CPG (Lincoln Park, N.J.)
for beads with a glass matrix and a variety of surface bonded
organics. For applications where it is anticipated that the beads
will be washed into and out of reaction chambers, each bead will
preferably have a diameter of less than about 1 mil, more
preferably, less than about 0.5 mil, which diameter facilitates
entry and exit through the channels by which material is inserted
or evacuated from the reaction chamber 160. For applications where
the beads are anticipated to remain in the reaction chamber 160, in
one embodiment of the invention, the diameter is preferably
sufficiently large to preclude their entry into these channels. The
entrances to such channels within a reaction chamber 160 are
preferably positioned or designed so as to minimize the chance that
a channel will be blocked by a bead that settles over the channel's
entryway.
In a preferred embodiment, the beads are locked in place using
magnetic fields. To generate sufficient movement among the beads,
it has been determined that the magnet used should preferably
generate a sufficient magnetic field gradient within a reaction
chamber 160. Such magnets can be constructed by forming sharp edges
on highly magnetic permanent magnets, such as those formed of rare
earths, such as the neodymium-iron-boron class of permanent
magnets. Such a permanent magnet is available from, for example,
Edmund Scientific (Barrington, N.J.). Sharp edges of dimensions
suitable for a particular reaction chamber 160 are, for example,
formed by abrasive grinding of the magnetic material. An example of
such a shaped magnet 1100 is shown in FIG. 16, where the magnet has
a roof-shape at one of the poles. The illustration shows a
preferred embodiment where there are two roof shapes and
illustrates that the magnet can be brought adjacent to or can be
removed from a cassette such as cassette 100 or cassette 200. In
the illustration, lower auxiliary block 1600B has slots (not
visible) that allow the magnet 1100 to be placed adjacent to
cassette 100 or 200. This magnet suitably has dimensions such that
the length of the peak of the roof-shape matches the
cross-sectional size of a reaction chamber 160. To maximize the
field gradient acting on the paramagnetic beads, the peak 1101 of
the magnet 1100 is placed adjacent to the reaction chamber or other
structure in which the beads are located. The paramagnetic beads
are held in place by leaving the peak 1101 adjacent to the beads.
By moving the magnet with its peak 1101 adjacent to the beads, the
beads are impelled to move with the magnet. Another way in which
high magnetic field gradients can be achieved is to make uniform
slices of a magnetic material and use an adhesive to join the
slices in alternating N to S orientations. Such alternating slice
magnets have high magnetic field gradients at the junctions of the
slices.
The sharp-edged magnets described above are effective in adhering
the paramagnetic beads in one place and in moving beads located,
for instance, in a fluid exchange channel or in a reaction chamber,
from one location to another. Such magnets thus can help retain the
paramagnetic beads in one place, for instance when a fluid in a
reaction chamber 160 is being removed from that chamber but it is
desirable to leave the beads in the chamber. Magnets with locations
having high magnetic field gradients that are particularly suitable
for use in this context are described in U.S. Provisional patent
application Ser. No. 60/006,202, filed Nov. 3, 1995, titled
"Magnet," Docket No. DSRC 11904P, which is incorporated herein in
its entirety by reference.
Various cell binding beads (e.g., beads having bound antibodies
specific for a certain subset of cells) can be used to adhere
selected cells from a population of cells. The beads can be locked
in place, for instance magnetically if the beads are paramagnetic,
while non-adherent cells and fluids are washed away. Thus,
cell-binding beads can be used to concentrate small sub-populations
of cells.
In synthetic chemistry applications, the beads suitably have
attachment sites for coupling the building blocks of chemicals or
polymers.
Septum Manufacture
A septum 131 can be fixed in place in inlet 130 using heated die
1200, as illustrated in FIGS. 17A and 17B. The die 1200 is heated
sufficiently so that the angled, sharp edges 1201 cut into body 105
and move melted material 132 such that it locks the septum 131 in
place.
Controller
The controller 960 typically will be a microprocessor. However, it
can also be a simpler device comprised of timers, switches,
solenoids and the like. The important feature of controller 460 is
that it directs the activation of the means for impelling a fluid,
the valves and the heating and cooling device, according to a
pre-set or programmable schedule that results in the operation of
an assay protocol, such as the protocol outlined below. Preferably,
the controller 460 receives input indicating the temperature of the
reaction chambers of the parallel reaction device and is capable of
adjusting its control signals in response to this input.
PCR Procedures Using the Assay System of the Invention
Often an important variable in PCR reactions is the amount of
interfering cellular debris, including membrane fragments and
cellular chemicals such as enzymes, fats and non-target nucleic
acid, present in the sample to be assayed. Ideally, only highly
purified nucleic acid is used as the sample subjected to a PCR
amplification. However, such purification is not practical with the
small amounts of tissue or fluid available for a diagnostic assay.
Further, given the sensitivity of the assay to contamination by
environmental sources of nucleic acid, a nucleic acid purification
step can increase the likelihood of getting a false positive
result. In some areas of diagnostic or forensic PCR this concern
about interference by cellular debris has been eased somewhat by
improvements in the characterization of PCR reaction conditions,
such that often much cruder nucleic acid samples can be used
without adverse effect. See Rolfs et al., PCR: Clinical Diagnostics
and Research, Springer Lab, 1992 (particularly Chapter 4 et seq.).
See, also, the literature available with such commercial products
as GeneReleaser (BioVentures, Inc., Murphreesboro, Tenn.), Pall
Leukosorb.TM. media (Pall, East Hills, N.Y.) and Dynbeads.RTM. DNA
Direct.TM. (Dynal, Lake Success, N.Y.). (On PCR procedures, see
generally, Ausubel et al., Current Protocols in Molecular Biology,
John Wiley & Sons, New York and PCR: A Practical Approach, IRL
Press, 1991.) Nonetheless, it is desirable to have the capability
of at least removing the cellular debris associated with the cell
membranes of the cells that may be present in the sample. Such a
technique for use in conjunction with the parallel reaction device
of the invention is described below. Such a cleanup step can be
applied when needed to achieve the needed level of sensitivity or
accuracy, or omitted if not needed.
It is preferable to conduct parallel control PCR reactions when
conducting PCR. One control omits sample from the reaction or uses
a sample previously characterized as negative. Another control
introduces a known amount of a purified nucleic acid that is known
to contain the sequence or sequences that the PCR reaction is
designed to amplify. These types of controls can be accomplished on
multiple parallel reaction devices or, more preferably, in separate
reaction flow-ways on the same parallel reaction device whereby
each reagent is distributed from a single source to all of the
reaction flow-ways.
Another control technique used in PCR is to design the PCR reaction
so that it will amplify multiple nucleic acid segments, each of
which can be indicative of a disease or a genetic circumstance or
marker. The different segments can be amplified in multiple
reactions or in the same reaction chamber. If amplified in the same
chamber, that binding competition between the various primers can
necessitate extending the time, in each amplification cycle, spent
at the replication temperature.
One method for removing cellular debris from a sample involves
binding the cells in the sample to a bead that has attached thereto
an antibody specific for a cell surface molecule found on the
cells. Beads that bind to the CD2 white blood cells or to E. coli
bacteria (such as the 0157E strain) are available from Dynal (Lake
Success, N.Y.). An ever-growing family of cell-surface molecules
found on mammalian cells, bacterial cells, viruses and parasites
has been characterized and antibodies against the majority of these
molecules have been developed. See, e.g., Adhesion Molecules, C. D.
Wegner, ed., Academic Press, New York, 1994. Many of these
antibodies are available for use in fabricating other types of
cell-affinity beads (for instance, from Sigma Chemical Co., St.
Louis, Mo.). The cells can be adhered to the antibodies on the
beads and lysed to release their nucleic acid content. The lysis
fluid together with the released nucleic acid can be moved to a
separate compartment for further processing, leaving behind the
beads and their adherent cellular debris.
The lysis fluid used to release nucleic acid from the sample cells
can also interfere with the PCR reaction. Thus, in some protocols
it is important to bind the nucleic acid to a substrate so that the
lysis fluid can be washed away. One such support is provided by
beads that bind to DNA, such as glass beads that bind to DNA by
ionic or other interactions such as Van der Waals interactions and
hydrophobic interactions. Suitable beads, with surfaces chemically
treated to maximize the number of interaction sites, are available
from, for example, BioRad (Hercules, Calif.). Paramagnetic beads
with a number of DNA binding surfaces, such as nitrocellulose or
nylon-coated surfaces, can be useful in operating the invention. In
some embodiments, it is desirable for the beads to be paramagnetic
so that they can be manipulated using magnetic forces. Paramagnetic
glass beads are manufactured by CPG (Lincoln Park, N.J.). Once the
nucleic acid is bound to the beads, the lysis fluid can be washed
from the beads. The nucleic acid can be amplified with the beads
present.
The lysis fluid used to release nucleic acid from the cells in a
sample typically includes a detergent, preferably nonionic, and a
buffer, usually the buffer used in the PCR amplification reaction.
The pH of the lysis fluid is preferably from about pH 7.8 (for
protease K-containing lysis fluids, for example) to about pH 8.0
(for phenol-mediated lysis, for example), typically about pH 8.0.
Suitable detergents include, without limitation, Sarkosyl and
Nonidet P-40. Other components can includes salts, including
MgCl.sub.2, chelators and proteases such as proteinase K.
Proteinase K can be inactivated by heating, for instance, to about
100.degree. C. for about 10 minutes. Depending on the composition
of the lysis buffer, it can be more or less important to wash the
lysis buffer away from the nucleic acid prior to conducting the
amplification assay.
The amplification buffer used to support the amplification reaction
will typically include the four deoxynucleotide triphosphates
(NTPs) (e.g., at a concentration of from about 0.2 mM each), a
buffer (e.g., Tris.HCl, about 10 mM), potassium chloride (e.g.,
about 50 mM) and magnesium chloride (e.g., about 1 to 10 mM,
usually optimized for a given PCR assay scheme). The pH is
preferably from about pH 8.0 to about pH 9.0, typically about pH
8.3. Other components such as gelatin (e.g., about 0.01% w/v) can
be added. The individual primers are typically present in the
reaction at a concentration of about 0.5 .mu.M. The amount of
sample nucleic acid needed varies with the type of nucleic acid and
the number of target nucleic acid segments in the nucleic acid
sample. For genomic DNA, where each cell in the sample has about 2
copies of target nucleic acid, a concentration of about 10 .mu.g/ml
is desirable.
For simplicity, the polymerase used in the procedure is a
heat-resistant DNA polymerase such as Taq polymerase, recombinate
Taq polymerase, Tfl DNA polymerase (Promega Corp., Madison, Wis.),
or Tli DNA polymerase (Promega Corp., Madison, Wis.). Heat
stability allows the PCR reaction to proceed from cycle to cycle
without the need for adding additional polymerase during the course
of the reaction process to replace polymerase that is irreversibly
denatured when the reaction vessel is brought to a DNA strand
separation temperature. Preferably, the DNA polymerase used has the
increased accuracy associated with the presence of a proofreading,
3' to 5' exonuclease activity, such as the proofreading activity of
the Tli DNA polymerase.
Blood provides one of the more convenient samples for diagnostic or
genetic PCR testing. For most genetic testing, from about 10 to
about 50 .mu.l of blood is sufficient to provide enough sample DNA
to allow for PCR amplification of specific target segments. For
fetal cell analysis, however, as much as about 20 mls, which may
contain as few as about 400 fetal cells, can be required. Such
large sample volumes require concentration, for instance, using the
methods described above. For testing for microbial diseases, the
concentration of target nucleic acid in the sample can be quite low
(e.g., no more than about 2-5 fg per bacterial genome). Thus, when
using the parallel reaction device to test for such microbes,
concentration methods may again be required.
To specifically amplify RNA, it is necessary to first synthesize
cDNA strands from the RNA in the sample using a reverse
transcriptase (such as AMV reverse transcriptase available from
Promega Corp., Madison, Wis.). Methods for conducting a PCR
reaction from an RNA sample are described, for example, in Ausubel
et al., Current Protocols in Molecular Biology, John Wiley &
Sons, New York and PCR: A Practical Approach, IRL Press, 1991. To
prepare RNA for this purpose, a facile procedure uses a lysis
buffer containing detergent (such as 0.5% Nonidet P-40), buffer
(e.g., pH 8.3) and suitable salts that has been, immediately prior
to use, mixed 1:1000 with a 1:10 diethylpyrocarbonate solution in
ethanol. After sample cells have been lysed with this solution, a
supernate containing RNA is separated away from a pellet of nuclei
by centrifugation. Primer, which is generally the same as one of
the primers used in the subsequent PCR cycling reaction, is
annealed to the RNA by heating (e.g., to about 65.degree. C.) and
subsequently reducing the temperature to, generally, about
37.degree. C. The reverse transcriptase, nucleotide triphosphates
and suitable buffer (if not already present) are then added to
initiate cDNA synthesis. Generally, a small volume (e.g., about 1.0
to about 2.0 .mu.l) of material from the cDNA synthesis is added to
a solution (e.g., about 50 to about 100 .mu.l) containing the
buffer, DNA polymerase, nucleotide triphosphates and primers needed
for the PCR amplification. The temperature cycling program is then
initiated.
Hybridization Procedures
The advantages of the parallel reaction device as it relates to
conducting PCR reactions also substantially apply to conducting
hybridization procedures. The ability of the valves of the parallel
reaction device to accommodate elevated temperatures allows the
system to be used in hybridization protocols. While hybridization
reactions are not as sensitive to contamination as PCR reactions,
these reactions are nonetheless very sensitive to contamination,
the risk of which is substantially reduced with the disposable
system of the invention.
Procedures for conducting hybridizations are well known in the art.
See, for example, Ausubel et al., Current Protocols in Molecular
Biology, John Wiley & Sons, New York and Sambrook et al.,
Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor
Press, 1989. In these procedures, a nucleic acid such as (a) a
sample source of nucleic acid containing a target sequence, or (b)
a probe nucleic acid is bound to a solid support and, after this
binding, the remaining binding sites on the support are
inactivated. Then, the other species of nucleic acid, which has
bound to it a detectable reporter molecule, is added under
appropriate hybridization conditions. After washing, the amount of
reporter molecule bound to (i.e. hybridized with) the nucleic acid
on the solid support is measured.
For instance, a hybridization can be conducted in a reaction
chamber in the parallel reaction device, where the reaction chamber
contains a nitrocellulose membrane (or another membrane that binds
nucleic acid) to which RNA has been bound (for instance, by
electrophoretic or capillary blotting from a separation gel,
followed by baking). A Northern prehybridization solution can then
be introduced into the reaction chamber from one of the fluid
chambers. (The recipes for Northern prehybridization solution (p.
A1-40), Northern hybridization solution (p. A1-39), SSC (p. A1-53,
20X recipe) and Denhart's solution (p. A1-14, 100X recipe) of
Ausubel et al., Short Protocols in Molecular Biology, 2nd Edition,
John Wiley & Sons, 1992 are incorporated herein by reference to
more fully exemplify the hybridization methods that can be
conducted in the parallel reaction device; note that the salmon
sperm DNA recited in two of these recipes, which DNA serves as a
competitor to reduce nonspecific hybridizations, is typically
sheared prior to use.) The membrane and prehybridization solution
are incubated overnight at a temperature between about 37.degree.
C. and about 42.degree. C., depending on the melting temperature
for the interaction between target sequence and the probe sequence.
Note that these incubation temperatures are in the range that is
generally appropriate given the presence of 50% formamide in the
prehybridization and hybridization solutions; for hybridizations
conducted without formamide, incubation temperatures are typically
higher, such as about 55.degree. C. to about 70.degree. C. The
membrane is then exposed to Northern hybridization solution
containing melted probe and incubated overnight at the same
temperature used in the prehybridization. Following hybridization,
the hybridization solution is pushed out of the reaction chamber,
the reaction chamber is brought to about 25.degree. C. and a first
wash solution (1X SSC, 0.1% w/v sodium dodecyl sulfate) is
introduced. After 15 minutes, the wash is repeated. After an
additional 15 minute wash, a third and final wash is conducted
using 0.25X SSC, 0.1% w/v sodium dodecyl sulfate.
The above outlined hybridization method is exemplary only. Numerous
other hybridization methods can be conducted in the assay system,
including those described in the following sections of Ausubel et
al., Short Protocols in Molecular Biology, which are incorporated
herein by reference: Unit 2.9, pp. 2-24 to 2-30 and the recipes of
Appendix 1 referred to therein; Unit 6.3, pp. 6--6 to 6-7 and the
recipes of Appendix 1 referred to therein; and Unit 13.12, p. 13-44
and the recipes of Appendix 1 referred to therein.
Using the parallel reaction device of the invention, the elevated
temperatures required for hybridization reactions can be handled in
an automated apparatus. For instance, hybridizations can be
conducted at a temperature defined by the melting temperature
T.sub.m. T.sub.m values for any hybridization probe can be
calculated using commercially available software such as Oligo TM
v4.0 from National Biosciences, Inc., Plymouth, Minn.
Immunological Procedures Using the System of the Invention
In immunoassay procedures, the antibody-antigen binding reactions
are generally conducted at room temperature or at a reduced
temperature, such as about 4.degree. C. After the binding
reactions, positive results are generally indicated by an enzymic
reaction, typically mediated by the enzyme alkaline phosphatase,
which enzyme reaction is generally conducted at a temperature
between about 20.degree. C. and about 40.degree. C. The parallel
reaction device of the invention allows these assays to be
automated in a system that allows fast and reliable temperature
regulation in the temperature range between about 0.degree. C. and
about 40.degree. C.
Typically, modern antibody-based screening procedures use a solid
support to which an "antigen" (which is a substance that when
injected into an animal, often in the presence of "adjuvants" known
to enhance antibody production, can cause the animal to manufacture
antibodies specific for the antigen) or an antibody has been
attached. Alternatively, the antigen is found on the surface of a
cell, such as a bacteria or eukaryotic cell, and the cell can
function as a solid support.
In one assay (indirect ELISA), the antigen is bound to the support
and a sample which may contain a first antibody specific for the
antigen and produced by a first animal species is incubated with
the bound antigen. After appropriate washing steps, a second
antibody from a second animal species, which antibody is specific
for antibodies of the first species and is attached to a detectable
moiety (such as alkaline phosphatase), is incubated with the
support. If the sample contained the first antibody, the second
antibody will bind and be detectable using the detectable moiety.
For instance, if the detectable moiety is alkaline phosphatase,
detection can be conducted by adding a chemical, such as
p-nitrophenyl phosphate, that develops a detectable characteristic
(such as color or light emission) in the presence of a developing
reagent such as a phosphatase enzyme. This assay can, for instance,
be used to test blood for the presence of antibodies to the AIDS
virus.
In another assay (direct competitive ELISA) that uses a support
with bound antigen, a sample which may contain an antigen is
incubated with the support together with a limiting amount of an
antibody specific for the antigen, which antibody has an attached
detectable moiety. Due to competition between the solution phase
antigen and the support-bound antigen, the amount of antigen in the
sample correlates with reduced amounts of antibody that bind to the
support-bound antigen and a weaker signal produced by the
detectable moiety.
Another assay (antibody-sandwich ELISA) uses a first antibody
specific for an antigen, which antibody is bound to the support. A
sample which may contain the antigen is then incubated with the
support. Following this, a second antibody that binds to a second
part of the antigen, and which has an attached detectable moiety,
is incubated with the support. If the sample contained the antigen,
the antigen will bind the support and then bind to the detectable
second antibody. This is the basis for the home pregnancy test,
where the antigen is the pregnancy-associated hormone chorionic
gonadotropin.
In another assay (double antibody-sandwich ELISA) that uses a
support with bound antibody, a sample which may contain a first
antibody from a first species is incubated with a support that has
bound to it a second antibody from a second species that is
specific for antibodies of the first species. The antigen for the
first antibody is then incubated with the support. Finally, a third
antibody specific for a portion of the antigen not bound by the
first antibody is incubated with the support. The third antibody
has an attached detectable moiety. If the sample contained the
first antibody, the detectable third antibody will bind to the
support.
These and other immunoassays are described in Units 11.1 and 11.2
of Ausubel et al., Short Protocols in Molecular Biology (pp. 11-1
to 11-17), which text and the recipes of Appendix 1 cited therein,
are incorporated herein by reference.
The following examples further illustrate the present invention
but, of course, should not be construed as in any way limiting its
scope.
EXAMPLE 1
Cassette Fabrication
The following example illustrates fabrication methods used in
constructing cassettes for a microfluidics device of the present
invention.
Various cassettes have been fabricated containing components that
are shown in FIG. 1. Cassette bodies have been made from
high-density polyethylene, both by machining and by molding. The
methods of fabrication and demonstrated performance include the
following:
Membrane embossing
The membrane covering the cassette body and forming the reaction
chambers was embossed prior to sealing to the body. The membrane
was stretched on a frame and embossed between positive and negative
hot dies. For membranes of polyester/polyethylene laminate, the
dies were heated to a temperature of above 140.degree. C. Since
this is above the melting point of the polyethylene, the die in
contact with the polyethylene was made of polytetrafluoroethylene,
to prevent adhesion. A preferable material for embossing is a
fluoropolymer/polyethylene laminate which can be given a more
permanent deformation at a lower temperature and which has a lower
water permeability.
Heat sealing
The membrane was sealed to the cassette body using a hot aluminum
die with raised lands corresponding to the heat seal areas. A
pressure, corresponding to approximately 150 to 300 psi over the
actual seal area, was applied for 1 to 2 seconds. Following
application of the pressure, the die was either rapidly quenched by
water channels running through the die block, or the die was
lifted. Superior results were obtained by quenching the die. With a
2 mil thick membrane of a polyester/low-density polyethylene
laminate, sealed to a body of high-density polyethylene, a die
temperature of 156.degree. C. was used. A blister 0.5" in diameter
sealed in this manner withstood internal pressures in excess of 50
psi.
To preserve uniformity of seal over a cassette of extended size,
the cassette regions at the seal were formed into a raised ridge,
about 0.01" high. Variation in the amount the die deforms the base
material, originating from small variations in cassette thickness,
can then occur with a minimum variation in the volume of base
material displaced. This ridge structure was found to reduce the
extruded material in regions such as the well surrounding a
valve.
Bursapak.TM. structure
The outer seal of the Bursapak.TM. was made as described above. The
center seal was made using a die heated to temperature of about
156.degree. C. This die contained small independently sprung steel
pins which contacted the center seal. The lower conductivity of the
steel and the air gap between the pins and the die were designed to
restrict the amount of heat available for sealing. When the seal
was formed at the center in this way, melting of the cassette base
material was minimal, although the low-density polyethylene of the
membrane was above its melting point. This seal was demonstrated to
withstand an internal excess pressure of about 16 psi. Above this
pressure, it ruptured as required by the design and released the
contents of the Bursapak.TM. through the central port.
Liquid fill
Liquid fill of both Bursapaks and storage vessels similar to the
"waste vessel" of FIG. 1 was achieved. The input needle was
connected to a 2-way valve which could be switched between a vacuum
pump and a syringe supplying the fill liquid. Following exhaustion
of the vessel by the pump, for a few seconds, the valve was
switched and the vessel filled by the syringe. The filled vessel
then contained no air bubbles. Both a septum, as shown in FIG. 1,
and a simple entry port were used for filling. Sealing of the entry
channel was achieved by a hot rod, as indicated in FIG. 2, which
melted the channel closed but kept the polyester component of the
membrane sufficiently intact.
Valve operation
Valves, constructed as in FIGS. 1, 5 and 6, were fabricated
according to the above descriptions. A molded polyethylene body and
polyester/polyethylene membrane was used. Functioning was tested
using pneumatically operated steel plungers. With a plunger force
of approximately 0.8 lb. and a water pressure of 20 psi, the
leakage rate was less than 0.1 microliter per minute.
EXAMPLE 2
PCR Amplification Reaction
The following example illustrates one embodiment of the present
invention whereby a PCR amplification reaction is conducted in the
context of a cassette in a microfluidics device.
A PCR assay is conducted using the cassette 200 illustrated in
FIGS. 4A-4E, the device having alpha through delta first reaction
chambers 262A-D, which are used for lysing the cells in the
samples, and alpha through delta second reaction chambers 262A-D,
with each first reaction chamber 261- second reaction chamber 262
pair forming a separate reaction flow-way 265. The parallel
reaction device has a set of one upper auxiliary block, e.g. 1500A
and one lower auxiliary block, e.g. 1500B (not shown), for each of
first reaction chamber 261 and each second reaction chamber 262.
The cassette 200 has pumps for moving fluid from one chamber to
another chamber. For instance, the gas pressure flow control means
or the foot-pad pumps described above can be used to empty chambers
and push the fluid therefrom into another chamber. Valves located
between the various chambers contained in the device regulate this
flow of fluids between and among the chambers. The reaction
protocol is as follows:
1. Each of the four first reaction chambers 261 receives from a
connected first supply chamber 251 a suspension in 160 .mu.l of
paramagnetic DNA-binding beads having a diameter of 2-4 mils, that
can be used in the cell lysis stage to bind the DNA released from
the lysed cells (these beads are, e.g., Dynabeads.RTM. DNA
Direct.TM., available from Dynal, Lake Success, N.Y.). The beads
are locked in place in the lysing reaction chambers 261 using the
magnet 1100 and suspending liquid is drained into first waste
chamber 271. Alpha first reaction chamber 261A receives a fluid (40
.mu.l) from alpha fifth supply chamber 255A containing purified DNA
that includes the amplification sequence being tested for in an
amount sufficient to generate a positive result, thereby creating a
positive control. Beta first reaction chamber 261B receives from
beta fifth supply chamber 255B buffer solution or a biological
sample known to not contain the target sequence (40 .mu.l) in place
of the sample or positive control, and therefore serves as a
negative control. Blood sample (40 .mu.l), stored in sixth supply
chamber 256, is drawn into each of gamma and delta first supply
chambers 261C and 261D. The first reaction chambers 261 are then
filled with a lysis solution (100 .mu.l) that is drawn from alpha,
gamma, epsilon and eta third supply chambers 253A, C, E and F,
respectively. The lysis solution is a solution of amplification
buffer supplemented with 1.0% v/w Tween 20 (Sigma Chemical Co., St.
Louis, Mo.). (The lysis solution can be substituted with the
solution provided by Dynal.) The temperature of first reaction
chambers 261 is now maintained at 56.degree. C.
2. After 10 minutes, the lysis solution is emptied into first waste
chamber 271. The lysis solution which exits from alpha and beta
first reaction chambers 261A and 261B, respectively, contains the
cellular and serum residue of the blood sample. The DNA-binding
beads, to which the cellular DNA is bound, remain in first reaction
chambers 261.
3. Wash solution (100 .mu.l) composed of amplification buffer (40
mM NaCl, 20 mM Tris-HCl, pH 8.3, 5 mM MgSO.sub.4, 0.01% w/v
gelatin, 0.1% v/v Triton X-100, Sigma Chemical Co., St. Louis, Mo.)
is now introduced into first reaction chambers 261 from the
connected second supply chambers 252.
4. After 10 minutes, the wash solution is transferred out of first
reaction chambers 261 into first waste chamber 271.
5. Steps 3 and 4 are repeated.
6. Solutions (volume 30 .mu.l) containing appropriate primers for
amplifying the target sequence (0.5 .mu.M) are then drawn into
first reaction chambers 261 from the connected beta, delta, zeta
and theta third supply chambers 253B, 253D, 253F and 253H.
Solutions (volume 30 .mu.l) containing the needed nucleotide
triphosphates (0.2 mM each), are introduced from the connected
alpha, gamma, epsilon and eta fourth supply chambers 254A, 254C,
254E and 254F. Solutions (volume 30 .mu.l) containing Taq
polymerase (2 Units, available from Promega Corp., Madison, Wis.)
are introduced from the connected beta, delta, zeta and theta
fourth supply chambers 254B, 254D, 254F and 254H. The contents of
each of first reaction chambers 261 are then transferred to the
corresponding one of alpha through delta second reaction chambers
262A-D.
7. The controller then initiates a temperature program modelled on
the protocol described by Wu et al., Proc. Natl. Acad. Sci. USA 86:
2752-2760, 1989. The program first heats second reaction chambers
262 to a temperature of 55.degree. C. and maintains that
temperature for 2 minutes. Next, the controller cycles the
temperature between a replication temperature of 72.degree. C.
(maintained for 3 minutes) and a DNA strand separation temperature
of 94.degree. C. (maintained for 1 minute). After the replication
temperature incubation has been conducted 25 times, the material in
reaction chambers 262 is analyzed for the presence of the proper
amplified sequence.
While this invention has been described with an emphasis upon a
preferred embodiment, it will be obvious to those of ordinary skill
in the art that variations in the preferred composition and method
may be used and that it is intended that the invention may be
practiced otherwise than as specifically described herein.
Accordingly, this invention includes all modifications encompassed
within the spirit and scope of the invention as defined by the
following claims.
* * * * *