U.S. patent application number 15/137983 was filed with the patent office on 2016-10-27 for fluidic test cassette.
The applicant listed for this patent is Mesa Biotech, Inc.. Invention is credited to Martin Bouliane, Hong Cai, Robert B. Cary, Conrad Lindberg, Mark Nowakowski, Donald J. Thomas, Michael Wang.
Application Number | 20160310948 15/137983 |
Document ID | / |
Family ID | 57144321 |
Filed Date | 2016-10-27 |
United States Patent
Application |
20160310948 |
Kind Code |
A1 |
Nowakowski; Mark ; et
al. |
October 27, 2016 |
Fluidic Test Cassette
Abstract
A disposable cassette for detecting nucleic acids or performing
other assays. The cassette can be inserted into a base station
during use. The cassette has numerous features to ensure correct
operation of the device under gravity, such as vent pockets for
enabling the flow of sample fluid from one chamber to the next when
the vent pocket is unsealed. The vent pockets have protrusions to
help prevent accidental resealing. The cassette also can have a
gasket to ensure free air movement between open vent pockets. A
flexible circuit with patterned metallic electrical components
disposed on a heat stable material can be in direct contact with
fluid in the chambers and has resistive heating elements aligned
with the vent pockets and the chambers. The detection chamber,
which houses a lateral flow detection strip can have a space below
the strip that has sufficient capacity to accommodate an entire
volume of the sample fluid entering the detection chamber at a
height that enables the fluid to flow up the detection strip by
capillary action without flooding or otherwise bypassing regions of
the detection strip. The space can also contain detection
particles. Recesses in in the cassette channels or chambers can
have structures such as ridges or grooves to direct fluid flow to
enhance rehydration of lyophilized reagents disposed in the recess.
Flow diverters in the chambers can reduce the flow velocity of the
sample fluid and increase the effective fluid flow path length,
enabling more accurate control of fluid flow in the cassette.
Inventors: |
Nowakowski; Mark; (San
Diego, CA) ; Wang; Michael; (San Diego, CA) ;
Cary; Robert B.; (Santa Fe, NM) ; Cai; Hong;
(Los Alamos, NM) ; Lindberg; Conrad; (Fallbrook,
CA) ; Bouliane; Martin; (Carlsbad, CA) ;
Thomas; Donald J.; (Vista, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Mesa Biotech, Inc. |
San Diego |
CA |
US |
|
|
Family ID: |
57144321 |
Appl. No.: |
15/137983 |
Filed: |
April 25, 2016 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
62152724 |
Apr 24, 2015 |
|
|
|
62322738 |
Apr 14, 2016 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 2200/147 20130101;
B01L 2300/0825 20130101; B01L 2400/0406 20130101; B01L 2400/0677
20130101; B01L 3/50273 20130101; B01L 7/52 20130101; B01L 3/5023
20130101; B01L 2300/123 20130101; B01L 2200/0689 20130101; B01L
2400/0694 20130101; B01L 3/502715 20130101; B01L 2300/1827
20130101; C12Q 1/6816 20130101; B01L 2300/0883 20130101; B01L
2400/0457 20130101; B01L 3/502723 20130101; B01L 3/502738 20130101;
B01L 2200/0621 20130101; B01L 2200/16 20130101; C12Q 1/6816
20130101; C12Q 2565/629 20130101 |
International
Class: |
B01L 7/00 20060101
B01L007/00; C12Q 1/68 20060101 C12Q001/68; B01L 3/00 20060101
B01L003/00 |
Claims
1. A cassette for detecting a target nucleic acid, the cassette
comprising: a plurality of chambers; a plurality of vent pockets
connected to said chambers; and a heat labile material for sealing
one or more of said vent pockets; wherein at least one said vent
pocket comprises a protrusion.
2. The cassette of claim 1 wherein said protrusion comprises a
dimple or an asperity.
3. The cassette of claim 1 wherein said protrusion sufficiently
prevents molten heat labile material from attaching to a heat
stable material disposed adjacent to said heat labile material to
prevent resealing of said vent pocket after said heat labile
material is ruptured.
4. A cassette for detecting a target nucleic acid, the cassette
comprising: a plurality of chambers; a plurality of vent pockets
connected to said chambers; a heat labile material for sealing one
or more of said vent pockets; a heat stable material; and a gasket
disposed between said heat labile material and said heat stable
material, said gasket comprising an opening encompassing said
plurality of vent pockets.
5. The cassette of claim 4 wherein said gasket is sufficiently
thick to provide a sufficient air volume to equilibrate pressures
and ensure free air movement between open vent pockets.
6. The cassette of claim 4 further comprising a flexible circuit,
said flexible circuit comprising patterned metallic electrical
components disposed on said heat stable material.
7. The cassette of claim 6 wherein said gasket comprises a second
opening, or is limited in dimension, such that said flexible
circuit will be in direct contact with fluid in at least one of the
chambers.
8. The cassette of claim 6 wherein said electrical components
comprise resistive heating elements or conductive traces.
9. The cassette of claim 8 wherein said resistive heating elements
are aligned with said vent pockets and said chambers.
10. The cassette of claim 4 comprising one or more ambient
temperature sensors for adjusting a heating temperature, heating
time, and/or heating rate of one or more of said chambers.
11. A cassette for detecting a target nucleic acid, the cassette
comprising: a vertically oriented detection chamber; a lateral flow
detection strip disposed in said detection chamber oriented such
that a sample receiving end of said detection strip is at the
bottom end of said detection strip; and a space in said detection
chamber below said lateral flow detection strip for receiving fluid
comprising amplified target nucleic acids; said space comprising
sufficient capacity to accommodate an entire volume of the fluid at
a height that enables the fluid to flow up the detection strip by
capillary action without flooding or otherwise bypassing regions of
the detection strip.
12. The cassette of claim 11 wherein said space comprises detection
particles.
13. The cassette of claim 12 wherein said detection particles are
selected from the group consisting of dye polystyrene microspheres,
latex, colloidal gold, colloidal cellulose, nanogold, and
semiconductor nanocrystals.
14. The cassette of claim 12 wherein said detection particles
comprise oligonucleotides complementary to a sequence of the
amplified target nucleic acids or ligands capable of binding to the
amplified target nucleic acids.
15. The cassette of claim 14 wherein the ligands are selected from
the group consisting of biotin, streptavidin, a hapten or an
antibody.
16. The cassette of claim 12 wherein the detection particles have
been dried, lyophilized, or present on at least a portion of the
interior surface as a dried mixture of detection particles in a
carrier to facilitate resuspension of the detection particles.
17. The cassette of claim 16 wherein the carrier comprises a
polysaccharide, a detergent, or a protein.
18. The cassette of claim 12 wherein a capillary pool of the fluid
forms in the space, providing improved mixing and dispersion of the
detection particles to facilitate comingling of the detection
particles with the amplified target nucleic acid.
19. The cassette of claim 11 for performing an assay having a
volume less than about 200 .mu.L.
20. The cassette of claim 19 wherein the assay has a volume less
than about 60 .mu.L.
21. A cassette for detecting a target nucleic acid, the cassette
comprising one or more recesses for containing at least one
lyophilized or dried reagent, at least one of said recesses
comprising one or more structures for directing fluids to
facilitate rehydration of the at least one dried or lyophilized
reagent, said recesses disposed in one or more channels connected
to said chambers or in one or more of said channels.
22. The cassette of claim 21 wherein said structures comprise
ridges, grooves, dimples, or combinations thereof.
23. A cassette for detecting a target nucleic acid, the cassette
comprising at least one chamber comprising a feature to prevent
fluid vertically entering a top of said chamber from flowing
directly into an outlet of said chamber.
24. The cassette of claim 23 wherein said feature deflects the
fluid to the side of said chamber opposite from said outlet.
25. The cassette of claim 23 wherein a resulting flow path of the
fluid comprises a horizontal component, thereby sufficiently
increasing an effective length of the flow path and sufficiently
decreasing a flow velocity of the fluid to restrict an amount of
fluid exiting said outlet.
26. The cassette of claim 23 wherein said feature creates a
swirling of fluid within said chamber, thereby increasing mixing of
reagents within the fluid.
27. The cassette of claim 23 wherein said feature is triangular or
trapezoidal in shape.
28. The cassette of claim 23 wherein said outlet is tapered.
29. The cassette of claim 23 wherein a channel located downstream
of said outlet comprises turns for increasing an effective length
of said channel.
30. The cassette of claim 23 wherein said feature is located near
or at a bottom of said chamber or near a middle of said
chamber.
31. A method of controlling vertical flow of a fluid through a
chamber in a cassette for detecting a target nucleic acid, the
method comprising deflecting a flow of fluid entering a top of the
chamber, thereby preventing the fluid from flowing directly into an
outlet of the chamber.
32. The method of claim 31 comprising reducing a flow velocity of
the fluid, thereby reducing a distance the fluid flows down a
channel connected to the outlet before the fluid stops.
33. The method of claim 31 comprising dividing a flow of the fluid
into the chamber into a first fluid flow that contacts a wall of
the chamber and is directed upward, and a second fluid flow that
enters the outlet.
34. The method of claim 33 further comprising creating a swirling
of the fluid in the first fluid flow in the chamber, thereby
increasing mixing of reagents within the fluid.
35. The method of claim 33 wherein the second fluid flow forms a
meniscus and travels through a channel connected to the outlet, the
meniscus increasing pressure in closed air space in the channel
downstream of the fluid until the pressure stops the flow of fluid
in the channel.
36. The method of claim 35 wherein the outlet is tapered, thereby
increasing compressible air volume at the entrance to the
outlet.
37. The method of claim 31 comprising providing turns in a channel
connected to the outlet, thereby increasing an effective path
length of the channel and reducing a flow velocity of fluid in the
channel.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to and the benefit of
filing of U.S. Provisional Patent Application Ser. No. 62/152,724,
entitled "Fluidic Test Cassette", filed on Apr. 24, 2015, and U.S.
Provisional Patent Application Ser. No. 62/322,738, entitled
"Fluidic Test Cassette", filed on Apr. 14, 2016, the specifications
and claims of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention (Technical Field)
[0003] Embodiments of the present invention relate to an integrated
device and related methods for detecting and identifying nucleic
acids. The device may be fully disposable or may comprise a
disposable portion and a reusable portion.
[0004] 2. Background Art
[0005] Note that the following discussion may refer to a number of
publications and references. Discussion of such publications herein
is given for more complete background of the scientific principles
and is not to be construed as an admission that such publications
are prior art for patentability determination purposes.
[0006] As the public health impact and awareness of infectious and
emerging diseases, biothreat agents, genetic diseases and
environmental reservoirs of pathogens has increased, the need for
more informative, sensitive and specific point-of-use rapid assays
has increased the demand for polymerase chain reaction (PCR)-based
tools. Nucleic acid-based molecular testing by such methods as
PCR-based amplification is extremely sensitive, specific and
informative. Unfortunately, currently available nucleic acid tests
are unsuitable or of limited utility for field use because they
require elaborate and costly instrumentation, specialized
laboratory materials and/or multiple manipulations dependent on
user intervention. Consequently, most samples for molecular testing
are shipped to centralized laboratories, resulting in lengthy
turn-around-times to obtain the required information.
[0007] To address the need for rapid point-of-use molecular
testing, prior efforts have focused on product designs employing a
disposable cartridge and a relatively expensive associated
instrument. The use of external instrumentation to accomplish fluid
movement, amplification temperature control and detection
simplifies many of the engineering challenges inherent to
integrating the multiple processes required for molecular testing.
Unfortunately, dependence upon elaborate instrumentation imposes
tremendous economic barriers for small clinics, local and state
government and law enforcement agencies. Further, dependence upon a
small number of instruments to run tests could cause unnecessary
delays during periods of increased need, as occurs during a
suspected biowarfare agent release or an emerging epidemic. Indeed,
the instrument and disposable reagent cartridge model presents a
potentially significant bottleneck when an outbreak demands surge
capacity and increased throughput. Additionally, instrumentation
dependence complicates ad hoc distribution of test devices to
deployment sites where logistic constraints preclude transportation
of bulky associated equipment or infrastructure requirements are
absent (e.g. reliable power sources).
[0008] Gravity has been described as a means of fluid movement in
existing microfluidic devices. However, the typical device does not
allow for programmable or electronic control of such fluid
movement, or the mixing of more than two fluids. Also, some devices
utilize a pressure drop generated by a falling inert or
pre-packaged fluid to induce a slight vacuum and draw reactants
into processing chambers when oriented vertically, which increases
storage and transport complexities to ensure stability of the
pre-packaged fluids. Existing devices which teach moving a fluid in
a plurality of discrete steps require frangible seals or valves
between chambers, which complicates operation and manufacture.
These devices do not teach the use of separate, remotely located
vents for each chamber.
[0009] Typical microfluidic devices make use of smaller reaction
volumes than are employed in standard laboratory procedures. PCR or
other nucleic acid amplification reactions such as loop mediated
amplification (LAMP), nucleic acid based sequence amplification
(NASBA) and other isothermal and thermal cycling methods are
typically conducted in testing and research laboratories using
reaction volumes of 5 to 100 microliters. These reaction volumes
accommodate test specimen volumes sufficient to ensure the
detection of scarce assay targets in dilute specimens. Microfluidic
systems that reduce reaction volumes relative to those employed in
traditional laboratory molecular testing necessarily also reduce
the volume of specimen that can be added to the reaction. The
result of the smaller reaction volume is a reduction in capacity to
accommodate sufficient specimen volume to ensure the presence of
detectable amounts of target in dilute specimens or where assay
targets are scarce.
SUMMARY OF THE INVENTION
[0010] The present invention is a cassette for detecting a target
nucleic acid, the cassette comprising a plurality of chambers, a
plurality of vent pockets connected to the chambers, and a heat
labile material for sealing one or more of the vent pockets,
wherein at least one the vent pocket comprises a protrusion. The
protrusion preferably comprises a dimple or an asperity and
preferably sufficiently prevents molten heat labile material from
attaching to a heat stable material disposed adjacent to the heat
labile material to prevent resealing of the vent pocket after the
heat labile material is ruptured.
[0011] The present invention is also a cassette for detecting a
target nucleic acid, the cassette comprising a plurality of
chambers, a plurality of vent pockets connected to the chambers, a
heat labile material for sealing one or more of the vent pockets, a
heat stable material, and a gasket disposed between the heat labile
material and the heat stable material, the gasket comprising an
opening encompassing the plurality of vent pockets. The gasket is
preferably sufficiently thick to provide a sufficient air volume to
equilibrate pressures and ensure free air movement between open
vent pockets. The cassette preferably comprises a flexible circuit,
the flexible circuit comprising patterned metallic electrical
components disposed on the heat stable material. The gasket
preferably comprises a second opening, or is limited in dimension,
such that the flexible circuit will be in direct contact with fluid
in at least one of the chambers. The electrical components
preferably comprise resistive heating elements or conductive
traces. The resistive heating elements are preferably aligned with
the vent pockets and the chambers. The cassette preferably
comprises one or more ambient temperature sensors for adjusting a
heating temperature, heating time, and/or heating rate of one or
more of the chambers.
[0012] The present invention is also a cassette for detecting a
target nucleic acid, the cassette comprising a vertically oriented
detection chamber, a lateral flow detection strip disposed in the
detection chamber oriented such that a sample receiving end of the
detection strip is at the bottom end of the detection strip, and a
space in the detection chamber below the lateral flow detection
strip for receiving fluid comprising amplified target nucleic
acids, the space comprising sufficient capacity to accommodate an
entire volume of the fluid at a height that enables the fluid to
flow up the detection strip by capillary action without flooding or
otherwise bypassing regions of the detection strip. The space
preferably comprises detection particles such as dye polystyrene
microspheres, latex, colloidal gold, colloidal cellulose, nanogold,
or semiconductor nanocrystals. The detection particles preferably
comprise oligonucleotides complementary to a sequence of the
amplified target nucleic acids or ligands, such as biotin,
streptavidin, a hapten or an antibody, capable of binding to the
amplified target nucleic acids. The detection particles have
preferably been dried, lyophilized, or present on at least a
portion of the interior surface as a dried mixture of detection
particles in a carrier, such as a polysaccharide, a detergent, or a
protein, to facilitate resuspension of the detection particles. A
capillary pool of the fluid preferably forms in the space,
providing improved mixing and dispersion of the detection particles
to facilitate comingling of the detection particles with the
amplified target nucleic acid. The cassette optionally performs an
assay having a volume less than about 200 .mu.L, and preferably
less than about 60 .mu.L.
[0013] The present invention is also a cassette for detecting a
target nucleic acid, the cassette comprising one or more recesses
for containing at least one lyophilized or dried reagent, at least
one of the recesses comprising one or more structures for directing
fluids to facilitate rehydration of the at least one dried or
lyophilized reagent, the recesses disposed in one or more channels
connected to the chambers or in one or more of the channels. The
structures preferably comprise ridges, grooves, dimples, or
combinations thereof.
[0014] The present invention is also a cassette for detecting a
target nucleic acid, the cassette comprising at least one chamber
comprising a feature to prevent fluid vertically entering a top of
the chamber from flowing directly into an outlet of the chamber.
The feature preferably deflects the fluid to the side of the
chamber opposite from the outlet. The resulting flow path of the
fluid preferably comprises a horizontal component, thereby
sufficiently increasing the effective length of the flow path and
sufficiently decreasing the flow velocity of the fluid to restrict
the amount of fluid exiting the outlet. The feature preferably
creates a swirling of fluid within the chamber, thereby increasing
mixing of reagents within the fluid. The feature is preferably
triangular or trapezoidal in shape. The outlet is optionally
tapered. A channel located downstream of the outlet optionally
comprises turns for increasing an effective length of the channel.
The feature is preferably located near or at a bottom of the
chamber or near a middle of the chamber.
[0015] The present invention is also a method of controlling
vertical flow of a fluid through a chamber in a, the method
comprising deflecting a flow of fluid entering a top of the
chamber, thereby preventing the fluid from flowing directly into an
outlet of the chamber. The method preferably comprises reducing a
flow velocity of the fluid, thereby reducing a distance the fluid
flows down a channel connected to the outlet before the fluid
stops. The method preferably comprises dividing a flow of the fluid
into the chamber into a first fluid flow that contacts a wall of
the chamber and is directed upward, and a second fluid flow that
enters the outlet. The first fluid flow preferably swirls in the
chamber, thereby increasing mixing of reagents within the fluid.
The second fluid flow preferably forms a meniscus and travels
through a channel connected to the outlet, the meniscus increasing
pressure in closed air space in the channel downstream of the fluid
until the pressure stops the flow of fluid in the channel. The
outlet is optionally tapered, thereby increasing compressible air
volume at the entrance to the outlet. The method optionally
comprises providing turns in a channel connected to the outlet,
thereby increasing an effective path length of the channel and
reducing a flow velocity of fluid in the channel.
[0016] Objects, advantages and novel features, and further scope of
applicability of the present invention will be set forth in part in
the detailed description to follow, taken in conjunction with the
accompanying drawings, and in part will become apparent to those
skilled in the art upon examination of the following, or may be
learned by practice of the invention. The objects and advantages of
the invention may be realized and attained by means of the
instrumentalities and combinations particularly pointed out in the
appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The accompanying drawings, which are incorporated into and
form a part of the specification, illustrate embodiments of the
present invention and, together with the description, serve to
explain the principles of the invention. The drawings are only for
the purpose of illustrating certain embodiments of the invention
and are not to be construed as limiting the invention. In the
drawings:
[0018] FIG. 1A is a drawing illustrating an embodiment of a test
cassette of the present invention.
[0019] FIG. 1B is an exploded view of one embodiment of the test
cassette revealing the sliding seal, sample port, sample cup and
internal region of the expansion chamber.
[0020] FIG. 2A is a representation of the fluidic network in one
embodiment of a test cassette of the invention.
[0021] FIGS. 2B-2C are schematic representations prior to and after
vent opening, respectively, of how a heat triggered vent can be
employed to vent to an expansion chamber to accomplish fluid flow
control in the context of a hermetically sealed test cassette.
[0022] FIG. 2D is a drawing of one embodiment of a disposable test
cassette showing the placement of the printed circuit assembly
(PCA) comprising resistive heating elements and temperature
sensors.
[0023] FIG. 2E is a photograph of an injection molded plastic test
cassette that includes the features described in FIG. 2A.
[0024] FIG. 3A is a representation of the operating principle of an
embodiment of the expansion chamber.
[0025] FIG. 3B is a cross-section of the piston-based expansion
chamber prior to gas expansion within the test cassette.
[0026] FIG. 3C is a cross-section of the piston-based expansion
chamber after gas expansion within the test cassette.
[0027] FIG. 4A is an illustration of an approach to forming an
expansion chamber wherein an expandable bladder is employed to
provide an expanding internal volume.
[0028] FIG. 4B is a cross-section of the bladder-based expansion
chamber prior to gas expansion within the test cassette.
[0029] FIG. 4C is a cross-section of the bladder-based expansion
chamber after gas expansion within the test cassette.
[0030] FIG. 5A is an illustration of an approach to forming an
expansion chamber wherein an expandable bellows is employed to
provide an expanding internal volume.
[0031] FIG. 5B is a cross-section of the bellows-based expansion
chamber prior to gas expansion within the test cassette.
[0032] FIG. 5C is a cross-section of the bellows-based expansion
chamber after gas expansion within the test cassette.
[0033] FIG. 6A illustrates the use of a semi-permeable barrier,
membrane or material that allows gas to pass freely while particles
such as bacteria, viruses, or large molecules such as DNA or RNA
are retained within the device.
[0034] FIG. 6B is a cross-section of the semi-permeable barrier
employed in lieu of an expansion chamber to equalize internal
pressures with ambient pressures or to reduce internal
pressure.
[0035] FIG. 7 is an exploded view of a test cassette design wherein
an expansion chamber is created by a spacer between a layer of
biaxially oriented polystyrene (BOPS) film.
[0036] FIG. 8A is a drawing of an embodiment of a flexible circuit
comprising resistive heating elements for two fluid chambers, a
detection strip chamber and three vents and electrical contact
pads.
[0037] FIG. 8B is an embodiment of a flexible circuit comprising
resistive heating elements for two fluid chambers, a detection
strip chamber and three vents and electrical contact pads for
energizing the resistive heating elements.
[0038] FIG. 8C is an exploded view of an embodiment of a test
cassette.
[0039] FIG. 8D is a view of the assembled test cassette of FIG.
8C.
[0040] FIG. 9 depicts lateral flow strips from devices with and
without a capillary pool at the sample receiving end of the strip.
More uniform distribution of detection particles and more uniform
signal across the strip is observed when a capillary pool is
present.
[0041] FIG. 10 is a diagram illustrating a hierarchical approach to
sample splitting.
[0042] FIG. 11 is an illustration of a multiple channel fluidic
network for multiplexing and sample subdivision showing the fluid
flow path for each test. Additional fluidic paths or channels can
be incorporated into the network to further increase the number of
parallel tests that can be performed simultaneously in a single
disposable test cassette.
[0043] FIG. 12 is a representation of the fluidic network in one
embodiment of a test cassette of the invention wherein a sample is
split following introduction to the sample cup via the sample port
to enable parallel independent tests on the same input sample. A
bifurcating fluid path from the sample cup allows sample solution
to be split into two distinct fluidic channels or paths of the test
cassette to allow simultaneous tests to run in parallel on the
split sample.
[0044] FIG. 13A is a drawing of an assembled sample preparation
subsystem showing the internal component arrangement.
[0045] FIG. 13B is an exploded view of the sample preparation
subsystem showing components of the nucleic acid purification
apparatus configured for integration with a test cassette.
[0046] FIG. 14 is a cross-section through the sample preparation
subsystem illustrating the movements of components occurring in the
course of processing a sample.
[0047] FIG. 15 is an exploded view drawing showing the sample
preparation subsystem with hermetic seal components, injection
molded fluidic subsystem, corresponding cassette backing and
PCA.
[0048] FIG. 16 is a photograph of a test cassette embodiment with
an integrated sample preparation subsystem.
[0049] FIG. 17A is an exploded view drawing showing a sample
preparation subsystem with hermetic seal components and injection
molded fluidic subsystem design.
[0050] FIG. 17B is a drawing of the test cassette embodiment with
integrated sample preparation subsystem shown interfaced to the
PCA.
[0051] FIG. 17C is a cutaway drawing of the test cassette
embodiment depicting the fluidic paths, interfaced electronics and
sample preparation components.
[0052] FIG. 18A is a drawing of an embodiment of a docking unit of
the present invention shown with the lid in the open position and a
test cassette inserted.
[0053] FIG. 18B is a drawing of the docking unit shown with the lid
in the closed position.
[0054] FIG. 19 is a photograph of one embodiment of the docking
unit shown with the lid in the open position and a test cassette
inserted. The LCD display indicates detection of the insertion of
an influenza A/B test cassette.
[0055] FIG. 20 illustrates an embodiment of a cassette sealing
mechanism of the present invention.
[0056] FIG. 21A is a drawing of the cassette seal sensor placement
within the docking unit with an inserted cassette with the seal in
the open position.
[0057] FIG. 21B is a cutaway drawing of the cassette seal sensor
placement within the docking unit with an inserted cassette with
the seal in the open position.
[0058] FIG. 21C is a drawing of the cassette seal sensor placement
within the docking unit with an inserted cassette with the seal in
the closed position.
[0059] FIG. 21D is a cutaway drawing of the cassette seal sensor
placement within the docking unit with an inserted cassette with
the seal in the closed position.
[0060] FIG. 22 is a drawing of an embodiment of the cassette
sealing mechanism wherein a drive gear is employed to mediate seal
closure using a rotating valve.
[0061] FIG. 23A is a drawing illustrating an embodiment of the test
cassette wherein the lid is a hinged lid comprising an o-ring seal
and a vacant air volume that serves as an expansion chamber. In
this drawing the lid is in the open position.
[0062] FIG. 23B is a drawing showing the lid in the closed
position, where the o-ring forms a hermetic seal with the rim of
the sample port.
[0063] FIG. 24A is an exploded view of the heater board and test
cassette holder components of the docking unit forming the test
cassette receiving subassembly.
[0064] FIG. 24B is a drawing of an embodiment of the test cassette
receiving subassembly of the docking unit.
[0065] FIG. 25 is a slide view of the test cassette holder and
heater board mounting system in the engaged and disengaged
positions.
[0066] FIG. 26 is a drawing depicting the placement of infrared
temperature sensors in one embodiment of the docking unit to
monitor the temperature of first and second heated fluidic
chambers.
[0067] FIG. 27A is a drawing showing optical sensor placement
within an embodiment of the docking unit to allow reading of a
barcode located near the bottom of the test cassette.
[0068] FIG. 27B is a detail of FIG. 27A.
[0069] FIGS. 28A and 28B are exploded and assembled drawings,
respectively, of a double heat board configuration wherein the test
cassette is sandwiched between two heater board assemblies.
[0070] FIGS. 29A and 29B are solid and transparent drawings,
respectively, of a docking unit embodiment wherein a pivoting door
is used to receive a test cassette. Closure of the pivoting door
brings the rear of the test cassette into contact with the heater
board mounted within the docking unit.
[0071] FIGS. 30A and 30B are front and side cutaway views
respectively of the internal components of a docking unit
comprising servo motors for actuating sample preparation and an
optical system for test results collection.
[0072] FIGS. 31A and 31B are front and side view photographs of an
optical subsystem for an embodiment of the docking unit that
incorporates a test reader.
[0073] FIGS. 32A and 32B are photographs of a docking unit
embodiment with a pivoting test cassette receiving door in the open
position and closed position, respectively.
[0074] FIG. 33 shows a reusable subassembly for a docking unit of
the present invention.
[0075] FIG. 34 shows test results obtained in Example 1 described
herein.
[0076] FIG. 35 shows test results obtained in Example 2 described
herein.
[0077] FIG. 36 shows test results obtained in Example 3 described
herein.
[0078] FIG. 37A is a perspective view of a cassette comprising
three chambers.
[0079] FIG. 37B is an exploded view of the cassette of FIG.
37A.
[0080] FIG. 38 is a transparent view of the cassette of FIG. 37A
showing fluidic features.
[0081] FIG. 39 shows an embodiment of a chamber of the present
invention comprising a triangular protruding flow feature and a
tapered outlet.
[0082] FIG. 40 shows an embodiment of a chamber of the present
invention comprising a triangular protruding flow feature and a
parallel outlet.
[0083] FIG. 41 shows an embodiment of a chamber of the present
invention comprising a trapezoidal protruding flow feature and a
parallel outlet.
[0084] FIG. 42 shows an embodiment of a chamber of the present
invention comprising stacked triangular flow features and a
parallel outlet.
[0085] FIG. 43 shows an embodiment of a chamber of the present
invention comprising a protruding flow feature in approximately the
middle of the chamber.
[0086] FIG. 44 shows a reagent recess comprising internal features
for directing fluid flow.
[0087] FIG. 45 shows an embodiment of a vent pocket of the present
invention comprising a dimple structure.
DETAILED DESCRIPTION OF THE INVENTION
[0088] An embodiment of the present invention is a sealable
disposable platform for detecting a target nucleic acid, the
disposable platform preferably comprising a sample chamber for
receiving a sample comprising the target nucleic acid, an
amplification chamber connected via a first channel to the sample
chamber and connected via a second channel to a first vent pocket,
a labeling chamber connected via a third channel to the
amplification chamber and connected via a fourth channel to a
second vent pocket, a detection subsystem connected to the labeling
chamber via a fifth channel and connected via a sixth channel to a
third vent pocket, a plurality of resistive heating elements, and
one or more temperature measuring devices, wherein the vent pockets
are each sealed from communication with an air chamber by a heat
labile material in a suitable form, such as a membrane, a film, or
a plastic sheet located in a vicinity of one or more of the
resistive heating elements. The disposable platform optionally
comprises a seal to seal the platform prior to the initiation of
the detection assay. The disposable platform preferably comprises
recesses along channels between chambers to accommodate the
incorporation of dried or lyophilized reagents into the disposable
platform. These recesses may optionally comprise structures on one
or more of the surfaces facing the reagent(s) to assist with
directing fluids, preferably using capillarity or surface tension
effects, to the enclosed dried reagents to facilitate rehydration
of the dried reagents. Such features may comprise ridges, such as
ridge 7001 of FIG. 44, grooves, dimples or other structures to
direct fluids to the internal space of the recess as the fluid
passes through the recess, or otherwise assist in fluid flow to the
internal space of the recess during fluid flow. Alternatively, a
recess may be directly located within one (or more) of the
chambers.
[0089] The disposable platform optionally further comprises a
sample preparation stage comprising an output in direct fluid
connection with an input of the sample chamber. Dimensions of a
substantially flat surface of the amplification chamber are
preferably approximately the same as dimensions of a substantially
flat surface of a resistive heating element in thermal contact with
the amplification chamber. The amplification chamber optionally
contains an amplification solution and a recess in the channel from
the sample chamber to the amplification chamber optionally
comprises a lyophilized amplification reagent mix, and there is
preferably a recess in the channel from the amplification chamber
to the labeling chamber comprising dried or lyophilized detection
particles. The amplification and labeling chambers are preferably
heatable using resistive heating elements. The detection subsystem
preferably comprises a lateral flow strip that comprises detection
particles. The chambers, the channels, and the vent pockets are
preferably located on a fluid assembly layer, and the electronic
elements of the device are preferably located on a separate layer
comprising a printed circuit board, the separate layer bonded to
the fluid assembly layer or placed in contact with the fluid
assembly layer by a docking unit. The detection subsystem is
preferably located on the fluid assembly layer or optionally on a
second fluid assembly layer. The volume of at least one of the
chambers is preferably between approximately 1 microliter and
approximately 150 microliters. The disposable platform preferably
further comprises a connector for docking the disposable platform
with a docking unit or docking unit, which preferably maintains the
disposable platform in a vertical or tilted orientation and
optionally provides electrical contacts, components and/or a power
supply.
[0090] An embodiment of the present invention is a method for
detecting one or more target nucleic acids, the method preferably
comprising dispensing a sample comprising the target nucleic acid
in a sample chamber of a disposable platform; orienting the
disposable platform vertically or at a tilt; opening a first vent
pocket connected to an amplification chamber to an enclosed air
volume, thereby enabling the sample to flow into the amplification
chamber, reacting the sample with a previously lyophilized
amplification reagent mix located in a recess of the channel
between sample chamber and amplification chamber, amplifying the
target nucleic acid in the amplification chamber, opening a second
vent pocket connected to a labeling chamber to an enclosed air
volume, thereby enabling the amplified target nucleic acid to flow
into the labeling chamber, labeling the amplified target nucleic
acid using detection particles in a recess in the channel between
the amplification chamber and the labeling chamber, opening a third
vent pocket connected to a detection subsystem to an enclosed air
volume, thereby enabling the labeled target nucleic acid to flow
into the detection subsystem, and detecting the amplified target
nucleic acid. The amplifying step preferably comprises amplifying
the target nucleic acid using a resistive heating element located
within the disposable platform in a vicinity of the amplification
chamber. The method preferably further comprises passively cooling
the amplification chamber. The method preferably further comprises
heating the labeling chamber during the labeling step using a
resistive heating element located within the disposable platform in
a vicinity of the labeling chamber. The method preferably further
comprises controlling operation of the disposable platform by using
a docking unit which is not an external instrument.
[0091] Embodiments of the present invention comprise a disposable
platform which integrates external instrument-independent means of
conducting all requisite steps of a nucleic acid molecular assay
and complements current immuno-lateral flow rapid assays with a new
generation of nucleic acid tests offering more informative and
sensitive analyses. Embodiments of the present invention facilitate
the broader use of rapid nucleic acid testing in small clinics and
austere or remote settings where infectious disease, biothreat
agent, agriculture and environmental testing are the most likely to
have the greatest impact. Certain embodiments of the present
invention are completely self-contained and disposable which
enables "surge capacity" in times of increased demand by allowing
parallel tests to be run without external instrument-imposed
bottlenecks. Additionally, in those application areas where a low
cost disposable cartridge coupled with an inexpensive
battery-powered or AC adapter energized docking unit is preferable,
an embodiment of the invention where a simple docking unit is
employed further reduces test costs by placing reusable components
in a reusable yet inexpensive base. The platform technology
disclosed herein offers sensitivity similar to laboratory nucleic
acid amplification-based methods, minimal user intervention and
training requirements, sequence specificity imparted by both
amplification and detection, multiplex capacity, stable reagents,
compatibility with low-cost large-scale manufacturing, battery or
solar powered operation to allow use in austere settings, and a
flexible platform technology allowing the incorporation of
additional or alternative biomarkers without device redesign.
[0092] Embodiments of the present invention provide systems and
methods for low-cost, point-of-use nucleic acid detection and
identification suitable to perform analyses in locations remote
from a laboratory environment where testing would ordinarily be
performed. Advantageously, nucleic acid amplification reaction
volumes can be in the same volume range commonly used in
traditional laboratory testing (e.g. 5-150 .mu.L). The reaction
conducted in embodiments of the present invention is thus directly
comparable to accepted laboratory assays, and allows the
accommodation of the same specimen volumes typically employed in
traditional molecular testing. Furthermore, the amplification of
nucleic acids preferably takes place in a hermetically sealed test
cassette that is preferably permanently sealed prior to the
initiation of amplification. Retaining amplified nucleic acids
within a sealed system prevents contamination of the testing
environment and surrounding areas with amplification products and
therefore reduces the likelihood subsequent tests will generate
false positive results. The integration of a sealing system into
the test cassette enables the use of a corresponding seal
engagement system in the docking unit to enforce the formation of a
seal at the time of assay initiation. In an embodiment of the
invention, a rack and pinion mechanism is employed to slide a test
cassette integrated sealing mechanism into place to ensure seal
closure prior to amplification. A sensor placed in the docking unit
interrogates the test cassette to confirm the seal has been formed
prior to initiating the test reaction.
[0093] Embodiments of the present invention may be produced using
injection molding processes and ultrasonic welding to achieve
high-throughput manufacture and low cost disposable components. In
some embodiments one or more recesses are provided in the fluidic
component to each accommodate a dried reagent pellet. The recesses
enable the use of lyophilized or otherwise dried materials to be
present in the fluidic component during final assembly when
ultrasonic welding may be used without disruption of the pellet by
any energy introduced to the system during the welding.
[0094] Embodiments of the present invention may be used to detect
the presence of a target nucleic acid sequence or sequences in a
sample. Target sequences may be DNA such as chromosomal DNA or
extra-chromosomal DNA (e.g. mitochondrial DNA, chloroplast DNA,
plasmid DNA, etc.) or RNA (e.g. rRNA, mRNA, small RNAs, or viral
RNA). Similarly, embodiments of the invention may be used to
identify nucleic acid polymorphisms including single nucleotide
polymorphisms, deletions, insertions, inversions and sequence
duplications. Further, embodiments of the invention may be used to
detect gene regulation events such as gene up- and down-regulation
at the level of transcription. Thus, embodiments of the invention
may be employed for such applications as: 1) the detection and
identification of pathogen nucleic acids in agricultural, clinical,
food, environmental and veterinary samples; 2) detection of genetic
biomarkers of disease; and 3) the diagnosis of the presence of a
disease or a metabolic state through the detection of relevant
biomarkers of the disease or metabolic state, such as gene
regulation events (mRNA up- or down regulation or the induction of
small RNAs or other nucleic acid molecules generated or repressed
during a disease or metabolic state) that occur in response to the
presence of a pathogen, toxin, other etiologic agent, environmental
stimulus or metabolic state.
[0095] Embodiments of the present invention comprise a means of
target nucleic acid sample preparation, amplification, and
detection upon addition of a nucleic acid sample, comprising all
aspects of fluid control, temperature control, and reagent mixing.
In some embodiments of the invention, the device provides a means
of performing nucleic acid testing using a portable power supply
such as a battery, and is fully disposable. In other embodiments of
the invention, a disposable nucleic acid test cartridge works in
conjunction with a simple reusable electronic component which can
perform all of the functions of laboratory instrumentation such as
an external instrument typically required for nucleic acid testing
without requiring the use of such laboratory instrumentation or
external instrument.
[0096] Embodiments of the present invention provide for a nucleic
acid amplification and detection device comprising, but not limited
to, a housing, a circuit board, and a fluidic or microfluidic
component. In certain embodiments, the circuit board may contain a
variety of surface-mount components such as resistors, thermistors,
light-emitting diodes (LEDs), photo-diodes, and microcontrollers.
In certain embodiments the circuit board may comprise a flexible
circuit board comprising a heat stable substrate such as polyimide.
Flexible circuits may, in some embodiments, comprise copper or
other conductive coatings or layers deposited onto or bonded to the
heat stable substrate. These coatings can be etched or otherwise
patterned to so as to comprise the resistive heating elements used
for biochemical reaction temperature control and/or conductive
traces to accommodate such heaters and/or surface mount components,
such as resistors, thermistors, light-emitting diodes (LEDs),
photo-diodes, and microcontrollers. The fluidic or microfluidic
component is the device portion which receives, contains, and moves
aqueous samples and may be made from a variety of plastics and by a
variety of manufacturing techniques, including ultrasonic welding,
bonding, fusing or lamination, laser cutting, water-jet cutting,
and/or injection molding. The fluidics and circuit board components
are held together either reversibly or irreversibly, and their
thermal coupling may be enhanced by heat conducting materials or
compounds. The housing preferably serves in part as a cosmetic and
protective sheath, hiding the delicate components of the
microfluidic and circuit board layers, and may also serve to
facilitate sample input, buffer release, nucleic acid elution, seal
formation and the initiation of processes required for device
functionality. For example, the housing may incorporate a sample
input port, a mechanical system for the formation or engagement of
a seal, a button or similar mechanical feature to allow user
activation, buffer release, sample flow initiation, nucleic acid
elution, and thermal or other physical interface formation between
electronic components and fluidic components.
[0097] In some embodiments of the invention, the fluidic or
microfluidic component comprises a series of chambers in controlled
fluid communication where the chambers are optionally
temperature-controlled, thereby subjecting the fluid contained
therein to programmable temperature regimens. In some embodiments
of the invention, the fluidic or microfluidic component comprises
five chambers, preferably including an expansion chamber, a sample
input chamber, a reverse transcription chamber, an amplification
chamber, and a detection chamber. The sample input chamber
preferably comprises a conduit to the expansion chamber, a sample
input orifice where a nucleic acid containing sample may be added,
a first recess wherein dried materials may be placed during
manufacture for mixing with the input sample, an egress conduit
leading to a second recess wherein dried materials may be placed
during manufacture and a conduit leading therefrom to the reverse
transcription chamber. In other embodiments functions of two or
more of the chambers are consolidated into a single chamber,
enabling the use of fewer chambers.
[0098] The first and second recesses may also comprise lyophilized
reagents that may include, for example, suitable buffers, salt,
deoxyribonucleotides, ribonucleotides, oligonucleotide primers, and
enzymes such as DNA polymerase and reverse transcriptase. Such
lyophilized reagents are preferably solubilized upon entrance of
the nucleic acid sample into the recess. In some embodiments of the
invention the first recess comprises salts, chemicals and buffers
useful for the lysis of biological agents and/or the stabilization
of nucleic acids present in the input sample. In some embodiments
of the invention the input sample is heated in the sample input
chamber to accomplish the lysis of cells or viruses present in the
sample. In some embodiments of the invention the second recess
comprises lyophilized reagents and enzymes such as reverse
transcriptase useful for the synthesis of cDNA from RNA. In an
embodiment of the invention the second recess is sufficiently
isolated from the sample input chamber to allow materials within
the second recess to maintain a lower temperature than the
temperature of the sample input chamber during heating. In some
embodiments of the invention the reverse transcription chamber
comprises a conduit comprising a third recess comprising
lyophilized reagents for the amplification of nucleic acids. The
sample input chamber, the reverse transcription chamber, the
amplification chamber and the detection chamber are preferably
situated in register with and in sufficient proximity to the heater
elements on the heater circuit board to provide thermal conduction
when mounted to the heater board either directly or through
insertion of the fluidic or microfluidic component or cassette into
a docking unit. Similarly, electronic components present on the
heater circuit board are preferably placed in physical contact or
proximity to vent pockets in the fluidic component to enable
electronic control by opening of the vent. The heater circuit board
physical layout is designed to provide registration with elements
of the fluidic or microfluidic component such that resistive
heating elements of the heater circuit board for lysis, reverse
transcription, amplification, hybridization, and/or fluid flow
control are situated to form a thermal interface with elements of
the fluidic component with which they interact.
[0099] In some embodiments of the invention the fluidic or
microfluidic component preferably comprises five chambers,
including a sample input chamber, a lysis chamber, a reverse
transcription chamber, an amplification chamber, and a detection
chamber and recesses for dried or lyophilized reagents located
along the channels between each chamber. In this embodiment reverse
transcription of RNA to cDNA and the amplification of cDNA occur in
separate chambers. In this embodiment, a first recess, located
along the conduit leading from the sample input cup to the lysis
chamber, comprises salts, chemicals (e.g. dithiothreitol) and
buffers (e.g. to stabilize, increase, or decrease pH) useful for
the lysis of biological agents and/or the stabilization of nucleic
acids present in the input sample. In some embodiments of the
invention the input sample is heated in the heat lysis chamber
having first flowed from the sample input cup through the first
recess wherein the sample has optionally comingled with the
substances that comprise the first recess. In other embodiments of
the invention, lysis is accomplished by means of chemical treatment
resulting from the comingling of the sample with chemicals in the
first recess and the incubation of the sample in the presence of
these chemicals in the lysis chamber.
[0100] After substantial completion of treatment in the lysis
chamber, the sample solution is released by means of electronic
control of a heater that non-mechanically ruptures a vent to allow
the sample solution to flow via a channel through a second recess
and into the reverse transcription chamber. Said second recess may
optionally comprise lyophilized reagents that may include suitable
buffers, salt, deoxyribonucleotides, ribonucleotides,
oligonucleotide primers, and enzymes such as DNA polymerase and/or
reverse transcriptase required to accomplish the reverse
transcription of RNA in the sample into cDNA. Following the
substantial completion of a reverse transcription reaction, a
second vent is opened to release the sample solution to flow
through a channel and third recess comprised of reagents required
for nucleic acid amplification such as lyophilized reagents that
may include suitable buffers, salt, deoxyribonucleotides,
ribonucleotides, oligonucleotide primers, and enzymes such as DNA
polymerase and into an amplification chamber.
[0101] Following the substantial completion of nucleic acid
amplification in the amplification chamber a third vent is opened
to release the sample solution to a channel leading to the
detection chamber. Said channel may optionally but preferably
comprise a fourth recess comprising dried or lyophilized detection
reagents such as chemicals and/or detection particle conjugates
useful for the detection of nucleic acids in the detection chamber.
The detection chamber preferably comprises a capillary pool,
reagents for the detection of the amplified nucleic acid and a
lateral flow detection strip. The capillary pool preferably
provides a space of sufficient capacity to accommodate the entire
volume of fluid in the detection chamber at a height that enables
the fluid to flow up the detection strip by capillary action
without flooding or otherwise bypassing the regions of the
detection strip designed to receive the fluid for correct capillary
migration up the detection strip. In some embodiments of the
invention the detection reagents are lyophilized reagents. In some
embodiments of the invention the detection reagents comprise dyed
polystyrene microspheres, colloidal gold, semiconductor
nanocrystals, or cellulose nanoparticles. The sample solution
comingles with the detection reagents in the detection chamber and
flows by capillary action up the detection strip. Microheaters in
register with the detection chamber may optionally be employed to
control the temperature of the solution as it migrates up the
detection strip.
[0102] In some embodiments of the invention the amplification
reaction is an asymmetric amplification reaction wherein one primer
of each primer pair in the reaction is present at a concentration
different from the other primer of a given pair. Asymmetric
reactions can be useful for the generation of single-stranded
nucleic acid for the facilitation of detection by hybridization.
Asymmetric reactions can also be useful for generating amplicons in
a linear amplification reaction allowing quantitative or
semi-quantitative analysis of target levels in a sample.
[0103] Other embodiments of the invention comprise a nucleic acid
reverse transcription, amplification and detection device that is
integrated with a sample preparation device. Embodiments including
the sample preparation device provide a means for the communication
of fluids between sample preparation subsystem output ports or
valves and the input port or ports of the fluidic or microfluidic
components of the device.
[0104] Other embodiments of the invention comprise a means of
splitting the input sample into two or more fluid paths in the
fluidic or microfluidic component. A means of splitting the input
sample comprises a branched conduit to carry input fluids to a
metering chamber of a volume designed to divide the fluid across
multiple fluid paths. Each metering chamber comprises a channel
conduit to a vent pocket and a channel conduit to the next chamber
in the fluid path, for example a lysis chamber or a reverse
transcription chamber or an amplification chamber.
[0105] Unless otherwise defined, all terms of art, notations and
other scientific terminology used herein are intended to have the
meanings commonly understood by those of skill in the art to which
this invention pertains. The techniques and procedures described or
referenced herein are generally well understood and commonly
employed using conventional methodologies by those skilled in the
art, such as, for example, the widely utilized molecular cloning
methodologies described in Sambrook et al., Molecular Cloning: A
Laboratory Manual 3rd. edition (2001) Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. and Current Protocols in Molecular
Biology (Ausbel et al., eds., John Wiley & Sons, Inc. 2001. As
appropriate, procedures involving the use of commercially available
kits and reagents are generally carried out in accordance with
manufacturer defined protocols and/or parameters unless otherwise
noted.
[0106] As used throughout the specification and claims, the terms
`target nucleic acid` or `template nucleic acid` mean a
single-stranded or double-stranded DNA or RNA fragment or sequence
that is intended to be detected.
[0107] As used throughout the specification and claims, the terms
`microparticle` or `detection particle` mean any compound used to
label nucleic acid product generated during an amplification
reaction, including fluorescent dyes specific for duplex nucleic
acid, fluorescently modified oligonucleotides, and
oligonucleotide-conjugated quantum dots or solid-phase elements
such as a polystyrene, latex, cellulose or paramagnetic particles
or microspheres.
[0108] As used throughout the specification and claims, the term
`chamber` means a fluidic compartment where fluid resides for some
period of time. For example, a chamber may be the sample chamber,
amplification chamber, labeling chamber, or the detection
chamber.
[0109] As used throughout the specification and claims, the term
"cassette" is defined as a disposable or consumable cassette,
housing, component, or cartridge used in performing an assay or
other chemical or biochemical analysis. A cassette may be single
use or multiple use.
[0110] As used throughout the specification and claims, the term
`pocket` means a compartment that serves as a venting mechanism. A
pocket is preferably adjacent or overlaid to a resistor or other
mechanism to open the pocket. For example, unlike fluidic chambers
as described above, a pocket created in the fluidic component of
the cassette may have one open face that aligns with a resistor on
the PCA. This open face is preferably covered by a thin membrane,
film, or other material to create a sealed cavity that is easily
ruptured by energizing the underlying resistor.
[0111] As used throughout the specification and claims, the term
`channel` means a narrow conduit within the fluidic assembly which
typically connects two or more chambers and/or pockets or
combinations thereof, including, for example, an inlet, outlet, or
a vent channel. In the case of an inlet or outlet channel, fluid
sample migrates through the channel. In the case of a vent channel,
the conduit preferably remains clear of fluid and connects a
fluidic chamber to a vent pocket.
[0112] As used throughout the specification and claims, the term
"external instrument" means a reusable instrument that has one or
more of the following characteristics: performs a mechanical action
on a disposable assay or cassette other than sealing the cassette,
including but not limited to piercing buffer packets and/or pumping
or otherwise actively providing a transport force for fluids,
comprises moving parts to control valves and other components for
fluid flow control in the cassette or disposable assay, controls
fluid flow other than by selective heating of the assay, or
requires periodic calibration.
[0113] As used throughout the specification and claims, the term
"docking unit" means a reusable device that controls assays but
does not have any of the characteristics listed above for external
instruments.
[0114] Embodiments of the present invention are devices for
low-cost, point-of-use nucleic acid testing suitable to perform
analyses in locations remote from a laboratory environment where
testing would ordinarily be performed. Certain devices comprise
fluidic and electronic components or layers, optionally encased by
a protective housing. In embodiments of the present invention, the
fluidic component is composed of plastic and comprises a series of
chambers and pockets connected by narrow channels in which chambers
are oriented vertically with respect to one another during
operation. The fluidic component is overlaid or otherwise placed in
physical contact with electronic components, preferably controlled
via a microcontroller, such as a printed circuit board containing
off-the-shelf surface mount devices (SMDs), and/or a flexible
circuit comprising etched conductive material to form resistive
heating elements and optionally containing SMDs. In some
embodiments of the device, the entire assembly is disposable. In
other embodiments, the fluidic and physically bonded electronic
layers are disposable, while a small inexpensive controlling unit
is reusable. In another embodiment, the fluidic component is
disposable, and a small controlling docking unit or docking unit is
reusable. For all embodiments, the present invention may be
integrated with a nucleic acid sample preparation device such as
that described in International Publication No. WO 2009/137059 A1,
entitled "Highly Simplified Lateral Flow-Based Nucleic Acid Sample
Preparation and Passive Fluid Flow Control" (incorporated herein by
reference), and/or use methods described therein.
[0115] Embodiments of the present invention comprise an integrated
nucleic acid testing device that can be manufactured inexpensively
with established manufacturing processes. The invention provides
molecular test data while retaining the simplicity from the
end-user perspective of widely accepted hand-held immunoassays,
overcoming the challenges of regulating fluid temperatures within
the device, transporting small sample volumes in sequential steps,
reagent addition, reagent mixing, detecting nucleic acids. In some
embodiments of the invention subsystems for collecting,
interpreting, reporting and/or transmitting assay results are
incorporated into the invention. Embodiments of the present
invention are uniquely adapted to utilize off-the-shelf electronic
elements that may be constructed by standard assembly techniques,
and requires no or few moving parts. Furthermore, the fluid layer
design enables the use of readily available plastics and
manufacturing techniques. The result is an inexpensive, disposable,
and reliable device capable of nucleic acid isolation,
amplification, and detection without the need for a dedicated
laboratory infrastructure.
[0116] Existing nucleic acid testing devices generally use
sophisticated heating elements such as deposited film heaters and
Peltier devices that add significant cost and/or require
specialized manufacturing methods. In embodiments of the invention,
heating of the reaction solution is preferably accomplished by use
of simple resistive surface-mount devices that may be purchased for
pennies or less and are assembled and tested by common
manufacturing standards. By layering fluidic chambers over these
resistive elements and associated sensor elements, the fluid
temperature of the reaction solutions may be conveniently
regulated. The broad use of SMD resistors and flexible circuits in
the electronics industry ensures that the present invention is
amenable to well established quality control methods. In other
embodiments of the invention, resistive heating is realized using
heating elements formed by patterns fabricated in the conductive
layer of a flexible circuit substrate. Many nucleic acid
amplification techniques, such as PCR, require not only rapid
heating of the reaction solution but rapid cooling as well.
Reaction chambers in the present invention are preferably heated on
one side and the ambient temperature across the opposite face is
used to help reduce fluid temperature. In addition, vertical
orientation of embodiments of the device allows for more rapid
cooling by passive convection than if the device was oriented
horizontally, thus, reducing the thermal cycle period without the
use of costly devices such as Peltier devices. In some embodiments
of the invention a fan is used to facilitate cooling.
[0117] Fluid control is another challenge associated with low-cost
nucleic acid test device designs. Devices known in the art
generally employ electromechanical, electrokinetic, or
piezoelectric pumping mechanisms to manipulate fluids during device
operation. These pumping elements increase both device complexity
and cost. Similarly, valves making use of elaborate micromechanical
designs or moving parts can increase fabrication costs and reduce
reliability due to complications such as moving part failure or
bio-fouling. Unlike previously described nucleic acid testing
devices, embodiments of the present invention utilize hydrostatic
pressure under microcontroller control together with capillary
forces and surface tension to manipulate fluid volumes. The
vertical orientation of some embodiments of the present invention
allows for the reaction solution to cascade from chamber to chamber
under microcontroller control to accommodate required manipulations
of the assay. Fluid may be held in individual reaction chambers
through a balance of channel size, hydrostatic pressure and surface
tension, where surface tension and hydrostatic pressure prohibits
fluid advancement by gas displacement. A sample advances to the
lower chamber preferably only after activation of a simple venting
mechanism under microcontroller control. Once open, the vent allows
fluid to move from a first chamber to a second chamber by means of
providing a path for displaced air to escape from the second
chamber as fluid enters. Each chamber (or each channel between
chambers) within the fluidic component preferably connects to a
sealed vent pocket through a narrow vent channel. The vent pocket
is preferably sealed on one face with a thin, heat labile plastic
membrane or sheet that is easily ruptured by heating a small
surface mount resistor underlying, near, or adjacent to the
membrane or sheet. Once the vent of a lower chamber is opened,
fluid advancement proceeds, even under low hydrostatic
pressures.
[0118] As more specifically described below, the fluidic or
microfluidic vent mechanism used in some embodiments of the present
invention preferably employs a heating element in thermal and
(optional) physical contact with a heat labile seal to enable
electronic control of fluid movement by means of venting a chamber
of lower elevation to allow a fluid from a chamber of higher
elevation to flow into the lower chamber. In one embodiment, a
resistor is mounted on a printed circuit board, using widely used
and well-established electronics manufacturing methods, and placed
in physical contact with a channel seal comprising a heat labile
material. When energized the surface mount resistor generates
sufficient heat to rupture the seal, which results in the venting
of the chamber to allow equilibration of pressure in the region or
chamber where fluid is being moved with the region or chamber where
fluid is resident prior to venting. The equilibration of the
pressure between the chambers allows the movement of fluid from a
chamber of higher elevation to a chamber of lower elevation. A
direct seal between higher and lower elevation chambers is
preferably not employed. The channel and vent seal may be located
remotely from the fluid chambers, thus facilitating fluidic device
layout in configurations efficient for manufacture. The seal
material may comprise any material that can seal the vent channel
and be ruptured from heating as described, for example a thin
plastic sheet. This approach to fluid movement control in the
apparatus benefits from low materials costs, suitability for
manufacture using established manufacturing techniques while
providing the capacity to move fluids through a series of chambers
under the control of electronic control circuits such as
microprocessors or microcontrollers. The use of vents, a heat
labile material to seal the vents (and not to seal the fluid
chambers or fluid microchannels themselves) and an electronic means
of breaking said seal with heat provides a means of controlling
fluid flow through the device to enable movement of fluid at
predetermined times or following the completion of specific events
(for example, attaining a temperature, a temperature change or a
series of temperature changes, or the completion of an incubation
time or times or other events). In some embodiments, a blockage may
be introduced to the channel between chambers when gas phase water
must be isolated from a chamber connected by said channel. The
blockage may be a soluble material that dissolves upon contact with
liquid water following vent opening or a readily melted material
such as paraffin that can be removed by the introduction of heat to
the site of blockage.
[0119] In addition, the vent approach has a number of advantages
over sealing the fluid chambers themselves. Vent pockets can be
located anywhere on the fluidics layout and simply communicate with
the chamber they regulate via a vent channel. From a manufacturing
standpoint, vent pockets can be localized so that only a single
sealing membrane for all vent pockets (which may comprise a vent
pocket manifold) is affixed to the fluidic component, preferably by
well established methods such as adhesives, heat lamination,
ultrasonic welding, laser welding etc. In contrast, directly
sealing a fluid chamber requires that the seal material be placed
at different locations corresponding to each chamber location,
which is more difficult to manufacture. This presents a more
challenging scenario during manufacture compared to a single vent
pocket manifold sealed by a single membrane. Additionally, if
chambers are directly sealed, melted sealing material can remain in
the channels between chambers, occluding flow. The viscosity of the
sealing material may require more pressure in the fluid column than
is obtained in a miniaturized gravity driven apparatus.
[0120] In embodiments of the present invention, reagent mixing
requires no more complexity than other systems. Reagents necessary
for nucleic acid amplification such as buffers, salts,
deoxyribonucleotides, oligonucleotide primers, and enzymes are
preferably stably incorporated by use of lyophilized pellets or
cakes. These lyophilized reagents, sealed in a fluidic chamber, a
recess in a fluidic chamber or a recess in a channel, may be
readily solubilized upon contact with aqueous solution. In the case
that additional mixing is required, the vertical orientation of
embodiments of the present invention offers opportunities for novel
methods of mixing solutions. By utilizing heaters underlying
fluidic chambers, gas may be heated, delivering bubbles to the
reaction solution in the chamber above when the solution contains
thermally-sensitive components. Alternatively, heaters may be used
to directly heat a solution to the point that boiling occurs, in
the case that the solution contains no thermally-sensitive
components. The occurrence of air bubbles is often undesirable in
previously disclosed fluidic and microfluidic devices, as they may
accumulate in fluidic chambers and channels and displace reaction
solutions or impede fluid movement within the device. The vertical
design of embodiments of the invention presented herein allows
bubbles to rise to the fluid surface, resulting in only minimal and
transient fluid displacement, effectively ameliorating any
disadvantageous impacts of bubbles on the fluidic or microfluidic
system. Mixing by boiling is also convenient with this vertical
design as fluid displaced during the process simply returns to the
original fluidic chamber by gravity after the heating elements are
turned off.
[0121] In embodiments of the invention, a colorimetric detection
strip is used to detect amplified nucleic acids. Lateral flow
assays are commonly used in immuno-assay tests due to their ease of
use, reliability, and low cost. The prior art contains descriptions
of the use of lateral flow strips for the detection of nucleic
acids using porous materials as a sample receiving zone which is at
or near a labeling zone also comprised of a porous material and
placed at or near one end of the lateral flow assay device. In
these prior inventions labeling moieties are in the labeling zone.
The use of porous materials as the sample receiving zone and the
labeling zone results in the retention of some sample solution as
well as detection particles in the porous materials. Although
labeling zones comprising porous materials having reversibly
immobilized moieties required for detection may be used in
embodiments of the present invention, embodiments of the present
invention preferably utilize detection particles or moieties held
in a region of the device distinct from the sample receiving zone
of the lateral flow strip and comprising nonporous materials with
low fluid retention characteristics. This approach allows nucleic
acid target containing samples to be labeled prior to introduction
to the porous components of the sample receiving end of the lateral
flow component of the device and thereby eliminates the retention
and/or loss of sample material and detection particles in a porous
labeling zone. This method further enables the use of various
treatments of the sample in the presence of detection moieties,
such as treatment with high temperatures, to accomplish
denaturation of a double-stranded target or secondary structures
within a single-stranded target without concern for the impacts of
temperature on porous sample receiving or labeling zone materials
or the lateral flow detection strip materials. Additionally, the
use of a labeling zone not in lateral flow contact with the sample
receiving zone but subject to the control of fluidic components
such as vents allows target and label to remain in contact for
periods of time controlled by fluid flow control systems. Thus
embodiments of the present invention can be different than
traditional lateral flow test strips wherein sample and detection
particle interaction times and conditions are determined by the
capillary transport properties of the materials. By incorporating
the detection particles in a temperature-regulated chamber,
denaturation of duplex nucleic acid is possible allowing for
efficient hybridization-based detection. In alternative
embodiments, fluorescence is used to detect nucleic acid
amplification using a combination of LEDs, photodiodes, and optical
filters. These optical detection systems can be used to perform
real-time nucleic acid detection and quantification during
amplification and end-point detection after amplification.
[0122] Embodiments of the invention comprise a low cost,
point-of-use system is provided wherein a nucleic acid sample may
be selectively amplified and detected. Further embodiments include
integration with a nucleic acid sample preparation device such as
that described in International Publication No. WO 2009/137059 A1,
entitled "Highly Simplified Lateral Flow-Based Nucleic Acid Sample
Preparation and Passive Fluid Flow Control". An embodiment of the
device preferably comprises both a plastic fluidic component and a
printed circuit assembly (PCA) and/or flexible circuit, and is
optionally encased in a housing that protects the active
components. Temperature regulation, fluid and reagent mixing are
preferably coordinated by a microcontroller. The reaction cassette
is preferably oriented and run vertically so that gravity,
hydrostatic pressure, capillary forces and surface tension, in
conjunction with microcontroller triggered vents, control fluid
movement within the device.
[0123] In embodiments of the present invention, prepared or crude
sample fluid enters a sample port and fills or partially fills a
sample cup. Sample may be retained, for varying periods of time, in
the sample cup where dried or lyophilized reagents can mix with the
sample. Such reagents as positive control reagents, control
templates, or chemical reagents beneficial to the performance of
the test may be introduced to the sample solution by inclusion in
dry, liquid or lyophilized form in the sample cup. Other treatments
such as controlled temperature incubations or heat lysis of
bacterial or viral analytes may optionally be accomplished in the
sample cup my means of an underlying microheater and temperature
sensor system interfaced to temperature control electronics. A
fluid network comprises the sample port through which sample is
introduced to the cassette either manually by the user or via an
automated system, e.g. a subsystem integral to the docking unit or
a sample processing subsystem; the sample cup wherein sample is
held to facilitate accumulation during sample introduction and to
add reagents, components to perform treatments required prior to
further movement of the sample into the downstream portions of the
fluid network (e.g. heat treatment to perform lysis of a bacterial
cell or virus); a recirculation vent passage for the equilibration
of air, gas or solution pressures of the fluidic channels and/or
chambers with the pressure of the expansion chamber of the
cassette; a bead recess wherein a reagent bead (e.g. a bead or
pellet of material, reagent, chemicals, biological agents,
proteins, enzymes or other substances or mixes of these substances)
in a dried/desiccated or lyophilized or semidry state may be
rehydrated by the sample solution or a buffer solution introduced
to the cassette prior to the addition of sample to rehydrate the
bead or pellet contained therein and thus comingle the materials
therein to the sample solution; a set of one or more vents that can
be opened to control fluid movement within the cassette; a first
chamber where the sample can be subjected to a regimen of
temperatures; an optional barrier within the fluidic channel
connecting the first chamber with a second chamber to preclude
premature invasion of liquids and/or gases into the second chamber
or to temporally control the movement of solution or gases into the
second chamber; a second chamber wherein the sample solution may be
subjected to further temperature regimens optionally following
addition of reagents from an optional reagent bead recess
optionally located between first and second chambers; a test strip
recess forming a chamber wherein a test strip is mounted to detect
an analyte or a reporter molecule or other substance indicative of
the presence of an analyte. In some embodiments the cassette is
inserted into a docking unit which performs the functions of
sealing the cassette, elution, detection, and data transmission.
Preferably no user intervention is required once the cassette is
inserted into the docking unit, the sample is loaded, and the lid
is closed.
[0124] Referring to the representative drawings of cassette 2500 in
FIGS. 1-2, a nucleic acid sample is added to the sample cup 10 in
fluidic component 5 through the sample port 20. Sliding seal 91 is
moved to the closed position by closure of the docking unit lid at
the time of assay initiation. Cover 25 holds slide 91 in place in
order to seal expansion chamber 52. The nucleic acid sample may
derive from an online (i.e. integrated nucleic acid preparation
sub-system), a separate nucleic acid preparation process (such as
one of many commercially available methods, e.g. spin-columns)
followed by addition of the purified nucleic acid to the device by
pipette, or an unprocessed nucleic acid containing sample. Already
present in the sample cup, or preferably in recess 13 within or
adjacent to the sample cup, is reagent mix 16, which may be in
liquid or dry form, containing components useful for facilitating
cell and virus lysis and/or stabilize liberated nucleic acids. For
example, dithiothreitol and/or pH buffering reagents may be
employed to stabilize nucleic acids and inhibit RNases. Similarly,
reagents to accomplish acid or base mediated lysis may be used. In
some embodiments the reagent mix is lyophilized to form lyophilized
reagents. In some embodiments a positive control such as a virus,
bacteria or nucleic acid is present in the reagent mix.
Introduction of the sample to the sample cup causes reagents and
samples to commingle such that the reagents act upon the sample. An
optional bubble-mixing step to further mix the sample with the
reagents or re-suspend the reagents may optionally be performed.
Fluid is then optionally heated in the sample cup 10 to lyse cells
and virus particles. Fluid is then preferably directed through
channel 40 to a first chamber 30 that resides below the sample cup
when the device is in the vertical orientation. Reagent recess 15
is preferably situated along the inlet channel such that fluid
passes through the recess to commingle with dried or lyophilized
reagents contained therein prior to entering the first chamber 30.
In embodiments wherein the first chamber is a reverse transcription
chamber, preferably present in reagent recess 15 are all components
necessary of a reverse transcription reaction such as buffering
reagents, dNTPs, oligonucleotide primers, and/or enzymes (e.g.
reverse transcriptase) in dried or lyophilized form. The reverse
transcription chamber is preferably in contact with heater elements
to provide a means for the temperature regulation necessary to
support the reverse transcription of RNA into cDNA. Channel 35
connects chamber 30 to reagent recess 37. Following cDNA synthesis
in chamber 30, vent 50 is opened to allow the reverse transcription
reaction to flow via channel 35 into reagent recess 37. Dried or
lyophilized reagents present reagent recess 37 commingle with fluid
as it passes through the recess to second chamber 90 via inlet 39
such that the reagents act upon the sample in the second chamber,
which is preferably an amplification chamber. Preferably present in
reagent recess 37 are all components necessary for the
amplification reaction, such as buffering agents, salts, dNTPs,
rNTPs, oligonucleotide primers, and/or enzymes. In some embodiments
the reagent mix is lyophilized to form lyophilized reagents. To
facilitate multiplexed tests, wherein multiple amplicons are
generated, multiplexed amplification can be accomplished by
deposition of multiple primer sets within the amplification
chamber(s) or preferably within reagent recesses upstream of said
amplification chamber(s). Additionally, circuit board and fluidic
designs in which multiple amplification and detection chambers are
incorporated into the device support multiple parallel
amplification reactions that may be single-plex or multiplex
reactions. This approach reduces or eliminates the complications
known to those skilled in the art that result from multiplexed
amplification using multiple pairs of primers in the same reaction.
Moreover, the use of multiple amplification reaction chambers
allows simultaneous amplification under different temperature
regimens to accommodate requirements for optimal amplification,
such as different melting or annealing temperatures required for
different target and/or primer sequences.
[0125] Following nucleic acid amplification, vent pocket 150 is
opened to allow the amplification reaction product to flow via
channel 135 into chamber 230. Detection strip 235 situated in
chamber 230 enables the detection of target nucleic acids labeled
by detection particles located on a region of detection strip 235
or optionally in capillary pool 93.
[0126] Fluid movement from the sample cup 10 to first chamber 30
occurs because chamber 30 is vented to expansion chamber 52 via
opening 51. Fluid movement from the first chamber to the second
chamber of the device is preferably accomplished by the opening of
a vent connected to the second chamber. When fluid enters first
chamber 30, vent pocket 50, connected to the downstream chamber, is
sealed, and thus fluid will not pass through channel 35 connecting
the two chambers. Referring now to FIG. 2A, movement of fluid from
chamber 30 to chamber 90 can be accomplished by allowing air within
chamber 90 to communicate with air in expansion chamber 52 by
rupturing a seal overlying vent pocket 50. Rupture of the seal at
vent pocket 50 allows communication of air in chamber 90 via vent
channel 60 with air in expansion chamber 52, which is connected via
opening 51 to vent pocket 54. The seal at vent pocket 54 is
preferably open, or was previously ruptured, as shown in FIG. 2B.
As shown in FIG. 2C, rupture of the seal of vent pocket 50 allows
vent pocket 50 (and thus chamber 90) to communicate with vent
pocket 54 (and thus expansion chamber 52). This method of fluid
movement is preferably embodied within a hermetically sealed space
to contain bio-hazardous samples and amplified nucleic acids within
the test cassette. To enable a hermetically sealed cassette,
selectively heat resistant and heat labile materials are layered in
the manner represented schematically in cross section in FIGS.
2B-2C. Referring now to FIGS. 2B-2C, heat source 70, which
preferably comprises a resistor, on printed circuit board or PCA 75
is placed in register with vent pockets 50, 54 and in proximity to
heat labile vent pocket seal material 80. The vent pocket seal may
comprise a heat labile material such as polyolefin or polystyrene.
A heat stable material (such as polyimide) 72 is preferably
disposed between heat source 70 and heat labile vent pocket seal
material 80 to form a hermetic barrier. In some embodiments the
sealed space 55 between or surrounding vent pockets is augmented by
the inclusion of an optional gasket or spacer 56 comprising an
adhesive layer that bonds heat stable material 72 to heat labile
material 80 and/or fluidic component 5 and maintains a hermetic
seal of the test cassette in the region of the vents after one or
more of the vents are opened, while preferably also providing an
air gap for the communication of air between opened vents and/or
the optional expansion chamber. In this embodiment, heat is
transferred from heat source 70 through heat stable material 72 and
sealed space 55 to the heat labile vent pocket seal material 80,
rupturing it and opening vent pocket 50. A microcontroller is
preferably responsible for sending electrical current to heat
source 70. Vent pocket 50 preferably opens to an enclosed space
such that the gas within the test cassette may remain sealed with
respect to the environment outside of the test cassette. The
enclosed space may comprise the air within the test cassette,
optionally including a vacant air chamber to allow for gas
expansion, such as an expansion chamber. As shown in FIG. 2C,
opening of vent pocket 50 results in the communication of gases in
the vented fluid chamber with the gas of the expansion chamber,
since vent pocket 54 was previously ruptured by heat source 71, and
vent pocket 54 is in gaseous communication with the expansion
chamber. The resulting reduced pressure in the vented fluid chamber
allows fluid to flow by gravity into the vented chamber from a
chamber situated above. Other embodiments of the vent pocket may
comprise seals other than a heat-sensitive membrane, and may
utilize other methods of breaking the seals, such as puncturing,
tearing, or dissolving. A photograph of such a cassette is shown in
FIG. 2E.
[0127] The face opposite the open face of the vent pocket may
optionally comprise a dimple, protrusion, asperity, or other
similar structure, such as dimple 7004 of FIG. 45, to facilitate
the formation of an opening during rupture of the vent seal
material. Such a structure also preferably prevents resealing of
the vent after rupture of the seal. This can occur in embodiments
comprising a circuit board with surface mount components. In such
embodiments the surface mount resistors can stretch the polyimide
film, pushing it into the opening in the gasket and against the
heat labile material. Once the seal ruptures, the molten seal
material can form a secondary seal with that polyimide, thereby
closing the vent. In embodiments with a flex circuit comprising
metallic traces forming heating elements, the heater can cause the
polyimide flex circuit to deform locally, often forming a
protrusion (often comprising the heater material) extending into
the opening in the gasket, possibly occluding the vent opening due
to the molten seal material. Dimple 7004 can help prevent these
occurrences.
[0128] Sealed space 55 optionally provides a conduit to other
vents, vent pockets or chambers (such as expansion chamber 52).
Following vent opening, fluidic component 5 remains sealed from the
external environment 59. Expansion chamber 52 preferably
accommodates gas expansion during heating by buffering the
air/water vapor volume either by providing a sufficiently large
volume so that gas expansion from temperature changes does not
significantly impact the pressure of the system, or by
accommodating gas expansion by displacement of a piston (FIG. 3), a
flexible bladder (FIG. 4), a bellows (FIG. 5), or a hydrophobic
barrier that allows gas but not macromolecules to pass free across
the barrier (FIG. 6). In FIG. 3 the expansion chamber makes use of
a piston that is displaced by increasing pressure within the sealed
fluidic system. The expansion chamber serves to reduce or eliminate
the accumulation of pressure within the sealed system. Displacement
of the piston occurs in response to increased pressure within a
hermetically sealed test cassette, reducing internal pressure
within the test cassette resulting from such processes as gas
expansion during heating. In FIG. 4, deflection of the bladder
occurs in response to increased pressure within a hermetically
sealed test cassette. Displacement of the bladder reduces internal
pressure within the test cassette resulting from such processes as
gas expansion during heating. In FIG. 5, stretching of the bellows
occurs in response to increased pressure within a hermetically
sealed test cassette. Stretching of the bellows reduces internal
pressure within the test cassette resulting from such processes as
gas expansion during heating.
[0129] Expansion chambers may be incorporated as a vacant air
volume, such as the included volume shown in expansion chamber 52
at the top of the test cassette illustrated in FIG. 1. As
illustrated in FIG. 7, to facilitate the fabrication of a cassette
of minimum thickness, expansion chambers may also be incorporated
into air gap 440 formed by a suitably designed gasket 420 when
sealed to heat labile material 410 and heat stable material 430 to
form the backing of fluidic component 400. Minimization of test
cassette physical dimensions is desirable to reduce shipping costs,
reduce thermal mass and provide an aesthetically pleasing and
convenient design. In addition to forming air volume for gas
expansion, gasket 420 generates a space between the heat stable
material 430 and heat labile material 410 to facilitate free
movement of air through open vents while maintaining a sealed
system to prevent exposure to the environment. Gasket 420 is
preferably thick enough to provide a sufficient air gap to
equilibrate pressures between open vents, but is also sufficiently
thin to not substantially impact the interface between the heaters
and the corresponding vent pockets or the sealing of the cassette
by the heat stable material. In embodiments of the present
invention which comprise a flex circuit, the flex circuit may
comprise a heat stable material such as polyimide, in which case
the separate sheet of heat stable material 430 is not required, for
example as shown in FIG. 8C. The use of an expansion chamber to
reduce or equilibrate pressure within the sealed test cassette
ensures that pressure disequilibria do not result in unfavorable or
premature solution movements within the test cassette and that
pressure accumulation does not adversely impact desired fluid
movement, such as movement between chambers or through channels.
This pressure control, i.e. the establishment of designated
pressure distributions throughout the device, enables the system to
work as designed regardless of atmospheric pressure. The expansion
chamber therefore enables controlled fluid movements which are
dependent upon stable pressure within the system to be employed,
and also enables use of a hermetically sealed test cassette,
thereby avoiding the disadvantages of the venting the test cassette
to atmosphere, for example the potential release of amplicon to the
atmosphere. Furthermore, the method of enabling fluid flow by
reducing pressure downstream of a fluid, such as by opening a vent
to the expansion chamber, eliminates the need for pumps, such as
those that create a positive pressure upstream of the fluid, or
other devices with moving parts. Similar advantages are possible by
venting an area downstream of a fluid to a relatively larger
reservoir (such as the expansion chamber) at substantially the same
pressure as the downstream area, thereby enabling the fluid to flow
under the force of gravity (provided the device is in the
appropriate orientation). The size of the expansion chamber is
preferably sufficiently large to accommodate reaction vapors
produced during the assay without increasing the pressure of the
system to a point where it overcomes the capillary force or
gravitational force necessary for the fluid to flow.
[0130] In embodiments where the second chamber is an amplification
chamber, the chamber is preferably in contact with heater elements
to provide a means for the temperature regulation necessary to
support nucleic acid amplification. In some embodiments of the
invention, the amplification chamber may contain oligonucleotides
on at least a portion of the interior surface. At the interface
between wall 95 of chamber 30 and one or more heating elements 100,
as illustrated in FIG. 2D, it may be advantageous to place a
thermally conductive material such as a thermal grease or compound.
A microcontroller preferably modulates current to the resistive
heating element(s), preferably by means of metal oxide
semiconductor field effect transistors (MOSFETs), based upon data
collected from temperature sensor 110 on PCA 75, using simple
on/off or proportional integral derivative (PID) temperature
control methods or other algorithmic temperature control known to
those skilled in the art.
[0131] Placing the heating elements, and in some embodiments the
corresponding temperature sensor(s), on the disposable component
enables the manufacture of highly reproducible thermal coupling
between the temperature control subsystem and the amplification and
detection chambers to which they interface. This approach enables a
highly reliable means of coupling the fluidic subsystem to the
electronic thermal control subsystem by forming the thermally
conductive interface during manufacture. The resulting superior
thermal contact between the electronic temperature control
components and the fluidic subsystem results in rapid temperature
equilibration, and therefore rapid assays. The use of a flexible
circuit to provide disposable resistive heating elements that are
fused to the rear of the fluidic component backing either directly
or with an intervening gasket, allows for a low cost means of
attaining excellent thermal contact, rapid temperature cycling and
reproducible manufacture. Resistive heating elements for reverse
transcription, amplification and fluid flow vent control can be
formed directly on the flex circuit by etching the conductive layer
of the flex circuit to form geometries exhibiting the required
resistance. This approach eliminates the need for additional
electronic components and simplifies manufacture while reducing
cost.
[0132] In an embodiment of the present invention, flexible circuit
799 for resistive heating and vent opening is shown in FIG. 8. The
use of a flexible heater as a component of disposable cassette
allows the cassette backing to be configured to enable fluid in the
heated fluid chambers to make direct contact with the material
comprising the flexible heater circuit. For example, as shown in
FIG. 8C, windows 806 in thermally labile material 807 (which
preferably comprises BOPS) that forms the rear of the cassette may
be situated over the fluid chambers to allow direct contact of
fluid with flexible circuit 799. Direct contact between the
flexible circuit layer and the fluid to be temperature controlled
by heaters on the flexible circuit provides for a low thermal mass
system capable of rapid temperature changes. To enable collection
of temperature data for use in temperature regulation a temperature
sensor may be optionally incorporated into the flexible circuit,
and/or a non-contact means of temperature monitoring such as an
infrared sensor may be employed. Resistive heating elements, such
as heating element 800, in a flexible circuit can be utilized for
vent rupture when they are situated in register with a vent pocket.
Electrical pads 812 provide current to heating elements 800.
Similarly, the flexible circuit or circuits may comprise resistive
heating elements 802 and 803 for heating the fluid chambers, and
optional resistive heating element 804 for regulating the
temperature of the detection strip.
[0133] In this embodiment flexible circuit 799 also preferably
serves as a heat stable seal to maintain a hermetically sealed
cassette, similar to heat stable material 72 described above.
Optionally an additional heat stable layer (for example comprising
polyimide) can be placed between flexible circuit 799 and rear
housing or panel 805. A spacer or gasket 808 is preferably placed
around vent resistors 800 between thermally labile material 807 and
flexible circuit 799 to ensure free air movement through open vents
while maintaining a sealed cassette. Rear housing or panel 805
preferably comprises thin plastic and is preferably placed over the
exposed surface of the flexible circuit to protect it during
handling. Rear housing or panel 805 may comprise windows over the
heater elements on flexible circuit 799 to facilitate cooling and
temperature monitoring. Electrical contact with controlling
electronics of the docking unit (described below) may optionally be
provided by a set of electrical pads 810, preferably comprising an
edge connector or connector pins such as spring loaded pins.
[0134] Embodiments of the test cassette chambers preferably
comprise materials capable of withstanding repeated heating and
cooling to temperatures in the range of approximately 30.degree. C.
to approximately 110.degree. C. Even more preferably, the chambers
comprise materials capable of withstanding repeated heating and
cooling to temperatures in the range of approximately 30.degree. C.
to approximately 110.degree. C. at a rate of temperature change on
the order of approximately 10.degree. C. to approximately
50.degree. C. per second. The chambers are preferably capable of
maintaining solutions therein at temperatures suitable for heat
mediated lysis and biochemical reactions such as reverse
transcription, thermal cycling or isothermal amplification
protocols, preferably controlled by programming of the
microcontroller. In some nucleic acid amplification applications,
it is desirable to provide an initial incubation at an elevated
temperature, for example a temperature between approximately
37.degree. C. and approximately 110.degree. C. for a period of 1
second to 5 minutes, to denature the target nucleic acid and/or to
activate a hot start polymerase. Subsequently, the reaction
solution is held at the amplification temperature in the
amplification chamber for isothermal amplification or, for
thermocycling-based amplification, is varied in temperature between
at least two temperatures including, but not limited to, a
temperature that results in nucleic acid duplex denaturation and a
temperature suitable to primer annealing by hybridization to the
target and extension of the primer through polymerase catalyzed
nucleic acid polymerization. The duration of incubations at each
requisite temperature in a thermal cycling regimen may vary with
the sequence composition of the target nucleic acid and the
composition of the reaction mix, but is preferably between
approximately 0.1 seconds and approximately 20 seconds. Repeated
heating and cooling is typically performed for approximately 20
cycles to approximately 50 cycles. In embodiments involving
isothermal amplification methods, the temperature of the reaction
solution is maintained at a constant temperature (in some cases
following an initial incubation at an elevated temperature) for
between approximately 3 minutes and approximately 90 minutes
depending on the amplification technique used. Once the
amplification reaction is complete, the amplification reaction
solution is transported, by opening the vent that is in
communication with a chamber below the chamber employed for
amplification, to the lower chamber to accomplish further
manipulations of the amplified nucleic acids. In some embodiments
of the invention manipulations comprise denaturation of the
amplified nucleic acids and hybridization to detection
oligonucleotides conjugated to detection particles. In some
embodiments of the invention, amplified nucleic acids are
hybridized to detection oligonucleotides conjugated to detection
particles and to capture probes immobilized on a detection
strip.
[0135] In some embodiments, additional biochemical reactions may be
conducted in the amplification chamber prior to, during, or after
the amplification reaction. Such processes may include but are not
limited to reverse transcription wherein RNA is transcribed into
cDNA, multiplexing wherein multiple primer pairs simultaneously
amplify multiple target nucleic acids, and real time amplification
wherein amplification products are detected during the
amplification reaction process. In the case of the latter, the
amplification chamber may not contain a valve or outlet channel,
and the amplification chamber would preferably comprise an optical
window or otherwise configured to enable interrogation of amplicon
concentration during the amplification reaction process. In one
real-time amplification embodiment, either fluorescently labeled
oligonucleotides complementary to the target nucleic acid or
fluorescent dyes specific for duplex DNA are monitored for
fluorescence intensity by means of an excitation light source such
as LEDs or diode laser(s) and a detector such as a photodiode, and
appropriate optical components including but not limited to optical
filters.
Detection
[0136] Embodiments of the detection chamber 230 preferably provide
for the specific labeling of amplified target nucleic acids
generated in the amplification chamber. As shown in FIG. 2A,
detection chamber 230 preferably comprises a capillary pool or
space 93 and a detection strip 235. Detection particles comprising
dye polystyrene microspheres, latex, colloidal gold, colloidal
cellulose, nanogold, or semiconductor nanocrystals are preferably
present in the capillary pool 93. Said detection particles may
comprise oligonucleotides complementary to the target analyte or
may comprise ligands capable of binding to the amplified target
nucleic acid such as biotin, streptavidin, a hapten or an antibody
directed against a label such as a hapten present on the target
amplified nucleic acids. Detection chamber 230 may contain
detection particles that are dried, lyophilized, or present on at
least a portion of the interior surface as a dried mixture of
detection particles in a carrier such as a polysaccharide,
detergent, protein or other compound known to those skilled in the
art to facilitate resuspension of the detection particles. In some
embodiments the lateral flow detection strip may comprise detection
particles. In other embodiments a reagent recess channel 135
leading to the detection chamber my comprise detection particles.
The detection chamber may be capable of being heated and/or
cooled.
[0137] Suitable detection particles include but are not limited to
fluorescent dyes specific for duplex nucleic acid, fluorescently
modified oligonucleotides, or oligonucleotide-conjugated dyed
microparticles or colloidal gold or colloidal cellulose. Detection
of amplicon involves a `detection oligonucleotide` or other
`detection probe` that is complementary or otherwise able to bind
specifically to the amplicon to be detected. Conjugation of a
detection oligonucleotide to a microparticle may occur by use of
streptavidin coated particles and biotinylated oligonucleotides, or
by carbodiimide chemistry whereby carboxylated particles are
activated in the presence of carbodiimide and react specifically
with primary amines present on the detection oligonucleotide.
Conjugation of the detection oligonucleotide to the detectable
moiety may occur internally or at the 5' end or the 3' end.
Detection oligonucleotides may be attached directly to the
microparticle, or more preferably through a spacer moiety such as
ethyleneglycol or polynucleotides. In some embodiments of the
invention, detection particles may bind to multiple species of
amplified nucleic acids resulting from such processes as
multiplexed amplification. In these embodiments the specific
detection of each species of amplified nucleic acid can be realized
by detection on the detection strip using a method specific for
each species to be detected. In such an embodiment, a tag
introduced to the target nucleic acids during amplification may be
used to label all amplified species present while subsequent
hybridization of the labeled nucleic acids to species specific
capture probes immobilized on the detection strip is employed to
determine which specific species of amplified DNA are present.
[0138] In the case of a duplex DNA amplification product, heating
the reaction solution following introduction to the detection
chamber may facilitate detection. Melting duplex DNA or denaturing
the secondary structure of single stranded DNA and then cooling in
the presence of detection oligonucleotide results in the
sequence-specific labeling of the amplified target nucleic acid.
The heating element underlying the detection chamber may be used to
heat the fluid volume for approximately 1 to approximately 120
seconds to initiate duplex DNA melting or denaturation of single
stranded DNA secondary structure. As the solution is allowed to
cool to room temperature, the amplified target nucleic acid may
specifically hybridize to detection microparticles. The reaction
volume is then preferably directed to a region of the detection
chamber below the labeling chamber by opening the vent of the
detection chamber.
[0139] For efficient labeling to occur, the solubilized detection
particles are preferably well mixed with the reaction solution. In
embodiments of the invention, detection particles may be localized
in capillary pool 93 at the outlet of channel 135 to facilitate
mixture with solution as it enters chamber 230. Detection particles
in capillary pool 93 may optionally be lyophilized detection
particles. The capillary pool provides improved mixing and
dispersion of particles to facilitate comingling of the detection
particles with the nucleic acids to which the detection particles
bind. The capillary pool also increases the uniformity of particle
migration on the detection strip, as shown in FIG. 9. A capillary
pool is especially advantageous for low volume assays, such as
those less than 200 .mu.L, or more specifically less than about 100
.mu.L, or even more particularly less than about 60 .mu.L, or even
more particularly about 40 .mu.L in volume.
[0140] Embodiments of the detection chamber of the present
invention provide for the specific detection of amplified target
nucleic acids. In certain embodiments of the invention, detection
is accomplished by capillary wicking of solution containing labeled
amplicon through an absorbent strip comprised of a porous material
(such as cellulose, nitrocellulose, polyethersulfone,
polyvinylidine fluoride, nylon, charge-modified nylon, or
polytetrafluoroethylene) patterned with lines, dots, microarrays,
or other visually discernable elements comprising a binding moiety
capable of specifically binding to the labeled amplicon either
directly or indirectly. In some embodiments, the absorbent strip
component of the device comprises up to three porous substrates in
physical contact: a surfactant pad comprising amphipathic reagents
to enhance wicking, a detection zone comprising a porous material
(such as cellulose, nitrocellulose, polyethersulfone,
polyvinylidine fluoride, nylon, charge-modified nylon, or
polytetrafluoroethylene) to which at least one binding moiety
capable of selectively binding labeled amplicon is immobilized,
and/or an absorbent pad to provide additional absorbent capacity.
Although detection particles may optionally be incorporated within
the lateral flow porous materials in the detection chamber, unlike
previously described lateral flow detection devices the detection
particles preferably are instead held upstream in a capillary pool
where substantially enhanced the formation of binding complexes
between amplicon and detection particles may be conducted prior to
or concomitant with the introduction of the resultant labeled
nucleic acids to the porous components of the device.
[0141] A `capture oligonucleotide` or `capture probe` is preferably
immobilized to the detection strip element of the device by any of
a variety of means known to those skilled in the art, such as UV
irradiation. The capture probe is designed to capture the labeled
nucleic acid as solution containing the labeled nucleic acid wicks
through the capture zone resulting in an increased concentration of
label at the site of capture probe immobilization, thus producing a
detectable signal indicative of the presence of the labeled target
nucleic acid amplicon(s). A single detection strip may be patterned
with one or multiple capture probes to enable multiplexed detection
of multiple amplicons, determination of amplicon sequence,
quantification of an amplicon by extending the linearity of the
detection signal, and assay quality control (positive and negative
controls).
Fluidic Component
[0142] Embodiments of the fluidic component preferably comprise
plastic, such as acrylic, polycarbonate, PETG, polystyrene,
polyester, polypropylene, and/or other like materials. These
materials are readily available and able to be manufactured by
standard methods. Fluidic components comprise both chambers and
channels. Fluidic chambers comprise walls, two faces, and connect
to one or more channels such as an inlet, an outlet, a recess, or a
vent. Channels can connect two fluidic chambers or a fluidic
chamber and a recess, and comprise of walls and two faces. Fluidic
chamber design preferably maximizes the surface area to volume
ratio to facilitate heating and cooling. The internal volume of a
chamber is preferably between approximately 1 .mu.L and
approximately 200 .mu.L. The area of a chamber face in contact with
solution preferably corresponds with the area to which heating
elements are interfaced to ensure uniform fluid temperature during
heating. The shape of the fluidic chambers may be selected to mate
with heating elements and to provide favorable geometries for
solution ingress and egress. In some embodiments, the volume of the
chamber may be larger than the fluid volume in order to provide a
space for bubbles that appear during the course of device
operation. Fluidic chambers may have enlarged extensions leading to
vent channels, to ensure that fluid does not encroach upon the
channel by capillary action or otherwise block the venting
mechanism.
[0143] In some embodiments, it may be desirable to reduce or
eliminate the invasion of liquid or gas phase water into a chamber
prior to the time of solution release. The elevated temperatures
employed in processes of some embodiments generate vapors (e.g. gas
phase water) that can result in premature invasion of moisture into
a channel, chamber or recess. Reduction of liquid phase or gas
phase invasion may be desirable to retain, for example, the dried
state of dried reagents or lyophilized reagents present in a
chamber or recess. In some embodiments, channels may be temporarily
blocked, completely or partially, with a material that can be
removed by external forces such as heat, moisture, and/or pressure.
Materials suitable for the temporary blockage of channels include
but are not limited to latex, cellulose, polystyrene, hot glue,
paraffin, waxes, and oils.
[0144] In some embodiments, the test cassette comprises a
preferably injection molded fluidic component comprising sample
cup, chambers, channels, vent pockets, and energy directors. The
injection molded test cassette fluidic component is preferably
comprised of a plastic suitable for ultrasonic welding to a backing
plastic of similar composition. In one embodiment of the invention
the test cassette fluidic component comprises a single injection
molded piece that is ultrasonically welded to a backing material.
The energy directors are optional features of the fluidic component
that direct the ultrasonic energy to only those areas of the heat
labile layer which are intended to bond to the fluidic component.
The injection molded fluidic component may optionally be housed in
a housing. FIG. 7 illustrates a cassette comprising a preferably
injection molded fluidic component 400 (preferably comprising a
polymer such as high-impact polystyrene (HIPS), polyethylene,
polypropylene, or NAS 30, a styrene acrylic copolymer), a heat
labile material 410 (comprising, for example, BOPS, which has
relatively low melting temperature of about 239.degree. C. and a
glass transition temperature of about 100.degree. C., which is
sufficient to withstand the elevated temperatures during
denaturation, or polycarbonate, which has a melting temperature of
265.degree. C. and glass transition temperature of 150.degree. C.),
adhesive spacer 420 (comprising, for example, a silicone transfer
adhesive that preferably does not incorporate a carrier, an acrylic
adhesive with a polyester carrier, or any adhesive that can
withstand the elevated temperatures of the device), and heat
resistant layer 430. Heat labile material 410 ruptures via heat
which is preferably transmitted through overlying heat resistant
layer 430 (comprising, for example, polyimide or another polymer
having high heat resistance). Melting of the heat labile material
over the vent features of the cassette opens the vent and vent
channel to the expansion chamber, thereby allowing pressure
equalization within the cassette. Overlying heat resistant layer
430 preferably remains intact, thereby enabling the cassette to
maintain a hermetic seal after vent opening.
[0145] In some embodiments, the adhesive spacer comprises a vacant
region 440 that may serve as an expansion chamber to buffer the
expansion of gases during heating to reduce the internal pressure
of a sealed cassette. Heat labile layer 410 is bonded to fluidic
component 400 by a bonding method or process such as ultrasonic
welding or employing adhesive. The resulting part is then bonded to
a spacer and a heat resistant layer. In some embodiments the heat
resistant layer is constructed in such a manner that it is not
present over heated chambers. In other embodiments, the heat
resistant layer is present over heater chambers. In yet other
embodiments, the adhesive spacer and heat resistant layers are
present only over a region that is in register with the vent pocket
features of the fluidic component. In this embodiment a heat
resistant layer may optionally be placed directly over the heat
labile material in the regions in register with the heated
chambers.
[0146] In some embodiments of the invention the thickness of the
fluidic chambers and channel walls are in the range of
approximately 0.025 mm to approximately 1 mm, and preferably in the
range of approximately 0.1 mm to approximately 0.5 mm. This
thickness preferably meets requirements of both structural
integrity of the fluidic component and to support sealing of the
closed chamber under high temperatures and associated pressures.
The thickness of channel walls, particularly vent channel walls,
are preferably less than that of the chambers and in the range of
approximately 0.025 mm to approximately 0.25 mm. The width of inlet
and outlet channels is preferably chosen to enhance capillarity. A
shallow vent channel imparts improved rigidity to the fluidic
component with no adverse effect on venting. Plastic forming faces
of the fluidic component is preferably thinner than that forming
the walls in order to maximize heat transfer. Optional thermal
breaks cut through some components of the fluidic component and
surround the amplification and detection chambers, contributing to
the thermal isolation of the temperature-controlled chambers.
[0147] In some embodiments of the invention, before the fluidic
component 400 is bonded to the heat labile backing material 410
additional components of the test cassette such as lyophilized
reagents 16, detection strip assembly 230, and detection particles
may be incorporated. In some embodiments, the components may be
laminated by applying pressure to ensure good adhesion. In some
embodiments the components may be bonded by a combination of
methods such as pressure sensitive adhesives and ultrasonic
welding. Adhesives known or found to negatively impact performance
of nucleic acid amplification reactions must be avoided. Acrylic-
or silicon-based adhesives have been successfully used in the
invention. One preferred adhesive film is SI7876 supplied by
Advanced Adhesives Research. Other adhesives may be used if found
to be chemically compatible with employed buffers, plastics and
reaction chemistries while providing robust sealing over the
temperatures encountered during device operation.
[0148] Referring to FIGS. 2 and 7, vent pockets are preferably
differentiated from other chambers in their construction. After
construction of the fluidic component as described above, vent
pockets possess an open face on the side of the fluidic component
that will meet with PCA 75 either directly or in some embodiments
indirectly through an intervening air gap 420 or vent pocket 54 and
heat resistant material 430. To form the vent pocket, an additional
plastic component is bonded to seal the chamber, preferably
comprising a thin, heat labile membrane 410 adjacent to vent
resistor 70 of the PCA. Film 410 comprises a material suitable for
ultrasonic welding to the injection molded fluidic component such
as polystyrene although other similar materials may be used. This
film is well suited to both seal the vent pocket and allow for easy
perforation and, thus, venting to a lower pressure chamber when
current is passed through the vent resistor generating a rapid
temperature increase. Preferably, the film is sufficiently stable
when heated so that the material can withstand the temperatures
employed in other operations of the test cassette such as heat
lysis, reverse transcription and nucleic acid amplification. Use of
a material with stability in the temperature ranges employed for
denaturation, labeling, reverse transcription, nucleic acid
amplification, and detection but with a melting temperature readily
attained by resistor 70 allows a single material to be employed for
the backing of the injection molded fluidic component 400 to serve
as both a face of the chambers and a face of the vent pocket. In
some embodiments, additional temperature stability in the areas of
the temperature controlled chambers can be realized by an overlying
film of heat resistant material such as polyimide. In other
embodiments of the invention, a window in the heat labile film is
in register with the temperature controlled chambers to allow
direct contact between fluids in the chamber and the substrate of a
flexible circuit fused to the rear of the test cassette.
Additional Components of the Fluidic Component
[0149] As described above, several additional components are
preferably incorporated within the fluidic component of the present
invention before final bonding. Reagents including buffers, salts,
dNTPs, NTPs, oligonucleotide primers, and enzymes such as DNA
polymerase and reverse transcriptase may be lyophilized, or
freeze-dried, into pellets, spheres or cakes prior device assembly.
Reagent lyophilization is well known in the art and involves
dehydration of frozen reagent aliquots by sublimation under an
applied vacuum. By adding specific formulations of lyoprotectants
such as sugars (di- and polysaccharides) and polyalcohols to the
reagents prior to freezing, the activity of enzymes may be
preserved and the rate of rehydration may be increased. Lyophilized
reagent pellets, spheres, or cakes are manufactured by standard
methods and, once formed, are reasonably durable and may be easily
placed into specific chambers of the fluidic component prior to
laminating the final face. More preferably, recesses are
incorporated into the fluidic network to allow pellets, spheres, or
cakes of lyophilized reagents to be placed in the fluidic component
prior to bonding of the fluidic component to the backing material.
By selecting the fluidic network geometry and recess location and
order, the sample can react with the desired lyophilized reagent at
the desired time to optimize performance. For instance, by
depositing lyophilized (or dried) reverse transcription (RD and
amplification reagent spheres into two separate recesses in the
flow paths of RT reaction chamber and amplification chamber enables
optimal reverse transcription reaction without the interference of
amplification enzymes. In addition, to minimize the interference of
RT enzymes to subsequent amplification reaction, RT enzymes post RT
reaction presented in the RT reaction could be heat inactivated
prior introduction to amplification reagents to minimize their
interference to amplification. Optionally, other salt, surfactants
and other enhancing chemicals could be added to different recesses
to modulate the performance of a assay. Moreover, these recesses
facilitate comingling of the lyophilized reagents with liquids as
they pass through the recess and also serve to isolate the
lyophilized materials from ultrasonic energy during ultrasonic
welding and to isolate lyophilized reagents from temperature
extremes during heating steps of a test prior to their
solubilization. In addition, the recesses ensure that the
lyophilized pellets aren't compressed or crushed during
manufacture, enabling them to remain porous to minimize rehydration
times.
[0150] In some embodiments of the invention, detection
microparticles are another additional component of the fluidic
component. In some embodiments, these microparticles may be
lyophilized as described for the reaction reagents above. In other
embodiments, microparticles in liquid buffer may be directly
applied to an interior face of a fluidic chamber and dried before
final assembly of the test cassette. The liquid buffer containing
the microparticles preferably also comprises sugars or polyalcohols
that aid in rehydration. Incorporation of microparticles in aqueous
buffer directly into the fluidic component prior to drying may
simplify and reduce the final cost of manufacturing, and complete
comingling of lyophilized particles with reaction solution and the
denaturation of double-stranded nucleic acids or double-stranded
regions of a nucleic acid into single-stranded nucleic acid may be
facilitated by heating or nucleate boiling. In some embodiments,
lyophilized detection particles are placed in recesses in the
fluidic network. In other embodiments, lyophilized or dried
detection particles are placed in a space 93 directly below the
detection strip. In other embodiments detection particles are dried
or lyophilized into a bibulous substrate in capillary communication
with the detection strip or are dried or lyophilized directly on
the detection strip. Capillary communication may be direct physical
contact of the said bibulous substrate with the detection strip or
indirect wherein capillary communication is over an intervening
distance comprised of a channel or chamber region through which
capillary transport is achieved to transport fluid from the
detection particle laden bibulous substrate to the detection
strip.
[0151] In some embodiments of the present invention, a lateral flow
detection strip assembly is also incorporated into the fluidic
component. The detection strip preferably comprises a membrane
assembly comprised of at least one porous component and optionally
may comprise an absorbent pad, a detection membrane, a surfactant
pad, and a backing film. The detection membrane is preferably made
of nitrocellulose, cellulose, polyethersulfone, polyvinylidine
fluoride, nylon, charge-modified nylon, or polytetrafluoroethylene
and may be backed with a plastic film. As described above, capture
probe may be deposited and irreversibly immobilized on the
detection membrane in lines, spots, microarrays or any pattern that
can be visualized by the unaided human eye or an automated
detection system such as an imaging system. Deposited
oligonucleotides may be permanently immobilized by UV-irradiation
of the detection membrane following capture probe deposition. The
surfactant pad may comprise a porous substrate, preferably with
minimal nucleic acid binding and fluid retention properties, that
permits unobstructed migration of the nucleic acid product and
detection microparticles. The surfactant pad may comprise materials
such as glass fiber, cellulose, or polyester. In embodiments of the
invention, formulations including at least one amphipathic reagent
are dried on the surfactant pad to allow uniform migration of
sample through the detection membrane. The absorbent pad may
comprise any absorbent material, and helps to induce sample wicking
through the detection membrane assembly. Using an adhesive backing
film, such as a double-sided adhesive film as a base, the detection
membrane component is assembled by first placing the detection
membrane, followed by optional absorbent pad and/or surfactant pad
in physical contact with the detection membrane with between
approximately 1 mm and approximately 2 mm overlap. In some
embodiments of the invention, the detection membrane may be in
indirect capillary communication with the surfactant pad wherein
there is a physical separation between the surfactant pad and the
detection pad with the intervening space comprised of a capillary
space wherein fluids may traverse the space by means of capillary
action. In some embodiments, the surfactant pad or a region of the
surfactant pad may comprise detection particles, dried detection
particles or lyophilized detection particles.
Three Chamber Cassette
[0152] In some embodiments of the invention, additional reaction
chambers and/or additional recesses for dried or lyophilized
reagents may be incorporated. In some embodiments such a design
facilitates tests in which it is desirable to provide for an
initial separate lysis reaction prior to reverse transcription and
amplification. As shown in FIGS. 37A, 37B, and 38, cassette 5000
comprises cover 5020 sealing expansion chamber 5021, flexible
heater circuit 5022 preferably disposed in intimate contact with
fluidic component 5023, and rear cover 5024 which conceals the
circuitry from the user. A sample containing nucleic acids is
introduced into sample cup 5002 through sample port 5001. The
sample flows freely into recess 5003 where it reconstitutes the
first lyophilized bead 5004, preferably comprising lysis reagents,
prior to flowing down channel 5005 into first reaction chamber
5006. This free flow is facilitated by vent channel 5007 which
connects to the top of the sample cup 5002. Vent channel 5007 may
additionally connect to expansion chamber 5021 via hole 5008. The
sealed air space below first reaction chamber 5006 pressurizes
slightly due to the fluid flow and causes the flow to stop just
below first reaction chamber 5006. First reaction chamber 5006 is
then preferably heated to a temperature to facilitate proper
reaction with the lysis reagents, lysing the biological particles
and/or cells in the sample, exposing any nucleic acids present
therein.
[0153] Opening of the vent valve 5009 connected to the top of
second reaction chamber 5011 then facilitates sample flow into a
second recess where second lyophilized bead 5010, preferably
comprising reagents for reverse transcription, is reconstituted.
The fluid then enters second reaction chamber 5011 where it's flow
stops as a result of increased air pressure in the closed air
volume below the flow. Second reaction chamber 5011 is then
preferably subsequently heated to an appropriate temperature to
facilitate the reverse transcription process.
[0154] Opening of the next vent valve 5009 connected to the top of
third reaction chamber 5013 initiates flow of the sample from
second reaction chamber 5011 through a third recess where
lyophilized bead 5012, preferably comprising lyophilized PCR
amplification reagents, is reconstituted. The sample then flows
into third reaction chamber 5013, where it undergoes thermal
cycling to amplify targeted analytes present in the sample.
[0155] Subsequently, opening of the final vent valve 5009 connected
to the far end of lateral flow strip 5014 enables the sample which
now contains amplified analytes to flow to lateral flow strip 5014
for detection of the analytes as previously described.
Flow Control Features
[0156] The design of the fluidic component may optionally comprise
flow control features within, or at the outlets of, the reaction
chambers. These features deflect the flow entering the chamber to
the side of the chamber opposite from the outlet, prior to the flow
entering the outlet. As a result the flow enters the outlet channel
at a lower velocity, reducing the distance the fluid flows down the
channel before it stops. Furthermore, the horizontal component of
the flow path adds length to the channel without adding vertical
spacing between the chambers, increasing the effective length of
the flow path so it is sufficient to stop the flow at the desired
location based on the reduced velocity of the flow. This enables
closer vertical spacing between chambers of the cassette since less
vertical channel is required. In addition, the redirection of the
flow across the reaction chamber creates a swirling action in the
flow within the chamber, improving mixing of the reagents with the
sample fluid. The flow control feature may comprise any shape.
[0157] In the embodiment shown in FIG. 39, the fluid enters
reaction chamber 4003 from inlet channel 4002 and flows to the
bottom of the reaction chamber, where it is redirected to the side
of reaction chamber 4003 opposite the opening to inlet channel 4002
by triangular flow control feature 4001. As the flow proceeds to
opposite corner 4004, the flow divides, with some entering outlet
4005, while the rest contacts the wall and is directed upward,
creating a swirling effect which improves mixing. The flow into the
outlet preferably forms a meniscus and travels through outlet
channel 4006 towards the next reaction chamber or lyophilized bead
recess. Since outlet channel 4006 is sealed below reaction chamber
4003, as the fluid travels along outlet channel 4006 the air
pressure increases in the channel below the flow until reaching
equilibrium with the fluidic pressure head, thus stopping the flow.
In this embodiment outlet 4005 tapers from reaction chamber 4003 to
outlet channel 4006 in order to effectively form a meniscus which
can subsequently increase pressure in the closed air space
downstream of the flow. This larger opening to the outlet channel
preferably provides increased compressible air volume so that a
meniscus may be reliably formed at the wider opening.
[0158] In the embodiment shown in FIG. 40, the fluid enters
reaction chamber 4103 from inlet channel 4102 and flows to the
bottom of the reaction chamber, where it is redirected to the side
of reaction chamber 4103 opposite the opening to inlet channel 4102
by triangular flow control feature 4101. As the flow proceeds to
opposite corner 4104, the flow divides, with some entering outlet
4105, while the rest contacts the wall and is directed upward,
creating a swirling effect which improves mixing. The flow into the
outlet preferably forms a meniscus and travels through outlet
channel 4106 towards the next reaction chamber or lyophilized bead
recess. Since outlet channel 4106 is sealed below reaction chamber
4103, as the fluid travels along outlet channel 4106 the air
pressure increases in the channel below the flow until reaching
equilibrium with the fluidic pressure head, thus stopping the flow.
In this embodiment outlet 4105 and outlet channel 4106 have uniform
width. In this embodiment formation of a meniscus at the reaction
chamber may be somewhat more reliable as a result of the narrower
channel. The meniscus subsequently increases pressure in the closed
air space downstream of the flow.
[0159] In the embodiment shown in FIG. 41, the fluid enters
reaction chamber 4103 from inlet channel 4202 and flows to the
bottom of the reaction chamber, where it is redirected to the side
of reaction chamber 4203 opposite the opening to inlet channel 4202
by trapezoidal flow control feature 4201. As the flow proceeds to
opposite corner 4204, the flow divides, with some entering outlet
4205, while the rest contacts the wall and is directed upward,
creating a swirling effect which improves mixing. In this
embodiment outlet 4205 is oriented substantially vertically. The
flow into the outlet preferably forms a meniscus and travels
through outlet channel 4206 towards the next reaction chamber or
lyophilized bead recess. Since outlet channel 4206 is sealed below
reaction chamber 4203, as the fluid travels along outlet channel
4206 the air pressure increases in the channel below the flow until
reaching equilibrium with the fluidic pressure head, thus stopping
the flow. In this embodiment outlet 4205 and outlet channel 4206
have uniform width. In this embodiment formation of a meniscus at
the reaction chamber may be somewhat more reliable as a result of
the narrower channel. The meniscus subsequently increases pressure
in the closed air space downstream of the flow.
[0160] In the embodiment shown in FIG. 42, the fluid enters
reaction chamber 4305 from inlet channel 4304 and flows to the
bottom of the reaction chamber, where it is redirected to the side
of reaction chamber 4305 opposite the opening to inlet channel 4304
by triangular flow control feature 4303. As the flow proceeds to
opposite corner 4306, the flow divides, with some entering outlet
4307, while the rest contacts the wall and is directed upward,
creating a swirling effect which improves mixing. The flow into the
outlet preferably forms a meniscus and travels through outlet
channel 4306 towards the next reaction chamber or lyophilized bead
recess. In this embodiment outlet channel 4306 travels through
stacked serial flow control features 4303, 4302, and 4301 which
provide a tortuous route for the fluid to flow, providing an
increased outlet channel length in a small vertical space.
[0161] In the embodiment shown in FIG. 43, the fluid enters
reaction chamber 4403 from inlet channel 4402 and is redirected to
the side of reaction chamber 4403 opposite the opening to inlet
channel 4402 by flow control feature 4401 disposed above the bottom
of the reaction chamber, preferably approximately halfway along the
length of reaction chamber 4403. In contrast to the previous
embodiments, flow control feature 4401 does not form the outlet of
reaction chamber 4403. Flow control feature 4401 deflects the flow
away from outlet channel 4405 into opposite corner 4404, thereby
reducing the flow velocity prior to exiting the chamber. Similar to
the previous embodiments the fluidic redirection promotes
turbulence and reagent mixing.
Multiplexing of Assays
[0162] In some embodiments of the invention, multiple independent
assays may be performed in parallel by employing a fluidic design
that enables splitting an input fluid sample into two or more
parallel fluidic paths through the device. FIG. 10 is a schematic
representation of splitting a fluid volume, for example 80 uL, in
two sequential steps into first two separate 40 uL volumes and
subsequently into four 20 uL volumes. The illustrated scheme is
useful to enable separate independent manipulations such as
biochemical reactions to be conducted on the split volumes. Such
configurations are useful for increasing the number of analytes
that can be detected in a single device by facilitating the
multiplexed detection of multiple targets such as nucleic acid
sequences in multiplexed nucleic acid reverse transcription and/or
amplification reactions. Similarly, the use of multiple detection
strips at the end of the independent fluid paths can afford
enhanced readability of strips for the detection of multiple
targets or distinguishing sequence differences or mutations in
nucleic acid analytes. Furthermore, providing additional detection
strips for independent interrogation of multiple amplification
reaction products can enhance specificity by reducing the
likelihood of spurious cross-reactivity such as cross hybridization
during the detection step of the test. FIG. 11 illustrates a test
cassette comprising two fluid paths in a single test cassette. Each
fluid path may be independently controlled with respect to timing,
reaction type, etc. Referring now to FIG. 12, a sample introduced
to sample cup 1000 is divided into approximately equal volumes and
flows into volume splitting chambers 1001 and 1002, flow into which
is regulated by vent valves 1003 and 1004. Splitting chambers 1001
and 1002 control the volume of sample in each test path by
passively equilibrating the amount of sample in each chamber. After
volume splitting, solution is allowed to flow through reagent
recesses 1007 and 1008 by opening of vents 1005 and 1006. Reagents
such as lyophilized reagents are disposed in recesses 1007, 1008
and comingled with the sample as it flows through the recesses and
into a first set of preferably temperature controlled chambers 1009
and 1010. Reactions such as heat lysis, reverse transcription,
and/or nucleic acid amplification are conducted in each of the
first set of heated chambers facilitated by reagents provided in
the reagent recesses 1007, 1008. Such reagents may include but are
not limited to lyophilized positive control agent (e.g. nucleic
acid, virus, bacterial cells, etc.), lyophilized reverse
transcriptase and associated accessory reagents such as
nucleotides, buffers, DTT, salts, etc. required for reverse
transcription of RNA to DNA, and/or DNA amplification using
lyophilized DNA polymerase or thermostable lyophilized DNA
polymerase and required accessory reagents such as nucleotides,
buffers, and salts.
[0163] Following the completion of biochemical reactions such as
reverse transcription, nucleic acid amplification or concomitant
reverse transcription and nucleic acid amplification (e.g. single
tube reverse transcription-polymerase chain reaction (RT-PCR) or
one-step RT-PCR or one-step RT-Oscar) in the first set of chambers,
seals for vent pockets 1011 and 1012 are ruptured to allow fluid to
flow from the first set of chambers through a second set of reagent
recesses 1013 and 1014 and into a second set of preferably
temperature controlled chambers 1015 and 1016. Reagents such as
lyophilized reagents may be disposed in recesses 1013 and 1014 such
that they comingle with the sample solution as fluid flows from
chambers 1009 and 1010 to chambers 1015 and 1016. Reagents such as
lyophilized reagents for nucleic acid amplification or dried or
lyophilized detection particles such as probe conjugated dyed
polystyrene microspheres or probe conjugated colloidal gold may
optionally be placed in reagent recesses 1013 and/or 1014.
Following completion of reactions or other manipulations such as
binding or hybridization to probe conjugated detection particles in
heated chambers, solution is allowed to flow into detection strip
chambers 1017 and 1018 by opening vent valves 1019 and 1020. In
some embodiments, a third set of reagent recesses may be placed in
the fluid paths from chambers 1015 and 1016 such that additional
reagents, such as detection reagents comprising detection
particles, salts and/or surfactants and other substances useful to
facilitate hybridization or other detection modalities, may be
comingled with the solution flowing into strip chambers 1017 and
1018. Detection strip chambers 1017 and 1018 may be heated and
preferably comprise detection strips such as lateral flow strips
for the detection of analytes such as amplified nucleic acids.
Detection strips may comprise a series of absorbent materials doped
or patterned with dried or lyophilized detection reagents such as
detection particles (e.g. dyed microsphere conjugates and/or
colloidal gold conjugates), capture probes for the capture of
analytes such as hybridization capture oligonucleotides for the
capture of nucleic acid analytes by sequence specific
hybridization, ligands such as biotin or streptavidin for the
capture of appropriately modified analytes, and absorbent materials
to provide an absorbent capacity sufficient to ensure complete
migration of the sample solution volume through the detection strip
by such means as capillary action or wicking.
Sample Preparation
[0164] In some embodiments of the invention, it may be desirable to
incorporate a sample preparation system into the cassette. A sample
preparation system, such as a nucleic acid purification system, may
comprise encapsulated solutions for accomplishing sample
preparation and elution of purified molecules such as purified DNA,
RNA or proteins into the test cassette. FIG. 13 depicts a nucleic
acid sample preparation subsystem 1300 designed for integration
with a test cassette. The sample preparation subsystem comprises a
main housing 1302 and housing lid 1301 to house components of the
subsystem. A solution compartmentalization component 1303 comprises
crude sample reservoir 1312 which is preferably open on the upper
face but sealed underneath by lower seal 1305. Solution
compartmentalization component 1303 also preferably comprises
reservoir 1314 containing a first wash buffer and reservoir 1315
containing a second wash buffer, both of which are preferably
sealed by means of upper seal 1304 and lower seal 1305. A nucleic
acid binding matrix 1306 is placed in the solution capillary flow
path provided by absorbent materials 1307 and 1308. Glass fiber or
silica gel exhibiting nucleic acid binding properties and wicking
properties are examples of materials suitable for use as binding
matrix 1306. A wide range of absorbent materials may comprise the
absorbent materials 1307 and 1308, including polyester, glass
fiber, nitrocellulose, polysulfone, cellulose, cotton or
combinations thereof as well as other wicking materials provided
they offer adequate capillarity and minimal binding to the
molecules to be purified by the subsystem. Any readily ruptured or
frangible material capable of being sealed to solution
compartmentalization component 1303 and chemically compatible with
the encapsulated solutions is suitable for use as seal material
1304 and 1305. Seal material 1305 comes in contact with sample or
sample lysate and must additionally be chemically compatible with
the sample or sample lysate solution. Examples of suitable seal
material are heat sealable metallic film and plastic film. Seal
material 1305 is ruptured at the time of use by displacement of the
solution compartmentalization component 1303 such that the seal
1305 is pierced by structures 1311 present in housing 1302. Crude
sample or crude sample mixed with a lysis buffer such as a buffer
comprised of a chaotropic agent is introduced at the time of use to
the sample reservoir 1312 via the sample port 1309 in lid 1301. In
some embodiments, lysis buffer may optionally be encapsulated in
reservoir 1312 by extending seal 1304 to cover the upper orifice of
reservoir 1312. In such embodiments it may be desirable to include
a tab or other means for the partial removal of that region of seal
1304 covering reservoir 1312 to allow the addition of crude sample
to reservoir 1312 such that crude sample may comingle or mix with
the lysis buffer contained therein. Sample solution or lysate
containing sample material is introduced to the sample addition
port 1309 and retained in sample reservoir 1312 of buffer reservoir
1303 until the initiation of the sample preparation process.
[0165] At the time of sample preparation initiation, solution
compartmentalization component 1303 is pushed onto the seal
piercing structures 1311 resulting in the simultaneous release of
sample solution or lysate in reservoir 1312 and first and second
wash buffers in reservoirs 1314 and 1315 respectively. Mechanical
displacement of component 1303 may be accomplished manually or by
the use of an actuator or actuators present in a reusable
instrument into which the disposable test cassette is placed at the
time of use. Actuator access or manual displacement mechanism
access to reservoir 1303 is preferably provided through access port
1310 of housing lid 1301. Sample or lysate solution and first and
second wash buffers are moved through materials 1307, 1306 and 1308
by capillary action. The physical arrangement of the reservoirs and
the geometric configuration of absorbent material 1307 ensure
sequential flow of the crude lysate, first wash buffer and second
wash buffer through the binding matrix 1306. Additional absorbent
capacity to ensure continued capillary transport of all solution
volumes through the system is provided by absorbent pad 1313 placed
in contact with wick 1308. At the completion of solution transport
through the absorbent materials, spent solutions come to rest in
absorbent pad 1313. Following exhaustion of capillary transport of
all solutions through the system, purified nucleic acids are bound
to binding matrix 1306, from which the nucleic acids may be eluted
into the sample cup 1402 of the integrated test cassette, as shown
in FIG. 15.
[0166] Movement of the sample preparation subsystem components
occurring during the sample preparation process are shown in FIG.
14, which depicts the sample preparation subsystem embodiment in
cross-section before and after sample processing. Elution is
preceded by displacement of binding matrix 1306 out of the
capillary flow path and through seal component 1316 by the action
of an actuator in the associated reusable test instrument. Seal
component 1316 forms a seal with a portion of elution buffer
conduit 1318 to allow the injection of elution buffer through
binding matrix 1306 and into sample cup 1402 without solution loss
to the capillary flow path of the sample preparation subsystem.
Conduit 1318 is attached to or part of the elution buffer injector
component comprised of elution buffer reservoir 1317 and plunger
1319. Plunger 1319 may optionally comprise o-rings to facilitate
sealing of elution buffer within reservoir 1317. During elution of
purified nucleic acid, an actuator moves elution reservoir
component 1317 such that attached conduit 1318 forms a seal with
seal component 1316 and displaces binding matrix 1306 into chamber
1321. Mechanical access to depress elution reservoir 1317 is
provided through actuator access port 1320. Following displacement
of binding matrix 1306 out of the main capillary solution flow path
of the sample preparation subsystem, binding matrix 1306 resides in
elution chamber 1321. Elution of purified nucleic acid into sample
cup 1402 is accomplished by forcing elution buffer from elution
buffer reservoir 1317 by an actuator acting through actuator port
1322 to move plunger 1319 through reservoir 1317 in a syringe-like
action. Elution buffer proceeds via conduit 1318 through binding
matrix 1306, resulting in the injection of elution buffer
containing eluted purified nucleic acids into sample cup 1402.
[0167] Referring now to FIG. 15, sample preparation subsystem 1300
is preferably bonded to fluidic component 1403 of cassette 1500 by
widely used manufacturing methods such as ultrasonic welding to
form an integrated single use sample-to-result test cassette. In
some embodiments, it is desirable to hermetically seal the test
cassette fluidics following the introduction of eluate containing
purified nucleic acid in order to reduce the likelihood of
amplified nucleic acid escape from the cassette. A sliding seal
1404 may optionally but preferably be placed between the sample
preparation subsystem and the test cassette fluidic housing 1403 to
seal the cassette at the entrance to sample cup 1402. Sliding seal
1404 is moved to the sealed position by the action of an actuator
to form a hermetic seal comprising o-ring 1405. Cassette backing
1406 is bonded to the fluidic housing following the introduction of
dried reagents and test strips. As described above for the backing
of other test cassette embodiments, backing 1406 comprises
materials for vent functionality, hermetic seal maintenance,
thermal interface, expansion chamber(s) and may optionally comprise
a printed circuit board or flexible circuit layer carrying fluid
and temperature control electronic components. Electronic
components may optionally be housed in a reusable docking unit. PCA
1501 comprising electronic components is preferably constructed of
low thermal mass materials and surface mount electronic components.
Arrays of surface mount resistors and proximally situated
temperature sensors provide one means of regulating chamber
temperatures in the test cassette. Surface mount resistors and
temperature sensor arrays of PCA 1501 are situated to be in
register with the test cassette when the test cassette in loaded
into the docking unit. A sample-to-result integrated cassette is
shown in FIG. 16. FIG. 17 illustrates the integrated cassette with
an underlying electronics layer based on traditional printed
circuit board and surface mount components. In some embodiments a
flexible circuit may be bonded to the rear of the test
cassette.
Electronics
[0168] In some embodiments it is desirable to place electronic
components in a reusable component such that heaters, sensors and
other electronics are interfaced to the disposable test cassette by
a means capable of establishing a favorable thermal interface and
accurate registration of electronics with overlying elements of the
disposable test cassette with which they must interface. In other
embodiments it is desirable to use a combination of reusable and
disposable components for temperature control. For example,
stand-off temperature monitoring can be accomplished with infrared
sensors placed in a reusable docking unit, while resistive heaters
for temperature control and fluidics control are placed in a
flexible circuit integrated into the disposable test cassette.
[0169] In some embodiments, the printed circuit board (PCB)
comprises a standard 0.062 inch thick FR4 copper clad laminate
material, although other standard board materials and thicknesses
may be used. Electronic components such as resistors, thermistors,
LEDs, and the microcontroller preferably comprise off-the-shelf
surface mount devices (SMDs) and are placed according to industry
standard methodology.
[0170] In alternative embodiments, the PCA could be integrated with
the cassette wall and comprise a flexible plastic circuit. Flex
circuit materials such as PET and polyimide may be used as shown in
FIG. 8. The use of flexible plastic circuitry is well known in the
art. In another embodiment, heating elements and temperature
sensors may be screen printed onto the plastic fluidic component
with technology developed by companies such as Soligie, Inc.
[0171] In some embodiments of the invention, the PCB thickness as
well as the amount and placement of copper in regions surrounding
the resistive heaters are tailored for thermal management of the
reaction solution in the fluidic component. This can be
accomplished by use of standard manufacturing techniques already
mentioned.
[0172] In some embodiments of the invention, the resistor is a
thick film 2512 package, although other resistors may be used.
Heating chambers in the fluidic component are preferentially of
dimensions similar to those of the resistor to ensure uniform
heating throughout the chamber. A single resistor of this size is
sufficient to heat approximately 15 .mu.L of solution, assuming a
fluidic component thickness of 0.5 mm. The drawing in FIG. 2D shows
two resistors 100 forming a heater sufficient to heat approximately
30 .mu.L of solution, assuming a fluidic component thickness of 0.5
mm. In this case, the resistors are preferably 40 ohm each and
arranged in a parallel configuration.
[0173] In some embodiments of the invention, temperature sensor 110
preferably comprises a thermistor, such as a 0402 NTC device, or a
temperature sensor such as the Atmel AT30TS750, each of which has a
height similar to that of the 2512 resistor package. The thermistor
is preferably aligned either adjacent to or in between the resistor
heaters in the case of a one resistor or two resistor set-up,
respectively. By closely aligning these electronic elements, only a
very thin air gap results between them. Furthermore, application of
a thermal compound before assembling the fluidic with the
electronic layer ensures good thermal contact between the fluidic
component, resistor, and thermistor.
[0174] In some embodiments of the invention, vent resistors 70, 71
comprise a thick film 0805 package, although similar resistors may
be used. In place of a resistor, a small gauge nichrome wire
heating element, such as a 40 gauge nichrome wire may also be
used.
[0175] In some embodiments of the invention, the microcontroller is
a Microchip Technologies PIC16F1789. The microcontroller is
preferably matched to the complexity of the fluidic system. For
example, with multiplexing, the number of individual vents and
heaters is commensurate with the number of microcontroller I/O
lines. Memory size can be chosen to accommodate program size.
[0176] In certain embodiments of the invention, N-channel MOSFETs
in the SOT-23 package operating in an ON-OFF mode are used to
modulate current load to vent and heater resistors. Modulation
signals are sent via the microcontroller. In alternative
embodiments, a pulse-width-modulation scheme and/or other control
algorithms could be used for more advanced thermal management of
fluidics. This would typically be handled by the microcontroller
and may require additional hardware and/or software features known
to those skilled in the art.
[0177] Depending on the application, some embodiments comprise a
device in which a small controlling docking unit or docking unit
operates a smaller disposable unit comprising fluidic systems which
come in contact with biological materials, referred to as the test
cassette. In one such embodiment, the docking unit comprises the
electronic components. Elimination of electric components from the
disposable test cassette reduces costs and in some cases
environmental impact. In another embodiment, some electronic
components are included in both the docking unit and the test
cassette. In this particular embodiment, the test cassette
preferably comprises a low cost PCA or preferably a flexible
circuit to provide some electrical functions such as temperature
control, fluid flow control and temperature sensing, which are
energized, controlled and/or interrogated by the docking unit
through an appropriate interface. As described above, the
electronic functions of such a device is preferably split into two
separate subassemblies. Disposable cassette 2500 preferably
comprises a rear surface designed to interface with resistive
heating and sensing elements of the docking unit. Materials
comprising the rear face of the test cassette are preferably
selected to provide suitable thermal conductivity and stability
while enabling fluid flow control via vent rupture. In some
embodiments, the rear face of the test cassette or a portion
thereof comprises a flexible circuit manufactured on a substrate
such as polyimide. Flexible circuits can be employed to provide low
cost resistive heating elements with low thermal mass. Flexible
circuit substrates may preferably be placed in direct contact with
solutions present in the fluid network of the test cassette to
enable highly efficient and rapid heating and cooling. Connector
810 as shown in FIG. 8 preferably provides current to the resistive
heaters along with a power and signal line to the optional
thermistor(s).
[0178] If flexible circuit 799 is used, one or more IR sensors
located in the docking unit can monitor the temperature of the
heated chambers (e.g. amplification or detection chambers) by
reading the signal through a window in backing 805 or directly off
the rear of flexible circuit 799. Optionally, thermistors on the
PCA or flexible circuit 799 can be used to monitor the
temperatures. Optionally performing a weighted average of the
outputs of the IR sensors and thermistors improves the correlation
between the readings and the fluid temperature in the cassette. In
addition, sensors can also detect ambient temperature, enabling the
system to correct for it to ensure that the sample fluid
equilibrates rapidly to the desired temperatures.
[0179] Referring now to FIG. 33, the docking unit preferably
comprises a reusable component subassembly 3980 comprising the
microcontroller, MOSFETs, switches, power supply or a power jack
and/or battery, optional cooling fan 903, optional user interface,
infrared temperature sensors 901, 902 and connector 900 compatible
with connector 810 of cassette 2500. When the subassemblies are
mated via connectors 810 and 900, the docking unit preferably
supports disposable cassette 2500 in a substantially vertical or
near-vertical orientation. Although a substantially vertical
orientation is preferable in some of the embodiments described
herein, similar results may be obtained if the device is operated
at a tilt, especially if certain pathways are coated to reduce the
wetting angle of solutions used.
[0180] Another embodiment of the device may be used in order to
minimize the operational costs, by reducing the cost of the
consumable part of the system by eliminating all electronic
circuitry located on the disposable part. The microcontroller,
heaters, sensors, power supply, and all other circuitry are located
on multiple PCA's and electrically connected to each other via high
conductor count industry standard ribbon cables. A display may also
be added to aid the user in operation of the device. An optional
serial control port may also be utilized in order to allow the user
to upload changes in test parameters, and to monitor the progress
of any testing. One version of this embodiment comprises five
different PCA's. The Main Board PCA contains the control circuitry,
serial port, power supply, and connectors to connect to the other
boards in the system. The Heater Board PCA contains the heating
resistor elements, temperature sensors, and vent burn heating
elements. In order to facilitate the thermal interface between this
heater board and the disposable fluidic cassette, this board is
mounted on a spring loaded carrier which is moved towards the
backside of the fluidic cassette by the closing action of the lid,
until contact with the fluidic cassette is made. A thin thermally
conductive heating pad is affixed on top of the chamber heater
resistors and temperature sensor, improving heat transfer between
the heater board and the fluidic cassette. A durable vent burning
heating element may be realized using nichrome wire wrapped around
a small ceramic carrier. The IR sensor board PCA is mounted some
small distance from the opposite side of the cassette and is used
for monitoring the heating chamber temperatures. This allows closed
loop temperature control of the heating and cooling process, and
accommodates ambient temperature variations. Also mounted on the IR
sensor board are multiple reflective sensing optical couplers which
allow the sensing of the presence of the cassette, and may be used
to identify the type of cassette denoted by the configurable
reflective pattern located on the cassette. A Display Board PCA may
be located approximately behind the IR Board to allow the user to
see the display from the front of the device. A final PCA, the
shutter board is located across from the top edge of the cassette
and contains a switch and reflective optical coupler which is used
to sense whether or not the cassette has already been used, and
when the lid closes, holding the cassette in place for testing.
[0181] System cooling is optionally augmented using a fan such as a
muffin style fan which is turned on by the microcontroller only
during the cooling phase of testing. A system of vents is
preferably used to direct cooler outside air against the heating
chambers and expel it out the sides of the device.
[0182] In order to provide a complete sample-to-result molecular
test, any of the above embodiments of the invention may be
interfaced to a sample preparation system 1300 that provides
nucleic acids as output to sample chamber 1402. This has been
demonstrated using the sample preparation technology described in
International Publication No. WO 2009/137059 A1, entitled "Highly
Simplified Lateral Flow-Based Nucleic Acid Sample Preparation and
Passive Fluid Flow Control". An embodiment of the resulting
integrated device is illustrated in FIG. 15 and FIG. 16.
Docking Unit
[0183] The reusable docking unit comprises requisite electronic
components to achieve test cassette functionality. Various docking
unit embodiments have been invented to interface with corresponding
variations in test cassette design. In one embodiment, the docking
unit, shown in FIG. 18 and FIG. 19, comprises all electronic
components required to run a test, eliminating the need for
electronic components in the test cassette. Referring now to FIG.
18, prior to sample addition, cassette 2500 is inserted into
docking unit 2501. Docking unit 2501 comprises a display such as
LCD display 2502 to communicate information such as test protocols
and test status to the user. Following cassette insertion into
docking unit 2501, sample is introduced to sample port 20 of
cassette 2500 and docking unit lid 2503 is closed to initiate the
test. A docking unit with inserted test cassette is shown in FIG.
19, and Docking unit 2501 with lid in the closed position is
illustrated in FIG. 18B.
[0184] In some embodiments of the docking unit, a mechanism is
incorporated into the hinge of lid 2503 which moves sliding seal 91
of the test cassette to the closed position. A sealed test cassette
is helpful to ensure amplified nucleic acids remain contained
within the test cassette. Referring now to FIG. 20, either a manual
or an automated method may be employed to slide a valve over the
sample port to seal the cassette. In some embodiments the slide
seals the sample port by engaging an o-ring. The expansion chamber
cover holds the valve slide in place above the sample port o-ring.
The seal is moved into position by a servo motor or by a manual
action such as closing the reusable docking unit lid, which in turn
actuates a mechanism to close the cassette seal. In the pictured
embodiment rack and pinion mechanism 2504 employs slide seal
actuator 3979 to move sliding seal 91 to the closed position. Rack
and pinion mechanism 2504 may be motorized or moved by the action
of closure of docking unit lid 2503 through a mechanical coupling
to lid hinge. Optionally, a sensor such as optical sensor 2505 may
be situated to interrogate the position of sliding seal 91 to
ensure proper seal placement prior to assay initiation as
illustrated in FIG. 21. The optical sensor detects the state (i.e.
position) of the cassette sample port seal. The optical sensor
allows the docking unit to be programed to detect accidental
insertion of a previously used test cassette and to detect the
successful closure of the test cassette seal. An error message
indicating seal malfunction may be displayed on display 2502 and
the test program aborted should sensor 2505 fail to detect seal
closure. In other embodiments of the test cassette and docking
unit, the sealing mechanism may comprise other means of
mechanically sealing the chamber such as a rotating valve as
illustrated in FIG. 22. In yet another embodiment, a test cassette
seal may be placed in a hinged cassette lid placed such that
insertion into the docking unit is not possible without first
closing the cassette lid and thus seating the seal. In this
embodiment, sample is added to the test cassette prior to insertion
into the docking unit. A test cassette comprising a hinged lid with
seal is illustrated in FIG. 23. In general, after the cassette is
inserted into the docking unit and the sample is loaded into the
cassette, it is preferable that closing the lid of the docking unit
both seals the cassette automatically and initiates the assay,
preferably without the use of servos or other mechanical
devices.
[0185] In some docking unit embodiments a set of components
preferably facilitate proper test cassette insertion while ensuring
electronic components that must interface with the test cassette do
not physical interfere with cassette insertion, yet form a reliable
thermal interface during testing. These components form a mechanism
for holding PCA 75 away from the cassette insertion path until
closure of lid 2503. Referring now to FIG. 24, within the docking
unit the heater board is mounted on PCA holder 2506, which
preferably serves as a low thermal mass scaffold, while the test
cassette is loaded into a low thermal mass cassette holder 2507,
wherein rails 2509 guide the cassette into the docking unit and
hold it in the correct position, such as parallel to the heater
board surface, for interfacing with PCA 75 mounted on PCA holder
2506. In the lid open position, prominences 2508 on cassette holder
2507 interfere with PCA holder 2506 to maintain an open path along
rails 2509 for cassette insertion. Preferably, a sloped surface
spans the distance between the surface of prominences 2508 and the
lower elevation of component 2507 to facilitate smooth movement of
prominences 2508 into depressions 2511 on PCA holder 2506 during
closure of lid 2503. Upon closure of the docking unit lid,
prominences 2508 engage with depressions 2511, thereby moving the
heater board mount closer to the rear surface of test cassette
2500. Closure of lid 2503 exerts downward force on cassette holder
2507 thereby moving cassette holder 2507 to a position where
prominences 2508 come to rest in depressions 2511 resulting in
movement of PCA holder 2506 such that PCA 75 is pressed against the
rear of cassette 2500. Preferably PCA holder 2506 is under constant
force, such as spring force, to enable the exertion of reproducible
pressure against the rear of the cassette by PCA 75 after lid
closure. Placement of PCA 75 against the rear of cassette 2500
forms the thermal interface which conducts heat from resistive
heater elements on the PCA to the temperature controlled chambers
and vents of the test cassette. Preferably components 2506 and 2507
are constructed to contribute minimal thermal mass to the system
and provide access to test cassette surfaces for cooling apparatus,
such as fans, and temperature monitoring by sensors, such as
infrared sensors. After lid closure the heater board is thus
preferably pressed firmly against the rear of the test cassette,
forming a thermal interface that enables the microheaters on the
heater board to heat solutions in the fluid chambers of the test
cassette and to melt the thermally labile vent films of the test
cassette, preferably in accordance with microcontroller or
microprocessor control. FIG. 25 illustrates the cassette-PCA
interfacing mechanism in cross-section in both disengaged (lid
open) and engaged (lid closed) positions.
[0186] In some embodiments, the docking unit comprises additional
sensors for such applications as temperature sensing, detecting the
presence of or removal of a test cassette and detecting specific
test cassettes for enabling automated selection of testing
parameters. Referring now to FIG. 26, infrared sensors 2600 detect
the temperature of the test cassette in regions overlying
temperature controlled chambers, such as chambers 30 and 90. The
sensors enable the collection of temperature data in addition to or
in lieu of temperature data collected by PCA 75 localized
temperature sensors, such as sensor 110. Optical sensors may
optionally but preferably be employed to detect specific test
cassettes to identify cassettes for specific diseases or conditions
and allow automated selection of temperature profiles suitable for
a specific test. Referring now to FIGS. 27A and 27B, an optical
sensor or optical sensor array such as optical sensor array 2601
may be employed in conjunction with barcode or barcode-like
features 2602 on the test cassette to determine the type of test
cassette and to confirm complete insertion and correct seating of
the test cassette. Sensor array 2602 in concert with sensor 2505
may be employed to detect the insertion of a previously used test
cassette by detecting a closed seal prior to lid closure. The
docking unit may comprise sensors to detect the type of test
cassette inserted into the docking unit and/or to confirm the
correct insertion, positioning, and alignment of the cassette
within the docking unit. Detection of an influenza A/B test
cassette is illustrated in the docking unit and test cassette
system depicted in FIG. 19. The docking unit can preferably also
read a barcode or other symbol on each cassette and change its
programming in accordance with stored programs for different
assays.
[0187] In some embodiments of the invention it is desirable to heat
both sides of a test cassette. A dual heater PCA configuration
wherein the test cassette is inserted between two heater PCAs is
depicted in FIGS. 28A and 28B.
[0188] In another embodiment, the docking unit comprises servo
actuators, an optical subsystem for automated result readout, a
wireless data communication subsystem, a touch screen user
interface, a rechargeable battery power source, and a test cassette
receiver which accepts a test cassette comprising an integrated
sample preparation subsystem. Referring now to FIGS. 29A, 29B, and
30, docking unit 2700 accepts test cassette 1500 and places the
test cassette in thermal contact with PCA 1501 to enable
temperature control and fluid flow control of the test cassette.
Test cassette 1500 is inserted into cassette receiver slot 3605 of
pivoting docking unit door 2702. Following the addition of crude
sample or lysate to the test cassette, closure of docking unit door
places the rear of the test cassette in register and in contact
with PCA 1501 and in alignment with servo actuators. Servo actuator
3602 is situated to access solution compartmentalization component
1303 through actuator port 1310 of cassette 1500 and provide
mechanical force required to rupture sealing material 1305. Rupture
of mechanical seal 1305 releases crude lysate and wash buffers to
flow through the sample preparation capillary materials of the
sample preparation subsystem as described above. Following
completion of capillary fluid transport, servo actuator 3601 which
is situated to access elution reservoir 1317 through actuator port
1320 of cassette 1500 provides mechanical force to move component
1317 such that attached conduit 1318 forms a seal with seal 1316
and displaces binding matrix 1306 into elution compartment 1321.
Servo actuator 3604 situated to access elution plunger 1319 through
actuator port 1322 of cassette 1500 then provides mechanical force
to plunger 1319 to expel elution buffer from elution reservoir 1317
though binding matrix 1306, resulting in the elution of nucleic
acids into sample cup 1402 of cassette 1500. Servo actuator 3603
seals the cassette after elution as described above. Actuator
control is preferably provided by a microcontroller or
microprocessor on control electronics PCA 3606 according firmware
or software instructions. Similarly, temperature and fluid flow
control within the test cassette is according to instructions
provided in firmware or software routines stored in microcontroller
or microprocessor memory. Optical subsystem 3607 comprising LED
light source 3608 and CMOS sensor 3609, shown in FIG. 31, digitizes
detection strip signal data. Collected detection strip images are
stored into memory within the docking unit where result
interpretation can be accomplished using an on-board processor and
reported to LCD display 2701. A ring of LEDs provides uniform
illumination during image collection with a CMOS-based digital
camera. Images collected with the postage stamp-sized device can
provide high-resolution data (5 megapixels, 10 bit) suitable for
colorimetric lateral flow signal analysis. A preferably low profile
design (.about.1 cm) together with short working distance optics
enables the system to be integrated into a thin device housing.
[0189] Optionally, digitized results may be transmitted for
off-line analysis, storage and/or visualization via a wireless
communication system incorporated into the docking unit employing
either standard WiFi or cellular communications networks.
Photographs of this docking unit embodiment are shown in FIGS. 32A
and 32B.
EXAMPLES
Example 1
Method of Multiplexed Amplification and Detection of Purified Viral
RNA (infA/B) and an Internal Positive Control Virus
[0190] An influenza A and B test cassette was placed into the
docking unit. 40 .mu.L of a sample solution was added to the sample
port. Sample solutions comprised either purified A/Puerto Rico
influenza RNA at a concentration equivalent to 5000 TCID.sub.50/mL,
purified B/Brisbane influenza RNA at a concentration equivalent to
500 TCID.sub.50/mL or molecular grade water (no template control
sample). Upon entering the sample port, the 40 .mu.L sample
comingles with a lyophilized bead as it flows to a first chamber of
the test cassette. The lyophilized bead was comprised of MS2 phage
viral particles as a positive internal control and DTT. In the
first chamber of the cassette the sample was heated to 90.degree.
C. for 1 minute to promote viral lysis then cooled to 50.degree. C.
prior to opening the vent connected a second chamber. Opening the
vent connected to the second chamber allows the sample to flow into
the second chamber by enabling the displacement of the air in the
second chamber to an expansion chamber. As the sample moved to the
second chamber it coming led with oligonucleotide amplification
primers to influenza A, influenza B and MS2 phage, and reverse
transcription and nucleic acid amplification reagents and enzymes
present as a lyophilized pellet in a recess of the fluid path
between first and second chambers.
[0191] The amplification chamber was heated to 47.degree. C. for 6
minutes, during which time RNA template was reverse transcribed
into cDNA. After completion of reverse transcription, 40 cycles of
thermal cycle amplification was conducted in the second chamber.
After thermal cycling was complete, a vent connected to a third
chamber was opened to allow the reaction solution to flow into the
third chamber. The third chamber comprised a test strip and a
lyophilized bead comprising three blue-dyed polystyrene microsphere
conjugates employed as detection particles. Conjugates were
comprised of 300 nm polystyrene microspheres covalently linked to
oligonucleotide probes complementary to amplified sequences of
influenza A, or influenza B or MS2 phage. The solution
reconstituted the lyophilized detection particles as it flowed into
the third chamber. Three capture lines were immobilized on the
lateral flow membrane, from the bottom of the device they were: A
negative control oligonucleotide not complementary to any assayed
targets; a capture probe complementary to the amplification product
of influenza B; a capture probe complementary to the amplification
product of influenza A; and a oligonucleotide complementary to the
amplification product of MS2 phage. The lateral flow strip was
allowed to develop for six minutes prior to visual interpretation
of the results. Upon development of the lateral flow strip,
influenza A positive samples displayed the formation of blue test
lines at the influenza A and MS2 phage positions, influenza B
positive samples displayed the formation of blue test lines at the
influenza B and MS2 phage positions, negative samples displayed the
formation of blue test lines only at the MS2 phage position as
shown in FIG. 34.
Example 2
Method of Multiplexed Amplification and Detection of Viral Lysate
in Buffer and an Internal Positive Control Virus
[0192] An influenza A and B test cassette was placed into the
docking unit. 40 .mu.L of a sample solution was added to the sample
port. Sample solutions comprised either A/Puerto Rico influenza
virus at a concentration equivalent to 5000 TCID.sub.50/mL,
B/Brisbane influenza virus at a concentration equivalent to 500
TCID.sub.50/mL or molecular grade water (no template control
sample). Upon entering the sample port, the 40 .mu.L sample
comingles with a lyophilized bead as it flows to a first chamber of
the test cassette. The lyophilized bead was comprised of MS2 phage
viral particles as a positive internal control and DTT. In the
first chamber of the cassette the sample was heated to 90.degree.
C. for 1 minute to promote viral lysis then cooled to 50.degree. C.
prior to opening the vent connected a second chamber. Opening the
vent connected to the second chamber allows the sample to flow into
the second chamber by enabling the displacement of the air in the
second chamber to an expansion chamber. As the sample moved to the
second chamber it coming led with oligonucleotide amplification
primers to influenza A, influenza B and MS2 phage, and reverse
transcription and nucleic acid amplification reagents and enzymes
present as a lyophilized pellet in a recess of the fluid path
between first and second chambers.
[0193] The amplification chamber was heated to 47.degree. C. for 6
minutes, during which time RNA template was reverse transcribed
into cDNA. After completion of reverse transcription, 40 cycles of
thermal cycle amplification was conducted in the second chamber.
After thermal cycling was complete, a vent connected to a third
chamber was opened to allow the reaction solution to flow into the
third chamber. The third chamber comprised a test strip and a
lyophilized bead comprising three blue-dyed polystyrene microsphere
conjugates employed as detection particles. Conjugates were
comprised of 300 nm polystyrene microspheres covalently linked to
oligonucleotide probes complementary to amplified sequences of
influenza A, or influenza B or MS2 phage. The solution
reconstituted the lyophilized detection particles as it flowed into
the third chamber. Three capture lines were immobilized on the
lateral flow membrane, from the bottom of the device they were: A
negative control oligonucleotide not complementary to any assayed
targets; a capture probe complementary to the amplification product
of influenza B; a capture probe complementary to the amplification
product of influenza A; and a oligonucleotide complementary to the
amplification product of MS2 phage. The lateral flow strip was
allowed to develop for six minutes prior to visual interpretation
of the results. Upon development of the lateral flow strip,
influenza A positive samples displayed the formation of blue test
lines at the influenza A and MS2 phage positions, influenza B
positive samples displayed the formation of blue test lines at the
influenza B and MS2 phage positions, negative samples displayed the
formation of blue test lines only at the MS2 phage position as
shown in FIG. 35.
Example 3
Method of Multiplexed Amplification and Detection of Influenza
Virus (Purified) Spiked into Negative Clinical Nasal Samples and an
Internal Positive Control Virus
[0194] Nasal swab samples collected from human subjects were placed
into 3 mL of a 0.025% Triton X-100, 10 mM Tris, pH 8.3 solution and
tested for the presence of influenza A and influenza B using an FDA
approved real-time RT-PCR test. Samples were confirmed to be
negative for influenza A and influenza B prior to use in this
study. Confirmed influenza negative nasal sample was spiked with
A/Puerto Rico influenza virus at a concentration equivalent to 5000
TCID.sub.50/mL or employed without the addition of virus as a
negative control. 40 .mu.L of the resulting spiked or negative
control samples were added to the sample port of a influenza A and
B test cassette. Upon entering the sample port, the 40 .mu.L sample
comingles with a lyophilized bead as it flows to a first chamber of
the test cassette. The lyophilized bead was comprised of MS2 phage
viral particles as a positive internal control and DTT. In the
first chamber of the cassette the sample was heated to 90.degree.
C. for 1 minute to promote viral lysis then cooled to 50.degree. C.
prior to opening the vent connected a second chamber. Opening the
vent connected to the second chamber allows the sample to flow into
the second chamber by enabling the displacement of the air in the
second chamber to an expansion chamber. As the sample moved to the
second chamber it comingled with oligonucleotide amplification
primers to influenza A, influenza B and MS2 phage, and reverse
transcription and nucleic acid amplification reagents and enzymes
present as a lyophilized pellet in a recess of the fluid path
between first and second chambers.
[0195] The amplification chamber was heated to 47.degree. C. for 6
minutes, during which time RNA template was reverse transcribed
into cDNA. After completion of reverse transcription, 40 cycles of
thermal cycle amplification was conducted in the second chamber.
After thermal cycling was complete, a vent connected to a third
chamber was opened to allow the reaction solution to flow into the
third chamber. The third chamber comprised a test strip and a
lyophilized bead comprising three blue-dyed polystyrene microsphere
conjugates employed as detection particles. Conjugates were
comprised of 300 nm polystyrene microspheres covalently linked to
oligonucleotide probes complementary to amplified sequences of
influenza A, or influenza B or MS2 phage. The solution
reconstituted the lyophilized detection particles as it flowed into
the third chamber. Three capture lines were immobilized on the
lateral flow membrane, from the bottom of the device they were: A
negative control oligonucleotide not complementary to any assayed
targets; a capture probe complementary to the amplification product
of influenza B; a capture probe complementary to the amplification
product of influenza A; and a oligonucleotide complementary to the
amplification product of MS2 phage. The lateral flow strip was
allowed to develop for six minutes prior to visual interpretation
of the results. Upon development of the lateral flow strip,
influenza A positive samples displayed the formation of blue test
lines at the influenza A and MS2 phage positions, negative control
samples displayed the formation of blue test lines only at the MS2
phage position as shown in FIG. 36.
[0196] Although the invention has been described in detail with
particular reference to the disclosed embodiments, other
embodiments can achieve the same results. Variations and
modifications of the present invention will be obvious to those
skilled in the art and it is intended to cover all such
modifications and equivalents. The entire disclosures of all
patents and publications cited above are hereby incorporated by
reference.
* * * * *