U.S. patent application number 17/017965 was filed with the patent office on 2020-12-31 for devices and methods for molecular diagnostic testing.
This patent application is currently assigned to Visby Medical, Inc.. The applicant listed for this patent is Visby Medical, Inc.. Invention is credited to Boris ANDREYEV, Victor BRIONES, Jesus CHING, Steven CHU, Brian CIOPYK, Adam DE LA ZERDA, Helen HUANG, Colin KELLY, Gregory LONEY, Keith E. MORAVICK, David SWENSON.
Application Number | 20200406256 17/017965 |
Document ID | / |
Family ID | 1000005080053 |
Filed Date | 2020-12-31 |
United States Patent
Application |
20200406256 |
Kind Code |
A1 |
ANDREYEV; Boris ; et
al. |
December 31, 2020 |
DEVICES AND METHODS FOR MOLECULAR DIAGNOSTIC TESTING
Abstract
A hand-held molecular diagnostic test device includes a housing,
an amplification (or PCR) module, and a detection module. The
amplification module is configured to receive an input sample, and
defines a reaction volume. The amplification module includes a
heater such that the amplification module can perform a polymerase
chain reaction (PCR) on the input sample. The detection module is
configured to receive an output from the amplification module and a
reagent formulated to produce a signal that indicates a presence of
a target amplicon within the input sample. The amplification module
and the detection module are integrated within the housing.
Inventors: |
ANDREYEV; Boris; (Foster
City, CA) ; MORAVICK; Keith E.; (Mountain View,
CA) ; CIOPYK; Brian; (Santa Clara, CA) ;
BRIONES; Victor; (Gilroy, CA) ; LONEY; Gregory;
(Los Altos, CA) ; DE LA ZERDA; Adam; (Palo Alto,
CA) ; CHING; Jesus; (Saratoga, CA) ; CHU;
Steven; (Menlo Park, CA) ; SWENSON; David;
(Santa Clara, CA) ; HUANG; Helen; (San Pablo,
CA) ; KELLY; Colin; (San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Visby Medical, Inc. |
San Jose |
CA |
US |
|
|
Assignee: |
Visby Medical, Inc.
San Jose
CA
|
Family ID: |
1000005080053 |
Appl. No.: |
17/017965 |
Filed: |
September 11, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15999820 |
Aug 23, 2018 |
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17017965 |
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15586780 |
May 4, 2017 |
10052629 |
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15999820 |
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15474083 |
Mar 30, 2017 |
10124334 |
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15586780 |
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14984573 |
Dec 30, 2015 |
9623415 |
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15474083 |
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62098769 |
Dec 31, 2014 |
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62213291 |
Sep 2, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 2400/0478 20130101;
B01L 2200/028 20130101; B01L 2200/0647 20130101; B01L 3/5027
20130101; B01L 2300/0672 20130101; B01L 7/525 20130101; B01L
2300/0681 20130101; B01L 2300/0654 20130101; B01L 3/502715
20130101; B01L 2400/0611 20130101; B01L 3/5029 20130101; B01L
2300/1822 20130101; B01L 2400/0457 20130101; B01L 2300/0627
20130101; B01L 3/527 20130101; B01L 2400/0487 20130101; B01L
2400/0605 20130101; B01L 2200/10 20130101; B01L 2300/18 20130101;
B01L 2300/0867 20130101; B01L 2300/0883 20130101; B01L 2400/0644
20130101; B01L 2200/025 20130101; B01L 7/52 20130101; B01L
2300/1844 20130101; B01L 2200/0689 20130101; B01L 2200/0684
20130101 |
International
Class: |
B01L 3/00 20060101
B01L003/00; B01L 7/00 20060101 B01L007/00 |
Claims
1. A single-use molecular diagnostic test device, comprising: a
housing including a sample input port; a sample preparation module
within the housing, the sample preparation module defining a
receiving volume in fluid communication with the sample input port,
the receiving volume configured to receive a biological sample, the
sample preparation module configured to react the biological sample
with a lysis reagent to produce an input sample; a sample lid
coupled to the housing, the sample lid configured to move from a
first position to a second position, the sample input port being
accessible when the sample lid is in the first position, the sample
input port being sealed closed when the sample lid is in the second
position, the sample lid configured to irreversibly engage a
portion of the housing when the sample lid is moved to the second
position to lock the sample lid in the second position; an
amplification module within the housing, the amplification module
configured to receive the input sample from the sample preparation
module, the amplification module defining a reaction volume and
including a heater, the amplification module configured to amplify
a target nucleic acid within the input sample to produce an
amplified output containing a target amplicon; and a detection
module within the housing, the detection module defining a
detection channel and a detection surface within the detection
channel, the detection channel configured to receive the amplified
output from the amplification module and react the target amplicon
within the amplified output with a detection reagent to produce a
signal from the detection surface that indicates a presence of the
target amplicon.
2. The single-use molecular diagnostic test device of claim 1,
wherein the signal is a non-fluorescent signal.
3. The single-use molecular diagnostic test device of claim 2,
wherein: the housing defines a detection window; the signal is a
visible signal; and the visible signal is viewable via the
detection window of the housing.
4. The single-use molecular diagnostic test device of claim 3,
wherein the detection surface includes a capture probe associated
with the target amplicon.
5. The single-use molecular diagnostic test device of claim 3,
wherein the detection surface includes an absorbent member
formulated to receive the amplified output produced by the
amplification module, the visible signal being produced from the
absorbent member.
6. The single-use molecular diagnostic test device of claim 3,
further comprising: a solid positive control that is nonpathogenic
to humans and is mixed with the biological sample.
7. The single-use molecular diagnostic test device of claim 6,
wherein the solid positive control is within the receiving volume,
the solid positive control being rehydrated and mixed with the
input sample when the input sample is conveyed from the receiving
volume toward the amplification module.
8. The single-use molecular diagnostic test device of claim 3,
wherein the amplification module is configured to heat the input
sample through a plurality of thermal cycles to amplify the target
nucleic acid.
9. The single-use molecular diagnostic test device of claim 3,
wherein the amplification module includes a flow member that
defines the reaction volume, the flow member constructed from at
least one of a cyclic olefin copolymer or a graphite-based material
and having a thickness of less than about 0.5 mm.
10. The single-use molecular diagnostic test device of claim 3,
wherein: the housing defines a status indicator, the status
indicator configured to produce a status signal associated with a
status of the single-use molecular diagnostic test device.
11. The single-use molecular diagnostic test device of claim 10,
wherein the status includes at least one of whether a tab has been
removed from the housing, whether a diagnostic test is in process,
whether an error state is present, and whether the diagnostic test
is completed.
12. The single-use molecular diagnostic test device of claim 3,
wherein the sample preparation module includes an indicator to
allow verification that a volume of the biological sample has been
received within the receiving volume.
13. A single-use molecular diagnostic test device, comprising: a
housing including a sample input port; a sample preparation module
within the housing, the sample preparation module defining a
receiving volume in fluid communication with the sample input port,
the receiving volume configured to receive a biological sample, the
sample preparation module including an indicator to allow
verification that a volume of the biological sample has been
received within the receiving volume; a sample lid coupled to the
housing, the sample lid configured to move from a first position to
a second position, the sample input port being accessible when the
sample lid is in the first position, the sample input port being
sealed closed when the sample lid is in the second position, the
sample lid configured to irreversibly engage a portion of the
housing when the sample lid is moved to the second position to lock
the sample lid in the second position; an amplification module
within the housing, the amplification module configured to receive
the biological sample from the sample preparation module, the
amplification module defining a reaction volume and including a
heater, the amplification module configured to amplify a target
nucleic acid within the biological sample to produce an amplified
output containing a target amplicon; and a detection module within
the housing, the detection module defining a detection channel and
a detection surface within the detection channel, the detection
channel configured to receive the amplified output from the
amplification module and react the target amplicon within the
amplified output with a detection reagent to produce a signal from
the detection surface that indicates a presence of the target
amplicon.
14. The single-use molecular diagnostic test device of claim 13,
wherein the signal is a non-fluorescent signal produced without an
excitation light source within the housing.
15. The single-use molecular diagnostic test device of claim 14,
wherein: the housing defines a detection window; the signal is a
visible signal; and the visible signal is viewable via the
detection window of the housing.
16. The single-use molecular diagnostic test device of claim 15,
wherein the visible signal remains visible via the detection window
for at least about 30 minutes after first being produced.
17. The single-use molecular diagnostic test device of claim 15,
wherein the detection surface includes a capture probe associated
with the target amplicon.
18. The single-use molecular diagnostic test device of claim 15,
wherein the detection surface includes an absorbent member
formulated to receive the amplified output produced by the
amplification module, the visible signal being produced from the
absorbent member.
19. The single-use molecular diagnostic test device of claim 13,
wherein the amplification module is configured to heat the
biological sample through a plurality of thermal cycles to amplify
the target nucleic acid.
20. The single-use molecular diagnostic test device of claim 13,
wherein: the housing defines a status indicator, the status
indicator configured to produce a status signal associated with a
status of the single-use molecular diagnostic test device.
21. A system for detecting the presence of a target nucleic acid,
comprising: a single-use molecular diagnostic test device including
a housing, a sample lid coupled to the housing, a sample
preparation module, an amplification module and a detection module,
the sample preparation module, the amplification module, and the
detection module each being within the housing, the housing
including a sample input port; the sample lid coupled to the
housing, the sample lid configured to move from a first position to
a second position, the sample input port being accessible when the
sample lid is in the first position, the sample input port being
sealed closed when the sample lid is in the second position, the
sample lid configured to irreversibly engage a portion of the
housing when the sample lid is moved to the second position to lock
the sample lid in the second position; the sample preparation
module defining a receiving volume in fluid communication with the
sample input port, the receiving volume configured to receive a
biological sample, the sample preparation module configured to
react the biological sample with a lysis reagent to produce an
input sample; the amplification module configured to receive the
input sample from the sample preparation module, the amplification
module defining a reaction volume and including a heater, the
amplification module configured to amplify the target nucleic acid
within the input sample to produce an amplified output containing a
target amplicon; and the detection module defining a detection
channel and a detection surface within the detection channel, the
detection channel configured to receive the amplified output from
the amplification module and react the target amplicon within the
amplified output with a detection reagent to produce a signal from
the detection surface that indicates a presence of the target
amplicon; and a power supply unit configured to supply power to the
single-use molecular diagnostic test device, the power supply unit
configured to be removably coupled to the single-use molecular
diagnostic test device.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/999,820, entitled "Devices and Methods for Molecular
Diagnostic Testing," filed Aug. 23, 2018, which is a continuation
of U.S. application Ser. No. 15/586,780, entitled "Devices and
Methods for Molecular Diagnostic Testing," filed May 4, 2017, now
U.S. Pat. No. 10,052,629, which is a divisional of U.S. application
Ser. No. 15/474,083, entitled "Devices and Methods for Molecular
Diagnostic Testing," filed Mar. 30, 2017, now U.S. Pat. No.
10,124,334, which is a divisional of U.S. application Ser. No.
14/984,573, entitled "Devices and Methods for Molecular Diagnostic
Testing," filed Dec. 30, 2015, now U.S. Pat. No. 9,623,415, which
claims priority to U.S. Provisional Application No. 62/098,769,
entitled "Molecular Diagnostic Device," filed Dec. 31, 2014 and
U.S. Provisional Application No. 62/213,291, entitled "Devices and
Methods for Molecular Diagnostic Testing," filed Sep. 2, 2015, the
entire disclosure of each of which is incorporated herein by
reference in its entirety.
BACKGROUND
[0002] The embodiments described herein relate to methods and
devices for molecular diagnostic testing. More particularly, the
embodiments described herein relate to disposable, self-contained
devices and methods for molecular diagnostic testing.
[0003] There are over one billion infections in the U.S. each year,
many of which are treated incorrectly due to inaccurate or delayed
diagnostic results. Many known point of care (POC) tests have poor
sensitivity (30-70%), while the more highly sensitive tests, such
as those involving the specific detection of nucleic acids or
molecular testing associated with a pathogenic target, are only
available in laboratories. Thus, approximately ninety percent of
the current molecular diagnostics testing is practiced in
centralized laboratories. Known devices and methods for conducting
laboratory-based molecular diagnostics testing, however, require
trained personnel, regulated infrastructure, and expensive, high
throughput instrumentation. Known laboratory instrumentation is
often purchased as a capital investment along with a regular supply
of consumable tests or cartridges. Known high throughput laboratory
equipment generally processes many (96 to 384 and more) samples at
a time, therefore central lab testing is done in batches. Known
methods for processing typically include processing all samples
collected during a time period (e.g., a day) in one large run, with
a turn-around time of hours to days after the sample is collected.
Moreover, such known instrumentation and methods are designed to
perform certain operations under the guidance of a skilled
technician who adds reagents, oversees processing, and moves sample
from step to step. Thus, although known laboratory tests and
methods are very accurate, they often take considerable time, and
are very expensive.
[0004] There are limited testing options available for testing done
at the point of care ("POC"), or in other locations outside of a
laboratory. Known POC testing options tend to be single analyte
tests with low analytical quality. These tests are used alongside
clinical algorithms to assist in diagnosis, but are frequently
verified by higher quality, laboratory tests for the definitive
diagnosis. Thus, neither consumers nor physicians are enabled to
achieve a rapid, accurate test result in the time frame required to
"test and treat" in one visit. As a result, doctors and patients
often determine a course of treatment before they know the
diagnosis. This has tremendous ramifications: antibiotics are
either not prescribed when needed, leading to infections; or
antibiotics are prescribed when not needed, leading to new
antibiotic-resistant strains in the community. Moreover, known
systems and methods often result in diagnosis of severe viral
infections, such as H1N1 swine flu, too late, limiting containment
efforts. In addition, patients lose much time in unnecessary,
repeated doctor visits.
[0005] Thus, a need exists for improved devices and methods for
molecular diagnostic testing. In particular, a need exists for an
affordable, easy-to-use test that will allow healthcare providers
and patients at home to diagnose infections accurately and quickly
so they can make better healthcare decisions.
SUMMARY
[0006] A molecular diagnostic test device includes a housing, an
amplification module and a detection module. The amplification
module is configured to receive an input sample, and defines a
reaction volume. The amplification module includes a heater such
that the amplification module can perform a polymerase chain
reaction (PCR) on the input sample. The detection module is
configured to receive an output from the amplification module and a
reagent formulated to produce a signal that indicates a presence of
a target amplicon within the input sample. The amplification module
and the detection module are integrated within the housing such
that the molecular diagnostic test device is a handheld device.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1 is a schematic illustration of a molecular diagnostic
test device, according to an embodiment.
[0008] FIG. 2 is a schematic illustration of a molecular diagnostic
test device, according to an embodiment.
[0009] FIGS. 3 and 4 are schematic illustrations of a molecular
diagnostic test device, according to an embodiment in a first
configuration and a second configuration, respectively.
[0010] FIGS. 5 and 6 are schematic illustrations of a molecular
diagnostic test device, according to an embodiment in a first
configuration and a second configuration, respectively.
[0011] FIG. 7 is a schematic illustration of a molecular diagnostic
test device, according to an embodiment.
[0012] FIG. 8 is a diagram illustrating an enzyme linked reaction
according to an embodiment conducted on the device of FIG. 7,
resulting in the production a colorimetric result.
[0013] FIG. 9 is a schematic illustration of a molecular diagnostic
test device, according to an embodiment.
[0014] FIGS. 10 and 11 are perspective views of a molecular
diagnostic test device, according to an embodiment.
[0015] FIG. 12 is a perspective view of a top portion of a housing
of the molecular diagnostic test device shown in FIGS. 10 and
11.
[0016] FIG. 13 is a perspective view of a bottom portion of a
housing of the molecular diagnostic test device shown in FIGS. 10
and 11.
[0017] FIG. 14 is a perspective view of the molecular diagnostic
test device shown in FIGS. 10 and 11, with the top portion of the
housing removed to show the internal components.
[0018] FIG. 15 is a perspective view of the molecular diagnostic
test device shown in FIGS. 10, 11 and 14, with the top portion of
the housing, the amplification module, and the detection module
removed to show the internal components.
[0019] FIG. 16 is a front perspective view of a sample input module
of the molecular diagnostic test device shown in FIGS. 10 and
11.
[0020] FIG. 17 is a perspective cross-sectional view of the sample
input module shown in FIG. 16 taken along the line X-X in FIG.
16.
[0021] FIG. 18 is a side perspective view of the sample input
module of the molecular diagnostic test device shown in FIGS. 10
and 11.
[0022] FIG. 19 is a perspective cross-sectional view of the sample
input module shown in FIG. 18 taken along the line X-X in FIG.
18.
[0023] FIG. 20 is a side perspective view of a sample actuator of
the molecular diagnostic test device shown in FIGS. 10 and 11.
[0024] FIG. 21 is a side cross-sectional view of the sample input
module shown in FIGS. 10 and 11 in an actuated configuration.
[0025] FIG. 22 is a front perspective view of a wash module of the
molecular diagnostic test device shown in FIGS. 10 and 11.
[0026] FIG. 23 is a perspective cross-sectional view of the wash
module shown in FIG. 22 taken along the line X-X in FIG. 22.
[0027] FIG. 24 is a side perspective view of a wash actuator of the
molecular diagnostic test device shown in FIGS. 10 and 11.
[0028] FIGS. 25 and 26 are a front perspective view and a rear
perspective view, respectively, of an elution module and a reagent
module of the molecular diagnostic test device shown in FIGS. 10
and 11.
[0029] FIG. 27 is a rear perspective view of the elution module and
the reagent module shown in FIGS. 25 and 26, with a top portion
removed.
[0030] FIG. 28 is a perspective cross-sectional view of the elution
module and the reagent module shown in FIGS. 25 and 26, with a top
portion removed.
[0031] FIGS. 29 and 31 are perspective cross-sectional the reagent
module shown in FIGS. 25 and 26, in a first (or ready)
configuration and a second (or actuated) configuration,
respectively.
[0032] FIG. 30 is a side perspective view of an elution and reagent
actuator of the molecular diagnostic test device shown in FIGS. 10
and 11.
[0033] FIGS. 32 and 34 are front perspective views of a filter
assembly of the molecular diagnostic test device shown in FIGS. 10
and 11, in a first (ready) configuration and a second (actuated)
configuration, respectively.
[0034] FIGS. 33 and 35 are a front exploded view and a rear
exploded view, respectively, of the filter assembly shown in FIGS.
32 and 34.
[0035] FIG. 36 is a side perspective view of an inactivation
chamber of the molecular diagnostic test device shown in FIGS. 10
and 11.
[0036] FIG. 37 is an exploded view of the inactivation chamber
shown in FIG. 36.
[0037] FIGS. 38 and 39 are a front exploded view and a rear
exploded view, respectively, of a mixing assembly of the molecular
diagnostic test device shown in FIGS. 10 and 11.
[0038] FIG. 40 is a front perspective view of a fluid transfer
module of the molecular diagnostic test device shown in FIGS. 10
and 11.
[0039] FIG. 41 is a cross-sectional view of the fluid transfer
module shown in FIG. 40 taken along the line X-X in FIG. 40.
[0040] FIG. 42 is an exploded view of the fluid transfer module
shown in FIG. 40.
[0041] FIG. 43 is an exploded view of an amplification module of
the molecular diagnostic test device shown in FIGS. 10 and 11.
[0042] FIG. 44 is a top view of a flow member of the amplification
module shown in FIG. 43.
[0043] FIG. 45 is an exploded perspective view of the amplification
module shown in FIG. 43 and a detection module of the molecular
diagnostic test device shown in FIGS. 10 and 11.
[0044] FIG. 46 is an exploded perspective view of the detection
module of the molecular diagnostic test device shown in FIGS. 10
and 11.
[0045] FIG. 47 is bottom perspective view of the detection module
shown in FIG. 46.
[0046] FIG. 48 is a side cross-sectional view of a portion of the
detection module shown in FIG. 46.
[0047] FIG. 49 is a top view of a portion of the detection module
shown in FIG. 46.
[0048] FIGS. 50 and 51 are a front perspective view and a rear
perspective view, respectively, of rotary valve assembly of the
molecular diagnostic test device shown in FIGS. 10 and 11.
[0049] FIGS. 52 and 53 are a front exploded view and a rear
exploded view, respectively, of the rotary valve assembly shown in
FIGS. 50 and 51.
[0050] FIGS. 54 through 61 are front views of the rotary valve
assembly shown in FIGS. 50 and 51 in each of eight different
operational configuration.
[0051] FIG. 62 is a side cross-sectional view of a sample transfer
portion of the molecular diagnostic test device shown in FIGS. 10
and 11 in a first configuration, and an external transfer device
according to an embodiment.
[0052] FIG. 63 is a perspective view of the molecular diagnostic
test device shown in FIGS. 10 and 11 in a second (sample actuated)
configuration.
[0053] FIG. 64 is a perspective view of the molecular diagnostic
test device shown in FIGS. 10 and 11 in a third (wash actuated)
configuration.
[0054] FIG. 65 is a perspective view of the molecular diagnostic
test device shown in FIGS. 10 and 11 in a fourth (elution and
reagent actuated) configuration.
[0055] FIG. 66 is a perspective view of the molecular diagnostic
test device shown in FIGS. 10 and 11 in a fifth (read)
configuration.
[0056] FIG. 67 is a graph of power usage and power source voltage
when the device shown in FIGS. 10 and 11 is used to conduct a test
protocol according to an embodiment.
[0057] FIGS. 68A-68C show a flow chart of a test process flow for a
diagnostic test, according to an embodiment.
[0058] FIG. 69 shows a flow chart of a method of diagnostic
testing, according to an embodiment.
[0059] FIG. 70 is a perspective view of a molecular diagnostic test
device, according to an embodiment.
[0060] FIG. 71 is a perspective view of the molecular diagnostic
test device shown in FIG. 70, with the top portion of the housing
removed to show the internal components.
[0061] FIG. 72 is a perspective view of the molecular diagnostic
test device shown in FIG. 70, with the top portion of the housing,
the amplification module, and the detection module removed to show
the internal components.
[0062] FIGS. 73 and 74 are perspective views of a reagent module of
the molecular diagnostic test device shown in FIG. 70.
[0063] FIG. 75 is a perspective view of an apparatus for diagnostic
testing, according to an embodiment.
[0064] FIG. 76 is a top view of the apparatus of FIG. 75.
[0065] FIG. 77 is a side view of the apparatus of FIG. 75.
[0066] FIG. 78 is an illustration of use of a sample input port of
the apparatus of FIG. 75.
[0067] FIG. 79 is an illustration of use of plungers of the
apparatus of FIG. 75.
[0068] FIG. 80 is an illustration of use of a pull-out tab of the
apparatus of FIG. 75.
[0069] FIG. 81 is an illustration of a detachable battery of the
apparatus of FIG. 75.
[0070] FIG. 82 is an illustration of a rechargeable battery of the
apparatus of FIG. 75.
[0071] FIG. 83 is a top view of a molecular diagnostic test device,
according to an embodiment.
[0072] FIG. 84 is a perspective view of the molecular diagnostic
test devices shown in FIG. 83, in an unpackaged configuration.
[0073] FIGS. 85-87 are various views of the molecular diagnostic
test devices shown in FIG. 83, in various stages of operation.
[0074] FIGS. 88-89 are schematic illustrations of a sample transfer
device according to an embodiment, in a first configuration and a
second configuration, respectively.
[0075] FIG. 90 is a perspective exploded view of components of a
sample preparation module, according to an embodiment.
[0076] FIG. 91 is a schematic illustration of the wash reagent
storage and dispensing assembly shown in FIG. 90.
[0077] FIG. 92 is a schematic illustration of the elution reagent
storage and dispensing assembly shown in FIG. 90.
[0078] FIG. 93 is perspective view of an amplification module,
according to an embodiment.
[0079] FIG. 94 is a schematic illustration of a heat sink of the
amplification module shown in FIG. 93.
[0080] FIG. 95 is an exploded view of components of the
amplification module shown in FIG. 93.
[0081] FIG. 96 is perspective cross-sectional view of a fluid
transfer module, according to an embodiment.
[0082] FIGS. 97-99 are perspective cross-sectional views of the
fluid transfer module shown in FIG. 96, in various stages of
operation.
DETAILED DESCRIPTION
[0083] In some embodiments, an apparatus is configured for a
disposable, portable, single-use, inexpensive, molecular diagnostic
approach. The apparatus can include one or more modules configured
to perform high quality molecular diagnostic tests, including, but
not limited to, sample preparation, nucleic acid amplification
(e.g., via polymerase chain reaction or PCR), and detection. In
some embodiments, sample preparation can be performed by isolating
the target pathogen/entity and removing unwanted PCR inhibitors.
The target entity can be subsequently lysed to release target
nucleic acid for PCR amplification. A target nucleic acid in the
target entity can be amplified with a polymerase undergoing
temperature cycling to yield a greater number of copies of the
target nucleic acid sequence for detection.
[0084] Detection can occur, in some embodiments, through a
colorimetric reaction in a read lane. Multiple nucleic acid targets
can be read in the lane, permitting for multiplexed
detection/testing. The apparatus can also contain on-board reagent
storage, fluidic pumping, valving and electronics to properly
sequence test steps and control operation. Further, the apparatus
can be battery powered, allowing the diagnostic test(s) to be run
without A/C power, and at any suitable location (e.g., outside of a
laboratory and/or at any suitable "point of care").
[0085] In some embodiments, the apparatus can be configured to
detect pathogens commonly associated with sexually transmitted
infections (STI) including, but not limited to, Chlamydia
trachomatis (CT), Neisseria gonorrhea (NG) and Trichomonas
vaginalis (TV), through nucleic acid detection. In some
embodiments, the apparatus includes on-board positive and negative
controls to ensure that the diagnostic test(s) are functioning
properly.
[0086] In some embodiments, the apparatus is optimized for
disposable and portable operation. For example, in some
embodiments, the power module can be operated by a small battery
(e.g., a 9V battery), and can include a controller to control the
timing and/or magnitude of power draw to accommodate the capacity
of the battery. In other embodiments, the apparatus can include any
number of features, such as safety locks, configured to minimize
the chances of user error.
[0087] In some embodiments, a hand-held molecular diagnostic test
device includes a housing, an amplification (or PCR) module, and a
detection module. The amplification module is configured to receive
an input sample, and defines a reaction volume. The amplification
module includes a heater such that the amplification module can
perform a polymerase chain reaction (PCR) on the input sample. The
detection module is configured to receive an output from the
amplification module and a reagent formulated to produce a signal
that indicates a presence of a target amplicon within the input
sample. The amplification module and the detection module are
integrated within the housing.
[0088] In some embodiments, an apparatus includes a housing, a
sample preparation module, an amplification (or PCR) module, and a
detection module. The sample preparation module is disposed within
the housing and is configured to receive an input sample. The
amplification module is disposed within the housing and is
configured to receive an output from the sample preparation module.
The amplification module includes a flow member and a heater, with
the flow member defining a serpentine flow path. The heater is
coupled to the flow member. The amplification module is configured
perform a polymerase chain reaction (PCR) on the output from the
sample preparation module. The detection module is disposed within
the housing and is configured to receive an output from the
amplification module. The detection module is configured to receive
a reagent formulated to produce a colorimetric signal that
indicates a presence of a target organism in said input sample. The
sample preparation module, amplification (or PCR) module, and
detection module are collectively configured for one-time use. In
some embodiments, the apparatus is disposable via standard waste
procedures after use.
[0089] In some embodiments, an apparatus includes an amplification
(or PCR) module, and a detection module. The amplification module
is configured to receive an input sample, and defines a reaction
volume. The amplification module includes a heater such that the
amplification module can perform a polymerase chain reaction (PCR)
on the input sample. The detection module is configured to receive
an output from the amplification module and a reagent formulated to
produce a signal that indicates a presence of a target organism
within the input sample. The apparatus is configured to produce the
signal in a time of less than about 25 minutes.
[0090] In some embodiments, an apparatus includes a housing, an
amplification (or PCR) module, and a detection module. The
amplification module is configured to receive an input sample, and
defines a reaction volume. The amplification module includes a
heater such that the amplification module can perform a polymerase
chain reaction (PCR) on the input sample. The detection module is
configured to receive an output from the amplification module and a
reagent formulated to produce a signal that indicates a presence of
a target organism within the input sample. The target organism is
associated with a disease. The amplification module and the
detection module are integrated within the housing and collectively
have a sensitivity of at least about 93 percent and a specificity
of at least about 95 percent for the detection of the disease.
[0091] In some embodiments, an apparatus includes a housing, an
amplification (or "PCR") module, a reagent module, and a detection
module. The housing includes a sample input port and defines a
detection opening. The PCR module is disposed within the housing,
and includes a flow member and a heater. The flow member defines a
PCR flow path having an inlet portion in fluid communication with
the sample input port. The heater is fixedly coupled to the flow
member such that the heater and the PCR flow path intersect at
multiple locations. The reagent module is disposed within the
housing, and contains a substrate formulated to catalyze the
production of an optical signal by a signal molecule associated
with a target amplicon. The detection module defines a detection
channel in fluid communication with an outlet portion of the PCR
flow path and the reagent module. The detection module includes a
detection surface within the detection channel that is configured
to retain the target amplicon. The detection module is disposed
within the housing such that the detection surface is visible via
the detection opening of the housing.
[0092] In some embodiments, the detection channel has a width of at
least about 4 mm. In some embodiments, the housing includes a mask
portion configured to surround at least a portion of the detection
surface. The mask portion can be configured to enhance visibility
of the detection surface through the detection opening.
[0093] In some embodiments, an apparatus includes a housing, an
amplification module, a reagent module, and a detection module. The
amplification module is disposed within the housing and is
configured to receive an input sample. The amplification module
defines a reaction volume and includes a heater such that the
amplification module can perform a polymerase chain reaction (PCR)
on the input sample. The reagent module is disposed within the
housing, and defines a reagent volume within which at least one of
a sample wash, an elution buffer, a PCR reagent, a detection
reagent or a substrate is contained. The reagent module is actuated
by a reagent actuator configured to convey the reagent from the
volume when the reagent actuator is moved from a first position to
a second position. The reagent actuator is configured to remain
locked in the second position. The detection module is disposed
within the housing and is configured to receive an output from the
amplification module. The detection module is configured to receive
the detection reagent from the reagent module, the detection
reagent being formulated to produce a colorimetric signal that
indicates a presence of a target organism in the input.
[0094] In some embodiments, the apparatus also includes a power
source disposed within the housing. In some embodiments, the power
source has a nominal voltage of about 9V and a capacity of less
than about 1200 mAh. In some embodiments, the apparatus also
includes a controller disposed within the housing, where the
controller is implemented in at least one of a memory or a
processor. In some embodiments, the controller includes at least a
thermal control module configured to produce a thermal control
signal to adjust an output of the heater.
[0095] In some embodiments, an apparatus includes a housing, an
amplification module, a reagent module, a detection module, and a
power source. The amplification module is disposed within the
housing and is configured to receive an input sample. The
amplification module includes a flow member defining a reaction
volume. The amplification module includes a heater coupled to the
flow member such that the amplification module can perform a
polymerase chain reaction (PCR) on the input sample. The reagent
module is disposed within the housing and defines a reagent volume
within which at least one of a sample wash, an elution buffer, a
PCR reagent, a detection reagent or a substrate is contained. The
reagent module includes a reagent actuator configured to convey the
reagent from the volume when the reagent actuator is moved from a
first position to a second position. The detection module is
configured to receive an output from the amplification module and
the detection reagent. The detection reagent is formulated to
produce a signal that indicates a presence of a target amplicon
within the input sample. The detection module includes a detection
surface from which the signal is produced, and which is visible via
the detection opening. The power source is electrically isolated
from at least one of a processor or the amplification module when
the reagent actuator is in the first position. The power source is
electrically coupled to at least one of the processor or the
amplification module when the reagent actuator is in the second
position.
[0096] In some embodiments, an apparatus includes a flow member and
a heater assembly. The flow member defines a serpentine flow path
having at least 30 amplification flow channels. The heater assembly
is coupled to the flow member to define three heating zones within
each amplification flow channel. The heater assembly and the flow
member are collectively configured to maintain a temperature of a
first portion of the flow member associated with the first heating
zone at a first temperature. The heater assembly and the flow
member are collectively configured to maintain a temperature of a
second portion of the flow member associated with the second
heating zone at a second temperature. The heater assembly and the
flow member are collectively configured to maintain a temperature
of a third portion of the flow member associated with the third
heating zone at the first temperature. The heater assembly is
coupled to a first side the flow member via an adhesive bond.
[0097] In some embodiments, a method includes conveying a sample
into a sample preparation module of a diagnostic device. The sample
preparation module is disposed within a housing of the diagnostic
device. The method also includes actuating the diagnostic device to
extract, within the sample preparation module, a target molecule.
The method also includes actuating the diagnostic device to flow a
PCR solution containing the target molecule within a PCR flow path
defined by a PCR module, such that the PCR solution is thermally
cycled by a heater coupled to the PCR module. The method also
includes actuating the diagnostic device to convey the PCR solution
from an outlet of the PCR module into a detection channel of a
detection module. The detection module includes a detection surface
within the detection channel, the detection surface configured to
retain the target molecule. The method also includes actuating the
diagnostic device to convey a reagent into the detection channel,
such that when the reagent reacts with a signal molecule associated
with a target amplicon, a visible optical signal associated with
the detection surface is produced. The method also includes viewing
the detection surface via a detection opening of the housing.
[0098] As used herein, the term "about" when used in connection
with a referenced numeric indication means the referenced numeric
indication plus or minus up to 10% of that referenced numeric
indication. For example, the language "about 50" covers the range
of 45 to 55.
[0099] As used in this specification and the appended claims, the
words "proximal" and "distal" refer to direction closer to and away
from, respectively, an operator of the diagnostic device. Thus, for
example, the end of an actuator depressed by a user that is
furthest away from the user would be the distal end of the
actuator, while the end opposite the distal end (i.e., the end
manipulated by the user) would be the proximal end of the
actuator.
[0100] As used in this specification and the appended claims, the
term "reagent" includes any substance that is used in connection
with any of the reactions described herein. For example, a reagent
can include an elution buffer, a PCR reagent, an enzyme, a
substrate, a wash solution, or the like. A reagent can include a
mixture of one or more constituents. A reagent can include such
constituents regardless of their state of matter (e.g., solid,
liquid or gas). Moreover, a reagent can include the multiple
constituents that can be included in a substance in a mixed state,
in an unmixed state and/or in a partially mixed state. A reagent
can include both active constituents and inert constituents.
Accordingly, as used herein, a reagent can include non-active
and/or inert constituents such as, water, colorant or the like.
[0101] The term "fluid-tight" is understood to encompass hermetic
sealing (i.e., a seal that is gas-impervious) as well as a seal
that is only liquid-impervious. The term "substantially" when used
in connection with "fluid-tight," "gas-impervious," and/or
"liquid-impervious" is intended to convey that, while total fluid
imperviousness is desirable, some minimal leakage due to
manufacturing tolerances, or other practical considerations (such
as, for example, the pressure applied to the seal and/or within the
fluid), can occur even in a "substantially fluid-tight" seal. Thus,
a "substantially fluid-tight" seal includes a seal that prevents
the passage of a fluid (including gases, liquids and/or slurries)
therethrough when the seal is maintained at pressures of less than
about 5 psig, less than about 10 psig, less than about 20 psig,
less than about 30 psig, less than about 50 psig, less than about
75 psig, less than about 100 psig, and all values in between. Any
residual fluid layer that may be present on a portion of a wall of
a container after component defining a "substantially-fluid tight"
seal are moved past the portion of the wall are not considered as
leakage.
[0102] The term "opaque" is understood to include structures (such
as portions of a device housing) that are not transparent and/or
that do not permit an object to be clearly or distinctly seen
through the structure. The term "opaque" or "substantially opaque"
or "semi-opaque" when used in connection with the description of a
device housing or any other structure described herein is intended
to convey that objects cannot be clearly seen through the housing.
A housing (or portion thereof) described as being "opaque" or
"substantially opaque" or "semi-opaque" is understood to include
structures that may have a blocking color, or that may not have a
color, but that are otherwise hazy, blurry, smeared, textured or
the like.
[0103] Unless indicated otherwise, the terms apparatus, diagnostic
apparatus, diagnostic system, diagnostic test, diagnostic test
system, test unit, and variants thereof, can be interchangeably
used.
[0104] FIG. 1 is a schematic illustration of a handheld molecular
diagnostic test device 1000 (also referred to as a "test device")
according to an embodiment. The test device 1000 includes a housing
1010, an amplification module 1600 and a detection module 1800. The
housing 1010 can be any structure within which the amplification
module 1600 and the detection module 1800 are contained to form a
handheld device. Similarly stated, the molecular diagnostic test
device 1000 has a size, shape and/or weight such that the device
can be carried, held, used and/or manipulated in a user's hands. In
this manner, the user can conduct a molecular diagnostics test for
rapid, accurate detection of disease without a large, expensive
instrument. Moreover, this arrangement portable, self-contained
molecular diagnostics test for rapid, accurate detection of
disease. In some embodiments, the test device 1000 (and any of the
test devices described herein) can have an overall volume of less
than about 260 cm.sup.3 (or about 16 cubic inches; e.g., a length
of about 10.2 cm, a width of about 10.2 cm and a thickness of about
2.5 cm). In some embodiments, the test device 1000 (and any of the
test devices described herein) can have an overall volume of less
than about 200 cm.sup.3 (or about 12.25 cubic inches; e.g., a
length of about 8.9 cm, a width of about 8.9 cm and a thickness of
about 2.5 cm). In some embodiments, the test device 1000 (and any
of the test devices described herein) can have an overall volume of
less than about 147 cm.sup.3 (or about 9 cubic inches; e.g., a
length of about 7.6 cm, a width of about 7.6 cm and a thickness of
about 2.5 cm). In some embodiments, the test device 1000 (and any
of the test devices described herein) can have an overall volume of
about 207 cm.sup.3 (or about 12.6 cubic inches; e.g., a length of
about 9.0 cm, a width of about 7.7 cm and a thickness of about 3.0
cm).
[0105] The amplification module 1600 is configured to receive an
input sample S1 that may contain a target organism associated with
a disease state. The sample S1 (and any of the input samples
described herein) can be, for example, blood, urine, male urethral
specimens, vaginal specimens, cervical swab specimens, and/or nasal
swab specimens gathered using a commercially available sample
collection kit. The sample collection kit can be a urine collection
kit or swab collection kit. Non-limiting examples of such sample
collection kits include Copan Mswab or BD ProbeTec Urine
Preservative Transport Kit, Cat #440928, neat urine. In some
embodiments, the sample S1 can be a raw sample obtained from the
source, and upon which limited preparation (filtering, washing, or
the like) has been performed. In some embodiments, for example, the
device 1000 can include a sample input module and/or a sample
preparation module of the types shown and described herein.
[0106] The amplification module 1600 defines a reaction volume 1618
and includes a heater 1630 such that the amplification module 1600
can perform a polymerase chain reaction (PCR) on the input sample
S1. In some embodiments, the reaction volume 1618 can be a central
volume within which the sample S1 is maintained while the heater
1630 repeatedly cycles the sample S1 through a series of
temperature set points to amplify the target organism and/or
portions of the DNA of the organism. In other embodiments, the
reaction volume 1618 can be a volume through which the sample S1 is
flowed, and that has various portions maintained at different
temperatures by the heater 1630. In this manner, the amplification
module 1600 can perform a "flow through" PCR. In some embodiments,
the reaction volume can have a curved, "switchback," and/or
serpentine shape to allow for a high flow length while maintaining
the overall size of the device within the desired limits.
[0107] The heater 1630 can be any suitable heater or collection of
heaters that can perform the functions described herein to amplify
the sample S1. For example, in some embodiments, the heater 1630
can be a single heater that is thermally coupled to the reaction
volume 1618 and that can cycle through multiple temperatures set
points (e.g., between about 60 C and about 90 C). In other
embodiments, the heater 1630 can be a set of heaters, each of which
is thermally coupled to the reaction volume 1618 and that is
maintained at a substantially constant set point. In this manner,
the heater 1630 and the reaction volume 1618 can establish multiple
temperature zones through which the sample S1 flows and/or can
define a desired number of amplification cycles to ensure the
desired test sensitivity (e.g., at least 30 cycles, at least 34
cycles, at least 36 cycles, at least 38 cycles, or at least 40
cycles). The heater 1630 (and any of the heaters described herein)
can be of any suitable design. For example, in some embodiments,
the heater 1630 can be a resistance heater, a thermoelectric device
(e.g. a Peltier device), or the like.
[0108] The detection module 1800 receives an output S7 from the
amplification module 1800 and a reagent R. The reagent R is
formulated to produce a signal OP1 that indicates a presence of a
target amplicon and/or organism within the input sample S1. In this
manner, the stand-alone device 1000 can provide reliable molecular
diagnosis within a point-of-care setting (e.g., doctor's office,
pharmacy or the like) or at the user's home. The signal OP1 can be
any suitable signal that alerts the user regarding whether or not
the target organism is present. Similarly stated, the signal OP1
can be any suitable signal to detect a disease associated with the
target amplicon and/or organism. The signal OP1 can be, for
example, a visual signal, an audible signal, a radio frequency
signal or the like.
[0109] In some embodiments, the signal OP1 is a visual signal that
can viewed by the user through a detection opening (not shown in
FIG. 1) defined by the housing. The visual signal can be, for
example, a non-fluorescent signal. This arrangement allows the
device 1000 to be devoid of a light source (e.g., lasers,
light-emitting diodes or the like) and/or any light detectors
(photomultiplier tube, photodiodes, CCD devices, or the like) to
detect and/or amplify the signal OP1. In some embodiments, the
signal OP1 is a visible signal characterized by a color associated
with the presence of the target amplicon and/or organism. Said
another way, in some embodiments, the device 1000 can produce a
colorimetric output signal that is visible to the user. In such
embodiments, the detection module 1800 (and any of the detection
modules described herein) can produce a chemiluminescent signal
that results from the introduction of the reagent R and/or any
other substances (e.g., a substrate to catalyze the production of
the signal OP1, and the like). In some embodiments, the reagent is
formulated such that the visible signal OP1 remains present for at
least about 30 minutes. The reagent R and any other compositions
formulated to produce the signal OP1 can be any suitable
compositions as described herein. In some embodiments, the reagent
R can be stored within the housing 1010 in any manner as described
herein (e.g., in a sealed container, a lyophilized form or the
like).
[0110] In some embodiments, the device 1000 (and any of the other
devices shown and described herein) can be configured to produce
the signal OP1 in a time of less than about 25 minutes from when
the sample S1 is received. In other embodiments, the device 1000
(and any of the other devices shown and described herein) can be
configured to produce the signal OP1 in a time of less than about
20 minutes from when the sample S1 is input, less than about 18
minutes from when the sample S1 is input, less than about 16
minutes from when the sample S1 is input, less than about 14
minutes from when the sample S1 is input, and all ranges
therebetween.
[0111] Similarly stated, the device 1000 and the components therein
can be configured to conduct a "rapid" PCR (e.g., completing at
least 30 cycles in less than about 10 minutes), and rapid
production of the signal OP1. Similarly stated, the device 1000
(and any of the other devices shown and described herein) can be
configured to process volumes, to have dimensional sizes and/or be
constructed from materials that facilitates a rapid PCR or
amplification in less than about 10 minutes, less than about 9
minutes, less than about 8 minutes, less than about 7 minutes, less
than about 6 minutes, or any range therebetween, as described
herein.
[0112] In some embodiments, the device 1000 (and any of the other
devices shown and described herein) can be disposable and/or
configured for a single use. Similarly stated, the device 1000 (and
any of the other devices shown and described herein) can be
configured for one and only one use. For example, in some
embodiments, the amount of the reagent R can be sufficient for only
one use. In other embodiments, the device 1000 can include an
on-board power source (e.g., a DC battery) to power the
amplification module 1600 and/or any sample preparation or fluid
transfer modules that may be present (not shown in FIG. 1) that has
a capacity sufficient for only one test. In some embodiments, the
device 1000 can include a power source (not shown in FIG. 1) having
a capacity of less than about 1200 mAh.
[0113] Another example of a device configured for a single use is
shown in FIG. 2, which shows a molecular diagnostic test device
2000 (also referred to as a "test device" or "device"), according
to an embodiment. The test device 2000 includes a housing 2010, a
sample preparation module 2200, an amplification module 2600 and a
detection module 2800. The housing 2010 can be any structure within
which the sample preparation module 2200, the amplification module
2600 and the detection module 2800 are contained. In some
embodiments, the test device 2000 have a size, shape and/or weight
such that the device can be carried, held, used and/or manipulated
in a user's hands (i.e., it can be a "handheld" device). In other
embodiments, the test device 2000 can be a self-contained,
single-use device that has an overall volume greater than about 260
cm.sup.3 (or about 16 cubic inches). In some embodiments, the test
device 2000 (and any of the test devices described herein) can have
an overall volume of about 207 cm.sup.3 (or about 12.6 cubic
inches; e.g., a length of about 9.0 cm, a width of about 7.7 cm and
a thickness of about 3.0 cm).
[0114] The sample preparation module 2200 is disposed within the
housing 2010, and is configured receive an input sample S1 via an
input portion 2162 of the housing 2010. As described herein, the
sample preparation module 2200 is configured to process the sample
S1 to facilitate detection of an organism therein that is
associated with a disease. For example, in some embodiments, the
sample preparation module 2200 can be configured to concentrate and
lyse cells in the sample S1, thereby allowing subsequent extraction
of DNA to facilitate amplification and/or detection. In some
embodiments, the processed/lysed sample is pushed and/or otherwise
transferred from the sample preparation module 2200 to other
modules within the device 2000 (e.g., the amplification module
2600, a mixing module (not shown), or the like). By eliminating the
need for external sample preparation and a cumbersome instrument,
the device 2000 is suitable for use within a point-of-care setting
(e.g., doctor's office, pharmacy or the like) or at the user's
home, and can receive any suitable sample S1. The sample S1 (and
any of the input samples described herein) can be, for example,
blood, urine, male urethral specimens, vaginal specimens, cervical
swab specimens, and/or nasal swab specimens gathered using a
commercially available sample collection kit.
[0115] The sample preparation module 2200 includes a filter
assembly 2230 through which the sample S1 flows during a "dispense"
or "sample actuation" operation. Although not shown in FIG. 2, in
some embodiments, the sample preparation module 2200 includes a
waste reservoir to which the waste product from the filtering
operation is conveyed. In some embodiments, the sample preparation
module 2200 includes components and/or substances to follow the
"sample dispense" operation with a wash operation. In some
embodiments, the sample preparation module 2200 is configured for a
back-flow elution operation to deliver captured particles from the
filter membrane and deliver the eluted volume to the target
destination (e.g., towards the amplification module 2600). In some
embodiments, the sample preparation module 2200 is configured so as
not cause the output solution to be contaminated by previous
reagents (e.g., like the sample or wash).
[0116] The amplification module 2600 includes a flow member 2610
and a heater 2630, and is configured to perform a polymerase chain
reaction (PCR) on the input sample S6 that is output by the sample
preparation module 2200. The flow member 2610 defines a
"switchback" or serpentine flow path 2618 through which the
prepared sample S6 flows. Similarly stated, the flow member 2610
defines a flow path 2618 that is curved such that the flow path
2618 intersects the heater 2630 at multiple locations. In this
manner, the amplification module 2600 can perform a "flow through"
PCR where the sample S6 flows through multiple different
temperature regions.
[0117] The heater 2630 can be any suitable heater or collection of
heaters that can perform the functions described herein to amplify
the sample S6. Specifically, the heater 2630 is coupled to the flow
member 2610, and is configured to establish multiple temperature
zones through which the sample S6 flows and/or can define a desired
number of amplification cycles to ensure the desired test
sensitivity (e.g., at least 30 cycles, at least 34 cycles, at least
36 cycles, at least 38 cycles, or at least 40 cycles). The heater
2630 (and any of the heaters described herein) can be of any
suitable design. For example, in some embodiments, the heater 2630
can be a resistance heater, a thermoelectric device (e.g. a Peltier
device), or the like. In some embodiments, the heater 2630 can be
one or more linear "strip heaters" arranged such that the flow path
2618 crosses the heaters at multiple different points. In other
embodiments, the heater 2630 can be one or more curved heaters
having a geometry that corresponds to that of the flow member 2610
to produce multiple different temperature zones in the flow path
2618.
[0118] The detection module 2800 receives an output S7 from the
amplification module 2800 and a reagent R. The reagent R is
formulated to produce a signal OP1 that indicates a presence of a
target amplicon and/or organism within the input sample S1. In this
manner, the stand-alone device 2000 can provide reliable molecular
diagnosis within a point-of-care setting (e.g., doctor's office,
pharmacy or the like) or at the user's home. The signal OP1 can be
any suitable signal that alerts the user regarding whether or not
the target organism is present. Similarly stated, the signal OP1
can be any suitable signal to detect a disease associated with the
target amplicon and/or organism. The signal OP1 can be, for
example, a visual signal, an audible signal, a radio frequency
signal or the like.
[0119] In some embodiments, the signal OP1 is a visual signal that
can viewed by the user through a detection opening (not shown in
FIG. 2) defined by the housing. The visual signal can be, for
example, a non-fluorescent signal. This arrangement allows the
device 2000 to be devoid of a light source (e.g., lasers,
light-emitting diodes or the like) and/or any light detectors
(photomultiplier tube, photodiodes, CCD devices, or the like) to
detect and/or amplify the signal OP1. In some embodiments, the
signal OP1 is a visible signal characterized by a color associated
with the presence of the target amplicon and/or organism. Said
another way, in some embodiments, the device 2000 can produce a
colorimetric output signal that is visible to the user. In such
embodiments, the detection module 2800 (and any of the detection
modules described herein) can produce a chemiluminescent signal
that results from the introduction of the reagent R and/or any
other substances (e.g., a substrate to catalyze the production of
the signal OP1, and the like). In some embodiments, the reagent is
formulated such that the visible signal OP1 remains present for at
least about 30 minutes. The reagent R and any other compositions
formulated to produce the signal OP1 can be any suitable
compositions as described herein. In some embodiments, the reagent
R can be stored within the housing 2010 in any manner as described
herein (e.g., in a sealed container, a lyophilized form or the
like).
[0120] In some embodiments, the device 2000 (and any of the other
devices shown and described herein) can be configured to produce
the signal OP1 in a time of less than about 25 minutes from when
the sample S1 is received. In other embodiments, the device 2000
(and any of the other devices shown and described herein) can be
configured to produce the signal OP1 in a time of less than about
20 minutes from when the sample S1 is input, less than about 18
minutes from when the sample S1 is input, less than about 16
minutes from when the sample S1 is input, less than about 14
minutes from when the sample S1 is input, and all ranges
therebetween.
[0121] Similarly stated, the device 2000 and the components therein
can be configured to conduct a "rapid" PCR (e.g., completing at
least 30 cycles in less than about 20 minutes), and rapid
production of the signal OP1. Similarly stated, the device 2000
(and any of the other devices shown and described herein) can be
configured to process volumes, to have dimensional sizes and/or be
constructed from materials that facilitates a rapid PCR or
amplification in less than about 10 minutes, less than about 9
minutes, less than about 8 minutes, less than about 7 minutes, less
than about 6 minutes, or any range therebetween, as described
herein.
[0122] As described above, the device 2000 is configured as a
single-use device that can be used in a point-of-care setting
and/or in a user's home. Similarly stated, in some embodiments, the
device 2000 (and any of the other devices shown and described
herein) can be configured for use in a decentralized test facility.
Further, in some embodiments, the device 2000 (and any of the other
devices shown and described herein) can be a CLIA-waived device
and/or can operate in accordance with methods that are CLIA waived.
Similarly stated, in some embodiments, the device 2000 (and any of
the other devices shown and described herein) is configured to be
operated in a sufficiently simple manner, and can produce results
with sufficient accuracy to pose a limited likelihood of misuse
and/or to pose a limited risk of harm if used improperly. In some
embodiments, the device 2000 (and any of the other devices shown
and described herein), can be operated by a user with minimal (or
no) scientific training, in accordance with methods that require
little judgment of the user, and/or in which certain operational
steps are easily and/or automatically controlled.
[0123] For example, in some embodiments, the sample preparation
module 2200 of the single-use molecular diagnostic test device 2000
can be fixedly coupled within the housing 2010. In this manner, the
risk of improperly positioning a removable cartridge within the
housing (such risk being present with known cartridge-based
systems) is eliminated. More particularly, in some embodiments, the
device 2000 can include a sample transfer module (not shown in FIG.
2) configured to generate fluid pressure, fluid flow and/or
otherwise convey the input sample S1 through the modules of the
device. Such a sample transfer module can be a single-use module
that is configured to contact and/or receive the sample flow. The
single-use arrangement eliminates the likelihood that contamination
of the fluid transfer module and/or the sample preparation module
2200 will become contaminated from previous runs, thereby
negatively impacting the accuracy of the results.
[0124] As another example, in some embodiments, the device 2000
(and any of the other devices shown and described herein), can
include a variety of lock-out devices that prevent the user from
conducting certain operational steps out of the desired order.
Moreover, the device 2000 (and any of the other devices shown and
described herein), can include a variety of lock-out devices that
prevent the user from reusing the device after an initial use has
been attempted and/or completed. In this manner, the device 2000
(and any of the other devices shown and described herein), can be
specifically configured for a single-use operation and can pose a
limited risk of misuse. For example, in some embodiments, the
device 2000 can include a sample actuator (not shown in FIG. 2)
configured to produce a force to convey the input sample S1 through
the filter assembly 2230 when the sample actuator is moved relative
to the housing 2010. The sample actuator can further be configured
with protrusions, recesses and/or other features such that the
sample actuator will remain locked in the actuated position after a
single use.
[0125] As yet another example, in some embodiments, a device can
include on board reagents and a single-use reagent module
configured to dispense the reagents in a manner that can be
operated by a user with minimal (or no) scientific training, in
accordance with methods that require little judgment of the user.
In some embodiments, a device including a reagent module can
include a lock-out device that prevents the user from actuating the
module out of the desired order and/or prevents the user from
reusing the device after an initial use has been attempted and/or
completed. For example, FIGS. 3 and 4 show a molecular diagnostic
test device 3000 (also referred to as a "test device" or "device"),
according to an embodiment. The test device 3000 includes a housing
3010, a reagent module 3700, an amplification module 3600 and a
detection module 3800. The housing 3010 can be any structure within
which the reagent module 3700, the amplification module 3600 and
the detection module 3800 are contained. In some embodiments, the
test device 3000 have a size, shape and/or weight such that the
device can be carried, held, used and/or manipulated in a user's
hands (i.e., it can be a "handheld" device). In other embodiments,
the test device 3000 can be a self-contained, single-use device
that has an overall volume greater than about 260 cm.sup.3 (or
about 16 cubic inches). In some embodiments, the test device 3000
(and any of the test devices described herein) can have an overall
volume of about 207 cm.sup.3 (or about 12.6 cubic inches; e.g., a
length of about 9.0 cm, a width of about 7.7 cm and a thickness of
about 3.0 cm).
[0126] The reagent module 3700 is disposed within the housing 3010,
and defines a reagent volume 3710 within which at least one reagent
is contained. Although FIGS. 3 and 4 show the reagent volume 3710
containing a reagent R and a reagent R1, and being fluidically
coupled to the amplification module 3600 and the detection module
3800, in other embodiments, a reagent module can contain any
suitable reagents and can be fluidically coupled to and/or can
convey such reagents to any suitable module within the device. For
example, in some embodiments, the reagent volume can contain any of
a sample wash, an elution buffer, one or more PCR reagents, a
detection reagent and/or a substrate.
[0127] As shown by the arrow AA in FIG. 4, the reagent module 3700
is actuated by a reagent actuator 3080 to convey the reagent
(indicated as reagent R and reagent R1) from the reagent volume
3710. Specifically, the reagent actuator 3080 is moved from a first
position (FIG. 3) to a second position (FIG. 4) to convey the
reagent(s) from the reagent volume 3710. The reagent actuator 3080
is configured to remain locked in the second position to prevent
reuse of the device 3000. In some embodiments, the reagent actuator
3080 can include protrusions, recesses and/or other features that
interface with the housing 3010 and/or other portions of the device
to maintain the actuator 3080 in the second position. Similarly
stated, the reagent actuator 3080 can include any suitable
structure to maintain the reagent module 3700 in its second (or
actuated) configuration. In this manner, the device 3000 (and any
of the other devices shown and described herein), can be
specifically configured for a single-use operation and can pose a
limited risk of misuse.
[0128] Although the reagent actuator 3080 is shown as being a moved
in a linear direction to convey the reagents, in other embodiments,
the reagent actuator 3080 can be configured to rotate to develop
the pressure and/or flow of reagent(s). Moreover, in some
embodiments, the reagent actuator 3080 (and any of the reagent
actuators described herein) can be an automatic actuator (i.e., an
electronic actuator, an actuator that is moved and/or actuated with
limited human interaction, and/or an actuator that is moved and/or
actuated with no direct human interaction). In other embodiments,
the reagent actuator 3080 (and any of the reagent actuators
described herein) can be a manual actuator (e.g., a non-electronic
actuator that is manipulated directly by a user). This arrangement
allows the reagent actuator 3080 to be actuated without the need
for electronic power and/or before the device 3000 is powered on.
In some embodiments, the movement of the actuator 3080 can also
initialize a power-on sequence of the device 3000. In this manner,
the device can limit any power use prior to beginning of the test,
thereby limiting the likelihood of misuse and/or an inaccurate test
(e.g., due to an unexpected dead battery).
[0129] The amplification module 3600 defines a reaction volume
3618, includes a heater 3630, and is configured to perform a
polymerase chain reaction (PCR) on the input sample S1. The input
sample S1, can be any suitable sample as described herein, and can
be conveyed to the amplification module via an input portion 3162
of the housing 3010. In some embodiments, the reaction volume 3618
can be a central volume within which the sample S1 is maintained
while the heater 3630 repeatedly cycles the sample S1 through a
series of temperature set points to amplify the target organism
and/or portions of the DNA of the organism. In other embodiments,
the reaction volume 3618 can be a volume through which the sample
S1 is flowed, and that has various portions maintained at different
temperatures by the heater 3630. In this manner, the amplification
module 3600 can perform a "flow through" PCR. In some embodiments,
the reaction volume can have a curved, "switchback," and/or
serpentine shape to allow for a high flow length while maintaining
the overall size of the device within the desired limits.
[0130] The heater 3630 can be any suitable heater or collection of
heaters that can perform the functions described herein to amplify
the sample S1. For example, in some embodiments, the heater 3630
can be single heater that is thermally coupled to the reaction
volume 3618 and that can cycle through multiple temperatures set
points (e.g., between about 60 C and about 90 C). In other
embodiments, the heater 3630 can be a set of heaters, each of which
is thermally coupled to the reaction volume 3618 and that is
maintained at a substantially constant set point. In this manner,
the heater 3630 and the reaction volume 3618 can establish multiple
temperature zones through which the sample S1 flows and/or can
define a desired number of amplification cycles to ensure the
desired test sensitivity (e.g., at least 30 cycles, at least 34
cycles, at least 36 cycles, at least 38 cycles, or at least 40
cycles). The heater 3630 (and any of the heaters described herein)
can be of any suitable design. For example, in some embodiments,
the heater 3630 can be a resistance heater, a thermoelectric device
(e.g. a Peltier device), or the like.
[0131] As shown in FIG. 4, the detection module 3800 receives an
output S7 from the amplification module 3800 and a reagent R from
the reagent module 3700. The reagent R is a detection reagent
formulated to produce and/or catalyze the production of a signal
OP1 that indicates a presence of a target amplicon and/or organism
within the input sample S1. In this manner, the stand-alone device
3000 can provide reliable molecular diagnosis within a
point-of-care setting (e.g., doctor's office, pharmacy or the like)
or at the user's home. The signal OP1 can be any suitable signal
that alerts the user regarding whether or not the target organism
is present. Similarly stated, the signal OP1 can be any suitable
signal to detect a disease associated with the target amplicon
and/or organism. The signal OP1 can be, for example, a visual
signal, an audible signal, a radio frequency signal or the
like.
[0132] In some embodiments, the signal OP1 is a visual signal that
can viewed by the user through a detection opening (not shown in
FIGS. 3 and 4) defined by the housing. The visual signal can be,
for example, a non-fluorescent signal. This arrangement allows the
device 3000 to be devoid of a light source (e.g., lasers,
light-emitting diodes or the like) and/or any light detectors
(photomultiplier tube, photodiodes, CCD devices, or the like) to
detect and/or amplify the signal OP1. In some embodiments, the
signal OP1 is a visible signal characterized by a color associated
with the presence of the target amplicon and/or organism. Said
another way, in some embodiments, the device 3000 can produce a
colorimetric output signal that is visible to the user. In such
embodiments, the detection module 3800 (and any of the detection
modules described herein) can produce a chemiluminescent signal
that results from the introduction of the reagent R and/or any
other substances (e.g., a substrate to catalyze the production of
the signal OP1, and the like). In some embodiments, the reagent is
formulated such that the visible signal OP1 remains present for at
least about 30 minutes. The reagent R and any other compositions
formulated to produce the signal OP1 can be any suitable
compositions as described herein. In some embodiments, the reagent
R can be stored within the housing 3010 in any manner as described
herein (e.g., in a sealed container, a lyophilized form or the
like).
[0133] In some embodiments, the device 3000 (and any of the other
devices shown and described herein) can be configured to produce
the signal OP1 in a time of less than about 25 minutes from when
the sample S1 is received. In other embodiments, the device 3000
(and any of the other devices shown and described herein) can be
configured to produce the signal OP1 in a time of less than about
20 minutes from when the sample S1 is input, less than about 18
minutes from when the sample S1 is input, less than about 16
minutes from when the sample S1 is input, less than about 14
minutes from when the sample S1 is input, and all ranges
therebetween.
[0134] Similarly stated, the device 3000 and the components therein
can be configured to conduct a "rapid" PCR (e.g., completing at
least 30 cycles in less than about 10 minutes), and rapid
production of the signal OP1. Similarly stated, the device 3000
(and any of the other devices shown and described herein) can be
configured to process volumes, to have dimensional sizes and/or be
constructed from materials that facilitates completion of a rapid
PCR or amplification in less than about 10 minutes, less than about
9 minutes, less than about 8 minutes, less than about 7 minutes,
less than about 6 minutes, or any range therebetween, as described
herein.
[0135] As described above, the device 3000 is configured as a
single-use device that can be used in a point-of-care setting
and/or in a user's home. Similarly stated, in some embodiments, the
device 3000 (and any of the other devices shown and described
herein) can be configured for use in a decentralized test facility.
Further, in some embodiments, the device 3000 (and any of the other
devices shown and described herein) can be a CLIA-waived device
and/or can operate in accordance with methods that are CLIA waived.
Similarly stated, in some embodiments, the device 3000 (and any of
the other devices shown and described herein) is configured to be
operated in a sufficiently simple manner, and can produce results
with sufficient accuracy to pose a limited likelihood of misuse
and/or to pose a limited risk of harm if used improperly. In some
embodiments, the device 3000 (and any of the other devices shown
and described herein), can be operated by a user with minimal (or
no) scientific training, in accordance with methods that require
little judgment of the user, and/or in which certain operational
steps are easily and/or automatically controlled.
[0136] For example, in some embodiments, the reagent module 3700 of
the molecular diagnostic test device 3000 can include seals such
that the reagent volume 3710 is a sealed reagent volume within
which the reagent(s) are stored. In such embodiments, the reagent
actuator 3080 is configured to puncture the seal that fluidically
isolates the reagent volume 3710 when moved. In this manner, the
molecular diagnostic test device 3000 can be configured for long
term storage in a manner that poses a limited likelihood of misuse
(spoilage of the reagent(s), expiration of the reagents(s), leakage
of the reagent(s), or the like). In some embodiments, the reagent
module 3700 and/or any area in fluid communication therewith (or
any other reagent modules described herein) can include a
desiccant, seals or other compositions or components to maintain
stability for long term storage. In some embodiments, the molecular
diagnostic test device 3000 is configured to be stored for up to
about 36 months, up to about 32 months, up to about 26 months, up
to about 24 months, up to about 20 months, up to about 18 months,
or any values there between.
[0137] In some embodiments, the device 3000 (or any of the devices
shown herein) can include an on-board power source (e.g., a DC
battery, a capacitor, or the like) to power the amplification
module 3600 and/or any sample preparation or fluid transfer modules
that may be present (not shown in FIGS. 3 and 4). Moreover, the
power source can have a capacity sufficient for only one test. In
this manner, the likelihood of misuse of the device is limited.
Moreover, by including a power source with a limited capacity, the
risk of re-use or improper use (e.g., after an erroneous "power on"
event) is limited or reduced. In some embodiments, the device 3000
can include a power source (not shown in FIG. 1) having a capacity
of less than about 1200 mAh. In some embodiments, the device 3000
(or any other devices shown and described herein) can include a
switch, isolation member or the like that facilitates electrically
coupling of the power source to a processor (not shown in FIGS. 3
and 4), the amplification module or any other module within the
device 3000 to actuation of sample preparation module, reagent
module or the like.
[0138] For example, FIGS. 5 and 6 show a molecular diagnostic test
device 4000 (also referred to as a "test device" or "device"),
according to an embodiment that includes a power source 4905. The
test device 4000 also includes a housing 4010, a reagent module
4700, an amplification module 4600 and a detection module 4800. The
housing 4010 can be any structure within which the reagent module
4700, the amplification module 4600, the detection module 4800, and
the power source 4905 are contained. In some embodiments, the test
device 4000 have a size, shape and/or weight such that the device
can be carried, held, used and/or manipulated in a user's hands
(i.e., it can be a "handheld" device). In other embodiments, the
test device 4000 can be a self-contained, single-use device that
has an overall volume greater than about 260 cm.sup.3 (or about 46
cubic inches). In some embodiments, the test device 4000 (and any
of the test devices described herein) can have an overall volume of
about 207 cm.sup.3 (or about 12.6 cubic inches; e.g., a length of
about 9.0 cm, a width of about 7.7 cm and a thickness of about 3.0
cm).
[0139] The reagent module 4700 is disposed within the housing 4010,
and defines a reagent volume 4710 within which at least one reagent
is contained. Although FIGS. 5 and 6 show the reagent volume 4710
containing a reagent R and a reagent R1, and being fluidically
coupled to the amplification module 4600 and the detection module
4800, in other embodiments, a reagent module can contain any
suitable reagents and can be fluidically coupled to and/or can
convey such reagents to any suitable module within the device. For
example, in some embodiments, the reagent volume can contain any of
a sample wash, an elution buffer, one or more PCR reagents, a
detection reagent and/or a substrate.
[0140] As shown by the arrow BB in FIG. 6, the reagent module 4700
is actuated by a reagent actuator 4080 to convey the reagent
(indicated as reagent R and reagent R1) from the reagent volume
4710. Specifically, the reagent actuator 4080 is moved from a first
position (FIG. 4) to a second position (FIG. 4) to convey the
reagent(s) from the reagent volume 4710. Although the reagent
actuator 4080 is shown as being a moved in a linear direction to
convey the reagents, in other embodiments, the reagent actuator
4080 can be configured to rotate to develop the pressure and/or
flow of reagent(s). Moreover, the reagent actuator 4080 is a manual
actuator (e.g., a non-electronic actuator that is manipulated
directly by a user). This arrangement allows the reagent actuator
4080 to be actuated without the need for electronic power and/or
before the device 4000 is powered on. Further, as described in more
detail below, the movement of the actuator 4080 can also initialize
a power-on sequence of the device 4000. In this manner, the device
4000 can limit any power use prior to beginning of the test,
thereby limiting the likelihood of misuse and/or an inaccurate test
(e.g., due to an unexpected dead battery).
[0141] The amplification module 4600 includes a heater 4630 and a
flow member 4610 that defines a reaction volume 4618, and is
configured to perform a polymerase chain reaction (PCR) on the
input sample S1. The input sample S1, can be any suitable sample as
described herein, and can be conveyed to the amplification module
via an input portion 4162 of the housing 4010. In some embodiments,
the reaction volume 4618 can be a central volume within which the
sample S1 is maintained while the heater 4630 repeatedly cycles the
sample S1 through a series of temperature set points to amplify the
target organism and/or portions of the DNA of the organism. In
other embodiments, the reaction volume 4618 can be a volume through
which the sample S1 is flowed, and that has various portions
maintained at different temperatures by the heater 4630. In this
manner, the amplification module 4600 can perform a "flow through"
PCR. In some embodiments, the reaction volume can have a curved,
"switchback," and/or serpentine shape to allow for a high flow
length while maintaining the overall size of the device within the
desired limits.
[0142] The heater 4630 can be any suitable heater or collection of
heaters that can perform the functions described herein to amplify
the sample S1. For example, in some embodiments, the heater 4630
can be single heater that is thermally coupled to the reaction
volume 4618 and that can cycle through multiple temperatures set
points (e.g., between about 60 C and about 90 C). In other
embodiments, the heater 4630 can be a set of heaters, each of which
is thermally coupled to the reaction volume 4618 and that is
maintained at a substantially constant set point. In this manner,
the heater 4630 and the reaction volume 4618 can establish multiple
temperature zones through which the sample S1 flows and/or can
define a desired number of amplification cycles to ensure the
desired test sensitivity (e.g., at least 30 cycles, at least 34
cycles, at least 36 cycles, at least 38 cycles, or at least 40
cycles). The heater 4630 (and any of the heaters described herein)
can be of any suitable design. For example, in some embodiments,
the heater 4630 can be a resistance heater, a thermoelectric device
(e.g. a Peltier device), or the like.
[0143] As shown in FIG. 6, the detection module 4800 receives an
output S7 from the amplification module 4800 and a reagent R from
the reagent module 4700. The reagent R is a detection reagent
formulated to produce and/or catalyze the production of a signal
OP1 that indicates a presence of a target amplicon and/or organism
within the input sample S1. In this manner, the device 4000 can
provide reliable molecular diagnosis within a point-of-care setting
(e.g., doctor's office, pharmacy or the like) or at the user's
home. The signal OP1 can be any suitable signal that alerts the
user regarding whether or not the target organism is present.
Similarly stated, the signal OP1 can be any suitable signal to
detect a disease associated with the target amplicon and/or
organism. The signal OP1 can be, for example, a visual signal, an
audible signal, a radio frequency signal or the like.
[0144] In some embodiments, the signal OP1 is a visual signal that
can viewed by the user through a detection opening (not shown in
FIGS. 5 and 6) defined by the housing. The visual signal can be,
for example, a non-fluorescent signal. This arrangement allows the
device 4000 to be devoid of a light source (e.g., lasers,
light-emitting diodes or the like) and/or any light detectors
(photomultiplier tube, photodiodes, CCD devices, or the like) to
detect and/or amplify the signal OP1. In some embodiments, the
signal OP1 is a visible signal characterized by a color associated
with the presence of the target amplicon and/or organism. Said
another way, in some embodiments, the device 4000 can produce a
colorimetric output signal that is visible to the user. In such
embodiments, the detection module 4800 (and any of the detection
modules described herein) can produce a chemiluminescent signal
that results from the introduction of the reagent R and/or any
other substances (e.g., a substrate to catalyze the production of
the signal OP1, and the like). In some embodiments, the reagent is
formulated such that the visible signal OP1 remains present for at
least about 30 minutes. The reagent R and any other compositions
formulated to produce the signal OP1 can be any suitable
compositions as described herein. In some embodiments, the reagent
R can be stored within the housing 4010 in any manner as described
herein (e.g., in a sealed container, a lyophilized form or the
like).
[0145] The device 4000 includes an electronic circuit system that
includes at least a processor 4950 and the power source 4905.
Although not shown in FIGS. 5 and 6, the electronic circuit system
(and any of the electronic circuit systems described herein) can
include any suitable electronic components, such as, for example,
printed circuit boards, switches, resistors, capacitors, diodes,
memory chips arranged in a manner to control the operation of the
device 4000. The processor 4950 (and any of the processors shown
herein) can be a commercially-available processing device dedicated
to performing one or more specific tasks. For example, in some
embodiments, the microprocessor 4950 can be a
commercially-available microprocessor, such as an 8-bit PIC
microcontroller. Alternatively, the processor 4950 can be an
application-specific integrated circuit (ASIC) or a combination of
ASICs, which are designed to perform one or more specific
functions, in yet other embodiments, the processor 4950 can be an
analog or digital circuit, or a combination of multiple
circuits.
[0146] The power source 4905 can be any suitable power source that
provides power to the electronic circuit system (including the
processor 4950) and any of the modules within the device 4000.
Specifically, the power source 4905 can provide power to the
amplification module 4600 and/or the heater 4630 to facilitate the
completion of the PCR on the input sample S1. In some embodiments,
the power source 4905 can be one or more DC batteries, such as, for
example, multiple 1.5 VDC cells (e.g., AAA or AA alkaline
batteries). In other embodiments, the power source 4905 can be a 9
VDC battery having a capacity of less than about 1200 mAh. In other
embodiments, the power source 4905 can be any suitable energy
storage/conversion member, such as a capacitor a magnetic storage
systems, a fuel cell or the like.
[0147] As shown in FIG. 5, power source 4905 is electrically
isolated from the processor 4950 and/or the amplification module
4600 when the reagent actuator 4080 is in the first position. In
this manner, the "power-up" event is tied to the movement of the
reagent actuator 4080. This arrangement limits the likelihood of
premature power drain from the power source 4905 during storage. As
shown in FIG. 6, the power source 4905 is electrically coupled to
the processor 4950 and/or the amplification module 4600 when the
reagent actuator 4080 is in the second position. This arrangement
allows for the device 4000 to be operated in a sufficiently simple
manner, and reduces the judgment of the user in the operation.
Specifically, no judgment is required regarding when to power-up
the device 4000, and the likelihood of a user powering up the
device 4000 and then delaying subsequent operation of the device
4000 (which can deplete the stored energy) is limited and/or
eliminated.
[0148] The reagent actuator 4080 can actuate the power source 1905
and/or place the power source 4905 in electrical connection with
the processor 4950 and/or the amplification module 4600 in any
suitable manner. For example, in some embodiments, the reagent
actuator 4080 can include a protrusion (not shown) that actuates a
switch to place the power source 4905 in electrical connection with
the processor 4950 and/or the amplification module 4600 when the
reagent actuator 4080 is moved from the first position to the
second position. In other embodiments, the reagent actuator 4080
can include and/or be coupled to an isolation member that, when
removed, places the power source 4905 in electrical connection with
the processor 4950 and/or the amplification module 4600 when the
reagent actuator 4080 is moved from the first position to the
second position.
[0149] In some embodiments, the device 4000 (and any of the other
devices shown and described herein) can be configured to produce
the signal OP1 in a time of less than about 25 minutes from when
the sample S1 is received. In other embodiments, the device 4000
(and any of the other devices shown and described herein) can be
configured to produce the signal OP1 in a time of less than about
20 minutes from when the sample S1 is input, less than about 18
minutes from when the sample S1 is input, less than about 16
minutes from when the sample S1 is input, less than about 14
minutes from when the sample S1 is input, and all ranges
therebetween.
[0150] Similarly stated, the device 4000 and the components therein
can be configured to conduct a "rapid" PCR (e.g., completing at
least 40 cycles in less than about 10 minutes), and rapid
production of the signal OP1. Similarly stated, the device 4000
(and any of the other devices shown and described herein) can be
configured to process volumes, to have dimensional sizes and/or be
constructed from materials that facilitates completion of a rapid
PCR or amplification in less than about 10 minutes, less than about
9 minutes, less than about 8 minutes, less than about 7 minutes,
less than about 6 minutes, or any range therebetween, as described
herein.
[0151] In some embodiments, the reagent actuator 4080 is configured
to remain locked in the second position to prevent reuse of the
device 4000. In this manner, the device 4000 (and any of the other
devices shown and described herein), can be specifically configured
for a single-use operation and can pose a limited risk of misuse.
For example, in some embodiments, the reagent module 4700 of the
molecular diagnostic test device 4000 can include seals such that
the reagent volume 4710 is a sealed reagent volume within which the
reagent(s) are stored. In such embodiments, the reagent actuator
4080 is configured to puncture the seal that fluidically isolates
the reagent volume 4710 when moved. In this manner, the molecular
diagnostic test device 4000 can be configured for long term storage
in a manner that poses a limited likelihood of misuse (spoilage of
the reagent(s), expiration of the reagents(s), leakage of the
reagent(s), or the like). In some embodiments, the reagent module
4700 and/or any area in fluid communication therewith (or any other
reagent modules described herein) can include a desiccant, seals or
other compositions or components to maintain stability for long
term storage. In some embodiments, the molecular diagnostic test
device 4000 is configured to be stored for up to about 46 months,
up to about 42 months, up to about 26 months, up to about 24
months, up to about 20 months, up to about 18 months, or any values
there between.
[0152] In some embodiments, a molecular diagnostic test device can
include a set of modules to produces an integrated test device that
can receive an input sample and deliver a signal indicative of
whether the sample contains an organism associated with a disease.
For example, in some embodiments, a molecular diagnostic test
device can include a sample input and/or preparation module, an
elution module, an amplification module, one or more reagent
modules and a detection module. Such devices can be, for example,
single-use devices that can be used in a point-of-care setting
and/or in a user's home. Further, in some embodiments, such devices
can be a CLIA-waived device and/or can operate in accordance with
methods that are CLIA waived.
[0153] An example of an integrated test device shown in FIG. 7,
which is a schematic block diagram of a molecular diagnostic system
5000 (also referred to as "system" or "test unit"), according to an
embodiment. The test unit 5000 is configured to manipulate a sample
to produce an optical indication associated with a target cell
according to any of the methods described herein. In some
embodiments, the test unit 5000 can be a single-use, disposable
device that can provide an optical output without need for any
additional instrument to manipulate or otherwise condition the test
unit 5000. Said another way, the test unit 5000 is an integrated
cartridge/instrument, and the entire unit can be used to perform a
diagnostic assay and then be disposed. The test unit 5000 includes
a sample transfer device 5100, a sample preparation module 5200, an
inactivation chamber 5300, a fluidic drive module 5400, a mixing
chamber 5500, an amplification module 5600, a reagent storage
module 5700, a detection module 5800, a power/electronics module
5900, and a control module 5950. A brief description of the major
subsystems of the test unit 5000 is provided below.
[0154] The sample transfer device 5100 is configured to transport a
sample such as, for example, a blood, urine, male urethral
specimens, vaginal specimens, cervical swab specimens, and/or nasal
swab specimens sample gathered using a commercially available
sample collection kit, to the sample preparation module 5200. The
sample collection kit can be a urine collection kit or swab
collection kit. Non-limiting examples of such sample collection
kits include Copan Mswab or BD ProbeTec Urine Preservative
Transport Kit, Cat #440928, neat urine. The sample transfer device
5100 dispenses and/or otherwise transfers an amount of sample or
sample/media to the sample preparation module 5200 through an input
port (not shown). The input port can then be capped. In some
embodiments, the sample transfer device 5100 can be locked and/or
fixedly coupled to the sample preparation module 5200 as a part of
the dispensing operation. In this manner, the interface between the
sample transfer device 5100 and the sample preparation module 5200
can be configured to prevent reuse of the test unit 5000, transfer
of additional samples, or the like. Although shown as including the
sample transfer device 5100, in other embodiments, the test unit
5000 need not include a sample transfer device.
[0155] In some embodiments, through a series of user actions or in
an automated/semi-automated matter, the sample preparation module
5200 is configured to process the sample. For example, the sample
preparation module 5200 can be configured to concentrate and lyse
cells in the sample, thereby allowing subsequent extraction of DNA.
In some embodiments, the processed/lysed sample is pushed and/or
otherwise transferred from the sample preparation module 5200 to
the inactivation chamber 5300, which is configured to inactivate,
in the lysed sample, the proteins used during lysing. In some
embodiments, the fluidic drive module 5400 is configured to
aspirate the sample from the inactivation chamber 5300, and is
further configured to convey the sample to the amplification module
5600. The fluidic drive module 5400 is also configured to convey
the sample and/or reagents (e.g., from the reagent storage module
5700) to perform any of the methods of diagnostic testing described
herein. Similarly stated, the fluidic drive module 5400 is
configured to generate fluid pressure, fluid flow and/or otherwise
convey the input sample S1 through the modules of the device. In
some embodiments, the fluidic drive module 5400, can be a
single-use module that is configured to contact and/or receive the
sample flow. The single-use arrangement eliminates the likelihood
that contamination of the fluid transfer module and/or the other
modules to which the fluidic drive module 5400 is fluidically
coupled will become contaminated from previous runs, thereby
negatively impacting the accuracy of the results.
[0156] The mixing chamber 5500 mixes the output of inactivation
chamber 5300 with the reagents necessary to conduct a PCR reaction.
In some embodiments, the mixing chamber 5500 can contain the PCR
reagents in the form of one or more lyophilized reagent beads that
contain the primers and enzymes necessary for PCR. In such
embodiments, the mixing chamber 5500 can be configured to hydrate
and/or reconstitute the lyophilized beads in a given input volume,
while ensuring even local concentrations of reagents in the
entirety of the volume. The mixing chamber 5500 can include any
suitable mechanism for producing the desired solution, such as, for
example, a continuous flow mixing channel, an active mixing element
(e.g., a stir rod) and/or a vibratory mixing element. The mixed
sample is then conveyed to the amplification module 5600 (e.g., by
the fluidic drive module 5400).
[0157] The amplification module 5600 is configured to run
polymerase chain reaction (PCR) on the sample to generate an
amplified sample, in any manner as described herein. After PCR, the
amplified sample is further pushed, transferred or conveyed to a
detection module 5800. In some embodiments, the detection module
5800 is configured to run and/or facilitate a colorimetric
enzymatic reaction on the amplified sample. In particular, a series
of reagents from the reagent storage module 5700 can be conveyed by
the fluidic drive module 5400 to facilitate the optical output from
the test. In some embodiments, all of the various
modules/subsystems of the main test unit 5000 are controlled and/or
powered by the power/electronics module 5900 and the control module
5950.
[0158] In some embodiments, the control module 5950 can include one
or more modules, and can automatically control the valves, pumps,
power delivery and/or any other components of the test unit 5000 to
facilitate the molecular testing as described herein. The control
module 5950 can include a memory, a processor, an input/output
module (or interface), and any other suitable modules or software
to perform the functions described herein.
[0159] FIG. 8 illustrates a portion of the operations and/or
features associated with an enzymatic reaction, according to an
embodiment, that can be conducted by or within the detection module
5800, or any other detection module described herein (e.g., the
detection module 6800 described below). In some embodiments, the
enzymatic reaction can be carried out to facilitate visual
detection of a molecular diagnostic test result using the device
5000, the device 6000, or any other devices or systems described
herein. The reaction, the detection module 5800 and/or the
remaining components within the test unit 5000 can be collectively
configured such that the test unit 5000 is a single-use device that
can be used in a point-of-care setting and/or in a user's home.
Similarly stated, in some embodiments, the test unit 5000 (and any
of the other devices shown and described herein) can be configured
for use in a decentralized test facility. Further, in some
embodiments, the reaction shown in FIG. 8 can facilitate the test
unit 5000 (and any of the other devices shown and described herein)
operating with sufficient simplicity and accuracy to be a
CLIA-waived device. Similarly stated, in some embodiments, the
reaction shown in FIG. 8 can provide the output signal OP1 in a
manner that poses a limited likelihood of misuse and/or that poses
a limited risk of harm if used improperly. In some embodiments, the
reaction can be successfully completed within the test unit 5000
(or any other device described herein) upon actuation by a user
with minimal (or no) scientific training, in accordance with
methods that require little judgment of the user, and/or in which
certain operational steps are easily and/or automatically
controlled.
[0160] As shown, the detection module 5800 includes a detection
surface 5821 within a read lane or flow channel. The detection
surface 5821 is spotted and/or covalently bonded with a specific
hybridizing probe 5870, such as an oligonucleotide. In some
embodiments, the hybridizing probe 5870 is specific for a target
organism and/or amplicon. The bonding of the hybridizing probe 5870
to the detection surface 5821 can be performed using any suitable
procedure or mechanism. For example, in some embodiments, the
hybridizing probe 5870 can be covalently bound to the detection
surface 5821. Reference S7 illustrates the biotinylated amplicon
that is produced from the PCR amplification step such as, for
example, by the amplification module 5600 of FIG. 7 (or any other
amplification modules described herein). The biotin can be
incorporated within the amplification operation and/or within the
amplification module 5600 in any suitable manner. As shown by the
arrow XX, the output from the amplification module, including the
biotinylated amplicon S7 is conveyed within the read lane and
across the detection surface 5821. The hybridizing probe 5870 is
formulated to hybridize to the target amplicon S7 that is present
within the flow channel and/or in proximity to the detection
surface 5821. The detection module 5800 and/or the detection
surface 5821 is heated to incubate the biotinylated amplicon S7 in
the read lane in the presence of the hybridizing probe 5870 for a
few minutes allowing binding to occur. In this manner, the target
amplicon S7 is captured and/or is affixed to the detection surface
5821, as shown. In some embodiments, a first wash solution (not
shown in FIG. 8) can be conveyed across the detection surface 5821
and/or within the flow channel to remove unbound PCR products
and/or any remaining solution.
[0161] As shown by the arrow YY, a detection reagent R4 is conveyed
within the read lane and across the detection surface 5821. The
detection reagent R4 can be, for example, a horseradish peroxidase
(HRP) enzyme ("enzyme") with a streptavidin linker. In some
embodiments, the streptavidin and the HRP are cross-linked to
provide dual functionality. As shown, the detection reagent is
bound to the captured amplicon S7. The detection module 5800 and/or
the detection surface 5821 is heated to incubate the detection
reagent R4 within the read lane in the presence of the biotinylated
amplicon S7 for a few minutes to facilitate binding. In some
embodiments, a second wash solution (not shown in FIG. 8) can be
conveyed across the detection surface 5821 and/or within the flow
channel to remove unbound detection reagent R4.
[0162] As shown by the arrow ZZ, a detection reagent R6 is conveyed
within the read lane and across the detection surface 5821. The
detection reagent R4 can be, for example, a substrate formulated to
enhance, catalyze and/or promote the production of the signal OP1
from the detection reagent R4. Specifically, the substrate is
formulated such that upon contact with the detection reagent R4
(the HRP/streptavidin) a colorimetric output signal OP1 is
developed where HRP attaches to the amplicon. The color of the
output signal OP1 indicates the presence of bound amplicon: if the
target pathogen, target amplicon and/or target organism is present,
the color product is formed, and if the target pathogen, target
amplicon and/or target organism is not present, the color product
does not form.
[0163] Similarly stated, upon completion of the reaction, if the
target pathogen, target amplicon and/or target organism is present
the detection module produces a signal OP1. In accordance with the
reaction described in FIG. 8, the signal OP1 is a non-fluorescent,
visual signal that can viewed by the user (e.g., through a
detection opening or window defined by a device housing). This
arrangement allows the device to be devoid of a light source (e.g.,
lasers, light-emitting diodes or the like) and/or any light
detectors (photomultiplier tube, photodiodes, CCD devices, or the
like) to detect and/or amplify the signal OP1.
[0164] Said another way, the reaction produces a colorimetric
output signal that is visible to the user, and that requires little
to no scientific training and/or little to know judgment to
determine whether the target organism is present. In some
embodiments, the reagents R4, R6 are formulated such that the
visible signal OP1 remains present for at least about 30 minutes.
In some embodiments, the reagents R4, R6 can be stored within a
housing (not shown in FIG. 8) in any manner as described herein
(e.g., in a sealed container, a lyophilized form or the like).
[0165] FIG. 9 is a schematic illustration of a molecular diagnostic
test device 6000 (also referred to as a "test device" or "device"),
according to an embodiment. The schematic illustration describes
the primary components of the test device 6000 as shown in FIGS.
10-66. As described below, the test device 6000 is an integrated
device (i.e., the modules are contained within a single housing)
that is suitable for use within a point-of-care setting (e.g.,
doctor's office, pharmacy or the like), decentralized test
facility, or at the user's home. In some embodiments, the device
6000 can have a size, shape and/or weight such that the device 6000
can be carried, held, used and/or manipulated in a user's hands
(i.e., it can be a "handheld" device). In other embodiments, the
test device 6000 can be a self-contained, single-use device.
Similarly stated, in some embodiments, the test device 6000 can be
configured with lock-outs or other mechanisms to prevent re-use or
attempts to re-use the device.
[0166] Further, in some embodiments, the device 6000 can be a
CLIA-waived device and/or can operate in accordance with methods
that are CLIA waived. Similarly stated, in some embodiments, the
device 6000 (and any of the other devices shown and described
herein) is configured to be operated in a sufficiently simple
manner, and can produce results with sufficient accuracy to pose a
limited likelihood of misuse and/or to pose a limited risk of harm
if used improperly. In some embodiments, the device 6000 (and any
of the other devices shown and described herein), can be operated
by a user with minimal (or no) scientific training, in accordance
with methods that require little judgment of the user, and/or in
which certain operational steps are easily and/or automatically
controlled. In some embodiments, the molecular diagnostic test
device 6000 can be configured for long term storage in a manner
that poses a limited likelihood of misuse (spoilage of the
reagent(s), expiration of the reagents(s), leakage of the
reagent(s), or the like). In some embodiments, the molecular
diagnostic test device 6000 is configured to be stored for up to
about 36 months, up to about 32 months, up to about 26 months, up
to about 24 months, up to about 20 months, up to about 18 months,
or any values there between.
[0167] The test device 6000 is configured to manipulate an input
sample S1 to produce on or more output signals OP1, OP2, OP3 (see
FIG. 66) associated with a target cell according to any of the
methods described herein (e.g., including the enzymatic reaction
described above with respect to FIG. 8). FIGS. 10 and 11 show
perspective views of the molecular diagnostic test device 6000. The
diagnostic test device 6000 includes a housing (including a top
portion 6010 and a bottom portion 6030), within which a variety of
modules are contained. Specifically, the device 6000 includes a
sample preparation module 6200, an inactivation module 6300, a
fluidic drive (or fluid transfer) module 6400, a mixing chamber
6500, an amplification module 6600, a detection module 6800, a
reagent storage module 6700, a rotary venting valve 6340, and a
power and control module 6900. A description of each module and/or
subsystem follows.
[0168] FIG. 14 shows the device 6000 with the top housing 6010
removed so that the placement of the modules can be seen. FIG. 15
shows the device 6000 with the top housing 6010, the actuation
buttons, the amplification module 6600, and the detection module
6800 removed so that underlying modules can be seen. As shown in
FIGS. 12 and 13, the device 6000 is includes a top housing 6010, a
lower housing 6030 and a bottom plate 6031. The top housing 6010
includes connection protrusions 6018, 6019 that correspond to
notches, slots and or openings defined by the lower housing 6030 to
facilitate assembly of the housing and/or the device. The top
housing further defines a series of detection (or "status")
openings that allow the user to visually inspect the output
signal(s) produced by the device 6000. Specifically, the top
housing 6010 defines a first detection opening 6011, a second
detection opening 6012, a third detection opening 6013, a fourth
detection opening 6014, and a fifth detection opening 6015. When
the top housing 6010 is coupled to the lower housing 6030, the
detection openings are aligned with the corresponding detection
surfaces of the detection module 6800 such that the signal produced
by and/or on each detection surface is visible through the
corresponding detection opening. Specifically, the first detection
opening 6011 corresponds to the first detection surface 6821 (see
FIG. 49), the second detection opening 6012 corresponds to the
second detection surface 6822, the third detection opening 6013
corresponds to the third detection surface 6823, the fourth
detection opening 6014 corresponds to the fourth detection surface
6824, and the fifth detection opening 6015 corresponds to the fifth
detection surface 6825.
[0169] In some embodiments, the top housing 6010 and/or the portion
of the top housing 6010 surrounding the detection openings is
opaque (or semi-opaque), thereby "framing" or accentuating the
detection openings. In some embodiments, for example, the top
housing 6010 can include markings (e.g., thick lines, colors or the
like) to highlight the detection openings. For example, in some
embodiments, the top housing 6010 can include indicia identifying
the detection opening to a particular disease (e.g., Chlamydia
trachomatis (CT), Neisseria gonorrhea (NG) and Trichomonas
vaginalis (TV)) or control. In other embodiments, the top housing
6010 can include a series of color spots having a range of colors
associated with a range of colors that is likely produced by the
signals OP1, OP2, OP3, CTL 1 and/or CTL 2 to assist the user in
determining the results of the test. In this manner, the housing
design can contribute to reducing the amount of user judgment
required to accurately read the test.
[0170] The lower housing 6030 defines a volume 6032 within which
the modules and or components of the device 6000 are disposed. As
shown in FIG. 13, the lower housing 6030 includes a sample input
portion 6160, a sample preparation portion 6023, a wash portion
6025, and an elution/reagent portion 6029. As shown in FIG. 62, the
sample input portion 6160 defines a receiving volume 6164, and
includes a movable cap 6152 and an input member 6162. The movable
cap 6152 can rotate about the lower housing 6030 to provide access
to the input member 6162 and/or the receiving volume 6164. The cap
6152 can include seals or other locking members such that it can be
securely fastened to the lower housing 6030 and/or closed during
shipping, after delivery of a sample thereto, or the like. In some
embodiments, the input port cap 6152 can include an irreversible
lock to prevent reuse of the device 6000 and/or the addition of
supplemental sample fluids. In this manner, the device 6000 can be
suitably used by untrained individuals.
[0171] The input member 6162 defines a passageway through which the
sample is conveyed into the receiving volume 6164. As shown, the
input member 6162 has a funnel shape and is configured to minimize
splash when transferring the sample from the transfer device 6110
(described below) into the receiving volume 6164. In some
embodiments, the sample input member 6162 can include a filter,
screen or the like.
[0172] The sample preparation portion 6023 receives at least a
portion of the sample input module 6170. As described in more
detail herein, the sample input module 6170 is actuated by the
sample actuator (or button) 6050. The sample preparation portion
6023 defines a notch or opening 6033 that receives a lock tab 6057
of the sample actuator 6050 after the actuator 6050 has been moved
to begin the sample preparation operation (see, e.g., FIGS. 20 and
21). In this manner, the sample actuator 6050 is configured to
prevent the user from reusing the device after an initial use has
been attempted and/or completed.
[0173] The wash portion 6025 receives at least a portion of the
wash module 6210. The wash module 6210 is actuated by the wash
actuator (or button) 6060. The wash portion 6025 defines a notch or
opening 6035 that receives a lock tab 6067 of the wash actuator
6060 after the actuator 6060 has been moved to begin the wash
operation (see, e.g., FIG. 64). In this manner, the wash actuator
6060 is configured to prevent the user from reusing the device
after an initial use has been attempted and/or completed.
[0174] The elution/reagent portion 6029 receives at least a portion
of the elution module 6260 and a portion of the reagent module
6700. The elution/reagent portion 6029 defines a notch or opening
6039 that receives a lock tab 6087 of the reagent actuator 6080
after the actuator 6080 has been moved to begin the elution and/or
reagent opening operation (see, e.g., FIG. 65). In this manner, the
reagent actuator 6080 is configured to prevent the user from
reusing the device after an initial use has been attempted and/or
completed. By including such lock-out mechanisms, the device 6000
is specifically configured for a single-use operation, and poses a
limited risk of misuse.
[0175] The lower housing 6030 of the device 6000 includes mounting
structure and features to retain the modules disposed therein. For
example, the lower housing 6030 includes mounting structure 6046
for retaining the fluid transfer module 6400. The lower housing
6030 also includes the waste reservoir 6205 within which waste
products and/or flow is stored.
Sample Transfer Device
[0176] In some embodiments, the diagnostic test device 6000 can
include and/or be packaged along with a sample transport device
6110 (see FIG. 62) configured to provide a sample into the device
6000 and/or the sample preparation module 6200. As shown in FIG.
62, the sample transfer device 6110 includes a distal end portion
6112 and a proximal end portion 6113, and can be used to aspirate
or withdraw a sample from a sample cup, container or the like, and
then deliver a desired amount of the sample to an input portion
6160 of the device 6000. Specifically, the distal end portion 6112
includes a dip tube portion defining a reservoir 6115 having a
desired volume. The proximal end portion 6113 includes an actuator
6117 or squeeze bulb that can be manipulated by the user to draw
the sample into the reservoir 6115. The sample transport device
6110 includes an overflow reservoir 6116 that receives excess flow
of the sample during the aspiration step. The overflow reservoir
6116 includes a valve member that prevents the overflow amount from
being conveyed out of the transfer device 6110 when the actuator
6117 is manipulated to deposit the sample into the input portion
6160 of the device 6000. This arrangement ensures that the desired
sample volume is delivered to the device 6000. Moreover, by
including a "valved" overflow reservoir 6116, the likelihood of
misuse during sample input is limited. This arrangement also
requires minimal (or no) scientific training and/or little judgment
of the user to properly deliver the sample into the device.
[0177] In some embodiments, the sample transfer device 6110, or any
other sample transfer devices herein, can be used to aspirate fluid
from a transfer tube or cup that is also included as part of a kit
within which the device 6000 is included. In some embodiments, the
sample transfer device 6110 can be any suitable, commercially
available transport pipette. For example, in some embodiments, the
sample transfer device 6110 can include the Alpha Industries, UK,
250 .mu.l Dual Bulb Pastette LW4790 (Pasteur Pipette), which
transfers a sample volume of 250 .mu.l+/-10%. The test system 6000
is configured to accommodate such variation (e.g., +/-10%) in
pipetted volume. Transfer pipettes holding and/or delivering 500
.mu.l and 1000 .mu.l can also be used with the device 6000. In some
embodiments, the sample transfer device 6110 (or any of the sample
transfer devices described herein) can deliver a sample volume of
between about 250 and about 500 .mu.l.
[0178] In some embodiments, the sample transfer device 6110 can
include a status window or opening through which the user can
visually check to see that adequate volume has been aspirated.
[0179] Although shown as being used in conjunction with and/or
packaged with an external sample transfer device (i.e., the sample
transfer device 6110), in other embodiments, the device 6000 can
include an integrated sample transfer portion or device.
Sample Preparation Module
[0180] The sample preparation module 6200 is disposed at least
partially within the sample preparation portion 6023 the lower
housing 6030, and is configured receive an input sample S1 from the
receiving volume 6164 of the sample input portion 6160. As
described herein, the sample preparation module 6200 is configured
to process the sample S1 to facilitate detection of an organism
therein that is associated with a disease. By eliminating the need
for external sample preparation and a cumbersome instrument, the
device 6000 is suitable for use within a point-of-care setting
(e.g., doctor's office, pharmacy or the like) or at the user's
home, and can receive any suitable sample S1. The sample S1 (and
any of the input samples described herein) can be, for example,
blood, urine, male urethral specimens, vaginal specimens, cervical
swab specimens, and/or nasal swab specimens gathered using a
commercially available sample collection kit.
[0181] In some embodiments, the sample preparation module 6200 is
configured to accept and allow for spill-proof containment of a
volume of liquid from the sample input portion 6160. As described
below, the sample preparation module 6200 is configured for onboard
storage of wash solution, elution solution, and/or a positive
control (e.g., Aliivibrio fischeri, N. subflava, or any other
suitable organism). The positive control may be stored in liquid
form in the wash solution or stored as a lyophilized bead that is
subsequently hydrated by the wash solution. In some embodiments,
the sample preparation module 6200 is configured for dispensing the
majority of the sample liquid (e.g., about 80%) through a filter,
and storing the generated waste in a secure manner (i.e., within
the waste reservoir 6205). In some embodiments, the sample
preparation module 6200 is configured for following the sample
dispense operation with a wash dispense operation, thereby
dispensing the bulk of the stored liquid (e.g., about 80%). In some
embodiments, the sample preparation module 6200 is configured for
back-flow elution to occur to remove the desired target particles
from the filter membrane and deliver the bulk (e.g., about 80%) of
the eluted volume to the target destination (e.g., the inactivation
module 6300, the amplification module 6600 or the like). In some
embodiments, the sample preparation module 6200 is configured so as
not cause the output solution to be contaminated by previous
reagents (e.g., like the sample or wash). In some embodiments, the
sample preparation module 6200 is configured for ease of operation
by a lay user, requiring few, simple, non-empirical steps, and for
a low amount of actuation force.
[0182] The sample preparation module 6200 includes a sample input
module 6170 (FIGS. 16-21), a wash module 6210 (FIGS. 22-24), an
elution module 6260 (FIGS. 25-28), a filter assembly 6230 (FIGS.
32-35), and various fluidic conduits (e.g., tubes, lines, valves,
etc.) connecting the various components. Referring to FIGS. 16-21,
the sample input module 6170 includes a housing 6172 that defines a
sample volume 6174, and a piston 6180 that is movably disposed
within the sample volume 6174. The housing 6172 further defines a
sample input port 6175, a sample output port 6177, and a wash input
port 6176. In use, the input sample is conveyed from the sample
input portion 6160 into the sample volume 6174 via the sample input
port 6175. The sample can be conveyed by gravity feed or any other
suitable mechanism. As shown the sample input port 6175 is disposed
towards the top of sample volume 6174 such that after the piston
6180 moves downward to move the sample, the sample input port 6175
is blocked to prevent backflow of the sample back towards and/or
into the sample input portion 6160. In other embodiments, the
sample input port 6175 can include any suitable flow control
devices, such as check valves, duck-bill valves, or the like.
[0183] As shown in FIG. 21, when the piston 6180 is moved downward
within the sample volume 6174, the sample within the sample volume
6174 is conveyed towards the filter assembly 6230 via the sample
output port 6177. The flow of the input sample towards the filter
assembly 6230 is shown by the arrow S2 in FIG. 9. The sample output
port 6177 can include any suitable flow control devices, such as
check valves, duck-bill valves, or the like, to prevent flow from
the filter back into and/or towards the sample volume 6174.
[0184] The sample input module 6170 is actuated by the sample
actuator (or button) 6050. The sample actuator 6050 is movably
coupled to the sample preparation portion 6023 of the housing 6030,
and includes a side wall 6054 that defines an inner volume 6055
that can receive a portion of the sample input module 6170. The
sample actuator 6050 includes a protrusion 6056 that is aligned
with and can move the piston 6180 when the sample input module 6170
is actuated. The sample actuator 6050 further includes a lock tab
6057 that is fixedly received within the notch or opening 6033 to
fix the sample actuator 6050 in its second or "actuated" position,
as described above.
[0185] In use, after the input sample S1 has been placed into the
sample input portion 6160 and the desired portion of the sample has
been conveyed into the volume 6174, the sample input operation can
be initiated by the downward movement of the sample actuator 6050
relative to the lower housing 6030 (this is shown by the arrow PP
in FIG. 63; see also FIG. 21). Movement of the piston 6180 within
the volume 6174 increases the internal pressure, and thus cause the
sample therein to flow through the output port 6177 towards the
filter assembly 6230. The sample actuator 6050 is remains locked in
its second or "actuated" position by the interface between the lock
tab 6057 and the notch 6033. When the sample actuator 6050 is in
the locked position, the piston 6180 is spaced apart from the
bottom surface defining the sample volume 6174 to allow some amount
of "dead volume" through which the wash compositions can flow.
[0186] Referring to FIGS. 22-23, the wash module 6210 includes a
piston 6220 and housing 6212 that defines a wash volume 6214. As
shown by the dashed line in FIG. 23, the wash volume 6214 contains
a first wash composition W1 and a second wash composition W2. More
particularly, the first wash composition W1 is a gas (e.g.,
nitrogen, air, or another inert gas), and the second wash
composition W2 is a liquid wash. In this manner, the wash operation
can include an "air purge" of the filter assembly 6230, as
described in more detail herein.
[0187] The piston 6220 is movably disposed within the sample wash
volume 6214, and defines a wash output port 6216. The wash output
port 6216 is fluidically coupled to the wash input port 6176 of the
sample input module 6170. Moreover, the wash output port 6216 can
include any suitable flow control devices, such as check valves,
duck-bill valves, or the like to prevent flow back towards and/or
into the wash volume 6214. The arrangement of the wash output port
6216 allows the wash compositions (e.g., W1 and W2) to be conveyed
from the wash volume 6174 into the "dead volume" remaining of
sample volume 6174 and towards the filter assembly 6230 when the
wash actuator 6060 is actuated. More particularly, by including the
wash output port 6216 on the piston 6220, movement of the piston
6220 downward will produce a serial flow of the first wash
composition W1 followed by the second wash composition W2. By first
including a gas (or air) wash (the first wash composition W1), the
amount of liquid constituents from the input sample that has been
conveyed to the filter assembly 6230 (indicated by the flow S2 in
FIG. 9) can be reduced. Said another way, after delivery of the
input sample to the filter assembly 6230 by actuation of the sample
input module 6170, the filter assembly 6230 will retain the desired
sample cells and some amount of residual liquid. By forcing the
first, gaseous wash composition W1 through the filter (i.e., an
"air wash"), the amount of residual liquid can be minimized. This
arrangement can reduce the amount of liquid wash (e.g., the second
wash composition W2) needed to sufficiently prepare the sample
particles. Reducing the liquid volume contributes to the reduction
size of the device 6000 and also reduces the likelihood of
potentially harmful shearing stress when the liquid wash W2 is
flowed through the filter assembly.
[0188] The wash module 6210 is actuated by the wash actuator (or
button) 6060. The wash actuator 6060 is movably coupled to the wash
portion 6025 of the lower housing 6030, and includes a side wall
6064 that defines an inner volume 6065 that can receive a portion
of the wash module 6210. The wash actuator 6060 includes a
protrusion 6066 that is aligned with and can move the piston 6220
when the wash module 6210 is actuated. The wash actuator 6060
further includes a lock tab 6067 that is fixedly received within
the notch or opening 6035 to fix the wash actuator 6060 in its
second or "actuated" position, as described above.
[0189] In use, after the input sample S1 has been conveyed from the
sample input module 6170 to the filter assembly (indicated by the
arrow S2), the wash operation can be initiated by the downward
movement of the wash actuator 6060 relative to the lower housing
6030 (this is shown by the arrow QQ in FIG. 64). Movement of the
piston 6220 within the volume 6214 increases the internal pressure,
and thus cause the first wash composition W1 and the second wash
composition W2 to flow through the output port 6216 towards the
sample input module 6170, as indicated by the arrow S3 in FIG. 9.
The wash actuator 6060 is remains locked in its second or
"actuated" position by the interface between the lock tab 6067 and
the notch 6035.
[0190] As described above, as the piston 6220 moves downward, the
first wash composition W1 (i.e., the air wash) flows through the
"dead volume" remaining in the sample input module 6170, through
the sample output port 6177, and towards the filter assembly 6230.
The second wash composition W2 (i.e., the liquid wash) then flows
through the "dead volume" remaining in the sample input module
6170, through the sample output port 6177, and towards the filter
assembly 6230. The flow of the first and second wash is shown in
FIG. 9 by the arrow S3 shown through the filter assembly 6230. The
first wash composition W1, the second wash composition W2, and any
other waste products that pass through the filter assembly 6230 are
conveyed to the waste reservoir 6205. As described in more detail
below, the filter assembly 6230 includes a valve 6280 that controls
the flow of the sample and the wash through the filter assembly
6230.
[0191] In some embodiments, the wash actuator 6060 and/or the
sample actuator 6050 can be interconnected or can otherwise include
locking features that limit the movement of the actuators out of
order. For example, in some embodiments the sample actuator 6050
can include a protrusion that contacts a portion of the lock
protrusion 6067 of the wash actuator 6060, thereby preventing
movement of the lock actuator 6060 when the sample actuator 6050 is
in its first position. In this manner, the actuators can be
configured to reduce the likelihood of being actuated out of
order.
[0192] Although shown and described as including a first wash
composition W1 (i.e., a gas) and a second wash composition W2
(i.e., a liquid), in other embodiments, the wash module 6210 can
include only a single wash composition.
[0193] The filter assembly 6230 is shown in FIGS. 14, 15 and 32-35.
The filter assembly 6230 includes a filter housing assembly 6250, a
first valve plate 6233, a second valve plate 6243, and a valve body
6290. As described herein, the filter assembly 6230 is configured
to filter and prepare the input sample (via the sample input
operation and the sample wash operation), and to allow a back-flow
elution operation to deliver captured particles from the filter
membrane 6254 and deliver the eluted volume to the target
destination (e.g., towards the amplification module 6600).
[0194] The filter housing assembly 6250 includes a first plate
6251, a second plate 6252, and a filter membrane 6254. The first
plate 6251 defines an input/output port 6255 through which the
sample and wash solutions flow (towards the waste reservoir 6205),
as indicated by the arrow EE in FIG. 32, and through which the
elution solution and sample particles flow (towards the
inactivation chamber 6300), as indicated by the arrow FF in FIG.
34. The input/output port 6255 is selectively placed in fluid
communication with the valve openings 6237 and 6238 to control the
flow therethrough. The second plate 6252 defines an input/output
port 6256 through which the sample and wash solutions flow (towards
the waste reservoir 6205), as indicated by the arrow EE in FIG. 32,
and through which the elution solution and sample particles flow
(towards the inactivation chamber 6300), as indicated by the arrow
FF in FIG. 34. The input/output port 6256 is selectively placed in
fluid communication with the valve openings 6247 and 6248 to
control the flow therethrough.
[0195] The filter membrane 6254 captures the target organism/entity
while allowing the bulk of the liquid within the sample, the first
wash composition W1, and the second wash composition W2 to flow
through into the waste tank 6230. The filter membrane 6254 (and any
of the filter membranes described herein) can be any suitable
membrane and or combination of membranes. For example, in some
embodiments, the filter membrane 6254 is a woven nylon filter
membrane with a pore size of about 1 .mu.m (e.g., 0.8 .mu.m, 1.0
.mu.m, 1.2 .mu.m) enclosed between the first plate 6251 and the
second plate 6252 such that there is minimal dead volume. In such
embodiments, the particle capture can be achieved primarily through
a binding event. Such pore sizes and filter construction can lead
to reduced fluid pressure during the sample delivery, wash and the
elution operations. Such designs, however, may also allow target
organisms to flow through the filter membrane 6254, potentially
resulting in lower efficiency of capture. Furthermore the target
organism may be harder to remove on the elution step (e.g., the
backwash) due to the nature of the binding. However the resulting
eluent solution is "cleaner" as more of the unwanted material gets
washed away through the filter membrane 6254. Thus, the filter
member 6254 and size thereof can be selected to be complimentary to
and/or consistent with the target organism. For example, the filter
membrane 6254 can be constructed and/or formulated to capture
target specimens through either size exclusion (where anything
smaller than the target organism is allowed to flow through the
membrane), or via binding the target to the filter membrane through
a chemical interaction (and later removing the target from the
membrane with the elution solution).
[0196] For example, in some embodiments, the filter membrane 6254
can be a cellulose acetate filters with a pore size of
approximately 0.35 .mu.m, and can be constructed to achieve
particle capture by size exclusion. Such filter construction,
however, can tend to clog more easily, thus generating higher
pressures during sample delivery, wash and the elution operations.
In some embodiments, the internal pressures can be reduced by
altering the diameter of the filter membrane 6254 and/or reducing
the total volume of sample to be conveyed through the filter
assembly 6230.
[0197] The first valve plate 6233 defines a valve slot 6234 in
fluid communication with the input/output port 6255. Thus, the
first valve plate 6233 provides fluidic access to the filter
membrane 6254 (via the valve body 6290). The second valve plate
6243 defines a valve slot 6244 in fluid communication with the
input/output port 6256. Thus, the second valve plate 6244 provides
fluidic access to the filter membrane 6254 (via the valve body
6290).
[0198] The valve body 6290 includes an actuation portion 6291, a
first valve leg 6232, and a second valve leg 6242. The first valve
leg 6232 and the second valve leg 6242 are coupled to the actuation
portion 6291, such that the sliding movement of the actuation
portion 6291 causes the first valve leg 6232 to slide within the
slot 6243 and the second valve leg 6242 to slide within the slot
6244. The first valve leg 6232 includes the valve openings 6237 and
6238, and a pair of O-rings (not shown) that sealing surround each
of the openings. The second valve leg 6242 includes the valve
openings 6247 and 6248, and a pair of O-rings 6253 that sealing
surround each of the openings. Thus, depending on the position of
the valve body 6290 within the slots 6234, 6244, a pair of the
openings can be selectively aligned with the opening 6255 of the
second plate 6251 and the opening 6256 of the second plate 6252 to
either block a particular flow path, or allow fluid flow
therethrough. In this manner, the valve assembly 6230 can control
the fluid flow during the sample flow, wash flow and elution flow
operations.
[0199] FIG. 32 shows the filter assembly 6230 in its first (or
"sample wash") configuration. When in the first configuration, the
valve opening 6237 and the valve opening 6247 are both aligned with
the input/output port 6255 and with the input/output port 6256. The
valve opening 6237 receives flow of the sample from the sample
output port 6177, and the valve opening 6247 is fluidically coupled
to the waste reservoir 6205. Thus, when the filter assembly 6230 is
in its first configuration, the sample S2 can be conveyed through
the filter membrane 6254 (with the waste portion going to the waste
reservoir 6205) as shown by the arrow EE. Further, the wash
compositions S3 can be conveyed through the filter membrane 6254
(with the waste portion going to the waste reservoir 6205) as shown
by the arrow EE. Moreover, the sample and or wash flows (S2 and S3,
respectively) are prevented from flowing through the filter
membrane 6254 and towards the elution module 6260 because the valve
opening 6248 is sealed with the second valve leg 6242. This is
depicted by the arrow FF in FIG. 32. The sample and or wash flows
(S2 and S3, respectively) are also prevented from bypassing the
filter membrane 6254 and flowing towards the inactivation chamber
6300 because the valve opening 6238 is sealed with the first valve
leg 6232.
[0200] FIG. 34 shows the filter assembly 6230 in its second (or
"elution") configuration. When in the second configuration, the
valve opening 6238 and the valve opening 6248 are both aligned with
the input/output port 6255 and with the input/output port 6256. The
valve opening 6248 receives the elution flow from the elution
module 6260 (described below), and the valve opening 6238 is
fluidically coupled to the inactivation chamber 6300. Thus, when
the filter assembly 6230 is in its second configuration, the
elution flow (indicated by the arrow S4 in FIG. 9) can be conveyed
back through the filter membrane 6254 as shown by the arrow FF.
Moreover, the elution flow S4 is prevented from flowing through the
filter membrane 6254 and towards the sample input module 6170
because the valve opening 6237 is sealed with the first valve leg
6232. This is depicted by the arrow EE in FIG. 34. The elution flow
S4 is also prevented from bypassing the filter membrane 6254 and
flowing towards the waste reservoir 6205 because the valve opening
6247 is sealed with the second valve leg 6242.
[0201] As described below, the valve body 6290 is actuated by
movement of the reagent actuator 6080. In particular, the ramp 6088
defined by the protrusion 6086 of the reagent actuator 6080
contacts the actuation portion 6291 and moves the valve body 6290
inward, as shown by the arrow GG in FIG. 34 to move the filter
assembly 6230 from its first configuration (FIG. 32) to its second
configuration (FIG. 34).
[0202] The elution module (or assembly) 6260 of the sample
preparation module 6200 is shown in FIGS. 25-28. The elution module
6260 is contained, along with the reagent module 6700, in the
reagent portion 6029 of the housing. Moreover, the elution module
6260 and the initial actuation of the reagent module 6700 are both
actuated by movement of a single, manual actuator (the reagent
actuator 6080). The elution module 6260 is described immediately
below, whereas the reagent module 6700 is described in more detail
further below.
[0203] The elution module 6260 is contained within the reagent
housing 6740 (also referred to as the "tank body" or the "reagent
body"), and includes a piston 6270 (see FIG. 28). The reagent
housing 6740 defines an elution volume 6264 within which an elution
composition is stored. The elution composition can include
proteinase K, which allows for the release of any bound cells
and/or DNA from the filter membrane 6254. The reagent housing 6740
further defines an input (or fill) port 6265 and an elution output
port 6266. The elution output port 6266 is fluidically coupled to
the valve opening 6248 of the second valve leg 6242, and can be
selectively placed in fluid communication with the filter assembly
6230, as described above. The elution output port 6266 can include
any suitable flow control devices, such as check valves, duck-bill
valves, or the like to prevent flow back towards and/or into the
elution volume 6264.
[0204] The elution module 6210 is actuated by the reagent actuator
(or button) 6080 (see FIG. 30). The reagent actuator 6080 is
movably coupled to the reagent portion 6029 of the lower housing
6030, and includes a side wall 6084 that defines an inner volume
6065 that can receive a portion of the elution module 6260. The
inner volume 6065 also receives the top member 6735 of the reagent
module 6700, which includes a protrusion that is aligned with and
can move the piston 6270 when the reagent actuator 6080 is moved.
The reagent actuator 6080 further includes a lock tab 6087 that is
fixedly received within the notch or opening 6039 to fix the
reagent actuator 6080 in its second or "actuated" position, as
described above.
[0205] In use, the filter assembly 6230 recovers the target
organisms with a certain efficiency, from a given starting volume.
The wash operation then removes undesired material, without
removing the target organisms (which stay present on the filter
membrane 6254). The elution operation then removes the target
organism from the filter membrane 6254, diluting the total amount
of captured organisms in the volume of the elution solution, thus
comprising the eluent. By modifying the total output volume of
eluent, a higher or lower concentration of both target organism and
any potential inhibiting matter can be achieved. In some
embodiments, a further dilution can be achieved, if desired, by
mixing the eluent solution with another reagent after the initial
sample preparation. Given a known volume of eluent, and a known
volume of diluent, a correct dilution factor can be achieved,
through to maintain the reliability of the system very high
dilution factors are avoided.
Reagent Module
[0206] As described herein, the detection method includes
sequential delivery of the detection reagents (reagents R3-R6) and
other substances within the device 6000. Further, the device 6000
is configured to be an "off-the-shelf" product for use in a
point-of-care location (or other decentralized location), and is
thus configured for long-term storage. In some embodiments, the
molecular diagnostic test device 6000 is configured to be stored
for up to about 36 months, up to about 32 months, up to about 26
months, up to about 24 months, up to about 20 months, up to about
18 months, or any values there between. Accordingly, the reagent
storage module 6700 is configured for simple, non-empirical steps
for the user to remove the reagents from their long term storage
containers, and for removing all the reagents from their storage
containers using a single user action. In some embodiments, the
reagent storage module 6700 and the rotary selection valve 6340
(described below) are configured for allowing the reagents to be
used in the detection module 6800, one at a time, without user
intervention.
[0207] Specifically, the device 6000 is configured such that the
last step of the initial user operation (i.e., the depressing of
the reagent actuator 6080) results in dispensing the stored
reagents. As described below, this action crushes and/or opens the
sealed reagent containers present in the assembly and relocates the
liquid for delivery. A rotary venting selector valve 6340 (see
FIGS. 50-62) allows all of the reagent module 6700 to be vented for
this step, and thus allows for opening of the reagent containers,
but closes the vents to the tanks once this process is concluded.
The reagents remain in the reagent module 6700 until needed in the
detection module 6800. When a particular reagent is needed, the
rotary valve 6340 opens the appropriate vent path to the reagent
module 6700, and the fluidic drive module 6400 applies vacuum to
the output port of the reagent module 6700 (via the detection
module 6800), thus conveying the reagent from the reagent module
6700.
[0208] As illustrated in FIGS. 9 (schematically) and 25-31, the
reagent storage module 6700 stores packaged reagents, identified
herein as reagent R3 (a first wash solution), reagent R4 (an enzyme
reagent), reagent R5 (a second wash solution), and reagent R6 (a
substrate), and allows for easy un-packaging and use of these
reagents in the detection module 6800. As shown in FIGS. 15-17, the
reagent storage module 6700 includes a first reagent canister 6701
(containing the first reagent R3), a second reagent canister 6702
(containing the second reagent R4), and a fourth reagent canister
6704 (containing the fourth reagent R6), a reagent housing (or
tank) 6740, a top member (or lid) 6735, and bottom (or outlet)
member 6780. As described above, the reagent housing 6740 also
contains and/or forms a portion of the elution module 6260.
[0209] Each of the reagent canisters includes frangible seals on
the upper and lower ends thereof to define a sealed container
suitable for long-term storage of the substance therein. For
example, referring to FIG. 29, the second reagent canister 6702
includes a first (or top) frangible seal 6718 and a second (or
lower) frangible seal 6717. As described below, the frangible seals
are punctured upon actuation of the reagent module 6700 to
configure or "ready" the reagent within each canister for use
within the detection module 6800. The frangible seals can be, for
example, a heat-sealed BOPP film (or any other suitable
thermoplastic film). Such films have excellent barrier properties,
which prevent interaction between the fluids within the canister
and external humidity, but also have weak structural properties,
allowing the films to be easily broken when needed. When the
reagent canister is pushed into the crush feature or puncturers, as
described below, the BOPP film breaks, allowing liquid within the
canister to flow when vented. Each of the reagent canisters also
includes two O-ring seals that fluidically isolate the canister
within its bore of the reagent housing 6740. For example, as shown
in FIG. 29, the second reagent canister 6702 includes a first (or
upper) O-ring 6716 and a second (or lower) O-rings 6719. These
O-rings seal the second reagent canister 6702 within the bore 6746
of the reagent housing 6740.
[0210] The reagent housing 6740 defines a set of cylindrical bores
within which a corresponding reagent canister is movably contained.
As shown in FIG. 27, a first bore contains the first reagent
canister 6701, a second bore (which is identified as bore 6746 in
FIG. 29) contains the second reagent canister 6702, a third bore
contains the third reagent canister 6703, and a fourth bore
contains the fourth reagent canister 6704. The reagent housing 6740
includes a puncturer in the bottom portion of each bore configured
to pierce the second frangible seal of the respective canister when
the canister is moved downward within the reagent housing 6740.
Similarly stated, the reagent housing 6740 includes a set of
puncturers that each pierce a corresponding frangible seal to open
a reagent canister when the reagent module 6700 is actuated.
Further, each puncturer defines a flow path that places the
internal volume of the reagent canister in fluid communication with
an outlet port of the reagent module 6700 after the frangible seal
is punctured. For example, referring to FIGS. 29 and 31, the second
bore 6746 includes a puncturer 6747 that defines a puncturer flow
path 6748. The puncturer flow path 6748 is in fluid communication
with the second outlet port 6792 via the passageway 6782.
[0211] The reagent housing 6740 also defines the elution volume
6264 (described above) and a guide bore 6706. The guide bore 6706
receives the corresponding pin or protrusion 6737 of the top member
6735 to guide movement of the top member 6735 relative to the
reagent housing 6740.
[0212] The bottom member 6780 is coupled to the bottom portion of
the reagent housing 6740 and defines the reagent outlet ports that
are in fluid communication with each of the reagent bores.
Specifically, the bottom member 6780 defines a first outlet port
6791 that is in fluid communication with the first bore, and
through which the first reagent R3 can flow. The bottom member 6780
defines a second outlet port 6792 that is in fluid communication
with the second bore 6746, and through which the second reagent R4
can flow (via the puncturer flow path 6748 and the passageway 6782,
as shown in FIG. 29). The bottom member 6780 defines a third outlet
port 6793 that is in fluid communication with the third bore, and
through which the third reagent R5 can flow. The bottom member 6780
defines a fourth outlet port 6794 that is in fluid communication
with the fourth bore, and through which the fourth reagent R6 can
flow.
[0213] The top member 6735 is configured to move relative to the
reagent housing 6740 when the reagent module 6700 is actuated. The
top member 6735 includes a set of shoulders, each including a
puncturer, and each of which corresponds to one of the reagent
canisters. Similarly stated, the top member 6735 includes a set of
shoulders, each including a puncturer, and each of which is aligned
with and is configured to move at least partially within a
corresponding bore defined by the reagent housing 6740. Referring
to FIGS. 29 and 31, for example, the top member 6735 includes a
first shoulder 6762 that corresponds to the first reagent canister
6701 (and the first bore) and a second shoulder 6767 that
corresponds to the second reagent canister 6702 (and the second
bore 6746). The first shoulder 6762 includes a first puncturer 6761
and the second shoulder 6767 includes a second puncturer 6766.
Further, each puncturer of the top member 6735 defines a flow path
that places the internal volume of the reagent canister in fluid
communication with vent port of the reagent module 6700 after the
top frangible seal is punctured. For example, referring to FIGS. 29
and 31, the second puncturer 6766 that defines a puncturer flow
path 6732 that serves as the vent port 6732 (see the outlet vent
ports in FIG. 26). Specifically, the first canister 6701 and/or
first bore is vented via the first vent port 6731, the second
canister 6702 and/or second bore 6746 is vented via the second vent
port 6732, the third canister 6703 and/or third bore is vented via
the third vent port 6733, and the fourth canister 6704 and/or
fourth bore is vented via the fourth vent port 6734. As described
below, each of the vent ports is fluidically coupled to the rotary
valve 6340 to allow for selective and/or sequential venting of each
canister to control the flow of the reagents to the detection
module 6800.
[0214] The vent portion 6736 of the top member 6735 is also
configured to engage with the switch 6906 to actuate the power and
control module 6900 when the reagent module 6700 (and the elution
module 6260) is actuated. The top member 6735 further includes the
guide pin (or protrusion) 6737 that moves within the guide bore
6706 of the reagent housing 6740 during use.
[0215] When the reagent module 6700 is in its first (or storage)
configuration (e.g., FIG. 29), the frangible seals 6717, 6718
fluidically isolate the interior volume of the second canister
6702, thus maintaining the reagent R2 in condition for storage. The
reagent module 6700 is actuated when the top member 6735 is moved
downward from its first position (FIG. 29) to its second position
(FIG. 31). Specifically, the reagent module 6700 is actuated along
with the elution module 6210 by the reagent actuator (or button)
6080 (see FIG. 30). The reagent actuator 6080 allows the user to
manually actuate the system by depressing the actuator 6080
downward (see the arrow RR in FIG. 65).
[0216] As the reagent actuator 6080 and the top member 6735 move
downward relative to the reagent housing 6740, the top puncturers
pierce the top frangible seal of each of the reagent containers.
Specifically, as shown in FIG. 31, the second puncturer 6768
pierces the top frangible seal 6718 thereby placing the interior
volume of the second reagent canister 6702 in fluid communication
with the vent port 6732. Further downward movement of the top
member 6735 causes the shoulders of the top member 6735 to engage
each respective canister and move the canister downward in its
respective bore. This causes the lower puncturers (of the reagent
housing 6740) to pierce the lower frangible seals. Specifically, as
shown in FIG. 31, the shoulder 6767 pushes the second canister 6702
downward within the bore 6746, thus causing the puncturer 6747 to
pierces the lower frangible seal 6717. This places the interior
volume of the second reagent canister 6702 in fluid communication
with the outlet port 6792.
[0217] When the reagent module 6700 is in the second configuration,
the reagents are "readied" for use (i.e., they are released from
the sealed canisters). The reagents, however, remain within their
respective canister and/or bore until such time as they are
actuated by operation of the rotary valve assembly 6340, which
selectively opens the vent ports 6731, 6732, 6733, and 6734 to
allow the reagents to flow out of the reagent assembly 6700 via the
outlet ports 6791, 6792, 6793 and 6794.
[0218] The reagent module 6700 and the rotary valve 6340 allows for
the reagents to be prepared and sequentially conveyed into the
detection module 6800 in a simple manner, and by a user with
minimal (or no) scientific training, in accordance with methods
that require little judgment. More particularly, the preparation of
the reagents requires only the manual depression of a button (the
reagent actuator 6080). The sequential addition of the reagents is
controlled automatically by the rotary valve 6340. This arrangement
contributes to the device 6000 being a CLIA-waived device and/or
being operable in accordance with methods that are CLIA waived.
Inactivation Chamber
[0219] As shown by the arrow S4 in FIG. 9, the elution solution and
the captured cells and/or organisms are conveyed during the elution
operation back through the filter assembly 6230, and to the
inactivation module (or "chamber") 6300. The inactivation module
6300 is configured to be fluidically coupled to and receive the
eluted sample S4 from the sample preparation module 6200. In some
embodiments, the inactivation module 6300 is configured for lysis
of the received input fluid. In some embodiments, the inactivation
module 6300 is configured for de-activating the enzymes present in
input fluid after lysis occurs. In some embodiments, the
inactivation module 6300 is configured for preventing
cross-contamination between the output fluid and the input
fluid.
[0220] Referring to FIGS. 36 and 37, the inactivation module 6280
includes a housing 6310, a lid 6318, a heater 6330, as well as
fluidic and electrical interconnects (not shown) to other modules.
The housing 6310 defines an inactivation chamber 6311, an input
port 6212, an output port 6313, and a vent 6314. As shown in FIG.
37, the inactivation chamber 6311 is constructed to allow for being
filled with the sample from the sample preparation module 6200,
followed by heating the entirety of the liquid received. This is
accomplished by making the input port 6212 and the output port 6313
have more arduous and/or tortuous flow paths than that of the vent
port 6314. In this manner, when the liquid is manipulated, or when
the liquid expands from being heated, the flow of liquid goes
either towards or away from the vent port 63214, rather than into
any of the conduits connected to the input port 6212 and/or the
output port 6313.
[0221] As shown in FIG. 37, the lid 6318 (or cover) is coupled to
the housing 6310 by an adhesive layer 6319. In other embodiments,
the inactivation module 6300 can be constructed using any suitable
mechanism.
[0222] The heater assembly 6330 can be any suitable heater
construct, and can include electrical connections 6332 to
electrically couple the heater 6330 to the controller 6950, power
supply 6905 or the like. In some embodiments, the heater 6330 can
integrate a simple heat spreader and a resistive heater layer with
an integrated temperature sensor (not shown). The lid 6318 of the
housing 6310 is constructed of a thin plastic membrane, and the
heater assembly 6330 can be attached thereto by any suitable
mechanism. This direct coupling arrangement allows for good thermal
conduction from the heater assembly 6330 into the liquid inside the
inactivation chamber 6311. The heater 6330 is controlled by the
electronics module (e.g., the electronic controller 6950, or any
other suitable controller) to control and/or maintain the heater
6330 at a certain temperature. Through characterization of the
module, an offset from this control temperature is developed to the
temperature of the liquid inside the inactivation chamber 6311.
[0223] In use, the sample is deposited in/transferred to the
inactivation chamber 6311 from the sample preparation module 6200
via the input port 6312, as shown by the arrow HH. In some
embodiments, the permanently open hydrophobic vent port 6314 allows
the inactivation chamber 6311 to be filled passively; i.e., without
need for intervention from a user (e.g., a manual operation) or
control module (e.g., activating an addition piston pump). Once the
fill is complete and the inactivation module 6300 is powered on,
the heater assembly 6330 warms the liquid in the inactivation
chamber 6311 to allow for the lysis reagents contained in the
eluent to function at peak efficiency. This process lyses the
target organism cells captured in the sample preparation module
6200 and releases the DNA present in the target. In some
embodiments, the sample can be heated to about 56 C for about 1
minute. After an allotted amount of time, the heater 6330 heats the
liquid to a high temperature to deactivate the lysis enzymes as
well as any other enzymes present. In some embodiments, the sample
can be heated to about 95 C for about 3 minutes. Liquid is then
retained in the inactivation chamber 6311 until moved by the
fluidic drive module 6400.
Mixing Module
[0224] As illustrated in FIGS. 9 (schematically), 38, and 39, the
mixing module (also referred to as simply the mixing chamber) 6500
mixes the output of inactivation module 6300 with the reagents
(e.g., R1 and R2) to conduct a successful PCR reaction. Similarly
stated, the mixing module 6500 is configured to reconstitute the
two reagent R1 and R2 in a given input volume, while ensuring even
local concentrations of reagents in the entirety of the volume. In
some embodiments, the mixing chamber module 6500 is configured to
produce and/or convey a sufficient volume of liquid for the
amplification module 6600 to provide sufficient volume output to
the detection module 6800.
[0225] The mixing module 6500 includes a first housing 6520, a
second housing (or cover) 6570, and a lyophilized reagent bead
containing two reagents (identified as reagents R1 and R2). The
mixing module 6500 also includes the tubing, interconnects and
other components to couple the mixing module 6500 to the
inactivation chamber 6300, the fluid drive module 6400, and the
rotary valve assembly 6340. The first housing 6520 defines a mixing
reservoir 6530, an inlet port 6540, an outlet port 6550 and a vent
port 6556. The first housing 6520 also defines an opening 6523
within which the joining pin 6522 can be disposed to couple the
first housing 6520 to the second housing 6570.
[0226] The input (or fill) port 6540 is fluidically coupled to the
outlet port 6313 of the inactivation module 6300, and is configured
to receive a flow from the inactivation module 6300 as shown by the
arrow JJ in FIG. 38. The outlet port 6550 is fluidically coupled to
the fluid transfer module 6400, and is configured to produce a flow
to the fluidic drive module 6400 (and on to the amplification
module 6600), as shown by the arrow KK in FIG. 38. The input port
6540 and the outlet port 6550 can include any suitable flow control
devices, such as check valves, duck-bill valves, or the like to
control the flow into and/or out of the mixing reservoir 6530.
Although the mixing module 6500 is shown as being disposed upstream
of the fluid transfer module 6400, in other embodiments, the mixing
module 6500 can be disposed between the fluid transfer module 6400
and the amplification module 6500 (i.e., the mixing module 6500 can
be downstream of the fluid transfer module 6400).
[0227] The second housing 6570 defines a portion of the mixing
reservoir 6530, and is coupled to the first housing 6520 by the pin
6522 and any other sealing mechanism (such as the laminate 6524).
Thus, together the first housing 6520 and the second housing 6570
define the mixing reservoir 6530 having the desired geometry to
promote fluidic mixing, as described herein. Specifically, the
mixing reservoir 6530 and/or other portions of the mixing module
6500 are configured to increase the effect of diffusion of the
liquid by increasing the total contact area between segments of the
liquid with areas of low and high local concentrations of reagents.
This is accomplished by allowing the initial portion the liquid
entering the chamber to contact the back of other portions of
liquid and/or structure, and then maintaining the liquid in the
mixing reservoir 6530 for sufficient time for diffusion to even out
the concentration. In some embodiments, the first housing 6520
and/or the second housing 6570 can include on or more flow
structures, vanes or the like (not shown) configured to influence,
impact and/or change the fluid flow within the mixing reservoir
6520. Such flow structures and produce areas of recirculation,
turbulent flow areas, and the like.
[0228] Although the mixing module 6500 is shown as being a passive
module (i.e., relying solely on fluid flow to achieve the desired
mixing and diffusion), in other embodiments, a mixing module can
include an active mixing approach. For example, in some
embodiments, the mixing module can include a stir rod or vibration
mixer.
[0229] In use, when the fluid flows from the inactivation chamber
6300 (as a result of actuation of the fluid transfer module 6400),
the initial (or first portion) of the liquid drawn into the mixing
reservoir 6530 reconstitutes the lyophilized beads R1, R2 into the
total volume of the mixing reservoir 6530. In some embodiments, the
mixing reservoir can include structures that limit the fluid flow
out of the mixing reservoir 6530 for a period of time until it is
fully filled. In this manner, an overall concentration can be
achieved and/or maintained prior to the sample being conveyed to
the amplification module 6600. Structural features of the mixing
reservoir 6530, combined with controlled stop/start flow from the
fluid transfer module 6400 allow correct local concentrations to be
achieved before the liquid flows out of the mixing chamber into the
amplification module 6600.
[0230] The reagents R1 and R2 are each lyophilized pellets having a
substantially hemispherical shape, and are disposed together in the
spherical portion of the mixing reservoir 6530. This arrangement
allows the two pellets to be hydrated together and/or substantially
simultaneously when the flow of solution from the inactivate
chamber 6300 is received within the mixing reservoir 6530.
Similarly stated, the two lyophilized pellets are shaped to
matingly fit together within the mixing reservoir 6530. In other
embodiments, however, the two lyophilized pellets can each be
spherically shaped, and can be placed within the mixing reservoir
6530.
[0231] The mixing module 6500 is also a storage location for the
two lyophilized beads R1, R2, which, once reconstituted and mixed,
form the master mix for the subsequent amplification step. The
reagents R1 and R2 can be any suitable PCR reagents, such as the
primers, nucleotides (e.g., dNTPs), and the DNA polymerase. In some
embodiments, the reagent R1 and/or the reagent R2 can include the
KAPA2G Fast DNA Polymerase, which includes a hot start function.
This arrangement allows for very fast thermocycling and very little
primer dimer formation. In some embodiments, the reagent R1 and/or
the reagent R2 can include the PCR primers designed to target
Chlamydia trachomatis (CT), Neisseria gonorrhea (NG) and
Trichomonas vaginalis (TV). In addition, the reagent R1 and/or the
reagent R2 can include a primer set for a non-target gram negative
organism (Aliivibrio fischeri) to act as a positive control
organism. All of the primers are designed with Tm values of
approximately 60.degree. C. In this manner, the PCR reaction
conducted by the device is a multiplex reaction containing all four
sets of primers. Specifically, in some embodiments, the primer set
for C. trachomatis targets the 7.5 kb endogenous plasmid and
produces a 101 bp amplicon. In some embodiments, the primer set for
N. gonorrhoeae targets the opa gene and produces a 70 bp amplicon.
In some embodiments, the primer set for T. vaginalis targets a
repetitive DNA fragment in the genome and produces a 65 bp
amplicon. In some embodiments, the primer set for A. fischeri
targets the hvnC locus and produces a 107 bp amplicon.
[0232] In some embodiments, the reagent R1 can contain the primers
and raw base pairs for the reaction, and reagent R2 can include the
enzymes necessary for PCR amplification. Moreover, because the
device 6000 can be configured for single-use in a point-of-care
setting, the reagents R1 and R2 can be formulated for and/or
packaged within the mixing module 6500 to enhance long term
storage. Accordingly, in some embodiments, the reagents R1 and R2
and/or the device 6000 can be configured to have a shelf life of up
to about 36 months, up to about 32 months, up to about 26 months,
up to about 24 months, up to about 20 months, up to about 18
months, or any values therebetween.
[0233] For example, by separating the two major constituents of the
master mix solution (the primers and the enzymes) high shelf life
and reagent stability can be achieved. In other embodiments,
however, the mixing module 6500 can include any number of
lyophilized pellets or beads, each containing any suitable reagents
for the PCR reaction. Additionally, the first housing defines the
vent port 6556, which fluidically coupled to a desiccated area of
the device, and which is coupled to the vent line 6356 of the
rotary valve assembly 6340. In this manner, moisture can be drawn
away from the reagents R1, R2 during storage and shipping.
Specifically, as described in more detail below, when the rotary
valve assembly 6340 is in the "shipping" condition, the vent port
6556 is open to atmosphere, and the desiccant is in-line between
the vent port 6556 and the valve assembly 6340. During use, the
valve assembly 6340 closes the vent port 6556 to ensure proper
fluid flow and mixing, as described above.
Fluidic Drive Module
[0234] FIGS. 40-42 show the fluidic drive module 6400 (also
referred to as the fluid transfer module 6400). The fluid transfer
module 6400 can be any suitable module for manipulating the sample
within the device 6000. Similarly stated, the fluid transfer module
6400 is configured to generate fluid pressure, fluid flow and/or
otherwise convey the input sample S1, and all of the reagents
through the various modules of the device 6000. As described below,
the fluid transfer module 6400 is configured to contact and/or
receive the sample flow therein. Thus, in some embodiments, the
device 6000 is specifically configured for a single-use to
eliminate the likelihood that contamination of the fluid transfer
module 6400 and/or the sample preparation module 6200 will become
contaminated from previous runs, thereby negatively impacting the
accuracy of the results.
[0235] As described herein, the fluid transfer module 6400 is
configured to aspirate and dispense at constant rates with
extremely high accuracy and precision in a small, lightweight,
simply constructed, and inexpensively manufactured format.
Moreover, the fluid transfer module 6400 is designed to be
discarded after a single use, and allows for all of the components
to be disposed of in ordinary waste streams throughout the world
without the need to disassemble and remove specific components for
special treatment after use. The basic design employs a series of
individual piston pumps, each with a plunger and barrel assembly,
driven by a common actuator composed of a frame, motor and lead
screw, to move fluids into different modules within the diagnostic
test cartridge. Each stroke, whether aspiration or dispense,
combined with targeted positioning of passive valve elements, such
as check valves of the flapper, umbrella or duckbill types, moves
fluid so that no actuator motions go unused.
[0236] By selectively venting specific fluid paths total control
over all fluid movement is achieved during power strokes. The
advantages offered by using multiple pistons include the ability to
address a wide range of fluid volumes, the use of a single stroke
length for conveying a wide range of fluid volumes, the use of a
single actuator to drive multiple pistons, the ability to provide
flow via multiple fluid paths simultaneously, a reduction in the
valves between fluid paths, the ability to produce complex
differential and tunable pressure gradients within a single fluid
circuit (e.g., by placing multiple pistons in fluid communication
with each circuit).
[0237] As illustrated in FIG. 14, the fluid transfer module 6400 is
disposed within the housing 6030 and is configured to manipulate
the sample and any of the reagents described herein to convey, mix
and otherwise transfer the fluids within the device 6000, as
described herein. Referring to FIG. 40, the fluid transfer module
6400 includes a housing 6405 that includes a first barrel portion
6410 and a second barrel portion 6440. The fluid transfer module
6400 also includes a single drive motor 6910 and lead screw 6480
that is configured to actuate both of the barrel portions. The
fluid transfer module 6400 also includes various fluidic conduits
(e.g., tubes, lines, valves, etc.) connecting the fluid transfer
module 6400 to the mixing module 6500, the amplification module
6600, the detection module 6800 and any other components within the
device 6000.
[0238] The housing 6405 serves as the overall frame for the fluid
transfer module 6400 to anchor all of the components therein to the
housing 6030. The design of the housing 6405 (or frame) is "U"
shaped, and includes the first barrel portion 6410 disposed spaced
apart from the second barrel portion 6440, with the drive motor
6910 located therebetween. The housing 6405 includes a mounting
portion 6406 in the center of the "U" shape that includes
accommodations for seating a bearing assembly (not show) and a set
of mounting holes for the mounting of the drive motor 6910. The
mounting portion 6406 also defines an opening that provides passage
for the lead screw 6480. The housing 6405 can be constructed from
material that offers flexibility and compliance while being able to
hold tight tolerances and maintain rigidity. Moreover, because the
housing 6405 defines at least one bore (e.g., the lumen 6441 within
which the sample is contained during transfer, the housing 6405 is
also constructed from a biocompatible material. For example, in
some embodiments, the housing 6405 can be constructed from
polycarbonate, cyclic olefin copolymer (COC), or certain grades of
polypropylene.
[0239] The first barrel portion 6410 (also referred to as the first
barrel assembly) includes a first end portion 6413 and a second end
portion 6414 and defines a lumen 6411 (or bore) therein. The bore
6411 has a defined length and an inner surface of defined diameter,
and thus can define a "swept volume" for control of the flow of
sample and/or reagents. The second end portion 6414 of the bore
6411 has a reduced diameter portion, and is in fluid communication
with the inlet port 6420 and the outlet port 6430. The opposite end
of the bore 6411 receives the sealing portion 6417 of the first
piston plunger 6415. When the first piston plunger 6415 is inserted
into the cylinder, an inner chamber of variable volume is formed
which follows the formula:
V(z)=.pi.r.sup.2z
where z is the linear distance traveled by the first piston plunger
6415 and r is the radius of the bore 6411.
[0240] The second end portion 6414 of the first barrel portion 6410
includes the inlet port 6420 and the outlet port 6430. The inlet
port 6420 includes a fitting 6422, a valve 6424, and O-rings or
seals. The inlet port 6420 is configured to receive fluid flow into
the bore 6411 (e.g., from the mixing module 6500), as shown by the
arrow LL in FIG. 40. The valve 6424 can be any suitable valve
(e.g., duckbill valve, check valve or the like) that allows the
inlet flow when the first piston plunger 6415 moves out of the bore
6411 (a negative pressure cycle, as shown in FIG. 40), but prevents
fluid flow out when the first piston plunger 6415 moves into the
bore 6411 (a positive pressure cycle). The outlet port 6430
includes a fitting 6432, a valve 6434, and O-rings or seals. The
outlet port 6430 is configured to convey fluid flow out of the bore
6411 (e.g., to the amplification module 6600), as shown by the
arrow MM in FIG. 40 (see also the arrow CC in FIG. 9, showing the
flow to the amplification module 6600). The valve 6434 can be any
suitable valve (e.g., duckbill valve, check valve or the like) that
prevents any inlet (or reverse) flow when the first piston plunger
6415 moves out of the bore 6411 (a negative pressure cycle, as
shown in FIG. 40), but allows fluid flow out when the first piston
plunger 6415 moves into the bore 6411 (a positive pressure
cycle).
[0241] Although the input port and the output port are shown as
being two separate ports, in other embodiments, the first barrel
assembly 6410 can be equipped with an integrated flow control
module. Whether as separate ports (as shown) or as an integrated
unit, the flow control (e.g., the inlet port 6420 and the outlet
port 6430) are configured direct and/or control the fluid flow
direction during a negative or positive pressure cycle. A secondary
function of the inlet port 6420 and the outlet port 6430 is to
limit dead volume by reducing captured air. As pressure is either
increased or decreased on one side of a valve element relative to
the opposite side, fluid is made to pass through the element or
stay on a specified side of the element. Depending on the
orientation of the valve element and its position in the inlet port
6420 or the outlet port 6430, it may either act as a stop flow
valve during a pressure stroke or a pass through orifice.
[0242] The first barrel portion 6410 includes the first piston
plunger 6415 that is movable disposed within the bore 6411. The
first piston plunger 6415 has a long cylindrical shape, and
includes a first end portion (or "head") 6416, a central portion
(or "shaft"), and the second end portion (or "sealing tip") 6417.
The basic body structure can be made from any formable material
with the appropriate rigidity such as plastics or metals. The first
end portion 6416 is coupled to the drive plate 6472 which in turn
is attached to and/or driven by the lead screw 6480. In some
embodiments, the first end portion 6416 has a larger diameter than
that of the shaft and second end portion 6417. The shaft is smaller
in diameter than the sealing tip and is smaller in dimension than
the inner diameter of the bore 6411, to allow unrestricted passage.
The fit of the shaft diameter to the piston barrel is an important
parameter for properly guiding the plunger assembly during
operation. The sealing tip 6417 is located opposite the head 6416,
and is responsible for smoothly traversing the slightly drafted
inner diameter of the bore 6411 while maintaining a seal capable of
withstanding both negative and positive pressure conditions through
its full stroke. The sealing tip 6417 includes an elastomeric
material and has one or more surface that contact the inner
diameter of the bore 6411 to form the seal. The shape of the
sealing tip 6417 is designed to mate with the inner surfaces of the
first barrel assembly 6410 to provide minimal dead volume at end of
stroke.
[0243] The second barrel portion 6440 (also referred to as the
second barrel assembly) includes a first end portion 6443 and a
second end portion 6444 and defines a lumen 6441 (or bore) therein.
The bore 6441 has a defined length and an inner surface of defined
diameter, and thus can define a "swept volume" for control of the
flow of air, sample and/or reagents. The second end portion 6444 of
the bore 6441 has a reduced diameter portion, and is in fluid
communication with the flow port 6450. The opposite end of the bore
6441 receives the sealing portion 6447 of the second piston plunger
6445. When the second piston plunger 6445 is inserted into the bore
6441, an inner chamber of variable volume is formed which follows
the formula:
V(z)=.pi.r.sup.2z
where z is the linear distance traveled by the second piston
plunger 6445 and r is the radius of the bore 6441.
[0244] The second end portion 6444 of the second barrel portion
6440 includes the flow port 6450. The flow port 6450 includes a
fitting 6452 and O-rings or seals, and is configured to receive
fluid flow into and convey fluid flow out of the bore 6441 (e.g.,
producing a vacuum within the detection module 6800). In some
embodiments, the flow port 6450 can include any suitable valve
(e.g., duckbill valve, check valve or the like) that controls the
inlet flow when the second piston plunger 6445 moves out of the
bore 6441 (a negative pressure cycle, as shown in FIG. 40), and the
outlet fluid flow out when the second piston plunger 6445 moves
into the bore 6441 (a positive pressure cycle).
[0245] Additionally, the inlet flow can be controlled by the vent
line 6355, which can be selectively placed into fluid communication
with the atmosphere via the rotary valve assembly 6340, as
described below. In particular, the vent 6355 can opened thereby
allowing any air or fluid within the bore 6441 to be exhausted to
atmosphere during a positive pressure cycle, rather than flowing
through the detection module 6800. The vent 6355 can be closed
during the negative pressure cycle to pull a vacuum through the
detection module 6800, as shown by the arrow DD in FIG. 9.
[0246] The second barrel portion 6440 includes the second piston
plunger 6445 that is movable disposed within the bore 6441. The
second piston plunger 6445 has a long cylindrical shape, and
includes a first end portion (or "head") 6446, a central portion
(or "shaft"), and the second end portion (or "sealing tip") 6447.
The basic body structure can be made from any formable material
with the appropriate rigidity such as plastics or metals. The first
end portion 6446 is coupled to the drive plate 6472 which in turn
is attached to and/or driven by the lead screw 6480. In some
embodiments, the first end portion 6446 has a larger diameter than
that of the shaft and second end portion 6447. The shaft is smaller
in diameter than the sealing tip and is smaller in dimension than
the inner diameter of the bore 6441, to allow unrestricted passage.
The fit of the shaft diameter to the piston barrel is an important
parameter for properly guiding the plunger assembly during
operation. The sealing tip 6447 is located opposite the head 6446,
and is responsible for smoothly traversing the slightly drafted
inner diameter of the bore 6441 while maintaining a seal capable of
withstanding both negative and positive pressure conditions through
its full stroke. The sealing tip 6447 includes an elastomeric
material and has one or more surface that contact the inner
diameter of the bore 6441 to form the seal. The shape of the
sealing tip 6447 is designed to mate with the inner surfaces of the
first barrel assembly 6440 to provide minimal dead volume at end of
stroke.
[0247] The drive plate 6472 links the lead screw 6480 to the first
piston plunger 6415 and the second piston plunger 6445. In some
embodiments the drive plate 6572 can include a threaded bore or
captive drive nut (not shown) in engagement with lead screw 6480.
In this manner, the threaded bore or captive drive nut can convert
the rotational motion of the lead screw 6480 into linear motion.
The drive plate 6472 and any threaded portion or drive nut therein
can be constructed from materials and/or machined to a tolerance in
a way that minimize friction and binding during its transit. In
some embodiments, a captive drive nut (not shown) can be configured
for some rotational (or non-axial) motion to overcome the tendency
to bind under asymmetric forces derived from uneven loading of the
two pistons during operation.
[0248] The lead screw 6480 delivers the required thrust to
translate the drive plate 6472 and displace the first piston
plunger 6415 and the second piston plunger 6445. The lead screw is
secured to, or part of, the motor 6910. In some embodiments, the
distal end of the lead screw 6480 can include a mating feature
concentric to the longitudinal axis of the screw and designed to
provide constraint in both the axially and radial directions. Such
a mating feature on the lead screw can cooperatively function with
a bearing assembly (not shown). A variety of materials can be used
to make the lead screw including plastics and plastics with filler
materials to alter the bearing properties as well as various
metals. The thread pitch is predetermined and sets the fluid flow
rates of the fluid transfer module 6400.
[0249] In some embodiments, the fluid transfer module 6400 conveys
fluid throughout the device 6000 according to a prescribed protocol
that includes multiple parts. When initiated, the first part of the
protocol (the "mixing method") signals the motor 6910 to move in a
first direction that causes the first barrel assembly 6410 to
develop negative pressure in the inlet port 6420 thereby drawing in
fluid from the inactivation chamber 6300, which is at atmospheric
pressure, and towards the mixing module 6500. The flow rate and/or
dwell time of the sample within the mixing module 6500 can be
controlled by changing the rotational speed of the motor 6910
and/or including periods of dwell during the movement of the motor
6910. In this manner, the desired fluid flow characteristics within
the mixing module 6500 can be established or maintained to ensure
the desired mixing. The mixing method includes continued movement
of the motor 6910 in the first direction thereby drawing in fluid
from the mixing module 6500, through the inlet port 6420, and into
the bore 6411 of the first barrel assembly 6410. Fluid conveyance
continues into the bore 6411 until it has been filled as
prescribed. Once filled, the control module 6950 signals the motor
6910 to reverse rotational direction, causing the development of
positive pressure within the bore 6411 (the "fluid delivery
method"). The positive pressure acts on the valve 6424, effectively
closing the inlet port 6420. As positive pressure builds, fluid
flows through the outlet port 6430, through tubing and then onward
to the amplification module 6600. This is shown schematically by
the arrow CC in FIG. 9. Continued movement of the motor 6910 in the
second direction pushes the sample through the amplification module
6600 at the desired flow rate, and into the detection module 6800.
During this first part of the protocol (i.e., the mixing method and
the fluid delivery method), the bore 6441 within the second barrel
assembly 6440 is maintained at atmospheric pressure via the vent
line 6355, which is controlled through a series of venting actions
initiated by the control module 6950 and conducted by the rotary
venting valve 6340.
[0250] The second part of the fluid transfer protocol (the
"detection method") is initiated by an advancement of the rotary
vent valve 6340, which effectively exchanges control over the fluid
movements from the first barrel assembly 6410 to the second barrel
assembly 6440. The "detection method" protocol reverses the motor
direction for a second time (i.e., the motor 6910 begins rotating
in the first direction). This causes the drive plate 6472 and thus,
the second piston plunger 6445 to retract causing a negative
pressure to develop within the chamber of the second barrel
assembly 6440 (because the vent line 6355 is closed). The pressure
drop created between the detection module 6800 and the bore 6441
results in a higher pressure on the inlet side of the detection
module 6800 then on the outlet side of the detection module 6800
creating a preferred direction of flow into the bore 6441 of the
second barrel assembly 6440. This is shown schematically by the
arrow DD in FIG. 9. After the sample has been conveyed from the
amplification module 6600 through the detection module 6800,
continued retraction of the second piston plunger 6445 can be used,
in conjunction with the valve assembly 6340, to sequentially flow
detection reagents through the detection module 6800. The operation
of the valve assembly 6340 is described below.
[0251] The motor 6910 can be any suitable variable direction motor
to provide power to the fluid transfer module 6400 with adequate
torque to drive the lead screw 6480. The pitch of the lead screw
6480 is also determined to provide the desired accuracy and flow
rates. There are a multitude of factors and parameters requiring
consideration and control to maintain a balanced load during
extension and compression cycles, and to maintain the desired
precision and accuracy. A balanced load around the drive train
presents a significant challenge for a multi-piston system with a
single drive motor (e.g., motor 6910) as there is a continually
varying load due to changing head pressure (positive or negative)
as fluid flows through elements of the circuitry. To achieve a
balance in load, compression in the sealing tips within piston
barrels must be controlled, along with the amount of surface area
in contact with the barrel due to the compression. Additionally,
the amount of draft or taper in the manufacturing of the piston
barrels must be considered, as small and large diameter seals
behave differently under increasing and decreasing compression
conditions. Failure to consider the issue of load balancing results
in a non-uniform fluid velocity profile which intern will result in
inefficiencies during amplification and inconsistencies during
detection.
[0252] Component sizing for flow rate control and chamber volume in
a dual-piston fluid transfer module system are demonstrated in
Tables 1-5 below. From the calculation in the tables,
specifications for a motor and the control requirements for the
motor 6910 are determined. For example, in some embodiments,
consider that the first barrel assembly 6410 (also referred to as
the "amplification piston" in Table 1 below) has a requirement to
deliver fluid at a rate of between 0.3 .mu.l/sec and 0.5 .mu.l/sec.
Given the nominal diameter of 4.65 mm for the first bore 6411, a
full stroke of 60 mm, and a lead screw pitch of 0.5 mm/rev, the
motor 6910 is required to operate with sufficient torque between
about 2.12 rpm and about 6.53 rpm. The second barrel assembly 6440
(also referred to as the "detection piston" in Table 1 below) has a
requirement to deliver fluid at a rate between 15 .mu.l/sec and 60
.mu.l/sec. Given the nominal diameter of 8.5 mm for the second bore
6441, a full stroke of 60 mm, and a lead screw pitch of 0.5 mm/rev,
the motor 6910 is required to operate with sufficient torque
between about 61.7 rpm and about 63.44 rpm. The total range of
speeds for a motor 6910 to meet the requirements specified is
therefore about 2 rpm to about 64 rpm, with sufficient torque to
overcome both the back pressure from the fluid and the drag due to
the piston seals.
TABLE-US-00001 TABLE 1 Amplification Piston 1 Detection Piston 2
0.3 flow rate (min) ul/sec 15 flow rate (min) ul/sec 0.5 flow rate
(max) ul/sec 30 flow rate (max) ul/sec 4.65 barrel diameter mm 8.5
barrel diameter mm 30 piston stroke mm 30 piston stroke mm 0.5 lead
screw pitch mm/rev 0.5 lead screw pitch mm/rev 60 Total Rev/Stroke
60 Total Rev/Stroke Vol/Stroke 509.47 ul Vol/Stroke 1702.35 ul
Vol/Rev 8.49 ul Vol/Rev 28.37 ul flow rate (min) 0.04 rev/sec flow
rate (min) 0.53 rev/sec flow rate (min) 2.12 rev/min flow rate
(min) 31.72 rev/min flow rate (max) 0.06 rev/sec flow rate (max)
1.06 rev/sec flow rate (max) 3.53 rev/min flow rate (max) 63.44
rev/min
[0253] The amount of torque that is sufficient to achieve the
required flow rates can be determined through an evaluation of data
tabulated during repeated measurements of the linear force needed
to compress and extend the first piston plunger 6415 and the second
piston plunger 6445, as indicated below in Tables 2-4.
TABLE-US-00002 TABLE 2 Piston 1 Piston 1 Compression (Individual)
Extension (Individual) Meas Crack Drive Meas Crack Drive # (lbF)
(lbF) # (lbF) (lbF) 1 0.09 0.04 1 0.19 0.06 2 0.14 0.05 2 0.1 0.07
3 0.19 0.02 3 0.07 0.08 4 0.07 0.02 4 0.1 0.07 5 0.08 0.03 5 0.13
0.05 6 0.09 0.04 6 0.09 0.04 Average 0.11 0.03 Average 0.11
0.06
TABLE-US-00003 TABLE 3 Piston 2 Piston 2 Compression (Individual)
Extension (Individual) Meas Crack Drive Meas Crack Drive # (lbF)
(lbF) # (lbF) (lbF) 1 0.34 0.11 1 0.21 0.14 2 0.4 0.09 2 0.15 0.13
3 0.48 0.04 3 0.19 0.15 4 0.33 0.05 4 0.19 0.04 5 0.32 0.16 5 0.23
0.11 6 0.34 0.11 6 0.17 0.14 Average 0.37 0.09 Average 0.19
0.12
TABLE-US-00004 TABLE 4 Dual Piston Compression Dual Piston
Extension Meas Crack Drive Meas Crack Drive # (lbF) (lbF) # (lbF)
(lbF) 1 0.54 0.38 1 0.46 0.12 2 0.48 0.44 2 0.24 0.14 3 0.27 0.46 3
0.28 0.19 4 0.32 0.49 4 0.2 0.22 5 0.35 0.51 5 0.3 0.1 6 0.39 0.37
6 0.31 0.14 Average 0.39 0.44 Average 0.30 0.15
[0254] The motor torque required to achieve a specific linear drive
force in the lead screw 6480 coupled system is a function of
parameters including lead screw pitch and lead screw efficiency.
Lead screw efficiency is itself a function of many factors
including, rotation speed and material selection for both the
threaded portion of the drive plate 6472 (and any drive nut
therein) and lead screw 6480. The required motor torque can be
represented using the formula:
.tau.(F)=F(p/2.pi..eta.)
In the formula, .tau.(F) is the torque as a function of the force,
F is the measured force, p is the lead screw pitch, .pi. is the
constant "pi", and .eta. is the lead screw efficiency. From the
tabulated data the maximum torque required to carry out the fluid
transfer function can be determined. Additionally, these calculated
values are used to specify the motor 6910 such that it can handle
the maximum required to load that will be experienced during both
compression and extension strokes of the fluid transfer module
6400. For example, the maximum torque experienced during fluid
transfer occurs during the compression stroke of the combined dual
piston and its value is 0.211 oz-in (shown in Table 5).
TABLE-US-00005 TABLE 5 Motor Torque Calculations 40% lead screw
efficiency .eta. 0.5 lead screw pitch (mm) p 3.14 pi .pi. Force
Torque Torque (lbF) Measurement Description (kg-cm) (oz-in) 0.02
Piston 1 min. drive force-compression 0.109 0.008 0.19 Piston 1
max. crack force-compression 1.032 0.074 0.04 Piston 1 min. drive
force-extension 0.217 0.016 0.19 Piston 1 max. crack
force-extension 1.032 0.074 0.04 Piston 2 min. drive
force-compression 0.217 0.016 0.48 Piston 2 max. crack
force-compression 2.608 0.188 0.04 Piston 2 min. drive
force-extension 0.217 0.016 0.23 Piston 2 max. crack
force-extension 1.250 0.090 0.54 Dual Piston max. force-compression
2.934 0.211 0.46 Dual Piston max. force-extension 2.499 0.180 max
motor torque for fluid transfer 2.934 0.211
[0255] Any suitable motor can be used to drive the fluid transfer
module to achieve the desired flow rates and power consumption
targets as described herein. For example, based on the maximum and
minimum flow rates for the assay, a lead screw 6480 pitch can be
selected and the maximum required torque is calculated. In
embodiments, the motor 6910 can be the Pololu item #1596 (Source:
https://www.pololu.com/category/60/micro-metal-gearmotors). This
motor 6910 can deliver the desired performance (14 RPM, 70 oz-in
stall torque, 986.41:1 gear ratio).
Amplification Module
[0256] As illustrated in FIGS. 9 (schematically) and 43-45, the
amplification module 6600 is configured to perform a PCR reaction
on an input of target DNA mixed with required reagents (from the
mixing module 6500, described above). In some embodiments, the
amplification module 6600 is configured to conduct rapid PCR
amplification of an input target. In some embodiments, the
amplification module 6600 is configured to generate an output copy
number that reaches or exceeds the threshold of the sensitivity of
the detection module 6800.
[0257] The amplification module 6600 includes a flow member 6610, a
substrate 6614 and a lid (or cover) 6615. As shown in FIG. 45, the
amplification module also includes a heater assembly 6630 and
electrical interconnects (not shown) to connect amplification
module 6600 to the surrounding modules. The components of the
amplification module 6600 can be coupled together by any suitable
manner, such as, for example, by clamps, screws, adhesive or the
like. In some embodiments, the flow member 6610 is fixedly coupled
to the heater assembly 6630. Said another way, in some embodiments,
the flow member 6610 is not designed to be removed and/or decoupled
from the heater assembly 6630 during normal use. For example, in
some embodiments, the heater assembly 6630 is coupled to the flow
member 6610 by a series of clamps, fasteners and potting material.
In other embodiments, the heater assembly 6630 is coupled to the
flow member 6610 by an adhesive bond. This arrangement facilitates
a single-use, disposable device 6000.
[0258] The flow member 6610 includes an inlet port 6611, and outlet
port 6612, and defines an amplification flow path (or channel)
6618. As shown, the amplification flow path has a curved,
switchback or serpentine pattern. More specifically, the flow
member (or chip) 6610 has two serpentine patterns molded into
it--the amplification pattern and the hot-start pattern 6621. The
amplification pattern allows for PCR to occur while the hot-start
pattern 6621 accommodates the hot-start conditions of the PCR
enzyme.
[0259] The serpentine arrangement provides a high flow length while
maintaining the overall size of the device within the desired
limits. Moreover, the serpentine shape allows the flow path 6618 to
intersect heater assembly 6630 at multiple locations. This
arrangement can produce distinct "heating zones" throughout the
flow path 6618, such that the amplification module 6600 can perform
a "flow through" PCR when the sample flows through multiple
different temperature regions. Specifically, as shown in FIG. 44,
the heater assembly 6630 is coupled to the flow member 6610 to
establish three temperature zones identified by the dashed lines: a
first temperature zone 6622, a second (or central) temperature zone
6623, and a third temperature zone 6624. In use, the first
temperature zone 6622 and the third temperature zone 6624 can be
maintained at a temperature of about 60 degrees Celsius (and/or at
a surface temperatures such that the fluid flowing therethrough
reaches a temperature of about 60 degrees Celsius). The second
temperature zone 6623 can be maintained at a temperature of about
90 degrees Celsius (and/or at a surface temperatures such that the
fluid flowing therethrough reaches a temperature of about 90
degrees Celsius).
[0260] As shown, the serpentine pattern establishes 40 different
zones of "cold to hot to cold;" or 40 amplification cycles. In
other embodiments, however, the flow member 6610 (or any of the
other flow members described herein) can define any suitable number
of switchbacks or amplification cycles to ensure the desired test
sensitivity. In some embodiments, the flow member can define at
least 30 cycles, at least 34 cycles, at least 36 cycles, at least
38 cycles, or at least 40 cycles.
[0261] The dimensions of the flow channel 6618 in the flow member
6610 determine the temperature conditions of the PCR and dictate
the overall dimensions of the chip, and thus impact the overall
power consumption. For example, a deeper, narrower channel will
develop a larger gradient in temperature from the side closest to
the lid 6615 to the bottom (resulting in lower PCR efficiency).
This arrangement, however, requires less overall space since the
channels will take up less overall surface area facing the heater
assembly 6630 (and thus require less energy to heat). The opposite
holds true for a wide and shallow channel. In some embodiments, the
depth of the flow channel 6618 is about 0.15 mm and the width of
the flow channel 6618 is between about 1.1 mm and about 1.3 mm.
More particularly, in some embodiments, the flow channel 6618 has a
width of about 1.1 mm in the "narrow" sections (that are within the
first temperature zone 6622 and the third temperature zone 6624)
and about 1.3 mm in the "wide" section (that falls within the
second temperature zone 6623). In some embodiments, the overall
path length is about 960 mm (including both the amplification
portion and the hot start portion 6621). In such embodiments, the
total path length of the amplification portion is about 900 mm.
This produces a total volume of the flow channel 6618 of about 160
.mu.l (including the hot start portion 6621) and about 150 .mu.l
(without the hot start portion 6621). In some embodiments, the
separation between each parallel path is between about 0.4 mm and
about 0.6 mm.
[0262] As fluid passes through the serpentine flow channel 6618, it
is inherently mixed due to the "u-turns" in the pattern. The liquid
near the outside of the channel 6618 walls takes a long path of
travel, while the liquid on the inside of the turn takes a shorter
path. As flow moves towards the straight sections of the channel
6618, the two areas of liquid that were not previously adjacent can
become mixed. This prevents localized depletion of reagents as well
as homogenizing the concentrations of target DNA. If left
completely unmanaged this effect can also cause a portion of the
liquid to have a reduced cold dwell time--the liquid on the shorter
path does not spend as much time in the cold zone.
[0263] Creating a cold zone dwell that allows even the inside path
to maintain the minimum cold dwell is one solution to this problem.
The other is to "pinch" the turn areas in an attempt to force all
of the liquid to have the same distance to travel thus forcing all
of the liquid to have the same cold dwell time.
[0264] The flow member 6610 can be constructed from any suitable
material, and can have any suitable thickness. For example, in some
embodiments, the flow member 6610 (and any of the flow members
described herein) can be molded from COC (Cyclic Olefin Copolymer)
plastic, which has inherent barrier properties and low chemical
interactivity. In other embodiments, the flow member 6610 (and any
of the flow members described herein) can be constructed from a
graphite-based material (for improved thermal properties). The
overall thickness of the flow member 6610 can be less than about
0.5 mm, less than about 0.4 mm, less than about 0.3 mm or less than
about 0.2 mm.
[0265] The flow member 6610 is lidded with a thin plastic lid 6615
and a substrate 6614, which are attached with a pressure sensitive
adhesive (not identified in the figure). The lid 6615 allows for
easy flow of thermal energy from the heater assembly 6630. In some
embodiments, the flow member 6610 also contains features to allow
other parts of the assembly (e.g., the heater assembly 6630) to
correctly align with the features on the flow member 6610, as well
as features to allow the fluidic connections to be bonded
correctly. The adhesive used to attach the lid 6615 is selected to
be "PCR-safe" and is formulated to not deplete the reagent or
target organism concentrations in the PCR reaction.
[0266] In some embodiments, the output volume from the
amplification module 6600 is sufficient to fully fill the detection
chamber in the detection module 6800.
[0267] The heater 6630 (and any of the heaters described herein)
can be of any suitable design. For example, in some embodiments,
the heater 6630 can be a resistance heater, a thermoelectric device
(e.g. a Peltier device), or the like. In some embodiments, the
heater assembly 6630 can include one or more linear "strip heaters"
arranged such that the flow path 6618 crosses the heaters at
multiple different points to define the temperature zones as
described above.
[0268] In some embodiments, the heater assembly 6630 can include
multiple different heater/sensor/heat spreader constructs (not
shown). The configuration and mating alignment of these determines
the areas of the temperature zones 6622, 6623 and 6624 on the flow
member 6610. The individual heater constructs (or strip heaters)
can be controlled to a pre-determined set point by the electronics
module 1950. In some embodiments, each construct can include a
resistive heater with an integrated sensor element which, when
connected to the electronics module 1950, allows for the
temperature of the attached heat spreader to be regulated to the
correct set point.
[0269] In some embodiments, the amplification module 6800 is
configured to consume minimal power, thus allowing the device 6000
to be battery-powered by the power source 6905 (e.g., by a 9V
battery). In some embodiments, for example, the power source 6905
is a battery having a nominal voltage of about 9 VDC and a capacity
of less than about 1200 mAh.
[0270] In use, fluid is conveyed into the amplification module 6600
by the fluid transfer module 6400 as described above. The
amplification is accomplished by the movement of the fluid through
the serpentine flow path 6618 held in contact with the heater
assembly 6630, during which the fluid inside the chip passes
through alternating temperature zones. The flow rate and the
temperature of the zones, as well as the layout of the
amplification flow path 6618, can determine the intensity and the
duration of the various temperature conditions as well as the total
number of PCR cycles. After the flow path 6618 fills with liquid,
any liquid emerging from the output side has undergone PCR (as long
as the total volume of the liquid collected from the output is
lower or equal to the "output" volume). The output of the module
flows directly into the detection module 6800. In some embodiments,
for example, the flow rate through the amplification path 6618 can
be about 0.35 .mu.l/second, and the temperature zones can
fluctuation the temperature between about 95 C and about 60 C. The
length and/or flow areas can be such that the sample is maintained
at about 95 C for about 1.5 seconds, and can be maintained at about
60 C for about 7 seconds. In other embodiments, the flow rate
through the amplification path 6618 can be at least 0.1
.mu.l/second. In yet other embodiments, the flow rate through the
amplification path 6618 can be at least 0.2 .mu.l/second.
Detection Module
[0271] As illustrated in FIGS. 9 (schematically) and 46-49, the
detection module 6800 is configured to receive output from the
amplification module 6600 and reagents from the reagent module 6700
to produce a colorimetric change to indicate presence or absence of
target organism in the initial input sample. The detection module
6800 also produces a colorimetric signal to indicate the general
correct operation of the test (positive control and negative
control). As described herein, the detection module 6800 is
configured for enzyme linked detection reaction resulting in a
colorimetric change in the detection chamber. Thus, the outputs
(e.g., OP1, OP2, OP3 shown in FIG. 66) are non-fluorescent signals.
This arrangement allows the device 6000 to be devoid of a light
source (e.g., lasers, light-emitting diodes or the like) and/or any
light detectors (photomultiplier tube, photodiodes, CCD devices, or
the like) to detect and/or amplify the output produced by the
detection module. In some embodiments, color change induced by the
reaction is easy to read and binary, with no requirement to
interpret shade or hue.
[0272] In some embodiments, the readout of the detection module
6800 is easy to read and remains so for sufficient time. For
example, in some embodiments, the output signals OP1, OP2, and/or
OP3 shown in FIG. 66 can remain present for at least about 30
minutes. Moreover, in some embodiments, the device 6000 (and any of
the other devices shown and described herein) can be configured to
produce the signals OP1, OP2, and/or OP3 in a time of less than
about 25 minutes from when the sample S1 is received. In other
embodiments, the device 6000 (and any of the other devices shown
and described herein) can be configured to produce the signal OP1,
OP2, and/or OP3 in a time of less than about 20 minutes from when
the sample S1 is input, less than about 18 minutes from when the
sample S1 is input, less than about 16 minutes from when the sample
S1 is input, less than about 14 minutes from when the sample S1 is
input, and all ranges therebetween.
[0273] The detection module 6600 includes a detection flow cell (or
"housing") 6810, a viewing window (or lid) 6802, a heater/sensor
assembly 6840, and fluidic and electrical interconnects (not
shown). The detection flow cell 6810 defines a detection
chamber/channel 6812 having a first inlet portion 6813, a second
inlet portion 6817, a detection portion 6820 and an outlet portion
6828. The first inlet portion 6813 includes a first inlet port
6814, a second inlet port 6815 and a third inlet port 6815. The
first inlet port 6814 is fluidically coupled to the outlet of the
amplification module 6600 and receives the amplified sample
(indicated by the arrow S7 in FIG. 47). The second inlet port 6815
is fluidically coupled to the reagent module 6700 and receives the
first reagent, which is a first wash (indicated by the arrow R3 in
FIG. 47). The third inlet port 6816 is fluidically coupled to the
reagent module 6700 and receives the second reagent, which can be,
for example, a horseradish peroxidase (HRP) enzyme with a
streptavidin linker (indicated by the arrow R4 in FIG. 47).
[0274] The second inlet portion 6817 includes a fourth inlet port
6818, and a fifth inlet port 6819. The fourth inlet port 6818 is
fluidically coupled to the reagent module 6700 and receives the
third reagent, which is a second wash (indicated by the arrow R5 in
FIG. 47). The fifth inlet port 6819 is fluidically coupled to the
reagent module 6700 and receives the fourth reagent, which can be,
for example, a substrate formulated to enhance, catalyze and/or
promote the production of the signal from the detection reagent R4
(indicated by the arrow R6 in FIG. 47). In some embodiments, for
example, the reagent R4 can be a tetramethylbenzidine (TMB)
substrate. The second inlet portion 6817 is separate from the first
inlet portion 6813 to ensure that any downstream area within the
path 6810 into which the substrate (reagent R6) might flow has been
thoroughly washed of enzyme (reagent R4). Similarly stated, the
second inlet portion 6817 is separate from the first inlet portion
6813 to minimize interaction between the substrate and the enzyme.
Undesired interaction could cause color change, and potentially
false positives results.
[0275] The detection portion (or "read lane") 6820 of the detection
channel 6812 is defined, at least in part by, and/or includes a
detection surface. Specifically, the detection portion 6820
includes a first detection surface (or spot) 6821, a second
detection surface (or spot) 6822, a third detection surface (or
spot) 6823, a fourth detection surface (or spot) 6824, and a fifth
detection surface (or spot) 6825. Each of the detection surfaces
are chemically modified to contain hybridization probes (i.e.,
single stranded nucleic acid sequences that capture complementary
strand of target nucleic acid.) to capture complementary strands of
the amplified nucleic acid. The first detection surface 6821
includes a hybridization probe specific to Neisseria gonorrhea
(NG). The second detection surface 6822 includes a hybridization
probe specific to Chlamydia trachomatis (CT). The third detection
surface 6823 includes a hybridization probe specific to Trichomonas
vaginalis (TV). The fourth detection surface 6824 includes a
hybridization probe for a positive control (A. fischeri, N.
subflava, or the like). The fifth detection surface 6825 includes a
non-target probe for a negative control.
[0276] The positive control surface 6824 includes any suitable
organism, such as, for example, Aliivibrio fischeri. This organism
is suitable because it is gram negative, nonpathogenic, bio safety
level 1, not harmful to the environment, and is extremely unlikely
to be found on a human. The positive control surface 6824 contains
capture probes for both the control organism (e.g., A. fischeri) as
well as each of the target organisms. This arrangement ensures that
the positive control surface 6824 always produces color if the
device functions correctly. If only the control organism were
present, a very strong positive for one of the target organisms
could "swamp out" or "outcompete" the amplification of the control
organism during PCR. Under such circumstances, the positive control
spot would not produce a color change which would be confusing for
the user. This arrangement facilitates the detection method and the
device 6000 being operated by a user with minimal (or no)
scientific training, in accordance with methods that require little
judgment.
[0277] The positive control portion of the assay is designed to be
sensitive to inhibition. More particularly, this is accomplished by
optimizing the number of control organisms added to the system
(e.g., via the lyophilized reagents or other suitable delivery
vehicle), as well as the concentration of the primers used to
amplify the control organism. In this manner, the control organism
should not be amplified if there is enough PCR inhibition to
prevent a target organism from amplification. If a weak positive
signal for one of the target organisms has been inhibited then the
system should register an "Invalid" run (due to no signal from the
positive control spot) rather than being read as a false negative.
The ordering of the capture probe surfaces ensures that a positive
control signal is valid because the target spots must first have
been exposed to the same reagents.
[0278] The negative control surface 6825 includes a non-target
probe and should always appear white (no color). The placement of
the negative control surface 6825 as the last spot is preferred
because this arrangement shows whether the reagent volumes, fluidic
movement, and wash steps were working properly.
[0279] The area of the spots on the detection surfaces (and thus
the width of the detection surfaces within the flow channel 6812)
is selected based on ease of manipulation as the area has little
effect on the visibility of the spot (to a certain lower limit at
which creating the spot becomes an issue). The volume of the liquid
above the spot (i.e., the depth of the flow channel 6812), however,
does affect the intensity of the color generated. A larger volume
(or depth) will generate a deeper color, while a lower volume will
generate a paler color. After the flow of sample and reagents,
diffusion can take place, and color from the spots can migrate into
areas outside the designated spot. The total amount of time
required for color from one spot to migrate and make the
neighboring spot appear positive affects the maximum read period of
the test. A larger volume flow cell, with more intense colors also
makes the migrating colors more intense. Since larger volume also
makes the amplification module take more time to complete its
process, a lower volume flow channel is preferable. In some
embodiments, the depth of the detection portion 6820 is between
about 0.135 mm and about 0.165 mm.
[0280] The lid (or "viewing window") 6802 allows the spot locations
within the flow channel 6812 be seen through the main housing 6010
of the device 6000. Specifically, as shown in FIG. 66, each of the
detection surfaces is aligned with and/or is viewable through the
corresponding detection openings defined by the top housing member
6010. The viewing window 6802 is a simple colored piece of plastic
providing contrast to the spot locations on the detection surfaces
and obscuring any non-spot location in the detection channel 6812.
The viewing window 6820 can have a simple molded plastic optic to
allow the viewer to see the spot from any angle, and to make the
result easier to read.
[0281] The flow cell 6810 can be constructed from any suitable
material. For example, in some embodiments, the flow cell 6810 can
be molded in COC plastic, then coupled to the lid 6802 to form flow
channel 6812. COC plastic is used for the construction of the
detection flow cell due to its barrier and chemical properties. The
barrier properties are necessary to maintain the chemistry stored
on the surface of the part over time. COC plastic is sufficiently
chemically active to accept the chemical modification necessary to
spot the detection zones, but not active enough to induce
non-specific binding of the reagents. In some embodiments, the
molded flow cell 6810 can include flash traps or other geometric
constructs to facilitate mounting of the lid 6802 to the flow cell
6810 (see e.g., FIG. 48). Moreover, the detection channel 6812 is
shaped to allow liquid to fill it evenly and without forming air
bubbles as liquid is introduced to the chamber.
[0282] The heater construct 6840 is a resistive heater with an
integrated sensor. The heater 6840 is attached to the detection
flow cell 6810 to allow for easy flow of thermal energy into the
fluid contained in the channel 6812. The heater 6840 is
electrically connected to the electronics module to allow it to
control to desired set temperatures.
[0283] In use, the post-amplification solution is flowed into the
detection flow cell 6810 from the amplification module 6600. After
the sample is in the flow cell 6810, DNA strands in the
post-amplification solution bind to complimentary pre-spotted zones
on the detection surfaces 6821, 6822, 6823, 6824, and 6825. The
pre-spotted zones are configured and/or formulated to bind only
their specific DNA targets, which are different for each zone based
on the target organism that the zone represents. Once sufficient
amount of time has passed, the amplicon solution is flushed from
the flow cell 6810 with a wash solution (reagent R3), and an enzyme
solution (reagent R4) is flowed into and maintained within the flow
channel 6812. During the dwell time, the enzyme binds to any DNA
strands still remaining in the flow cell (which are now attached to
specific detection surfaces 6821, 6822, 6823, 6824, and 6825).
After the enzyme binding has occurred the flow cell 6810 is flushed
with a second wash (reagent R5), and is then refilled with a
substrate solution (reagent R6). The enzymes (also attached to the
specific detection surfaces 6821, 6822, 6823, 6824, and 6825)
interact with the substrate, which causes the substrate to change
color. Because enzymes are bound locally to only some areas, the
color change is also localized to the specific detection surfaces
6821, 6822, 6823, 6824, and 6825. The viewing window 6802 and/or
the detection openings of the top housing 6010 limit the view of
the user to only show the specific detection surfaces 6821, 6822,
6823, 6824, and 6825 highlighting the results of the test. The
heater construct 6840 mediates the temperature in the flow cell to
allow for higher enzyme activity levels, and thus lower requisite
dwell times.
Rotary Valve
[0284] As described herein, the detection method includes
sequential delivery of the detection reagents (reagents R3-R6) and
other substances within the device 6000. Further, the device 6000
is configured to be an "off-the-shelf" product for use in a
point-of-care location (or other decentralized location), and is
thus configured for long-term storage. In some embodiments, the
molecular diagnostic test device 6000 is configured to be stored
for up to about 36 months, up to about 32 months, up to about 26
months, up to about 24 months, up to about 20 months, up to about
18 months, or any values there between. Accordingly, the reagent
storage module 6700 is configured for simple, non-empirical steps
for the user to remove the reagents from their long term storage
container, and for removing all the reagents from their storage
containers using a single user action. In some embodiments, the
reagent storage module 6700 is configured for allowing the reagents
to be used in the detection module, one at a time, without user
intervention.
[0285] The sequential addition of the detection reagents and/or
wash (including the amount each respective reagent and the timing
of addition of each reagent) is controlled automatically by the
rotary vent valve 6340. In this manner, the detection method and
the device 6000 can be operated by a user with minimal (or no)
scientific training, in accordance with methods that require little
judgment.
[0286] The rotary vent valve 6340 is shown in FIGS. 9
(schematically) and 50-53. FIGS. 54-61 show the rotary vent valve
6340 in each of eight different operational configurations. The
rotary vent valve 6340 includes a vent housing 6342, a valve body
(or disk) 6343, a drive member 6344, a retainer 6345 and a motor
6930. The vent housing 6342 defines a valve pocket 6358 within
which the valve disk 6343 is rotatably disposed. The vent housing
6342 includes a flow path portion 6360 that defines seven vent flow
paths. The flow path portion 6360 is shown with the end cover
removed so that each of the vent paths can be easily seen. A
description of each vent path follow: the vent path 6357 is
fluidically coupled to the atmosphere). The vent path 6356 is
fluidically coupled to the vent port 6556 of the mixing module
6500. The vent path 6355 is fluidically coupled to the outlet port
6828 of the detection module 6800 and/or the outlet port 6450 of
the fluid transfer module 6400. The vent path 6354 is associated
with the fourth reagent R6, and is fluidically coupled to the
reagent vent port 6734 of the reagent module 6700. The vent path
6353 is associated with the third reagent R5, and is fluidically
coupled to the reagent vent port 6733 of the reagent module 6700.
The vent path 6352 is associated with the second reagent R4, and is
fluidically coupled to the reagent vent port 6732 of the reagent
module 6700. The vent path 6351 is associated with the first
reagent R3, and is fluidically coupled to the reagent vent port
6731 of the reagent module 6700. As shown in FIG. 51, the vent
housing 6342 includes a flow path portion 6350 that includes
connection portions where each of the vent paths can be coupled to
the respective modules via tubing, interconnects and the like (not
shown).
[0287] As shown in FIG. 53, each of the vents ports described above
opens into the valve pocket 6358. Specifically, each of the vent
ports has an opening within the valve pocket 6358 that is spaced
apart from the center of the valve pocket 6358 by a specific radius
and also at a different angular position. Specifically, the vent
path 6357 (to atmosphere) is located at the center. In this manner,
when the valve body 6343 rotates around the center of the valve
pocket 6358 (as shown by the arrow NN), the slot channel 6370 of
the valve body 6343 can connect the central port, atmospheric vent
path 6357 to the other ports depending on their radial and angular
position. The use of multiple radii allows not only a single port,
but multiple ports at once to be vented depending on the
configuration.
[0288] The valve body 6343 includes the slot channel 6370 and a
series of seals 6372. The slot channel 6370 is tapered, and thus
has a wide angular tolerance, allowing the valve to be operated in
a low-precision regiment. The seals 6372 are aligned with the vent
path openings within the valve pocket 6358 to maintain the seal
when those vents are not selected. The valve body 6343 is pressed
into the valve pocket 6358 by the retainer 6346, and is coupled to
the drive motor 6930 by the drive member 6344, which includes a
series of lugs 6345.
[0289] The valve assembly 6340 can be moved between eight different
configurations, depending on the angular position of the valve body
6343 within the valve pocket 6358. FIG. 54 shows the assembly in
the first configuration (with the valve body 6343 in "position 0").
In the zeroth configuration, no vents are open, and the valve body
6343 rests against a hard stop. The zeroth configuration is used
for homing the valve only. FIG. 55 shows the assembly in the first
configuration (with the valve body 6343 in "position 1"). In the
first configuration, all vents are open, and thus, the reagent
actuator 6080 can be manually depressed allowing the reagent
canisters to be punctured, as described above. The first
configuration also allows for the dry reagents (e.g., the reagents
R1 and R2 within the mixing chamber 6500) to be properly
desiccated. The first configuration is the "shipping" and storage
configuration.
[0290] After the device 6000 is "powered-on" by actuation of the
switch 6906 when the reagent actuator 6080 is depressed, the power
and control module 6900 can incrementally move the valve body 6343.
FIG. 56 shows the assembly in the second configuration (with the
valve body 6343 in "position 2"). In the second configuration, the
vent 6355 (to outlet port 6828 of the detection module 6800 and/or
the outlet port 6450 of the fluid transfer module 6400) is open.
Further the vent 6356 to the mixing module 6500 is closed. Thus,
the inactivation chamber 6311 and the mixing module 6500 can be
emptied and filled as described above. The sample can also be
conveyed via the fluid transfer module 6400 into the PCR module
6600. FIG. 57 shows the assembly in the third configuration (with
the valve body 6343 in "position 3"). In the third configuration,
the vent 6351 (to the first reagent R3) is open. Thus, when the
fluid transfer module 6400 produces a vacuum through the detection
module 6800, the first reagent R3 (the wash) can move freely
through the detection module 6800 when the assembly is in the third
configuration. Because the other reagent vent ports are sealed, the
remaining reagents R4, R5 and R6 are not conveyed through the
detection module 6800 when the valve assembly 6340 is in the third
configuration.
[0291] FIG. 58 shows the assembly in the fourth configuration (with
the valve body 6343 in "position 4"). In the fourth configuration,
the vent 6352 (to the second reagent R4) is open. Thus, when the
fluid transfer module 6400 produces a vacuum through the detection
module 6800, the second reagent R4 (the enzyme) can move freely
through the detection module 6800 when the assembly is in the
fourth configuration. Because the other reagent vent ports are
sealed, the remaining reagents R3, R5 and R6 are not conveyed
through the detection module 6800 when the valve assembly 6340 is
in the fourth configuration.
[0292] FIG. 59 shows the assembly in the fifth configuration (with
the valve body 6343 in "position 5"). In the fifth configuration,
the vent 6352 (to the third reagent R5) is open. Thus, when the
fluid transfer module 6400 produces a vacuum through the detection
module 6800, the third reagent R5 (the second wash) can move freely
through the detection module 6800 when the assembly is in the fifth
configuration. Because the other reagent vent ports are sealed, the
remaining reagents R3, R4 and R6 are not conveyed through the
detection module 6800 when the valve assembly 6340 is in the fifth
configuration.
[0293] FIG. 60 shows the assembly in the sixth configuration (with
the valve body 6343 in "position 6"). In the sixth configuration,
the vent 6352 (to the fourth reagent R6) is open. Thus, when the
fluid transfer module 6400 produces a vacuum through the detection
module 6800, the fourth reagent R6 (the substrate) can move freely
through the detection module 6800 when the assembly is in the sixth
configuration. Because the other reagent vent ports are sealed, the
remaining reagents R3, R4 and R5 are not conveyed through the
detection module 6800 when the valve assembly 6340 is in the sixth
configuration.
[0294] FIG. 61 shows the assembly in the seventh configuration
(with the valve body 6343 in "position 7"). In the seventh
configuration, all vents are closed. This is the disposal
configuration.
[0295] By including a vent valve to control the reagent flow, the
number of moving parts is minimized, and thus the simplicity of the
device 6000 is improved. Moreover, this approach eliminates the
possibility of valve contamination because only air, and no fluid,
ever passes through the valve.
Power Management and Control
[0296] The system 6000 (or any other systems shown and described
herein) includes a control module 6900 that includes a power source
6905, a processor (which can be similar to the processor 4950 shown
and described above), and an electronic circuit system. The
electronic circuit system (not shown) can include any suitable
electronic components, such as, for example, printed circuit
boards, switches, resistors, capacitors, diodes, memory chips or
the like arranged in a manner to control the operation of the
device 6000, as described herein.
[0297] The power source 6905 can be any suitable power source that
provides power to the electronic circuit system (including the
processor) and any of the modules (e.g., heaters, motors, and the
like) within the device 6000. Specifically, the power source 6905
can provide power to the amplification module 6600 and/or the
heater 6630 to facilitate the completion of the PCR on the input
sample S1. In some embodiments, the power source 6905 can be one or
more DC batteries, such as, for example, multiple 1.5 VDC cells
(e.g., AAA or AA alkaline batteries). In other embodiments, the
power source 6905 can be a 9 VDC battery having a capacity of less
than about 1200 mAh. In some embodiments, the power source 6905 can
be an alkaline battery (e.g. a 9 VDC alkaline battery), which
exhibits a high energy density at a low cost. This arrangement
facilitates the device 6000 being a hand-held, disposable,
single-use diagnostic test. These energy sources are considered
depleted when terminal voltages drop below 5V, a common logic level
voltage. By regulating the digital controller signal directly from
the battery, a stable control voltage throughout the lifetime of
the battery is possible.
[0298] The primary consumer of power in the system 6000 will be the
resistive heaters shown and described above (e.g., for the
inactivation module 6300, the amplification module 6600, and the
detection module 6800). By specifying the resistance of the
inactivation heater and detection heater to be low enough such that
the required power density can be obtained from a nearly depleted
battery, these heaters can be powered from the unregulated battery
source.
[0299] The processor used to control the device 6000 (and any of
the processors shown herein) can be a commercially-available
processing device dedicated to performing one or more specific
tasks. For example, in some embodiments, the device 6000 can
include and be controlled by an 8-bit PIC microcontroller, which
will control the power delivered to various components of the
system. This microcontroller can also contain code for and/or be
configured to minimize the instantaneous power requirements on the
battery. The highest power consumption occurs when amplification
heater 6630, the inactivation heater 6330, and detection heater
6840 are being raised to temperature. By scheduling these warmup
times during periods of low power consumption, the power
requirements on the battery 6905 are reduced at the expense of
increased energy consumption. With the high energy density of the
alkaline battery, this is a favorable tradeoff. When multiple loads
require power simultaneously, the controller contain code for
and/or be configured to ensure that each load receives the
necessary average power while minimizing the time in which multiple
loads are powered simultaneously. This is achieved by interleaving
the PWM signals to each load such that the periods in which both
signals are in an on state is kept to a minimum.
[0300] For example, in some embodiments, the control and power
module 6900 can regulate the modules within the device 6000 to
perform within a power budget that is sufficient to allow the
device to be powered by the power source 6905 that is a 9 VDC
battery having a capacity of about 1200 mAh. FIG. 67 shows a graph
of a power budget as a function of elapsed time for the device 6000
running a test protocol according to an embodiment. As shown, the
line identified as 6990 indicates the power output of the power
source 6905 (i.e., the 9 VDC battery) in mW. The line identified as
6991 indicates a threshold of the minimum allowable voltage (in mV)
of the battery 6905. The line identified as 6992 indicates voltage
drawn (in mV) during three test runs. As shown, because the voltage
drawn does not drop below the minimum allowable voltage (line
6991), the tests were successfully completed using the 9 VDC
battery as the power source 6905.
[0301] In some embodiments, the total electric charge consumed by
one cycle of operation can be about 550 mAh. In such embodiments,
the device 6000 can include as the power source 6905 a 9 VDC
battery having a capacity of less than about 1200 mAh, which can
allow for a margin of safety of about 650 mAh. In particular, Table
6 lists the approximate charge consumption for each major operation
in the detection process.
TABLE-US-00006 TABLE 6 Module/Operation Approx Charge Consumed
(mAh) Sample prep 100 Amplification 300 Detection 50
Motors/microcontroller 100 TOTAL 550
[0302] Although the system 6000 is shown and described as including
a 9 volt alkaline battery 6905, in other embodiments, the device
6000 can include multiple power sources and/or energy storage
devices. For example the power and control module 6900 can include
supercapacitors in parallel with the battery 6905 to deliver
additional power. In such embodiments, the capacitor will be
charging continuously during periods of low power consumption and
will assist the battery 6905 in delivering power throughout the
run. Increasing this capacitance increases the stored energy and,
hence, the time during which the system can operate at elevated
power levels. Supercapacitors require large inrush currents, so
this capacitor will be current limited to prevent the battery
voltage from dropping below the required logic level voltage while
the capacitor is charging, resulting in a reset of the
microcontroller.
[0303] As described above, the system 6000 requires control of
brushed DC motors 6910 and 6930, which is can be accomplished, in
some embodiments, using rotary encoders (not shown). In other
embodiments, the processor can include code to and/or be configured
to implement a closed loop method of tracking motor position by
monitoring the current draw of motors 6910 and 6930. More
particularly, due to the reactive nature of motor coils, the
current draw by a brushed DC motor is not constant. By monitoring
current through a low impedance shunt resistor, the processor can
detect a DC component superimposed with an AC component. The DC
component represents the power required to actuate the motor under
its current load and the AC component is due to the self-inductance
of each motor coil, the mutual inductance between motor coils, and
the changing resistance of the rotor windings as the brushes move
across the armature windings during rotation. This changing
resistance is the primary contributor to this alternating current
and is directly related to the angular position of the motor.
[0304] In some embodiments, the electronic circuit system and/or
processor can determine and isolate this small AC component, filter
this component, and then amplify it to a logic level signal. The
processor can include a motor control module that keeps track of
the time between each pulse. These time values can be filtered
(e.g., using a single pole IIR digital filter) and then used as an
input for a PID controller within the motor control module. The PID
controller controls the input power to the motor, regulating the
power such that the time between motor pulses maintains a
predetermined value based on the desired flowrate. By counting the
number of pulses coming from this feedback circuit, a brushed DC
motor can aspirate or dispense a known volume from the drive
syringe or move the rotary valve to known positions.
[0305] As described herein, the device 6000 (and any of the other
devices shown and described herein) can be configured to produce
the signals OP1, OP2, and/or OP3 in a time of less than about 25
minutes from when the sample S1 is received. In other embodiments,
the device 6000 (and any of the other devices shown and described
herein) can be configured to produce the signal OP1, OP2, and/or
OP3 in a time of less than about 20 minutes from when the sample S1
is input, less than about 18 minutes from when the sample S1 is
input, less than about 16 minutes from when the sample S1 is input,
less than about 14 minutes from when the sample S1 is input, and
all ranges therebetween.
[0306] More particularly, the device 6000, the control module 6900
and the other modules within the device 6000 are collectively
configured to produce sample flow rates and overall sample volumes
in amounts and in a manner to achieve the power consumption and
delivery time specifications set forth herein. In this manner, the
device 6000 can be operated in a sufficiently simple manner, and
can produce results with sufficient accuracy to pose a limited
likelihood of misuse and/or to pose a limited risk of harm if used
improperly. For example, in some embodiments, the device 6000 is
configured to produce the volumes at each operation as set forth in
Table 6 below. The nominal time for each operation is also included
in Table 7.
TABLE-US-00007 TABLE 7 Re- Initial sulting vol. vol. Time Step Task
(ml) (ml) Yield (min) 1 Add sample 0.5-1.0 -- 2 Wash filter 0.5 --
0.1 3 Back-flush/elution 0.2 0.2 0.5 0.1 4 Inactivation (37 C.)
0.15 0.15 1 5 Inactivation (95 C.) 0.15 0.15 0.2 5 6 Master mix and
sample 0.085 0.085 0.16 prep eluent 7 40 cycle PCR 0.085 0.085
10.sup.10 8 8 Ready for Detection 9 Flow amplicon 0.075 0.075 n/a
0.5 10 Amplicon hybridization 0.075 0.075 .sup. 10.sup.-3 3 11
First wash 0.5 0.5 n/a 0.5 12 Flow enzyme 0.1 0.1 80 2 and incubate
13 Second wash 0.5 0.5 n/a 0.5 14 Flow substrate 0.1 0.1 1.2
.times. 10.sup.5 3 and incubate 15 Ready to read
Methods of Use
[0307] FIGS. 68A-C illustrate a detailed process flow chart of a
method 6000' for a diagnostic test according to an embodiment, such
as one executed/run by the diagnostic test device 6000 (or any
other system described herein). At step 6010', the method 6000'
includes dispensing a sample into an input port of the test system.
The input port is capped and the sample is pushed through a filter,
followed by a wash buffer, at step 6020'. In some embodiments, this
button as a last action opens a valve to allow elution of the
sample from the filter at step 6030'. At step 6040', an elution
lysing buffer is pushed through the filter to backflush the
contents off the filter, filling an inactivation chamber. In some
embodiments, the method 6000' further includes, at the step 6040',
opening a series of reagent tanks for use later in the method. At
step 6050', the method includes powering on/activating electronics
and heaters contained within the test system, such as, for example,
by an operator attaching a battery pack to the test system. In some
embodiments, the power on operation can be performed automatically
and/or in conjunction with a reagent opening step (e.g., operation
6040'). Alternatively, if the battery is stored within the
cartridge/system, in some embodiments, the operator can push a
power button to start electronics and heaters contained in the test
system. In some embodiments, a light indicator on the test system
lights to notify the operator that the test is operating.
[0308] At step 6060A', once the test is powered on, the
inactivation and/or lysis heater is powered and allowed to rise to
its set-point temperature. This heater is controlled by a digital
circuit (e.g., similar to the electronic control module 6900
described above) to ensure that the set-point temperature or
temperatures is held within tolerance, at step 6070'. Substantially
simultaneously, at step 6060B', control electronics continually
monitor the test system to ensure that a fault condition has not
occurred. A fault condition might include, for example, an
out-of-temperature condition, out-of-voltage condition,
out-of-pressure condition, etc. If a fault condition is detected at
step 6080', in some embodiments, an indicator light changes state
to notify the operator, and the method proceeds to step 6300'
(described later). In some embodiments, the presence of a fault
condition will render the device inoperable (e.g., will cause the
cartridge/system to cease operations), thereby minimizing the risk
that a user will receive an inaccurate result.
[0309] Once the inactivation chamber temperature set-point(s) have
been achieved, the elution lysing volume, which has now undergone
cell lysis, is incubated to inactivate the PK enzyme/lysing reagent
at step 6090'. This incubation time, in some embodiments, can be on
the order of about 5 minutes. Once this incubation has been
completed, the inactivation heater is turned off at 6100', and at
6110', a syringe pump is activated to aspirate the eluent ready for
dispense to a mixing chamber. At step 6120', the rotary valve is
actuated to vent the PCR fluidic circuit and detection flow
channel. In this manner, the fluidic pathways are prepared to allow
transfer of the desired fluids therethrough, as described below. At
step 6130A', the syringe pump reverses direction to dispense the
eluent into a mixing chamber where it hydrates a lyophilized
pellet/bead holding the primers and enzymes necessary for PCR. In
some embodiments, this hydration occurs over about 2 minutes to
allow complete mixing of reagents. In some embodiments, the mixing
operation can occur before and/or upstream of the syringe pump.
[0310] At step 6130B', PCR heaters are turned on and raised to
their set-point temperatures. In some embodiments, the PCR heaters
can be activated at substantially the same time as when the syringe
pump dispenses the eluent into the mixing chamber. At step 6140',
control electronics ensure that the PCR heaters are controlled to
within their set-point tolerances. At steps 6150A' and 6160', the
detection heater is turned on and allowed to warm up for subsequent
use. Substantially simultaneously, at step 6150B', the syringe pump
continues to push fluid from the mixing chamber to the PCR fluidic
circuit, where the mixed lysed sample and polymerase are thermally
cycled between about 59 degree C. and about 95 degree C. for 40
cycles. When the desired amplified volume is produced, the heaters
are shut off to conserve power at step 6170. The syringe pump
continues to push fluid from the PCR module to the detection
module. At step 6180', the amplicon is incubated in a flow channel
for about 6-5 minutes to perform amplicon hybridization. The flow
channel is heated to about 65 degree C. for this incubation step.
At step 6190', the detection heater is turned off.
[0311] The rotary valve of the test system is actuated to the first
wash position at step 6200'. The rotary valve can be any suitable
rotary valve, such as those described herein. The syringe pump
reverses direction and a vacuum is pulled on the wash reagent, and
at step 6210', the unbound amplicon is washed from the channel
followed by a volume of air. At step 6220', the rotary valve is
actuated to the HRP enzyme position. At step 6230', HRP enzyme is
conveyed within the flow channel and, at step 6240, is incubated
for 6-5 minutes. The enzyme is removed followed by an air slug. At
step 6250', the rotary valve is actuated to the wash 2 position. At
step 6260', wash buffer is pulled over the flow channel to wash
away unbound enzyme followed by a volume of air. At step 6270', the
rotary valve is actuated to the substrate position. At step 6280',
substrate is pulled into the flow channel and parked. At step
6290', the rotary valve is actuated to the "all ports closed"
position. In some embodiments, a light indicator is illuminated to
notify the operator the test results are ready. At step 6300', all
heaters and motors are stopped and/or shut down.
[0312] At step 6310', it is determined if an error was detected
such as, for example, if a fault occurs at step 6080'. If a fault
is detected, the appropriate error code is indicated on error LEDs
of the test system at 6320'. If no error is detected, then at step
6330, when the read frame expires after approximately 20 minutes,
the "test ready" light indicator is shut off to indicate the read
frame has elapsed and testing is complete at step 6340'.
[0313] The operations described above can be performed by the
diagnostic test system 6000 (or any other system described herein).
In some embodiments, the test system (or unit) can include a series
of modules configured to interact with the other modules to
manipulate the sample to produce a diagnostic test.
[0314] FIG. 69 is a flowchart of a method 10 of molecular
diagnostic testing, according to an embodiment. The method 10 can
be conducted on the device 6000 or any other device and/or system
shown and described herein. The method includes conveying a sample
into a sample preparation module disposed within a housing of a
diagnostic device, at 12. The sample can be any sample as described
herein, and can be conveyed into the device using any method as
described herein (e.g., using a transfer device such as the device
6100). The method then includes actuating the device, at 14, to: A)
extract, within the sample preparation module, a target molecule
(at 15); B) flow a solution containing the target molecule within
an amplification flow path defined by an amplification module such
that the solution is thermally cycled by a heater coupled to the
amplification module (at 16); C) convey the solution from an outlet
of the amplification module into a detection channel of a detection
module, the detection module including a detection surface within
the detection channel, the detection surface configured to retain
the target molecule (at 17); and D) convey a reagent into the
detection channel such that when the reagent reacts with a signal
molecule associated with a target amplicon a visible optical signal
associated with the detection surface is produced (at 18). The
method includes viewing the detection surface via a detection
opening of the housing, at 19.
Applications
[0315] The diagnostic test/test system 6000 (and all other devices
and systems described herein) is a platform for detection of
infectious disease from biological fluids. In some embodiments, the
diagnostic system detects targeted infectious agents (e.g.,
bacteria and viruses) by changing the types of primers inside the
consumable platform to amplify and detect the desired nucleic acid
sequence of interest. While the diagnostic system 6000 has been
designed for sample collection of either urine or swab sample and
detection of a 4-plex STI panel (i.e., a 3-plex plus a positive
control), in other embodiments, the diagnostic system 6000 (or any
of the other devices shown and described herein) can easily be
extended to other diagnostic panels. For example, consider a
urinary tract infection panel which allows detection of E. coli,
Staphylococcus saprophyticus, Enterococcus faecalis, Klebsiella
pneumoniae, Proteus, and P. aeruginosa. The sample prep module has
been shown to isolate the desired pathogen and lyse these organisms
with the addition of reagents (e.g., lysozyme and proteinase K) and
heat. Subsequently, pathogen specific primers would need to be
added to the mixing chamber to allow amplification of these target
pathogen gene sequences. Finally, the hybridizing probe bound to
the read lane in the detection module would need to change to bind
these new specific amplified targets. All other aspects of the test
cartridge can remain unchanged.
[0316] In some embodiments, a device (such as the device 6000, or
any of the other devices shown and described herein) can be
configured to detect a universal reagent immunoabsorbent assay
(URI). In some embodiments, a device (such as the device 6000, or
any of the other devices shown and described herein) can be
configured to detect a hemagglutination inhibition test (HAI).
[0317] For viral targets, the sample preparation module 6200 (and
any of the sample preparation modules described herein) can be
modified in any suitable manner. For example, in some embodiments,
a sample preparation module can be configured to isolate viruses
from biological fluids using a solid phase material such as a
filter of specific chemisorbent material with a pore size conducive
to flow and capture of viral particles. The captured viral particle
are washed and eluted from the filter into a heated chamber where
the viral particles are lysed and any PCR inhibitors are
neutralized. Pathogen specific primers and master mix are added to
the viral nucleic acid for amplification. For viral RNA targets,
reverse transcription takes place in the heating chamber prior to
PCR). After PCR amplification, the amplicons are captured by
sequence specific hybridizing probes in the read lane for
detection.
[0318] Although the molecular diagnostic system 6000 is shown and
described above as including certain modules disposed within a
housing in a particular arrangement, in other embodiments, a device
need not include all of the modules identified in the device 6000.
Moreover, in some embodiments, the functions described as being
performed by two modules can be performed by a single device and/or
structure. For example, in some embodiments a device need not
include a separate mixing module, but instead can perform the
mixing operation described above with respect to the mixing module
6500 within another module (such as the inactivation module or the
fluid transfer module). Moreover, in other embodiments, a device
can include the modules disposed within a housing in any suitable
arrangement. For example, FIGS. 70-72 show perspective views of a
molecular diagnostic test device 7000 according to an embodiment.
The diagnostic test device 7000 includes a housing (including a top
portion 7010 and a bottom portion 7030), within which a variety of
modules are contained. Specifically, the device 7000 includes a
sample preparation module 7200, an inactivation module 7300, a
fluidic drive (or fluid transfer) module 7400, a mixing chamber
7500, an amplification module 7600, a detection module 7800, a
reagent storage module 7700, a rotary venting valve 7340, and a
power and control module 7900. The device 7000 can be similar to
the device 6000, and thus the internal components and functionality
is not described in detail herein.
[0319] FIG. 71 shows the device 7000 with the top housing 7010
removed so that the placement of the modules can be seen. FIG. 72
shows the device 7000 with the top housing 7010, the actuation
buttons, the amplification module 7600, and the detection module
7800 removed so that underlying modules can be seen. As shown, the
device 7000 is includes a top housing 7010 and a lower housing
7030. The top housing 7010 defines a detection (or "status")
opening 7011 that allows the user to visually inspect the output
signal(s) produced by the device 7000. When the top housing 7010 is
coupled to the lower housing 7030, the detection opening 7011 is
aligned with the corresponding detection surfaces of the detection
module 7800 such that the signal produced by and/or on each
detection surface is visible through the corresponding detection
opening.
[0320] In some embodiments, the top housing 7010 and/or the portion
of the top housing 7010 surrounding the detection opening 7011 is
opaque (or semi-opaque), thereby "framing" or accentuating the
detection openings. In some embodiments, for example, the top
housing 7010 can include markings (e.g., thick lines, colors or the
like) to highlight the detection openings. For example, in some
embodiments, the top housing 7010 can include indicia identifying
the detection opening to a particular disease (e.g., Chlamydia
trachomatis (CT), Neisseria gonorrhea (NG) and Trichomonas
vaginalis (TV)) or control.
[0321] The lower housing 7030 defines a volume within which the
modules and or components of the device 7000 are disposed. For
example, the sample preparation portion receives at least a portion
of the sample input module 7170. The sample input module 7170 is
actuated by the sample actuator (or button) 7050. The housing
defines a notch or opening 7033 that receives a lock tab 7057 of
the sample actuator 7050 after the actuator 7050 has been moved to
begin the sample preparation operation. In this manner, the sample
actuator 7050 is configured to prevent the user from reusing the
device after an initial use has been attempted and/or
completed.
[0322] The wash portion of the housing receives at least a portion
of the wash module 7210. The wash module 7210 is actuated by the
wash actuator (or button) 7060. The housing defines a notch or
opening 7035 that receives a lock tab 7067 of the wash actuator
7060 after the actuator 7060 has been moved to begin the wash
operation. In this manner, the wash actuator 7060 is configured to
prevent the user from reusing the device after an initial use has
been attempted and/or completed.
[0323] The elution portion of the housing receives at least a
portion of the elution module 7260. The elution module 7260 is
actuated by the elution actuator (or button) 7070. The housing
defines a notch or opening 7037 that receives a lock tab 7077 of
the elution actuator 7070 after the actuator 7070 has been moved to
begin the wash operation. In this manner, the elution actuator 7070
is configured to prevent the user from reusing the device after an
initial use has been attempted and/or completed.
[0324] The reagent portion of the housing receives at least a
portion of the reagent module 7700. The housing defines a notch or
opening 7039 that receives a lock tab 7087 of the reagent actuator
7080 after the actuator 7080 has been moved to begin the reagent
opening operation. In this manner, the reagent actuator 7080 is
configured to prevent the user from reusing the device after an
initial use has been attempted and/or completed. By including such
lock-out mechanisms, the device 7000 is specifically configured for
a single-use operation, and poses a limited risk of misuse.
[0325] As shown in FIGS. 73 and 74, the reagent module 7700 can
include a holding tank 7740 that defines a series of bores 7741
within which the reagent canisters are stored, and also a series of
holding reservoirs 7761 to which the reagents flow upon actuations.
The reagent module includes a top member 7735 that includes a
series of vent ports that function similar to the vent ports
described above with respect to the reagent module 6700.
[0326] FIGS. 75-82 illustrate an embodiment of an apparatus 8000
for diagnostic testing that can be structurally and/or functionally
similar to the apparatus 6000 and/or the apparatus 7000. As best
illustrated in FIG. 75, the apparatus 8000 includes a housing 8010,
a sample input port 8020 (including a cap), three plungers
8030/4040/4050, a pull tab 8060, status indicators/lights 8070, a
read lane (and/or detection openings) 8080, a battery housing 8090,
and a label 8110.
[0327] As illustrated in FIG. 76, in some embodiments, the overall
dimensions of the apparatus 8000 in a front view can be about 101
mm (dimension A').times.about 73 mm (dimension B'), or any suitably
scaled value. One dimension of the plungers 8030, 8040, 8050, and
the tab 8060 can be about 22 mm (dimension C'), or any suitable
scaled value relative to the rest of the apparatus 8000. As best
illustrated in FIG. 77, in some embodiments, the dimensions of the
apparatus 8000 in a side view can be about 82 mm (dimension
D').times.about 26 mm (dimension E'), or any suitably scaled value.
In some embodiments, the housing 8010 includes a clear top surface
for ease of viewing by the user. In some embodiments (not shown),
the housing 8010 can include a prepare module and a read module.
The prepare module (not shown) is configured to intuitively guide
the user in preparing a sample for analysis/testing, while the read
module (not shown) is configured to intuitively guide the user in
reading out the test results.
[0328] In some embodiments, as illustrated, the input port 8020,
the plungers 8030/4040/4050, and the pull tab 8060 have indicators
"1", "2", etc., to guide a user in the correct sequence of steps
for use of the apparatus 8000. In some embodiments, during use, the
sample input port 8020 is configured to receive a sample, such as a
patient sample (see FIG. 78). In some embodiments, the cap is
tethered to the port and/or any other part of the apparatus 8000 to
prevent it from being misplaced. In some embodiments, the port 8020
is configured for use with standard pipettes. In some embodiments,
the port 8020 can hold up to about 700 .mu.L of sample. In some
embodiments, the port and cap structure can withstand up to 50 psi
of pressure. In some embodiments (not shown), the port 8020
includes one or more visual indicators (e.g., LEDs) to verify the
correct volume has been dispensed.
[0329] In some embodiments, as best illustrated in FIG. 79, the
plunger 8030 is configured to push the sample in the port 8020
through a filter, similar to the operation of the sample
preparation module 6200 described earlier. The plunger 8030 is also
configured to convey a volume of air, followed by a wash buffer,
through the filter. In some embodiments, the plunger 8030 locks
into place once the user depresses it substantially completely. In
some embodiments, the locking of the plunger 8030 is
irreversible.
[0330] In some embodiments, the plunger 8040 is configured to flush
the filter with eluent, similar to the operation of the sample
preparation module 1200 described earlier. The plunger 8040 is also
configured to push eluent into the inactivation chamber. In some
embodiments, the plunger 8040 locks into place once the user
depresses it substantially completely. In some embodiments, the
locking of the plunger 8040 is irreversible.
[0331] In some embodiments, the plunger 8050 "bursts" the reagent
tank, or releases reagents from the reagent tank, similar to the
operation of the reagent module 6700 described earlier. In some
embodiments, the plunger 8050 locks into place once the user
depresses it substantially completely. In some embodiments, the
locking of the plunger 8060 is irreversible.
[0332] In some embodiments, as best illustrated in FIG. 80, the tab
8060 is configured such that, when pulled by the user, an internal
electrical circuit is completed, which begins one or more
diagnostic tests on the sample, such as by, for example, initiating
operation of the amplification module (which can be similar to the
amplification module 6600. In some embodiments, the tab 8060 is
detachable and disposable, such that a user can dispose the tab
8060 after removal from the apparatus 8000.
[0333] In some embodiments, the input port 8020, the plungers
8030/4040/4050, and the pull tab 8060 are configured for
irreversible operation. Said another way, each of these elements is
configured to "lock" and/or disable reversal once properly deployed
by the user. In this manner, a user is prevented from improperly
using the device. In some embodiments, the input port 8020, the
plungers 8030/4040/4050, and the pull tab 8060 include one or more
lock out mechanisms to prevent the user from completing steps/using
the apparatus 8000 out of order.
[0334] In some embodiments, the status lights 8070 are visual
indicators, such as LED lights, that are configured for providing
feedback to the user on one or more states of the apparatus 8000
including, but not limited to, when the tab 8060 is removed, when
the diagnostic test is processing (after the tab 8060 is pulled),
when the diagnostic test is ready for the user to review, when an
error state is present, and/or the like. For example, in some
embodiments, some variation in number of lit LEDs, the pattern of
lighting of LEDs, the duration of lighting of LEDs, and/or the
color of the lit LEDs, can be employed to represent each state of
the apparatus 8000.
[0335] In some embodiments, the read lane and/or detection opening
8080 is configured to permit interpretation of the test results by
the user. In some embodiments, the read lane 8080 includes a
substrate that produces a color indicator, in accordance with the
methods described herein (e.g., the enzymatic reaction described
above with reference to FIG. 8). In other embodiments, the read
lane 8080 includes color strip or absorbent paper configured to
produce a colorimetric output associated with a target. In some
embodiments, the housing 8010 partially masks the read lane 8080.
In this manner, housing 8010 can be labeled for the convenience of
the user. In some embodiments, as seen in FIG. 75, the read lane
8080 can include one or more dots or "spots." In some embodiments,
some dots are configured to indicate test results, while some dots
are configured to indicate control results. FIG. 75 illustrates an
example scenario with three dots as a test panel and two dots as a
control panel for user analysis.
[0336] As best illustrated in FIGS. 75 and 81, the battery housing
8090 is configured to hold a battery source, such as, for example,
a 9V battery, for powering the apparatus 8000. A button 8100 is
configured to permit a user to removably detach an attached
battery, such as, for example, for replacement and/or disposal. As
best illustrated in FIG. 82, in some embodiments, the apparatus
8000 can be configured for use with a rechargeable battery unit
8120. In this manner, instead of disposing the entire apparatus
8000 after use, the user retains the battery unit 8120 for
recharging and reuse with a new cartridge (i.e., where a
"cartridge," for purposes of this example embodiment, is the
apparatus 8000 without the battery unit 8120).
[0337] In other embodiments, the power source in any of the devices
shown and described herein can be any suitable energy
storage/conversion member, such as a capacitor, a magnetic storage
systems, a fuel cell or the like. In yet other embodiments, any of
the devices shown and described herein, including the device 6000,
can be configured to operate on AC power. Thus, in some
embodiments, a device can include a plug configured to be disposed
within an AC outlet. In such embodiments, the power and control
module (e.g., the module 6900) can include the necessary voltage
and/or power converters to supply the appropriate power to each of
the modules therein. In some embodiments, the AC plug can also
serve as a mechanism to ensure that the device is properly oriented
(e.g., in a level and flat orientation) during use.
[0338] Although the device 6000 is shown as including a separate
fluid transfer device 6110, in other embodiments, a device can
include a sample transfer device that engages with and/or is
removably coupled to the overall housing. For example, FIGS. 83-87
show a molecular diagnostic test device 9000 according to an
embodiment. The diagnostic test device 9000 is contained with a
housing 9010, and includes a variety of modules. Specifically, the
device 9000 includes a sample preparation module (similar to the
sample preparation module 6200), an inactivation module (similar to
the inactivation module 6300), a fluidic drive (or fluid transfer)
module (similar to the fluid transfer module 6400), a mixing
chamber (similar to the mixing module 6500), an amplification
module (similar to the amplification module 6600), a detection
module (similar to the detection module 6800), a reagent storage
module (similar to the reagent module 6700), a valve module
(similar to the valve module 6340), and a power and control module
(similar to the power and control module 6900). The device 9000 can
be similar to the device 6000, and thus the internal components and
functionality is not described in detail herein. The device 9000
differs from the device 6000, however, in that the device 9000
includes an interlocking transfer member 9110, as described
below.
[0339] FIG. 83 shows a top view of the device 9000, and illustrates
the housing 9010 and the sample transfer device 9110 coupled to
and/or disposed within the housing 9010. The housing 9010 defines a
detection (or "status") opening 9011 that allows the user to
visually inspect the output signal(s) produced by the device 9000.
The opening 9011 is aligned with and allows viewing of five
detection surfaces of the detection module contained therein. In
particular the opening 9011 allows viewing of a signal produced by
a first detection surface 9821, a second detection surface 9822, a
third detection surface 9823, a fourth detection surface 9824, and
a fifth detection surface 9825. These detection surfaces can
produce signals for detection of a disease in a similar manner as
described above with respect to the detection module 6800.
[0340] The housing 9010 and/or the portion of the housing 9010
surrounding the detection opening 9011 is opaque (or semi-opaque),
thereby "framing" or accentuating the detection openings. In some
embodiments, the housing 9010 can include markings (e.g., thick
lines, colors or the like) to highlight the detection openings.
Additionally, the housing 9010 can include indicia 9017 identifying
the detection opening to a particular disease (e.g., Chlamydia
trachomatis (CT), Neisseria gonorrhea (NG) and Trichomonas
vaginalis (TV)) or control. The housing 9010 also includes a bar
code 9017'.
[0341] The device 9000 is packaged along with and/or incudes a
sample transport device 9110 configured to convey a sample S1 into
the device 9000 and/or the sample preparation module therein. As
shown in FIG. 84, the sample transfer device 9110 includes a distal
end portion 9112 and a proximal end portion 9113, and can be used
to aspirate or withdraw a sample S1 from a sample cup 9101. The
sample transfer device 9110 then delivers a desired amount of the
sample S1 to an input portion 9160 of the device 9000.
Specifically, the distal end portion 9112 includes a dip tube
portion, and in some embodiments, can define a reservoir having a
desired and/or predetermined volume.
[0342] The proximal end portion 9113 includes a housing 9130 and an
actuator 9117. The actuator 9117 can be manipulated by the user to
draw the sample into the distal end portion 9112. The housing 9130
includes a status window 9131 or opening through which the user can
visually check to see that adequate volume has been aspirated. In
some embodiments, the sample transport device 9110 includes an
overflow reservoir that receives excess flow of the sample during
the aspiration step. The overflow reservoir includes a valve member
that prevents the overflow amount from being conveyed out of the
transfer device 9110 when the actuator 9117 is manipulated to
deposit the sample into the input portion 9160 of the device 9000.
This arrangement ensures that the desired sample volume is
delivered to the device 9000. Moreover, by including a "valved"
sample transfer device 9110, the likelihood of misuse during sample
input is limited. This arrangement also requires minimal (or no)
scientific training and/or little judgment of the user to properly
deliver the sample into the device.
[0343] In use, the sample transfer device 9110 is removed from the
housing 9010 and the distal end portion 9112 is disposed within the
sample cup 9101. The actuator 9117 is manipulated to withdraw a
portion of the sample S1 into the sample transfer device 9110.
During use, the operator can inspect the status window 9131 to
ensure that the sample S1 is visible, thereby indicating that the
sample aspiration operation was successful. As shown in FIG. 86,
the sample transfer device 9110 is then placed into the receiving
portion 9160 of the housing 9010, as indicated by the arrow SS. In
some embodiments, the sample transfer device 9110, the housing 9130
and/or the housing 9010 can include locking mechanisms, such as
mating protrusions, recesses and the like that prevent removal of
the sample transfer device 9110 after it has been locked in
place.
[0344] To initiate a test, the actuator 9117 is the moved as shown
by the arrow TT in FIG. 87, to push the sample into the sample
preparation module of the device 9000.
[0345] Although the device 6000 is shown as including a wash module
6210 that is included within the housing, and that is separate from
the sample transfer device 6110, in other embodiments, a device can
include a sample transfer device that includes the wash therein. In
such embodiments, movement of an actuator to deliver the sample
(e.g., to convey the sample through a filter within the device) can
also be used to convey a wash solution (including an air wash)
contained within the sample transfer device through the filter. For
example, FIGS. 88 and 89 are schematic illustrations of a sample
transfer device 9110' according to an embodiment. The sample
transfer device 9110' can be used in conjunction with any of the
molecular diagnostic test devices shown and described herein.
[0346] The sample transfer device 9110' includes a housing 9130'
having distal end portion and a proximal end portion, and can be
used to aspirate or withdraw a sample from a sample cup (not
shown). The sample transfer device 9110' then delivers a desired
amount of the sample to an input portion of a molecular diagnostic
test device of the types shown and described herein. The housing
9130' defines a sample reservoir 9115' (for receiving a sample),
and a wash reservoir 9214' (that contains a wash solution). The
sample reservoir 9115' and the wash reservoir 9214' are separated
by (and or fluidically isolated from each other by) a septum (or
elastomeric stopper) 9132'.
[0347] The distal end portion of the housing includes a dip tube
9112'. The proximal end portion of the housing includes an actuator
9117'. In use, the actuator 9117' is moved and/or manipulated by
the user to draw the sample through the dip tube 9112' and into the
sample reservoir 9115'. To transfer the sample to the device (not
shown), the dip tube 9112' and/or a portion of the housing 9130' is
placed into and/or adjacent the device, and the actuator 9117' is
moved distally (as indicated by the arrow in FIG. 89). Movement of
the actuator 9117' pushes the sample out of the dip tube 9112', and
also moves the septum 9132' down towards the puncture 9133'. After
the sample has been dispensed, the puncture 9133' pierces the
septum 9132' thereby allowing the wash solution to flow from the
wash reservoir 9214' to the sample reservoir 9115' and/or out of
the dip tube 9112'.
[0348] Although the device 6000 is shown and described as including
a wash module 6210 that is separate from (and/or in a different
housing from) the elution module 6260, in other embodiments, any of
the sample transfer, sample input, wash and/or elution modules
described herein can be constructed together as integral units, or
maintained as distinct components. Similarly stated, any of the
components in any of the sample preparation modules described
herein can be in any suitable form. For example, in some
embodiments Individual components can include modifications and
changes. For example, in some embodiments a sample preparation
module can include a sample delivery portion, a wash portion, an
elution portion and a filter portion e (including a flow valve
assembly) within a common housing. FIGS. 90-92 show a sample
preparation module 10200 according to an embodiment. As illustrated
in FIG. 90, the sample preparation module 10200 is configured to
receive an input sample in connection with any suitable device
(such as the diagnostic test devices 6000, 7000, 8000, 9000 or any
other devices shown and described herein), and process the sample
for use in the subsequent modules. The sample preparation module
10200 includes a reservoir 10210 for receiving and containing the
sample, a filter assembly 10220, waste tank 10230, a normally
closed valve 10240, two storage and dispensing assemblies (10250
and 10260, see also FIGS. 91 and 92, respectively), and various
fluidic conduits (e.g., the output conduit 10241) connecting the
various components.
[0349] In some embodiments, the sample preparation module 10200 is
configured to accept and allow for spill proof containment of a
volume of liquid from the sample transfer module (not shown). In
some embodiments, the sample preparation module 10200 is configured
for onboard storage of wash solution, elution solution, and a
positive control. The positive control may be stored in liquid form
in the wash solution or stored as a lyophilized bead that is
subsequently hydrated by the wash solution. In some embodiments,
the sample preparation module 10200 is configured for dispensing
the bulk of the sample liquid (.about.80%) through a filter, while
storing the generated waste in a secure manner. In some
embodiments, the sample preparation module 10200 is configured for
following the sample dispense with a wash dispense, thereby
dispensing the bulk of the stored liquid (e.g., about 80%). In some
embodiments, the sample preparation module 10200 is configured for
back-flow elution to occur off the filter membrane and deliver the
bulk (e.g., about 80%) of the eluted volume to the target
destination. In some embodiments, the sample preparation module
10200 is configured so as not cause the output solution to be
contaminated by previous reagents (e.g., like the sample or wash).
In some embodiments, the sample preparation module 10200 is
configured for ease of operation by a lay user, requiring few,
simple, non-empirical steps, and for a low amount of actuation
force.
[0350] The sample preparation module 10200 first accepts an input
sample through input port 10211. A sample input port cap 10212 is
placed over the input port 10211 to contain the sample in its
reservoir 10210, to disallow spillage, and to allow accurate
manipulation. In some embodiments, the input port cap 10212 can
include an irreversible lock to prevent reuse of the device and/or
the additional of supplemental sample fluids. In this manner, the
sample preparation module 10200 and/or the device within which the
module is included can be suitably used by untrained
individuals.
[0351] To actuate the sample preparation module 10200, the end user
pushes down on a handle 10251, which is portion of a wash reagent
storage and dispensing assembly 10250. The assembly 10250 moves the
entire plunger assembly towards the bottom of the sample reservoir
10210 and thus forces the sample through a series of conduits into
a filter assembly 10220. A filter membrane 10221 captures the
target organism/entity while allowing the remaining liquid to flow
through into the waste tank 10230. Once substantially all of the
sample is emptied from the sample reservoir 10210, the wash
solution is flowed through the filter assembly 10220 by the
continuing motion of the storage and dispensing assembly 10250. The
wash solution removes as much as possible of the remaining
non-target material from the filter membrane 10221 and flows into
the waste tank 10230. After the completion of the wash, a
push-valve 10240 is actuated to open an output conduit 10241. The
second storage and dispensing assembly 10260 is then actuated using
the handle 10261. The initial motion closes the conduit connecting
the filter assembly 10220 to the waste tank 10230, and the
continuing motion flows the elution solution through the filter
10220 and removes the target organism from the filter membrane
10221, outputting the solution into an output conduit 10241
connected to a subsequent module (e.g., an inactivation module, not
shown).
[0352] Referring to FIGS. 90 and 91, in some embodiments, the wash
reagent storage and dispensing assembly 10250 includes two seal
disks 10253 (top seal disk), 10254 (bottom seal disk) housed in a
cylindrical bore 10252 to form a sealed reservoir. An opening
formed as a fill port 10255 in the side of the bore between the two
seals allows the reservoir to be filled. The opening/port 10255 is
sealed with a heat seal film (not shown) after the reservoir is
filled. Another opening formed as an output port 10257 below the
seal disks 10253, 10254 serves as the output for the stored
reagent. A handle 10251 is placed on top of the top seal disk
10253, so that when the handle 10251 is actuated downward both of
the seals 10253, 10254 (and the liquid trapped between them) are
moved downward in a bore 10252 due to the incompressibility of the
liquid. Once the bottom seal disk 10254 moves past the output port
10257, however, a new path for the liquid to escape is opened, and
instead of the whole assembly moving downward, the top seal disk
10253 is moved, thus compressing the liquid reservoir, and forcing
the liquid into the output port 10257.
[0353] Referring to FIGS. 90 and 92, the eluent reagent storage and
dispensing assembly 10260 contains at least some of the same
components as the wash reagent storage and dispensing assembly
10250, but differs at least in the sense that the assembly 10260
stores the eluent reagent downstream of the filter assembly 10220.
The lower disk seal (10254' on the elution side of the assembly
10260 also acts as a normally open valve for the filter to waste
fluidic conduit. Once this lower seal is moved past the output port
10241' in its bore 10252', it serves to segregate the fluidic path
between the output conduit and the waste location further in the
bore.
[0354] Through manipulation of the initial starting positions of
the disk seals (10253', 10254'), the total volume of each of the
reagent reservoirs can be modified. Manipulation of the fill volume
for each of the reagents, and of the volume transferred by the
sample preparation module can also allow for either minimizing or
maximizing the volume of air in the reservoir. Combined with the
orientation of the module during operation, this can be used to
create an "air purge" of the filter 10221 at any desired step, or
be used to substantially eliminate air interaction with the filter
10221.
[0355] In some embodiments, the module 10200 can be operated with
the fill opening/sample input port 10211 facing upward, so that any
air remaining in the sample input reservoir 10210 is trapped in the
top of the input cavity when the module is operated. The volume of
reagents dispensed into the storage reservoirs can be calibrated to
leave as little air volume in those chambers as possible. In this
manner, the sample preparation module 10200 can be used in a manner
to minimize air volume.
[0356] In other embodiments (e.g., those directed to maximizing air
volume), the module 10200 can be used with the operating handles
10251 facing upward (sample can still be input from any
orientation). With the volumes involved, this would force the air
to the top of each of the reagent reservoirs, and thus allow for
substantially all of the reagent to be dispensed first before an
air slug would be pushed through. For the stored reagents, the fill
volume would be adjusted to leave an appropriate amount of air
volume in the reservoir.
[0357] Referring to FIG. 90, the filter assembly 10220 includes any
suitable membrane 10221. The membrane can be any suitable membrane
material, and can be constructed in any manner as described here.
In some embodiments, the housings 10222, 10223 can be
ultrasonically welded together to correctly tension the filter
membrane 10221. The housings 10222, 10223 are also configured to
spread the liquid out over the whole area of the filter membrane
10221, rather than allowing the liquid to flow directly through the
center. The upper housing 10223 includes a conduit (not shown) to
return the liquid back to the plane of the lower housing upon
passing through the filter membrane 10221.
[0358] Although the heater assembly 6630 of the amplification
module 6600 is described above a including a single member or
construct (that can include any number heating elements to produce
the desired heating zones as described above), in other
embodiments, a heater assembly can be constructed of multiple
heaters, clamps, heat spreaders, fasteners or the like. For
example, FIGS. 93-95 show an amplification module 10600 according
to an embodiment. The amplification module 10600 can receive an
input sample in connection with any suitable device (such as the
diagnostic test devices 6000, 7000, 8000, 9000 or any other devices
shown and described herein), and amplify the sample for use in the
subsequent modules.
[0359] As illustrated in FIGS. 93-95, the amplification module
10600 is configured to perform a PCR reaction on an input of target
DNA mixed with required reagents. The amplification module 10600
includes a serpentine pattern fluidic chip 10610, a hot plate
construct 10620, a heat sink construct 10630, support and clamping
structure 10640 to mount all the components, and fluidic and
electrical interconnects (not shown) to connect to the surrounding
modules.
[0360] In some embodiments, the amplification module 10600 is
configured to conduct rapid PCR amplification of an input target.
In some embodiments, the amplification module 10500 is configured
to generate an output copy number that reaches or exceeds the
threshold of the sensitivity of the detection module 10600, as
described herein. In some embodiments, the output volume is
sufficient to fully fill the detection chamber in the detection
module 10600. In some embodiments, the amplification module 10600
employs a constant set point control scheme--for example, heaters
are powered on to control to a set point and set point does not
change through the process. Amplification is conducted as long as
the reagents are present and the input flow rate is correct. In
some embodiments, the amplification module 10600 consumes minimal
power, allowing the overall device 10000 to be battery powered
(e.g., by a 9V battery), similar to the device 6000 described
above.
[0361] In use, amplification is accomplished by the movement of the
fluid through a serpentine fluidic chip 10610 held in contact with
a hot plate construct 10620 during which the fluid inside the chip
passes through alternating temperature zones. In some embodiments,
the serpentine fluidic chip 10610 is in fixed contact with the hot
plate construct 10620, while in other embodiments, the serpentine
fluidic chip 10610 is in removable contact with the hot plate
construct 10620.
[0362] The hot plate construct 10620 heats the zones to the correct
temperatures, while the heat sink construct 10630 draws thermal
energy away from the areas next to the hot zones, thus allowing the
liquid to cool upon exit. Once the chip 10610 fills with liquid,
any liquid emerging from the output side has undergone PCR (as long
as the total volume of the liquid collected from the output is
lower or equal to the "output" volume). The output of the module
flows directly into the detection module (e.g., the detection
module 6800 described above).
[0363] As with the flow member 6610 described above, the serpentine
fluidic chip 10610 has two serpentine patterns molded into it--the
amplification pattern and the hot-start pattern. The chip 10610 is
lidded with a thin plastic lid 10613 ("serpentine chip lid") which
is attached with a pressure sensitive adhesive (not identified in
the figure). The lid 10613 allows for easy flow of thermal energy
from the hot plate 10620. The chip 10610 also contains features to
allow other parts of the assembly (like the hot plate) to correctly
align with the features on the chip, as well as features to allow
the fluidic connections to be bonded correctly.
[0364] The hot plate assembly 10620 is made up from four different
heater/sensor/heat spreader constructs 10621 (one construct), 10622
(one construct), 10623 (two constructs). The configuration and
mating alignment of these determines the areas of the temperature
zones on the fluidic chip 10610. The individual heater constructs
are controlled to a pre-determined set point by the electronics
module. Each construct has a resistive heater with an integrated
sensor element which, when connected to the electronics module,
allows for the temperature of the attached heat spreader to be
regulated to the correct set point. There are two "hot"
constructs--the hot start zone construct 10621, and the center zone
construct 10622, and two "cold" constructs--the two identical side
zones constructs 10623.
[0365] The heat sink construct 10630 includes pieces of conductive
material bonded to the side of the serpentine chip opposite the hot
plate. As best illustrated in the schematic illustration of FIG.
94, these allow for some of the thermal energy that the liquid
carries from the center hot zone to be dissipated, thus allowing
the temperature in the "side cold" zones to be regulated.
[0366] Although the fluid transfer module 6400 is shown and
described above as including two barrel portions within a
monolithically constructed housing, in other embodiments, a fluid
transfer module can include two separately constructed barrel
assemblies that are coupled together via a frame member. In yet
other embodiments, a fluid transfer module can include a single
barrel design, in which the single barrel functions to move the
sample through the mixing and amplification modules, and also
functions to draw the vacuum through the detection module (as
described above). For example, FIGS. 96-99 show a fluid transfer
module 11400 according to an embodiment. The fluid transfer module
11400 operates to aspirate a fluid sample, store the fluid during a
heated incubation period, remove residual gas from the syringe
barrel, and then dispense the fluid (e.g., to an amplification
module) at a constant rate against varying head pressure.
[0367] In use, a linear actuator is connected to the plunger 11415
or the flange 11462 to drive the "piston" in and out of the barrel
11410. The sequence of actions for using the device is as follows:
Initially, the piston 11415 is disposed into the syringe barrel
11410. When the piston 11415 retracted, a vacuum is created inside
the syringe barrel 11410 causing fluid to enter through the sample
inlet port 11420 from a mixing chamber, an inactivation chamber, a
filter or any other upstream portion of the sample preparation
module. Once the piston 11415 is fully retracted (see FIG. 98) and
the barrel 11410 is filled with sample, motion stops. In some
embodiments, the chamber heater 11495 brings the sample to 95C
effectively inactivating the lysing enzyme. After incubation, the
heat is turned off and the linear actuator (not show) changes
direction and the piston 11415 moves back into the syringe barrel
11410. The plunger head 11417 pushes on the fluid in the barrel
11410 and any trapped gas therein is forced through a low cracking
pressure flapper type check valve 11491 and exits through a
hydrophobic vent filter 11492 mounted in the filter valve housing
11464. As soon as fluid enters the filter 11492, the hydrophobic
nature of the material prevents the liquid from passing though and
effectively becomes blocked. As the piston 11415 is driven further
into the barrel 11410 (see FIG. 99), all of the gas within the
sample is pushed out and liquid sample is now forced through the
higher cracking pressure duckbill check valve 11424 mounted inside
of the plunger head 11417, and exits the syringe through the hollow
piston drive shaft 11415 and into the PCR tube connector 11430 and
on to the amplification module (not shown).
[0368] Following the PCR dispense cycle, the fluid transfer module
11400 is again used to produce a vacuum directed at moving fluids
through the detection module (not shown), in a similar manner as
described above. To redirect the vacuum to the detection module,
the normally closed dog-bone slide valve 11454 is opened at the
vacuum inlet port 11450. This port stays open for the remainder of
the test. As described above, a valve system (e.g., the valve
system 6340) can sequentially apply the vacuum to the reagents to
produce the desired flow through the detection module.
[0369] While various embodiments have been described above, it
should be understood that they have been presented by way of
example only, and not limitation. Where methods and/or schematics
described above indicate certain events and/or flow patterns
occurring in certain order, the ordering of certain events and/or
flow patterns may be modified. While the embodiments have been
particularly shown and described, it will be understood that
various changes in form and details may be made.
[0370] The devices and methods described herein are not limited to
performing a molecular diagnostic test on human samples. In some
embodiments, any of the devices and methods described herein can be
used with veterinary samples, food samples, and/or environmental
samples.
[0371] Although the fluid transfer assemblies are shown and
described herein as including a piston pump (or syringe), in other
embodiments, any suitable pump can be used. For example, in some
embodiments any of the fluid transfer assemblies described herein
can include any suitable positive-displacement fluid transfer
device, such as a gear pump, a vane pump, and/or the like.
[0372] Although the filter assembly 6230 shown and described above
includes an integral control valve (e.g., including the valve arm
6290), in other embodiments, a device can include a filter assembly
and a valve assembly that are separately constructed and/or are
spaced apart.
[0373] Some embodiments described herein relate to a computer
storage product with a non-transitory computer-readable medium
(also can be referred to as a non-transitory processor-readable
medium) having instructions or computer code thereon for performing
various computer-implemented operations. The computer-readable
medium (or processor-readable medium) is non-transitory in the
sense that it does not include transitory propagating signals per
se (e.g., a propagating electromagnetic wave carrying information
on a transmission medium such as space or a cable). The media and
computer code (also can be referred to as code) may be those
designed and constructed for the specific purpose or purposes.
Examples of non-transitory computer-readable media include, but are
not limited to: magnetic storage media such as hard disks, floppy
disks, and magnetic tape; optical storage media such as Compact
Disc/Digital Video Discs (CD/DVDs), Compact Disc-Read Only Memories
(CD-ROMs), and holographic devices; magneto-optical storage media
such as optical disks; carrier wave signal processing modules; and
hardware devices that are specially configured to store and execute
program code, such as Application-Specific Integrated Circuits
(ASICs), Programmable Logic Devices (PLDs), Read-Only Memory (ROM)
and Random-Access Memory (RAM) devices.
[0374] Examples of computer code include, but are not limited to,
micro-code or microinstructions, machine instructions, such as
produced by a compiler, code used to produce a web service, and
files containing higher-level instructions that are executed by a
computer using an interpreter. For example, embodiments may be
implemented using imperative programming languages (e.g., C,
Fortran, etc.), functional programming languages (Haskell, Erlang,
etc.), logical programming languages (e.g., Prolog),
object-oriented programming languages (e.g., Java, C++, etc.) or
other suitable programming languages and/or development tools.
Additional examples of computer code include, but are not limited
to, control signals, encrypted code, and compressed code.
[0375] The positive control organism can be stored in any suitable
portion of any of the devices shown and described herein. For
example, referring to the device 6000, in some embodiments, the
positive control organism can be a lyophilized bead that is located
in the sample volume 6174 and rehydrated as sample is added. In
such embodiments, the control organism is not used to verify sample
adequacy. Rather, the sample adequacy would be checked visually by
the user verifying the volume of sample in the transfer pipette
1110, as described above. In other embodiments, the positive
control organism pellet can be located in a fluidic path that leads
out of the sample volume 6174 at a specific location. In such
embodiments, if more than a desired amount of sample (e.g., about
300 .mu.L) is present then a portion of sample will rehydrate the
control pellet properly. If, however, less than the desired amount
of sample (e.g., about 300 .mu.L) is present then the control
pellet will not be rehydrated and will result in an invalid signal
(no color on the positive control spot) at the end of the run
(unless one of the target organisms is detected). In this manner,
the location of the control organism can verify sample volume
adequacy. In yet other embodiments, the control organism pellet can
be located in a sample transfer device (e.g., the device 1100) in
such a manner or position that if less than a desired amount of
sample (e.g., about 300 .mu.L) is transferred the pellet will not
be sufficiently rehydrated. This arrangement will also result in an
invalid signal (no color on the positive control spot) at the end
of the run (unless one of the target organisms is detected).
[0376] Although various embodiments have been described as having
particular features and/or combinations of components, other
embodiments are possible having a combination of any features
and/or components from any of embodiments as discussed above.
[0377] For example, any of the devices shown and described herein
can include a processor (such as the processor 4950 shown and
described above), and can include a memory device configured to
receive and store information, such as a series of instructions,
processor-readable code, a digitized signal, or the like. The
memory device can include one or more types of memory. For example,
the memory device can include a read only memory (ROM) component
and a random access memory (RAM) component. The memory device can
also include other types of memory suitable for storing data in a
form retrievable by the processor, for example,
electronically-programmable read only memory (EPROM), erasable
electronically-programmable read only memory (EEPROM), or flash
memory.
[0378] As another example, any of the devices shown and described
herein can include an indicator light, such as the LED indicator
light shown and described above with respect to the device 8000.
The light indicator can include, for example, two LEDs (a green and
a red) that illuminate to indicate various operations, including a
successful "power on" event, notification that the test is in
process; notification that the test is complete and/or that the
device can be read; and/or an error message.
* * * * *
References