U.S. patent application number 11/380327 was filed with the patent office on 2007-01-18 for systems and methods for multiple analyte detection.
This patent application is currently assigned to Applera Corporation. Invention is credited to Nigel P. Beard, Yuh-Min Chiang, Alexander Dromaretsky, Sergey V. Ermakov, Ian A. Harding, Dennis Lehto, David M. Liu, Mark F. Oldham, Joy Roy, Maryam Shariati, Umberto Ulmanella, John Van Camp, Charles S. Vann, Joon Mo Yang, Min Yue.
Application Number | 20070014695 11/380327 |
Document ID | / |
Family ID | 37215516 |
Filed Date | 2007-01-18 |
United States Patent
Application |
20070014695 |
Kind Code |
A1 |
Yue; Min ; et al. |
January 18, 2007 |
Systems and Methods for Multiple Analyte Detection
Abstract
Systems and methods for multiple analyte detection include a
system for distribution of a biological sample that includes a
substrate, wherein the substrate includes a plurality of sample
chambers, a sample introduction channel for each sample chamber,
and a venting channel for each sample chamber. The system may
further include a preloaded reagent contained in each sample
chamber and configured for nucleic acid analysis of a biological
sample that enters the substrate and a sealing instrument
configured to be placed in contact with the substrate to seal each
sample chamber so as to substantially prevent sample contained in
each sample chamber from flowing out of each sample chamber. The
substrate can be constructed of detection-compatible and
assay-compatible materials.
Inventors: |
Yue; Min; (Belmont, CA)
; Liu; David M.; (Los Altos, CA) ; Chiang;
Yuh-Min; (Foster City, CA) ; Yang; Joon Mo;
(Redwood City, CA) ; Lehto; Dennis; (Santa Clara,
CA) ; Vann; Charles S.; (El Granada, CA) ;
Beard; Nigel P.; (Redwood City, CA) ; Harding; Ian
A.; (San Mateo, CA) ; Van Camp; John; (San
Ramon, CA) ; Dromaretsky; Alexander; (Davis, CA)
; Ermakov; Sergey V.; (Hayward, CA) ; Oldham; Mark
F.; (Los Gatos, CA) ; Roy; Joy; (San Jose,
CA) ; Shariati; Maryam; (Sunnyvale, CA) ;
Ulmanella; Umberto; (Foster City, CA) |
Correspondence
Address: |
MILA KASAN, PATENT DEPT.;APPLIED BIOSYSTEMS
850 LINCOLN CENTRE DRIVE
FOSTER CITY
CA
94404
US
|
Assignee: |
Applera Corporation
Foster City
CA
|
Family ID: |
37215516 |
Appl. No.: |
11/380327 |
Filed: |
April 26, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60674750 |
Apr 26, 2005 |
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60674876 |
Apr 26, 2005 |
|
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60674750 |
Apr 26, 2005 |
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60696157 |
Jun 30, 2005 |
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Current U.S.
Class: |
422/400 |
Current CPC
Class: |
B01L 2400/0481 20130101;
B01L 2300/041 20130101; B01L 2400/0655 20130101; B01J 2219/00317
20130101; Y10T 436/2575 20150115; B01L 3/502707 20130101; B01L
2200/0684 20130101; B01L 2200/0689 20130101; B01L 2300/0864
20130101; B01L 2200/16 20130101; B01J 2219/00722 20130101; B01L
2300/0816 20130101; B01L 3/502723 20130101; B01L 2400/0487
20130101; G01N 35/028 20130101; B01L 2300/0829 20130101; B01L
3/50853 20130101; B01L 2400/0694 20130101; B01L 2300/048 20130101;
B01L 2300/0636 20130101; C12Q 1/6806 20130101; B01L 2300/087
20130101 |
Class at
Publication: |
422/100 |
International
Class: |
B01L 3/00 20070101
B01L003/00 |
Claims
1. A system for distribution of a biological sample, the system
comprising: a substrate, wherein the substrate comprises a
plurality of sample chambers, a sample introduction channel for
each sample chamber, and a venting channel for each sample chamber;
a preloaded reagent contained in each sample chamber and configured
for nucleic acid analysis of a biological sample that enters the
substrate; and a sealing instrument configured to be placed in
contact with the substrate to seal each sample chamber so as to
substantially prevent sample contained in each sample chamber from
flowing out of each sample chamber, wherein the substrate is
constructed of detection-compatible and assay-compatible
materials.
2. The system of claim 1, wherein the sealing instrument is
configured to be placed in contact with an exterior portion of the
substrate.
3. The system of claim 1, wherein the sealing instrument is
configured to be placed in at least one of pressure contact and
thermal contact with the substrate.
4. The system of claim 1, wherein the sealing instrument is
configured to seal the sample introduction channels and venting
channels for each sample chamber.
5. The system of claim 1, wherein the sealing instrument is
configured to be placed in contact with the substrate at least one
of before a reaction process that occurs in the sample chambers and
during a reaction process that occurs in the sample chambers.
6. The system of claim 5, wherein the sealing plate is configured
to be placed in contact with the substrate before a reaction
process that occurs in the sample chambers to seal each sample
chamber.
7. The system of claim 5, wherein the sealing plate is configured
to be placed in contact with the substrate during a reaction
process that occurs in the sample chambers to seal each sample
chamber.
8. The system of claim 7, further comprising a thermal block,
wherein the thermal block comprises the sealing plate.
9. The system of claim 1, wherein the sealing instrument comprises
one of a plate and a roller.
10. The system of claim 1, wherein the substrate comprises a film
layer and wherein a portion of the film layer fills the sample
introduction channels and the venting channels upon contact of the
sealing instrument with the substrate.
11. The system of claim 1, wherein the sealing instrument comprises
a plurality of sealing protrusions.
12. The system of claim 11, wherein the sealing protrusions are
configured to seal the sample introduction channels and venting
channels for each sample chamber.
13. The system of claim 11, further comprising an additional
sealing instrument, wherein the plurality of sealing protrusions
comprises a first plurality of sealing protrusions associated with
the sealing instrument and a second plurality of the sealing
protrusions associated with the additional sealing instrument, the
first plurality and second plurality of sealing protrusions being
configured to seal differing groups of sample chambers when the
sealing instrument and the additional sealing instrument are
respectively placed in contact with the substrate.
14. The system of claim 13, wherein the first plurality of sealing
protrusions is configured to seal the sample introduction channels
for each sample chamber and the second plurality of sealing
protrusions are configured to seal the venting channels for each
sample chamber.
15. The system of claim 11, wherein each sealing protrusion is
configured to contact a respective sample introduction channel and
venting channel for each sample chamber when the sealing instrument
is placed in contact with the substrate.
16. The system of claim 11, wherein the plurality of sealing
protrusions are configured to respectively align with the plurality
of sample chambers.
17. The system of claim 16, wherein each sample chamber is
configured to receive a respective sealing protrusion such that the
sealing protrusions seal the sealing chambers substantially at an
inner peripheral surface defining each sample chamber.
18. The system of claim 11, wherein the sealing instrument further
comprises a plurality of optical apertures configured to provide
optical access to the sample chambers.
19. The system of claim 18, wherein the sealing protrusions are
configured to pierce the substrate and enter the introduction and
venting channels for each sample chamber.
20. The system of claim 11, wherein the sealing protrusions
comprise one of pins, disks, blades, and bumps.
21. The system of claim 1, wherein the substrate further comprises
a film layer and a base, and at least one venting mechanism
configured to permit gas to escape the substrate.
22. The system of claim 21, wherein the substrate further comprises
a sample supply channel in fluid communication with a plurality of
the sample introduction channels.
23. The system of claim 22, wherein the sample supply channel
comprises a serpentine portion.
24. The system of claim 22, wherein the sample supply channel
comprises a bifurcation.
25. The system of claim 22, wherein the sample supply channel
comprises a U-shape portion.
26. The system of claim 21, wherein the substrate further comprises
a plurality of sample supply channels, each sample supply channel
being in flow communication with a differing group of sample
introduction channels and corresponding sample chambers.
27. The system of claim 26, wherein the substrate further comprises
a plurality of sample inlet ports, each sample inlet port being in
flow communication with a differing sample supply channel.
28. The system of claim 21, wherein the at least one venting
mechanism comprises a plurality of venting mechanisms respectively
corresponding to the plurality of sample chambers.
29. The system of claim 21, wherein the substrate further comprises
a venting chamber in flow communication with each venting
channel.
30. The system of claim 21, wherein the substrate further comprises
at least one overfill chamber corresponding to the at least one
venting mechanism.
31. The system of claim 30, further comprising a detector
configured to detect sample in the overfill chamber.
32. The system of claim 31, wherein the detector comprises one of
an optical sensor, a refractive sensor, and a capacitance
sensor.
33. The system of claim 30, further comprising an additional
venting mechanism disposed upstream of the at least one vent
mechanism corresponding to the overfill chamber.
34. The system of claim 21, wherein the substrate further comprises
a venting chamber for each sample chamber, the venting chambers
being in flow communication with the sample chambers via the
venting channels, wherein the at least one venting mechanism
comprises a plurality of venting mechanisms each corresponding to a
respective venting chamber.
35. The system of claim 21, wherein the substrate further comprises
a main venting channel in flow communication with the venting
channels.
36. The system of claim 35, wherein the at least one venting
mechanism comprises a porous fiber disposed in the main venting
channel.
37. The system of claim 36, wherein the fiber is a hollow fiber or
a solid fiber.
38. The system of claim 21, wherein the at least one venting
mechanism comprises a gas-permeable or porous membrane.
39. The system of claim 38, wherein the substrate further comprises
a venting chamber for each sample chamber, the venting chambers
being in flow communication with the sample chambers via the
venting channels, each venting chamber being configured to receive
a gas-permeable or porous membrane.
40. The system of claim 38, wherein the gas-permeable or porous
membrane is in contact with the film layer at a surface of the film
facing away from the base.
41. The system of claim 40, wherein the gas-permeable or porous
membrane has a sheet-like or strip-like configuration.
42. The system of claim 41, wherein the substrate further comprises
a venting chamber for each sample chamber, the venting chambers
being in flow communication with the sample chambers via the
venting channels, and wherein the at least one gas-permeable or
porous membrane corresponds to a plurality of venting chambers.
43. The system of claim 42, wherein the at least one gas-permeable
or porous membrane comprises a plurality of membranes, each
membrane corresponding to differing groups of venting chambers.
44. The system of claim 40, wherein the substrate further comprises
an additional film layer, the film layers being disposed on
opposing surfaces of the base.
45. The system of claim 44, wherein the additional film layer and
the base together define the sample chambers, sample introduction
channels, and venting channels.
46. The system of claim 21, wherein the at least one venting
mechanism comprises at least one through hole in the substrate.
47. The system of claim 46, wherein the at least one through hole
is provided in one of the base and the film layer.
48. The system of claim 47, wherein the at least one through hole
is provided in the film layer and is configured to prevent sample
from passing therethrough via capillarity.
49. The system of claim 21, wherein the at least one venting
mechanism is configured to substantially prevent sample from
passing through the at least one venting mechanism and out of the
substrate.
50. A method for distribution of a biological sample, the method
comprising: providing a base, wherein the base comprises a
plurality of sample cavities containing a preloaded reagent
therein, a sample introduction trench for each sample cavity, and a
venting trench for each sample cavity, and wherein the base is
constructed of detection-compatible and assay-compatible materials;
providing a film to adhere to the base to form a substrate, wherein
the film and base form a plurality of sample chambers from each
sample cavity, a sample introduction channel from each sample
introduction trench, and a venting channel from each sample venting
trench; supplying the biological sample to the substrate; filling
the sample chambers with the biological sample via the sample
introduction channels; passing gas out of the substrate via at
least one venting mechanism in the substrate; and sealing the
sample chambers to substantially prevent sample in the sample
chambers from flowing out of the sample chambers.
51. The method of claim 50, wherein the sealing the sample chambers
comprises placing a sealing instrument in contact with the
substrate.
52. The method of claim 50, wherein filling the sample chambers
comprises at least one of spinning the substrate to provide
centrifugal force, sizing the sample introductory channels to
provide capillary force, and aspirating the sample through the vent
channels.
53. The method of claim 50, wherein filling the sample chambers
comprises providing positive pressure to the sample.
54. The method of claim 53, further comprising venting gas in the
sample chamber through the venting channels and the at least one
venting mechanism.
55. The method of claim 54, wherein the venting mechanism comprises
at least one gas-permeable or porous membrane.
56. The method of claim 50, further comprising substantially
preventing the sample from passing through the at least one venting
mechanism and out of the substrate.
57. The method of claim 56, wherein preventing the sample from
passing through the at least one venting mechanism comprises
holding the sample in the venting mechanism via capillarity.
58. A system for distribution of a biological sample, the system
comprising: means for distributing the sample to a plurality of
sample chambers containing a preloaded reagent; means for venting
each of the sample chambers; and means for sealing each of the
sample chambers.
59. A device for testing a biological sample, the device
comprising: a substrate defining a plurality of distribution
portions configured to distribute biological sample throughout the
substrate, the plurality of distribution portions comprising a
plurality of sample chambers, a sample introduction channel for
each sample chamber, and a venting channel for each sample chamber;
and a substance disposed in at least one of the distribution
portions, the substance being configured to seal each sample
chamber so as to substantially prevent sample disposed in each
sample chamber from flowing out of each sample chamber during
testing of the biological sample.
60. The device of claim 59, wherein the substance comprises one of
an adhesive, a gas, a substance configured to dissolve into a gas
upon contact with the biological sample, a fluid immiscible with
the biological sample, an absorbent material, and porous
hydrophobic material.
61. The device of claim 59, wherein the substance is disposed in
the sample introduction and venting channels for each sample
chamber.
62. The device of claim 59, wherein the plurality of distribution
portions further comprises a vent chamber in flow communication
with each venting channel and wherein the substance is disposed in
each vent chamber.
63. The device of claim 59, wherein the plurality of distribution
portions further comprises a main fluid supply channel in flow
communication with the sample introduction channels, and wherein
the substance is disposed in the main fluid supply channel.
64. The device of claim 59, wherein the substance is configured to
seal each sample chamber upon sample contacting the substance.
65. The device of claim 59, wherein the substance is configured to
seal each sample chamber in response to elevating a temperature of
the substrate during testing of the biological sample.
Description
CROSS-REFERENCE TO COPENDING APPLICATIONS
[0001] This application claims the benefits of priority to U.S.
Provisional Application No. 60/674,750, filed on Apr. 26, 2005,
which is incorporated by reference in its entirety herein.
[0002] This application makes cross-reference to U.S. Provisional
Application No. 60/674,876, entitled "System for Population
Security and Epidemiological Analysis," concurrently filed with
U.S. Provisional Application No. 60/674,750, and later filed
60/696,157 of the same title, both of which are also incorporated
by reference herein in their entirety.
FIELD
[0003] The present teachings relate to systems and methods for
multiple analyte detection.
BACKGROUND
[0004] Biochemical testing for research and diagnostic applications
can require simultaneous assays including a large number of
analytes in conjunction with one or a few samples and can include
extended sample manipulation, multiple test substrates, multiple
analytical instruments, and other steps. It is desirable to provide
a method for analyzing one or a few biological samples using a
single test device with a large number of analytes while requiring
a small amount of sample. It is desirable to provide a device that
is small in size while providing high-sensitivity detection for the
analytes of interest with minimal sample manipulation. It is
desirable to provide a method of loading the sample(s) into
chambers on the substrate and individually sealing each chamber. It
is further desirable to provide a mechanism for venting of the
substrate during filling, while also avoiding and/or minimizing
leakage of fluid (e.g., biological sample and/or reagents) from the
test device.
SUMMARY
[0005] In various embodiments, the present teachings can provide a
system for distribution of a biological sample including a
substrate with a plurality of sample chambers, a sample
introduction channel for each sample chamber, and a venting channel
for each sample chamber, wherein the substrate is constructed of
detection-compatible and assay-compatible materials, and a sealing
plate with sealing protrusions for sealing the sample introduction
channels and the venting channels for each sample chamber.
[0006] In various embodiments, the present teachings can provide a
method for distribution of a biological sample including providing
an injection molded base, wherein the base includes a plurality of
sample cavities, a sample introduction trench for each sample
cavity, and a venting trench for each sample cavity, and wherein
the base is constructed of detection-compatible and
assay-compatible materials, providing a film to adhere to the base
forming a substrate, wherein film includes a plurality of vents,
and wherein the film forms a plurality of sample chambers from each
sample cavity, a sample introduction channel for each sample
introduction trench, and a venting channel for sample venting
trench, providing the biological sample to the substrate, forcing
the biological sample to the sample chambers through the sample
introduction channels, providing a sealing plate comprising sealing
protrusions for each sample chamber, heating the sealing plate,
sealing the sample chambers by contacting the sealing protrusions
with the sample introductory channels and the venting channels of
each sample chamber.
[0007] In various embodiments, the present teachings may provide a
system for distribution of a biological sample that includes a
substrate, wherein the substrate includes a plurality of sample
chambers, a sample introduction channel for each sample chamber,
and a venting channel for each sample chamber. The system may
further include a preloaded reagent contained in each sample
chamber and configured for nucleic acid analysis of a biological
sample that enters the substrate, and a sealing instrument
configured to be placed in contact with the substrate to seal each
sample chamber so as to substantially prevent sample contained in
each sample chamber from flowing out of each sample chamber. The
substrate can be constructed of detection-compatible and
assay-compatible materials.
[0008] In still further various embodiments, a method for
distribution of a biological sample may include providing a base,
wherein the base includes a plurality of sample cavities containing
a preloaded reagent therein, a sample introduction trench for each
sample cavity, and a venting trench for each sample cavity, and
wherein the base is constructed of detection-compatible and
assay-compatible materials. The method also may include providing a
film to adhere to the base to form a substrate, wherein the film
and base form a plurality of sample chambers from each sample
cavity, a sample introduction channel from each sample introduction
trench, and a venting channel from each sample venting trench. The
method may further include supplying the biological sample to the
substrate, filling the sample chambers with the biological sample
via the sample introduction channels, passing gas out of the
substrate via at least one venting mechanism in the substrate, and
sealing the sample chambers to substantially prevent sample in the
sample chambers from flowing out of the sample chambers.
[0009] According to yet other embodiments, the present teachings
may provide a system for distribution of a biological sample that
includes means for distributing the sample to a plurality of sample
chambers containing a preloaded reagent, means for venting each of
the sample chambers, and means for sealing each of the sample
chambers.
[0010] In various embodiments, the present teachings also can
provide a device for testing a biological sample that includes a
substrate defining a plurality of distribution portions configured
to distribute biological sample throughout the substrate, the
plurality of distribution portions comprising a plurality of sample
chambers, a sample introduction channel for each sample chamber,
and a venting channel for each sample chamber. The device also may
include a substance disposed in at least one of the distribution
portions, the substance being configured to seal each sample
chamber so as to substantially prevent sample disposed in each
sample chamber from flowing out of each sample chamber during
testing of the biological sample.
[0011] Additional embodiments are set forth in part in the
description that follows, and in part will be apparent from the
description, or may be learned by practice of the various
embodiments described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] Various embodiments of the present teachings are exemplified
in the accompanying drawings. The teachings are not limited to the
embodiments depicted, and include equivalent structures and methods
as set forth in the following description and known to those of
ordinary skill in the art. In the drawings:
[0013] FIG. 1 illustrates a perspective view of a substrate base
for biological analysis according to various embodiments of the
present teachings;
[0014] FIG. 2A illustrates a top view of a substrate for biological
analysis according to various embodiments of the present
teachings
[0015] FIG. 2B illustrates an exploded perspective view of a
substrate for biological analysis according to various embodiments
of the present teachings;
[0016] FIGS. 3A-3B illustrate a perspective view with magnified
section of a sealing plate according to various embodiments of the
present teachings;
[0017] FIG. 3C is a partial perspective view of another sealing
plate and substrate according to various embodiments of the present
teachings;
[0018] FIG. 3D is a partial view of the sealing plate of FIG. 3C
from the protrusion side of the plate;
[0019] FIG. 4 illustrates an exploded perspective view of a
substrate for biological analysis with a sealing plate according to
various embodiments of the present teachings;
[0020] FIG. 5 illustrates top view of a substrate for biological
analysis according to various embodiments of the present
teachings;
[0021] FIG. 6 illustrates a perspective view of an instrument for
biological sample preparation including six fluidic cartridges
according to various embodiments of the present teachings;
[0022] FIG. 7 illustrates a cut-away perspective view of a
cartridge for biological sample preparation according to various
embodiments of the present teachings;
[0023] FIG. 8A illustrates a perspective view of a substrate for
biological analysis according to various embodiments of the present
teachings;
[0024] FIGS. 8B and 8C illustrate cross-sectional views of FIG. 8A
taken through line 8C-8C according to various embodiments of the
present teachings;
[0025] FIGS. 9A-9B illustrate perspective views of exemplary
embodiments for sealing the substrate of FIGS. 8A-8C and
10A-10C;
[0026] FIG. 10A illustrates a perspective view of a substrate for
biological analysis according to various embodiments of the present
teachings;
[0027] FIGS. 10B and 10C illustrate cross-sectional views of FIG.
10A taken through line 10C-10C according to various embodiments of
the present teachings;
[0028] FIG. 11 illustrates a cross-sectional view of a substrate
for biological analysis according to various embodiments of the
present teachings;
[0029] FIGS. 12A-12B are cross-sectional views of filling and
sealing the substrate of FIG. 11 according to various embodiments
of the present teachings;
[0030] FIG. 13 is a perspective view of a substrate for biological
analysis according to various embodiments of the present
teachings;
[0031] FIG. 14 is a perspective, isometric view of another
substrate for biological analysis according to various embodiments
of the present teachings;
[0032] FIG. 15 is a perspective, isometric view of yet another
substrate for biological analysis according to various embodiments
of the present teachings;
[0033] FIG. 16 is a perspective, isometric view of a substrate for
biological analysis according to various embodiments of the present
teachings;
[0034] FIG. 17 is a perspective, isometric view of yet another
substrate for biological analysis according to various embodiments
of the present teachings;
[0035] FIGS. 18A-18C illustrate perspective views of vent holes
formed in various materials via Oxford Laser, Inc. instruments;
[0036] FIG. 19 is a perspective view of yet another substrate for
biological analysis according to various embodiments of the present
teachings;
[0037] FIGS. 20A and 20B illustrate perspective views of sample
chambers, venting channels, and venting chambers according to
various embodiments of the present teachings;
[0038] FIGS. 21A-21C schematically illustrate cross-sectional views
of exemplary steps of filling a feature of a substrate for
biological analysis;
[0039] FIGS. 22A-22C schematically illustrate cross-sectional views
of exemplary steps of filling a feature of a substrate for
biological analysis according to various embodiments of the present
teachings;
[0040] FIGS. 23A and 23B schematically illustrate cross-sectional
views of exemplary steps of filling a feature of a substrate for
biological analysis;
[0041] FIG. 24 illustrates a top view of yet another substrate for
biological analysis according to various embodiments of the present
teachings;
[0042] FIG. 25 illustrates a perspective view of yet another
substrate for biological analysis according to various embodiments
of the present teachings;
[0043] FIG. 26 illustrates a top view of yet another substrate for
biological analysis according to various embodiments of the present
teachings;
[0044] FIG. 27 illustrates a top view of yet another substrate for
biological analysis according to various embodiments of the present
teachings;
[0045] FIG. 28 is a partial cross-sectional view of a sample
chamber and venting chamber provided with a vent through hole
according to various embodiments of the present teachings;
[0046] FIGS. 29 and 30 are top and perspective views of a sealing
roller for sealing a substrate according to various embodiments of
the present teachings;
[0047] FIG. 31 is a perspective view of another sealing roller
according to various embodiments of the present teachings;
[0048] FIGS. 32 and 33 are partial, cross-sectional views of a
substrate and a thermal block for sealing a substrate according to
various embodiments of the present teachings;
[0049] FIGS. 34, 35, and 35A are partial, cross-sectional views of
thermal blocks for sealing a substrate according to various
embodiments of the present teachings;
[0050] FIG. 36 is a top view of a sample chamber with an escape
channel according to various embodiments of the present
teachings;
[0051] FIG. 37 is a partial cross-sectional view of a substrate
that uses capacitance overfill detection in accordance with various
embodiments of the present teachings;
[0052] FIG. 38 is a perspective view of a substrate for biological
analysis according to various embodiments of the present
teachings;
[0053] FIGS. 39A-39C are perspective views of steps of filling and
sealing the substrate of FIG. 38 according to various embodiments
of the present teachings;
[0054] FIG. 40A is a partial, perspective, isometric view of a
substrate for biological analysis according to various embodiments
of the present teachings;
[0055] FIGS. 40B and 40C are partial cross-sectional views of the
substrate of FIG. 40A showing the substrate before and after being
filled with sample, respectively;
[0056] FIGS. 41A and 41B are partial perspective views of a
substrate for biological analysis according to various embodiments
of the present teachings;
[0057] FIGS. 42A-42C are partial perspective views of a substrate
for biological analysis according to various embodiments of the
present teachings;
[0058] FIGS. 43A-43C are partial perspective views of a substrate
for biological analysis according to various embodiments of the
present teachings;
[0059] FIG. 44 is a perspective view of a substrate for biological
analysis according to various embodiments of the present
teachings;
[0060] FIG. 44A is a close-up of section 44A of the substrate of
FIG. 44;
[0061] FIG. 45 is a perspective view of a device for inserting
porous fibers in substrates for biological analysis according to
various embodiments of the present teachings;
[0062] FIGS. 46A-46D show exemplary steps for making the substrate
of FIG. 44;
[0063] FIGS. 47A and 47B are a partial perspective and
cross-sectional view of a substrate for biological analysis
according to various embodiments of the present teachings;
[0064] FIGS. 48A-48C are perspective views of yet another substrate
for biological analysis according to various embodiments of the
present teachings;
[0065] FIG. 49 is a partial cross-sectional view of the substrate
of FIGS. 48A-48C;
[0066] FIG. 50 is a partial perspective view of the substrate of
FIGS. 48A-48C;
[0067] FIGS. 51A and 51B are perspective views of yet another
substrate for biological analysis according to various embodiments
of the present teachings;
[0068] FIG. 52 is a perspective view of a system for packaging a
plurality of substrates for biological analysis according to
various embodiments of the present teachings;
[0069] FIGS. 53A and 53B are a perspective and cross-sectional view
of a thermocycler in accordance with various embodiments of the
present teachings;
[0070] FIG. 54 is a perspective view of a holding fixture and
subcards in accordance with various embodiments of the present
teachings;
[0071] FIG. 55 is a perspective view of a holding fixture and
subcards in accordance with various embodiments of the present
teachings;
[0072] FIG. 56 is a perspective view of another holding fixture and
subcards in accordance with various embodiments of the present
teachings; and
[0073] FIGS. 57-61 are perspective views of substrate carriers in
accordance with various embodiments of the present teachings.
[0074] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are intended to provide a further
explanation of the various embodiments of the present
teachings.
DESCRIPTION OF VARIOUS EMBODIMENTS
[0075] In this application, the use of the singular includes the
plural unless specifically stated otherwise. In this application,
the use of "or" means "and/or" unless stated otherwise.
Furthermore, the use of the term "including", as well as other
forms, such as "includes" and "included", is not limiting. Also,
terms such as "element" or "component" encompass both elements and
components comprising one unit and elements and components that
comprise more than one subunit unless specifically stated
otherwise. Wherever possible, the same reference numbers will be
used throughout the drawings to refer to the same or like
parts.
[0076] The section headings used herein are for organizational
purposes only, and are not to be construed as limiting the subject
matter described. All documents cited in this application,
including, but not limited to patents, patent applications,
articles, books, and treatises, are expressly incorporated by
reference in their entirety for any purpose.
[0077] The term "sample chamber" as used herein refers to any
structure that provides containment to a sample. The chamber can
have any shape including circular, rectangular, cylindrical, etc.
Multi-chamber arrays can include 12, 16, 24, 36, 48, 96, 192, 384,
3072, 6144, or more sample chambers. The term "channel" as used
herein refers to any structure that is smaller than a chamber. A
channel can have any shape. It can be straight or curved, as
necessary, with cross-sections that are shallow, deep, square,
rectangular, concave, or V-shaped, or any other appropriate
configuration. A "distribution portion" of a substrate may refer to
any portion of the substrate configured to contain, flow, receive,
or otherwise hold sample and/or gas in the substrate. Examples of
distribution portions include main fluid supply channels, venting
channels, venting chambers, sample chambers, sample introduction
channels, overfill chambers, and virtually any other channels
and/or chambers of the substrate.
[0078] The term "biological sample" as used herein refers to any
biological or chemical substance, typically in an aqueous solution
with luminescent dye that can produce emission light in relation to
nucleic acid present in the solution. The biological sample can
include one or more nucleic acid sequence(s) to be incorporated as
a reactant in a polymerase chain reaction (PCR) and other reactions
such as ligase chain reactions, antibody binding reactions,
oligonucleotide ligations assay, hybridization assays, and invader
assays (e.g., for an isotherm reaction). The biological sample can
include one or more nucleic acid sequences to be identified for DNA
sequencing.
[0079] The term "luminescent dye" as used herein may refer to
fluorescent or phosphorescent dyes that can be excited by
excitation light or chemiluminscent dyes that can be excited
chemically. As used herein, "luminescent dye" may also include the
use of energy transfer pairs (intramolecular or intermolecular).
For example, excitation of one energy transfer pair member and
emission of the other member may expand the range of emission
wavelengths for multiplexing. Quenchers selected should be suitable
for both members of the energy transfer pair. Luminescent dyes can
be used to provide different colors depending on the dyes used.
Several dyes will be apparent to one skilled in the art of dye
chemistry, including, for example, intercalating dyes. One or more
colors can be collected for each dye to provide identification of
the dye or dyes detected. The dye can be a dye-labeled fragment of
nucleotides. The dye can be a marker triggered by a fragment of
nucleotides. The dye can provide identification of a nucleic acid
sequence in the biological sample by association, for example,
bonding to or reacting with a detectable marker, for example, a
respective dye and quencher pair. The respective identifiable
component can be positively identified by the luminescence of the
dye. The dye can be normally quenched, and then can become
unquenched in the presence of a particular nucleic acid sequence in
the biological sample. The fluorescent dyes can be selected to
exhibit respective and, for example, different, excitation and
emission wavelength ranges. The luminescent dye can be measured to
quantitate the amount of nucleic acid sequences in the biological
sample. The luminescent dye can be detected in real-time to provide
information about the identifiable nucleic acid sequences
throughout the reaction. Examples of fluorescent dyes with
desirable excitation and emission wavelengths can include
5-FAM.TM., TET.TM., and VIC.TM.. The term "luminescence" as used
herein refers to low-temperature emission of light including
fluorescence, phosphorescence, electroluminescence, and
chemiluminescence.
[0080] In various embodiments, sample chambers can be dimensioned
to hold from 0.0001 .mu.L to 10 .mu.L of sample per chamber, or
between 0.001 .mu.L and 2 .mu.L. Conveniently, the volume of each
detection chamber is between 0.001 .mu.L and 1 .mu.L. For example,
a chamber having a volume of 0.2 .mu.L may have dimensions of 1
mm.times.1 mm.times.0.2 mm, where the last dimension is the
chamber's depth. As a further example, a chamber may have a
substantially cylindrical shape having a diameter of about 1.96 mm
and a depth of about 0.5 mm and a volume of about 1.35 .mu.L.
[0081] In various embodiments, the sample introduction channels can
be dimensioned to facilitate rapid delivery of sample to the sample
chambers, while occupying as little volume as possible. For
example, cross-sectional dimensions for the channels can range from
0.5 .mu.m to 250 .mu.m for both the width and depth. In some
embodiments, the channel path lengths to the sample chambers can be
minimized to reduce the total channel volume. For example, the
network can be substantially planar, i.e., the sample introduction
channels and sample chambers in the substrate intersect a common
plane.
[0082] "Venting mechanisms" as used herein may refer to any
mechanism configured and arranged to permit gas to escape
therethrough and leave the substrate. Venting mechanisms may be the
last structure the gas passes through prior to leaving the
substrate or be a structure that passes the escaping gas
therethrough to another structure that is the last structure before
the gas escapes the substrate. According to various embodiments,
venting mechanisms may permit the passage of gas while
substantially preventing the passage of liquid. Also, venting
mechanisms may be used in combination with one another to permit
gas to escape the substrate. Examples of venting mechanisms
include, but are not limited to, gas-permeable or porous membranes,
vent through holes either in a film layer, a base or both, porous
fibers, and hydrophobic liquid stops.
[0083] In various embodiments, there are a variety of means for
distributing the biological sample to the plurality of sample
chambers. All of these include applying a force. The force can be a
pulling force or a pushing force, depending on whether it provides
a negative (pulling) or positive (pushing) force relative to the
direction of fluid flow. Examples of forces and how the force is
enacted upon the biological sample include spinning the substrate
to provide centrifugal force to push the liquid, sizing the sample
introductory channels to provide capillary force to pull the
liquid, aspirating the sample through the vents to pull the liquid,
evacuating the sample chamber to pull the liquid, and/or providing
pressure, such as by pumping, compressing, plunging, etc. to push
the liquid. In each of these configurations, the venting channels
and vents can be used to accommodate the displaced venting gas,
whether air or other gas such as nitrogen, that is pushed out by
the sample or the venting channels and vents can be used to
evacuate the gas in the sample chambers to create a vacuum for the
sample or aspirate sample itself.
[0084] In various embodiments, the substrate that defines the
sample-distribution network can be constructed from any solid
material that is suitable for conducting analyte detection.
Materials that can be used will include various plastic polymers
and copolymers, such as polypropylenes, polystyrenes, polyimides,
COP, COC, and polycarbonates. Inorganic materials such as glass and
silicon are also useful. Silicon is especially advantageous in view
of its high thermal conductivity, which facilitates rapid heating
and cooling of the substrate if necessary. The substrate can be
formed from a single material or from a plurality of materials.
Examples of this are described at U.S. Pat. No. 6,126,899.
[0085] In various embodiments, the sample-distribution network
including cavities and trenches, for example, formed in a substrate
base portion, can be formed by any suitable method known in the
art. For plastic materials, injection molding can be suitable to
form sample cavities and connecting channels having a desired
pattern. For silicon, standard etching, RIE, DRIE, and wet-etching
techniques from the semiconductor industry can be used as known in
the art of photo-lithography.
[0086] In various embodiments, the substrate can be prepared from
two or more laminated layers. The term "detection-compatible
material" as used herein refers to the optical detection with a
substrate that includes one or more layers which provide an optical
transparency for each sample chamber. By way of example, the
optical transparency may permit detection of a luminescent dye.
Silica-based glasses, quartz, polycarbonate, or an optically
transparent plastic layer may be used, for example. Selection of
the particular detection-compatible material depends in part on the
optical properties of the material and the detection mechanism. For
example, in luminescent dye-based assays, the material should have
low fluorescence emission at the wavelength(s) being measured. The
detection-compatible material should also exhibit minimal light
absorption for the signal wavelengths of interest. However, in some
cases, for example, to minimize cross-talk, it may be desirable to
provide a substrate material that has relatively high absorption.
Examples of such materials are described at U.S. Pub. No.
2005/0226779A1.
[0087] In various embodiments, other layers in the substrate can be
formed using the same or different materials. The term
"assay-compatible material" as used herein refers to the
interaction of assay reagents and assay conditions (heat, pressure,
pH, etc.) with the substrate material (hydrophobic, hydrophilic,
inert, etc.). For example, the layer or layers, such as a film
defining the sample chambers can be formed predominantly from a
material that has high heat conductivity, such as silicon, a
heat-conducting metal, or carbon fill in plastic. The silicon
surfaces that contact the sample can be coated with an oxidation
layer or other suitable coating, to render the surface more inert
and make it an assay-compatible material. Similarly, where a
heat-conducting metal is used in the substrate, the metal can be
coated with an assay-compatible material, such as a plastic
polymer, to prevent corrosion of the metal and to separate the
metal surface from contact with the sample. The suitability of a
particular surface should be verified for the selected assay as
known by the conditions and reagents used in the assay.
[0088] In various embodiments, for optical detection, the opacity
or transparency of the substrate material defining the sample
chambers, for example, the base, can have an effect on the
permissible detector geometries used for signal detection. For the
following discussion, references to the "upper wall" of a detection
chamber may refer to the chamber surface or wall through which the
optical signal is detected, and references to the "lower wall" of a
chamber may refer to the chamber surface or wall that is opposite
the upper wall. For example, the upper wall can be formed by the
base or the film, and the lower wall by the other,
respectively.
[0089] In various embodiments, in fluorescence detection the
substrate material defining the lower wall of the sample chambers
can be optically opaque, and the sample chambers can be illuminated
and optically scanned through the same surface (i.e., the top
surfaces of the chambers which are optically transparent). Thus,
for fluorescence detection, the opaque lower wall material can
exhibit low reflectance properties so that reflection of the
illuminating light back toward the detector can be minimized.
Opacity also may prevent collection of background signals from a
thermal cycler block or other instrumentation used to test the
substrates. In other cases, the substrate lower wall of the sample
chambers may be reflective so that more fluorescent signal is
collected.
[0090] In various embodiments, in fluorescence detection the
substrate material defining the upper wall of the sample chambers
can be optically clear, the chambers can be illuminated with
excitation light through the sides of the chambers (in the plane
defined collectively by the sample chambers in the substrate), or
more typically, diagonally from above (e.g., at a 45 degree angle),
and emitted light is collected from above the chambers (i.e.,
through the upper walls, in a direction perpendicular to the plane
defined by the detection chambers). The upper wall material can
exhibit low dispersion of the illuminating light in order to limit
Rayleigh scattering.
[0091] In various embodiments, in fluorescence detection the
substrate material defining the entirety of the substrate can be
optically clear, or at least the upper and lower walls of the
chambers can be optically clear, the chambers can be illuminated
through either wall (upper or lower), and the emitted or
transmitted light is measured through either wall as appropriate.
Illumination of the chambers from other directions can also be
possible as already discussed above.
[0092] In various embodiments, in chemiluminescence detection,
where light of a distinctive wavelength is typically generated
without illumination of the sample by an outside light source, the
absorptive and reflective properties of the substrate can be less
important, provided that the substrate provides at least one
optically transparent window for detecting the signal.
[0093] In various embodiments, the substrate can be designed to
provide a vacuum-tight environment within the sample-distribution
network for sample loading, and also to provide sample chambers
having carefully defined reaction volumes. It is desirable to
ensure that the network and associate sample chambers do not leak.
Accordingly, lamination of substrate layers to one another can be
accomplished so as to ensure that all chambers and channels are
well sealed.
[0094] In various embodiments, the substrate layers can be sealably
bonded in a number of ways. A suitable bonding substance, such as a
glue or epoxy-type resin, can be applied to one or both opposing
surfaces that will be bonded together. The bonding substance may be
applied to the entirety of either surface, so that the bonding
substance (after curing) can come into contact with the sample
chambers and the distribution network. In this case, the bonding
substance is selected to be compatible with the sample and
detection reagents used in the assay. Alternatively, the bonding
substance can be applied around the distribution network and
detection chambers so that contact with the sample can be minimal
or avoided entirely. The bonding substance may also be provided as
part of an adhesive-backed tape or membrane, which is then brought
into contact with the opposing surface. In yet another approach,
the sealable bonding is accomplished using an adhesive gasket
layer, which is placed between the two substrate layers. In any of
these approaches, bonding may be accomplished by any suitable
method, including pressure-sealing, ultrasonic welding, and heat
curing, for example.
[0095] In various embodiments, a pressure-sensitive adhesive (PSA)
can be used in constructing the substrate. PSA films which can be
applied to a surface and adhered to that surface are obtained by
applying pressure to the film. Normally, pressure is applied
throughout the whole film, so that the whole film can adhere to the
surface. PSA films can have threshold pressure in order to activate
the adhesion. The threshold pressure can be very low. By applying
pressure to some selected regions, the bonding can be limited to
those regions only, thus allowing obtaining a bonding pattern. This
way channels and chambers can be defined. The elastic properties of
the film can then be used to pressure-drive a fluid through the
unbonded regions, since the film would deform under the liquid
pressure, thus opening up a channel. Eventually, the channel could
be sealed by applying pressure on the portion of the film defining
the channel. PSA films can be either hydrophobic or hydrophilic.
PSA films can have hydrophobic and hydrophilic areas on the same
film to provide areas of different wetting characteristics,
properly patterned, to provide, for example fluid flow in sample
introduction channels and gas venting in venting channels. Thus, by
providing differing regions of hydrophobicity and hydrophilicity of
the films, control over fluid flow through a device may be
achieved. In various embodiments, PSA films that are hydrophilic
can have the hydrophilic properties deteriorate in a matter of
days. The lack of stability (hydrophilic film turning into
hydrophobic) can provide controllable, irreversible or reversible,
changes (upon temperature change, heat addition, UV exposure, or
just time delay after curing) in the wetting nature of the film. In
various embodiments, PSA films can have different porosities and
permeabilities to a gas. A highly permeable PSA film can be more
advantageous than a low-permeability one for instance to vent the
sample chambers. Further, a PSA film whose permeability/porosity
can be modified in a reversible fashion with temperature change,
and/or in an irreversible fashion by heat addition or UV exposure
can be used for sample distribution and then sealed for sample
processing. In various embodiments, PSA films can be hydrophilic,
provide solvent resistance, maintain the adhesion characteristics
at a high temperature (95-100 degree Celsius), and can be optically
clear with low auto-fluorescence. In various embodiments, PSA films
can be thermally expandable to swell at desired locations and close
off channels.
[0096] In various embodiments, the substrate of the present
teaching can be adapted to allow rapid heating and cooling of the
sample chambers to facilitate reaction of the sample with the
analyte-detection reagents, including luminescent dyes. In one
embodiment, the substrate can be heated or cooled using an external
temperature-controller. The temperature-controller is adapted to
heat/cool one or more surfaces of the substrate, or can be adapted
to selectively heat the sample chambers themselves. To facilitate
heating or cooling with this embodiment, the substrate can be
formed of a material that has high thermal conductivity, such as
copper, aluminum, or silicon. Alternatively, bases can be formed
from a material having moderate or low thermal conductivity, while
the film can be formed form a conductive material such that the
temperature of the sample chambers can be conveniently controlled
by heating or cooling the substrate through the film, regardless of
the thermal conductivity of the base. For example, the film can be
formed of an adhesive copper-backed tape.
[0097] In various embodiments, the sample chambers of the substrate
can be pre-loaded with detection reagents that are specific for the
selected analytes of interest. By way of example, such reagents may
be deposited in the sample chambers in liquid form and dried (e.g.,
lyophilized). For example, reagents for nucleic acid analysis of a
biological sample may be preloaded in the substrate, for example,
in the sample chambers. In such embodiments, the substrate may then
be loaded with sample when biological testing is desired to be
performed by supplying sample to the substrate containing the
pre-loaded reagent(s). The detection reagents can be designed to
produce an optically detectable signal via any of the optical
methods known in the field of detection. It will be appreciated
that although the reagents in each detection chamber can contain
substances specific for the analyte(s) to be detected in the
particular chamber, other reagents for production of the optical
signal for detection can be added to the sample prior to loading,
or may be placed at locations elsewhere in the network for mixing
with the sample. Examples of such reactions are described in U.S.
Pub. No. 2005/0260640A1. Whether particular assay components are
included in the detection chambers or elsewhere will depend on the
nature of the particular assay, and on whether a given component is
stable to drying. Pre-loaded reagents added in the detection
chambers during manufacture of the substrate can enhance assay
uniformity and minimize the assay steps conducted by the
end-user.
[0098] In various embodiments, the analyte to be detected may be
any substance whose presence, absence, or amount is desirable to be
determined. The detection means can include any reagent or
combination of reagents suitable to detect or measure the
analyte(s) of interest. It will be appreciated that more than one
analyte can be tested for in a single detection chamber, if
desired.
[0099] In one embodiment, the analytes are selected-sequence
polynucleotides, such as DNA or cDNA, RNA, and the analyte-specific
reagents include sequence-selective reagents for detecting the
polynucleotides. The sequence-selective reagents include at least
one binding polymer that is effective to selectively bind to a
target polynucleotide having a defined sequence. The binding
polymer can be a conventional polynucleotide, such as DNA or RNA,
or any suitable analog thereof, which has the requisite sequence
selectivity. Other examples of binding polymers known generally as
peptide nucleic acids may also be used. The binding polymers can be
designed for sequence specific binding to a single-stranded target
molecule through Watson-Crick base pairing, or sequence-specific
binding to a double-stranded target polynucleotide through
Hoogstein binding sites in the major groove of duplex nucleic acid.
A variety of other suitable polynucleotide analogs are also known
in the art of nucleic acid amplification. The binding polymers for
detecting polynucleotides are typically 10-30 nucleotides in
length, with the exact length depending on the requirements of the
assay, although longer or shorter lengths are also
contemplated.
[0100] The present teachings can find utility in a wide variety of
amplification methods, such as PCR, Reverse Transcription PCR
(RT-PCR), Ligation Chain Reaction (LCR), Nucleic Acid Sequence
Based Amplification (NASBA), self-sustained sequence replication
(3SR), strand displacement activation (SDA), Q (3replicase) system,
isothermal amplification methods, and other known amplification
method or combinations thereof. Additionally, the present teachings
can find utility for use in a wide variety of analytical
techniques, such as ELISA; DNA and RNA hybridizations; antibody
titer determinations; gene expression; recombinant DNA techniques;
hormone and receptor binding analysis; and other known analytical
techniques.
[0101] In one embodiment, the analyte-specific reagents include an
oligonucleotide primer pair suitable for amplifying, by polymerase
chain reaction, a target polynucleotide region of the selected
analyte that is flanked by 3'-sequences complementary to the primer
pair. In practicing this embodiment, the primer pair is reacted
with the target polynucleotide under hybridization conditions which
favor annealing of the primers to complementary regions of opposite
strands in the target. The reaction mixture is then thermal cycled
through several, and typically about 20-40, rounds of primer
extension, denaturation, and primer/target sequence annealing,
according to well-known polymerase chain reaction (PCR) methods.
Typically, both primers for each primer pair are pre-loaded in each
of the respective sample chambers. The primer also may be loaded
along with the standard nucleotide triphosphates, or analogs
thereof, for primer extension (e.g., ATP, CTP, GTP, and TTP), and
any other appropriate reagents, such as MgCl.sub.2 or MnCl.sub.2. A
thermally stable DNA polymerase, such as Taq, Vent, or the like,
may also be pre-loaded in the chambers, or may be mixed with the
sample prior to sample loading. Other reagents may be included in
the detection chambers or elsewhere as appropriate. Alternatively,
the detection chambers may be loaded with one primer from each
primer pair, and the other primer (e.g., a primer common to all of
sample chambers) can be provided in the sample or elsewhere. If the
target polynucleotides are single-stranded, such as single-stranded
DNA, cDNA, or RNA, the sample is preferably pre-treated with a DNA-
or RNA-polymerase prior to sample loading, to form double-stranded
polynucleotides for subsequent amplification. Also, a reverse
transcription enzyme may be used to pretreat RNA to cDNA. This
pre-treatment can be provided in the cartridge.
[0102] In various embodiments, the presence and/or amount of target
polynucleotide in a sample chamber, as indicated by successful
amplification, is detected by any suitable means. For example,
amplified sequences can be detected in double-stranded form by
including an intercalating or crosslinking dye, such as ethidium
bromide, acridine orange, or an oxazole derivative, such as, Cyber
Green, for example, which exhibits a fluorescence increase or
decrease upon binding to double-stranded nucleic acids. The level
of amplification can also be measured by fluorescence detection
using a fluorescently labeled oligonucleotide. In this embodiment,
the detection reagents include a sequence-selective primer pair as
in the more general PCR method above, and in addition, a
sequence-selective oligonucleotide (FQ-oligo) containing a
fluorescer-quencher pair. The primers in the primer pair are
complementary to 3' regions in opposing strands of the target
analyte segment which flank the region which is to be amplified.
The FQ-oligo is selected to be capable of hybridizing selectively
to the analyte segment in a region downstream of one of the primers
and is located within the region to be amplified. The
fluorescer-quencher pair can include a fluorescent dye and a
quencher which are spaced from each other on the oligonucleotide so
that the quencher is able to significantly quench light emitted by
the fluorescer S at a selected wavelength, while the quencher and
fluorescer are both bound to the oligonucleotide. The FQ-oligo can
include a 3'-phosphate or other blocking group to prevent terminal
extension of the 3' end of the oligo. The fluorescer and quencher
dyes may be selected from any dye combination having the proper
overlap of emission (for the fluorescer) and absorptive (for the
quencher) wavelengths while also permitting enzymatic cleavage of
the FQ-oligo by the polymerase when the oligo is hybridized to the
target. Suitable dyes, such as rhodamine and fluorscein
derivatives, and methods of attaching them, are well known in the
art of nucleic acid amplification.
[0103] In another embodiment, the detection reagents include first
and second oligonucleotides effective to bind selectively to
adjacent, contiguous regions of a target sequence in the selected
analyte, and which can be ligated covalently by a ligase enzyme or
by chemical means as known in the art of oligonucleotide ligation
assay, (OLA). In this approach, the two oligonucleotides (oligos)
can be reacted with the target polynucleotide under conditions
effective to ensure specific hybridization of the oligonucleotides
to their target sequences. When the oligonucleotides have
base-paired with their target sequences, such that confronting end
subunits in the oligos are base-paired with immediately contiguous
bases in the target, the two oligos can be joined by ligation,
e.g., by treatment with ligase. After the ligation step, the
detection wells are heated to dissociate unligated probes, and the
presence of a ligated, target-bound probe is detected by reaction
with an intercalating dye or by other means. The oligos for OLA may
also be designed so as to bring together a fluorescer-quencher
pair, as discussed above, leading to a decrease in a fluorescence
signal when the analyte sequence is present. In the above OLA
ligation method, the concentration of a target region from an
analyte polynucleotide can be increased, if necessary, by
amplification with repeated hybridization and ligation steps.
Simple additive amplification can be achieved using the analyte
polynucleotide as a target and repeating denaturation, annealing,
and ligation steps until a desired concentration of the ligated
product is achieved.
[0104] In another embodiment, the ligated product formed by
hybridization and ligation can be amplified by ligase chain
reaction (LCR). In this approach, two sets of sequence-specific
oligos are employed for each target region of a double-stranded
nucleic acid. One probe set includes first and second
oligonucleotides designed for sequence-specific binding to
adjacent, contiguous regions of a target sequence in a first strand
in the target. The second pair of oligonucleotides is effective to
bind (hybridize) to adjacent, contiguous regions of the target
sequence on the opposite strand in the target. With continued
cycles of denaturation, reannealing and ligation in the presence of
the two complementary oligo sets, the target sequence is amplified
exponentially, allowing small amounts of target to be detected
and/or amplified.
[0105] In various embodiments, it will be appreciated that since
the selected analytes in the sample can be tested for under
substantially uniform temperature and pressure conditions within
the substrate, the detection reagents in the various sample
chambers should have substantially the same reaction kinetics. This
can be accomplished using oligonucleotides and primers having
similar or identical melting curves, which can be determined by
empirical or experimental methods as are known in the art. In
another embodiment, the analyte is an antigen, and the
analyte-specific reagents in each detection chamber include an
antibody specific for a selected analyte-antigen. Detection may be
by fluorescence detection, agglutination, or other homogeneous
assay format. As used herein, "antibody" is intended to refer to a
monoclonal or polyclonal antibody, an Fc portion of an antibody, or
any other kind of binding partner having an equivalent function.
For fluorescence detection, the antibody may be labeled with a
fluorescent compound such that specific binding of the antibody to
the analyte is effective to produce a detectable increase or
decrease in the compound's fluorescence, to produce a detectable
signal (non-competitive format). In an alternative embodiment
(competitive format), the detection means includes (i) an
unlabeled, analyte-specific antibody, and (ii) a fluorescer-labeled
ligand which is effective to compete with the analyte for
specifically binding to the antibody. Binding of the ligand to the
antibody is effective to increase or decrease the fluorescence
signal of the attached fluorephore. Accordingly, the measured
signal can depend on the amount of ligand that is displaced by
analyte from the sample. In a related embodiment, when the analyte
is an antibody, the analyte-specific detection reagents include an
antigen for reacting with a selected analyte antibody which may be
present in the sample. The reagents can be adapted for a
competitive or non-competitive type format, analogous to the
formats discussed above. Alternatively, the analyte-specific
reagents can include a mono- or polyvalent antigen having one or
more copies of an epitope which is specifically bound by the
antibody-analyte, to promote an agglutination reaction which
provides the detection signal.
[0106] In various embodiments, the selected analytes can be
enzymes, and the detection reagents include enzyme substrate
molecules which are designed to react with specific analyte enzymes
in the sample, based on the substrate specificities of the enzymes.
Accordingly, detection chambers in the device may each contain a
different substrate or substrate combination, for which the analyte
enzyme(s) may be specific. This embodiment is useful for detecting
or measuring one or more enzymes which may be present in the
sample, or for probing the substrate specificity of a selected
enzyme. Examples of detection reagents include chromogenic
substrates such as NAD/NADH, FAD/FADH, and various other reducing
dyes, for example, useful for assaying hydrogenases, oxidases, and
enzymes that generate products which can be assayed by hydrogenases
and oxidases. For esterase or hydrolase (e.g., glycosidase)
detection, chromogenic moieties such as nitrophenol may be used,
for example.
[0107] In various embodiments, the analytes are drug candidates,
and the detection reagents include a suitable drug target or an
equivalent thereof, to test for binding of the drug candidate to
the target. It will be appreciated that this concept can be
generalized to encompass screening for substances that interact
with or bind to one or more selected target substances. For
example, the assay device can be used to test for agonists or
antagonists of a selected receptor protein, such as the
acetylcholine receptor. In a further embodiment, the assay device
can be used to screen for substrates, activators, or inhibitors of
one or more selected enzymes. The assay may also be adapted to
measure dose-response curves for analytes binding to selected
targets. The assays also may be immunoassays.
[0108] Reference will now be made to various exemplary embodiments,
examples of which are illustrated in the accompanying drawings.
Wherever possible, the same reference numbers are used in the
drawings and the description to refer to the same or like
parts.
[0109] In various embodiments, as illustrated in FIG. 1, the
substrate 10 has an array of features providing parallel processing
of several samples. The substrate can have dimensions, for example,
127.0 millimeters by 85.7 millimeters providing 384 sample
chambers. FIG. 2A illustrates a portion of substrate 10 showing the
features. The top view is through the film 20 shown in ghost lines.
FIG. 2B illustrates the exploded view showing film 20 with vents 40
aligning with base 30 and gas-permeable membranes 50. Sample
chambers 80 form a regularly spaced array. Sample introduced in
sample ports 60 flows to main channels 70 and from there to sample
introduction channels 110 into sample chambers 80. Each sample
chamber 80 is connected to venting channel 100 which joins venting
chamber 90 with sample chamber 80. Venting chamber 90 contains
gas-permeable membranes 50 and aligns with vents 40. The membrane
typically has a burst pressure of greater than 6 psi. In various
embodiments, the film 20 can be a PSA film with laser or
mechanically punched vent 40. A membrane layer can be bound to the
PSA film 20 and dye cut portions of the membrane layer can be
removed leaving gas-permeable membranes 50. The base 30 can be
injection molded or etched. The PSA film 20 with gas-permeable
membranes 50 attached can then be aligned with the base 30 and
laminated together. In various embodiments, the substrate can be
mated with a plate providing a plurality of contacts to provide
uniform pressure across the substrate where the contacts do not
provide substantial thermal transfer between the substrate and the
plate relative to the thermal transfer at the surface of the
substrate opposite to the plate. The plate can have through holes
to permit light to pass from the sample chambers to a detector for
detection. The plate is described in U.S. Pat. Pub. No.
2001/0029794.
[0110] Examples of suitable membranes include gas-permeable
membranes and/or porous membranes. For example, suitable porous
membranes may include Gortex.RTM. and other similar materials known
in the art of micro-porous membranes. Suitable gas permeable
membranes may include, for example, PDMS membranes. The membrane
materials also can be liquid impermeable to prevent a sponging
effect of the liquid that can reduce the volume in the sample
chamber. Some examples of suitable porous membrane materials are
described in U.S. Pat. No. 5,589,350 and some examples of suitable
gas-permeable membrane materials are described in U.S. Pat. Pub.
No. 2005/0164373 Titled "Diffusion-Aided Loading System for
Microfluidic Devices," the entire disclosures of both of which are
incorporated by reference herein.
[0111] In various embodiments, as illustrated in FIGS. 3A-3B, the
sealing plate 120 has a plurality of sealing protrusions 130 such
that each protrusion can align with a sample chamber. The sealing
plate 120 can be a thermal transfer die to isolate each of the
sample chambers. The sealing protrusions can have dimensions of
about, for example, 2.5 millimeters. FIG. 4 illustrates the
alignment with substrate 10. Sealing plate 120 seals sample
introduction channels 110 and venting channels 100 as shown in FIG.
5 by gaps 140.
[0112] FIGS. 3C and 3D show another sealing plate according to
exemplary embodiments of the teachings herein. FIG. 3C shows an
isometric perspective view of the sealing plate 320 in alignment
for sealing a substrate 310. FIG. 3D shows a view of the surface of
the sealing plate 320 having the protrusions 330 thereon. The
sealing plate 320 has a plurality of sealing protrusions 330 in the
form of substantially arc-shaped pins. The sealing protrusions 330
are configured and arranged on the plate 320 such that when the
plate 320 is aligned with the substrate 310, the protrusions 330
intersect the sample introduction and outlet (venting) channels 375
and 376 to seal the sample chambers 380. Rather than encircling the
entire sample chambers 380, the sealing protrusions 330 extend just
enough to contact the area of the substrate 310 around the sample
chambers 380 in the regions of the channels 375 and 376 to perform
the sealing function. Because the protrusions 330 contact
relatively small, focused regions of the substrate 310 during
sealing, less force on the sealing plate 320 may be required.
[0113] FIGS. 29 and 30 depict an exemplary embodiment of another
instrument that may be useful for sealing the sample chambers 2980
of a substrate 2910 (e.g., staking the substrate) and may be used
instead of a sealing plate like that shown in FIGS. 3A-3B. The
sealing instrument embodiment of FIGS. 29 and 30 includes a roller
2920 provided with a plurality of sealing protrusions in the form
of circumferential disks 2930 spaced from each other along the
longitudinal axis of the roller 2920. Sealing of the substrate
chambers 2980, for example, in a manner similar to that shown in
FIG. 5 by the gaps 140, may occur by rolling the disks 2930 across
the substrate 2910 using a sufficient force. For example, the force
may be enough to force an adhesive (e.g., a PSA) into the inlet and
outlet channels 2975 and 2976 and/or otherwise deform the channels
2975 and 2976 at the locations that the disks 2930 cross over the
channels 2975 and 2976 to prevent flow communication between the
channels 2975, 2976 and the sample chambers 2980.
[0114] The number and positioning (spacing) of the disks 2930 may
be selected such that the roller 2920 may pass over the substrate
2910 once to isolate all of the sample chambers 2980, sealing both
inlet and outlet channels 2975 and 2976 simultaneously. The number
and positioning of the disks 2930 may thus depend on a variety of
factors, including but not limited to, for example, the number of
sample chambers, the arrangement of the sample chambers, and the
arrangement of the inlet channels and outlet channels. According to
various exemplary embodiments, the number of disks may be reduced
by staking the shared main fluid channels 2970 and 2971 between two
lines of chambers 2980 in the embodiment of FIGS. 29 and 30 instead
of the inlet channels 2975 of each of the chambers 2980. In various
embodiments, it may be desirable to provide a main fluid channel
(or main fluid channels) with a zig-zag configuration so that it
can be intersected and sealed (staked) multiple times, for example,
with one pass of the roller 2920 over the substrate 2910. Further,
in the case of shared outlet (e.g., venting) channels between
adjacent rows of chambers, the number of disks also may be
reduced.
[0115] With reference now to FIG. 31, another exemplary embodiment
of a sealing instrument in the form of a roller 3120 is depicted.
In the embodiment of FIG. 31, the roller 3120 includes a plurality
of sealing protrusions in the form of circular pins 3130 provided
around the outer circumference of the roller 3120. The pins 3120
may be aligned over a row of chambers 3180 in a substrate 3110 and
seal the chambers 3180 in a manner similar to that depicted by the
gaps 140 in FIG. 5. However, rather than sealing all of the
chambers 3180 of the substrate 3110 at the same time, like the
sealing plate of FIGS. 3A and 3B, a few chambers 3180 get sealed as
the roller 3120 passes over the substrate 3110. This requires less
force to seal the chambers, since not all of the chambers 3180 are
sealed at once. Although each chamber 3180 in a row of chambers
3180 (e.g., x-direction in FIG. 31) may be sealed simultaneously,
it is also envisioned that the roller 3120 may be moved such that
less than all chambers in a row are sealed at the same time as the
roller 3120 passes over the substrate 3110. For example, the roller
3120 of FIG. 31 may be aligned over a portion of the substrate 3110
(e.g., shifted in the x direction shown in FIG. 31) so as to seal
only some chambers 3180 in each row sealed with a pass of the
roller 3120. The roller may then be shifted again over another
portion of the substrate 3110 and passed over the substrate 3110
again to seal the remaining chambers 3180 in each row.
[0116] It should be understood that the protrusions provided on the
rollers in the embodiments of FIGS. 29-31 may have a variety of
different sizes, shapes, and arrangements and those of ordinary
skill would understand how to make rollers with sealing protrusions
of other configurations and arrangements to perform desired sealing
of the sample chambers of a substrate. By way of example and not
limitation, it is envisioned that the sealing protrusions 3130 of
FIG. 31 may be replaced with the sealing protrusions 330 of FIG.
3C, with the arrangement of the protrusions 330 on the roller being
selected so as to seal the sample introduction and venting channels
from flow communication with the sample chambers of a
substrate.
[0117] In the exemplary embodiments of FIGS. 3A-3D and 29-31, the
force on the sealing plates 120 and 320 and rollers 2920 and 3120
required to effect sealing of the substrate 10, 310, and 2910 and
3110 may be relatively low, for example, due to the relatively
small contact area between the roller and the substrate during
sealing. Further, to reduce the force applied to achieve sealing,
plural plates 120 and 320 or rollers 2910 and 3110 may be used,
with less sealing protrusions provided on each of the plural
rollers or plates. In such a case, the positioning and/or shape of
the protrusions on each of the plural rollers or plates used to
perform complete sealing of all of the chambers of the substrate
may be selected so as to achieve sealing of some portions of the
substrate with a first roller or plate and other portions of the
substrate with a second roller or plate, etc. By way of example,
the sealing protrusions on a first roller or plate may seal the
outlet (e.g., venting channels) leading from the sample chambers,
while the sealing protrusions on a second roller or plate may seal
the inlet channels leading to the sample chambers. Those having
ordinary skill in the art would understand a variety of numbers and
configurations of rollers and/or plates and sealing protrusions on
those rollers and/or plates to accomplish desired sealing of the
substrate with a desired force applied.
[0118] In addition, it should be understood that in the embodiments
of FIGS. 29-31, the rolling of the rollers 2920 and 3120 over the
substrates 2910 and 3110 is intended to refer to relative motion
between the rollers 2920 and 3120 and the substrates 2910 and 3310.
Thus, either the rollers 2920 and 3120 can move while the
substrates 2910 and 3110 remain stationary, vice versa, or both the
rollers 2920 and 3120 and the substrates 2910 and 3110 may move. In
another exemplary aspect, the rollers 2920 and 3120 may be idle or
can drive the motion of the substrates 2910 and 3110. Likewise, in
the embodiments of FIGS. 3A-3D and 4, the movement of the plates
120 and 320 and the substrates 10 and 310 is relative.
[0119] It also is envisioned that, in the case of an integrated
instrument where the substrate passes through various stations, the
sealing rollers or plates of the may be placed in the transfer path
of the substrate. For example, the sealing rollers or plates may be
placed at a location after the substrate has been filled (e.g., at
a filling station) and before the next station, such as, for
example, a thermocycling block or instrument delivery port.
[0120] The exemplary sealing roller embodiments of FIGS. 29-31 may
be relatively easy to manufacture. They also may facilitate
appropriate alignment of the roller with the substrate during
sealing, as alignment is necessary only along the longitudinal axis
of the roller and not along the longitudinal axis of the
substrate.
[0121] In various embodiments, sample preparation instrument 150
can take raw biological sample from syringe 170 and prepare the
sample for introduction into substrate 10. Preparation of sample
can include extraction of nucleic acids and pre-treatment for
detection as described above. The instrument 150 docks with several
cartridges 160 that provide preparation. FIG. 7 illustrates the
cartridge 160. Sample syringe inlet 190 introduces the raw
biological sample into the cartridge. Pre-filled reagent reservoirs
180 provide the analyte-specific reagents for the assay to be
performed on substrate 10. The back of cartridge housing 200 is
open to permit interconnection of a flex circuit PCB device 210
with instrument 150.
[0122] Various other exemplary embodiments may provide mechanisms
for sealing, venting, controlling pressure, sample preparation,
mixing, and/or other features useful in multiple analyte detection
in a substrate in accordance with teachings of the disclosure and
are described in further detail below.
[0123] As discussed above, once the sample chambers of a substrate
have been filled, it may be desirable to seal filled chambers from
flow communication with each other and the various distribution
channels. Such sealing may be desirable, for example, before
various operations, such as, for example, PCR, may be performed,
and to prevent cross-contamination between wells. It also may be
desirable to provide a mechanism for sealing that is relatively
easily performed by a user of the biological testing device.
Further, it may be desirable to provide a mechanism for sealing the
substrate that does not require the use of sensors, heaters, and/or
other components that may be relatively difficult and costly to
implement.
[0124] Referring to FIGS. 8A-8C, an exemplary embodiment of a
substrate 810 is depicted. It should be understood that the
substrate 810 depicted in FIGS. 8A-8C is schematic for purposes of
simplifying the drawings. Thus, FIG. 8A shows only three sample
chambers 880 having inlet channels 875 and outlet (venting)
channels 876 connected to main fluid channels 870. The arrows in
FIG. 8A illustrate the direction of flow of sample for filling the
sample chambers 880. It should be understood that the substrate 810
could include an array of sample chambers 880 connected by fluid
distribution channels and may also include venting chambers (not
shown in FIG. 8A). By way of example, the substrate 810 may include
features similar to that shown in FIG. 1 or may have other
configurations in accordance with the teachings herein.
[0125] The substrate 810 may include a base 830, which may have a
configuration like the bases described above. The base 830 may be
covered with an adhesive-backed film 820. In various embodiments,
the adhesive-backed film 820 may be, for example, a PSA film. With
reference to the cross-section of the substrate 810 shown in FIG.
8B taken through line 8C-8C in FIG. 8A, prior to filling the
substrate 810 with biological sample, the adhesive-backed film 820
may be loosely applied over the base 830 such that the channels 875
and 876 and sample chambers 880 are in flow communication, thereby
permitting sample to be injected into the channels 875, 876, and
870 to fill the sample chambers 880. After the sample chambers 880
have been filled and it is desired to seal the chambers 880,
pressure may be applied to the film 820 so as to cause the adhesive
822 of the adhesive-backed film 820 to be forced into the channels
870, 875 and 876 and partially into the chambers 880, as depicted
in FIG. 8C. Forcing the adhesive into the channels 870, 875, and
876 closes the channels 890, thereby preventing flow communication
between the chambers 880 and between the channels 875, 876, and
870, and chambers 880.
[0126] By way of example, in the case of a consumable product, the
adhesive-backed film 820 may be loosely applied during
manufacturing and a user of the substrate 810 may apply the
pressure required for sealing after loading the substrate with
sample. Various mechanisms may be used to apply pressure, for
example, a substantially uniform pressure, over substantially the
entire adhesive-backed film. For example, the pressure could be
applied by the user's hand pressing on the film 820. Other
techniques for applying the pressure include using a motorized
stepping plate 825 (as schematically depicted in FIG. 9A) or a
motorized roller 826 (as schematically depicted in FIG. 9B). The
plate 825 and/or motorized roller 826 may be provided as part of
separate instrumentation or as a separate mechanism to be used with
the substrates, and may be used to seal numerous substrates. Those
having ordinary skill in the art would understand various
mechanisms that may be used to apply a sufficient force across the
film layer 820.
[0127] According to various exemplary embodiments, in addition or
as an alternative to providing pressure to force the adhesive 822
to fill the channels 875, 876, and 870, heat also may be used to
facilitate the filling of the channels with the adhesive 822.
However, it is envisioned that the use of heat is not necessary.
The appropriate thickness of the adhesive layer 822 may be selected
so as to perform adequate channel closing without injecting too
much adhesive into the chambers 880. By way of example, the
thickness of the adhesive layer 822 may be such that the entire
depths of the channels are filled with adhesive, leaving no air
pockets within the channels. Also, if sample is displaced during
sealing, it may be desirable to provide an adhesive thickness that
results in a substantially consistent amount of fluid being
displaced, while also minimizing the amount of wasted sample due to
displacement.
[0128] FIGS. 10A-10C illustrate another exemplary embodiment that
uses the adhesive of an adhesive-backed film to seal the channels
from the chambers in a biological testing device. With reference to
FIG. 10A, a protruding portion 850 is provided in a portion of each
of the inlet and outlet channels 875 and 876 that lead to and from
the chambers 880. As shown in the cross-sectional view of FIG. 10B,
the adhesive-backed film 820 may be lightly applied prior to
filling the substrate 810 with the sample, such that the channels
875, 876 and 870 may be in flow communication with the chambers
880, as described above with reference to FIG. 8B. Once the
substrate 810 has been filled with fluid (e.g., biological sample)
as desired, a pressure may be applied, for example, substantially
uniformly over substantially the entire surface of the film 820.
The pressure may be applied via any mechanism, including those
described above with reference to the embodiments of FIGS. 8 and 9,
and, optionally, heat may be applied. As a result of the pressure
on the film 820, the adhesive 822 of the film 820 will be forced
into the channels 875, 876 and 870. However, due to the presence of
the protruding portions 850, the adhesive 822 may come into contact
with the protruding portions 850 to seal the channels 875 and 876
from flow communication with the chamber 880 without filling the
entire channels 875 and 876. The adhesive 822 may make
substantially uniform contact over substantially the entire surface
of the protruding portion 850, as shown in FIG. 10C. Due to the
adhesive 822 making contact with the protruding portion 850 and not
filling the entire channel depth, sealing may be implemented with
much of the sample remaining in the channels 875 and 876 since a
relatively small amount of sample will may be displaced due to the
entry of the adhesive throughout the channels 875 and 876. For
example, the relatively small amount of sample that is displaced
may be so as to slightly deform the film layer 820. Further,
sealing may be relatively easy to accomplish since the adhesive
layer may only need to contact the protruding portions 850, rather
than filling the entire channels 875 and 876.
[0129] According to various exemplary embodiments, the protruding
portions 850 may be made of the same material as the base 830 and
may be formed via injection molding of the base 830. Other
materials and techniques for forming the protruding portions 850
also may be used and would be understood to those having ordinary
skill in the art. By way of example, and not limitation, the
protruding portions 850 may have a height equal to about one-half
the depth of the channels 875 and 876, and may span across the
width of the channels (e.g., in the left to right direction shown
in FIG. 10A).
[0130] In various exemplary embodiments, for example, when using a
motorized roller 826 such as that depicted in FIG. 9B to apply
pressure to the adhesive-backed film 820, the roller 826 may be
oriented such that its longitudinal axis (e.g., its axle) is
nonparallel (for example, perpendicular) to the channels 875 and
876 in which the protruding portions 850 are placed when performing
sealing. With such a nonparallel orientation to a channel during
sealing, the roller 826 may contact only a relatively small part of
the channels 875 and 876 at a time, thereby displacing a relatively
small amount of fluid (e.g., sample), if at all, from the channels.
In an exemplary embodiment, the roller 826 may be placed at a 45
degree angle to both axes of the substrate. Placing the roller 826
in a parallel orientation to a channels 875 and 876 during sealing
may cause a relatively large portion of fluid (e.g., sample) in the
channels 875 and 876 to be displaced therefrom and may potentially
increase the pressure to a sufficient amount so as to break the
seal formed between the film 820 and the base 830.
[0131] In various embodiments, a stationary rotating cam may be
used, for example, instead of the motorized plate or roller of
FIGS. 9A and 9B, to apply a pressure to the adhesive film layer of
a substrate in order to effect sealing of the sample chambers, for
example, of a stationary line of sample chambers. In conjunction
with such a motorized rotating cam, a member that applies pressure
to substantially the entire film layer prior to the sealing by the
cam may be used. By applying pressure to the film layer, a small
amount of sample in the sample chambers may move into adjacent
channels (e.g., sample introduction (inlet) and venting (outlet)
channels). As the rotating cam comes into contact with the
substrate to perform the sealing function, the pressure on the film
layer applied by the member may be removed at a rate substantially
proportional to the application of the pressure exerted by the cam
and the volume of the channels that will be reduced due to the
adhesive entering the channels. This may allow for accommodation of
increased pressure in the sample chambers caused by the sealing
operation and provide a defined sample volume in each sample
chamber, which may make thermocycling more efficient.
[0132] FIGS. 11, 12A, and 12B schematically depict a side
cross-sectional view of another exemplary embodiment of an
instrument useful for sealing fluid sample chambers in a biological
testing device. As shown, the device may include a substrate 1110
that includes a base 1130 and a cover 1135 (e.g., a PSA film layer)
for the base 1130 that together define a plurality of channels 1190
in flow communication with a plurality of sample chambers 1180 such
that the channels 1190 can deliver sample fluid to and from the
sample chambers 1180. For purposes of simplification, the schematic
depiction in FIGS. 11, 12A, and 12B show only sample chambers 1180
and introduction and outlet channels 1190 in flow communication
with those chambers 1180. It should be understood, however, that
the substrate 1110 may include venting chambers, main fluid
channels, sample introduction channels, venting channels, a fluid
inlet port for supplying fluid to the substrate, etc., in
accordance with the teachings herein.
[0133] The biological testing device may further include a sealing
carrier 1120, having a plate-like structure, that includes a
plurality of staking blades 1122 on a side of the carrier 1120
facing the substrate 1110. Prior to filling the substrate 1110, the
carrier 1120 may be separated from the substrate 1110 via a
temporary mechanical mechanism, such as, for example, a film hinge,
or may be separate form the substrate 1110 with no connection. The
carrier 1120 may be brought into contact with the substrate 1110
when sealing is desired. In various embodiments, the carrier 1120
and substrate 1110 may be provided with mating pins and holes or
other fastening mechanisms that are configured for insertion in one
direction but prevent separation in the opposite direction. The
carrier 1120 may be separated such that the blades 1122 are above
and at a distance from the upper surface (as shown in FIGS. 11 and
12) of the substrate 1110. In this separated configuration, the
channels 1190 are in flow communication with the sample chambers
1180 and a biological sample may be loaded into the substrate 1110
via a suitable inlet, as depicted by the arrow in FIG. 12A. It
should also be understood that an inlet for fluid supply may be
provided on the upper or lower surface of the substrate 1110 via a
port (not shown) that is in flow communication with the channel
1190 toward the right hand side of the figures.
[0134] Once the sample chambers 1180 of the substrate 1110 have
been filled, as shown in FIG. 12A, a force may be applied to the
carrier 1120 and/or the substrate 1110 so as to move the carrier
1120 toward the substrate 1110 (e.g., as shown by the arrows in
FIG. 12B). The force may be applied via a variety of mechanisms,
including but not limited to, for example, a motorized plate, a
motorized roller, a clamp, a user's hand, or other suitable
mechanisms. The force may be sufficient to bring the carrier 1120
into contact with the substrate 1110 (for example, by breaking or
deforming the mechanical mechanism that initially separates the
carrier 1120 from the substrate 1110). With the carrier 1120 and
substrate 1110 in the contacting position, as shown in FIG. 12B,
the staking blades 1122 are driven into the substrate 1110 at a
location of the channels 1190 proximate the chambers 1180. In
various embodiments, each channel 1190 may be aligned with a
differing blade 1122. In other embodiments, a single blade 1122 may
be aligned with a plurality of channels 1190, for example, the
blades 1122 in FIGS. 11, 12A and 12B may extend into the drawing
sheet to seal differing channels positioned along a direction into
the drawing sheet.
[0135] The blades 1122 may pierce, deform, or otherwise alter the
structure of the substrate 1110 at the locations so as to prevent
flow communication between the chambers 1180 and between the
channels 1190 and the chambers 1180. By way of example, the blades
1122 may pierce through film layer 1135 and enter the channels 1190
so as to block flow between the channels 1190 and corresponding
chambers 1180. In addition to blocking flow communication between
the channels 1190 and the chambers 1180, the carrier 1120 may be
configured to provide a seal (e.g., prevent flow communication)
between the substrate 1110 and the exterior, for example, through
the fill port in the substrate 1110.
[0136] The staking blades 1122 may be positioned relative to the
carrier 1120 such that they are properly aligned with the channels
1190 as desired to prevent flow communication between the channels
1190 and the sample chambers 1180 when the carrier 1120 is placed
into the contacting position with the substrate 1110 via the
applied clamping force. Providing the blades 1122 as part of the
carrier 1120 (e.g., an integral part of the carrier 1120) may
facilitate manufacturing and alignment of the blades 1122, as the
appropriate alignment can be assured prior to sealing the substrate
1110 with the carrier 1120. The appropriate alignment of the blades
1122 with the channels 1190 may ensure reliable sealing of the
substrate 1110 and may permit the use of relatively small staking
blades 1122. Relatively small staking blades in turn may require
less force to drive the blades into the substrate 1110, for
example, as compared to larger staking blades. The shape, size, and
material of the blades may be selected based on the thickness,
material, and other properties of the cover 1135.
[0137] The carrier 1120 also may include optical apertures 1123,
for example, windows, that are in substantial alignment with the
sample chambers 1180 when the carrier 1120 is in the sealing
position, as depicted in FIG. 12B. The optical apertures 1123 may
thus permit optical detection of the sample chambers 1180 during
biological testing/analysis. Other sealing plates according to
embodiments of the teachings herein also may include such
apertures.
[0138] In accordance with various exemplary embodiments, when using
the device of FIGS. 11, 12A, and 12B to perform PCR, the substrate
1110 and carrier 1120 may be placed between a clamp and a thermal
block of a PCR instrument. When the instrument applies a clamping
force via the clamp to the carrier 1120, for example, after the
substrate 1110 has been filled with sample as desired, the carrier
1120 may move toward the substrate 1110. As the carrier 1120 moves
toward the substrate, any mechanical mechanism that separates the
carrier 1120 and the substrate 1110 may fail (e.g., break or
deform) such that the carrier 1120 moves into a contacting position
with the substrate 1110 and the blades 1122 are driven into the
substrate 1110, as depicted in FIG. 12B. The device may be placed
between the clamp and thermal block of the PCR instrument either
prior to or after filling of the substrate 1110 with sample,
however, the clamping force will be applied after filling. It also
may be possible to use a clamping device that applies force to one
or more sections of the substrate and/or carrier 1120 at a time to
reduce the force required for the blades 1122 to penetrate and seal
the substrate 1110. Further, in various embodiments, as suggested
above, retaining clips or other mechanical connection means may be
provided to secure the carrier to the substrate in addition to the
blades 1122 themselves holding the carrier 1120 and substrate 1110
together.
[0139] In various embodiments, for example, in the case of PCR, the
sealing of the substrate sample chambers may be implemented using
the thermal block that is placed in contact with the substrate to
perform thermocycling. Using the thermal block to perform the
sealing function reduces a step in the processing of the substrate,
allowing the step of thermocycling and sealing to be performed at
the same time. Further, as will be explained, using the thermal
block to perform the sealing function may enhance thermal contact
and heat transfer between the thermal block and the sample
chambers, which may thereby reduce thermocycling times.
[0140] FIGS. 32 and 33 show schematic cross-sectional views of a
substrate 3210 including a base 3230 and film layer 3220. The base
3230 may define a plurality of features that, together with the
film layer 3220 form a fluid distribution network of fluid
distribution channels and chambers, as discussed herein. In the
view of FIGS. 32 and 33, for ease of illustration, a single sample
chamber 3280 is illustrated with channels 3275 and 3276 leading to
and from the chamber 3280. The substrate 3210 may be made of a
variety of materials in accordance with the teachings herein.
[0141] To perform the sealing function, the thermal block 3250 is
provided with a plurality of sealing protrusions (e.g., bumps) 3260
(which may be in the form of an array), only one of which is
depicted in FIGS. 32 and 33, that are configured and arranged to
align with the sample chambers 3280. To perform thermal cycling,
the thermal block 3250 is brought into contact with the film layer
3220 of the substrate 3210 and force is applied to move the
substrate 3210 and thermal block 3250 together, for example, via an
optical detection mechanism acting on the side of substrate 3210
opposite to the side the thermal block 3250 is in contact with, as
shown in FIG. 33. The protrusions 3260 may be configured such that
the protrusions 3260 deform the film layer 3220 and partially enter
the chamber 3280, such that the film layer 3220 contacts the inner
periphery of the opening of the chamber 3280, sealing the chamber
3280 from the channels 3275 and 3276, as shown in FIG. 33. Thus,
the protrusions 3260 on the thermal block 3250 permit direct
isolation of the sample in the sample chambers 3280, rather than
sealing portions of the channels 3275 and 3276.
[0142] The sealing protrusions 3260 of the thermal block 3250 may
have various configurations. In an exemplary embodiment, the
dimensions of the protrusions 3260 should be such that sufficient
contact is made to seal the chambers 3280 from the channels 3275
and 3276. By way of example, the sealing protrusions 3260 may have
a substantially circular configuration with a radius that is
slightly larger than the radius of the chambers 3280. The top of
the sealing protrusions 3260 may be substantially flat, as shown by
protrusions 3260a and 3260b in FIG. 34, or may be rounded, as shown
by protrusion 3260c in FIG. 34. Likewise, the sides of the sealing
protrusions 3260 may be rounded, as shown by protrusions 3260a and
3260c, or substantially flat, as shown by protrusion 3260b. In the
case of a sealing protrusion having a flat top and rounded sides,
like 3260a, a rounded protrusion may be formed with its top cut off
so as to be flat. A rounded configuration may provide a slightly
larger alignment tolerance with the sample chambers by permitting a
sliding movement to be implemented during positioning the thermal
block in contact with the substrate.
[0143] According to exemplary embodiments, the sealing protrusions
3260 may be machined directly on the thermal block 3250 or may be
formed on a metal insert secured to the thermal block 3250.
[0144] Due to the sealing protrusions of a thermal block entering
the sample chambers to perform sealing, as discussed above, it may
be desirable to permit a relatively small amount of displaced
sample contained in the chambers to escape. FIGS. 35 and 36
illustrate exemplary embodiments that permit the escape of a small
amount of displaced sample from the sample chambers when the
sealing protrusions of a thermal block enter the sample chambers to
seal the chambers.
[0145] Referring first to FIG. 35, a small recess 3261 (e.g.,
dimple) may be formed in the sealing protrusions 3260 of the
thermal block 3250 in order to fill with displaced fluid as a
result of reduction of the sample chamber volume during sealing. In
various exemplary embodiments, the recess 3261 may form a hole
through the sealing protrusions 3260 from one side to another to
permit a small amount of fluid displaced from the chamber to escape
if needed so as to avoid the potential for over-pressurization of
the chamber and potential adhesion failure of the film layer to the
base during thermal cycling. FIG. 35A depicts another exemplary
shape of a sealing protrusion 3260d on a thermal block 3250 for
performing sealing and also permitting displaced sample in a sample
chamber to escape. The sealing protrusion 3260d includes raised
perimeter portions 3263 and a recessed center portion 3264. The
raised perimeter portions 3263 may enter the sample chamber
proximate the edge (periphery) of the sample chamber and seal the
sample chamber. The recessed center portion 3264 may permit any
displaced sample from the chamber to escape during sealing.
[0146] FIG. 36 represents another exemplary embodiment that may be
used to protect against over-pressurization and/or adhesion failure
when the thermal block sealing protrusions 3260 enter the chambers
3280. In FIG. 36, a side escape channel 3278 that is slightly
deeper than the feed channels 3275 and 3276 may be provided in flow
communication with each sample chamber 3280. A main fluid channel
3270 that is used to supply sample to the inlet feed channel 3275
also is depicted in the exemplary embodiment of FIG. 36. During
filling, a small displaced amount of sample may escape through the
escape channel 3278, and may also leave a small amount of air at
the end of the escape channel 3278, which may end in a venting
chamber 3279 similar to the venting chambers 3290 at the end of the
vent (outlet) channel 3276, as are described herein. By forming the
escape channel 3278 with a greater depth than the channels 3275 and
3276, when the sealing protrusions of the thermal block enter the
chamber to seal off the channels 3275 and 3276, for example, as
depicted in FIG. 33, the sealing protrusions 3260 may not fully
seal the channel 3278 due to its greater depth. This may permit any
displaced fluid in the chamber 3280 to pass into the escape channel
3278 during sealing and thermocycling, which in turn, may reduce
the potential for over-pressurization in the chamber 3280 and
adhesion failure (e.g., leakage) of the film layer 3220.
[0147] Although the embodiments of FIGS. 32-36 above described a
thermal block having sealing protrusions (sealing protrusions 3260)
that mate with sample chambers to perform sealing, it is envisioned
that protrusions on a thermal block also may be configured and
arranged to achieve a sealing pattern similar to that shown by the
gaps 140 of FIG. 5. In other words, the thermal block and sealing
protrusions thereon may contact and seal the substrate at locations
of the sample introduction and venting channels in flow
communication with each sample chamber. Those of ordinary skill in
the art would understand how to configure and arrange sealing
protrusions on a thermal block to accomplish this type of
sealing.
[0148] FIGS. 38 and 39A-39C depict yet another exemplary approach
for achieving sealing of sample chambers in a substrate for
biological testing. Referring to FIG. 38, a schematic
representation of a substrate 3910 is shown, showing only three
sample chambers 3980 for ease of illustration. In accordance with
the teachings herein, the substrate 3910 may comprise a base 3930
and a film layer covering the base to form the sample distribution
network shown. In the exemplary embodiment, each sample chamber
3980 is in flow communication with a main fluid supply channel 3970
via a sample introduction (inlet) channel 3975. A venting channel
3976 leads from each sample chamber 3980 to a venting chamber 3990.
A through hole (not shown) leads through the substrate 3910 from
each venting chamber 3990 to a corresponding venting chamber 3992
provided in a main fluid outlet channel 3972 that connects to the
main fluid supply channel 3970, for example, in a U-shaped bend as
shown. The main fluid outlet channel 3972 terminates in an overfill
chamber 3995. A dissolvable plug of material 3900 is positioned in
the main fluid channel 3970 just downstream of the last
introduction channel 3975 and corresponding chamber 3980 and
upstream of the venting chamber 3992 corresponding to that chamber
3980. For example, the dissolvable plug 3900 may be positioned
before the U-shaped junction of the main fluid supply channel 3970
and the main fluid outlet channel 3972. The plug 3900 may be made
of a material that can fill the channel 3970 to block fluid flow
temporarily as the material dissolves at a controlled rate. By way
of example, the plug 3900 may be made of polyethylene glycol.
[0149] FIGS. 39A-39C illustrate various exemplary steps to fill and
seal the substrate 3910. In FIG. 39A, sample S (e.g., a biological
sample) is introduced to the substrate 3910. The sample S may be
pumped through the main fluid supply channel 3970, the introduction
channels 3975, and into the sample chambers 3980 via pressure from
a volume of oil (not shown in FIG. 39A), or other substance that is
immiscible with the sample, that is pumped behind the sample S. As
shown in FIG. 39A, the sample S that is introduced is sufficient to
fill the sample chambers 3980, the introduction and venting
channels 3975 and 3976, the venting chambers 3990, and the main
fluid supply channel 3970 from the inlet of the substrate 3910 (at
the right hand side in FIG. 39A) up to the plug 3900. The plug 3900
prevents the sample S from advancing past the plug 3900 until the
sample S has a chance to fill the various chambers and channels, as
shown in FIG. 39A.
[0150] Once the substrate 3910 has been filled with sample S, as
depicted in FIG. 39A, the plug 3900 may begin to dissolve, thus
allowing any remaining supply of sample S to the substrate 3910 and
the oil O behind it to flow in the main fluid supply channel 3970
past the location of the plug 3900, as shown in FIG. 39B. The oil O
may continue to be supplied to the substrate 3910 such that it
fills the main fluid supply channel 3970, the main fluid outlet
channel 3972, the venting chambers 3992, and reaches the overfill
chamber 3995, as shown in FIG. 39C. Due to the immiscibility of the
oil O and sample S, once the oil fills the portions of the
substrate 3910 described above and shown in FIG. 39C, the oil acts
to seal the inlet and outlet of each of the sample chambers 3980,
for example, so that further processing of the sample in the
chambers 3980 may occur. The total volume of sample S and oil O
that are supplied to the substrate 3910 may be selected so as not
to fill the overfill chamber 3995 completely. According to various
embodiments, the main outlet channel 3972 may have a volume that is
larger than the main fluid supply channel 3970 since the channel
3972 fills with oil and does not affect the waste ratio of the
sample, assuming the time required to pump the oil through the main
fluid supply channel 3970 is not too great.
[0151] In an exemplary aspect, the pumping of the sample into the
substrate 3910 may be at a substantially constant pressure so that
the pressure is not excessive so as to burst the seals during the
time between filling the last well and breaking through the plug
3900. To allow the sample S to reach the plug 3900, a vent hole
(not shown) that permits gas (e.g., air) to escape the substrate
3910 may be provided. According to various embodiments, a
mechanical sealing mechanism may be desired at the inlet and outlet
of channels 3970 and 3972 to prevent oil from being pumped out due
to potentially expanding sample S, for example, during PCR and/or
thermocycling. In various embodiments, it also may be desirable to
exert pressure on the oil to pressurize the fluids to reduce bubble
formation. In an exemplary aspect, such force may be placed on the
film layer covering the base.
[0152] According to various embodiments, the oil sealing approach
described above may include variations. By way of example, and not
limitation, instead of the dissolvable plug 3900, a burst valve or
a Timavo valve can be used. In this case, rather than waiting for
the plug to dissolve, the sample slug can be immediately followed
with an oil slug. Also, rather than utilizing the vents described
above, membranes (porous or gas permeable) can be used between the
venting channel and the main fluid outlet channel. In yet another
exemplary embodiment, in place of the vents described with
reference to FIGS. 38 and 39A-39C, a hydrophobic stop can be used
between the venting channel and the main fluid channel. Further,
the configuration of the main channels may be more along the lines
of FIG. 8A, where sample is introduced along one main channel, and
venting occurs along the other main channel, without the two
channels being connected in the U-shaped junction of the embodiment
of FIGS. 38 and 39A-39C. The main venting channel could either
terminate in a large overfill chamber or use a valve to a waste
port. The sample can be followed directly with oil in the main
fluid supply channel. After the sample has been filled, oil can be
introduced into the main venting channel, either at the same time
as the oil is introduced into the main fluid supply channel, or
either one can precede the other. This configuration can be
combined with any of the venting approaches in accordance with the
teachings herein.
[0153] FIGS. 40A-40C depict yet another exemplary embodiment of an
approach to seal sample chambers in a substrate. The embodiment of
the substrate 4010 shown in the partial views of FIGS. 40A-40C is
similar in design to the substrate 3910 of FIGS. 38 and 39A-39C
with respect to the main fluid supply channel 4070, sample
introduction channels 4075, sample chambers 4080, and main outlet
channel 4072. In the embodiment of FIGS. 40A-40C, the sample
chambers 4080 are in flow communication with venting chambers 4090
via venting channels 4076 that are tunneled through the base 4030,
as shown best in FIGS. 40B and 40C. The venting chambers 4090 are
in flow communication with the main fluid outlet channel 4072 via
connection channels 4073, as shown in FIGS. 40A-40C, which may be
formed at the upper surface of the base 4030, rather than through
the base 4030 like channels 4076. For simplicity, FIGS. 40A-40C
show only a partial view of the substrate 4010 depicting one sample
chamber 4080. It should be understood, however, that an array of
such sample chambers 4080 and corresponding introduction and
venting channels and chambers are provided.
[0154] The exemplary embodiment of FIGS. 40A-40C includes a plug
(e.g., bead) 4000 of super-absorbent material disposed in the
venting chambers 4090. Such a super-absorbent material may be
configured so as to absorb many times the bead's volume in water
relatively rapidly and retain the water under relatively high
pressure so that water is prevented from filtering through the bead
and exiting therefrom. Examples of such super-absorbent materials
that may be used to form the bead 4000 include, but are not limited
to, polymers, such as, for example, cross-linked polyacrylate.
[0155] Prior to filling the substrate 4010 with sample, the bead
4000 may be positioned within the venting chamber 4090 such that it
does not occupy the entire volume of the venting chamber 4090, as
depicted in FIG. 40B. After sample S is introduced into the
substrate 4010 and fills the chambers 4080, as depicted in FIG.
40C, the sample S exits through the venting channels 4076 and into
the venting chambers 4090 in contact with the beads 4000, causing
the beads 4000 to absorb the sample S and swell. The swelling of
the beads 4000 in turn occupies the venting chambers 4090 and
blocks the venting channels 4076, thereby sealing the chambers 4080
so that sample therein cannot escape and further processing, such
as PCR, may be performed. In various embodiments, the sample
introduction channels 4075 may be sealed via a fluid that is
immiscible with the sample, such as, for example, oil, in a manner
similar to that described with reference to FIGS. 38 and 39A-39C.
Other sealing mechanisms in accordance with the present teachings
also may be used to seal the sample introduction channels 4075 and
would be understood by those skilled in the art based on the
present teachings.
[0156] The beads 4000 may be configured so as not to block the
connection channels 4073 when they have absorbed the sample S,
thereby permitting escape of gas (e.g., air) into the main outlet
(vent) channel 4072. In various embodiments, it may not be
necessary to permit gas to escape the venting chambers and out of
the substrate, however, since even at elevated temperatures, the
super-absorbent beads 4000 may retain water without the tendency
for the water to evaporate. This may be especially true when the
beads are used for a relatively small fraction of their absorptive
capacity.
[0157] In various embodiments, the beads 4000 may be substantially
spherical and have a diameter of about 1 mm prior to absorbing
sample. It is envisioned, however, that other shapes and sizes of
the beads 4000 may be used. In particular, the shape and size of
the beads 4000, as well as the configuration of the venting chamber
4090, may be selected such that the beads may swell and deform to
substantially match the surface of the opening of the venting
channel 4076 to the venting chamber 4090. Further, venting chamber
4090 may be small enough so that the bead 4000 may only expand to a
limited extent to prevent the bead 4000 from absorbing more than a
predetermined amount of sample. In the exemplary embodiment of
FIGS. 40A-40C, the venting chamber 4090 may have a substantially
egg-shaped configuration, narrowing toward the end where the
venting channel 4076 enters the chamber 4090.
[0158] According to various other embodiments, the venting channel
4076 may be provided in the surface of the base 4030, as previously
described herein, rather than having the cylindrical configuration
shown in FIGS. 40A-40C. With such a configuration, it may be more
difficult to ensure that the beads 4000 expand so as to conform to
the relatively square profile defined by the film layer covering
the base. However, it may be possible for the expansion of the
beads 4000 to cause enough pressure on the film layer 4020 to
create a rounded top surface. Further, the sealing may not need to
be complete if a secondary sealing mechanism also is employed, such
as, for example, the oil sealing described with reference to FIGS.
38 and 39A-39C.
[0159] In yet further exemplary embodiments, the bead 4000 may be
in the form of a superporous hydrogel bead that acts to absorb
sample and permit passage of gas.
[0160] FIGS. 41A and 41B show an embodiment of a substrate 4110
that includes the use of a porous, hydrophobic pellet 4100 inserted
into a venting chamber 4190 for both sealing the sample chambers
4180 and also permitting venting of gas from the substrate. Such a
pellet 4100 may be relatively easy to manipulate and insert into
the individual venting chambers 4190. Further, placing the pellets
4100 substantially in the same plane as the chambers 4180, as
described below, may be advantageous during thermocycling, for
example, to provide more efficient heat transfer and/or a more
effective thermal contact between a thermal block and the
substrate.
[0161] The substrate 4110 may have a configuration similar to that
described in the embodiment of FIGS. 40A-40C, with the exception
that the venting channel 4176 is not formed through the substrate
4110, though it may be if desired, but rather on the surface of the
substrate 4110. In the embodiment of FIGS. 41A-41B, the venting
chamber 4190 may have a substantially square edge at the side of
the chamber 4190 proximate the venting channel 4176 and a tapered
edge at the side proximate the connection channel 4173. A
substantially cylindrical porous hydrophobic pellet 4100 may be
inserted into the venting chamber 4190 into contact with the
tapered side first and then pushed forward against the square side,
as depicted in FIG. 41B. The top surface of the pellet 4100 may sit
slightly above the surface of the base 4130, as shown in FIG. 41B,
and may be pushed down so as to be substantially flush with the
surface of the base 4130 when the film layer 4120 is adhered to the
base 4130.
[0162] After filling the substrate 4110 with sample S, the pellet
4100 may prevent the sample from flowing past it, as shown in FIG.
41B, but could allow for the passage of air due to its porous
nature. According to various embodiments, the pellets 4100 for each
venting chamber 4190 may be formed from a coil of material, similar
to a coil of string, and cut into small pieces and placed in a
consistent orientation so as to be properly positioned in each
venting chamber 4190.
[0163] In various embodiments, the sample introduction channels
4175 leading to the sample chambers 4180 may be sealed via a fluid
that is immiscible with the sample, such as, for example, oil, in a
manner similar to that described with reference to FIGS. 38 and
39A-39C. Other sealing mechanisms in accordance with the present
teachings also may be used to seal the sample introduction channels
4175 and would be understood by those skilled in the art based on
the present teachings.
[0164] According to still further embodiments, a material capable
of breaking down to a gas, for example, with elevated temperatures
may be used to seal sample chambers of a substrate. With reference
to FIG. 42A, a partial perspective view of a substrate 4210 is
depicted. The view in FIG. 42A shows a main fluid supply channel
4270 that is in flow communication with two sample introduction
channels 4275 that lead to sample chambers (not shown). A material
4200 that is configured to break down into a gas at elevated
temperatures is placed at the junction between the main fluid
supply channel 4270 and the introduction channels 4275. According
to various exemplary embodiments, the material 4200 may be
predeposited in the substrate 4210. The material 4200 may be or may
be made insoluble in water so it does not dissolve upon contact
with the sample S as the sample S fills the substrate 4210, as
depicted in FIG. 42B. For example, the material may be deposited
with an organic solvent.
[0165] The material 4200 may break down into a gas, like a blowing
agent, at elevated temperatures, for example, at temperatures
associated with PCR and/or thermocycling. By way of example, the
material 4200 may turn to gas at temperatures of about 90.degree.
C. Thus, as shown in FIG. 42C, the material 4200 may turn into a
gas, for example, after the substrate 4210 has been heated in the
first step of a PCR process. This creates a bubble B at the
junction that prevents the sample S from migrating from one sample
chamber to another, thereby sealing the sample chambers. Although
FIGS. 42A-42C depict the use of the material 4200 at the junction
between a main fluid supply channel 4270 and introduction channels
4275, it should be understood that this approach also may be used
to seal the sample chambers at their outlet (e.g., vent) sides. In
various embodiments, the interior channel surfaces may be
relatively hydrophobic in order to prevent sample from wicking
around the bubble B.
[0166] FIGS. 43A-43C depict yet another exemplary approach for
sealing the sample chambers of a substrate for biological testing
in accordance with the present teachings. Again, for ease of
discussion, only one sample chamber 4380 of the substrate 4310 is
depicted in FIGS. 43A-43C. With reference to FIG. 43A, the
substrate 4310 includes a small blind chamber 4382 in flow
communication with the sample introduction channel 4375 upstream of
the sample chamber 4380 between the sample chamber 4380 and a main
fluid supply channel 4370. Assuming that pressure filling is used
to supply sample to the substrate, the sample S progresses through
the main fluid supply channel 4370, into the introduction channel
4375 and sample chamber 4380, and into the venting channel 4376,
without substantially filling the blind chamber 4382, as shown in
FIG. 43B. This is due to the pressure resistance of the relatively
small blind chamber 4382 in comparison to that of the chambers
4380. Thus, the blind chamber 4382 contains trapped air after the
remainder of the substrate has been filled.
[0167] Upon further processing of the sample in the sample
chambers, for example, during thermocycling and/or PCR, elevating
the temperature of the substrate 4310 causes the trapped air in the
chamber 4382 to expand, introducing an air pocket P in the portion
of the introduction channel 4375 slightly upstream and downstream
of the chamber 4382, as shown in FIG. 43C. The air pocket P serves
to seal the chamber 4380. Skilled artisans would understand that a
blind chamber, similar to 4382, also may be provided on an outlet
side of the sample chamber 4280 in conjunction with the venting
channel 4376 to perform sealing.
[0168] Although FIGS. 43A-43C show the blind chamber 4382 being
completely filled with trapped air, it should be understood that a
small amount of sample S may enter the blind chamber 4382 during
filling. However, as the substrate 4310 is heated during biological
testing (e.g., PCR and/or thermocycling), the trapped air in the
chamber 4382 will expands, forcing out any sample in the chamber
4382.
[0169] The embodiment of FIGS. 2A and 2B provides an exemplary
configuration for achieving venting of gas via membranes from a
substrate of a biological testing device, while substantially
preventing leakage of the sample and/or other fluids that fill the
substrate. Various additional exemplary embodiments for achieving
venting in accordance with the disclosure are described below.
[0170] With reference to FIG. 13, a partial perspective isometric
view of an exemplary embodiment of a substrate 1310 including an
array of features providing parallel processing of several samples
for carrying out biological testing is illustrated. The substrate
1310 includes a base 1330 and a film layer 1320. Sample chambers
1380 may form a regularly spaced array, as depicted, for example,
in FIG. 1. Sample introduced to the substrate (e.g., via sample
ports like sample ports 60 in the embodiment of FIG. 1 and not
shown in FIG. 13) flows to main channel 1370 and from there to
sample introduction channels 1375 into sample chambers 1380. Each
sample chamber 1380 is connected to a venting channel 1300 which
joins a venting chamber 1390 with the sample chamber 1380.
[0171] The base 1330 and film layer 1320 may be made from any of
the materials described herein for the bases and film layers,
respectively. By way of example, the film layer 1320 may be a COP
film or a PSA film and may be thermally bonded to the base 1330,
which may be manufactured from a plastic material, for example,
such as COP. As described above with reference to the embodiment of
FIGS. 2A and 2B, the film layer 1320 may be provided with a
plurality of vent holes 1340 configured to be aligned with the
venting chambers 1390 when the film layer 1320 is attached to the
base 1330. Rather than providing a die-cut membrane in each of the
venting chambers 1390 like in the embodiment of FIGS. 2A and 2B,
however, in the exemplary embodiment of FIG. 13, a venting membrane
strip 1350 is provided on a side of the film layer 1320 that faces
away from the base 1330 (e.g., on the top surface of the film layer
1320). Providing such a strip configuration may facilitate
manufacturing of the device, for example, by permitting a single
strip to serve as the vent membrane for a plurality of chambers
1390 and/or permitting relatively simple manufacturing of the strip
1350 and manipulation of the strip 1350 into position due to its
relatively large size. The strip 1350 may be attached to the film
layer 1320 via adhesive and may be aligned with a row of venting
chambers 1390, as depicted in FIG. 13. Thus, a plurality of
membrane strips 1350 may be positioned on the top surface of the
film layer 1320 so as to align with a plurality of rows of venting
chambers 1390 of the substrate 1310.
[0172] As shown in FIG. 13, according to various exemplary
embodiments, the adhesive used to bond the membrane strip 1350 to
the film layer 1320 may also be in the form of a strip 1325, for
example, a PSA strip, provided on a bottom side of the membrane
strip 1350 and having a length and width substantially similar to
the membrane strip 1350. Vent holes 1326 may be provided through
the adhesive strip 1325 and in alignment with the vent holes 1340
of the film layer 1320 and the venting chambers 1390. The vent
holes 1326 and the vent holes 1340 may be formed via a laser,
mechanically punching, or other suitable technique for forming vent
holes. If PSA strips 1325 are used to bond the membrane strips 1350
to the film layer 1320, the film layer may be, for example, a COP
film layer thermally bonded to the base 1330.
[0173] The membrane strips 1350 may be gas-permeable or porous and
also liquid impermeable so as to prevent leakages of the sample
fluid from the substrate 1310. In various exemplary embodiments,
the membrane strips may be made of materials such as those
described above for the membranes 40 of the embodiment of FIGS. 2A
and 2B.
[0174] In various exemplary embodiments (not shown in the figures),
instead of providing adhesive strips 1325 to bond the membrane
strips 1350 to the film layer 1320, the film layer 1320 may be a
double-sided adhesive PSA layer such that adhesive on one side of
the layer 1320 is used to bond the film 1320 to the base 1330 and
adhesive on the opposite side is used to bond the membrane strips
1350 to the film layer 1320. In such an embodiment, the vent holes
1340 would be formed through the entire film layer 1320 including
both adhesive sides of the layer 1320.
[0175] According to various exemplary embodiments, when using
multi-chamber devices for parallel processing of plural fluid
samples, it may be desirable to cycle the device through various
temperatures. For example, it may be desirable to perform PCR,
which requires thermal cycling of the device over a range of
temperatures, for example from about 60.degree. C. to about
95.degree. C. In such cases, relatively precise temperature control
in the individual sample chambers of a substrate, as well as
temperature uniformity over the entire substrate area may be
desired. Further, as discussed above, the ability to isolate the
individual chambers after filling the chambers (e.g., to prevent
flow communication between the chambers and between the chambers
and channels that lead to and from each chamber, such as fluid
introduction and venting channels), may be desired in order to
prevent cross-contamination during a biological testing process
such as PCR.
[0176] In order to perform thermal cycling, in accordance with
various exemplary embodiments, a thermal block may be placed in
contact with the multi-chambered substrate. Typically, the thermal
block is placed in contact with the film layer of the substrate
that, together with the cavities formed in the base of the
substrate form the fluid distribution network made up of, for
example, main fluid channels, a plurality of sample chambers (e.g.,
in an array) in flow communication with the main fluid channel by a
plurality of sample introduction channels, and a plurality of
venting chambers in flow communication with the plurality of sample
chambers via a plurality of venting channels. In other words, the
thermal block may be positioned in contact with the substrate on
the side of the base of the substrate that defines the various
channel and chamber openings. For example, in the exemplary
embodiments of FIGS. 1, 2, and 13 the thermal block may be
positioned in contact with the film layer 20 and with the membranes
1350 and film layer 1320.
[0177] Placing the thermal block on the same side of the base of
the substrate that the membranes are located, however, may impair
the ability to achieve effective and uniform thermal conductivity
between the thermal block and the sample chambers. In particular,
the presence of membranes, whether disposed between the film layer
and the base or on the side of the film layer facing away from the
base, may cause an irregular surface (e.g., a "bumpy" surface).
Such an irregular surface may prevent uniform contact of the
thermal block with the substrate, and in some cases, it may be
desirable to remove the membranes and add a metal lamination layer
instead of the film layer to perform PCR after the substrate has
been loaded with sample.
[0178] Further membranes in the form of strips of material may have
a potential to leak around the borders of the strips.
[0179] With reference to FIGS. 14-16, various exemplary embodiments
are schematically illustrated that provide gas-permeable or porous
membranes for venting a substrate on a side of the substrate
opposite to the side that is placed in contact with a thermal block
for performing thermal cycling of the sample loaded into the
substrate. FIG. 14 is an isometric perspective view of an exemplary
embodiment of a substrate 1410 for which membrane strips 1450 are
positioned on a side of the substrate 1410 (the side facing up in
FIG. 14) that is opposite to the side (the side facing down in FIG.
14) of the substrate 1410 that the thermal block is placed in
contact with during thermal cycling.
[0180] In the embodiment of FIG. 14, the substrate 1410 may
comprise a base 1430 covered with a film layer 1420 that together
define a fluid distribution network. FIG. 14 shows the side of the
base 1430 looking through the film layer 1420. As shown in FIG. 14,
the base 1430 and film layer 1420 may together define a plurality
of sample chambers 1480 that form a regularly spaced array. Sample
may be introduced in sample ports 1460 and may flow to main fluid
supply channels 1470 and from there to sample introduction channels
1475 into sample chambers 1480. Each sample chamber 1480 may be
connected to a venting channel 1400 that joins a venting chamber
1490 with the sample chamber 1480. A venting through hole (not
shown) may be formed from each venting chamber 1490 and through the
base 1430 so as to open at the side of the base 1430 facing upward
in FIG. 14. Elements 1421, 1423 and 1431 are indexing holes
provided in each layer of the substrate 1410 to provide appropriate
alignment of the substrate 1410 with other instrumentation, if
needed, for example, during filling and/or sample analysis.
[0181] FIG. 28 shows an exemplary venting through hole 2800 that
may be used in conjunction with a venting chamber 2890 associated
with a sample chamber 2880. The venting through hole 2800 in FIG.
28 may be used in the base 1430 of FIGS. 14-16. As shown in the
exemplary embodiment of FIG. 28, the through hole 2800 may have a
conical shape with a smaller opening leading from the venting
chamber 2890 and a larger opening formed at the underside of the
base. By way of example only, the opening leading from the venting
chamber 2890 may be about 100 .mu.m in diameter and the opening at
the underside of the base may be about 500 .mu.m in diameter. The
conical configuration shown in FIG. 28 is exemplary only, and
venting through holes in accordance with the teachings herein may
have a variety of configurations, including, for example,
cylindrical. The shape and size of the vent through holes may be
selected based on various factors, including, for example, the
manufacturing technique used to form the base, desired venting, and
other such factors.
[0182] With reference again to FIG. 14, the film layer 1420 and the
base 1430 may be made of any of the materials described herein for
film layers and bases. In various embodiments, the film layer 1420
may be a metal or polymer PSA film and may be bonded to the base
1430 via the adhesive, for example, by applying pressure and/or
heat. Further, the base 1430 may be etched, stamped, hot-embossed,
or injection molded to form the various chambers and channels.
Using a metal PSA film for film layer 1420 may be desirable to
achieve good thermal conductivity.
[0183] As shown in FIG. 14, the substrate 1410 may further include,
on the side of the base 1430 opposite to the side on which the film
layer 1420 is placed, a film layer 1425 formed with a plurality of
vent holes 1426. The vent holes 1426 may be configured and arranged
so as to be substantially aligned with the vent through holes of
the base 1430 described above. The film layer 1425 may be made of
any of the materials described herein as useful for forming a film
layer. By way of example, the film layer 1425 may be a PSA film
layer and may be configured to be adhesively bonded and aligned
with the base 1430. In accordance with various exemplary
embodiments, the vent holes 1426 may be formed in the film layer
1425 via laser or via mechanical punching. In an alternative
embodiment, the film layer 1425 may formed of a porous hydrophobic
material and the vent holes 1426 may be eliminated.
[0184] Gas-permeable or porous membrane strips 1450 may be placed
in contact with the film layer 1425 and in alignment with the vent
holes 1426. Each membrane strip 1450 may be arranged and configured
so as to cover a row of vent holes 1426. Examples of porous
membranes include Gortex.RTM. and other similar materials known in
the art and examples of selectively permeable membrane materials
include, for example, PDMS. The membrane strips 1450 can be liquid
impermeable so as to prevent leakage of sample from the substrate
and to prevent a sponging effect of the liquid that can reduce the
volume in the sample chamber. Other suitable porous membrane
materials are described in U.S. Pat. No. 5,589,350 and other
suitable gas-permeable membrane materials are described in U.S.
Pat. Pub. No. 2005/0164373 entitled "Diffusion-Aided Loading System
for Microfluidic Devices," both of which are incorporated
herein.
[0185] According to various embodiments, the film layer 1425 may be
a double-sided adhesive film layer, for example, a double-sided PSA
polymer film, and the membrane strips 1450 may be adhered to the
film layer 1425 via the adhesive provided on the side of the film
layer 1425 facing the strips 1450. In alternative exemplary
embodiments (not shown) the film layer 1425 may be a PSA film
having only one adhesive layer facing the base 1430. The membrane
strips 1450 may be adhered to the opposite side of the film layer
1425 via adhesive strips (e.g., PSA strips) having substantially
the same length and width as the membrane strips 1450. Thus, for
example, the membrane strips 1450 may be adhered to the film layer
1425 via adhesive strips similar to adhesive strips 1325 shown and
described with reference to the embodiment of FIG. 13. Like the
adhesive strips 1325 of FIG. 13, adhesive strips used to adhere the
membrane strips 1450 to the film layer 1425 may be provided with
vent holes that align with the vent holes 1426 of the film layer
1425 and with the vent through holes (not shown) provided in the
base 1430. When using PDMS membrane strips, additional adhesive may
not be needed as PDMS is self-adhering.
[0186] With reference now to FIG. 15, another exemplary embodiment
of a substrate having membranes for venting positioned on a side of
the substrate opposite to the side of the sample chamber and
channel openings in the base is shown. The exemplary embodiment of
FIG. 15 includes components and materials similar to those
described above with reference to the exemplary embodiment of FIG.
14, with the reference labels of such components being the same as
those used in FIG. 14. In addition, in the exemplary embodiment of
FIG. 15, recesses 1530 are formed in the base 1430. The recesses
1530 have substantially the same dimensions as the membrane strips
1550 and are configured to receive the membrane strips 1550 and a
film layer attached to the membrane strips 1550 for bonding the
membrane strips 1550 to the base 1430. The recesses 1530 may
facilitate proper alignment of the membrane strips 1550 relative to
the vent holes 1426 and vent through holes (not shown) of the base
1430 during placement of the strips 1550 on the substrate 1410.
Also, providing recesses 1530 having a depth substantially equal to
the thickness of the membrane strips 1550 may permit the membrane
strips 1550 to be positioned flush with the upper surface of the
substrate 1410. As such, a thermal block may be positioned in
contact with the upper surface (e.g., the membrane side of the
substrate 1410) and may make substantially uniform thermal contact
with the upper surface, thereby enhancing uniform thermal
conductivity. It should be understood, however, that a thermal
block also may be positioned in addition or instead in contact with
the bottom surface of the substrate 1410 (e.g., in contact with the
film layer 1420), as described with reference to the embodiment of
FIG. 14.
[0187] As described above with reference to the exemplary
embodiment of FIG. 14, in various embodiments, the membranes 1550
of FIG. 15 may be bonded to the surface of a single sided adhesive
layer 1425 via adhesive strips (not shown), for example, PSA
adhesive strips, having substantially the same length and width as
the membranes 1550. Again, however, if self-adhering PDMS strips
are used, additional adhesive is not needed. Regardless of how the
membrane strips 1550 are attached within the recesses 1530, the
depth of the recesses 1530 may be selected so as to accommodate
both the thickness of the membrane strips 1550 and the thickness of
an adhesive layer such that membrane strips 1550 are substantially
flush with the top surface of the substrate 1410. In other words,
the top surface of the substrate 1410 and the membrane strips 1550
placed in position in the recesses should be substantially flat and
uniform.
[0188] Yet another exemplary embodiment of a substrate for parallel
processing of biological samples that utilizes a venting membrane
on the backside of the substrate is shown in FIG. 16. The exemplary
embodiment of FIG. 16 includes many of the same components and
materials as described above with reference to the exemplary
embodiment of FIG. 14, and illustrated components that are the same
as those in the exemplary embodiment of FIG. 14 are indicated by
the same reference labels. The exemplary embodiment of FIG. 16
differs from that of FIG. 14, however, in that the membrane strips
1450 are replaced with a single venting membrane layer 1650
configured and arranged to cover substantially the entire top
surface of the substrate 1410, as depicted in FIG. 16.
[0189] Providing a single membrane 1650 may facilitate positioning
and attaching of the membrane 1650 to the substrate 1410, may
reduce the number of components, and thus also may facilitate
manufacturing. The membrane 1650 also may include a plurality of
optical apertures 1655 configured and arranged to be substantially
aligned such that the sample chambers 1480 (shown in FIG. 14) can
be optically detected during biological testing. It should be noted
that optical detection of the chambers 1480 can occur through the
optical apertures 1655 by providing a transparent film layer 1425
and transparent base 1430. The optical apertures 1655 may be
substantially circular, although apertures also may have shapes
other than circular.
[0190] According to various embodiments, in a manner similar to
that described with reference to FIG. 15, the base 1430 of FIG. 16
may be provided with a single large recessed region (not shown in
the view of FIG. 16) configured to receive the film layer 1425 and
membrane 1650. This may permit the membrane 1650 to lie flush with
the upper surface of the base 1430.
[0191] By providing the venting membranes 1450, 1550, and 1650 on
the side of the substrate 1410 opposite to the side that is placed
in contact with the thermal block during thermal cycling, as shown
in the exemplary embodiments of FIGS. 14-16, it may be possible to
achieve a more uniform and effective thermal conduction between the
thermal block and the substrate 1410. Moreover, isolation of the
sample chambers may be facilitated. For example, if staking and/or
filling channels with adhesive is used to effect isolation of the
sample chambers (e.g., blocking flow communication between sample
chambers and between sample chambers and channels), such techniques
may be performed at the side of the substrate opposite to the side
on which the membranes are placed. Thus, a lower force may be
applied to deform and/or puncture the film layer 1420 than would be
required to deform and/or puncture both a film layer and membranes.
Alternatively, sealing could occur on the same side as the venting
membranes, especially in the embodiment of FIG. 16 if pressure is
applied at the apertures 1655. The various membrane embodiments of
FIGS. 14-16 also may facilitate sealing of the sample chambers of
the substrate via the thermal block itself, for example, as shown
and described with reference to the exemplary embodiments of FIGS.
32-36. The various membrane embodiments of FIGS. 14-16 also may
facilitate manufacturing of the device as the membranes are
relatively easily manipulated and installed. Moreover, the thermal
conductivity may be improved by using a metal film layer 1420 and
reducing thickness of that layer, which may be placed in contact
with the thermal block during thermocycling. In some embodiments,
where it may be desirable to heat the substrate from both sides
(e.g., place a thermal block in contact with the membrane side and
opposite side of the substrate), optical detection may occur via
illumination from the edges of the substrate, though chambers may
be restricted to locations around the perimeter of the
substrate.
[0192] According to various embodiments, a heated cover used for
processing (e.g., a thermal block in a thermocycler), if placed in
contact with the venting side of the substrate in FIGS. 14-16, for
example, may also include holes or porous areas that align with the
vent holes and venting chambers to permit gas to escape during
loading of the substrate while in place in a thermocycler.
[0193] In some circumstances, it may be desirable to eliminate
venting membranes at each of the venting chambers. For example, by
eliminating the need for such membranes, manufacturing may be
facilitated and less costly since handling and assembly of the
membranes is not needed. Further, precise alignment of the
membranes will not be required and the chances of misalignment of a
membrane and potential consequent leakage of sample may be avoided.
Also, isolation (e.g., sealing) of the sample chambers of the
substrate may be facilitated and improved due to a reduction in
force needed to deform and/or penetrate substrate layers to achieve
isolation, as removal of the membranes may provide less layers to
deform and/or penetrate. Finally, removal of such venting membranes
may improve thermal conductivity and thermal uniformity, for
example, during PCR thermal cycling, due to the provision of a
substantially flat surface with which a thermal block may be placed
in contact and/or a decrease in thickness of the layers of the
substrate that a thermal block must act on.
[0194] According to various embodiments, a multi-chambered
substrate may include a plurality of micro-sized vent holes in the
film layer that, together with the base, forms the fluid
distribution network (e.g., sample chambers, main fluid channel,
sample introduction channels, venting channels, and venting
chambers) in the substrate. The micro-sized vent holes may function
both as capillary stops to prevent leakage of sample from a filled
substrate and as vents to release gas from the substrate.
[0195] A partial, isometric, perspective view of a multi-chambered
substrate 1710 that eliminates the need to provide a membrane over
each venting chamber is depicted in FIG. 17. The substrate 1710
includes a base 1730 and a film layer 1720 that is adhered to the
base 1730. The base 1730 defines a plurality of features and, with
the layer 1720 placed in position over the base, defines a main
fluid channel 1770 configured to receive the sample supplied to the
substrate 1710 and distribute the sample to a plurality of sample
introduction channels 1775 that are in flow communication to in
turn supply the sample to a plurality of sample chambers 1780. Each
of the sample chambers 1780 is in flow communication with a venting
chamber 1790 via a venting channel 1700.
[0196] The film layer 1720 is provided with a plurality of
micro-sized vent holes 1742 configured and arranged to be aligned
with the venting chambers 1790 when the film layer 1720 is in
position on the base 1730. By way of example only, the film layer
1720 may be a PSA film layer with adhesive on one side used to
attach the film layer 1720 to the base 1730. The film layer 1720
may be, for example, a PSA polymer film or a PSA metal film. The
vent holes 1742 may be sized so as to allow gas to escape from the
substrate 1710 while creating a fluidic stop that prevents the
sample within the substrate from leaking through the holes 1742.
For example, capillary forces may prevent the sample from passing
through the holes 1742 and out of the substrate 1710. In various
exemplary embodiments, the vent holes 1742 may have a dimension
(e.g., a diameter) ranging from about 1 .mu.m to about 10 .mu.m,
for example, about 5 .mu.m. In some embodiments, areas surrounding
the vent holes 1742 may be substantially free of adhesive to
prevent adhesive from flowing (e.g., cold-flowing) into and
reducing the diameter of the vent holes.
[0197] In some cases, it may also be desirable to provide venting
at the end of the fill channel 1770. Thus, the exemplary embodiment
of FIG. 17 also includes a venting chamber 1795 and corresponding
vent membrane 1798 provided at the end of the main fill channel
1770. The membrane 1798 may be contained in the venting chamber
1795 between the film layer 1720 and the base 1730, for example,
similar to the membranes 50 discussed with reference to the
exemplary embodiment of FIGS. 2A and 2B. The membrane 1798 thus may
be sized and configured to substantially fill the venting channel
1795. The membrane 1798 may be made of any material described
herein as suitable for such porous or gas-permeable membranes. A
vent hole 1749, which may be formed in the same manner and may have
a similar structure as the vent holes 1742, may be provided in the
film layer 1720 in a position aligned with the membrane 1798 and
venting chamber 1795. The venting chamber 1795 is relatively large
compared to the venting chambers 1790. Although the exemplary
embodiment of FIG. 17 depicts the use of the venting chamber 1795,
the membrane 1798, and the vent hole 1749, a substrate like that in
FIG. 17 but that does not include those features is also considered
as within the scope of the invention. In such a case, sufficient
venting may be provided solely by the use of vent holes 1742
corresponding to each venting chamber 1790.
[0198] Various techniques may be used to provide the micro-sized
vent holes 1742 in the film layer 1720. According to various
embodiments, laser micro-machining (e.g., drilling) may be used to
form the holes 1742 in the film layer 1740. For example, a laser
micro-machining process may be used to drill holes through the film
layer 1740 after it has been attached to the base 1730, without
penetrating the base 1730. One exemplary laser micro-machining
process developed by Oxford Lasers, Inc. (Oxon, United Kingdom)
uses an ultraviolet cold laser process capable of drilling holes
having a dimension (e.g., diameter) ranging from about 5 .mu.m to
about 10 .mu.m in the film layer 1720. This process may form about
10 holes to several hundred holes per second, for example about 16
holes per second, in the film layer 1720 after the film layer 1720
has been bonded to the base 1730, without damage to the base
1730.
[0199] The laser drilling process can be applied to a variety of
materials, including, but not limited to, for example, silicon,
glass, metal, and/or polyimide. Examples of holes laser-drilled in
various materials using Oxford Lasers, Inc. instruments are shown
in FIGS. 18A-18C. In particular, FIG. 18A shows a laser-drilled
hole of about 5 .mu.m in diameter in steel, FIG. 18B shows 2 square
laser-drilled holes with each side being about 50 .mu.m formed in
silicon, and FIG. 18C shows several holes about 50 .mu.m in
diameter formed laser-drilled in Kapton.
[0200] A technique that permits vent holes 1742 to be formed in the
film layer 1720 after the film layer 1720 has been bonded to the
base 1730 eliminates the need to precisely align the film layer
with the base, which may thereby facilitate manufacturing. In other
words, in a film layer that has pre-formed holes, precise alignment
of the film layer with the base during bonding is needed to ensure
alignment of the pre-formed holes with the venting chambers.
Moreover, a micro-machining technique for forming the holes, such
as that described above, for example, permits the size (e.g.,
diameter) of the vent holes to be altered as desired and
progressively. This may permit control over the pressure gradient
along the fill path during filling of the substrate.
[0201] Although the exemplary embodiment of FIG. 17 depicts a
single vent hole 1742 corresponding to each venting chamber 1790,
it should be understood that one or more vent holes 1742 may be
provided in communication with each venting chamber 1790. The
number of vent holes per venting chamber may be selected based on a
variety of factors, including size of the vent holes, desired
venting of the gases in the substrate, minimization of leakage of
sample from the substrate, and other factors. Results of tests
performed for substrates having differing number of vent holes
associated with each venting chamber are provided below.
[0202] Filling tests were performed on substrates defining a 24
sample-chamber array having a configuration substantially as shown
in the partial schematic representation of the substrate 1910
depicted in FIG. 19. In FIG. 19, the arrangement of the sample
chambers 1980, inlet channels 1975, venting channels 1976, venting
chambers 1990, main fluid channel 1970, and sample supply inlet
1960 are shown. The substrate used for the tests also included a
film layer like the film layer 1720 with vent holes 1742 aligned
with the venting chambers 1990. The substrates 1910 were made of a
COP base covered with an aluminum PSA film layer that included a 5
mm thick aluminum layer with a 1.5 mm thick PSA laminate layer.
[0203] In a first test configuration, a single vent hole of
approximately 10 .mu.m in diameter was laser-drilled in the film
layer and aligned with each venting chamber. In a second test
configuration, three vent holes of approximately 10 .mu.m in
diameter were laser-drilled in the film layer and aligned with each
venting chamber. In a third test configuration, six vent holes of
approximately 10 .mu.m in diameter were laser-drilled in the film
layer and aligned with each venting chamber. A syringe pump was
used to supply a red dye fluid to each substrate at a pump speed of
40 .mu.l/minute. Red dye was used to assist in observing the flow
and filling in the substrate.
[0204] For the first test configuration using a single hole for
each venting chamber 1990, no leakage was observed during filling.
For the second test configuration using three holes for each
venting chamber 1990, single droplet leakage was observed for three
vent locations. For the third test configuration using six holes
for each venting chamber 1990, single droplet leakage was observed
in two vent locations. Those skilled in the art would understand
that the number and/or size of vent holes provided for each venting
chamber may vary based on a variety of factors, including, the
sample being introduced, the pressure in the substrate, and other
factors. Overall, the size and number of vent holes may be chosen
so as to substantially prevent leakage of sample through the one or
more vent holes and out of the device, while permitting gas (e.g.,
air) to escape through the one or more vent holes.
[0205] Various exemplary embodiments may utilize a hydrophobic,
porous filter, substantially in the form of a fiber-like
configuration, in lieu of a venting membranes described earlier, to
permit gas (e.g, air) to escape the substrate while preventing
sample leakage therethrough. Although the embodiments described
below use a hydrophobic, porous fiber member, it may also be
possible to utilize a porous or gas-permeable membrane material
formed into a fiber-like structure. With reference to FIG. 44, an
exemplary embodiment of a substrate 4410 for biological sample
analysis is depicted. The substrate 4410 includes a base 4430 and a
film layer 4420 covering the base 4430. The substrate 4410 defines
a sample distribution network including an array of sample chambers
4480 in flow communication with a plurality of main fluid supply
channels 4470 via sample introduction branch channels 4475. Each
sample chamber 4480 also is in flow communication with a main
venting channel 4472 via branch venting channels 4476. A
hydrophobic, porous fiber 4400 may be placed in the main venting
channel 4472, as shown in FIGS. 44 and 44A. Thus, rather than each
sample chamber 4480 terminating in an individual venting chamber,
as described in other embodiments herein, a group of sample
chambers 4480 terminates in a common venting channel 4472. As shown
in the close-up view of FIG. 44A, the film layer 4420 may be
provided with vent holes 4425 aligned with the main venting channel
4472. Each adjacent pair of chambers 4480 and corresponding venting
channels 4476 may be associated with a vent hole 4425, as depicted
in FIG. 44A. The vent holes 4425 may permit gas to escape through
the fibers 4400 and out of the substrate 4410. The vent holes 4425
may be formed via a variety of techniques, including the laser
process described above with reference to FIG. 17. In an
alternative embodiment, rather than providing vent holes 4425 in
the film layer 4420, vent through holes may be formed from the
venting channel 4472 through the depth of the base 4430, opening to
the bottom of the base 4430 shown in FIGS. 44 and 44A.
[0206] The hydrophobic porous fibers 4400 may have a configuration
similar to such fibers used in the filtration industry to filter
impurities from water pumped into the fiber at pressures higher
than the outside of the fiber. Such fibers permit impurities to
flow through the pores of the fiber wall while water is retained.
In various other exemplary embodiments, the fibers 4400 may be in
the form of a resilient fiber cord that has a porous hydrophobic
coating. Similarly, in the case of use with a substrate for
biological testing, the filter 4400 can permit gas (e.g., air) to
pass therethrough while retaining sample. Thus, with the fibers
4400 in place in the substrate 4110, gas may be permitted to pass
through the main venting channels 4172 and fibers 4400 and out of
the substrate 4410 through vent through holes.
[0207] FIG. 45 shows an exemplary technique for placing the fibers
4400 in an assembly-line fashion into a plurality of bases 4430 to
form substrates 4410. A fiber supply roller 4500 may supply a
plurality of separate fibers 4400, for example, corresponding to at
least the number of main venting channels 4172 in a substrate. The
fibers 4400 may be secured in position in the main venting channels
4172 in the first base 4430 and the first base 4430 may move down a
belt or other similar device, thereby pulling the fibers 4400 with
it. As the bases 4430 move to the right shown in FIG. 45, new bases
4430 to be supplied with fibers 4400 are added to the left end.
After a desired number of bases 4430 have been supplied with fibers
4400, a film layer may be adhered to the bases 4430 and the parts
cut away from each other in the spaces between the parts shown.
[0208] FIGS. 46A-46C show an exemplary embodiment for securing
hydrophobic porous fibers in place in venting channels of a base
portion of a substrate. For simplicity, the base 4630 depicted in
FIGS. 46A-46C shows only two rows of sample chambers 4680 in flow
communication with a common main fluid supply channel 4670 and
differing main venting channels 4672. FIG. 46A depicts the base
4630 prior to the placement of the fibers 4600 in the channels
4672. A plurality of weld spots 4650 are deposited along the length
of each of the main venting channels 4672 in positions between
where adjacent venting channels 4676 intersect the main venting
channels 4672. By way of example, the weld spots 4650 may be formed
from a low melting point polymer deposited in the channels 4672,
for example, via an ink-jet type of device. In another example, the
base 4630 may be molded with the weld spots 4650. Between the weld
spots 4650, vent through holes 4640 may be provided in the base
from the channels 4672 to the bottom of the base 4630 in order to
permit gas to escape the substrate 4610. Alternatively, such vent
through holes may be provided in the film layer that covers the
base 4630, as has been described herein.
[0209] As shown in FIG. 46B, the fibers 4600 may be placed in the
channels 4672, for example via a fiber supply tool as was described
with reference to FIG. 45 above. A heated pressing instrument 4655
may be used to press the fibers 4600 into the channels 4672,
preferably while the fibers 4600 are held in tension. At the same
time, the heated instrument 4655 melts the weld spots 4650 to fuse
the weld spots 4650 and fibers 4600 together at the locations of
the weld spots 4650, as shown in FIG. 46C. This melting process may
serve to block the paths between the sample chambers 4680 and thus
may serve as a sealing mechanism for sealing the chambers 4680. A
series of bases 4630 may be formed in this way using the assembly
line process discussed in FIG. 45, with the film layers being
applied and the substrates being separated from each other by
cutting the fibers as described above. In other embodiments, a
heated instrument may be used after the film layer 4620 has been
applied in order to fuse the weld spots 4650 and fibers 4600
together and at the same time bond the film layer 4620 to the base
4630.
[0210] In yet further various embodiments, the instead of the weld
spots 4650, a two layer laminated material may be used.
[0211] FIG. 46D depicts a partial cross-section of the completed
substrate 4610 with the film layer 4620 adhered to the base 4630.
The cross-section in FIG. 46D is taken through a vent through hole
4640. As shown in FIG. 46D, the main venting channel 4672 may have
a depth that is less than a diameter of the fiber 4600 such that
the film layer 4620 presses down on the top of the fiber 4600 to
hold the fiber against the bottom of the channel 4672 and seal off
the vent through hole 4640 to ensure that no sample leaks around
the fiber 4600 and escapes through the through hole 4640. If
needed, the substrate 4610 may be held against a flat plate or the
like during filling to prevent the film layer 4620 from bulging
rather than maintaining a tight seal like that shown in FIG.
46D.
[0212] To further improve sealing of the vent through holes 4640
with the fiber 4600, a circular seal ring 4642 may be provided
around the vent through hole opening in the channel 4672, as shown
in FIGS. 47A and 47B. The ring 4642 may have a raised surface
relative to the bottom surface of the channel 4672 and provide a
flat surface to press against the fiber 4600 rather than, for
example, a rounded surface of the bottom of the channel 4672.
Further, because the surface of the seal ring 4642 is slightly
raised relative to the bottom of the channel 4672 in the area of
the vent through hole 4642, a better seal may be achieved between
the fiber 4600 and the vent through hole 4642.
[0213] Although FIG. 47B depicts a hollow tubular fiber structure,
it should be understood that porous hydrophobic fibers in
accordance with the present teachings may have a variety of
cross-sectional shapes, including, but not limited to, for example,
a solid circular cross-section (e.g., a rod).
[0214] FIGS. 48-50 show another exemplary embodiment of a substrate
4810 that includes a porous, hydrophobic fiber 4800 for retaining
sample in the substrate while permitting gas to escape. FIGS. 48A
and 48B show opposite sides of the base portion 4830 of the
substrate 4810, while FIG. 48C shows the substrate 4810 including
film layer 4820 as viewed from the same side as in FIG. 48B. In the
embodiment of FIGS. 48A-48C, the base 4830 is transparent and the
film layer 4820 may be metallic, such as, for example, an aluminum
PSA film layer. However, it should be understood that the base 4830
and film layer 4820 may be made of any materials described herein
as suitable for making a base and film layer. To avoid confusion
between features of the substrate 4810 on the near and far sides,
FIGS. 48A and 48B are shown as being opaque.
[0215] With reference to FIG. 48A, the substrate 4810 may comprise
a base 4830 that, together with the film layer 4820, defines a
sample distribution network that includes a plurality of sample
chambers 4880 which all connect to a common main fluid supply
channel 4870. Each chamber 4880 is in flow communication with the
main fluid supply channel 4870 via a sample introduction channel
4875. Each chamber 4880 also is in flow communication with a
venting channel 4876 that leads to a main venting channel 4872
provided in the side of the base 4830 facing up in FIG. 48B, i.e.,
opposite to the side in which the other features discussed above
are provided. The substrate 4810 further includes a sample inlet
port 4860 in flow communication with the main fluid supply channel
4870. An initial portion 4865 of the main fluid supply channel 4870
may have a serpentine configuration so as to permit passive mixing
of the sample, for example, of an eluted sample, prior to
introducing the sample to the introduction channels 4875. A more
detailed explanation of using a serpentine channel to achieve
sample mixing is provided below.
[0216] A hydrophobic, porous fiber 4800 may be provided in the main
venting channel 4872 in a manner similar to that described above
with reference to the exemplary embodiments of FIGS. 44-47. As
shown in FIGS. 48C and the close up views of FIGS. 49 and 50, a
film layer 4820 may cover the side of the base 4830 shown in FIG.
48A and a portion of the film layer 4820 may wrap around the base
portion to cover and seal the channel 4872 and fiber 4800. Flow
communication between the venting channels 4876 and the main
venting channel 4872 may be provided via a vent through hole 4840
that leads from the end of the venting channels 4876 to the venting
channel 4872. According to various exemplary embodiments, in a
manner similar to that described above in FIGS. 47A and 47B, the
vent through holes 4840 may terminate in the main venting channel
4872 in a raised sealing rim 4842 that presses against the fiber
4800, as shown in partial cross-sectional view of FIG. 49.
[0217] Vented air may pass into the porous fiber 4800, which may be
in the form of a hollow tube as shown or may have other
configurations, as described above. The air may pass down the fiber
4800 and/or exit the fiber 4800 into the channel 4872 that the
fiber 4800 lies in. According to various embodiments, a single vent
hole 4825, shown in FIG. 50, may be provided in the film layer 4820
and aligned with the main venting channel 4872, permitting any air
leaving the substrate 4810 to pass therethrough. Providing a single
vent hole 4825 may limit the potential of sample escaping from the
substrate due, for example, to improper sealing of the vent
passages after filling the substrate 4810. However, it should be
understood that plural vent holes also may be formed in the film
layer 4820 to allow air to escape therethrough.
[0218] FIGS. 51A and 51B show the opposing sides of another
substrate provided with a porous, hydrophobic fiber venting member
according to various embodiments of the present teachings. The
substrate 5110 of FIGS. 51A and 51B has substantially the same
structure as the substrate 4810 described above, except that the
main fluid supply channel 5170 is provided in the same side of the
base 5130 as the main venting channel 5172. The two channels 5170
and 5172 are depicted in the view of FIG. 51B and are on a side of
the substrate opposite to the chambers 5180, sample introduction
channels 5175 and venting channels 5176. As with the main venting
channel 5172, flow communication between the introduction channels
5175 and the main fluid supply channel 5170 may be provided via
through holes (not shown) in the base 5130. The configuration of
FIGS. 51A and 51B may permit isolation of the sample chambers 5180
via a sealing (staking) mechanism on the detection side (e.g., the
side shown in FIG. 51B) of the substrate 5110. This may allow the
sealing mechanism to be provided on a portion of instrumentation
that is not part of a thermocycler and thus may be made of
materials that do not need to take thermal properties into
consideration.
[0219] Providing the sample chambers 4880 and 5180 in two rows, as
shown in the exemplary embodiments of FIGS. 48-51 may be
advantageous in that all of the chambers 4880 and 5180 are
positioned at an outer perimeter of the substrate 4810 and 5110.
This may avoid edge effects that may cause interior chambers of a
substrate to experience differing temperatures than temperatures of
chambers at a perimeter of the substrate. Thus, all of the chambers
may have a substantially uniform temperature, for example, during
thermocycling of the substrate. Even in the case where one row of
chambers is hotter than the other row of chambers if the
temperature difference is uniform between the two rows, the
temperature of the chambers may be uniform. Further, due to the
relatively small size of the substrates 4810 and 5110, for example,
in the configuration shown that includes 16 chambers, a smaller
thermal block may be used with reduced margin on either side, which
may decrease the overall size of the instrumentation used for
biological testing of the substrates 4810 and 5110.
[0220] Although the exemplary substrates 4810 and 5110 include an
array of two rows of chambers 4880 and 5180, the substrates may be
formed with any number of chamber rows. By way of example only, the
substrates may be formed with four rows of chambers, in which case
a main fluid supply channel may be positioned between a first pair
of chamber rows and a second pair of chamber rows. Two venting
channels may then be provided at the two opposite edges of the
substrate in conjunction with each of the pair of rows of chambers.
Any number of rows may be used, with the porous, hydrophobic
filters disposed inward from the edges of the substrate being
secured in position by a separate film or series of strips of film
on the underside of the substrate. The substrates also may include
negative template control sections, described in more detail with
reference to the embodiments of FIGS. 26 and 27, which according to
various embodiments may be provided substantially in the center of
the substrate array with corresponding sample inlet ports.
[0221] The substrates 4810 and 5110 may be assembled in a manner
similar to that described above with reference to FIG. 45, that is,
in a continuous fashion by tensioning the fibers 4800 and 5100 from
a roll and making plural substrates 4810 and 5110 in an assembly
line. According to various embodiments, after applying the film
layer to the bases, as described with reference to FIG. 45, the
substrates 4410, 4810, and 5110 may be left in a continuous
strip-like configuration (e.g., without separating the individual
substrates) and packaged in a reel 5200, as shown in the exemplary
embodiment of FIG. 52. The reel 5200 may have a cutter mechanism
5250, similar to a tape reel, in order to separate individual
substrates as desired. According to yet other exemplary
embodiments, the substrates may be left connected to one another
and supplied in an automated manner to a processing instrument,
such as, for example, a thermocycler or the like, in a continuous
and/or high throughput manner. This may permit processing of the
substrates without an operator handling each substrate
individually, which could potentially contaminate and/or damage
each substrate. Those having ordinary skill in the art would
understand how to package any of the substrate embodiments herein
in a continuous reel mechanism like that of FIG. 52.
[0222] In yet other embodiments, a thermocycler may be configured
to accommodate two sample substrates, for example, substrates 4810
or 5110. A perspective view of such a thermocycler 5300 is depicted
in FIG. 53A and a partial cross-sectional view is depicted in FIG.
53B. The substrates could be processed at both ends of the
thermocycler 5300 (e.g., the left and right sides) shown in FIG.
53A. As shown in FIG. 53B, the thermocycler 5300 may be provided
with a heated plate (thermal block) 5350 having a crowned profile
and pressure may be applied to the chamber array by providing
pressure on the outer edges of the substrate 5310 without
transmitting force through the open area (or window) 5305 directly
over the sample chambers of the substrates. A relatively thin,
narrow heated plate 5350 may be used and transmit sufficient
clamping force to the side walls of the substrates rather than
through a Peltier device or other component.
[0223] As discussed above, control over the pressure gradient along
the fill path during filling of the substrate may be provided, for
example, by controlling the size of vent holes, such as vent holes
1742 provided in the substrate 1710, as was described in relation
to the embodiment of FIG. 17. Other techniques also may be used,
either alone, in combination with the vent holes or other substrate
configurations in accordance with the disclosure, and/or in
combination with each other, to provide control over the pressure
gradient during filling of a substrate via positive pressure. It
may be desirable to control the pressure gradient by creating a
higher pressure in the venting channels so as to reduce the
potential for leakage from the substrate (e.g., through the vent
holes in a film layer).
[0224] By way of example, the hydrophobicity of the venting
channels of the substrate may be modified, for example increased,
to control the pressure gradient while filling the substrate with
sample. The hydrophobicity may be modified, for example, by adding
texture and/or increasing roughness (e.g., on a nano-scale level)
to the surface defining the venting channels. Such texturing and/or
increasing roughness may be introduced during the injection molding
process, for example, by texturing the mold as desired in the area
that forms the venting channels. Other techniques for modifying the
hydrophobicity of a surface defining the venting channels may
include providing a coating, or chemically treating the surface. By
way of example only, Kim et al., "Nanostructured Surfaces For
Dramatic Reduction Of Flow Resistance In Droplet-Based
Microfluidics," IEEE 2002, hereby incorporated by reference in its
entirety herein, teaches one technique for providing nanostructures
on a surface to alter hydrophobicity. It is envisioned that the
hydrophobicity of all or a portion of the venting channels may be
altered.
[0225] According to various embodiments, the venting channel
configuration (e.g., geometry) also may be modified in order to
control the pressure gradient during filling of the substrate. For
example, the venting channels may be provided with a region of
reduced cross-section so as to increase the pressure within the
venting channel and reduce the potential for leakage. With
reference to the exemplary embodiments of FIGS. 20A and 20B, a
sample chamber 2080 and corresponding venting channel 2000 in flow
communication with the chamber 2080 is depicted. As shown, the
venting channel 2000 may be provided with a reduced cross-section
R, for example, toward an end of the venting channel 2000 that
leads to the venting chamber 2090. In the exemplary embodiment of
FIG. 20A, the reduced cross-section R is achieved by narrowing the
side walls defining the channel 2000. In the exemplary embodiment
of FIG. 20B, the reduced cross-section R is achieved by raising the
bottom surface of the channel 2000 at the location R in comparison
to the remainder of the bottom surface of the channel. In other
words, the depth of the channel 2000 is less at the location than
the depth of the remainder of the channel.
[0226] According to various embodiments, for example, when filling
a multi-chambered substrate via positive pressure (e.g., via
pumping, syringe, etc.), it is desirable to know when to stop the
filling once the various chambers and channels have been filled in
order to control over-pressurization and/or sample leakage. An
exemplary mechanism for determining when to stop filling the
substrate includes providing optical sensors in association with
the sample chambers. The sensors, which in an exemplary embodiment
may be an optical sensor including a photodiode and LED, can detect
the presence of the sample by a difference in the index of
refraction and send a signal to stop the filling process (which may
occur either manually or automatically). Due to potential increased
costs and manufacturing complexity associated with such a
sensor/feedback mechanism, it may be desirable to provide a
relatively simple substrate design configured to passively and
automatically stop sample delivery so as to avoid
over-pressurization and/or leakage of the substrate.
[0227] FIGS. 21A-21C schematically depict exemplary steps of
filling a multi-chambered substrate that includes a venting
mechanism, such as, for example, either a vent membrane or vent
hole, as have been described above. It should be noted that FIGS.
21A-21C are simplified for the purposes of showing the principles
of filling channels and/or chambers of a substrate and leakage of
fluid that may occur due to over-pressurization. Thus, in the
figures, only a single channel is illustrated with a vent
positioned at a distal end of the channel.
[0228] Referring to FIG. 21A, a volume of sample S is delivered,
for example, via positive pressure, at an end of the channel 2190
(or chamber) opposite to an end at which vent membrane 2150 is
disposed. The dashed arrows in FIGS. 21A-21C indicate the direction
of sample delivery and movement through the channel 2190, with the
shaded area representing the sample S and the nonshaded area
representing gas (e.g., air). The channel 2190, in an exemplary
configuration, may be defined by a base portion 2130 and a film
layer 2120, with the film layer 2120 comprising a vent hole (not
shown) positioned beneath the membrane 2150 to permit gas to escape
therethrough. In FIG. 21A, as the sample S is forced via pressure
through the channel 2190, gas (e.g., air) residing in the channel
2190 is compressed and passed out of the channel 2190 through the
vent hole (not shown) and the gas-permeable membrane 2150, as shown
by the solid arrow proximate the membrane 2150. As the sample S
continues to move downstream in the channel 2190 (e.g. toward the
membrane 2150), gas that is between the sample S and the end of the
channel 2190 continues to be released through the vent membrane
2150, as shown in FIG. 21B.
[0229] Eventually, due to the continued positive pressure applied
at the end of the channel 2190 proximate the dashed arrow, as shown
in FIG. 21C, the sample S reaches a location in the channel 2190
corresponding to the vent hole (not shown) and membrane 2150 (e.g.,
the end of the channel 2190 opposite the end to which pressure is
applied). If further pressure is applied after the sample S reaches
the position shown in FIG. 21C, the channel 2190 becomes
over-pressurized and the sample S may leak out of the vent hole and
membrane 2150. For example, in the case of a gas-permeable, liquid
impermeable membrane 2150, the membrane may burst due to
over-pressurization and/or sample S may leak around the edges of
the membrane 2150, as depicted by the arrow in FIG. 21C.
[0230] In FIGS. 21A-21C, it should be understood that the membrane
2150 may be eliminated and a vent hole of micro-size may be used
instead, as described, for example, with reference to the
embodiment of FIGS. 17 and 19.
[0231] Leakage of sample out of the substrate may depend upon
various factors, including, for example, the configuration of the
substrate, the applied pressure, the method of pressure generation
(e.g., via syringe, pump, constant pressure source, etc.),
properties of the membrane material and/or configuration of vent
holes, and other factors that may influence the extent to which the
substrate becomes over-pressurized during filling with sample.
[0232] According to various embodiments, providing an additional
vent upstream of the vent 2150 of FIGS. 21A-21C may alleviate
over-pressurization and leakage. FIGS. 22A-22C schematically depict
the filling process that occurs for the channel 2190 of FIGS.
21A-21C when an additional, bypass venting mechanism 2250 is placed
upstream of the vent membrane 2150.
[0233] In FIG. 22A, like in FIG. 21A, sample S is delivered, for
example, via positive pressure, at an end of the channel 2190 (or
chamber) opposite to an end at which gas-permeable vent membrane
2150 is disposed. The dashed arrows in FIGS. 21A-21C indicate the
direction of sample delivery and movement through the channel 2190,
with the shaded area representing the sample S and the nonshaded
area representing gas (e.g., air). The channel 2190, in an
exemplary configuration, may be defined by a base portion 2130 and
a film layer 2120, with the film layer comprising a vent hole (not
shown) positioned beneath the membranes 2150 and 2250 to permit gas
to escape therethrough. Alternatively, as described with reference
to FIGS. 21A-21C above, the membranes 2150 and 2250 may be
eliminated, and a micro-sized vent hole in the film layer used
instead.
[0234] In FIG. 22A, as the sample S is forced via pressure through
the channel 2190, gas (e.g., air) residing in the channel 2190 is
compressed and passed out of the channel 2190 through the vent
holes (not shown) and the gas-permeable membranes 2150 and 2250, as
shown by the solid arrows proximate the membranes 2150 and 2250. As
the sample S continues to move downstream in the channel 2190 (e.g.
toward the membrane 2150), gas that is between the sample S and the
end of the channel 2190 continues to be released through the vent
membrane 2150, with no gas being released through the membrane 2250
while the sample S moves into the region of the channel 2190
aligned with the membrane 2250, as shown in FIG. 22B.
[0235] Eventually, due to the continued positive pressure applied
at the end of the channel 2190 proximate the dashed arrow, as shown
in FIG. 21C, the sample S reaches a location in the channel 2190
corresponding to the vent hole (not shown) and membrane 2150 (e.g.,
the end of the channel 2190 opposite the end to which pressure is
applied). In the additional upstream venting arrangement depicted
in FIG. 22, however, if further pressure is applied after the
sample S reaches the position shown in FIG. 22C, gas trapped in the
channel 2190 upstream of the sample S may be released through the
membrane 2250 and corresponding vent hole (not shown), as indicated
by the solid arrow proximate the membrane 2250. By releasing the
gas through the additional upstream venting mechanism (e.g.,
membrane 2250 in FIGS. 22A-22C), over-pressurization of the channel
2190 may be prevented and the sample movement in the channel 2190
will stop and the sample S will remain in the position shown in
FIG. 22C without leaking from the channel 2190.
[0236] In order to provide the proper functioning of the upstream
bypass venting mechanism, as described with reference to FIGS.
22A-22C, however, the location of the upstream venting mechanism
and the volume of the sample supplied must be selected so as not to
cause over-pressurization or incomplete sample delivery. Examples
of how over-pressurization and incomplete delivery may occur if the
sample volume and upstream venting position are not chosen
appropriately are schematically depicted in FIGS. 23A and 23B,
respectively.
[0237] Referring to FIG. 23A, incomplete sample delivery may occur
if the location of the upstream venting mechanism 2355 and the
volume of delivered sample S are not selected appropriately. In
other words, the sample S will not reach and fill the end portion
of the channel (or chamber) 2390 and gas (e.g., air) will become
trapped underneath the venting mechanism 2350 downstream of the
sample S. The situation in FIG. 23A may occur when the amount of
sample S supplied to the channel 2390 and the location of the
upstream bypass vent mechanism 2355 are such that the sample S
advances past the vent mechanism 2355 prior to reaching the vent
2350 at the end of the channel 2390 where it is desired to collect
the sample S. In this situation, as depicted in FIG. 23A, as
positive pressure is supplied to the channel 2390, shown by the
dashed arrow, the sample S is moved within the channel 2390 toward
the vent mechanism 2350. However, as the sample S moves past the
upstream vent mechanism 2355, the sample front has not yet reached
the vent mechanism 2350, but continued application of pressure
causes gas in front of the sample S (i.e., to the left of the
sample S in FIG. 23A) to escape through the upstream vent mechanism
2355. This upstream venting of gas results in the pressure becoming
equalized with the atmosphere despite the continued application of
pressure in the channel 2390. Due to the pressure equalization,
there is no pressure to cause further advancement of the sample S
within the channel 2390, thus resulting in incomplete delivery of
the sample S to the desired location (e.g., the end of the channel
beneath the vent mechanism 2350 in FIG. 23A).
[0238] On the other hand, as depicted in FIG. 23B, the location of
the upstream venting mechanism 2355 and the amount of sample S
delivered to the channel 2390 may be selected such that the sample
S completely blocks both venting mechanisms 2355 and 2350 once the
sample S has advanced through and reached the end of the channel
2390. In this situation, continued application of pressure to the
channel 2390, as indicated by the dashed arrow in FIG. 23B, may
result in sample S leaking from the venting mechanism 2350, as
shown by the solid arrow in FIG. 23B. Sample leakage through the
venting mechanism 2350 and/or 2355 may occur substantially as
described with reference to FIG. 21C.
[0239] Referring now to FIG. 24, an exemplary embodiment of a
multi-chamber substrate 2410 may include an upstream venting
mechanism that protects against over-pressurization and leakage, as
described in FIGS. 22A-22C, while also including features that
avoid the problems described in FIGS. 23A and 23B. As shown in FIG.
24, the substrate 2410, which may include a base and film layer in
accordance with various embodiments of the disclosure, defines a
plurality of sample chambers 2480 forming an array. The chambers
2480 are in flow communication with a plurality of sample
introduction chambers 2475, which distribute sample supplied to the
substrate 2410 via a main fluid channel 2470. The chambers 2480
also are in flow communication with venting chambers 2490 via
venting channels 2476. The venting chambers 2490 are associated
with venting mechanisms (not shown), such as, for example, the
various membrane embodiments or micro-sized vent holes described
herein.
[0240] Upstream of the chambers 2480, the substrate 2410 is
provided with a venting mechanism 2455, which may be, for example,
in the form of a vent hole in a film layer of the substrate 2410
covered with a venting membrane. This upstream venting mechanism
2455 may allow gas (e.g., air) to escape from the substrate 2410
after the various channels and chambers have been filled such that
over-pressurization and sample leakage out of the venting
mechanisms associated with the venting chambers do not occur.
[0241] In accordance with the exemplary embodiment of FIG. 24, the
substrate 2410 also defines an overfill channel 2475 and overfill
chamber 2495. The overfill channel 2475 leads from the downstream
end of the main fluid channel 2470 and terminates in the overfill
chamber 2495. The purpose of the overfill channel 2475 and overfill
chamber 2495 is to provide a collection reservoir for the sample so
as to ensure that complete delivery of the sample to the chambers
2480 and venting chambers 2490 occurs. Providing an overfill
chamber 2495 of sufficient size allows for a sufficient volume of
sample S to be loaded into the substrate 2410 to ensure complete
delivery of the sample S, without a risk of overfilling and/or
overpressurizing the substrate 2410 such that leakage may
occur.
[0242] The overfill channel 2475 may have a smaller cross-sectional
area than the main fluid channel 2470. The smaller cross-section
will increase the fluidic resistance (e.g., pressure) encountered
by the sample S as it fills the substrate 2410. As such, the
overfill chamber 2495 will fill only after the remaining chambers
2480 and 2490 and channels 2470, 2420, and 2400. In various
embodiments, rather than a straight overfill channel of reduced
cross-section, an overfill channel having a serpentine
configuration may be used as depicted in FIG. 24 and/or a
combination of serpentine configuration and reduced cross-section
may be used. The serpentine configuration can lengthen the overfill
channel in comparison to a straight overfill channel substantially
without increasing the overall size of the substrate. The
lengthening and serpentine configuration of the overfill channel
also may function to increase fluidic resistance encountered by the
sample such that the overfill chamber fills after the remaining
portions of the substrate.
[0243] According to various embodiments, the inlet sample volume
requirement may be calculated as follows to ensure that all of the
sample chambers 2480 are filled and the substrate is not
overpressurized. Assuming that the substrate 2480 has 24 sample
chambers, as shown in FIG. 24, the inlet sample volume=(the volume
of all 24 chambers 2480)+(the volume of all 24 venting chambers
2490)+(the volume of the main fluid channel 2470)+(the volume of
the sample introduction channels 2420)+((the volume of the overfill
chamber 2495)/2) Thus, the sample volume tolerance using the above
inlet sample volume is 1/2 the volume of the overfill chamber,
which will allow the volume to fill all of the sample chambers,
without over-pressurizing the device.
[0244] According to various embodiments, aspiration of sample into
the chambers can be assisted by moving the overfill chamber to the
inlet to provide protection against over-aspirating the sample.
[0245] As discussed above, in various embodiments, it may be
desirable to integrate sample preparation with a multi-chamber
array substrate. For example, it may be desirable to elute nucleic
acid from a membrane and supply the eluted sample volume directly
to a substrate. Before filling the sample chambers with the eluted
sample, however, it is desirable to ensure that the eluted sample
has been sufficiently mixed to substantially homogenize the
concentration of the sample prior to filling the sample chambers.
If the eluted sample is not sufficiently homogenized, a
concentration gradient may result in the substrate chambers. For
example, the concentration of nucleic acid may be higher in
upstream chambers than in downstream chambers of the substrate.
Such a concentration gradient in the substrate may impair
detection, quantization (e.g., for gene expression) and/or analysis
of the biological sample being tested.
[0246] To mix the eluted sample so as to obtain a substantially
homogenized concentration, an external mixing force, for example,
via a vortex or the like, may be applied to the collected sample
and the mixed sample may then be introduced into the substrate. In
an alternative embodiment, however, it may be desirable to provide
a mechanism for mixing eluted sample as part of the substrate
itself. FIG. 25 shows an exemplary embodiment of a substrate
configuration that provides for mixing of eluted sample within the
substrate itself prior to the eluted sample being loaded into the
sample chambers. In the exemplary embodiment of FIG. 25, passive
mixing of the sample may occur in the substrate via a relatively
simple design and without moving parts.
[0247] In the multi-chamber array substrate 2510 shown in FIG. 25,
a tube 2505 configured to collect eluted sample (e.g., a sample of
nucleic acid eluted from a membrane) is positioned in flow
communication with a sample inlet port 2560 of the substrate 2510.
The tube 2505 may collect eluted sample prior to any mixing, for
example, nucleic acid sample eluted directly from a membrane. The
substrate 2510 includes an array of sample chambers 2580, venting
chambers 2590, sample introduction channels 2575, venting channels
2576, and a main fluid channel 2570, similar to other substrate
embodiments described herein.
[0248] In addition to the various features listed above, the
substrate 2510 also defines a serpentine mixing channel 2565 that
connects the inlet port 2560 and the main fluid channel 2570 in
flow communication with each other. The serpentine channel 2565
serves to lengthen the distance the sample travels between being
supplied to the inlet port 2560 and filling the sample chambers
2580. By increasing the distance, and thus time, the sample travels
prior to filling the chambers 2580, diffusion may be increased in
the sample thereby mixing the sample and promoting homogenization
of the sample concentration. In other words, moving a plug of
liquid, such as a volume of eluted sample, through a channel of
sufficient length prior to introducing the sample into the sample
chambers may take advantage of the recirculation patterns that
occur along the axis of the microfluidic channel that results from
the plug of liquid having a parabolic velocity profile with
substantially flat menisci at both ends of the plug. With a long
enough mixing channel, and thus time, such recirculation patterns
may act to mix the sample plug and provide a substantially uniform
concentration prior to the sample being introduced into the sample
chambers. Enhanced mixing may also occur by providing the mixing
channel 2565 with sharp corners rather than rounded corners and/or
by applying various surface finishes configured to enhance
mixing.
[0249] Thus, the mixing channel 2565 provides a passive mixing
feature that is integral with the substrate 2510. This permits
direct loading of the substrate 2510 with eluted sample without
prior mixing, while ensuring that the sample filling the chambers
2580 will have a substantially uniform concentration for all of the
chambers 2580. The serpentine mixing channel 2565 may be relatively
large in comparison to the main fluid channel 2570. By way of
example only, the main fluid channel 2570 may be approximately 150
.mu.m wide by approximately 50 .mu.m deep, while the width of the
mixing channel 2565 may range from approximately 1 mm to
approximately 2 mm and have the same depth as the main fluid
channel 2570.
[0250] A straight mixing channel may be used rather than the
serpentine mixing channel shown in FIG. 25, however, using a
straight channel having approximately the same length of the
serpentine channel may increase the overall dimensions of the
substrate 2510. Thus, the serpentine configuration provides a
benefit of providing a sufficient length over which diffusion of
the eluted sample can occur, without substantially increasing the
overall dimensions of the substrate. Further, the bends in the
serpentine channel configuration may promote additional mixing of
the sample as it travels through the channel.
[0251] Yet another exemplary embodiment of a multi-chamber array
substrate 2610 is depicted in FIG. 26. The view of the substrate
2610 shows the various features of the base 2630 of the substrate
that, together with a film layer, as has been described for various
substrate embodiments above, form a sample fluid distribution
network including main fluid channels 2670a, 2670b, and 2671, inlet
channels 2672 leading from the main fluid channels to sample
chambers 2680, and venting channels 2600 leading from sample
chambers 2680 to venting chambers 2690. Thus, the view in FIG. 26
is through a film layer applied over the side of the base 2630 that
has openings defining the various features described above. Further
features of the substrate 2610 include sample inlet ports 2660 for
supplying sample to the substrate 2610 and to main fluid channels
2670a, 2670b, and 2671, overfill chambers 2695 and overfill
channels 2675 leading from main fluid channels 2670b and 2671 to
the overfill channels 2695. According to various exemplary
embodiments, the substrate 2610 also may include indexing holes
2621 to help align and position the substrate 2160 in various
biological testing apparatuses, such as, in conjunction with
thermal blocks and the like for performing PCR or other biological
analysis.
[0252] The substrate 2610 of FIG. 26 may be made of a variety of
materials for the base and film layers, including any of the
materials that have been discussed above. In particular, the
materials chosen may be PCR compatible materials. By way of
example, the base 2630 may be made molded from COP (e.g., ZEONOR
1420R) and the film layer that together with the base 2630 defines
the sample distribution network (e.g., the sample chambers,
channels, venting chambers, etc.) may be an aluminum PSA layer. In
addition, it is envisioned that various sealing, venting, and
mixing mechanisms that have been described above may be used in
combination with the substrate depicted in FIG. 26 and those having
skill in the art would understand based on the teachings herein how
to combine those mechanisms and/or various structural
configurations associated with those mechanisms with the embodiment
of FIG. 26. By way of example only, in various embodiments, the
substrate of FIG. 26 may be combined with any of the venting
mechanisms illustrated in FIGS. 14-16. That is, the base 2630 of
the embodiment of FIG. 26 may replace the base 1430 shown in FIGS.
14-16 and be combined with the other components shown in those
figures, including the film layer 1420, the film layer 1425, and
any of the membranes 1450, 1550, and 1650. Other structural aspects
of the substrate 2610 shown in FIG. 26 are discussed below in more
detail.
[0253] As noted above, the exemplary embodiment of FIG. 26 includes
two inlet fluid supply ports 2660. One of the ports 2660 is in flow
communication with main fluid supply channels 2670a and 2670b and
the other inlet port 2660 is in flow communication with main fluid
supply channel 2671. Main fluid supply channels 2670a and 2670b are
in parallel connection with each other and the inlet port 2660,
however, the inlet port 2660 also may supply fluid to main fluid
channels that are serially connected, for example, as shown in the
exemplary embodiment of FIG. 27. The main fluid supply channels
2670a and 2670b are configured to supply fluid (e.g., biological
sample) to a first group of sample chambers 2680 (e.g., 16 chambers
in FIG. 26) and the main fluid supply channel 2671 is configured to
supply sample to a second group of chambers 2680 (e.g., 8 chambers
in FIG. 26). The main fluid supply channels 2670a and 2670b and the
first group of chambers 2680 are not in flow communication with the
main fluid supply channel 2671 and the second group of chambers
2680.
[0254] This configuration of two inlet ports 2660 for supplying two
differing sample chamber networks that are not in flow
communication with each other permits two differing samples to be
supplied to the substrate 2610 and/or differing biological testing
(analysis) to be performed within the same substrate 2610.
Moreover, according to various embodiments, the dual fluid
distribution network provided in the substrate 2610 may provide a
negative template control mechanism in order, for example, to test
for false positives. By way of example, the inlet portion 2660
connected to the main fluid channels 2670a and 2670b may be
supplied with a biological sample for which PCR analysis may be
desired, while the inlet port 2660 in flow communication with the
main fluid supply channel 2671 may be supplied with a blank sample
(such as, for example, an elution buffer such as deionized water or
Tris HCl). Analysis, such as via optical detection (for example, by
detection of a fluorescent signal), of both groups of sample
chambers may be performed and if a signal is detected in the sample
chambers filled with the blank sample, this may indicate that the
substrate is contaminated or otherwise susceptible to giving a
false positive result.
[0255] Although the exemplary embodiment of FIG. 26 depicts 16
sample chambers in flow communication with one inlet port 2660 and
8 sample chambers in flow communication with the other inlet port
2660, any number of sample chambers may be provided in flow
communication with each inlet port. However, when using one group
of sample chambers and corresponding inlet port as a negative
template control, it may be desirable to provide less sample
chambers than for a group of chambers and corresponding inlet port
being used for analysis of biological sample. It also should be
understood that each inlet port may supply more than one main fluid
channel, which may be connected either in parallel or serially. In
addition, more than two sample inlet ports may be provided and thus
more than two groups of sample chambers may be supplied with
differing samples.
[0256] As mentioned above, for an inlet supply port supplying
sample to more than one main fluid channel, those main fluid
channels may be connected either serially or in parallel. The
exemplary embodiment of FIG. 27 depicts a substrate 2710 similar to
that of the substrate 2610 of FIG. 26, with the exception that the
main fluid channels 2770a and 2770b that supply the group of 16
sample chambers 2780 are connected in series rather than in
parallel. Like the substrate 2610, the substrate 2710 includes two
inlet supply ports 2760 that are configured to supply differing
fluids to two differing groups of sample chambers 2780, and thus
can provide a negative template control as discussed above. Based
on studies performed, providing the configuration of FIG. 26,
wherein the main fluid channels 2670a and 2670b are connected in
parallel rather than serially like the main fluid channels 2770a
and 2770b, permits faster filling of the substrate. For example,
for machined substrate prototypes having configurations similar to
the embodiments depicted in FIGS. 26 and 27, using an applied
pressure of 2 psi to perform the filling, the embodiment of FIG. 26
filled in 15 seconds while the embodiment of FIG. 27 filled in 4
minutes. At an applied pressure of 5 psi, the embodiment of FIG. 26
filled in 6 seconds and the embodiment of FIG. 27 filled in 22
seconds.
[0257] The exemplary embodiment of FIGS. 26 and 27 also include two
overfill chambers 2695 and 2795 and two overfill channels 2675 and
2775 associated with each group of sample chambers 2680 and 2780.
Each overfill channel 2675 leads respectively from the main fluid
supply channels 2670b and 2671 to the overfill chambers 2695. Each
overfill channel 2775 leads respectively from the main fluid supply
channels 2770b and 2771 to the overfill chambers 2695. In a manner
similar to that described above with reference to FIG. 24, the
overfill chambers 2695 and 2795 and overfill channels 2675 and 2775
act to protect against overfilling, and thus over-pressurization of
the substrate while ensuring sufficient filling of all of the
sample chambers 2680 and 2780 with fluid.
[0258] An upstream venting mechanism in conjunction with the inlet
supply ports 2660 and 2760 may be provided in order to protect
against overfill and over-pressurization of the substrate, as
discussed above with reference to FIGS. 22 and 24, for example. The
upstream venting mechanism may be in the form of any of the venting
mechanisms in accordance with the teachings herein, including, but
not limited to, the venting mechanism of the embodiment of FIGS. 2A
and 2B, the venting mechanism of the embodiment of FIG. 13, the
backside venting mechanisms discussed with reference to the
embodiments of FIGS. 15-16, and the venting mechanism of the
embodiment of FIG. 17. In an exemplary aspect, the venting
mechanism may be a vent hole provided in the film layer that covers
the openings of the various fluid distribution features of the base
(e.g., the film layer that together with the base forms the fluid
distribution network of chambers and channels) and a gas permeable
or porous membrane (e.g., hydrophobic membrane) situated over the
inlet ports 2660 and 2760. Further, rather than positioning the
upstream venting mechanism over the inlet ports 2660 and 2760, the
upstream venting mechanism could be provided in conjunction with
the fluid channels leading from the inlet ports 2660 and 2760 at a
location proximate the inlet ports 2660 and 2760.
[0259] Using an upstream venting mechanism, the sample volume that
may be used to fill the sample chambers 2680 and 2780 associated
with the main fluid supply channels 2670a, 2670b and 2770a, 2770b
may range from a minimum determined by adding the total volume of
the sample chambers (the 16 chambers in the case of FIGS. 26 and
27), the total volume of the venting chambers, the main fluid
supply channels, the inlet channels, and the venting channels
associated with those chambers, and the volume of vent through
holes (if any, for example, if the substrate of FIGS. 26 and 27 has
a configuration like those shown in one of FIGS. 14-16) associated
with the venting chambers and inlet supply port feeding the first
group of chambers. The sample volume maximum may be calculated by
adding the above volumes to the volume of the overfill chamber.
According to an exemplary embodiment, assuming that for the fluid
distribution networks associated with the group of 16 chambers of
FIGS. 26 and 27 that the volume of each sample chamber 2680 and
2780 is 1.35 .mu.L, the volume of the overfill chamber 2695 and
2795 is 5.09 .mu.L, the total volume of the main fluid channels
2670a, 2670b and 2770a, 2770b, the inlet channels 2670 and 2770 and
venting channels 2600 and 2700 associated with those main fluid
channels, and the venting chambers 2690 and 2790 associated with
the 16 sample chambers is 1.40 .mu.L, the sample supply volume may
range from 24.53 .mu.L to 29.62 .mu.L.
[0260] According to other exemplary embodiments, instead of or in
addition to providing an upstream venting mechanism to protect
against over-pressurization of the substrate, optical detection of
sample reaching the overfill chambers 2695 and 2795 may be
implemented. In an exemplary aspect, a dried, colored, fluorescence
dye (e.g., a red dye) may be deposited in the overfill chambers
2695 and 2705, for example, proximate an inlet of the chambers 2695
and 2795. Thus, when the sample begins filling the overfill
chambers 2695 and 2795, an optical detection mechanism may detect a
change in color in the overfill chamber 2695 and 2795 and a
feedback control mechanism may send a signal indicating to stop the
application of pressure used for filling the substrate 2610 and
2710. For example, a feedback signal may be sent to a
pressure-providing device (e.g., a pump, syringe, etc.) to
automatically stop the pressure being used to fill the substrate
and/or to an individual to manually stop the pressure.
[0261] The optical detection system may, for example, include an
optical cover over the substrates 2610 and 2710 such that only the
sample chambers 2680 and 2780, and the outlet portion of the
overfill chambers 2695 and 2795 are viewable by an optical reading
mechanism. The optical detection system may use, for example, an
LED beam to illuminate the overfill chambers 2695 and 2795 and the
optical reading mechanism may monitor the overfill chambers 2695
and 2795 near their respective outlets 2696 and 2697 for a change
in fluorescence. After all of the chambers 2680 and 2780 have been
filled, the sample will move into the overfill chambers 2695 and
2795, dissolve the predeposited dye and carry it to the outlets
2696 and 2796. The optical reading mechanism may then detect a
fluorescence signal change and send a feedback signal, for example,
to an operator or a filling device, to stop the application of
pressure for supplying sample to the substrate 2610 and 2710. In
other embodiments, the detector could detect the presence of an
internal standard in the mastermix mixed with sample (which may be
ROX), rather than spotting additional dye in the overfill
chambers.
[0262] It has been observed that flowing deionized water into an
empty chamber also causes a signal increase, which may be
contributed by air and water having differing optical background
signals and/or by the meniscus of the traveling water causing a
signal change through both reflection and diffraction effects.
Thus, in various exemplary embodiments, rather than using a red
dye, LED beam, and fluorescence detecting mechanism in the overfill
chamber, an optical sensor configured to detect the presence of
liquid may be used. By way of example, a refractive index sensor
may be used to detect liquid filling the overfill chamber. Because
the refractive index of water (e.g., sample) differs from that of
air, the light is deflected in a way that differs when the sample
enters an overfill chamber and can be recognized by the detector.
When the detector senses the change, a signal can be sent to stop
the application of pressure and supply of sample.
[0263] Yet further exemplary embodiments for detection of the
presence of sample in an overfill chamber include the use of a
capacitance sensor or the use of an infrared sensor. Regarding the
former, a capacitance sensor may be used to measure the capacitance
between the film layer (e.g., an aluminum film layer) covering an
overfill chamber and the opposite side of the substrate at the
location of the overfill chamber. Since the dielectric constant of
water is much greater than air, when sample fills the overfill
chamber, the sensed capacitance may change and the capacitance
sensor may send a signal indicating to stop filling (e.g., pressure
application to) the substrate. FIG. 37 depicts an exemplary
embodiment of using a capacitance sensor to detect the presence of
sample in an overfill chamber 3895 in a substrate 3810. In FIG. 37,
an electrode 3801 may be positioned on a side of the substrate 3810
opposite to the side of a film layer 3820, for example, an aluminum
film layer. The electrode 3801 may be disposed on the substrate
3810 or may be part of an instrument cover or the like that clamps
the substrate 3810 during filling. The aluminum film layer 3820 may
be connected to a voltage supply to serve as a second electrode. In
the case where the film layer is not a metal, another electrode
could be positioned underneath the film layer 3820 similar
electrode 3801. A voltage may be applied between the two electrodes
(e.g., 3801 and the aluminum film layer 3820 in FIG. 37) and a
capacitance of the chamber 3895 may be sensed. In various
embodiments, the voltage may be applied as an AC field, and the
capacitance may be detected as a phase shift, as well as permitting
multiple readings, instead of the single change that would be
registered with a DC field. An additional, optional electrode 3802
may be positioned adjacent, e.g., downstream, of the electrode 3801
and over the overfill chamber 3895 to use as a reference and a
differential measurement may be made to increase the accuracy of
the capacitance measurement. According to various embodiments, a
conductivity detector can be used where the sample liquid is
permitted to come into direct contact with the electrodes.
[0264] In various other embodiments, an infrared sensor may be used
to detect sample filling of an overfill chamber. Because water and
air have differing absorbance in the infrared range, infrared
absorbance and/or reflection may be measured at the overfill
chamber to detect the presence of sample (which contains water)
entering the chamber. For example, in the case of an aluminum film
layer covering the base of a substrate, infrared reflection off the
aluminum layer over an overfill chamber may be detected.
[0265] Those having skill in the art will recognize various other
detection mechanisms that may be used to detect the presence of the
sample filling the overfill chamber, and the exemplary embodiments
above should not be construed as limiting. Also, it should be
understood that the various optical detection mechanisms described
with reference to the embodiments of FIGS. 26 and 27 may also be
applied to the exemplary embodiment of FIG. 24.
[0266] Using an optical detection mechanism, the volume of sample
that may be supplied to the sample distribution networks associated
with the first group of sample chambers 2680 and 2780 (the 16
chamber group) of the substrates 2610 and 2710 may range from a
minimum determined by adding the total volume of the sample
chambers (the 16 chambers in the case of FIGS. 26 and 27), the
total volume of the venting chambers, the main fluid supply
channels, the inlet channels, and the venting channels associated
with those chambers, the total volume of the vent through holes (if
any) associated with the venting chambers and inlet supply port
feeding the first group of chambers, and half of the volume of the
overfill chamber associated with the first group of chambers. The
sample volume maximum may be determined in the same manner as the
sample volume maximum when using the upstream venting mechanism
approach, discussed above. Thus, assuming the various volume values
as discussed above, the sample volume supplied to the first group
of chambers 2680 and 2780 using optical detection as an
over-pressurization/overfill protection mechanism may range from
27.08 .mu.l to 29.62 .mu.l.
[0267] Whether implementing the upstream venting mechanism approach
or the optical detection approach, using an overfill chamber and
channel, as described in the embodiments of FIGS. 24, 26, and 27
helps to ensure that sufficient filling of the sample chambers
occurs without over-pressurization, and potential leakage, of the
substrate. Further, this protection against underfilling and
over-pressurization can occur with a relatively large tolerance of
the input sample volume. In other words, a precise amount of sample
does not need to be determined and used to fill the substrate such
that all of the sample chambers are filled, but over-pressurization
does not occur. Rather, there is a volume range that may be used
while still protecting against underfilling of the chambers and
over-pressurization of the substrate.
[0268] Although various exemplary substrate embodiments described
above and shown in the figures depicted partial views, schematic
views, or substrates defining a 12-chamber, 16-chamber, or
24-chamber array, it should be understood that the various
configurations and features of those embodiments can be applied to
substrates of varying sizes and chamber arrays, including, for
example, substrates defining multi-chamber arrays including various
number of chambers, including, but not limited to, 12, 24, 36, 48,
96, 192, 384, 3072, 6144, or more sample chambers. In an exemplary
configuration, the various substrates described above can have
dimensions of, for example, about 127.0 millimeters by about 85.7
millimeters and define 384 sample chambers.
[0269] In some cases, for example when performing biological
testing using a substrate defining a chamber array that differs
from a conventional substrate, it may be desirable to use existing
instrumentation for such conventional substrate configurations to
perform biological analysis on substrates of other sizes. For
example, at least some substrates in accordance with the present
teachings may define 12-, 16-, or 24-chamber arrays, which have
fewer chambers, and thus require fewer assays, than are available
in a conventional low density array substrate (e.g., a substrate
including a 96-chamber or 384-chamber array). When performing
biological testing, such as, for example, real-time PCR, on the
substrates with fewer chambers, a solution may be to perform
testing on more than one substrate and thereby enable analysis of
multiple samples during the same testing step. However, this may
result in a mismatch between the number of samples presenting for
analysis and the number of positions available on a substrate.
Further, using a low density array substrate that does not use all
of the chambers available may not be desirable since once subject
to biological analysis, the substrates may not be suitable for
further use. Also, in some cases, it may be desirable to subject
one or more samples to differing assay panels (e.g., biological
analysis) during a single processing routine (e.g., a single real
time PCR assay step).
[0270] In order to achieve at least some of the desirable features
above and in accordance with various embodiments of the present
teachings, substrates that define a subset of chambers (e.g.,
"subcards") of a standard low density array substrate (e.g., 96- or
384-chamber array) may be provided that are configured to be
combined and positioned into a holding fixture compatible for use
with standard low density array testing instrumentation. FIGS.
54-56 show schematic perspective views of various holding fixtures
that may be used to hold one or more subcards in accordance with
exemplary embodiments.
[0271] The holding fixtures 5400 and 5600 of FIGS. 54-56 are in the
form of a 4-sided frame with an array of openings 5401 and 5601
that correspond to locations of sample chambers on subcards. One or
opposing sides of the holding fixtures 5400 and 5600 may define a
slot configured to receive one or more subcards to be inserted into
the fixture when performing biological testing, for example,
real-time PCR. In the holding fixture of FIG. 54, 4 16-chamber
array substrates (subcards) 5410 are shown inserted into the
holding fixture 5400. The holding fixture 5400 is configured to
hold a number of substrates that combined total a 96-chamber array.
That is, the holding fixture 5400 includes an array of 96 openings
5401. FIG. 55 shows an exemplary embodiment of the use of the
holding fixture 5400 to hold 4 16-chamber array subcards 5510 that
include sample processing modules 5550 connected to the subcards
5510. In various exemplary embodiments, the sample processing
modules 5550 may be detachable from the subcards 5510 prior to
performing biological testing.
[0272] FIG. 56 depicts an exemplary embodiment of a holding fixture
5600 configured to hold subcards 5610 including 24-chamber arrays,
with each subcard 5610 including 4 chambers across the substrate
and 6 chambers down the substrate. The holding fixture 5600 may
define slots at opposing ends to receive 4 subcards having a
configuration like subcards 5610. The subcards 5610 shown in FIG.
56 include sample processing modules 5650, which may be detachable.
It should be understood that the subcards without such sample
processing modules also may be used.
[0273] The configurations of the subcards and holding fixtures of
FIGS. 54-56 are exemplary only and not limiting. Various holding
fixture and subcard configurations having differing number and
arrangement of arrays may be used in accordance with the present
teachings. Further, the subcards that are placed in the holding
fixtures need not be of the same size and/or arrangement. For
example, a subcard having a configuration like the subcards 5510
may be provided in the same holding fixture as a subcard having a
configuration like the subcards 5610. Those having skill in the art
would recognize that other combinations of subcards and
configurations of subcards and holding fixtures may be used based
on the present teachings.
[0274] In various embodiments, when performing biological testing,
differing numbers of substrates maybe processed in each testing
run. For example, one, two, three, four, or more cards may be
processed (e.g., thermocycled) during a given biological testing
run. This may be particularly true in benchtop testing of
substrates.
[0275] In this situation, it is desirable to achieve substantial
chamber-to-chamber thermal uniformity within and between substrates
being processed. Moreover, it may be desirable to provide
information to the testing instrumentation regarding the number of
substrates being processed in a given run.
[0276] With reference to FIGS. 57-60, a set of four differing
substrate (e.g., card) carriers are depicted. Each carrier is
configured to hold a differing number of substrates. For example,
the carrier 5700 in FIG. 57 defines a single cavity 5705 configured
to hold a single substrate. The carrier 5800 in FIG. 58 defines two
cavities 5805 each configured to hold a substrate. The carrier 5900
in FIG. 59 is configured to hold three substrates in three cavities
5905 defined by the carrier, and the carrier 6000 in FIG. 60
defines four cavities 6005 configured to hold four substrates.
[0277] Each of the differing carriers in FIGS. 57-60 may include
indicators readable by the instrumentation used for processing to
indicate how many substrates the carrier is configured to hold. By
way of example, each carrier 5700, 5800, 5900, and 6000 may include
one or more fluorescent scan marks 5715, 5815, 5915, and 6015 that
indicate how many substrates the carrier is configured to hold
(e.g., 1 scan mark for carrier 5700, 2 scan marks for 5800, 3 scan
marks for 5900, and 4 scan marks for 6000). The carriers 5700,
5800, 5900, and 6000 also may include an indicator 5720, 5820,
5920, and 6020 readable by an individual (e.g., an Arabic numeral 1
through 4) to indicate how many substrates the carrier is
configured to hold. Indicators other than Arabic numerals and/or
fluorescent scan marks, such as, for example, bar codes and RFID
identifiers, also may be appropriate identification mechanisms.
Those having ordinary skill in the art would recognize numerous
ways to achieve identification of the carriers, both by operators
and by instrumentation.
[0278] To use the carriers of FIGS. 57-60, a user installs the
appropriate number of sample-filled substrates (e.g., one to four
in the exemplary embodiments of FIGS. 57-60) into a carrier, places
the carrier into the testing instrumentation, and begins testing
(e.g., thermocycling). The testing instrumentation reads the
indicators on the carrier to determine how many substrates are
being processed and may be configured to control the processing
steps (such as the thermocycling temperatures, times, locations of
applied heat, etc.) based on that information. This may ensure that
the testing processes are compatible with the number of substrates
being tested so as to promote thermal uniformity.
[0279] FIG. 61 depicts another exemplary embodiment of a substrate
carrier that may be useful when performing biological testing on
differing numbers of substrates in a given testing run. The carrier
6100 of FIG. 61 defines four cavities 6105 that are configured to
receive a substrate for biological testing or a mockup card 6120.
The mockup card 6120 may be removable from a cavity 6105 and
replaced with a substrate for biological testing or may be placed
in a cavity 6105 when the cavity 6105 is not being used to hold a
substrate for biological testing. Each mockup card 6120 may be
provided with an indicator 6115 readable by the instrumentation
used for processing to indicate that a mockup card 6120 rather than
a sample substrate is in place in a cavity. By being able to sense
the number and locations of mockup cards versus sample substrates
held by the carrier 6100 during a testing run, the testing
processes may be adjusted (e.g., thermocycling temperatures, times,
and locations of applied heat, etc.).
[0280] The present teachings provide a variety of structural
arrangements, techniques, and/or methodology useful for performing
biological analysis, including multiple analyte detection. It
should be understood that although in some cases the embodiments
described herein may focus on a particular aspect, various
embodiments may be combined to form a system and/or substrate
configuration useful for multiple analyte detection. By way of
example only, various sealing approaches may be combined with
various venting approaches. The various embodiments described
herein are not intended to be mutually exclusive.
[0281] For the purposes of this specification and appended claims,
unless otherwise indicated, all numbers expressing quantities,
percentages or proportions, and other numerical values used in the
specification and claims, are to be understood as being modified in
all instances by the term "about." Accordingly, unless indicated to
the contrary, the numerical parameters set forth in the following
specification and attached claims are approximations that may vary
depending upon the desired properties sought to be obtained by the
present invention. At the very least, and not as an attempt to
limit the application of the doctrine of equivalents to the scope
of the claims, each numerical parameter should at least be
construed in light of the number of reported significant digits and
by applying ordinary rounding techniques.
[0282] Notwithstanding that the numerical ranges and parameters
setting forth the broad scope of the invention are approximations,
the numerical values set forth in the specific examples are
reported as precisely as possible. Any numerical value, however,
inherently contains certain errors necessarily resulting from the
standard deviation found in their respective testing measurements.
Moreover, all ranges disclosed herein are to be understood to
encompass any and all subranges subsumed therein. For example, a
range of "less than 10" includes any and all subranges between (and
including) the minimum value of zero and the maximum value of 10,
that is, any and all subranges having a minimum value of equal to
or greater than zero and a maximum value of equal to or less than
10, e.g., 1 to 5.
[0283] It is noted that, as used in this specification and the
appended claims, the singular forms "a," "an," and "the" include
plural referents unless expressly and unequivocally limited to one
referent. Thus, for example, reference to "a layer" includes two or
more different layers. As used herein, the term "include" and its
grammatical variants are intended to be non-limiting, such that
recitation of items in a list is not to the exclusion of other like
items that can be substituted or added to the listed items.
[0284] Various embodiments of the teachings are described herein.
The teachings are not limited to the specific embodiments
described, but encompass equivalent features and methods as known
to one of ordinary skill in the art. Other embodiments will be
apparent to those skilled in the art from consideration of the
present specification and practice of the teachings disclosed
herein. It is intended that the present specification and examples
be considered as exemplary only.
* * * * *