U.S. patent application number 13/784736 was filed with the patent office on 2014-02-13 for microfluidic devices and methods.
This patent application is currently assigned to Fluidigm Corporation. The applicant listed for this patent is Fluidigm Corporation. Invention is credited to Geoff Facer, Brian Fowler, Martin Pieprzyk, Timothy Woudenberg.
Application Number | 20140045184 13/784736 |
Document ID | / |
Family ID | 42729929 |
Filed Date | 2014-02-13 |
United States Patent
Application |
20140045184 |
Kind Code |
A1 |
Pieprzyk; Martin ; et
al. |
February 13, 2014 |
Microfluidic Devices and Methods
Abstract
Embodiments of the present invention provide improved
microfluidic devices and related apparatus, systems, and methods.
Methods are provided for reducing mixing times during use of
microfluidic devices. Microfluidic devices and related methods of
manufacturing are provided with increased manufacturing yield
rates. Improved apparatus and related systems are provided for
supplying controlled pressure to microfluidic devices. Methods and
related microfluidic devices are provided for reducing dehydration
of microfluidic devices during use. Microfluidic devices and
related methods are provided with improved sample to reagent
mixture ratio control. Microfluidic devices and systems are
provided with improved resistance to compression fixture pressure
induced failures. Methods and systems for conducting temperature
controlled reactions using microfluidic devices are provided that
reduce condensation levels within the microfluidic device. Methods
and systems are provided for improved fluorescent imaging of
microfluidic devices.
Inventors: |
Pieprzyk; Martin; (Menlo
Park, CA) ; Facer; Geoff; (Lane Cove NSW, AU)
; Woudenberg; Timothy; (Moss Beach, CA) ; Fowler;
Brian; (San Mateo, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Fluidigm Corporation |
South San Francisco |
CA |
US |
|
|
Assignee: |
Fluidigm Corporation
South San Francisco
CA
|
Family ID: |
42729929 |
Appl. No.: |
13/784736 |
Filed: |
March 4, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13295028 |
Nov 11, 2011 |
8389960 |
|
|
13784736 |
|
|
|
|
12688462 |
Jan 15, 2010 |
8058630 |
|
|
13295028 |
|
|
|
|
61145459 |
Jan 16, 2009 |
|
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Current U.S.
Class: |
435/6.12 |
Current CPC
Class: |
B01L 2300/123 20130101;
Y10T 137/7793 20150401; B01L 2400/0481 20130101; B01L 7/52
20130101; B01L 3/502738 20130101; G01N 2021/0346 20130101; B01L
2200/16 20130101; B01L 2400/0655 20130101; B01L 2300/0874 20130101;
G01N 21/05 20130101; Y10T 436/2575 20150115; C12Q 1/686 20130101;
G01N 1/28 20130101; B33Y 80/00 20141201; G01N 21/6452 20130101;
B01L 2300/0867 20130101 |
Class at
Publication: |
435/6.12 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method for reducing mixing times associated with using a
microfluidic device, the method comprising: providing a
microfluidic device having a reaction cell, the reaction cell
including a sample chamber having a first volume and a reagent
chamber having a second volume, the sample chamber and the reagent
chamber being in fluid communication through an interface channel
having an interface valve for controlling fluid communication
between the sample chamber and the reagent chamber; introducing a
sample fluid into the sample chamber so as to pressurize the sample
fluid in the sample chamber to a first pressure; introducing a
reagent fluid into the reagent chamber so as to pressurize the
reagent fluid in the reagent chamber to a second pressure; and
mixing sample fluid from the sample chamber with reagent fluid from
the reagent chamber by opening the interface valve, wherein the
first pressure and the second pressure are sufficiently different
to cause the mixing to occur at least in part by fluid injection.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/295,028, filed Nov. 11, 2011, which is a
continuation of U.S. patent application Ser. No. 12/688,462, filed
Jan. 15, 2010 (now U.S. Pat. No. 8,058,630), which claims the
benefit of U.S. Provisional Application No. 61/145,459, filed Jan.
16, 2009, the entire contents of which are incorporated herein by
reference for all purposes.
BACKGROUND OF THE INVENTION
[0002] The present invention relates generally to microfluidic
devices, which provide for precise control and manipulation of
fluids that are geometrically constrained to a small, typically
sub-millimeter, scale. In particular, the invention provides
microfluidic devices and related apparatus, systems, and
methods.
[0003] Recently, there have been concerted efforts to develop and
manufacture microfluidic systems to perform various chemical and
biochemical analyses and syntheses, both for preparative and
analytical applications. The goal to make such devices arises
because of the significant benefits that can realized from
miniaturization with respect to analyses and syntheses conducted on
a macro scale. Such benefits include a substantial reduction in
time, cost and the space requirements for the devices utilized to
conduct the analysis or synthesis. Additionally, microfluidic
devices have the potential to be adapted for use with automated
systems, thereby providing the additional benefits of further cost
reductions and decreased operator errors because of the reduction
in human involvement. Microfluidic devices have been proposed for
use in a variety of applications, including, for instance,
capillary electrophoresis, gas chromatography and cell
separations.
[0004] Microfluidic devices adapted to conduct nucleic acid
amplification processes are potentially useful in a wide variety of
applications. For example, such devices could be used to determine
the presence or absence of a particular target nucleic acid in a
sample, as an analytical tool. Examples of utilizing microfluidic
device as an analytical tool include:
[0005] testing for the presence of particular pathogens (e.g.,
viruses, bacteria or fungi);
[0006] identification processes (e.g., paternity and forensic
applications);
[0007] detecting and characterizing specific nucleic acids
associated with particular diseases or genetic disorders (e.g.,
fetal diagnostics);
[0008] detecting gene expression profiles/sequences associated with
particular drug behavior (e.g., for pharmacogenetics, i.e.,
choosing drugs which are compatible/especially efficacious for/not
hazardous with specific genetic profiles); and
[0009] conducting genotyping analyses and gene expression analyses
(e.g., differential gene expression studies).
[0010] Alternatively, the devices can be used in a preparative
fashion to amplify nucleic acids, producing an amplified product at
sufficient levels needed for further analysis. Examples of these
analysis processes include sequencing of the amplified product,
cell-typing, DNA fingerprinting, and the like. Amplified products
can also be used in various genetic engineering applications. These
genetic engineering applications include (but are not limited to)
the production of a desired protein product, accomplished by
insertion of the amplified product into a vector that is then used
to transform cells into the desired protein product.
[0011] While currently available microfluidic devices and related
apparatus, systems, and methods provide for a wide range of uses,
further improvements are desirable. In particular, it would be
beneficial to reduce costs associated with the production and use
of microfluidic devices. It would also be beneficial to provide
improved processes for using microfluidic devices.
BRIEF SUMMARY OF THE INVENTION
[0012] Embodiments of the present invention provide improved
microfluidic devices and related apparatuses, systems, and methods.
Methods are provided for reducing mixing times during use of
microfluidic devices. Microfluidic devices and related methods of
manufacturing are provided with increased manufacturing yield
rates. Improved apparatuses and related systems are provided for
supplying controlled pressure to microfluidic devices. Methods and
related microfluidic devices are provided for reducing dehydration
of microfluidic devices during use. Microfluidic devices and
related methods are provided with improved sample to reagent
mixture ratio control. Microfluidic devices and systems are
provided with improved resistance to compression fixture pressure
induced failures. Methods and systems for conducting temperature
controlled reactions using microfluidic devices are provided that
reduce condensation levels within the microfluidic device. Methods
and systems are provided for improved fluorescent imaging of
microfluidic devices. Reduced mixing times lowers both time and
related costs for the analysis being conducted. Microfluidic
devices with increased manufacturing yield rates lower costs and
help to minimize waste. Improved apparatus and systems for
supplying controlled pressure to microfluidic devices help to
simplify microfluidic device processing equipment, thereby reducing
costs. Decreased microfluidic device dehydration helps to minimize
detrimental impacts that occur when water escapes from reaction
fluids, especially from sample fluids that contain significant
relative amounts of water. Improved sample to reagent mixture ratio
control helps to increase processing effectiveness. Lower rates of
compression fixture pressure induced failure help to increase the
success rate achieved during temperature controlled reactions.
Improved fluorescent imaging helps to increase the certainty by
which analysis results can be determined.
Reduced Mixing Times
[0013] Thus, in one aspect, embodiments of the present invention
provide methods for reducing mixing time for a mixture of a sample
fluid and a reagent fluid. A method can, for example, involve a
microfluidic device having a reaction cell that includes a sample
chamber and a reagent chamber that are in fluid communication
through an interface channel having an interface valve. Sample
fluid can be introduced into the sample chamber at a first pressure
and reagent fluid can be introduced into the reagent chamber at a
second pressure. The sample fluid and the reagent fluid can be
mixed by opening the interface valve. The first pressure and the
second pressure can be sufficiently different to cause the mixing
to occur at least in part by fluid injection. In some embodiments,
the sample chamber pressure is greater than the reagent chamber
pressure, and can be greater by 1.0 psi. In other embodiments, the
reagent chamber pressure is greater than the sample chamber
pressure, and can be greater by 1.0 psi. Often, a microfluidic
device reaction cell will be configured with a sample chamber
volume being greater than a reagent chamber volume, but can also be
configured with a reagent chamber volume being equal to or greater
than a sample chamber volume.
Increased Manufacturing Yield Rates
[0014] In another aspect, embodiments of the present invention
provides microfluidic devices with increased manufacturing yield
rates, and related methods for manufacturing. A device can include,
for example, a configuration that allows for the isolation of
defective reaction cells. During the manufacture of a microfluidic
device, manufacturing defects may result in one or more defective
reaction cells. Often, these defective cells can be identified
during quality control inspections. Embodiments of the present
invention provide microfluidic devices that can be configured so as
to isolate one or more reaction cells, including one or more
identified defective reaction cells. The device can include a first
flow channel for introduction of a fluid via an inlet to the first
reaction cell and a second flow channel for introduction of a fluid
via the inlet to the second reaction cell. A microfluidic device
can be configurable to have the inlet in fluid communication with
the first flow channel, and fluidically separated from the second
flow channel. Often, the device will also be configurable to have
the inlet in fluid communication with the second flow channel and
fluidically separated from the first flow channel. In some
embodiments, each of the first flow channel and the second flow
channel is initially in fluid communication with the inlet, and the
device includes a first fusible isolation feature and a second
fusible isolation feature, the first fusible isolation feature
being configurable to fluidically separate the first flow channel
from the inlet, and the second fusible isolation feature being
configurable to fluidically separate the second flow channel from
the inlet. In some embodiments, each of the first and second
fusible isolation features is a containment valve that is fusible
by an exposure of ultraviolet light. The first and second flow
channels can be for the introduction of a sample fluid.
Alternatively, the first and second flow channels can be for the
introduction of a reagent fluid.
[0015] In further embodiments, a microfluidic device with increased
manufacturing yield rate can include both a sample inlet and
associated first and second sample flow channels, and a reagent
inlet and associated first and second reagent flow channels. The
first sample flow channel can be for introducing a sample fluid via
the sample inlet into a first reaction cell, and the second sample
flow channel can be for introducing a sample fluid via the sample
inlet into a second reaction cell. Likewise, the first reagent flow
channel can be for introducing a reagent fluid into the first
reaction cell via the reagent inlet, and the second reagent flow
channel can be for introducing a reagent fluid into the second
reaction cell via the reagent inlet. The device can be configurable
to have the sample inlet in fluid communication with the first
sample flow channel and fluidically separated from the second
sample flow channel. Likewise, the device can be configurable to
have the reagent inlet in fluid communication with the first
reagent flow channel and fluidically separated from the second
reagent flow channel. Often, a device will also be configurable to
have the sample inlet in fluid communication with the second sample
flow channel and fluidically separated from the first sample flow
channel. Likewise, a device will often be configurable to have the
reagent inlet in fluid communication with the second reagent flow
channel and fluidically separated from the first reagent flow
channel. In some embodiments, each inlet can be in initial fluid
communication with two reaction cells by way of separate flow
channels, and the device can include fusible isolation features
configured to fluidically separate an inlet from a reaction cell.
In some embodiments, a fusible isolation feature includes a
containment valve that is fusible by an exposure of ultraviolet
light or other electromagnetic radiation.
[0016] In further embodiments, an increased manufacturing yield
rate microfluidic device for reacting M number of samples with N
number of different reagents can include a plurality of sample
inlets, a plurality of sample flow channels, a plurality of reagent
inlets, and a plurality of reagent flow channels. A device can be
configurable to selectively cause each of the plurality of sample
inlets to be in fluid communication with a first of a unique pair
of the plurality of sample flow channels and fluidically separated
from a second of the unique pair of the plurality of sample flow
channels. Likewise, a device can be configurable to selectively
cause each of the plurality of reagent inlets to be in fluid
communication with a first of a unique pair of the plurality of
reagent flow channels and fluidically separate from a second of the
unique pair of the plurality of reagent flow channels. Often, a
device can be configurable to selectively cause each of the
plurality of sample inlets to be in fluid communication with a
second of a unique pair of the plurality of sample flow channels
and fluidically separated from a first of the unique pair of the
plurality of sample flow channels. Likewise, a device can often be
configurable to selectively cause each of the plurality of reagent
inlets to be in fluid communication with a second of a unique pair
of the plurality of reagent flow channels and fluidically separate
from a first of the unique pair of the plurality of reagent flow
channels. In some embodiments, each of the plurality of sample
inlets is in initial fluid communication with a unique pair of the
plurality of sample flow channels, and the device further comprises
a plurality of fusible sample isolation features, each fusible
sample isolation feature being configurable to fluidically separate
one of the plurality of sample inlets from one of the plurality of
sample flow channels. In some embodiments, each of the plurality of
reagent inlets is in initial fluid communication with a unique pair
of the plurality of reagent flow channels, and the device further
comprises a plurality of fusible reagent isolation features, each
fusible reagent isolation feature being configurable to fluidically
separate one of the plurality of reagent inlets from one of the
plurality of reagent flow channels. In some embodiments, a fusible
isolation feature includes a containment valve that is fusible by
an exposure to ultraviolet light or other electromagnetic
radiation.
[0017] Related methods for manufacturing a microfluidic device
having increased manufacturing yield rate can include the
identification of defective reaction cells and configuring the
device to isolate the identified defective reaction cells. In some
embodiments, a method for manufacturing a microfluidic device can
include: fabricating a microfluidic device according to any of the
above described embodiments; performing an inspection for defects
in the microfluidic device; and configuring the device so to
isolate defects.
Supplying Controlled Pressure
[0018] In another aspect, embodiments of the present invention
provide apparatus and systems for supplying controlled pressure to
a microfluidic device. An apparatus can include, for example, a
holder configured to couple with a microfluidic device, a plurality
of accumulators for supplying controlled pressure to the
microfluidic device, and a pressure regulator for selectively
regulating pressure supplied to each of the plurality of
accumulators. In some embodiments, the pressure regulator includes
an accumulator selector valve. In some embodiments, the pressure
regulator employs rotary motion. In some embodiments, an apparatus
for supplying controlled pressure includes one or more first supply
outlet selector valves for selectively placing a first supply
outlet in fluid communication with one of the plurality of
accumulators. In some embodiments, an apparatus for supplying
controlled pressure includes one or more second supply outlet
selector valves for selectively placing a second supply outlet in
fluid communication with one of the plurality of accumulators. In
some embodiments, a first supply outlet selector valve includes a
rotary valve. In some embodiments, a second supply outlet selector
valve includes a rotary valve. A system can include, for example,
any of the above described apparatus and a control unit for
controlling the operation of the pressure regulator. In some
embodiments, the system can control one or more accumulator
selector valves, and one or more supply outlet selector valves.
Reduced Dehydration
[0019] In another aspect, embodiments of the present invention
provide methods for reducing dehydration of a microfluidic device,
and related microfluidic devices. A method can include, for
example, providing a microfluidic device having one or more vent
channels, introducing a sample fluid and a reagent fluid into the
device, and introducing a substantially non-permeable fluid into at
least one of the vent channels after the introduction of the sample
fluid and the reagent fluid so as to inhibit dehydration of the
device. In some embodiments, a method can include connecting two or
more vent channels. In some embodiments, the substantially
non-permeable fluid is perfluoropolyether oil. In some embodiments,
each vent channel is in fluid communication with a vent channel
port for introduction of the substantially non-permeable fluid into
the vent. In some embodiments, introduction of substantially
non-permeable fluid forces air to emerge from a vent channel vent.
A related microfluidic device can include, for example, a plurality
of reaction cells, each reaction cell for reacting a sample fluid
with a reagent; and a plurality of vent channels, each vent channel
adapted to vent the device during the introduction of fluid into
the device, and each vent line being in fluid communication with a
vent channel port.
Improved Mixture Ratio Control
[0020] In another aspect, embodiments of the present invention
provide microfluidic devices having improved sample to reagent
mixture ratio control, and related methods. A microfluidic device
can include, for example, a sample chamber for containing a sample
fluid; a reagent chamber for containing a reagent fluid; a reaction
chamber for receiving sample fluid from the sample chamber and
reagent fluid from the reagent chamber; a sample channel for
transferring sample fluid from the sample chamber to the reaction
chamber; the sample channel including a sample channel restriction
for controlling the flow rate of sample fluid, and a sample
interface valve associated therewith for controlling fluid
communication between the sample chamber and the reaction chamber;
and a reagent channel for transferring reagent fluid from the
reagent chamber to the reaction chamber; the reagent channel
including a reagent channel restriction for controlling the flow
rate of reagent fluid, and a reagent interface valve associated
therewith for controlling fluid communication between the reagent
chamber and the reaction chamber. In some embodiments, the device
includes an interface control channel for controlling actuation of
the sample interface valve and the reagent interface valve. In some
embodiments, the device includes a sample interface control channel
for controlling actuation of the sample interface valve and a
reagent control channel for controlling actuation of the reagent
interface valve. In some embodiments, the sample interface valve is
disposed between the sample channel restriction and the reaction
chamber. In some embodiments, the reagent interface valve is
disposed between the reagent channel restriction and the reaction
chamber. In some embodiments, the sample channel restriction and
the reagent channel restriction are adapted so that the amount of
sample fluid received by the reaction chamber exceeds the amount of
reagent fluid received by the reaction chamber. In some cases, the
ratio of sample fluid received to reagent fluid received is
approximately ten to one.
[0021] In further embodiments, a microfluidic devices having
improved sample to reagent mixture ratio control can include: a
plurality of sample chambers, each of the sample chambers being
adapted to contain a sample fluid; a plurality of reagent chambers,
each of the reagent chambers being adapted to contain a reagent
fluid; a plurality of reaction chambers, each of the reaction
chambers being adapted to receive sample fluid from one of the
sample chambers and to receive reagent fluid from one of the
reagent chambers; a plurality of sample channels, each of the
sample channels being adapted to transfer sample fluid from one of
the sample chambers to one of the reaction chambers, at least one
of the sample channels including a sample channel restriction for
controlling the flow rate of sample fluid, and a sample interface
valve associated therewith for controlling fluid communication
between the sample chamber and the reaction chamber; and a
plurality of reagent channels, each of the reagent channels being
adapted to transfer reagent fluid from one of the reagent chambers
to one of the reaction chambers, at least one of the reagent
channels including a reagent channel restriction for controlling
the flow rate of reagent fluid, and a reagent interface valve
associated therewith for controlling fluid communication between
the reagent chamber and the reaction chamber. In some embodiments,
a sample channel restriction is configured differently than another
sample channel restriction so as to compensate for variations in
sample chamber pressure and/or sample channel flow impedance. In
some embodiments, a reagent channel restriction is configured
differently than another reagent channel restriction so as to
compensate for variations in reagent chamber pressure and/or
reagent channel flow impedance. In some embodiments, a microfluidic
device can include a plurality of interface control channels, each
of which is for controlling actuation of one of the sample
interface valves and one of the reagent interface valves so as to
control the flow of sample fluid and reagent fluid into one of the
reaction chambers. In some embodiments, the microfluidic device can
include a plurality of sample interface control channels each of
which is for controlling actuation of a sample interface valve, and
can include a plurality of reagent interface control channels each
of which is for controlling actuation of a reagent interface valve.
In some embodiments, one or all of the sample interface valves or
reagent interface valves can be disposed between a channel
restriction and one of the reaction chambers. In some embodiments,
the channel restrictions are adapted so that the amount of sample
fluid received by a reaction chamber exceeds the amount of reagent
received. In some embodiments, the ratio of sample received to
reagent received by a reaction chamber is approximately ten to
one.
[0022] Related methods for conducting a reaction between a sample
and a reagent with improved sample to reagent mixture ratio control
can include, for example, providing a microfluidic device in
accordance with any of the above described embodiments having
channel restrictions; introducing a sample fluid into a sample
chamber with the sample chamber interface valve closed; introducing
a reagent fluid into a reagent chamber with the reagent interface
valve closed; opening the sample interface valve to transfer sample
fluid to the reaction camber; opening the reagent interface valve
to transfer reagent fluid to the reaction chamber; closing the
sample interface valve after the transfer of the sample fluid to
the reaction chamber; and closing the reagent interface valve after
the transfer of reagent fluid to the reaction chamber. In some
embodiments, a sample interface valve and a reagent interface valve
are opened or closed at substantially the same time.
Increased Resistance to Compression Fixture Induced Failure
[0023] In another aspect, embodiments of the present invention
encompasses microfluidic devices and related systems providing
increased resistance to compression fixture pressure induced
failure. A microfluidic device can include, for example, a
plurality of inlets formed in an elastomeric substrate; a plurality
of chambers formed in the elastomeric substrate, each one of the
chambers being in fluid communication with one of said plurality of
inlets through one of a plurality of flow channels, each flow
channel having a control valve for controlling fluid communication
between said one of the plurality of inlets and said one of the
plurality of chambers; and a control channel formed in the
elastomeric substrate, the control channel having a first end and a
second end, the first end being in fluid communication with the
second end through a restriction feature for preventing the flow of
control fluid from the second end to the first end, the second end
being coupled with at least one of said control valves for
actuation of said at least one control valve. In some embodiments,
the restriction feature is a check valve. In some embodiments, the
restriction feature is sealed by an application of ultraviolet
light. In some embodiments, the restriction feature is sealed
thermally. In some embodiments, the restriction feature is an
actuated element, which can be a guided pin or cam. In some
embodiments, a microfluidic device can include a compensation
feature adapted to increase fluid pressure in a control channel in
response to the microfluidic device being compressed. The
compensation feature can be in fluid communication with a control
channel, and can be a fluid filled structure.
[0024] In some embodiments, a microfluidic system having increased
resistance to compression fixture pressure induced failure can
include, for example, a microfluidic device having a control
channel coupled with at least one control valve, and a control
fluid introduction device in fluid communication with the control
channel. In some embodiments, the control fluid introduction device
can be adapted to prevent backflow of control fluid from the
microfluidic device. In some embodiments, the control fluid
introduction device includes an accumulator and is adapted to
eliminate the presence of gas within the accumulator. In some
embodiments, the control fluid introduction device includes a
backflow restriction feature, which can be a check valve, or can
include an actuated element such as a guided pin or cam.
[0025] In some embodiments, a microfluidic system having increased
resistance to compression fixture pressure induced failure can
include, for example, a preferential compression fixture for
applying pressure preferentially to areas of the microfluidic
device where control-fluid-filled structures exist so as to produce
increase control fluid pressure in a control channel in response to
the microfluidic device being compressed. In some embodiments, the
preferential compression fixture can include a pad or a pin for
applying pressure preferentially.
[0026] In some embodiments, a microfluidic system having increased
resistance to compression fixture pressure induced failure can
include, for example, a compensation device in fluid communication
with a control channel. The compensation device can produce
increased control fluid pressure in the control channel in response
to the microfluidic device being compressed by the compression
fixture. In some embodiments, the compensation device includes a
syringe plunger.
Temperature Controlled Reactions with Reduced Condensation
[0027] In another aspect, embodiments of the present invention
provide methods and systems for conducting a microfluidic
temperature controlled reaction using a thermal control device so
as to reduce condensation levels within a microfluidic device. A
method can include, for example, placing a microfluidic device in
thermal communication with a thermal control source using a
compression fixture and heating the compression fixture so that a
temperature of an elastomeric surface of the microfluidic device
contacted by the compression fixture is elevated above a
condensation threshold. In some embodiments, the compression
fixture is heated using a heat source other than the thermal
control source. In some embodiments, the temperature of an
elastomeric surface of the microfluidic device is elevated above
forty degrees centigrade, and can be elevated above seventy degrees
centigrade. Another method can include, for example, using a
compression fixture that includes a permeable portion adapted to be
held in contact with an elastomeric portion of the microfluidic
device. In some embodiments, the thermal control device includes
venting adapted to remove moisture from an elastomeric portion of a
microfluidic device. In some embodiments, the venting is forced. In
some embodiments, the thermal control device includes a dehydration
device for removing moisture from an elastomeric portion of a
microfluidic device.
[0028] In some embodiments, a system for conducting a microfluidic
temperature controlled reaction can include, for example, a thermal
control device that includes a thermal control source and a
compression fixture for holding a microfluidic device in thermal
communication with the thermal control source. The thermal control
device can be adapted to heat the compression fixture so that a
temperature of an elastomeric surface of the microfluidic device
contacted by the compression fixture is elevated above a
condensation threshold. The thermal control device can include a
heat source separate from the thermal control source, and the
separate heat source can supply heat to an upper region of an
elastomeric portion of a microfluidic device. In some embodiments,
the compression fixture includes a permeable portion adapted to be
held in contact with an elastomeric portion of a microfluidic
device. The thermal control device can include venting adapted to
remove moisture from an elastomeric portion of a microfluidic
device, and the venting can be forced. In some embodiments, the
thermal control device includes a dehydration device for removing
moisture from an elastomeric portion of the microfluidic
device.
Improved Fluorescent Imaging
[0029] In another aspect, the present invention provides methods
and systems for fluorescent imaging of a microfluidic device. A
method can include, for example, providing a microfluidic device
having a non-opaque portion that includes a plurality of processing
sites, a first plurality of flow channels primarily oriented along
a first feature direction, and a second plurality of flow channels
primarily oriented along a second feature direction. A method can
include illuminating the microfluidic device from an illumination
direction having a non-orthogonal azimuth relative to both the
first feature direction and the second feature direction, and
imaging the microfluidic device from an imaging direction. In some
embodiments, the azimuth can be non-orthogonal by at least 15
degrees, by at least 30 degrees, or by approximately 45 degrees. A
method can include determining an elevation for the illumination
direction so as to substantially minimize a ratio of reflections to
fluorescent signal in the imaging direction. A method can include
illuminating the microfluidic device from an illumination direction
so as to substantially minimize a ratio of reflections to
fluorescent signal in the imaging direction, and can include
determining the illumination direction. Determining the
illumination direction can include determining a ratio of
reflections to fluorescent signal for a plurality of different
azimuths and/or a plurality of different elevations.
[0030] In some embodiments, a system for fluorescent imagining of a
microfluidic device can include, for example, a microfluidic
device, an illumination subsystem adapted to illuminate the
microfluidic device with electromagnetic radiation, and an imaging
subsystem adapted to image the microfluidic device from an imaging
direction. The microfluidic device can have a non-opaque portion
that includes a plurality of processing sites, a first plurality of
flow channels primarily oriented along a first feature direction,
and a second plurality of flow channels primarily oriented along a
second feature direction. The illumination subsystem can illuminate
the microfluidic device from an illumination direction
non-orthogonal to both the first feature direction and the second
feature direction. The imaging subsystem can include a
charge-coupled device (CCD) camera array and/or a complementary
metal-oxide-semiconductor (CMOS) device. The illumination direction
can be adjustable, such as in azimuth and/or elevation. The
illumination subsystem can be adapted to illuminate the
microfluidic device from an azimuth that is non-orthogonal relative
to both the first feature direction and the second feature
direction by at least 15 degrees, by at least 30 degrees, or by
approximately 45 degrees. The illumination subsystem can be adapted
to illuminate the microfluidic device so as to substantially
minimize a ratio of reflections to fluorescent signal in the
imaging direction.
[0031] For a fuller understanding of the nature and advantages of
the present invention, reference should be made to the ensuing
detailed description and accompanying drawings. Other aspects,
objects and advantages of the invention will be apparent from the
drawings and detailed description that follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 depicts a perspective view of a unit cell of a
microfluidic device according to embodiments of the present
invention, showing flow paths for fluid introduction.
[0033] FIG. 1A shows an exploded perspective view of individual
layers of a unit cell of a microfluidic device according to
embodiments of the present invention.
[0034] FIGS. 1B to 1E show microfluidic molds according to
embodiments of the present invention.
[0035] FIG. 2 illustrates a perspective view of the unit cell of
FIG. 1, showing a flow path between two chambers.
[0036] FIG. 3 illustrates a perspective view of a microfluidic
device matrix having multiple unit cells according to embodiments
of the present invention, showing flow paths for fluid
introduction.
[0037] FIG. 4 illustrates a perspective view of the microfluidic
device matrix of FIG. 3, showing flow paths for fluid mixing.
[0038] FIG. 5A to 5C show cross-section views of a microfluidic
device unit cell according to embodiments of the present
invention.
[0039] FIGS. 6A to 6C show a microfluidic device according to
embodiments of the present invention.
[0040] FIG. 7 illustrates a microfluidic device according to
embodiments of the present invention.
[0041] FIG. 8 illustrates a microfluidic device according to
embodiments of the present invention.
[0042] FIGS. 9A and 9B illustrate a microfluidic device according
to embodiments of the present invention.
[0043] FIG. 10A is a simplified schematic diagram illustrating a
microfluidic device matrix having alternate fluid introduction flow
paths configurable to isolate device defects according to
embodiments of the present invention.
[0044] FIG. 10B is a simplified schematic diagram illustrating a
microfluidic device matrix having alternate fluid introduction flow
paths configurable to isolate device defects according to
embodiments of the present invention.
[0045] FIG. 11A is a simplified schematic diagram illustrating a
pressure control apparatus for use with a microfluidic device
according to embodiments of the present invention.
[0046] FIG. 11B is a simplified schematic diagram illustrating a
pressure control apparatus for use with a microfluidic device
according to embodiments of the present invention.
[0047] FIG. 11C is a simplified schematic diagram illustrating a
pressure control apparatus for use with a microfluidic device
according to embodiments of the present invention.
[0048] FIG. 11D is a simplified schematic diagram illustrating a
control system for a pressure control apparatus for use with
microfluidic devices according to embodiments of present
invention.
[0049] FIG. 12A shows a microfluidic matrix device having
peripheral vent channels according to embodiments of the present
invention.
[0050] FIG. 12B shows a close-up view of the upper left hand corner
of the microfluidic matrix device of FIG. 12A.
[0051] FIG. 12C is a simplified schematic diagram illustrating
microfluidic device vent lines connected with a vent port and a
vent according to embodiments of the present invention.
[0052] FIG. 13 shows a diagram illustrating a microfluidic device
configuration providing flow rate control of a sample and a reagent
introduced into a reaction chamber according to embodiments of the
present invention.
[0053] FIG. 14A shows a cross-section view of a microfluidic device
being held in thermal communication with a thermal control source
via a compression fixture.
[0054] FIG. 14B depicts a perspective view of a unit cell of a
microfluidic device according to embodiments of the present
invention.
[0055] FIG. 15 shows a cross-section view of a microfluidic device
unit cell being held in thermal communication with a thermal
control source via a compression fixture.
[0056] FIG. 16 shows a diagram of a control fluid introduction
device with a backflow prevention feature according to embodiments
of the present invention.
[0057] FIG. 17 depicts a perspective view of a unit cell of a
microfluidic device having a backflow prevention feature according
to embodiments of the present invention.
[0058] FIG. 18A depicts a perspective view of a unit cell of a
microfluidic device having a pressure compensation feature
according to embodiments of the present invention.
[0059] FIG. 18B shows a cross-section view of a microfluidic device
unit cell having a pressure compensation feature according to
embodiments of the present invention.
[0060] FIG. 19 shows a cross-section view of a microfluidic device
unit cell being held in thermal communication with a thermal
control source by way of a preferential compression fixture
according to embodiments of the present invention.
[0061] FIG. 20 shows a cross-section view of a microfluidic device
unit cell being held in thermal communication with a thermal
control source by way of a compression fixture coupled with a
pressure compensation device according to embodiments of the
present invention.
[0062] FIG. 21A shows a cross-section diagram of a microfluidic
device being heated by a thermal control source.
[0063] FIG. 21B shows a cross-section diagram of a microfluidic
device held in place during heating by a compression fixture.
[0064] FIG. 21C shows a cross-section diagram of a microfluidic
device held in place during heating by a compression fixture
according to embodiments of the present invention.
[0065] FIG. 22 is a simplified schematic diagram illustrating an
optical imaging system according to an embodiment of the present
invention.
[0066] FIG. 23A shows reflection test results using illumination in
accordance with embodiments of the present invention.
[0067] FIG. 23B shows additional reflection test results using
illumination in accordance with embodiments of the present
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0068] The present invention relates generally to microfluidic
devices and related apparatus, systems, and methods. In particular,
methods are provided for reducing mixing times during use of
microfluidic devices. Microfluidic devices and related methods of
manufacturing are provided with increased manufacturing yield
rates. Improved apparatus and related systems are provided for
supplying controlled pressure to microfluidic devices. Methods and
related microfluidic devices are provided for reducing dehydration
of microfluidic devices during use. Microfluidic devices and
related methods are provided with improved sample to reagent
mixture ratio control. Microfluidic devices and systems are
provided with improved resistance to compression fixture pressure
induced failure. Methods and systems for conducting temperature
controlled reactions using microfluidic devices are provided that
reduce condensation levels within the microfluidic device. Methods
and systems are provided for improved fluorescent imaging of
microfluidic devices.
Microfluidic Device Discussion
[0069] In the present application, references are made to certain
types of "reaction" chambers in a microfluidic device. In general,
these "reaction chambers" include processing sites, processing
chambers, and/or reaction sites, any combination of these, and the
like. These chambers may be closed, partially closed, open,
partially open, sealed, or combinations thereof, including any
temporary or transient conditions involving any of these states,
and the like. In some embodiments, the chambers are sealed, capable
of being sealed, closeable, isolated, capable of being isolated,
and combinations thereof, and any combination or single condition
of any temporary or transient conditions involving any of these
states, and the like. Therefore, use of the term reaction chamber
is not intended to limit the present invention, but to include
these other structures. Additionally, the term "chamber" is not
intended to limit the present invention, but should be used in its
ordinary meaning, unless specific features associated with the
chamber have been recited. Of course, there can be other
variations, modifications, and alternatives.
[0070] Moreover, through the present document, references are made
to fluorescent indications from a microfluidic device. Embodiments
herein are not limited to such fluorescent indications, but also
include luminescent indications, including chemiluminescent,
electroluminescent, electrochemiluminescent, and
phospholuminescent, bioluminescent, and other luminescent
processes, or any other processing involving any other type of
indications that may be detected using a detection device. As will
be evident to one of skill in the art, methods and systems operable
in the detection and analysis of these fluorescent and luminescent
indications are transferable from one indication to another.
Additionally, although some embodiments utilize spectral filters as
optical elements, this is not required. Some fluorescent and
luminescent applications do not utilize spectral filters in the
optical excitation path, the optical emission path, or both. As
described herein, other embodiments utilize spectral filters. One
of skill in the art will appreciate the differences associated with
particular applications.
[0071] In some embodiments, a variety of devices and methods for
conducting microfluidic analyses are utilized herein, including
devices that can be utilized to conduct thermal cycling reactions
such as nucleic acid amplification reactions. The devices can
differ from conventional microfluidic devices in that they can
include elastomeric components such as deflectable membranes that
can form valves; in some instances, much or all of the device is
composed of elastomeric material. For example, amplification
reactions can be linear amplifications, (amplifications with a
single primer), as well as exponential amplifications (i.e.,
amplifications conducted with a forward and reverse primer
set).
[0072] The methods and systems provided by some embodiments utilize
blind channel type devices in performing nucleic acid amplification
reactions. In these devices, the reagents that are typically
deposited within the reaction sites are those reagents necessary to
perform the desired type of amplification reaction. Usually this
means that some or all of the following are deposited: primers,
polymerase, nucleotides, metal ions, buffer, and cofactors, for
example. The sample introduced into the reaction site in such cases
is the nucleic acid template. Alternatively, however, the template
can be deposited and the amplification reagents flowed into the
reaction sites. As discussed in more detail throughout the present
specification, when a matrix device is utilized to conduct an
amplification reaction, samples containing nucleic acid template
are flowed through the vertical flow channels and the amplification
reagents through the horizontal flow channels or vice versa.
[0073] A variety of matrix or array-based devices are also utilized
in some embodiments. Certain of these devices include: (i) a first
plurality of flow channels formed in an elastomeric substrate, (ii)
a second plurality of flow channels formed in the elastomeric
substrate that intersect the first plurality of flow channels to
define an array of reaction sites, (iii) a plurality of isolation
valves disposed within the first and second plurality of flow
channels that can be actuated to isolate solution within each of
the reaction sites from solution at other reaction sites, and (iv)
a plurality of perimeter guard channels surrounding one or more of
the flow channels and/or one or more of the reaction sites to
inhibit evaporation of solution therefrom. The foregoing devices
can be utilized to conduct a number of different types of
reactions, including those involving temperature regulation (e.g.,
thermocycling of nucleic acid analyses).
[0074] Some of the microfluidic devices utilize a design typically
referred to herein as "blind channel" or "blind fill" and are
characterized in part by having a plurality of blind channels,
which are flow channels having a dead end or isolated end such that
solution can only enter and exit the blind channel at one end
(i.e., there is not a separate inlet and outlet for the blind
channel). These devices require only a single valve for each blind
channel to isolate a region of the blind channel to form an
isolated reaction site. Additionally, the blind channels can be
connected to an interconnected network of channels such that all
the reaction sites can be filled from a single, or limited number,
of sample inputs. Because of the reduction in complexity in inputs
and outputs and the use of only a single valve to isolate each
reaction site, the space available for reaction sites is increased.
Thus, the features of these devices means that each device can
include a large number of reaction sites (e.g., up to tens of
thousands) and can achieve high reaction site densities (e.g., over
1,000-4,000 reaction sites/cm.sup.2). Individually and
collectively, these features also directly translate into a
significant reduction in the size of these devices compared to
traditional microfluidic devices.
[0075] Some microfluidic devices utilize a matrix design.
Microfluidic devices of this type can utilize a plurality of
intersecting horizontal and vertical flow channels to define an
array of reaction sites at the points of intersection. Discrete
reaction chambers can also be fluidically coupled with the points
of intersection. A valve system referred to as a switchable flow
array can be used to control the flow through the flow channels.
Matrix devices can be constructed to analyze a large number of
samples under a limited number of conditions. Some microfluidic
devices can be hybrids that include both matrix and blind channel
features.
[0076] Other microfluidic devices are massively partitioning
devices (DID) such as described in PCT publication WO 2004/089810,
U.S. patent application Ser. No. 10/819,088 published as US
20050019792, copending commonly assigned patent application Ser.
No. PCT/U.S. 06/2141 entitled "Analysis using microfluidic
partitioning devices" filed Jun. 2, 2006 (attorney docket number
020174-012400), each of which is incorporated by reference in its
entirety for all purposes. Using massively partitioning devices, a
sample can be partitioned into a multitude of isolated reaction
chambers, and reactions carried out simultaneously in each
chamber.
[0077] The microfluidic devices that are described herein can be
made from various materials. For example, various components such
as flow channels, control channels, valves and/or pumps can be
fabricated from elastomeric materials. In some instances,
essentially the entire device can be made of elastomeric materials.
However, while some embodiments are described that include
components made from elastomeric materials, various other materials
can also be used.
[0078] The design of the devices enables them to be utilized in
combination with a number of different heating systems. Thus, the
devices are useful in conducting diverse analyses that require
temperature control. Additionally, those microfluidic devices
adapted for use in heating applications can incorporate a further
design feature to minimize evaporation of sample from the reaction
sites. Devices of this type may include a number of guard channels
and/or reservoirs or chambers formed within the device through
which water can be flowed to increase the water vapor pressure
within the material from which the device is formed, thereby
reducing evaporation of sample material from the reaction
sites.
[0079] In another embodiment, a temperature cycling device may be
used to control the temperature of the microfluidic devices.
Preferably, the microfluidic device would be adapted to make
thermal contact with the temperature cycling device. Where the
microfluidic device is supported by a substrate material, such as a
glass slide or the bottom of a carrier plate, such as a plastic
carrier, a window may be formed in a region of the carrier or slide
such that the microfluidic device, preferably a device having an
elastomeric block, may directly contact the heating/cooling block
of the temperature cycling device. In a preferred embodiment, the
heating/cooling block has grooves therein in communication with a
vacuum source for applying a suction force to the microfluidic
device, preferably a portion adjacent to where the reactions are
taking place. Alternatively, a rigid thermally conductive plate may
be bonded to the microfluidic device that then mates with the
heating and cooling block for efficient thermal conduction.
[0080] The array format of certain of the devices means the devices
can achieve high throughput. Collectively, the high throughput and
temperature control capabilities make the devices useful for
performing large numbers of nucleic acid amplifications (e.g.,
polymerase chain reaction (PCR)). Such reactions will be discussed
at length herein as illustrative of the utility of the devices,
especially of their use in any reaction requiring temperature
control. However, it should be understood that the devices are not
limited to these particular applications. The devices can be
utilized in a wide variety of other types of analyses or reactions.
Examples include analyses of protein-ligand interactions and
interactions between cells and various compounds. Further examples
are provided throughout the present specification.
[0081] The microfluidic devices disclosed herein are typically
constructed at least in part from elastomeric materials and
constructed by single and multilayer soft lithography (MSL)
techniques and/or sacrificial-layer encapsulation methods (see,
e.g., Unger et al. (2000) Science 288:113-116, and PCT Publication
WO 01/010 25, both of which are incorporated by reference herein in
their entirety for all purposes). Utilizing such methods,
microfluidic devices can be designed in which solution flow through
flow channels of the device is controlled, at least in part, with
one or more control channels that are separated from the flow
channel by an elastomeric membrane or segment. This membrane or
segment can be deflected into or retracted from the flow channel
with which a control channel is associated by applying an actuation
force to the control channels. By controlling the degree to which
the membrane is deflected into or retracted out from the flow
channel, solution flow can be slowed or entirely blocked through
the flow channel. Using combinations of control and flow channels
of this type, one can prepare a variety of different types of
valves and pumps for regulating solution flow as described in
extensive detail in Unger et al. (2000) Science 288:113-116, and
PCT Publication WO/02/43615 and WO 01/01025.
[0082] The devices provided herein incorporate such pumps and/or
valves to isolate selectively a reaction site at which reagents are
allowed to react. Alternatively, devices without pumps and/or
valves are utilized that use pressure driven flow or polymerization
processes to close appropriate channels and thereby selectively
isolate reaction sites. The reaction sites can be located at any of
a number of different locations within the device. For example, in
some matrix-type devices, the reaction site is located at the
intersection of a set of flow channels. In blind channel devices,
the reaction site is located at the end of the blind channel.
[0083] If the device is to be utilized in temperature control
reactions (e.g., thermocycling reactions), then, as described in
greater detail infra, the elastomeric device can be fixed to a
support (e.g., silicon wafer, plastic carrier, etc.). The resulting
structure can then be placed on a temperature control plate, for
example, to control the temperature at the various reaction sites.
In the case of thermocycling reactions, the device can be placed on
any of a number of thermocycling plates.
[0084] Because the devices can be made of materials that are
relatively optically transparent (e.g., elastomeric materials),
reactions can be readily monitored using a variety of different
detection systems at essentially any location on the microfluidic
device. Most typically, however, detection occurs at the reaction
site itself (e.g., an isolated chamber along a blind flow filled
device). The fact that the device can be manufactured from
substantially transparent materials also means that certain
detection systems can be utilized with the current devices that are
not usable with some traditional silicon-based microfluidic
devices. Detection can be achieved using detectors that are
incorporated into the device or that are separate from the device
but aligned with the region of the device to be detected.
[0085] Devices utilizing the matrix design can have a plurality of
vertical and horizontal flow channels that intersect to form an
array of junctions. Because a different sample and reagent (or set
of reagents) can be introduced into each of the flow channels, a
large number of samples can be tested against a relatively large
number of reaction conditions in a high throughput format. Thus,
for example, if a different sample is introduced into each of M
different vertical flow channels and a different reagent (or set of
reagents) is introduced into each of N horizontal flow channels,
then M.times.N different reactions can be conducted at the same
time. Matrix devices can include valves that allow for switchable
isolation of the vertical and horizontal flow channels. The valves
can be positioned to allow selective flow just through the vertical
flow channels or just through the horizontal flow channels. Because
devices of this type allow flexibility with respect to the
selection of the type and number of samples, as well as the number
and type of reagents, these devices can be used for conducting
analyses in which one wants to screen a large number of samples
against a relatively large number of reaction conditions. The
matrix devices can optionally incorporate guard channels to help
inhibit evaporation of sample and reactants.
[0086] Some high-density matrix designs utilize fluid communication
vias between layers of the microfluidic device to weave control
lines and fluid lines through the device. For example, by having a
fluid line in each layer of a two layer elastomeric block, higher
density reaction cell arrangements are possible. As will be evident
to one of skill in the art, multi-layer devices allow fluid lines
to cross over or under each other without being in fluid
communication. For example, in a particular design, a reagent fluid
channel in a first layer is connected to a reagent fluid channel in
a second layer through a via, while the second layer also has
sample channels therein, the sample channels and the reagent
channels terminating in sample and reagent chambers, respectively.
The sample and reagent chambers are in fluid communication with
each other through an interface channel that has an interface valve
associated therewith to control fluid communication between each of
the chambers of a reaction cell. In use, the interface is first
closed, then reagent is introduced into the reagent channel from
the reagent inlet and sample is introduced into the sample channel
through the sample inlet. Containment valves are then closed to
isolate each reaction cell from other reaction cells. Once the
reaction cells are isolated, the interface valve is opened to cause
the sample chamber and the reagent chamber to be in fluid
communication with each other so that a desired reaction may take
place. One of ordinary skill in the art would recognize many
variations, modifications, and alternatives.
[0087] Accordingly, a particular design for a microfluidic device
provides for a microfluidic device adapted to react M number of
different samples with N number of different reagents comprising: a
plurality of reaction cells, each reaction cell comprising a sample
chamber and a reagent chamber, the sample chamber and the reagent
chamber being in fluid communication through an interface channel
having an interface valve associated therewith for controlling
fluid communication between the sample chamber and the reagent
chamber; a plurality of sample inlets each in fluid communication
with the sample chambers; a plurality of reagent inlets each in
fluid communication with the reagent chambers; wherein one of the
sample inlets or reagent inlets is in fluid communication with one
of the sample chambers or one of the reagent chambers,
respectively, through a via. Certain embodiments include having the
reaction cells be formed within an elastomeric block formed from a
plurality of layers bonded together and the interface valve is a
deflectable membrane; having the sample inlets be in communication
with the sample chamber through a sample channel and the reagent
inlet in fluid communication with the reagent chamber through a
reagent channel, a portion of the sample channel and a portion of
the reagent channel being oriented about parallel to each other and
each having a containment valve associated therewith for
controlling fluid communication therethrough; having the valve
associated with the sample channel and the valve associated with
the reagent channel in operable communication with each other
through a common containment control channel; having the
containment common control channel located along a line about
normal to one of the sample channel or the reagent channel.
[0088] The microfluidic devices utilized in embodiments of the
present invention may be further integrated into the carrier
devices described in co-pending and commonly owned U.S. patent
application Ser. No. 11/058,106, filed on Feb. 14, 2005, which is
incorporated herein for all purposes. The carrier devices provide
on-board continuous fluid pressure to maintain valve closure away
from a source of fluid pressure, e.g., house air pressure. patent
application Ser. No. 11/058,106 further provides for an automated
system for charging and actuating the valves of the present
invention as described therein. An another preferred embodiment,
the automated system for charging accumulators and actuating valves
employs a device having a platen that mates against one or more
surfaces of the microfluidic device, wherein the platen has at
least two or more ports in fluid communication with a controlled
vacuum or pressure source, and may include mechanical portions for
manipulating portions of the microfluidic device, for example, but
not limited to, check valves.
[0089] Another device utilized in embodiments of the present
invention provides a carrier used as a substrate for stabilizing an
elastomeric block. Preferably the carrier has one or more of the
following features; a well or reservoir in fluid communication with
the elastomeric block through at least one channel formed in or
with the carrier; an accumulator in fluid communication with the
elastomeric block through at least one channel formed in or with
the carrier; and, a fluid port in fluid communication with the
elastomeric block, wherein the fluid port is preferably accessible
to an automated source of vacuum or pressure, such as the automated
system described above, wherein the automated source further
comprises a platen having a port that mates with the fluid port to
form an isolated fluid connection between the automated system for
applying fluid pressure or vacuum to the elastomeric block. In
devices utilized in certain embodiments, the automated source can
also make fluid communication with one or more accumulators
associated with the carrier for charging and discharging pressure
maintained in an accumulator. In certain embodiments, the carrier
may further comprise a region located in an area of the carrier
that contacts the microfluidic device, wherein the region is made
from a material different from another portion of the carrier, the
material of the region being selected for improved thermal
conduction and distribution properties that are different from the
other portion of the carrier. Preferred materials for improved
thermal conduction and distribution include, but are not limited to
silicon, preferably silicon that is highly polished, such as the
type of silicon available in the semiconductor field as a polished
wafer or a portion cut from the wafer, e.g., chip.
[0090] As described more fully below, embodiments of the present
invention utilize a thermal source, for example, but not limited to
a PCR thermocycler, which may have been modified from its original
manufactured state. Generally the thermal source has a thermally
regulated portion that can mate with a portion of the carrier,
preferably the thermal conduction and distribution portion of the
carrier, for providing thermal control to the elastomeric block
through the thermal conduction and distribution portion of the
carrier. In a preferred embodiment, thermal contact is improved by
applying a source of vacuum to a one or more channels formed within
the thermally regulated portion of the thermal source, wherein the
channels are formed to contact a surface of the thermal conduction
and distribution portion of the carrier to apply suction to and
maintain the position of the thermal conduction and distribution
portion of the carrier. In a preferred embodiment, the thermal
conduction and distribution portion of the carrier is not in
physical contact with the remainder of the carrier, but is
associated with the remainder of the carrier and the elastomeric
block by affixing the thermal conduction and distribution portion
to the elastomeric block only and leaving a gap surrounding the
edges of the thermal conduction and distribution portion to reduce
parasitic thermal effects caused by the carrier. It should be
understood that in many aspects of the invention described herein,
the preferred elastomeric block could be replaced with any of the
known microfluidic devices in the art not described herein, for
example devices produced such as the GeneChip.RTM. by
Affymetrix.RTM. of Santa Clara, Calif., USA, or by Caliper of
Mountain View, Calif., USA. U.S. patents issued to Soane, Parce,
Fodor, Wilding, Ekstrom, Quake, or Unger, describe microfluidic or
mesoscale fluidic devices that can be substituted for the
elastomeric block of the present invention to take advantage of the
thermal advantages and improvements, e.g., suction positioning,
reducing parasitic thermal transfer to other regions of the fluidic
device, which are described above in the context of using an
elastomeric block.
[0091] Utilizing systems and methods provided according to
embodiments of the present invention, throughput increases are
provided over 384 well systems. As an example, throughput increases
of a factor of 4, 6, 12, and 24 and greater are provided in some
embodiments. These throughput increases are provided while reducing
the logistical friction of operations. Moreover the systems and
methods of embodiments of the present invention enable multiple
assays for multiple samples. For example, in a specific embodiment
96 samples and 96 assays are utilized to provide a total of 9,216
data points. In a particular example, the 96 assays are components
of a TaqMan 5' Nuclease Assay.
[0092] Furthermore, embodiments of the present invention provide
reduced reaction volumes. In embodiments of the present invention,
reaction volumes ranging from 10 picoliters to 100 nanoliters are
utilized. In some embodiments, reaction volumes greater than 100
nanoliters can be utilized. Merely by way of example, in
embodiments, the methods and systems of the present invention can
be utilized with reaction volumes of 10 picoliters to 1 nanoliter.
In alternative embodiments, reaction volumes of 2 nanoliters to 100
nanoliters can be utilized.
[0093] Depending on the geometry of the particular microfluidic
device and the size of the microfluidic device and the arrangement
of the fluid communication paths and processing site, embodiments
of the present invention provide for a range of processing site (or
reaction chamber) densities. In some embodiments, the methods and
systems of the present invention are utilized with chamber
densities ranging from about 100 chambers per cm.sup.2 to about 1
million chambers per cm.sup.2. Merely by way of example,
microfluidic devices with chamber densities of 250, 1,000, 2,500,
10,000, 25,000, 100,000, and 250,000 chambers per cm.sup.2 are
utilized according to embodiments of the present invention. In some
embodiments, chamber densities in excess of 1,000,000 chambers per
cm.sup.2 are utilized, although this is not required by the present
invention.
[0094] Operating microfluidic devices with such small reaction
volumes reduces reagent usage as well as sample usage. Moreover,
some embodiments of the present invention provide methods and
systems adapted to perfoim real-time detection, when used in
combination with real-time quantitative PCR. Utilizing these
systems and methods, six orders of linear dynamic range are
provided for some applications as well as quantitative resolution
high enough to allow for the detection of sub-nanoMolar fluorophore
concentrations in 10 nanoliter volumes. One of ordinary skill in
the art would recognize many variations, modifications, and
alternatives.
[0095] Methods conducted with certain blind channel type devices
involve providing a microfluidic device that comprises a flow
channel formed within an elastomeric material; and a plurality of
blind flow channels in fluid communication with the flow channel,
with an end region of each blind flow channel defining a reaction
site or multiple reaction sites formed along the flow channel. At
least one reagent is introduced into each of the reaction sites,
and then a reaction is detected at one or more of the reaction
sites. The method can optionally include heating the at least one
reagent within the reaction site. Thus, for example, a method can
involve introducing the components for a nucleic acid amplification
reaction and then thermocycling the components to form amplified
product. As more fully described below, an optical imaging system
adapted to characterize reactions occurring in certain microfluidic
devices is provided according to embodiments of the present
invention.
[0096] It is understood that the invention is not limited to the
particular methodology, protocols, and reagents, etc., described
herein, as these may vary as the skilled artisan will recognize. It
is also to be understood that the terminology used herein is used
for the purpose of describing particular embodiments only, and is
not intended to limit the scope of the invention. It is also to be
noted that as used herein and in the appended claims, the singular
forms "a," "an," and "the" include the plural reference unless the
context clearly dictates otherwise. Thus, for example, a reference
to "a cell" is a reference to one or more cells and equivalents
thereof known to those skilled in the art.
[0097] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which the invention pertains. The
embodiments of the invention and the various features and
advantageous details thereof are explained more fully with
reference to the non-limiting embodiments and examples that are
described and/or illustrated in the accompanying drawings and
detailed in the following description. It should be noted that the
features illustrated in the drawings are not necessarily drawn to
scale, and features of one embodiment may be employed with other
embodiments as the skilled artisan would recognize, even if not
explicitly stated herein. Descriptions of well-known components and
processing techniques may be omitted so as to not unnecessarily
obscure the embodiments of the invention. The examples used herein
are intended merely to facilitate an understanding of ways in which
the invention may be practiced and to further enable those of skill
in the art to practice the embodiments of the invention.
Accordingly, the examples and embodiments herein should not be
construed as limiting the scope of the invention, which is defined
solely by the appended claims and applicable law. Moreover, it is
noted that like reference numerals reference similar parts
throughout the several views of the drawings.
[0098] Accordingly, provided immediately below is a "Definition"
section, where certain terms related to the invention are defined
specifically for clarity, but all of the definitions are consistent
with how a skilled artisan would understand these terms. Particular
methods, devices, and materials are described, although any methods
and materials similar or equivalent to those described herein can
be used in the practice or testing of the invention. All references
referred to herein are incorporated by reference herein in their
entirety.
DEFINITIONS
[0099] PNA is peptide nucleic acid
[0100] LNA is locked nucleic acid
[0101] DA is dynamic array
[0102] PCR is polymerase chain reaction
[0103] BSA is bovine serum albumin
[0104] FRET is fluorescence resonance energy transfer
[0105] GT is genotyping
[0106] PEG is polyethylene glycol
[0107] PLP is padlock probe
[0108] The term "analyte" as used herein, generally refers to a
nucleic acid molecule or mixture of nucleic acid molecules, defined
infra, that is to be detected or quantified using the methods of
the invention. The terms "target nucleic acid analyte" and "nucleic
acid analyte" are used interchangeably with the term "analyte" for
the purposes of this invention.
[0109] The terms "complementary" or "complementarity" as used
herein, may include the natural binding of polynucleotides under
permissive salt and temperature conditions by base-pairing. For
example, the sequence "A-G-T" binds to the complementary sequence
"T-C-A." Complementarity between two single-stranded molecules may
be "partial," in which only some of the nucleic acids bind, or it
may be complete when total complementarity exists between the
single stranded molecules. The degree of complementarity between
nucleic acid strands has significant effects on the efficiency and
strength of hybridization between nucleic acid strands. This is of
particular importance in amplification reactions, which depend upon
binding between nucleic acids strands and in the design and use of
molecules.
[0110] The term "dye" as used herein, generally refers to any
organic or inorganic molecule that absorbs electromagnetic
radiation at a wavelength greater than or equal 340 nm.
[0111] The term "fluorescent dye" as used herein, generally refers
to any dye that emits electromagnetic radiation of longer
wavelength by a fluorescent mechanism upon irradiation by a source
of electromagnetic radiation, such as a lamp, a photodiode, or a
laser.
[0112] The term "GT sample buffer," as used herein generally refers
to a buffer that is capable of blocking binding sites on the
surface of the reaction channels and chambers in a DA chip. The
buffer protects the reaction components from depletion during the
chip loading process or reaction. It may also reduce the usage of
additional Taq-Gold Polymerase by less than about 80% for reagent
costs. A 20.times.GT buffer may include a combination of betaine
(FW 117.15), BSA, Superblock.RTM. T20 (in PBS) (Thermo Scientific,
Rockford, Ill.), Superblock.RTM. (in PBS) (Thermo Scientific,
Rockford, Ill.), Superblock.RTM. (in TBS) (Thermo Scientific,
Rockford, Ill.), Superblock.RTM. T20 (in TBS) (Thermo Scientific,
Rockford, Ill.), glycerol, PEG 20,000, PEG MME550, PEG MME5000, and
Tween 20.
[0113] The term "homogenous assay" as used herein, generally refers
to a method to detect or quantify a nucleic acid analyte that
requires no post-assay processing to record the result of the
assay. The homogenous assays may be carried out in closed tubes or
microfluidic arrays where no further addition of reagents or
supplementary chemicals are necessary to record the result once the
assay is started. Homogenous assays allow recordation of the result
of the assay in real time, meaning that the result of the assay can
be continuously recorded as the assay progresses in time.
[0114] The term "hydrolysis probes" as used herein are generally
described in U.S. Pat. No. 5,210,015 incorporated herein by
reference in its entirety. Hydrolysis probes take advantage of the
5'-nuclease activity present in the thermostable Taq polymerase
enzyme used in the PCR reaction (TaqMan.RTM. probe technology,
Applied Biosystems, Foster City Calif.). The hydrolysis probe is
labeled with a fluorescent detector dye such as fluorescin, and an
acceptor dye or quencher. In general, the fluorescent dye is
covalently attached to the 5' end of the probe and the quencher is
attached to the 3' end of the probe, and when the probe is intact,
the fluorescence of the detector dye is quenched by fluorescence
resonance energy transfer (FRET). The probe may anneal downstream
of one of the primers that defines one end of the amplification
target site on the nucleic acid target analyte in the PCR reaction.
Using the polymerase activity of the Taq enzyme, amplification of
the target nucleic acid analyte is directed by one primer that is
upstream of the probe and a second primer that is downstream of the
probe but anneals to the opposite strand of the target nucleic
acid. As the upstream primer is extended, the Taq polymerase
reaches the region where the labeled probe is annealed, recognizes
the probe-template hybrid as a substrate, and hydrolyzes
phosphodiester bonds of the probe. The hydrolysis reaction
irrevocably releases the quenching effect of the quencher dye on
the reporter dye, thus resulting in increasing detector
fluorescence with each successive PCR cycle. In particular, the
hydrolysis probes of the invention may capable of detecting 8-mer
or 9-mer motifs that are common in the human and other
transcriptomes and may have a high T.sub.m of about 70.degree. C.
enabled by LNA analogs.
[0115] The term "label" as used herein refers to any atom or
molecule which can be used to provide a detectable and/or
quantifiable signal. In particular, the label can be attached to a
nucleic acid or protein. Labels may provide signals detectable by
fluorescence, radioactivity, colorimetric, X-ray diffraction or
absorption, magnetism, enzymatic activity, and the like.
[0116] The term "nucleic acid" as used herein generally refers to
cDNA, DNA, RNA, single-stranded or double-stranded and any chemical
modification thereof, such as PNA and LNA. LNAs are described in
U.S. Pat. Nos. 6,794,499, 6,670,461, 6,262,490, and 6,770,748
herein incorporated by reference in their entirety. Nucleic acids
may be of any size. Nucleic acid modifications may include addition
of chemical groups that incorporate additional charge,
polarizability, hydrogen bonding, electrostatic interaction, and
functionality to the individual nucleic acid bases or to the
nucleic acid as a whole. Such modifications may include modified
bases such as 2'-position sugar modifications, 5-position
pyrimidine modifications, 8-position purine modifications,
modifications at cytosine exocylcic amines, substitutions of
5-bromo-uracil, backbone modifications, methylations, unusual base
pairing combinations such as the isobases isocytidine and
isoguanidine and the like. The nucleic acid can be derived from a
completely chemical synthesis process, such as a solid phase
mediated chemical synthesis, or from a biological origin, such as
through isolation from almost any species that can provide nucleic
acid, or from processes that involve the manipulation of nucleic
acids by molecular biology tools, such as DNA replication, PCR
amplification, reverse transcription, or from a combination of
those processes.
[0117] The term "nucleic acid probe" as used herein is a nucleic
acid that carriers at least one covalently attached dye, such as a
fluorescent dye. In particular, the probe does not contain a
sequence complementary to sequences used to prime the PCR
reaction.
[0118] The term "padlock probe" or "PLP" as used herein, generally
refers to linear oligonucleotides having a length of about 100 base
pairs. The sequences at the 3' and 5' ends of the PLP are
complementary to adjacent sequences in the target nucleic acid
analyte. In the central, noncomplementary region of the PLP there
is a "tag sequence" that may be used to identify the specific PLP.
The tag sequence may be flanked by universal primer sites or unique
and/or specific primer sites, which allow PCR amplification of the
tag sequence. Upon hybridization to the target, the 5' and 3' ends
of the PLP are brought into close proximity and may be subsequently
ligated. The resulting product is a circular probe molecule
catenated to the target nucleic acid analyte. The tag regions of
circularized PLPs may be amplified and quantified and/or detected
using TAQMAN.RTM. Real Time PCR, for example. The presence and
amount of amplicon may be correlated with the presence and quantity
of target sequence in the sample. For descriptions of PLPs see,
e.g., Landegren et al., 2003, Padlock and proximity probes for in
situ and array-based analyses: tools for the post-genomic era,
Comparative and Functional Genomics 4:525-30; Nilsson et al., 2006,
Analyzing genes using closing and replicating circles Trends
Biotechnol. 24:83-8; Nilsson et al., 1994, Padlock probes:
circularizing oligonucleotides for localized DNA detection, Science
265:2085-8. The above references are incorporated by reference
herein in their entirety.
[0119] The term "PCR," as used herein, generally refers to a method
for amplifying, detecting, or quantifying a specific region of an
analyte. One skilled in the art appreciates that there are several
variations on the basic PCR technique such as allele-specific PCR,
assembly PCR or polymerase cycling assembly (PCA), colony PCR,
helicase-dependent amplification, hot start PCR,
intersequence-specific (ISSR) PCR, inverse PCR, ligation-mediated
PCR, methylation-specific PCR, multiplex ligation dependent probe
amplification, multiplex PCR, nested PCR, overlap-extension PCR,
quantitative PCR, quantitative real-time PCR, RT-PCR, thermal
asymmetric interlaces (TAIL) PCR, touchdown PCR, and PAN-AC.
Additionally, one skilled in the art would understand how to
practice these variations on the basic PCR technique.
[0120] The phase "preliminary amplification reaction" as used
herein, generally refers to processes for preparing the sample
prior to running the homogenous assay. The term "pre-amplified
sample" may be used interchangeably with the phrase "preliminary
amplification reaction" for the purposes of the invention
herein.
[0121] The term "purification," as used herein, generally refers to
any process by which proteins, polypeptides, or nucleic acids are
separated from other elements or compounds on the basis of charge,
molecular size, or binding affinity.
[0122] The term "quencher" as used herein, generally refers to dye
that reduces the emission of fluorescence of another dye.
[0123] The term "querying" as used herein, generally refers to
determining whether a target-specific probe is associated with
(e.g., bound to or cantenated with) the nucleic acid analyte, and
optionally quantifying the amount of target-specific probe in the
sample.
[0124] A "sample" as used herein, generally refers to a sample of
tissue or fluid from a human or animal including, but not limited
to plasma, serum, spinal fluid, lymph fluid, the external sections
of the skin, respiratory, intestinal and genitourinary tracts,
tears, saliva, blood cells, tumors, organs, tissue and sample of in
vitro cell culture constituents. In particular, the sample may be
single cells, paraffin embedded tissue samples, and needle
biopsies. Moreover, a sample may include environmental samples such
as lake water, and food samples.
[0125] The phrase "substantially purified," or "substantially
isolated," as used herein generally includes nucleic or amino acid
sequences that are removed from their natural environment, isolated
or separated, and are at least about 60% free, specifically at
least about 75% free, and most specifically at least about 90% free
from other components with which they may be associated with, and
includes recombinant or cloned nucleic acid isolates and chemically
synthesized analogs or analogs biologically synthesized by
systems.
[0126] Given the tremendous diversity of polymer chemistries,
precursors, synthetic methods, reaction conditions, and potential
additives, there are a huge number of possible elastomer systems
that could be used to make elastomeric blocks, layers, membranes,
microvalves, pumps, and the like. Variations in the materials used
may in some cases be driven by the need for particular material
properties, i.e., solvent resistance, stiffness, gas permeability,
or temperature stability. There are many, many types of elastomeric
polymers. A brief description of the most common classes of
elastomers is presented here, with the intent of showing that even
with relatively "standard" polymers, many possibilities for bonding
exist. Common elastomeric polymers include polyisoprene,
polybutadiene, polychloroprene, polyisobutylene,
poly(styrene-butadiene-styrene), the polyurethanes, and silicones
or polysiloxanes.
[0127] Polyisoprene, polybutadiene, and polychloroprene are all
polymerized from diene monomers, and therefore have one double bond
per monomer when polymerized. This double bond allows the polymers
to be converted to elastomers by vulcanization (generally, sulfur
is used to form crosslinks between the double bonds by heating).
This would easily allow homogeneous multilayer soft lithography by
incomplete vulcanization of the layers to be bonded; photoresist
encapsulation would be possible by a similar mechanism.
[0128] Pure polyisobutylene has no double bonds, but is crosslinked
to use as an elastomer by including a small amount (.about.1%) of
isoprene in the polymerization. The isoprene monomers give pendant
double bonds on the polyisobutylene backbone, which may then be
vulcanized as above.
[0129] Poly(styrene-butadiene-styrene) is produced by living
anionic polymerization (that is, there is no natural
chain-terminating step in the reaction), so "live" polymer ends can
exist in the cured polymer. This makes it a natural candidate for a
photoresist encapsulation system (where there will be plenty of
unreacted monomer in the liquid layer poured on top of the cured
layer). Incomplete curing would allow homogeneous multilayer soft
lithography (A to A bonding). The chemistry also facilitates making
one layer with extra butadiene ("A") and coupling agent and the
other layer ("B") with a butadiene deficit (for heterogeneous
multilayer soft lithography). SBS is a "thermoset elastomer",
meaning that above a certain temperature it melts and becomes
plastic (as opposed to elastic); reducing the temperature yields
the elastomer again. Thus, layers can be bonded together by
heating.
[0130] Polyurethanes are produced from di-isocyanates (A-A) and
di-alcohols or di-amines (B-B); since there are a large variety of
di-isocyanates and di-alcohols/amines, the number of different
types of polyurethanes is huge. The A vs. B nature of the polymers,
however, make them useful for heterogeneous multilayer soft
lithography just as RTV 615 is: by using excess A-A in one layer
and excess B-B in the other layer.
[0131] Allcock et al, Contemporary Polymer Chemistry, 2nd Ed.
describes elastomers in general as polymers existing at a
temperature between their glass transition temperature and
liquefaction temperature. Elastomeric materials exhibit elastic
properties because the polymer chains readily undergo torsional
motion to permit uncoiling of the backbone chains in response to a
force, with the backbone chains recoiling to assume the prior shape
in the absence of the force. In general, elastomers deform when
force is applied, but then return to their original shape when the
force is removed. The elasticity exhibited by elastomeric materials
may be characterized by a Young's modulus. Materials having a
Young's modulus of between about 1 Pa to about 1 TPa, or between
about 10 Pa to about 100 GPa, or between about 20 Pa to about 1
GPa, or between about 50 Pa to about 10 MPa, or between about 100
Pa to about 1 MPa are useful in accordance with embodiments of the
present invention, although materials having a Young's modulus
outside of these ranges could also be utilized depending upon the
needs of a particular application. In some cases, materials can
have a Young's modulus of about 100 MPA (megapascals) or less. In
other embodiments, the Young's modulus of the material is about 75
MPA or less, about 50 MPa or less, about 25 MPa or less, about 10
MPa or less, about 8 MPa or less, about 5 MPa or less, or about 2
MPa or less.
[0132] Embodiments of the present invention provide a microfluidic
device that includes features such as channels, valves, and
chambers, that are at least partially contained, embedded, or
formed by or within one or more layers and/or levels. Such layers
and/or levels can be made from various materials, such as an
elastomeric material. An exemplary microfluidic device has a
reagent flow channel, or reagent line, formed in a first layer. The
reagent flow channel includes a containment valve and a chamber
conduit. The microfluidic device may also have a control channel,
or containment line, formed in a second layer adjacent to the first
layer. Further, the microfluidic device may contain a sample flow
channel, or sample line, formed in a third layer adjacent to the
second layer. The sample flow channel may include a containment
valve and a chamber conduit. The control channel can be in
operative association with both the reagent flow channel
containment valve and the sample flow channel containment valve.
The microfluidic device can include a reagent chamber in fluid
communication with the reagent line, and a sample chamber in fluid
communication with the sample line. The reagent chamber and the
sample chamber may be in fluid communication with each other by way
of a reaction flow channel or reaction line, formed in the third
layer. The reaction line may include an interface valve. The
microfluidic device may also include an interface channel formed in
a fourth layer adjacent to the third layer. The interface channel
can be in operative association with the reaction flow channel
interface valve.
[0133] Embodiments of the present invention also encompass methods
of making and using the microfluidic devices disclosed herein. For
example, operation of a microfluidic device can involve opening one
or more isolation valves, closing one or more interface valves, and
flowing material past the isolation valves and into one or more
chambers, optionally under pressure. Techniques may also include
changing the pressure in a containment line to close the isolation
valves, so as to seal off the individual chambers, and changing the
pressure in an interface line, so as to open an interface valve. A
first material in a first chamber can flow past an open interface
valve and into a second chamber, where the first material mixes or
reacts with a second material contained therein.
Exemplarly Fabrication Methods
[0134] The methods used in fabrication of a microfluidic device may
vary with the materials used, and include soft lithography methods,
microassembly, bulk micromachining methods, surface micro-machining
methods, standard lithographic methods, wet etching, reactive ion
etching, plasma etching, stereolithography and laser chemical
three-dimensional writing methods, modular assembly methods,
replica molding methods, injection molding methods, hot molding
methods, laser ablation methods, combinations of methods, and other
methods known in the art or developed in the future. A variety of
exemplary fabrication methods are described in Fiorini and Chiu,
2005, "Disposable microfluidic devices: fabrication, function, and
application" Biotechniques 38:429-46; Beebe et al., 2000,
"Microfluidic tectonics: a comprehensive construction platform for
microfluidic systems." Proc. Natl. Acad. Sci. USA 97:13488-13493;
Rossier et al., 2002, "Plasma etched polymer microelectrochemical
systems" Lab Chip 2:145-150; Becker et al., 2002, "Polymer
microfluidic devices" Talanta 56:267-287; Becker et al., 2000,
"Polymer microfabrication methods for microfluidic analytical
applications" Electrophoresis 21:12-26; U.S. Pat. No. 6,767,706 B2,
e.g., Section 6.8 "Microfabrication of a Silicon Device"; Terry et
al., 1979, A Gas Chromatography Air Analyzer Fabricated on a
Silicon Wafer, IEEE Trans. on Electron Devices, v. ED-26, pp.
1880-1886; Berg et al., 1994, Micro Total Analysis Systems, New
York, Kluwer; Webster et al., 1996, Monolithic Capillary Gel
Electrophoresis Stage with On-Chip Detector in International
Conference On Micro Electromechanical Systems, MEMS 96, pp. 491496;
and Mastrangelo et al., 1989, Vacuum-Sealed Silicon Micromachined
Incandescent Light Source, in Intl. Electron Devices Meeting, IDEM
89, pp. 503-506. Each of these references are incorporated herein
by reference for all purposes.
[0135] In preferred embodiments, the device is fabricated using
elastomeric materials. Fabrication methods using elastomeric
materials and methods for design of devices and their components
have been described in detail in the scientific and patent
literature. See, e.g., Unger et al., 2000, Science 288:113-16; U.S.
Pat. No. 6,960,437 (Nucleic acid amplification utilizing
microfluidic devices); U.S. Pat. No. 6,899,137 (Microfabricated
elastomeric valve and pump systems); U.S. Pat. No. 6,767,706
(Integrated active flux microfluidic devices and methods); U.S.
Pat. No. 6,752,922 (Microfluidic chromatography); U.S. Pat. No.
6,408,878 (Microfabricated elastomeric valve and pump systems);
U.S. Pat. No. 6,645,432 (Microfluidic systems including
three-dimensionally arrayed channel networks); U.S. Patent
Application publication Nos. 2004/0115838, 2005/0072946;
2005/0000900; 2002/0127736; 2002/0109114; 2004/0115838;
2003/0138829; 2002/0164816; 2002/0127736; and 2002/0109114; PCT
patent publications WO 2005/084191; WO 05030822A2; and WO 01/01025;
Quake & Scherer, 2000, "From micro to nanofabrication with soft
materials" Science 290: 1536-40; Xia et al., 1998, "Soft
lithography" Angewandte Chemie-International Edition 37:551-575;
Unger et al., 2000, "Monolithic microfabricated valves and pumps by
multilayer soft lithography" Science 288:113-116; Thorsen et al.,
2002, "Microfluidic large-scale integration" Science 298:580-584;
Chou et al., 2000, "Microfabricated Rotary Pump" Biomedical
Microdevices 3:323-330; Liu et al., 2003, "Solving the
"world-to-chip" interface problem with a microfluidic matrix"
Analytical Chemistry 75, 4718-23," Hong et al, 2004, "A
nanoliter-scale nucleic acid processor with parallel architecture"
Nature Biotechnology 22:435-39; Fiorini and Chiu, 2005, "Disposable
microfluidic devices: fabrication, function, and application"
Biotechniques 38:429-46; Beebe et al., 2000, "Microfluidic
tectonics: a comprehensive construction platform for microfluidic
systems." Proc. Natl. Acad. Sci. USA 97:13488-13493; Rolland et
al., 2004, "Solvent-resistant photocurable "liquid Teflon" for
microfluidic device fabrication" J. Amer. Chem. Soc. 126:2322-2323;
Rossier et al., 2002, "Plasma etched polymer microelectrochemical
systems" Lab Chip 2:145-150; Becker et al., 2002, "Polymer
microfluidic devices" Talanta 56:267-287; Becker et al., 2000, and
other references cited herein and found in the scientific and
patent literature. Each of these references are incorporated herein
by reference for all purposes.
[0136] Embodiments of the present invention further encompass
aspects of microfluidic fabrication and production, as well as
microfluidic device operation and use, as disclosed in U.S. patent
application Ser. No. 12/018,138 filed Jan. 22, 2008, the content of
which is incorporated herein by reference for all purposes.
[0137] Any of a variety of ablation, etching, or similar techniques
can be used to form vias or passages in an elastomeric block,
membrane, or layer. Such etching procedures are well suited for
creating elastomeric layers having multiple holes or apertures, for
example. In an exemplary process, an elastomeric material is placed
on a wafer or mold, and allowed to cure. The elastomeric material
can include one or more polymers incorporating materials such as
chlorosilanes or methyl-, ethyl-, and phenylsilanes, and
polydimethylsiloxane (PDMS) such as Dow Chemical Corp. Sylgard 182,
184 or 186, or aliphatic urethane diacrylates such as (but not
limited to) Ebecryl 270 or In 245 from UCB Chemical may also be
used. In some cases, the elastomeric material is deposited on the
wafer or mold in a spin coating process, a spray coating process, a
dip coating process, a screen printing process, an inkjet
deposition process, or the like. The curing procedure can involve
baking or room temperature vulcanizing (RTV), photocuring, and the
like.
[0138] An elastomeric composition may include multiple parts, which
can be mixed together at various ratios to obtain desired bond
properties. For example, an elastomeric material may include a Part
A and a Part B, which when mixed together in prescribed amounts
facilitates the desired bond parameters. In some cases, the parts
may be mixed in a ratio within a range from about 3:1 to about
30:1. For example, an elastomeric PDMS composition is baked to
provide a 10:1 RTV layer.
[0139] In some cases, a photoresist material can be placed on the
cured elastomeric material. For example, an SU-8 resist (available
from MicroChem Corp., Newton Mass.) can be applied to the
elastomer. Exemplary SU-8 resists include SU-8 2000, SU-8 3000,
SU-8 2007, SU-8 3005, and the like. The photoresist can be
deposited on the elastomeric material in a spin coating procedure,
at a desired rotational speed and duration. In some cases, the spin
coating can be performed at a rotational speed within a range from
about 1000 to about 10,000 rpm, and for a duration within a range
from about 20 seconds to about 2,000 seconds. For example, the spin
coating can be performed at 5000 rpm for 200 seconds. Following
this deposition, the photoresist mask can have a thickness or depth
within a range from about 0.5 to about 50 microns. In some cases,
the thickness is about 5 microns. The thickness of the mask can be
selected for facile via opening formation, and the selected spin
time can eliminate or inhibit beading of the photoresist material.
The photoresist material can be used as an etch mask.
[0140] Additional procedures can be performed to prepare the
photoresist for lithography exposure. For example, the photoresist
can be processed at a selected temperature for a selected time
duration. In some embodiments, the photoresist is soft baked at a
temperature within a range from about 45.degree. C. to about
85.degree. C. Relatedly, the photoresist can be baked for a
duration within a range from about 1 minute to about 10 minutes. In
some cases, the soft bake is performed for 5 minutes at 65.degree.
C. Such preparation techniques can help to eliminate or inhibit
photoresist mask cracking at exposure. The preparation procedure
may also include cooling the photoresist. For example, the
photoresist may be cooled at room temperature or at a temperature
within a range from about 18.degree. C. to about 37.degree. C., and
for a duration within a range from about 3 minutes to about 300
minutes. In some cases, the photoresist is cooled for about 30
minutes.
[0141] The lithography procedure can involve multiple exposure
steps. For example, a first exposure step can be performed with a
first exposure mask, and a second subsequent exposure step can be
performed with a second exposure mask. An exposure step can involve
the application of radiation or energy, through an exposure mask,
toward a photoresist. Exposure radiation can include ultraviolet
light, near ultraviolet light, deep ultraviolet light, visible
light, infrared light, or energy at any desired wavelength along
the electromagnetic spectrum. In some cases, exposure radiation is
delivered at one or more wavelengths within a range from about 10
to about 10.sup.-9 cm. In some cases, the type of radiation or
energy is selected based on the composition of the photoresist. For
example, specific types of radiation or energy can be applied to
I-line photoresists, G-line photoresists, H-line photoresists, and
the like.
[0142] The use of multiple masks can help to prevent or inhibit the
effect of contaminants on a mask from replicating on the
photoresist. For example, if there is an unwanted particle on the
first mask at a certain location, exposure with a second mask can
help to ensure exposure of the photoresist at that location. The
exposure process can be followed with a post-exposure bake (PEB)
procedure. In some cases, a PEB procedure is performed for a
duration within a range from about 0 to about 200 minutes, and at a
temperature within a range from about 50.degree. C. to about
80.degree. C. For example, a PEB can be performed for 2 minutes at
65.degree. C. In some cases, the PEB can operate to cross-link the
photoresist mask material, rendering the material nonsoluble.
Thereafter, the exposed photoresist can be allowed to cool. An
exemplary cooling process is performed at a temperature within a
range from about 18.degree. C. to about 37.degree. C. for a
duration within a range from about 1 hour to about 40 hours. In
some cases, the exposed photoresist is cooled at room temperature
for about 18 hours.
[0143] A development process can be performed following exposure.
In some cases, the photoresist mask is developed for a duration
within a range from about 10 seconds to about 10 minutes. During
the development process a developer is applied to the exposed
photoresist. The developer can include, for example, an organic
solvent such as acetate. It is understood that the developer or
solvent may be selected based on the composition of the
photoresist. The developer can operate to dissolve or degrade areas
or locations of the photoresist layer that were unexposed or masked
during the exposure process. Following development, the photoresist
mask is subject to a drying procedure. For example, the mask can be
spin-dried. The mask may also be allowed to relax for a desired
period of time. In some cases, the mask is allowed to relax at or
near room temperature for a duration within a range from about 1
minute to about 48 hours.
[0144] Optionally, additional elastomeric layers can be spin-coated
or otherwise applied onto the developed photoresist. For example,
an RTV coating have a thickness or depth within a range from about
0.3 microns to about 30 microns can be deposited on the photoresist
mask. The elastomeric coating can be baked for a duration within a
range from about 5 minutes to about 3 hours, at a temperature
within a range from about 40.degree. C. to about 80.degree. C. In
some cases, a 3 micron RTV coating is spin-coated on an SU-8 mask,
and baked for 1 hour at 60.degree. C. Such techniques can help to
minimize lateral etch, and reduce via size non-uniformity. The RTV
layer can be deposited on a patterned photoresist layer, to help
prevent or inhibit the formation of pinholes in the underlying
elastomer.
[0145] Typically, etching involves removing certain areas of the
elastomeric material that are not protected by the photoresist
following development. In an exemplary procedure, etching can be
performed for a duration within a range from about 1 minute to
about 20 minutes and at a temperature within a range from about
50.degree. C. to about 90.degree. C. For example, etching can be
carried out in an 80% tetrabutylammonium fluoride (TBAF) etchant
solution for 6-8 minutes at 70 degrees .degree. C. In some cases,
etching is performed in an ultrasonic bath tank, optionally in
degas mode. Such procedures can help to ensure a uniform etching
depth, with minimum damage to a photoresist mask during the etching
procedure. Etching can be followed with a deionized water wash. In
some cases, a hot water wash is performed for three minutes. The
photoresist mask can be removed with adhesive tape. A deionized
water wash can be applied again, optionally for 3 minutes. Stacking
procedures can provide additional layers to the elastomer. In some
cases, adjacent layers adhere to one another by way of interlayer
bonding.
[0146] Turning now to the drawings, FIG. 1 depicts a perspective
view of a unit cell 100 of a microfluidic device, according to some
embodiments. Unit cell 100 includes a first channel 130, a first
isolation valve 132, a first chamber 140, a second channel 110, a
second isolation valve 112, a second chamber 120, a control channel
150, an interface channel 160, an interface valve 162, and a
reaction channel 170. Typically, these features are at least
partially contained, embedded, or formed by or within an
elastomeric block 180. As shown here, first channel 130 is at least
partially disposed within a first layer 181 of elastomeric block
180.
[0147] FIG. 1A shows an exploded perspective view of individual
layers of a unit cell 100 of a microfluidic device, according to
embodiments of the present invention. Each layer typically includes
an elastomeric membrane with one or more recesses, channels,
chambers, or the like. As depicted here, the first layer 181 of
unit cell 100 includes the first channel 130 in fluid communication
with the first chamber 140. The first layer 181 also includes the
second chamber 120. Second layer 182 includes the control channel
150, a first via 111, and a second via 131. The third layer 183
includes a second channel 110 and a reaction channel 170. As
further discussed elsewhere herein, unit cell 100 can be configured
so that second channel 110 and reaction channel 170 are in fluid
communication with second chamber 120, optionally by way of via
111. For example, creating a fluid passage that extends from second
channel 110 to reaction channel 170 can involve removing a portion
of second layer 182 that is disposed below second chamber 120.
Creation of this fluid passage can also involve removing a
corresponding portion of third layer 183 that is disposed below
second chamber 120. Similarly, unit cell 100 can be configured so
that reaction channel 170 is in fluid communication with first
chamber 140, optionally by way of via 131. For example, creating a
fluid passage that extends from first channel 130 to reaction
channel 170 can involve removing a portion of second layer 182 that
is disposed below first chamber 140. Creation of this fluid passage
can also involve removing a corresponding portion of third layer
183 that is disposed below first chamber 140. Fourth layer 184
includes an interface channel 160.
[0148] Hence, as shown here, first channel 130 is at least
partially contained within a first layer 181. Control channel 150,
via 111, and via 131 are each at least partially contained within a
second layer 182, where the second layer is adjacent to the first
layer. Second channel 110 and reaction channel 170 are at least
partially contained within a third layer 183, where the third layer
is adjacent to the second layer. As shown here, vias 131, 111 are
disposed in second layer 182. It is understood that corresponding
vias can be formed in third layer 183, so as to provide fluid
communication from chamber 140 through via 131 and into channel
170, and fluid communication from chamber 120 through via 111 and
into the intersection of channels 110 and 170. Interface channel
160 is at least partially contained within a fourth layer 184,
where the fourth layer is adjacent to the third layer. First
chamber 140 is at least partially contained within first layer 181.
First chamber 140 in some instances can also be least partially
contained within or in communication with passages located in
second layer 182 and third layer 183, thus providing fluid
communication between first chamber 140 and first channel 130, and
between first chamber 140 and reaction channel 170. Second chamber
120 is at least partially contained within first layer 181. In some
instances second chamber 120 can be at least partially contained
within or in communication with passages located in second layer
182 and third layer 183, thus providing fluid communication between
second chamber 120 and reaction channel 170.
[0149] With reference to the "A" arrows in FIG. 1, a first
material, such as an assay reagent, can flow through first channel
130, past first isolation valve 132, and into first chamber 140.
Similarly, with reference to the "B" arrows, a second material,
such as an assay sample, can flow through second channel 110, past
second isolation valve 112, through via 111, and into second
chamber 120. To allow flow into the reaction chambers 140, 120,
first and second isolation valves 132, 112, respectively, are both
in a normally open valve state. To prevent or inhibit flow between
first reaction chamber 140 and second reaction chamber 120 through
reaction channel 170, interface valve 162 is in a normally closed
valve state. Under such conditions, first channel 130 is in open
fluid communication with first reaction chamber 140, and second
channel 110 is in fluid communication with second reaction chamber
120, whereas fluid communication between the first and second
chambers is interrupted or inhibited. Reaction chamber sizes may
vary. In some embodiments, the volume of second reaction chamber
120 is different or greater than the volume of first reaction
chamber 140. For example, the volume of second reaction chamber 120
can be ten times greater than the volume of first reaction chamber
140. Materials can be loaded into their respective chambers under
pressure. Relatedly, materials can be loaded into chambers at
certain concentrations. In some cases, a reagent solution is loaded
into a chamber at a 10.times. concentration, and is then diluted
when reacted with a sample solution contained in another
chamber.
[0150] After the first and second materials have been loaded into
first and second reaction chambers 140, 120 respectively, the
control channel 150 can be activated, for example by pressurizing
the control channel, so as to transform each of first and second
isolation valves 132, 112 from an open valve state to a closed
valve state. In this way the materials can be confined, optionally
under pressure, within the reaction chambers. Hence, it is
understood that a single control channel, for example control
channel 150, can control flow of a first material into a first
reaction chamber, and can also control flow of a second material
into a second reaction chamber. Operation of a single control
channel can thus act to isolate a first volume of material or
solution within the first chamber via actuation of the first
isolation valve 132, and can also isolate a second volume of
material or solution within the second chamber via actuation of the
second isolation valve 112. Relatedly, operation of a single
control channel can cause a first deflection in a first direction
at first isolation valve 132, and a second deflection in a second
direction at second isolation valve 112, where first direction is
opposite to second direction. For example, the deflection in the
first isolation valve can be in the upward direction, and the
deflection in the second isolation valve can be in the downward
direction. Accordingly, control of more than one isolation valve
can be effected simultaneously by operation of the single control
channel. Materials can be confined within the reaction chambers
under any suitable amount of pressure. In some embodiments, the
pressure in the first reaction chamber 140 is different or greater
than the pressure in the second reaction chamber 120. For example,
a first material such as a reagent can be disposed in first
reaction chamber 140 at a first pressure that is within a range
from about 0 psi to about 15 psi. Relatedly, a second material such
as a sample can be disposed in second reaction chamber 120 at a
second pressure that is within a range from about 0 psi to about 10
psi. In some instances, material can be contained in the first
reaction chamber 140 at about 10 psi, and material can be contained
in the second reaction chamber 120 at about 0 psi. Often, loading
of the microfluidic device involves introducing material into first
channel 130 or second channel 110, or both, under pressure. A
pressurizing mechanism (as are know in the art) can be used to
drive materials into the chambers.
[0151] In some cases, embodiments are directed to systems and
methods for conducting one or more reactions at one or more
selected temperatures or ranges of temperatures over time. A
microfluidic system may include a plurality of separate reaction
chambers formed in a multi-layer elastomeric block. The system may
also include a thermal transfer device proximal to or near at least
one of the reaction chambers. The thermal transfer device can be
formed to contact a thermal control source. Reagents for carrying
out a desired reaction can be introduced into a microfluidic array
device or matrix. The array device or matrix can be contacted with
the thermal control device such that the thermal control device is
in thermal communication with the thermal control source so that a
temperature of the reaction in at least one of the reaction chamber
is changed or controlled as a result of a change in temperature of
the thermal control source. Exemplary thermal cycling techniques
are discussed in U.S. Patent Publication No. 2007/0196912, the
content of which is incorporated herein by reference. In some
embodiments, a microfluidic device or chip can be coupled with or
in operative association with an Integrated Heat Spreader (IHS).
Such heating mechanisms are discussed in U.S. Pat. No. 7,307,802,
the content of which is incorporated herein by reference.
[0152] In some cases, passages or vias can be formed between
channels or chambers at one layer and channels or chambers at
another layer. For example, it is possible to create a via 131
through second layer 182 to provide fluid communication between
first chamber 140 in first layer 181 and reaction channel 170 in
third layer 183. Similarly, it is possible to create a via 111
through second layer 182 to provide fluid communication between
second chamber 120 in first layer 181 and second channel 110 and
reaction channel 170 in third layer 183. In some instances,
creation of these vias can enlarge the volume of the reaction
chambers. In some cases, the vias can be formed by using a laser
punch to remove or ablate portions of elastomeric membrane. As
shown in FIG. 1A, for example, reaction chambers 120, 140 can have
an interior space that extends above a plane defined by the top of
first channel 130. This interior space can also extend above a
plane defined by the top of second channel 110, and above a plane
defined by the top of reaction channel 170. Hence, during loading
of the unit cell, fluid can flow through first channel 130 and
upward into the interior of first chamber 140. Similarly, during
loading, fluid can flow through second channel 110 and upward into
the interior of second chamber 120, optionally through a via formed
in the second layer. Relatedly, during a mixing operation, fluid
can flow from the interior of first chamber 140 and downward into
reaction channel 170, optionally through a via 131 formed in the
second layer. Similarly, during a mixing operation fluid can flow
from reaction channel 170 and upward into the interior of second
chamber 120, optionally through a via 111 formed in the second
layer.
[0153] According to the embodiment shown in FIG. 1, reaction
channel 170 and interface valve 162 are not located within the same
plane or layer as first chamber 140 and second chamber 120. For
example, reaction channel 170 is disposed in third layer 183,
interface valve 162 operates at or near the boundary or junction
between fourth layer 184 and third layer 183, and first and second
chambers 140, 120 are disposed in first layer 181. As the routing
passage or reaction channel 170 passes in a lower or different
layer than that of the chambers, this allows the chambers to be
located in close proximity with one another. In some embodiments, a
sidewall of first chamber 140 and a facing sidewall of second
chamber 120 are separated by a distance of about 120 microns. In
related embodiments, a distance between facing sidewalls of first
and second chambers is within a range from about 40 microns to
about 225 microns. For example, a first chamber sidewall and a
facing second chamber sidewall can be separated by a distance of
about 50 microns, about 60 microns, about 70 microns, or about 80
microns. Often, interface valve 162 is about 50 microns in width.
Hence, the distance between facing sidewalls of first and second
chambers can be less than, about the same as, or more than the
diameter or width of the interface valve which controls or
modulates flow between the chambers. Accordingly, microfluidic
devices employing such architecture can present extremely large
numbers of chambers within a given area. Such high densities may be
difficult to achieve in situations where a valve that controls flow
between two chambers is disposed in the same layer as the
chambers.
[0154] The chambers 140 and 120 may have varied dimensions and
volumes. In an embodiment, first chamber 140 may have a width
within a range from about 25 microns to about 75 microns, a length
within a range from about 80 microns to about 240 microns, and a
height within a range from about 30 microns to about 90 microns.
Relatedly, first chamber 140 may have a volume within a range from
about 0.1 nanoliters to about 10 nanoliters. For example, first
chamber 140 can have a width of 50 microns, a length of 162.5
microns, a height of 60 microns, and a volume of 0.49 nanoliters.
Second chamber 120 can have a width within a range from about 70
microns to about 210 microns, a length within a range from about 80
microns to about 240 microns, and a height within a range from
about 150 microns to about 450 microns. Relatedly, second chamber
120 can have a volume within a range from about 1 nanoliter to
about 20 nanoliters. For example, second chamber 120 can have a
width of 137.5 microns, a length of 162.5 microns, a height of 300
microns, and a volume of 6.7 nanoliters. A microfluidic device
according to embodiments of the present invention can provide a
center-to-center distance between first chamber 120 and second
chamber of about 300 microns. In some cases, this center-to-center
distance is within a range from about 250 microns to about 350
microns. Optionally, the center-to-center distance between the
first chamber and the second chamber is about 312.5 microns.
[0155] Microfluidic devices can be fabricated using a variety of
methods. The attached Appendix contains additional discussion of
related fabrication methods.
[0156] In some embodiments, a microfluidic device can include one
or more layers that have been prepared according to spin or pour
fabrication protocols. For example, a spin protocol can involve
placing a polymeric material on a patterned disc or mold, and
spinning the disc to create a layer of polymer across the disc.
Exemplary polymers include polymethylmethacrylate, polystyrene,
polypropylene, polyester, fluoropolymers, polytetrafluoroethylene,
polycarbonate, polysilicon, and polydimethylsiloxane (PDMS). A pour
protocol can involve pouring a PDMS material, for example, on a
patterned template or mold, which can result in a layer of PDMS
which can be peeled or pulled off the mold intact. Often, a layer
prepared by a pour fabrication technique is thicker than a layer
prepared by a spin fabrication technique. Elastomeric blocks can
include one or more pour or spin layers, in any desired
combination. In some embodiments, first layer 181 can be fabricated
according to a pour protocol. For example, PDMS can be poured onto
a mold that has raised portions corresponding to the various
desired fluid flow channels and chambers. FIG. 1B shows an
exemplary mold 190b which can be used to fabricate first layer 181
of FIG. 1A. After curing, the first layer can be peeled away from
the mold. First layer 181 can include openings, recesses, or other
voids that at least partially form or define first channel 130,
first chamber 140, and second chamber 120. To create second layer
182, PDMS can be placed onto a mold that has raised portions
corresponding to the various desired containment or control
channels. FIG. 1 C shows an exemplary mold 190c which can be used
to fabricate second layer 182 of FIG. 1A. Mold 190c can also
include, for example, raised or contoured portions 191c that form
corresponding marks in second layer 182. These marks can be used
during a laser ablation procedure, such that the laser ablation is
directed toward the marks during the ablation. Mold 190c can be
spun, so as to provide a thin layer of PDMS across the mold. Second
layer 182 can include openings, recesses, or other voids that at
least partially form or define control channel 150. In some cases,
second layer 182 can be exposed to one or more laser ablations. An
ablative laser beam directed to second layer 182 can form vias 111,
131. After second layer 182 is sufficiently cured, first layer 181
can be aligned and contacted with the second layer. The first layer
can adhere with the second layer, and both layers can be peeled off
mold 190c simultaneously. To create third layer 183, PDMS can be
placed onto a mold that has raised portions corresponding to the
various desired containment or control channels. FIG. 1D shows an
exemplary mold 190d which can be used to fabricate third layer 183
of FIG. 1A. The mold can be spun, so as to provide a thin layer of
PDMS across the mold. Third layer 183 can include openings,
recesses, or other voids that at least partially form or define
second channel 110 and reaction channel 170. After third layer 183
is sufficiently cured, the combined first layer 181 and second
layer 182 can be aligned and contacted with the third layer. The
third layer can adhere with the second layer, and all three layers
can be peeled off mold 190d simultaneously. To create fourth layer
184, PDMS can be placed onto a mold that has raised portions
corresponding to the various desired containment or control
channels. FIG. 1E shows an exemplary mold 190e which can be used to
fabricate fourth layer 184 of FIG. 1A. The mold can be spun, so as
to provide a thin layer of PDMS across the mold. Fourth layer 184
can include openings, recesses, or other voids that at least
partially form or define interface channel 160. After fourth layer
184 is sufficiently cured, the combined first layer 181, second
layer 182, and third layer 183 can be aligned and contacted with
the fourth layer. The fourth layer can adhere with the third layer,
and all four layers can be peeled off mold 190e simultaneously.
Optionally, the four layers can be placed on or contacted with a
fifth layer 186 as shown in FIG. 1A. The fifth layer 186 can
include a laminate or tape, or a similarly suitable material, which
operates to seal a recess in the fourth layer, so as to form or
seal interface channel 160. In this way, the combined first,
second, third, and fourth layers 181, 182, 183, 184 can then be
placed on or contacted with the fifth layer 186, which may be a
solid spin layer. The fifth layer 186 can act as a sealing layer.
According to some embodiments, the fifth layer 186 may include an
elastomeric material, such as PDMS. In some cases, the fifth layer
186 can include a rigid or hard material such as glass, silicon, or
a plastic such as polystyrene. The fifth layer 186 may, for
example, seal recesses formed in bottom of the fourth layer, so as
to provide channels in the fourth layer 184. The fifth layer 186
can include a film which may be attached to the fourth layer 184
via an adhesive. Hence, a sealing layer can form channels from
recesses molded or machined into an adjacent layer. A sealing layer
can be a transparent material, for example, polystyrene,
polycarbonate, or polypropylene. Relatedly, a sealing layer can be
flexible, such as an adhesive tape, and may be suitable for
attachment to a substrate by bonding, such as with adhesive or heat
sealing, or mechanically attached such as by compression. Often,
materials used to fabricate a sealing layer are compliant to form
fluidic seals with each recess to form a fluidic channel with
minimal leakage. A sealing layer may further be supported by an
additional support layer that is rigid (not shown). In some cases,
a sealing layer is rigid.
[0157] According to embodiments of the present invention, the
second, third, fourth, and fifth layers can be processed or laser
punched as part of a procedure that forms a loading passage to
first channel 130 in first layer 181. Relatedly, the fourth and
fifth layers can be processed or laser punched as part of a
procedure that forms a loading passage to second channel 110 in
third layer 183. Loading passages, vias, and the like can be formed
using a drilling or ablation mechanism. For example, a loading
passage or via can be fabricated by ablating a portion of the
elastomeric block. Excimer lasers are well suited for such ablation
techniques, as they can produce a laser beam which removes a
portion of the elastomeric block. In some cases, loading passages
or vias, or portions thereof, can be formed before one or more of
the individual layers are adhered together. For example, a portion
of a loading passage or via can be formed in a layer during the
molding process, or after the molding process and before the
adhesion process. Optionally, formation of at least a portion of
the loading passage or via can involve etching one or more
elastomeric layers prior to forming the complete multilayer
elastomeric block.
[0158] FIG. 2 illustrates another perspective view of unit cell
100. As shown here with reference to the "B" arrows, the first
material can flow from first reaction chamber 140, through via 131,
through reaction channel 170, past interface valve 162, through via
111, and into second reaction chamber 120, where the first material
can contact the second material. It is understood that in some
embodiments, the second material can flow from second reaction
chamber 120, through via 111, through reaction channel 170, past
interface valve 162, through via 131, and into first reaction
chamber. To allow such flow through reaction channel 170, the
interface channel 160 can be activated so as to transform interface
valve 162 from a closed valve state to an open valve state. Under
such conditions, the first and second reaction chambers 140, 120
are in open fluid communication by way of the reaction channel and
the vias. When, for example, material contained in first reaction
chamber 140 is more highly pressurized relative to material
contained in second reaction chamber 120, the pressure differential
can help to release or open interface valve 162. Relatedly, such a
pressure differential can facilitate mixing between the first
material and the second material, as the first material is
forcefully expelled from first chamber and into second chamber,
thus squirting a stream of first material into a second material
contained in the second chamber, where the first material can
diffuse into or permeate through the second material. Often, the
presence, absence, or extent of any reaction between the first and
second materials, or involving either or both of the first and
second materials, within the second reaction chamber can be
characterized, confirmed, detected, or quantified by inspection,
for example with a reader, sensor, or imaging device 190. An
imaging device 190 can include a camera, optionally having a
charge-coupled device (CCD), that detects or monitors energy that
emits from the chamber. In some cases, the imaging device can
detect emission intensity output. Exemplary imaging devices and
reader techniques suitable for use with embodiments of the present
invention are described in U.S. Pat. No. 7,307,802 issued Dec. 11,
2007, the content of which is incorporated herein by reference. In
some cases, a reaction within a chamber is facilitated by a thermal
cycler. Embodiments of the present invention encompass systems and
methods for mixing or reacting materials within chambers, where
such mixing or reacting procedures involve any of a variety of
desired thermal cycling heating protocols or thermal gradient
modalities.
[0159] FIG. 3 illustrates a perspective view of a matrix 300 having
four unit cells 300.sub.(1,1), 300.sub.(1,2), 300.sub.(2,1), and
300.sub.(2,2) arranged in two rows and two columns. Matrix 300
includes a plurality of first channels 330.sub.(1), 330.sub.(2), a
plurality of first isolation valves 332.sub.(1,1), 332.sub.(1,2),
332.sub.(2,1), 332.sub.(2,2), a plurality of first chambers
340.sub.(1,1), 340.sub.(1,2), 340.sub.(2,1), 340.sub.(2,2), a
plurality of second channels 310.sub.(1), 310.sub.(2), a plurality
of second isolation valves 312.sub.(1,1), 312.sub.(1,2),
312.sub.(2,1), 312.sub.(2,2), a plurality of second chambers
320.sub.(1,1), 320.sub.(1,2), 320.sub.(2,1), 320.sub.(2,2), a
plurality of control channels 350.sub.(1), 350.sub.(2), a plurality
of interface channels 360.sub.(1), 360.sub.(2), and a plurality of
reaction channels 370.sub.(1,1), 370.sub.(1,2), 370.sub.(2,1),
370.sub.(2,2). It is appreciated that the unit cell architecture
embodiments disclosed herein can be scaled to provide a matrix
having any number of desired unit cells. For example, a matrix can
include 9216 unit cells arranged in 96 rows and 96 columns. Hence,
embodiments of the present invention provide a high density format
for reacting a plurality of samples with a plurality of reagents,
for example, ninety-six (96) samples with ninety-six (96)
reagents.
[0160] Microfluidic device features such as channels, valves,
chambers, are often at least partially contained, embedded, or
formed by or within one or more layers of an elastomeric block 380.
As shown here with reference to the "A1" arrows, a first material
such as a reagent can flow through a first channel 330.sub.(1),
past or through a plurality of first isolation valves
332.sub.(1,1), 332.sub.(1,2), and into a plurality of first
chambers 340.sub.(1,1), 340.sub.(1,2), respectively. Likewise, with
reference to the "A2" arrows, a second material such as a reagent
can flow through a first channel 330.sub.(2), past or through a
plurality of first isolation valves 332.sub.(2,1), 332.sub.(2,2),
and into a plurality of first chambers 340.sub.(2,1),
340.sub.(2,2), respectively. In some embodiments, materials flow
through first channel 330.sub.(1) and first channel 330.sub.(2) in
the same direction. In some embodiments, material flowing through
first channel 330.sub.(1) travels in a direction opposite from
material flowing through first channel 330.sub.(2). With reference
to the "B1" arrows, a third material such as a sample can flow
through a second channel 310.sub.(1), past or through a plurality
of second isolation valves 312.sub.(1,1), 312.sub.(2,1), and into a
plurality of second chambers 320.sub.(1,1), 320.sub.(2,1),
respectively. Hence, embodiments of the present invention provide
microfluidic techniques whereby a material can be flowed through a
common passage or trunk of a channel, such as second channel
310.sub.(1), and into a plurality of individual branches stemming
from the common trunk, such as those branch channels which
individually feed into second chambers 320.sub.(1,2),
320.sub.(2,2). Similarly, with reference to the "B2" arrows, a
fourth material such as a sample can flow through a second channel
310.sub.(2), past or through a plurality of second isolation valves
312.sub.(1,2), 312.sub.(2,2), and into a plurality of second
chambers 320.sub.(1,2), 320.sub.(2,2), respectively. In some
embodiments, materials flow through second channel 310.sub.(1) and
second channel 310.sub.(2) in the same direction. In some
embodiments, material flowing through second channel 310.sub.(1)
travels in a direction opposite from material flowing through
second channel 310.sub.(2). As shown in FIG. 3, material flowing
through second channel 310.sub.(1) and material flowing through
second channel 310.sub.(2) travel in opposing directions "B1" and
"B2", respectively. Hence, when loading multiple samples into an
elastomeric layered block, some samples can be introduced through
routing lines on one side of the block, and some samples can be
introduced through routing lines on an opposing side of the block.
This allows for an even distribution or placement of sample loading
route lines on opposite sides of the block, instead of placing all
or most sample loading route lines on the same side of the block.
Because the sum total of the sample routing lines are divided
between different sides of the block, more sample routing lines can
be introduced into the block. Consequently, a greater number of
samples can be analyzed within the block during a single
procedure.
[0161] To allow flow from the plurality of first channels
330.sub.(1), 330.sub.(2) into the plurality of first reaction
chambers 340.sub.(1,1), 340.sub.(1,2), 340.sub.(2,1),
340.sub.(2,2), each of the plurality of first isolation valves
332.sub.(1,1), 332.sub.(1,2), 332.sub.(2,1), 332.sub.(2,2) is in an
open valve state. To allow flow from the plurality of second
channels 310.sub.(1), 310.sub.(2) into the plurality of second
reaction chambers 320.sub.(1,1), 320.sub.(1,2), 320.sub.(2,1),
320.sub.(2,2), each of the plurality of second isolation valves
312.sub.(1,1), 312.sub.(1,2), 312.sub.(2,1), 312.sub.(2,2) is in an
open valve state. To prevent or inhibit flow between each of the
plurality of first reaction chambers 340.sub.(1,1), 340.sub.(1,2),
340.sub.(2,1), 340.sub.(2,2) and their corresponding counterpart of
the plurality of second reaction chambers 320.sub.(1,1),
320.sub.(1,2), 320.sub.(2,1), 320.sub.(2,2) through their
corresponding counterpart of the plurality of reaction channels
370.sub.(1,1), 370.sub.(1,2), 370.sub.(2,1), 370.sub.(2,2),
respectively, each of the plurality of interface valves
362.sub.(1,1), 362.sub.(1,2), 362.sub.(2,1), 362.sub.(2,2),
respectively, is in a closed valve state. Under such conditions,
first channel 330.sub.(1) is in fluid communication with first
reaction chambers 340.sub.(1,1), 340.sub.(1,2), first channel
330.sub.(2) is in fluid communication with first reaction chambers
340.sub.(2,1), 340.sub.(2,2), second channel 310.sub.(1) is in
fluid communication with second reaction chambers 320.sub.(1,1),
320.sub.(2,1), and second channel 310.sub.(2) is in fluid
communication with second reaction chambers 320.sub.(1,2),
320.sub.(2,2). Fluid communication between first chambers
340.sub.(1,1), 340.sub.(1,2), 340.sub.(2,1), 340.sub.(2,2) and
second chambers 320.sub.(1,1), 320.sub.(1,2), 320.sub.(2,1),
320.sub.(2,2), respectively, is interrupted.
[0162] A first material can be loaded into first reaction chambers
340.sub.(1,1), 340.sub.(1,2) via first channel 330.sub.(1). A
second material can be loaded into first reaction chambers
340.sub.(2,1), 340.sub.(2,2) via first channel 330.sub.(2). A third
material can be loaded into second reaction chambers 320.sub.(1,1),
320.sub.(2,1) via second channel 310.sub.(1). A fourth material can
be loaded into second reaction chambers 320.sub.(1,2),
320.sub.(2,2) via second channel 310.sub.(2). Optionally, such
materials can be loaded into the chambers under a desired or
selected pressure. Control channel 350.sub.(1) can be activated so
as to transform each of first isolation valves 332.sub.(1,1),
332.sub.(2,1) and second isolation valves 312.sub.(1,1),
312.sub.(2,1) from an open valve state to a closed valve state.
Similarly, control channel 350.sub.(2) can be activated so as to
transform each of first isolation valves 332.sub.(1,2),
332.sub.(2,2) and second isolation valves 312.sub.(1,2),
312.sub.(2,2) from an open valve state to a closed valve state. In
this way the materials can be confined or maintained, optionally
under pressure, within the reaction chambers.
[0163] FIG. 4 illustrates another perspective view of matrix 300
having four unit cells 300.sub.(1,1), 300.sub.(1,2), 300.sub.(2,1),
300.sub.(2,2). As shown here with reference to the "A" arrows, the
first material can flow from first reaction chamber 340.sub.(1,1),
through reaction channel 370.sub.(1,1), past interface valve
362.sub.(1,1), and into second reaction chamber 320.sub.(1,1),
where the first material can contact the third material. To allow
such flow through reaction channel 370.sub.(1,1), the interface
channel 360.sub.(1) can be activated so as to transform interface
valve 362.sub.(1,1) from a closed valve state to an open valve
state. Under such conditions, first reaction chamber 340.sub.(1,1)
and second reaction chamber 320.sub.(1,1) are in fluid
communication via reaction channel 370.sub.(1,1). Often, the
presence, absence, or extent of any reaction between the first and
third materials within second reaction chamber 320.sub.(1,1) can be
confirmed, detected, or quantified by inspection, for example with
a reader or sensor 390.
[0164] With reference to the "B" arrows, the first material can
flow from first reaction chamber 340.sub.(1,2), through reaction
channel 370.sub.(1,2), past interface valve 362.sub.(1,2), and into
second reaction chamber 320.sub.(1,2), where the first material can
contact the fourth material. To allow such flow through reaction
channel 370.sub.(1,2), the interface channel 360.sub.(1) can be
activated so as to transform interface valve 362.sub.(1,2) from a
closed valve state to an open valve state. Under such conditions,
first reaction chamber 340.sub.(1,2) and second reaction chamber
320.sub.(1,2) are in fluid communication via reaction channel
370.sub.(1,2). Often, the presence, absence, or extent of any
reaction between the first and third materials within second
reaction chamber 340.sub.(1,2) can be confirmed, detected, or
quantified by inspection, for example with a reader or sensor
390.
[0165] With reference to the "C" arrows, the second material can
flow from first reaction chamber 340.sub.(2,1), through reaction
channel 370.sub.(2,1), past interface valve 362.sub.(2,1), and into
second reaction chamber 320.sub.(2,1), where the second material
can contact the third material. To allow such flow through reaction
channel 370.sub.(2,1), the interface channel 360.sub.(2) can be
activated so as to transform interface valve 362.sub.(2,1) from a
closed valve state to an open valve state. Under such conditions,
first reaction chamber 340.sub.(2,1) and second reaction chamber
320.sub.(2,1) are in fluid communication via reaction channel
370.sub.(2,1). Often, the presence, absence, or extent of any
reaction between the first and third materials within second
reaction chamber 320.sub.(2,1) can be confirmed, detected, or
quantified by inspection, for example with a reader or sensor
390.
[0166] With reference to the "D" arrows, the second material can
flow from first reaction chamber 340.sub.(2,2), through reaction
channel 370.sub.(2,2), past interface valve 362.sub.(2,2), and into
second reaction chamber 320.sub.(2,2), where the second material
can contact the fourth material. To allow such flow through
reaction channel 370.sub.(2,2), the interface channel 360.sub.(2)
can be activated so as to transform interface valve 362.sub.(2,2)
from a closed valve state to an open valve state. Under such
conditions, first reaction chamber 340.sub.(2,2) and second
reaction chamber 320.sub.(2,2) are in fluid communication via
reaction channel 370.sub.(2,2). Often, the presence, absence, or
extent of any reaction between the fourth and second materials
within second reaction chamber 320.sub.(2,2) can be confirmed,
detected, or quantified by inspection, for example with a reader or
sensor 390.
[0167] In some embodiments, the terms "isolation valve" and
"containment valve" may be used interchangeably. Similarly, the
terms "interface valve" and "reaction valve" may be used
interchangeably. Such valves can be actuated or activated or
otherwise controlled by any of a variety of valve operation methods
or configurations. Exemplary valve systems and techniques which are
well suited for use with embodiments of the present invention are
described, for example, in U.S. Pat. No. 6,408,878, the content of
which is incorporated herein by reference. Often, such valves
include an elastomeric portion that, when actuated, deflects into a
recess. For example, FIG. 5A shows a side view or cross section of
a microfluidic device unit cell 500. The unit cell includes a first
channel 530 and first sample chamber 540 in a first layer 581 of an
elastomeric block 580, control channel 550 and via 511a in a second
layer 582, and a reaction channel 570 in a third layer 583. An
isolation valve 532 can be actuated, so as to inhibit or prevent
flow through first channel 530. Actuation of isolation valve 532
can involve the deflection of an elastomeric portion 532a into a
recess 531 of first channel 530. Fourth layer 584 includes
interface channel 560. FIG. 5B shows another side view or cross
section of microfluidic device unit cell 500. The unit cell
includes a sample chamber 520 in a first layer 581, a control
channel 550 and via 531b in a second layer 582, and a second
channel 510 in a third layer 583. Isolation valve 512 can be
actuated, so as to inhibit or prevent flow through second channel
510. Actuation of isolation valve 512 can involve the deflection of
an elastomeric portion 512a into a recess 511 of second channel
510. FIG. 5C shows a side view or cross section which is orthogonal
to the side views of FIGS. 5A and 5B. As depicted here, actuation
of control channel 550 can operate to activate both isolation valve
532 and isolation valve 512. For example, by changing the pressure
of fluid within control channel or containment line 550, it is
possible to simultaneously deflect a first elastomeric portion
upward into first channel 530 and a second elastomeric portion
downward into second channel 510. Optionally this can result in the
containment or isolation of a first material within a first chamber
and a second material within a second chamber.
[0168] As shown in FIG. 5C, first channel 530 and second channel
510 each present a rectangular cross section. In some instances,
either or both of these channels can present an arcuate shaped
cross section, where the cross section is upright with regard to
first channel 530 and inverted with regard to second channel
510.
[0169] FIG. 6A illustrates a microfluidic device 699 according to
embodiments of the present invention. Materials can be delivered
from wells 605 toward elastomeric block 608 through passages or
routing lines 601. FIG. 6B depicts microfluidic device 699 in a
plan view. Microfluidic device 699 includes a substrate 600 with
integrated pressure accumulator wells 601 and 602, each having a
receptacle 603, 604 that contains a valve, such as a check valve.
Microfluidic device 699 also includes one or more wells 605 for
receiving materials such as samples or reagents, and one or more
channels or routing lines disposed between wells 605 and an
elastomeric block location 607 of substrate 600. An elastomeric
block 608 can be coupled with substrate 600 at elastomeric block
location 607. Elastomeric block 608 can include one or more layers
of elastomeric material having microfabricated recesses or channels
formed therein. Elastomeric block 608 can be coupled with substrate
600 in any of a variety of ways. For example, the elastomeric block
can be attached or bonded with the substrate. In some cases, the
block is directly bonded to the substrate. In some cases, the block
is coupled with the substrate without the use of an adhesive. In
some cases, the block is coupled with the substrate with an
adhesive. Within elastomeric block 608 are one or more channels in
fluid communication with one or more vias 614, which in turn
provide fluid communication between the elastomeric block channels
and the substrate channels. Hence, the substrate channels can
provide fluid communication between wells 605 and channels within
the elastomeric block.
[0170] Accumulator wells 601, 602 often include valves 611, 612,
respectively, which can be check valves for introducing and holding
gas of fluid under pressure into accumulator chambers 615 and 616.
Valves 611 and 612 are situated inside of receptacles 604 and 603,
respectively, which can keep liquid, when present in accumulator
chambers 615 and 616, from contacting valves 611 and 612. In some
embodiments, valves 611 and 612 may be mechanically opened by
pressing a shave, pin or the like, within a check valve to overcome
a self closing force of the check valve, thereby permitting release
of pressure from the accumulator chamber, or reducing fluid
pressure contained within the accumulator chamber.
[0171] Substrate 600 and associated components may be fabricated
from polymers, such as polypropylene, polyethylene, polycarbonate,
high-density polyethylene, polytetrafluoroethylene PTFE or
Teflon.RTM., glass, quartz, transparent materials, polysilicon,
metals, such as aluminum, or the like. Any of a variety of gases,
liquids, or fluids can be introduced into accumulator chambers 615
and 616. In some cases, valves 611 and 612 can be actuated to
release fluid pressure otherwise held inside of accumulator
chambers 615 and 616. Optionally, a portion of substrate 600
beneath the elastomeric block region 607 can be transparent. In
some cases, the portion may be opaque or reflective. Accumulator
chambers 601 and 602 can be in fluid communication with channels
contained in elastomeric block region 607, and ultimately, with
channels contained in elastomeric block 608. Accumulator operation
is described in U.S. Patent Publication No. 2007/0196912, the
content of which is incorporated herein by reference. In some
cases, operation of a channel, such as a control channel 150 as
shown in FIG. 1, can be modulated or controlled by an accumulator.
FIG. 6C illustrates further aspects of a microfluidic device in
accordance with embodiments of the present invention.
[0172] FIG. 7 illustrates a microfluidic device 700 according to
embodiments of the present invention. Device 700 includes a carrier
710 coupled with a chip or block 750. Carrier or frame 710 includes
a plurality of routing lines 712 configured to allow flow from
carrier wells (see wells 605 in FIG. 6B) toward chip 750. For
example, routing lines disposed on the "S" side of the carrier can
provide for the passage of sample, and routing lines disposed on
the "R" side of the carrier can provide for the passage of reagent.
In some cases, chip 750 can also include a plurality of routing
lines 752. For example, routing lines 752 on the chip 750 can
provide material transport on the chip from location A to location
B, and from location C to location D. In this way, a portion of the
samples loaded onto the carrier 710 can be transported to location
E of the chip, and another portion of the samples loaded onto the
carrier can be transported to locations D and B of the chip, such
that some sample is loaded at one side of the block, and some
sample is loaded at an opposing side of the block, as discussed
above with reference to FIG. 3.
[0173] FIG. 8 illustrates a microfluidic device 800 according to
embodiments of the present invention. Device 800 includes a carrier
810 coupled with a chip or block 850. Carrier or frame 810 includes
a plurality of routing lines 812 configured to allow flow from
carrier wells toward chip 850. For example, routing lines disposed
on the "S" side of the carrier can provide for the passage of
sample, and routing lines disposed on the "R" side of the carrier
can provide for the passage of reagent. As shown in this
illustration, 24 samples loaded into wells at zone S.sub.1 flow to
the left side of the chip (upper half), 24 samples loaded into
wells at zone S.sub.2 flow to the upper side of the chip (left
half), 24 samples loaded into wells at zone S.sub.3 flow to the
upper side of the chip (right half), and 24 samples loaded into
wells at zone S.sub.4 flow to the right side of the chip (upper
half). Thereafter, through a set of routing lines optionally
disposed at or within the elastomeric block, the S.sub.1 samples
flow to the left side of the chip (lower half) as shown by the
arrows, the S.sub.2 samples flow to the left side of the chip
(upper half), the S.sub.3 samples flow to the right side of the
chip (upper half), and the S.sub.4 samples flow to the right side
of the chip (lower half). Further, 24 reagent portions loaded into
wells at zone R.sub.1 flow to the left side of the chip (lower
half), 24 reagent portions loaded into wells at zone R.sub.2 flow
to the lower side of the chip (left half), 24 reagent portions
loaded into wells at zone R.sub.3 flow to the lower side of the
chip (right half), and 24 reagent portions loaded into wells at
zone R.sub.4 flow to the right side of the chip (lower half).
Thereafter, through another set of routing lines optionally
disposed at or within the elastomeric block, the R.sub.1 reagent
portions flow to the lower side of the chip (left half), the
R.sub.2 reagent portions flow to the lower side of the chip (left
half), the R.sub.3 reagent portions flow to the lower side of the
chip (right half), and the R.sub.4 samples flow to the lower side
of the chip (right half). Hence, routing lines on or in the chip
can provide material transport on or through the chip from one
location to another. In this way, a portion of the samples loaded
at one end of the carrier (e.g., the "S" end) can be transported
such that some sample is loaded at one side of the block, and some
sample is loaded at an opposing side of the block, as further
discussed herein with reference to FIG. 3.
[0174] In some embodiments, microfluidic devices may contain blind
flow channels which include a region that functions as a reaction
chamber or reaction site. Blind flow, or blind fill, can refer to
the filling of a dead-end tube or flow channel with a liquid where
a head of gas is pushed in front of the liquid bolus, and where
that head of gas is vented or otherwise released from the flow
channel, allowing the dead-end flow channel to fill fully with the
liquid. In some embodiments, polydimethylsiloxane (PDMS) can be
used as an elastomeric material. PDMS is sufficiently gas permeable
that liquid pressurized at a few psi can drive the gas out of the
channels, leaving them completely filled with liquid.
[0175] Table 1 provides an exemplary use of a microfluidic device
where various materials can be loaded or introduced into four unit
cells of the device. According to this table, sample can be flowed
through first channels and reagent can be flowed through second
channels. It is understood that alternatively, reagent can be
flowed through first channels and sample can be flowed through
second channels. Embodiments of the present invention encompass
techniques where sample is flowed through a set of first channels
and a set of second channels, and reagent is flowed through a set
of first channels and a set of second channels.
TABLE-US-00001 TABLE 1 channel material chamber first channel
330.sub.(1) DNA sample from person A first chambers 340.sub.(1, 1),
340.sub.(1, 2) first channel 330.sub.(2) DNA sample from person B
first chambers 340.sub.(2, 1), 340.sub.(2, 2) second channel
310.sub.(1) disease X gene primers/probes second chambers
320.sub.(1, 1), 320.sub.(2, 1) second channel 310.sub.(2) disease Y
gene primers/probes second chambers 320.sub.(1, 2), 320.sub.(2,
2)
[0176] Table 2 shows the mixtures occurring in the microfluidic
device reaction chambers, and the experimental inquiries which can
be answered, for example, by conducting a PCR reaction where the
sample contains patient DNA and the reagent contains an
oligonucleotide primer and probe set.
TABLE-US-00002 TABLE 2 reaction chamber materials mixed inquiry
second chamber DNA sample from person A person A has gene
320.sub.(1, 1) disease X gene primer/probe for disease X? second
chamber DNA sample from person A person A has gene 320.sub.(1, 2)
disease Y gene primer/probe for disease Y? second chamber DNA
sample from person B person B has gene 320.sub.(2, 1) disease X
gene primer/probe for disease X? second chamber DNA sample from
person B person B has gene 320.sub.(2, 2) disease Y gene
primer/probe for disease Y?
[0177] It is understood that any of a variety of materials may be
mixed or reacted in according to embodiments of the present
invention. For example, genotyping applications may involve
detecting the presence or absence of a target in a sample. Gene
expression applications may involve measuring or quantifying
amounts of materials contained in a sample. Such applications may
benefit from the enhanced mixing function provided by embodiments
of the present invention. Further, microfluidic devices and methods
can be used to crystallize a protein. In one embodiment a method
includes providing a microfluidic device having a first chamber
having a dimension between 1000 .mu.m and 1 .mu.m, a second chamber
having a dimension between 1000 .mu.m and 1 .mu.m, and one or more
flow or control channels each having a dimension between 1000 .mu.m
and 1 .mu.m. The first and second chambers can be in fluid
communication with each other through a channel. A valve can be
disposed along a channel which, when actuated to open or close,
controls fluid communication between the first and second chambers,
or into or out of the first or second chamber, or both. The method
can include introducing a crystallization reagent into the first
chamber, introducing the protein in a solution into the second
chamber, opening a valve so that the solution containing the
protein in the second chamber becomes in fluid communication with
the crystallization reagent in the first chamber, and closing the
valve after a period of time to interrupt fluid communication
between the first and second chambers.
[0178] In some embodiments, a valve can be under the control of an
automated valve actuating device, which in turn may be further
under control of a computer or processor. A multilayer microfluidic
device can include at least one elastomeric layer, and a valve can
include a deflectable membrane. In some cases, a deflectable
membrane of a valve can be deflectable into one or more channels to
control fluid movement along the channels. Multiple elastomeric
membranes may be bonded or adhered together to form an elastomeric
block. In some cases, portions of channels or chambers can overlap
with portions of other channels or chambers at an overlap region.
Such channels or chambers can be in fluid communication through a
via located at the overlap region.
Reduced Mixing Times
[0179] Embodiments of the present invention providing methods for
reducing microfluidic device mixing times will now be described
with reference to FIGS. 9A and 9B. FIG. 9A shows the unit cell 100
of FIG. 1. As discussed above with reference to FIG. 1, a first
material, such as an assay reagent, can be introduced into first
chamber 140 past open first isolation valve 132. Similarly, a
second material, such as an assay sample, can be introduced into
second chamber 120 past open second isolation valve 112. The first
and second materials can be introduced under pressure. Once the
first and second materials have been introduced, the first
isolation valve 132 and the second isolation valve 112 can be
closed via activation of control channel 150. In some embodiments,
the first material is disposed within first chamber 140 at a first
pressure and the second material is disposed within second chamber
120 at a second pressure. During introduction of the first material
and the second material, interface valve 162 can be in the closed
position so as to isolate the first material from the second
material.
[0180] FIG. 9B shows the unit cell 100 of FIG. 2. As discussed
above with reference to FIG. 2, the first material disposed within
first chamber 140 can be mixed with the second material in second
chamber 120 by opening interface valve 162. In instances where
little to no pressure differential exists between the first
material disposed in the first chamber 140 and the second material
disposed in the second chamber 120, opening interface valve 162
will typically result in mixing by diffusion. Mixing by diffusion
typically takes a significantly greater amount of time relative to
the time required for some non-diffusion types of mixing.
[0181] In embodiments of the present invention, the first and
second materials are introduced so as to be disposed within their
respective chambers at pressure levels that are sufficiently
different so that when interface valve 162 is opened, mixing
between the first material and the second material occurs at least
in part by fluid injection. For example, the first and second
materials can be introduced so that the first material is disposed
within first chamber 140 at a first pressure that is higher than a
second pressure at which the second material is disposed within
second chamber 120. Where this pressure differential is
sufficiently different, opening interface valve 162 will result in
some of the first material being injected into the second material.
Preferably, the pressure differential will be sufficiently
different such that some of the first material will be injected
into second chamber 120. Although various pressure differentials
can be used, it may be advantageous to use a pressure differential
of 1 psi or greater.
[0182] The extent to which mixing occurs due to fluid injection may
vary due to a number of factors, such as the materials involved,
the pressure differential involved, and the characteristics of the
microfluidic device involved. Less viscous materials may result in
higher levels of mixing by injection than more viscous materials.
Greater pressure differentials will typically result in higher
levels of mixing by injection than lower pressure differentials.
Microfluidic device characteristics, such as chamber volume,
chamber compliance, and channel impedance, can also influence the
amount of mixing that occurs by injection. When interface valve 162
is opened, material will flow from the higher pressure chamber
towards the lower pressure chamber until equilibrium is reached.
Larger and more compliant chambers may result in greater amounts of
material flow for a given pressure differential. Chamber volume
differentials can be used to vary the relationship between the two
chamber pressures and the resulting equilibrium pressure. For
example, where first chamber 140 is smaller than second chamber
120, the resulting equilibrium pressure may be closer to the
pressure within second chamber 120 prior to opening interface valve
162 than to the pressure within first chamber 140 prior to opening
interface valve 162. Greater amounts of pressure difference between
a high pressure chamber and the equilibrium pressure may result in
greater resulting amounts of material flow.
Increased Manufacturing Yield Rates
[0183] Embodiments of the present invention providing microfluidic
devices with increased manufacturing yield rates, and related
methods of manufacture, will now be described with reference to
FIGS. 10A and 10B. A device can include, for example, a
configuration that allows for the isolation of defective portions
of a microfluidic device. During manufacture of a microfluidic
device, manufacturing defects may result in one or more defective
portions of a microfluidic device. A defect may render one or more
fluid paths associated with a defective portion sufficiently
non-functional as a result. For example, a defect may result in a
sample channel or a reagent channel being rendered sufficiently
non-functional due to their association with the defective portion
of the microfluidic device. Embodiments of the present invention
provide microfluidic devices that can be configured so as to
isolate these defects.
[0184] Often, defects can be identified and located during quality
control inspection(s). Such defects can include dysfunctional
valves that may exist due to various causes (e.g., the presence of
particles, mold defects, etc.). A microfluidic device can be
non-destructively tested by filling the device with an inert
volatile material (e.g., FC72 made by the 3M Corporation) and
performing a microscopic inspection for defects. For example, the
microscopic inspection can detect flows of the inert material
indicative of failures (e.g., a flow indicative of a failed valve
such as a containment valve, etc.). Based on the microscopic
inspection, the defect can often be located.
[0185] FIG. 10A diagrammatically illustrates a microfluidic array
device that provides an ability to isolate defects. FIG. 10A shows
a pre-quality control configuration that has not yet been
configured to isolate any particular defect. Microfluidic array
device 400 includes a plurality of reagent inlets 402 and a
plurality of sample inlets 404. Each of the reagent inlets 402 is
in initial fluid communication with a unique pair of reagent flow
channels 406. Likewise, each of the sample inlets 404 is in initial
fluid communication with a unique pair of sample flow channels 408.
Each of the reagent flow channels 406 is for the introduction of a
reagent fluid into one or more reaction cells. Likewise, each
sample flow channel 408 is for the introduction of a sample fluid
into one or more reaction cells. While each reagent inlet 402 is
shown in initial fluid communication with a unique pair of reagent
flow channels 406, it should be appreciated that any particular
reagent inlet 402 may be in initial fluid communication with any
number of reagent flow channels 406, such as one, two, or more. The
same applies for each sample inlet 404, which may be in initial
fluid communication with any number of sample flow channels 408,
such as one, two, or more.
[0186] FIG. 10B diagrammatically illustrates a portion of the
microfluidic array device 400 of FIG. 10A. FIG. 10B shows a
post-quality control configuration that has been configured to
isolate a defect 414. Microfluidic array device 400 includes
fusible isolation features by which individual reagent flow
channels 406 and individual sample flow channels 408 can be
selectively fluidically closed, thereby isolating their associated
inlet from their associated one or more reaction cells. A fusible
isolation feature can be provided by a containment valve 412
located under each of the reagent flow channels 406 and under each
of the sample flow channels 408. All of the containment valves 412
can be actuated by one or more control channels 410. Once a
containment valve 412 selected for fusing has been closed using its
control channel 410, an exposure to a low frequency ultraviolet
light can be used to cause the exposed containment valve 412 to
fuse and remain closed once the control channel 410 pressure is
released. This selective fusing of containment valves 412 allows
for a selective approach in determining which reagent flow channels
406 and which sample flow channels 408 remain active. It should be
appreciated that a variety of different approaches can be used to
selectively determine which reagent flow channels and which sample
flow channels are active. For example, each flow channel may be
initially fluidically closed, and may be selectively opened, such
as by removing material so as to open the flow channel.
[0187] The ability to selectively choose which of two flow channels
will be connected with a particular inlet provides the ability to
isolate the inlet from a defective flow channel, while still having
the inlet connected with a functional flow channel. For example, a
defective flow channel can be isolated from two associated inlets,
while the two associated inlets can themselves be connected with
adjacent functional channels. By creating a design with built in
redundancy, the manufacturing yield rate can be significantly
improved. For example, a microfluidic array device 400 that
includes 48 reagent inlets 402 and 48 sample inlets 404 that are
collectively in initial fluid communication with 50 reagent flow
channels 406 and 50 sample flow channels 408 respectively can
tolerate up to two reagent flow channels 406 and two sample flow
channels 408 that cannot be used due to identified defects and
still maintain a functioning 48 by 48 microfluidic device array
400.
Supplying Controlled Pressure
[0188] Embodiments of the present invention providing apparatus and
systems for supplying controlled pressure to a microfluidic device
will now be described with reference to FIGS. 11A, 11B, 11C, and
11D. Microfluidic device processing equipment is often used to
supply pressurized fluids to a microfluidic device. For example,
these fluids can include one or more sample fluids, one or more
reagent fluids, and one or more control fluids. In certain
instances, it may be desirable to use multiple pressure levels,
such as greater than eight pressure levels, with any particular
microfluidic device.
[0189] FIG. 11A diagrammatically illustrates an embodiment of a
simplified controlled supply pressure apparatus 420 capable of
providing multiple supply pressures to multiple supply outlets 423.
Supply pressure apparatus 420 includes a single pressure
regulator/transducer 421 that can be used to selectively supply
pressure to each of a plurality of accumulators 422 (one through
n). Pressure regulator/transducer 421 can employ rotary motion to
couple its outlet with the desired accumulator 422. Each of the
accumulators 422 (one through n) can supply a specific supply
outlet 423 (one through n).
[0190] A key consideration regarding the supply of controlled
pressure to a microfluidic device is that these devices typically
require very little actual flow during the introduction of fluids,
or during valve actuation. Because very little volume is required,
it is not necessary to have a dedicated pressure
regulator/transducer 421 for each supply outlet 423. Instead, a
single pressure regulator/transducer 421 can periodically update
the pressure within each accumulator 422 (one through n) to the
extent necessary to account for any small amount of pressure change
that occurred since the previous pressure update.
[0191] FIG. 11B diagrammatically illustrates another embodiment of
a simplified controlled supply pressure apparatus capable of
providing multiple supply pressures to multiple supply outlets 423.
Supply pressure apparatus 430 includes a single pressure
regulator/transducer 431 that can be used to selectively supply
pressure to each of the accumulators 422 (one through n). In some
embodiments, an accumulator selector valve 432, such as a rotary
valve, can be used to selectively couple the output of pressure
regulator/transducer 431 with an accumulator 422. In some
embodiments, an integrated component can be used in place of
pressure regulator/transducer 431 and accumulator selector valve
432, such as pressure regulator/transducer 421 shown in FIG. 11A.
Each of the supply outlets 423 (one through m) can be supplied by
an associated supply outlet selector valve 434 (one through in)
that can selectively place a supply outlet 423 in fluid
communication with an accumulator 422. In some embodiments, each
accumulator 422 is used to supply a particular supply outlet 423,
thereby eliminating the need for supply outlet selector valves
434.
[0192] FIG. 11C diagrammatically illustrates another embodiment of
a simplified controlled supply pressure apparatus capable of
providing multiple supply pressures to multiple supply outlets 423.
Supply pressure apparatus 440 is similar to supply pressure
apparatus 430, but employs simple single inlet/outlet valves 441 in
place of the more complex selector valves used in the embodiment of
supply pressure apparatus 430 shown in FIG. 11B. In some
embodiments, each accumulator 422 is used to supply a particular
supply outlet 423, thereby eliminating the need for the associated
selector valves 441.
[0193] FIG. 11D diagrammatically illustrates an embodiment of a
control system 450 that can be used to control the operation of a
simplified controlled supply pressure apparatus. Control unit 452
typically includes at least one processor 454 which communicates
with a number of peripheral devices via bus subsystem 456. These
peripheral devices typically include a storage subsystem 460
(memory subsystem 462 and file storage subsystem 468), and a
control console 470.
[0194] Storage subsystem 460 maintains the basic programming and
data constructs that provide the functionality of the control unit
452. Software modules for implementing control of the pressure
regulator/tranducer and valves 472 discussed above are typically
stored in storage subsystem 460. Storage subsystem 460 typically
includes memory subsystem 462 and file storage subsystem 468.
[0195] Memory subsystem 462 typically includes a number of memories
including a main random access memory (RAM) 464 for storage of
instructions and data during program execution and a read only
memory (ROM) 466 in which fixed instructions are stored.
[0196] File storage subsystem 468 provides persistent
(non-volatile) storage for program and data files, and can include
a hard drive, a disk drive, or other non-volatile memory such as
flash memory. The disk drive can be used to input the software
modules discussed above. Alternatively, any other means known in
the art may be used to input the software modules, such as a USB
port.
[0197] Control console 470 can provide an interface with a user.
Control console 470 can be used to display information to the user
and receive input from the user.
[0198] In this context, the term "bus subsystem" is used
generically so as to include any mechanism for letting the various
components and subsystems communicate with each other as intended.
Bus subsystem 456 is shown schematically as a single bus, but a
typical system has a number of buses such as a local bus and one or
more expansion buses (e.g., ADB, SCSI, ISA, EISA, MCA, NuBus, or
PCI), as well as serial and parallel ports.
[0199] Control system 450 can include digital to analog interface
devices 474 if necessary so as to provide analog control signals to
the pressure regulator and assorted valves.
Reduced Dehydration
[0200] Embodiments of the present invention providing methods and
related microfluidic devices for reducing dehydration of a
microfluidic device will now be described with reference to FIGS.
12A, 12B, and 12C. Dehydration of a microfluidic device can occur,
for example, when the device is used to process a sample fluid that
includes water.
[0201] Microfluidic device vent channels may provide a low
impedance path for dehydration. Although microfluidic devices can
often be blind filled with control fluid, reagent fluid, and sample
fluid because air trapped within the device is able to escape
through the device material (e.g., elastomeric material), vent
channels are often established to facilitate faster loading. A vent
channel can be located such that the air escaping through the
device material can be collected by a vent channel, thereby
reducing the distance within device material through which the
escaping air travels. However, these vent channels provide a path
by which dehydration may occur, especially during elevated
temperature processing.
[0202] FIG. 12A shows a microfluidic device 480 that includes
vertical vent channels 482, horizontal vent channels 484, and a
ring of sacrificial chambers called dehydration chambers 486.
Dehydration chambers 486 can be used to provide a reservoir of
sample fluid that will evaporate first, thereby helping to reduce
the amount of dehydration that interior chambers may experience.
However, adjacently located vent channels 482 and 484 may provide
low impedance dehydration paths that serve to increase the amount
of dehydration that occurs. FIG. 12B is a close-up view of a
portion of microfluidic device 480 of FIG. 12A, and shows a number
of adjacently located vertical vent channels 482 and horizontal
vent channels 484.
[0203] To reduce dehydration, one or more vent channels can be
filled with substantially non-permeable fluid, such as a
perfluoropolyether oil. The substantially non-permeable fluid can
be introduced after control fluid, reagent, and sample are loaded
into the chip. At this point in time, the vent channels 482 and 484
have served their purpose, but remain as a dehydration
liability.
[0204] A vent channel 482 or 484 can be filled in a variety of
ways. One approach is to simply blind fill a vent channel 482
and/or 484 with a substantially impermeable fluid, such as control
fluid. With blind filling, any air trapped within the vent channel
can escape through the permeable elastomeric material. In
microfluidic device 480, vertical vent channels 482 are all
directly connected with each other and horizontal vent channels 484
are all directly connected with each other. However, vertical vent
channels 482 and horizontal vent channels 484 are on different
planes and communicate with each other only through the permeable
elastomeric material. A small modification can be made to
interconnect vertical vent channels 482 with horizontal vent
channels 484 by forming a few well placed vias in the 4 corners of
the microfluidic device 480. These vias can be formed in a variety
of ways, such as with a laser. An existing vent channel port that
provides an escape path for air during loading of the microfluidic
device 480 can be used as an introduction port for the
substantially non-permeable fluid.
[0205] Additional embodiments are best described with reference to
FIG. 12C, which diagrammatically illustrates microfluidic device
configuration 490 that includes interconnected vertical vent
channels 492 and horizontal vent channels 494 that are in fluid
communication with a vent channel port 496. These concentric vent
channels can be located so as to go almost entirely around a
microfluidic device. In one embodiment, a vent channel vent 498 is
provided that is not fluidically connected with vent channels 492
or 494, but is located within another plane of the microfluidic
device and is not connected by a via. A substantially non-permeable
fluid, such as a perfluoropolyether oil, can be injected into vent
channel port 496, thereby pushing air within vent channels 492 and
494 towards vent channel vent 498, where it escapes through the
permeable elastomeric material to emerge from vent channel vent
498. A vent channel vent 498 that is not directly connected with
vent channels 492 and 494 may provide better retention for the
substantially non-permeable fluid. In some embodiments, vent
channel vent 498 can be connected with vent channels 492 and 494,
thereby facilitating faster loading of the substantially
non-permeable fluid. However, this may result in reduced retention
of the substantially non-permeable fluid as compared to
configurations where vent channel vent 498 is not directly
connected with vent channels 492 and 494.
Improved Mixture Ratio Control
[0206] Embodiments of the present invention providing microfluidic
devices and related methods for improved sample to reagent mixture
ratio control will now be described with reference to FIG. 13.
Often, it may be important for sample fluids and reagent fluids to
be mixed within certain ratio limits.
[0207] FIG. 13 diagrammatically illustrates a microfluidic device
configuration 900 providing flow rate control of a sample fluid
and/or a reagent fluid introduced into a reaction chamber 902.
Reaction chamber 902 is in fluid communication with a sample
channel 904 through a sample interface valve 906 for controlling
fluid communication between reaction chamber 902 and sample channel
904. Sample channel 904 can be in fluid communication with a sample
chamber (not shown) for storing a sample fluid. Reaction chamber
902 is also in fluid communication with a reagent channel 908
through a reagent interface valve 910 for controlling fluid
communication between reaction chamber 902 and reagent channel 908.
Reagent channel 908 can be in fluid communication with a reagent
chamber (not shown) for storing a reagent fluid. Sample interface
valve 906 and reagent interface valve 910 can be actuated via a
control channel 912. Sample channel 904 can include a sample
channel restriction 914 for controlling the flow rate of sample
fluid into reaction chamber 902. Likewise, reagent channel 908 can
include a reagent channel restriction 916 for controlling the flow
rate of reagent fluid into reaction chamber 902. In the embodiment
shown, sample interface valve 906 is disposed between sample
channel restriction 914 and reaction chamber 902, and reagent
interface valve 910 is disposed between reagent channel restriction
916 and reaction chamber 902.
[0208] Various embodiments of a microfluidic device providing flow
rate control are possible. For example, sample channel restriction
914 can be disposed between sample interface valve 906 and reaction
chamber 902, and reagent channel restriction 916 can be disposed
between reagent interface valve 910 and reaction chamber 902. In
some embodiments, sample interface valve 906 can be actuated via a
separate sample interface control channel (not shown), and reagent
interface valve 910 can be actuated via a separate reagent
interface control channel (not shown). In some embodiments, a
microfluidic device can include a plurality of flow rate
controlling configurations. In some embodiments, a microfluidic
device can incorporate flow rate control for just a reagent fluid,
or even just a sample fluid. In some embodiments, the amount of
sample fluid received by a reaction chamber 902 can exceed the
amount of reagent fluid received. In some embodiments, the ratio of
sample fluid to reagent fluid received by a reaction chamber 902
can be approximately a specific ratio, such as ten to one.
[0209] Embodiments of the present invention provide related methods
for using a microfluidic device configured to provide flow rate
control. A method can include, for example, providing a
microfluidic device with flow rate control, introducing sample
fluid into a sample chamber with sample interface valve 906 closed,
introducing reagent fluid into a reagent chamber with reagent
interface valve 910 closed, opening sample interface valve 906 to
transfer sample fluid to reaction chamber 902, opening reagent
interface valve 910 to transfer reagent fluid to reaction chamber
902, closing sample interface valve 906 after the transfer of
sample fluid to reaction chamber 902, and closing reagent interface
valve 910 after the transfer of reagent fluid to reaction chamber
902. In some embodiments, sample interface valve 906 and reagent
interface valve 910 are opened at the same time, and closed at the
same time. In embodiments with a separate sample interface control
channel and/or a separate reagent interface control channel, sample
interface valve 906 and reagent interface valve 910 can be opened
at the same time or different times, and closed at the same time or
different times.
Increased Resistance to Compression Fixture Induced Failure
[0210] Embodiments of the present invention providing microfluidic
devices and systems with increased resistance to compression
fixture pressure induced failures will now be described with
reference to FIGS. 14A, 14B, 15, 16, 17, 18A, 18B, 19, and 20.
Compression fixtures are often used to hold a microfluidic device
in place during elevated temperature processing. A compression
fixture may cause increased pressure levels within a microfluidic
device, which may result in a sufficient pressure differential
across a closed isolation valve for valve failure, which may result
in failure of the microfluidic device.
[0211] FIG. 14A shows a cross-section view of a microfluidic device
920 being held in thermal communication with a thermal control
source 922 via a compression fixture 924. Compression fixture 924
may impart compressive forces on the top of an elastomeric portion
926 of microfluidic device 920. These compressive forces typically
result in internal compressive stresses within elastomeric portion
926, and typically result in increased pressure levels within fluid
filled structures of elastomeric portion 926, such as within
reaction sites 928 that typically contain a mixture of sample fluid
and reagent fluid being reacted at an elevated temperature.
[0212] FIG. 14B depicts a perspective view of an exemplary unit
cell 100 of a microfluidic device that can provide one of the
reaction sites 928 shown in FIG. 14A. Various fluid filled
structures that may be subject to increased pressure levels in
response to compression fixture 924 forces can be seen, such as
first channel 130, first isolation valve 132, first chamber 140,
second channel 110, second isolation valve 112, second chamber 120,
control channel 150, interface channel 160, interface valve 162,
and reaction channel 170. Compression fixture 924 may be used
during elevated temperature processing, which typically occurs only
after the microfluidic device has been loaded with sample fluid,
reagent fluid, and control fluid. At this point, first isolation
valve 132 and second isolation valve 112 are closed and are
maintained closed by pressure within control channel 150, and
interface valve 162 is open, thereby allowing mixing of sample and
reagent fluid. Compression fixture 924 forces may result in
increased pressure within the mixture of sample and reagent fluid
trapped between first isolation valve 132 and second isolation
valve 112. Often, compression fixture 924 forces do not result in
corresponding increases in pressure within portions of first
channel 130 and second channel 110 that are disposed upstream of
first isolation valve 132 and second isolation valve 112
respectively, due to the ability of fluid within these channels to
flow out of the microfluidic device. This may result in an
increased pressure differential across first isolation valve 132
and/or second isolation valve 112, which may reach a magnitude
necessary to overcome the actuation force provided by control
channel 150, thereby causing valve failure. The potential for valve
failure under these circumstances is aggravated because the
pressure within control line 150 may remain essentially constant
where control fluid within control line 150 is free to be pushed
out towards the accumulator. The entire volume of control fluid is
sufficiently small that, when some of the control fluid is pushed
into the accumulator, it causes a negligible change in gas volume
within the accumulator, and hence a negligible change in
accumulator pressure.
[0213] FIG. 15 shows a cross-section view of a microfluidic device
unit cell 100 being held in thermal communication with a thermal
control source 922 via a compression fixture 924. Compression
fixture 924 force results in downward deflection of the upper
portions of elastomeric portion 926, which typically results in
increased pressure within the fluid trapped between first isolation
valve 132 (not shown) and second isolation valve 112, as discussed
above. This increased pressure may result in a pressure
differential across one or both isolation valves sufficient in
magnitude to cause valve failure, so that the previously trapped
fluid is squeezed out of the unit cell 100.
[0214] Embodiments of the present invention can provide improved
resistance to compression fixture pressure induced failures by
restricting the backflow of control fluid. By restricting backflow
of control fluid, the pressure within control channel 150 is
elevated during compression fixture 924 use, thereby providing
increased actuation forces to first isolation valve 132 and second
isolation valve 112. With increased actuation forces, first
isolation valve 132 and second isolation valve 112 are capable of
resisting increased pressure differentials before failure.
[0215] FIG. 16 shows an embodiment of the present invention that
includes a control fluid introduction device 930 that restricts the
backflow of control fluid from a microfluidic device 932. In some
embodiments, a control fluid introduction device 930 can include a
backflow restriction feature 934 that restricts the backflow of
control fluid during compression fixture 924 use. A variety of
devices can be used as a backflow restriction feature 934, such as
a valve that is closed during compression fixture use, or a check
valve that provides for normal control channel use required to
actuate valves within the microfluidic device 932. In some
embodiments, control fluid introduction device 930 can be adapted
to eliminate the presence of gas within accumulator 936 during
compression fixture 924 use. Elimination of gas within accumulator
936 helps to reduce the backflow of control fluid during
compression fixture 924 use.
[0216] FIG. 17 depicts a perspective view of a unit cell 940 of a
microfluidic device having an on-chip backflow prevention feature
942 according to embodiments of the present invention. An on-chip
backflow prevention feature 942 can be, for example, located so as
to prevent backflow of control fluid within control channel 150
during compression fixture 924 use. In some embodiments, the
on-chip backflow prevention feature 942 can be a check valve, such
as check valves as described in co-pending and commonly owned
International Publication Number WO 2008/043046 A2, filed Oct. 4,
2007, which is incorporated herein for all purposes. In some
embodiments, the on-chip backflow prevention feature 942 can be a
valve that can be fused in a close position, such as by an
application of thermal energy or by an application of low-frequency
ultraviolet light and/or other electromagnetic radiation. In some
embodiments, a valve is fused in the closed position by way of
polymer cross-linking. In some embodiments, the polymer
cross-linking is induced by the application of ultraviolet light
(e.g., 200 to 240 nm wavelength light) and/or other electromagnetic
radiation. In some embodiments, backflow of control fluid within
control channel 150 can be restricted by sealing closed control
channel 150 either within or outside the elastomeric portion of the
microfluidic device (e.g., by application of ultraviolet light
and/or other electromagnetic radiation, by application of thermal
energy, or actuation of a guided pin or cam). In some embodiments,
control channel 150 may be self-sealed, such as by solidifying
control fluid within control channel 150.
[0217] FIG. 18A depicts a perspective view of a microfluidic device
unit cell 950 having a pressure compensation feature according to
embodiments of the present invention. A pressure compensation
feature can include, for example, a control fluid chamber 952 in
fluid communication with control channel 954. During compression
fixture 924 use, control fluid is squeezed out of control fluid
chamber 952, which may serve to increase the pressure within
control channel 954 by increasing the amount of control fluid that
must be accommodated within a limited volume elsewhere. The level
of pressure increase can be further increased through the use of
features that limit the backflow of control fluid, or that limit
the space available for the control fluid to escape to, or both.
FIG. 18B shows a cross-section view of a microfluidic device unit
cell 950 having a pressure compensation feature. It should be
appreciated that downward deflection of the top surface of the
microfluidic device that may occur during compression fixture use
may result in squeezing control fluid out of control fluid chamber
952, thereby increasing pressure within control channel 954 and
helping to resist against pressure induced valve failure.
[0218] FIG. 19 shows a cross-section view of a microfluidic device
unit cell being held in thermal communication with a thermal
control source by way of a preferential compression fixture 960
according to embodiments of the present invention. A preferential
compression fixture 960 can include, for example, a pad 962 for
applying pressure preferentially to areas of a microfluidic device
where control-fluid-filled structures exist so as to produce
increased control fluid pressure in control channel 964 in response
to compression by the preferential compression fixture 960. As
shown, pad 962 can be used to locally cause increased downward
deflection of the upper surface of the microfluidic device in
locations above control-fluid-filled structures, such as control
channel 964. This localized increased downward deflection creates
localized increased internal compressive strains that imposed
increased amounts of constriction upon control-fluid-filled
structures. This increased constriction may elevate the pressure
level within the control fluid, especially where backflow of
control fluid is restricted. As discussed above, increased control
fluid pressure levels provides increased resistance to valve
failure.
[0219] FIG. 20 shows a simplified diagrammatic cross-section view
of a microfluidic device unit cell being held in thermal
communication with a thermal control source by way of a compression
fixture 970 coupled with a pressure compensation device 972
according to embodiments of the present invention. A pressure
compensation device 972 can include, for example, a
control-fluid-pressurization device 974 that can be used to produce
increased control fluid pressure in response to compression fixture
970 force. The amount of pressure increase for a given amount of
compression fixture 970 force can be varied so as to provide
sufficient increases in interface valve actuation forces necessary
to counteract resulting pressure differentials across interface
valves. The control-fluid-pressurization device 974 can include a
syringe type plunger 976 that is subjected to an actuation force
when the compression fixture 970 is used. The
control-fluid-pressurization device 974 can provide control fluid
pressure increases proportional to the amount of compression
fixture 970 force. The control-fluid-pressurization device 974 can
be coupled with the compression fixture 970 via a mechanical
linkage 978 that provides a suitable level of actuation force to
control-fluid-pressurization device 974 in response to compression
fixture 970 force. The control-fluid-pressurization device 974 can
be independently actuated in response to a signal indicating the
amount of compression fixture 970 force, and the signal can be
generated using a force sensing transducer, which can include a
strain sensing gage.
Temperature Controlled Reactions with Reduced Condensation
[0220] Embodiments of the present invention providing microfluidic
methods and systems for conducting temperature controlled reactions
so as to reduce condensation levels within a microfluidic device
will now be described with reference to FIGS. 21A, 21B, and 21C.
Conducting elevated temperature controlled reactions using a
microfluidic device having an elastomeric portion sometimes results
in condensation within the elastomeric portion.
[0221] FIG. 21A shows a cross-sectional diagram of a microfluidic
device 980 being heated by a thermal control device 982. Thermal
control device 982 may include a carrier 984, an integrated heat
spreader 986, a thermal control source 988, and a compression
fixture 990 (shown in FIG. 21B). Microfluidic device 980 may
include an elastomeric portion 992, reaction sites 994 disposed
within elastomeric portion 992, and a device bottom layer 996.
During heating, temperatures of the reaction sites 994 are
elevated, which results in greater levels of evaporation of water
from reaction sites 994, and sample fluid and reagent structures.
Some of the evaporated water travels through permeable elastomeric
portion 992. Often, temperatures within regions of elastomeric
portion 992 permit condensation of the evaporated water, resulting
in a thin fogged layer 997. This thin fogged layer 997 is typically
located above reaction cells 994. A thin fogged layer 997 does not
necessarily prevent fluorescent imaging of the reaction sites.
[0222] FIG. 21B shows a cross-sectional diagram of the microfluidic
device 980 being held in contact with a thermal control source by
way of a non-permeable compression fixture plate 990. Using a
non-permeable compression fixture plate, such as the fixture plate
990, during thermal cycling often results in a thick fogged layer
998 with extensive amounts of condensation. The non-permeable
compression fixture plate 990 prevents the escape of moisture from
contacted surfaces of elastomeric portion 992. The increased
moisture content results in increased amounts of condensation. A
thick fogged layer 998 may interfere with fluorescent imaging of
the reaction sites.
[0223] FIG. 21C shows a cross-section diagram of a microfluidic
device held in place during heating by a compression fixture 1002
according to embodiments of the present invention. A thermal
control device 1000 can be adapted so as to reduce condensation
within elastomeric portion 992 in a variety of ways. In one
approach, compression fixture 1002 can be heated so as to elevate
the temperature of one or more points on elastomeric portion 992
contacted by the compression fixture 1002 above a condensation
threshold. When the temperature(s) of the contacted point(s) is/are
heated above a condensation threshold, internal temperatures within
the elastomeric portion 992 are elevated a sufficient extent to
decrease the size and intensity of the fogged layer (resulting in a
reduced fogged layer 1004 that is smaller and thus more transparent
than thick fogged layer 998) so as to permit fluorescent imaging of
the reaction sites. Preferably, the temperature(s) of the contacted
point(s) is/are elevated above forty degrees centigrade. More
preferably, the temperature(s) of the contacted point(s) is/are
elevated above seventy degrees centigrade. The optimum amount of
temperature elevation may depend on various factors (e.g., the
fluids involved, the temperature of the thermal control source,
etc.). A compression fixture 1002 can be heated with the thermal
control source 1004, or the thermal control device 1000 can include
a separate heat source for heating the compression fixture
1002.
[0224] In another approach, the thermal control device 1000 can be
adapted for the egress of moisture from elastomeric portion 992. A
compression fixture 1002 can, for example, include a permeable or
perforated portion that is held in contact with elastomeric portion
992 of the microfluidic device. The permeable or perforated portion
of compression fixture 1002 provides a path for moisture egress
from elastomeric portion 992. Moisture egress helps to reduce
moisture levels within elastomeric portion 992, which in turn helps
reduce the extent of any fogging. Thermal control device 1000 can
include venting adapted to remove moisture from elastomeric portion
992. The venting can be passive, or forced. Thermal control device
1000 can also include a dehydration device for removing moisture
from elastomeric portion 992. Thermal control device 1000 can
include a combination of compression fixture heating and adaptation
for the egress of moisture from elastomeric portion 992.
Improved Fluorescent Imaging
[0225] Embodiments of the present invention providing methods and
systems for improved fluorescent imaging of microfluidic devices
will now be described with reference to FIGS. 22, 23A, and 23B. The
present invention can provide improved imaging through the use of
improved illumination that reduces the amount of electromagnetic
radiation reflected in the imaging direction.
[0226] As illustrated in FIG. 22, optical imaging systems provided
according to some embodiments of the present invention include
fluorescence imaging systems coupled to thermal control modules.
Such systems are adapted to collect data from microfluidic chips
with N.times.M geometries. In some embodiments, N is equal to M.
For example, embodiments of the present invention utilize
microfluidic devices with 48.times.48 reaction chambers,
96.times.96 reaction chambers, and other geometries. In a
particular embodiment, 96 samples and 96 reagents are utilized in a
microfluidic device with a 96.times.96 reaction chamber geometry.
As will be evident to one of skill in the art, the methods and
systems provided according to embodiments of the present invention
enable one platform to perform multiple applications.
[0227] FIG. 22 is a simplified schematic diagram illustrating an
optical imaging system according to an embodiment of the present
invention. As illustrated in FIG. 22, an optical source 242 is
provided according to embodiments of the present invention. As will
be described more fully below, in some embodiments of the present
invention, light from optical source 242 is utilized to induce
fluorescence in a sample. In other embodiments, chemiluminescence
is utilized as a indicator. Depending on the embodiment, system
components will be added, removed, or used, as will be evident to
one of skill in the art. In various embodiments, optical sources
including light emitting diodes (LEDs), lasers, arc lamps,
incandescent lamps, and the like are utilized. These sources may be
polychromatic or monochromatic. In a particular embodiment, the
optical source is characterized by a first spectral bandwidth. In a
specific embodiment, the optical source is a white light source
producing optical radiation over a spectral range from about 400 nm
to about 700 nm. Merely by way of example, a Lambda LS 300W Xenon
Arc lamp, available from Sutter Instruments of Novato, Calif. is
utilized as an optical source is some embodiments of the present
invention. As will be evident to one of skill in the art, other
optical sources characterized by larger or smaller spectral
bandwidths are capable of being utilized in alternative
embodiments.
[0228] Excitation filter wheel 244 is illustrated in FIG. 22. In
some embodiments, for example, those in which the optical source is
polychromatic, the excitation filter wheel 244 is utilized to
spectrally filter the light emitted by the optical source 242. Of
course, multiple filters could also be used. As an example, in an
embodiment, the excitation filter wheel provides a number of
spectral filters each adapted to pass a predetermined wavelength
range as appropriate for exciting specific fluorescence from a
sample. As illustrated in FIG. 22, the excitation filter wheel 244
is coupled to computer 270, providing for computer control of the
filters. In a particular embodiment, the excitation filter wheel
provides a number of spectral filters:
[0229] Filter 1: A filter with a center wavelength of 485 nm and a
spectral bandwidth of 20 nm;
[0230] Filter 2: A filter with a center wavelength of 530 nm and a
spectral bandwidth of 20 nm; and
[0231] Filter 3: A filter with a center wavelength of 580 nm and a
spectral bandwidth of 20 nm.
[0232] As will be evident to one of skill in the art, embodiments
of the present invention are not limited to these particular
spectral filters, but will utilize spectral filters adapted for
fluorescence processes for particular samples. Moreover, although
the previous discussion related to the use of a filter wheel, this
is not required by the present invention. In alternative
embodiments, spectral filters are provided in geometries other than
a wheel. For example, spectral filters that drop into a filter
holder, electro-optic filters, filters placed into the optical path
by actuators, and the like are included according to embodiments of
the present invention. Moreover, in other embodiments, the optical
source is a tunable laser adapted to emit radiation at
predetermined wavelengths suitable for excitation of fluorescence.
One of ordinary skill in the art would recognize many variations,
modifications, and alternatives.
[0233] As illustrated in FIG. 22, excitation shutter 246 is
provided according to embodiments of the present invention. The
excitation shutter is operated under control of the computer 270 in
some embodiments, to block/pass the optical signal generated by the
optical source 242 and spectrally filtered by the excitation filter
wheel 244. Depending on the application, the excitation source is
blocked while samples are inserted and removed from the system as
well as for calibration operations. In some embodiments, the
excitation shutter is not utilized, for example, in embodiments
utilizing laser sources, which provide alternative means to
extinguish the optical source.
[0234] When the excitation shutter is operated in an open position,
the optical excitation signal passes through a fiber bundle 248 and
is directed so as to impinge on a microfluidic device 205 provided
in chip carrier 207. Other embodiments of the present invention
utilize quartz light guides, liquid light guides, other scrambling
systems, and the like to increase illumination homogeneity. As
illustrated in FIG. 22, the excitation optical signal is directed,
through reflection by optical illuminator 250, refraction, or
combinations thereof, to impinge on a surface of the microfluidic
device 205. As illustrated in FIG. 22, illumination of the
microfluidic device is via optical illuminator 250. In other
embodiments illumination maybe coupled to the microfluidic device
obliquely from one or more sides of device, via a ring light, or
via a portion of the collection optical train (the optical path
between the microfluidic device and the detector 260.
[0235] In some embodiments, the illumination of the microfluidic
device with light produced by the excitation source is provided
over a two-dimensional area of the sample. In these embodiments, a
large field of view is provided, which enables the performance of
fluorescence applications that involve imaging of time resolved
chemical processes and reactions. As an example, fluorescent
imaging of protein calorimetry and nucleic acid amplification
processes are time resolved processes that benefit from embodiments
of the present invention. In some of these processes,
simultaneously excitation of the fluorescent samples provided in a
number of reaction chambers and simultaneous collection of the
fluorescent signals produced by the reactions occurring in the
number of reaction chambers is desirable. In other processes, for
instance, fluorescence lifetime imaging, a brief excitation pulse
is followed by detection (and analysis) of the fluorescent signal
as it decays in time from an initial level. One of ordinary skill
in the art would recognize many variations, modifications, and
alternatives.
[0236] As an example, nucleic acid amplification processes
typically include the target DNA, a thermostable DNA polymerase,
two oligonucleotide primers, deoxynucleotide triphosphates (dNTPs),
a reaction buffer, and magnesium. Once assembled, the reaction is
placed in a thermal cycler, an instrument that subjects the
reaction to a series of different temperatures for varying amounts
of time. This series of temperature and time adjustments is
referred to as one cycle of amplification. Each cycle theoretically
doubles the amount of targeted sequence (amplicon) in the reaction.
Ten cycles theoretically multiply the amplicon by a factor of about
one thousand; 20 cycles, by a factor of more than a million in a
matter of hours. In some applications, it is desirable to acquire
fluorescent imaging data from a large area (e.g., on the order of
several cm.sup.2) in a time period ranging from seconds to
minutes.
[0237] In some embodiments of the present invention, the methods
and systems provided by embodiments of the present invention
facilitate image capture processes that are performed in a
predetermined time period. Merely by way of example, in an
embodiment of the present invention a method of imaging
microfluidic devices is provided. The method includes capturing an
image of a spatial region associated with at least a determined
number of chambers of a microfluidic device using an image
detection spatial region during a time frame of less than one
minute, whereupon the capturing of the image of the spatial region
is substantially free from a stitching and/or scanning process.
[0238] Embodiments of the present invention provide a variety of
time frames for image capture, ranging from 1 millisecond to 1
minute. In some embodiments, time frames for image capture are
greater than one minute. Depending on the emission properties
associated with the processes performed in the chambers of the
microfluidic device, the time frame for image capture will vary.
For example, in an embodiment, the time frame is 10 ms, 50 ms, 100
ms, 250 ms, 500 ms, 750 ms, or 1 second. In other embodiments, the
time frame is 2 seconds, 5 seconds, 10 seconds, 15 seconds, 20
seconds, 30 seconds, 40 seconds, 50 seconds, or 1 minute. Of
course, the time frame will depend on the particular
applications.
[0239] In some embodiments, the image capture process is performed
in a synchronous manner, capturing an image of a determined number
of chambers simultaneously. As an example, in an exemplary PCR
process, the microfluidic device is maintained at a temperature of
90.degree. C. for a time period of 15 seconds. Subsequently, the
microfluidic device is maintained at a temperature of 60.degree. C.
for 45 seconds. The heating and cooling cycle is repeated at a one
minute cycle period for a number of cycles. Utilizing embodiments
of the present invention, images of a determined number of chambers
present in the microfluidic device are acquired synchronously,
while the chambers are maintained at a uniform temperate as a
function of position. For example, a two-dimensional image of an
entire microfluidic device may be acquired utilizing a 30 second
exposure while the microfluidic device is maintained at the
temperature of 60.degree. C. One of skill in the art will
appreciate the benefits provided by the present invention over
raster scanning or stitching systems, in which images of chambers
in a first portion (e.g., an upper left quadrant) of the
microfluidic device are acquired prior to images of chambers in a
second portion (e.g., a lower right quadrant) of the microfluidic
device.
[0240] In other embodiments, multiple images are acquired of the
determined number of chambers during a time frame of less than one
minute. As an example of these embodiments, multiple images
associated with multiple fluorophores are acquired in a particular
embodiment. During the 45 second time period during which the
microfluidic device is maintained at the temperature of 60.degree.
C., three consecutive images utilizing exposures of 15 seconds may
be acquired for three different fluorophores, for example, Rox.TM.,
Vic.RTM., and Fam.TM.. Utilizing these multiple images,
differential fluorescence ratios can be calculated and analyzed. Of
course, depending on the strength of the fluorescent emissions, the
exposure times for the various fluorophores may be modified as
appropriate the particular application. In this way, embodiments of
the present invention provide for imaging of a microfluidic device
in multiple spectral bands while the microfluidic device is
maintained a constant temperature. The constant temperature, as
illustrated by the previous example, may be a portion of a PCR
process including cyclical temperature processes.
[0241] Embodiments of the present invention provide methods and
systems are also adapted to perform and analyze chemiluminescence
processes. In some of these processes, reactions occur on a first
time scale and an image of the chemiluminescence process is
acquired on a second time scale. In a particular process, the
second time scale is less than the first time scale. Thus,
embodiments of the present invention are adapted to capture
synchronous images of chemiluminescence processes when the samples
in the reaction chambers of interest have been reacting for an
equal amount of time. In some of these processes, temperature
control, including temperature cycling of the samples is provided,
whereas in other embodiments, the reaction chambers are maintained
at a constant temperature.
[0242] As illustrated in FIG. 22, a thermal controller, also
referred to as a temperature controller, 240 is provided according
to embodiments of the present invention. A number of different
options of varying sophistication are available for controlling
temperature within selected regions of the microfluidic device or
the entire device. Thus, as used herein, the term temperature
controller is meant broadly to refer to a device or element that
can regulate temperature of the entire microfluidic device or
within a portion of the microfluidic device (e.g., within a
particular temperature region or at one or more junctions in a
matrix of channels of a microfluidic device).
[0243] When microfluidic device 205 is illuminated, the
illumination radiation may be reflected by numerous features of
microfluidic device 205. Reflective features can include flow
channels, vents, valves, etc., which are typically primarily
oriented along two mutually perpendicular directions. Reflective
features can also include any other feature that reflects light to
any significant degree. Reflected illumination radiation may
interfere with the image capture process by adding undesirable
reflected illumination radiation to the desired fluorescent signal
as seen in the imaging direction. Typically, the imaging direction
is generally perpendicular to the microfluidic device, although
other imaging directions may be practiced.
[0244] Embodiments of the present invention provide methods and
systems with reduced imaging interference from reflected
illumination radiation. By reducing the amount of illumination
radiation that is reflected in the imaging direction, the image
capture process is improved. In some embodiments, the use of an
illumination direction that produces a significant amount of
reflected illumination in the imaging direction is avoided. This
can be accomplished by avoiding illumination directions that align
with primary feature directions of the microfluidic device 205.
This suppresses high-efficiency reflection paths, and effectively
reduces the brightness of unwanted clutter in fluorescent
images.
[0245] FIGS. 23A and 23B show results for reflection tests using
illumination in accordance with embodiments of the present
invention. Each of these figures shows test results for a
microfluidic device for four illumination directions. The images
shown in these figures were obtained by rotating the microfluidic
chip relative to the illumination direction by the indicated
amount, thereby producing illumination from an illumination
direction with an azimuth of the indicated amount relative to a
primary feature direction of the microfluidic device. The images
were obtained from the imaging direction. These images
qualitatively demonstrate the impact that illumination direction
has upon the amount of unwanted reflections as seen from the
imaging direction. As can be seen in image 1010 of FIG. 23A and
image 1050 of FIG. 23B, when microfluidic device 205 is positioned
at zero degree azimuth relative to the illumination direction, the
resulting images contain notable amounts of reflective clutter from
areas adjacent to the rectangular reaction sites. As can be seen in
image 1040 of FIG. 23A and image 1080 of FIG. 23B, when
microfluidic device 205 is positioned at 90 degree azimuth relative
to the illumination direction, the resulting images also contain
notable amounts of reflective clutter. As can be seen in image 1020
of FIG. 23A and image 1060 of FIG. 23B, when microfluidic device
205 is positioned at 15 degree azimuth relative to the illumination
direction, the resulting images contain reduced amounts of
reflective clutter as compared to the zero degree and 90 degree
azimuth images. As can be seen in image 1030 of FIG. 23A and image
1070 of FIG. 23B, when microfluidic device 205 is positioned at 45
degree azimuth relative to the illumination direction, the
resulting images contain the least amount of reflective clutter as
compared to the other images.
[0246] There are several ways to reduce the amount of reflective
clutter in the resulting image. For example, the illumination
direction can be varied in both azimuth and elevation relative to
the microfluidic device so as to reduce unwanted reflective clutter
in the resulting image. As a further example, microfluidic device
features can be designed with reflection characteristics in mind so
as to minimize the amount of reflections produced, and/or minimize
the amount of reflections directed in the imaging direction.
[0247] All publications and patent documents (patents, published
patent applications, and unpublished patent applications) cited
herein are incorporated herein by reference as if each such
publication or document was specifically and individually indicated
to be incorporated herein by reference. Citation of publications
and patent documents is not intended as an admission that any such
document is pertinent prior art, nor does it constitute any
admission as to the contents or date of the same.
[0248] It is understood that the examples and embodiments described
herein are for illustrative purposes and that various modifications
or changes in light thereof will be suggested to persons skilled in
the art and are to be included within the spirit and purview of
this application and the scope of the appended claims. Numerous
different combinations are possible, and such combinations are
considered to be part of the present invention.
* * * * *