U.S. patent application number 16/928225 was filed with the patent office on 2021-03-11 for microfluidic reporter cell assay methods and kits thereof.
The applicant listed for this patent is Berkeley Lights, Inc.. Invention is credited to Kevin T. Chapman, Xiao Guan, Jason M. McEwen, Christine E. Sun, Gang F. Wang, Xiaohua Wang, Mark P. White.
Application Number | 20210069698 16/928225 |
Document ID | / |
Family ID | 1000005222797 |
Filed Date | 2021-03-11 |
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United States Patent
Application |
20210069698 |
Kind Code |
A1 |
Guan; Xiao ; et al. |
March 11, 2021 |
MICROFLUIDIC REPORTER CELL ASSAY METHODS AND KITS THEREOF
Abstract
Functional assays using reporter cell assays are described which
probe the activity of at least one cell of interest. The ability to
probe at least one cell is provided by using the microfluidic
methods, devices and kits described herein. Assays combining both
reporter cell signaling as well as binding assay signaling for at
least one cell is also described herein.
Inventors: |
Guan; Xiao; (San Rafael,
CA) ; White; Mark P.; (San Francisco, CA) ;
McEwen; Jason M.; (El Cerrito, CA) ; Wang; Gang
F.; (Mountain View, CA) ; Chapman; Kevin T.;
(Santa Monica, CA) ; Wang; Xiaohua; (Albany,
CA) ; Sun; Christine E.; (Emeryville, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Berkeley Lights, Inc. |
Emeryville |
CA |
US |
|
|
Family ID: |
1000005222797 |
Appl. No.: |
16/928225 |
Filed: |
July 14, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15136481 |
Apr 22, 2016 |
10751715 |
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16928225 |
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62151363 |
Apr 22, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 2400/0424 20130101;
C12M 23/16 20130101; C12M 41/18 20130101; B01L 3/502715 20130101;
B01L 2300/161 20130101; C12M 41/32 20130101; C12Q 1/02 20130101;
C12M 41/48 20130101; B01L 2300/1822 20130101; B01L 2400/0454
20130101; C12M 23/20 20130101; B01L 2300/168 20130101; C12M 23/34
20130101; C12M 23/50 20130101; B01L 3/50273 20130101; C12M 23/58
20130101; G01N 33/5005 20130101 |
International
Class: |
B01L 3/00 20060101
B01L003/00; C12M 1/02 20060101 C12M001/02; C12M 1/34 20060101
C12M001/34; C12M 1/36 20060101 C12M001/36; C12M 1/00 20060101
C12M001/00; C12M 3/06 20060101 C12M003/06; C12Q 1/02 20060101
C12Q001/02; G01N 33/50 20060101 G01N033/50 |
Claims
1.-67. (canceled)
68. A method of assaying at least one biological cell for a
biological activity in a microfluidic device comprising: an
enclosure having an inner lower surface and an inner upper surface
spaced apart defining a chamber, comprising a flow region
configured to contain a flow of a first fluidic medium, and at
least one incubation chamber, each of which is disposed upon the
inner lower surface, wherein the at least one incubation chamber is
enclosed by walls extending from the inner lower surface to the
inner upper surface of the chamber and has a single lateral opening
to the flow region, and wherein the single lateral opening between
the flow region and the connection region is configured to
facilitate substantially only diffusion between the first fluidic
medium and the second fluidic medium in the isolation region when
the first fluidic medium is flowing through the flow region, the
method comprising: introducing the at least one biological cell
into the at least one incubation chamber; introducing at least one
reporter cell into the at least one incubation chamber, wherein the
at least one reporter cell is configured to produce a first
detectable signal when the at least one biological cell comprises
the biological activity; and analyzing the at least one reporter
cell for an activity stimulated by the presence of a biological
activity of the at least one biological cell.
69. The method of claim 68, wherein analyzing comprises incubating
the at least one biological cell and the one at least one reporter
cell in the at least one incubation chamber for a pre-determined
period of time, thereby allowing the one at least one reporter cell
to produce the first detectable signal.
70. The method of claim 69, wherein incubating further comprises
providing the one at least one reporter cell with one or more
reagents forming one or all of the components of the detectable
signal of the at least one reporter cell.
71. The method of claim 68, wherein analyzing further comprises
providing excitation light to excite a fluorophore of the first
detectable signal of the at least one reporter cell.
72. The method of claim 71, further comprising detecting the
excited fluorophore.
73. The method of claim 68, further comprising introducing at least
one capture micro-object into at least the flow region.
74. The method of claim 73, further comprising introducing one or
more visualization reagents which are configured to bind to the at
least one capture micro-object to produce a second detectable
signal.
75. The method of claim 74, further comprising detecting the second
detectable signal.
76. The method of claim 68, wherein introducing the at least one
biological cell into the at least one incubation chamber of the
microfluidic device comprises using a DEP force having sufficient
strength to move the at least one biological cell.
77. The method of claim 76, further comprising optically actuating
the DEP force.
78. The method of claim 68, wherein the at least one biological
cell is a mammalian cell.
79. The method of claim 68, wherein the at least one biological
cell is a hybridoma.
80. The method of claim 68, wherein the at least one biological
cell is a lymphocyte or a leukocyte.
81. The method of claim 68, wherein the at least one incubation
chamber further comprises: an isolation region having a single
opening and configured to contain a second fluidic medium; and a
connection region, comprising a distal opening to the isolation
region and a proximal opening comprising the single lateral opening
into the flow region.
82. The method of claim 81, further comprising introducing the at
least one reporter cell into an isolation region of the at least
one incubation chamber.
83. The method of claim 82, further comprising introducing the at
least one biological cell into the isolation region of the at least
one incubation chamber.
84. The method of claim 68, wherein the single lateral opening into
the microfluidic channel of the at least one incubation chamber has
a width ranging from about 20 microns to about 100 microns.
85. The method of claim 68, wherein a distance between the inner
lower surface and the inner upper surface of the enclosure defining
the chamber is from about 30 to about 200 microns.
86. The method of claim 68, wherein a distance between the inner
lower surface and the inner upper surface of the enclosure defining
the chamber is a substantially uniform distance.
87. The method of claim 68, wherein the at least one incubation
chamber further comprises at least one surface conditioned to
support cell growth, viability, portability, or any combination
thereof.
Description
[0001] This application is a non-provisional application claiming
the benefit under 35 U.S.C. 119(e) of U.S. Provisional Application
No. 62/151,363 filed on Apr. 22, 2015, which disclosure is herein
incorporated by reference in its entirety.
CROSS REFERENCE
[0002] This application cross-references U.S application Ser. No.
15/135,707, entitled "Microfluidic Cell Culture", filed on Apr. 22,
2016, which disclosure is herein incorporated by reference in its
entirety.
BACKGROUND
[0003] Reporter cell assays are useful probes of the biological
function of a cell, yielding information on the status of the cell.
This type of information is not easily obtainable from other
classes of assays, such as binding assays or cell surface stains.
While reporter cell assays have been performed within reaction well
format or flow cytometry format, investigation of the status of at
least one cell, is not readily performed. There is need for
improvement in this field in order to support basic biological
research, pharmaceutical research and development, medical
diagnostics and treatment as well as for bioproduction of cells
expressing useful biological/chemical species.
SUMMARY
[0004] In a first aspect, a system is provided for assaying at
least one biological cell of interest in a microfluidic device,
including a microfluidic device comprising a flow region configured
to contain a flow of a first fluidic medium and at least one
incubation chamber, and wherein the incubation chamber is
configured to contain at least one reporter cell and the at least
one biological cell of interest; and at least one reporter cell.
The at least one incubation chamber may include an isolation region
and a connection region, wherein the isolation region is
fluidically connected to the connection region and the connection
region comprises an opening directly into the flow region.
[0005] In various embodiments of the system, the at least one
incubation chamber may be configured to contain no more than a
single biological cell of interest. In some embodiments, the at
least one incubation chamber may be configured to contain a
plurality of biological cells of interest. The at least one
incubation chamber may be configured to isolate the at least one
reporter cell and the at least one biological cell of interest. the
incubation chamber is configured to locate the at least one
reporter cell and the at least one biological cell of interest at
spatially distinct locations within the incubation chamber. In some
embodiments, the microfluidic device may further include a flow
channel including at least a portion of the flow region, and
wherein the at least one incubation chamber includes a connection
region that opens directly into the flow channel. In some
embodiments, the isolation region of the at least one incubation
chamber may be fluidically connected to the flow channel via the
connection region and is configured to contain a second fluidic
medium, where when the flow region and the at least one incubation
chamber are substantially filled with the first and second fluidic
media respectively, then components of the second fluidic medium
diffuse into the first fluidic medium and/or components of the
first fluidic medium diffuse into the second fluidic medium; and
the first medium does not substantially flow into the isolation
region.
[0006] In various embodiments of the system, the at least one
reporter cell may be configured to provide a detectable signal. In
some embodiments, the detectable signal may be produced when the at
least one biological cell of interest includes a biological
activity of interest. In other embodiments, the at least one
reporter cell may be configured to produce a different detectable
signal when the at least one biological cells of interest does not
comprise the biological activity of interest. The detectable signal
of the at least one reporter cell may be colorimetric, fluorescent,
or bioluminescent.
[0007] In various embodiments of the system, the flow region of the
microfluidic device may further include one or more capture
micro-objects. In various embodiments, each of the one or more
capture micro-objects may include a binding substance configured to
specifically bind to a biological product of the at least one
biological cell of interest. In some embodiments, the biological
product may be a secreted biological product. In various
embodiments, the biological product may be bound to the binding
substance of the one or more capture micro-object thereby producing
a bound capture micro-object. In some embodiments, each of the one
or more capture micro-objects may be a bound capture micro-object.
The one or more bound capture micro-objects may be configured to be
detectable. The one or more bound capture micro-objects may be
indirectly or directly detectable. The detectable signal of the one
or more bound capture micro-objects may be colorimetric,
fluorescent, or chemiluminescent. In some embodiments, the one or
more capture micro-objects may include a bead. In some embodiments,
the one or more capture micro-objects may include a magnetic
bead.
[0008] In various embodiments of the system, the one or more
capture micro-objects may be in fluid connection with the one or
more biological cells of interest. In some embodiments, the one or
more capture micro-objects may be located in the connection region
of the incubation chamber or in the flow region proximal to the
incubation chamber. In other embodiments, the one or more capture
micro-objects may be located at a location within the microfluidic
device other than in the isolation region of the incubation
chamber.
[0009] In various embodiments of the system, the detectable signal
of the at least one reporter cells and the detectable signal of the
one or more bound capture micro-objects may be spectrally
distinct.
[0010] In various embodiments of the system, the microfluidic
device may further include at least one inlet port configured to
input the first or second fluidic medium into the flow region and
at least one outlet port may be configured to receive the first
medium as it exits from the flow region. In various embodiments,
the microfluidic device may be configured to perfuse the first
medium to maintain cell viability. In some embodiments, the
microfluidic device may be configured to perfuse the first medium
irregularly. In other embodiments, the microfluidic device may be
configured to perfuse the first medium periodically.
[0011] In various embodiments of the system, the microfluidic
device may include a substrate configured to generate a
dielectrophoresis (DEP) force, wherein a surface of the substrate
may form a surface of the incubation chamber and the flow region.
In various embodiments, the dielectrophoretic substrate is
optically actuated. In some embodiments, the substrate may include
a plurality of electrodes wherein the plurality of electrodes is
configured to generate a dielectrophoresis (DEP) force. In some
embodiments, the system may further include a selector control
module configured to activate and deactivate each of the plurality
of electrodes, wherein activation of an electrode generates a
dielectrophoresis (DEP) force sufficiently strong to move the at
least one biological cell into or out of the at least one
incubation chamber or the isolation region thereof. In some
embodiments, each of the plurality of electrodes may be optically
actuated. In other embodiments, the microfluidic device may further
include a substrate having an electrode connected to a plurality of
transistors, wherein a surface of the substrate may form a surface
of the incubation chamber and the flow region. In some embodiments,
each transistor of the plurality may be configured to generate a
dielectrophoresis (DEP) force. In some embodiments, the system may
further include a selector control module configured to activate
and deactivate each of the plurality of transistors, thereby
generating a dielectrophoresis (DEP) force sufficiently strong to
move at least one biological cell into or out of the at least one
incubation chamber or the isolation region thereof. In some
embodiments, each of the plurality of transistors may be optically
actuated. In various other embodiments, the microfluidic device may
further include a substrate having an electrode and a layer of
amorphous silicon, wherein a surface of the substrate may form a
surface of the incubation chamber and the flow region. In some
embodiments, the system may further include a selector control
module configured to activate and deactivate the virtual electrode
in the layer of amorphous silicon, thereby generating a
dielectrophoresis (DEP) force sufficiently strong to move at least
one biological cell into or out of the at least one incubation
chamber or the isolation region thereof. In some embodiments, the
layer of amorphous silicon may be optically activated. In various
embodiments, the DEP force may be produced by optoelectronic
tweezers (OET).
[0012] In various embodiments of the system, the at least one
reporter cell is moved into or out of the at least one incubation
chamber or the isolation region thereof by fluid flow and/or
gravity.
[0013] In various embodiments of the system, the at least one
incubation chamber of the microfluidic device may have at least one
surface conditioned to support cell growth, viability, portability,
or any combination thereof. In some embodiments, the at least one
conditioned surface of the incubation chamber may include a
polymer. In some embodiments, the polymer of the at least one
conditioned surface of the microfluidic device may include alkylene
oxide moieties, amino acid moieties or saccharide moieties. In
various embodiments, the at least one conditioned surface of the
microfluidic device may include a covalently linked conditioned
surface. In various embodiments, the covalently linked conditioned
surface may include alkylene ether moieties, alkyl moieties,
fluoroalkyl moieties, amino acid moieties, or saccharide moieties.
In some embodiments, the covalently linked conditioned surface may
be linked to the surface via a siloxy linking group. In various
embodiments, the conditioned surface may be a monolayer.
[0014] In various embodiments of the system, the microfluidic
device may include a plurality of incubation chambers. In various
embodiments, no more than one biological cell may be introduced
into each of the plurality of incubation chambers. In various
embodiments, the at least one biological cell may include a
mammalian cell. In some embodiments, the at least one biological
cell may include a hybridoma cell. In other embodiments, the at
least one biological cell may include a lymphocyte or a leukocyte.
In various embodiments, the at least one biological cell may
include a B cell, T cell, NK cell, dendritic cell, or macrophage.
In various embodiments, the at least one biological cell may
include an adherent cell.
[0015] In various embodiments of the system, the system may further
include a light source configured to provide excitation energy to a
moiety configured to be detectable by fluorescence. In various
embodiments of the system, the system may further include a
detector configured to capture an image of the at least one
incubation chamber and any biological cells contained therein. In
various embodiments, the detector may capture images under visible,
infrared, or ultraviolet wavelengths of light.
[0016] In another aspect, a method is provided for assaying at
least one biological cell for a biological activity in a system
comprising a microfluidic device having at least one incubation
chamber and a flow region, the method including the steps of
introducing the at least one biological cell into the at least one
incubation chamber; introducing one or more reporter cells into the
at least one incubation chamber; and analyzing the one or more
reporter cells for an activity stimulated by the presence of a
biological activity of the at least one biological cell. The at
least one incubation chamber may include an isolation region and a
connection region, wherein the isolation region may be fluidically
connected to the connection region and the connection region may
include an opening directly into the flow region.
[0017] In various embodiments of the method, the one or more
reporter cells may be configured to produce a detectable signal
when the at least one biological cell comprises the biological
activity. In some embodiments, the detectable signal of the one or
more reporter cells may be a colorometric, fluorescent, or
bioluminescent signal.
[0018] In various embodiments of the method, the step of
introducing the one or more reporter cells may be performed before
the step of introducing the at least one biological cell. In
various embodiments of the method, the step of introducing the one
or more reporter cells may be performed after the step of
introducing the at least one biological cell.
[0019] In various embodiments of the method, the method may further
include a step of introducing the one or more reporter cells into
an isolation region of the at least one incubation chamber. In
various other embodiments of the method, the method may further
include a step of introducing the at least one biological cell into
an isolation region of the at least one incubation chamber. In some
embodiments, a single biological cell may be introduced into the
isolation region of the incubation chamber. In various embodiments
of the method, the method may further include a step of introducing
the at least one biological cell to a spatially distinct region of
the isolation region from the location of the one or more reporter
cells.
[0020] In various embodiments of the method, the step of analyzing
may include incubating the at least one biological cell and the one
or more reporter cells in the at least one incubation chamber for a
pre-determined period of time, thereby producing the detectable
signal of the one or more reporter cells. In various embodiments of
the method, the step of incubating may further include providing
the one or more reporter cells with one or more reagents forming
one or all of the components of the detectable signal of the one or
more reporter cells. In various embodiments of the method, the step
of analyzing may further include analyzing the one or more reporter
cells at more than one time point during the incubation period. In
various embodiments of the method, the step of analyzing the one or
more reporter cells may further include providing excitation light
to excite a fluorophore of the detectable signal of the one or more
reporter cells.
[0021] In various embodiments of the method, the method may further
include a step of detecting the detectable signal of the one or
more reporter cells. In various embodiments of the method, the
method may further include a step of quantifying the detectable
signal of the one or more reporter cells, thereby quantifying the
presence of the biological activity.
[0022] In various embodiments of the method, the one or more
reporter cells may be configured to produce a second detectable
signal when the at least one biological cell does not comprise the
biological activity.
[0023] In various embodiments of the method, the step of incubating
the at least one biological cell and the one or more reporter cells
for the pre-determined period of time may include producing the
second detectable signal of the one or more reporter cells, thereby
indicating an absence of the biological activity. In various
embodiments of the method, the step of analyzing the one or more
reporter cells may further include providing excitation light to
excite a fluorophore of the second detectable signal of the one or
more reporter cells. In various embodiments of the method, the
method may further include a step of detecting the second
detectable signal of the one or more reporter cells. In various
embodiments of the method, the method may further include a step of
quantifying the detectable signal of the one or more reporter
cells, thereby quantifying the absence of the biological
activity.
[0024] In various embodiments of the method, the method may further
include a step of introducing at least one capture micro-object
into at least the flow region. In some embodiments, the step of
introducing the at least one micro-object may further include
disposing the at least one micro-object in a location adjacent to a
proximal opening of the incubation chamber in the flow region. In
some embodiments, introducing the at least one capture micro-object
may not include introducing the at least one capture micro-object
to the isolation region of the incubation chamber.
[0025] In various embodiments of the method, each of the one or
more capture micro-objects may include a binding substance
configured to specifically bind a biological product of the at
least one biological cells, thereby forming a bound capture
micro-object configured to be detectable. In some embodiments, the
binding substance may be covalently attached to each of the one or
more micro-objects. In other embodiments, the binding substance may
be noncovalently attached to each of the one or more micro-objects.
In some embodiments, the bound capture micro-object is configured
to be directly detectable. In some embodiments, the bound capture
micro-object is configured to be indirectly detectable. A
detectable signal of the bound capture micro-object may be a
colorimetric, fluorescent, or chemiluminescent signal. In some
embodiments, the at least one capture micro-object may be a bead.
In various embodiments, the biological product of the at least one
biological cell may be a secreted biological product.
[0026] In various embodiments of the method, the method may further
include a step of incubating the at least one capture micro-object
during the incubation period, thereby producing the at least one
bound capture micro-object. In various embodiments of the method,
the method may further include a step of introducing one or more
visualization reagents which may be configured to bind to the bound
capture micro-object to produce the detectable signal. In various
embodiments of the method, the method may further include a step of
providing excitation light to excite the detectable signal of the
bound capture micro-object. In various embodiments of the method,
the method may further include a step of detecting the detectable
signal of the bound capture micro-object. In various embodiments of
the method, the method may further include a step of quantifying
the detected signal of the binding substance. In various
embodiments of the method, the method may further include a step of
introducing the at least one capture micro-object using a magnetic
field. In various embodiments of the method, the system may be any
system as described herein.
[0027] In various embodiments of the method, the step of
introducing the at least one biological cell into the microfluidic
device, incubation chamber, isolation region or location within the
isolation region thereof, may include using a dielectrophoresis
(DEP) force having sufficient strength to move the biological cell.
In some embodiments, the step of using the DEP force includes
optically actuating the DEP force. In some embodiments, the step of
introducing the one or more reporter cells into the at least one
incubation chamber may include using fluid flow and/or gravity. In
some embodiments, the step of introducing the one or more capture
micro-objects into the flow region may include using fluid flow
and/or gravity.
[0028] In some embodiments of the method, the method may further
include a step of introducing a first fluidic medium into a flow
channel of the flow region of the microfluidic device. The rate of
introducing the first fluidic medium may not sweep the isolation
region of the incubation chamber. In some embodiments of the
method, the method may further include a step of perfusing the
first fluidic medium during the incubating step, wherein the first
fluidic medium is introduced via at least one inlet port of the
microfluidic device and is exported via at least one outlet of the
microfluidic device and further wherein the first fluidic medium
may optionally include components from the second fluidic medium.
In some embodiments, the perfusing may be non-continuous. In other
embodiments, the perfusing may be periodic. In some embodiments of
the method, the method may further include a step of perfusing the
first fluidic medium at a rate sufficient to permit components of
the second fluidic medium in the isolation region to diffuse into
the first fluidic medium in the flow region and/or components of
the first fluidic medium to diffuse into the second fluidic medium
in the isolation region; and at the rate wherein the first medium
does not substantially flow into the isolation region.
[0029] In some embodiments of the method, the at least one
biological cell may include a mammalian cell. In other embodiments
of the method, the at least one biological cell may include a
hybridoma cell. In yet other embodiments of the method, the at
least one biological cell may include a lymphocyte or a leukocyte.
In some other embodiments of the method, the at least one
biological cell may include a B cell, T cell, NK cell, dendritic
cell, or macrophage. In further embodiments of the method, the at
least one biological cell may include an adherent cell.
[0030] In some embodiments of the method, the method may further
include a step of replenishing the conditioned surface.
[0031] In another aspect, a composition is provided including a
biological cell and one or more reporter cells in an isolation
region of a microfluidic device, where the one or more reporter
cells are configured to detect a biological activity of the
biological cell when contacted by a first extracellular species
produced by the biological cell. In some embodiments, the
biological cell and one or more reporter cells cell may be at least
one biological cell and one or more reporter cells. The
microfluidic device of the compositions may have at least one
incubation chamber and a flow region, where the at least one
incubation chamber includes an isolation region and a connection
region, wherein the isolation region is fluidically connected to
the connection region and the connection region comprises an
opening directly into the flow region. The microfluidic device may
include at least one conditioned surface configured to support cell
growth, viability, portability or any combination thereof.
[0032] The at least one conditioned surface may include an alkylene
ether moiety configured to support cell growth, viability,
portability or any combination thereof. In other embodiments, the
at least one conditioned surface may include an alkyl or
fluoroalkyl (including perfluoroalkyl) moiety configured to support
cell growth, viability, portability or any combination thereof. In
some other embodiments, the at least one conditioned surface may
include a dextran moiety configured to support cell growth,
viability, portability or any combination thereof. In some
embodiments, the biological cell and one or more reporter cells may
be in contact with the at least one conditioned surface.
[0033] In some embodiments, a first extracellular species may be
produced by the biological cell contacts the one or more reporter
cells without the biological cell directly contacting any of the
one or more reporter cells. In some embodiments, when the one or
more reporter cells are contacted by the first extracellular
species, then the one or more reporter cells may be configured to
produce a first detectable signal. In some embodiments, the first
detectable signal of the one or more reporter cells may include a
colorimetric, fluorescent, bioluminescent or luminescent
signal.
[0034] In various embodiments, the composition may further include
at least one capture micro-object, wherein the at least one capture
micro-object may be configured to bind an extracellular species
produced by the biological cell, without physically contacting the
biological cell.
[0035] In some embodiments, the extracellular species produced by
the biological cell that binds to the at least one capture
micro-object may be different from the extracellular species
produced by the single biological cell that is detected by the one
or more reporter cells. In various embodiments, the at least one
capture micro-object may not be located within the isolation
region.
[0036] In various embodiments of the composition, the at least one
capture micro-object may be configured to form at least one
detectable bound capture micro-object when the extracellular
species binds to the at least one capture micro-object. In some
embodiments, the at least one bound capture micro-object may be
directly detectable. In other embodiments, the at least one bound
capture micro-object may be indirectly detectable. In various
embodiments, a detectable signal of the at least one bound capture
micro-object may be fluorescent or chemiluminescent.
[0037] In some embodiments of the composition, the biological cell
may include a mammalian cell. In other embodiments of the
composition, the biological cell may include a hybridoma cell. In
some embodiments of the composition, the biological cell may
include a lymphocyte or a leukocyte. In further embodiments, the
biological cell may include a B cell, T cell, NK cell, or
macrophage. In some other embodiments, the biological cell may
include an adherent cell.
[0038] In another aspect, a kit is provided, including a
microfluidic device comprising at least one incubation chamber and
a flow region; and one or more reporter cells configured to test
for a biological activity of a biological cell. In some
embodiments, the at least one incubation chamber of the
microfluidic device may include an isolation region and a
connection region, wherein the isolation region may be fluidically
connected to the connection region and the connection region may
include an opening directly into the flow region. In some
embodiments, the microfluidic device may further include a flow
channel comprising at least a portion of the flow region, and the
incubation chamber may include a connection region that opens
directly into the flow channel. In some embodiments, the at least
one incubation chamber may further include an isolation region. In
some embodiments, the isolation region may be fluidically connected
to the connection region and may be configured to contain a second
fluidic medium, wherein: when the flow region and the at least one
incubation chamber are substantially filled with the first and
second fluidic media respectively, then components of the second
fluidic medium may diffuse into the first fluidic medium and/or
components of the first fluidic medium may diffuse into the second
fluidic medium; and the first medium may not substantially flow
into the isolation region. In some embodiments, the at least one
incubation chamber may be a plurality of isolation chambers. In
various embodiments, the microfluidic device may further include at
least one inlet port configured to input the first or second
fluidic medium into the flow region and at least one outlet
configured to receive the first medium as it exits from the flow
region, wherein the first medium may optionally contain components
of the second fluidic medium.
[0039] In various embodiments of the kit, the microfluidic device
may further include a substrate having a dielectrophoresis (DEP)
configuration wherein a surface of the substrate may form a surface
of the incubation chamber and the flow region. The DEP
configuration may be optically actuated.
[0040] In some embodiments of the kit, the kit may further include
one or more micro-objects configured to bind a biological product
of a biological cell.
[0041] In some embodiments of the kit, the kit may further include
one or more reagents used to provide a detectable signal from the
reporter cells configured to test for a biological activity of the
biological cell.
[0042] In some embodiments of the kit, the microfluidic device may
further include at least one conditioned surface configured to
support cell growth, viability, portability or any combination
thereof. In some embodiments of the kit, the kit may further
include a reagent to replenish the conditioned surface. In some
embodiments, the at least one conditioned surface of the at least
one incubation chamber may include a polymer. In various
embodiments, the polymer of the at least one conditioned surface of
the microfluidic device may include alkylene oxide moieties, amino
acid moieties or saccharide moieties. In other embodiments, the at
least one conditioned surface of the microfluidic device may
include a covalently linked conditioned surface. In various
embodiments, the covalently linked conditioned surface may include
alkylene ether moieties, alkyl moieties, fluoroalkyl moieties,
amino acid moieties, or saccharide moieties. In some embodiments,
the covalently linked conditioned surface may be linked to the
surface via a siloxy linking group.
DETAILED DESCRIPTION
[0043] Reporter cell assays may be performed within a microfluidic
device as described herein, where the behavior of at least one cell
is examined. The ability to assay at least one biological cell of
interest within an isolation region and obtain both location
dependent and time dependent reporter signals provides more precise
and selective data. While the microfluidic environment provides the
ability to isolate one or more biological cells for investigation,
it also offers the opportunity to multiplex assays to probe sets of
individual cells of interest. The instant methods also offer the
opportunity to simultaneously employ capture agents having specific
binding partners in order to multiplex a reporter cell assay with
one or more binding assays. Particularly with the motive forces
available within the system used with the instant microfluidic
devices, improved methods are provided for probing and linking
assay data with specific cells of interest within a population of
cells introduced into the microfluidic device.
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] FIG. 1 illustrates an example of a system for use with a
microfluidic device and associated control equipment according to
some embodiments of the invention.
[0045] FIGS. 2A and 2B illustrate a microfluidic device according
to some embodiments of the invention.
[0046] FIGS. 2C and 2D illustrate incubation chambers according to
some embodiments of the invention.
[0047] FIG. 2E illustrates a detailed incubation chamber according
to some embodiments of the invention.
[0048] FIG. 2F illustrates a microfluidic device according to an
embodiment of the invention.
[0049] FIG. 3A illustrates a specific example of a system for use
with a microfluidic device and associated control equipment
according to some embodiments of the invention.
[0050] FIG. 3B illustrates an imaging device according to some
embodiments of the invention.
[0051] FIGS. 4A-4C show another embodiment of a microfluidic
device, including a further example of an incubation chamber used
therein.
[0052] FIG. 5 is an example of one embodiment of a process for
perfusing a fluidic medium in a microfluidic device.
[0053] FIG. 6 is an example of another embodiment of a process for
perfusing a fluidic medium in a microfluidic device
[0054] FIG. 7 is a schematic representation of a conditioned
surface providing enhanced support cell growth, viability,
portability, or any combination thereof.
[0055] This specification describes exemplary embodiments and
applications of the invention. The invention, however, is not
limited to these exemplary embodiments and applications or to the
manner in which the exemplary embodiments and applications operate
or are described herein. Moreover, the figures may show simplified
or partial views, and the dimensions of elements in the figures may
be exaggerated or otherwise not in proportion. In addition, as the
terms "on," "attached to," "connected to," "coupled to," or similar
words are used herein, one element (e.g., a material, a layer, a
substrate, etc.) can be "on," "attached to," "connected to," or
"coupled to" another element regardless of whether the one element
is directly on, attached to, connected to, or coupled to the other
element or there are one or more intervening elements between the
one element and the other element. Where reference is made to a
list of elements (e.g., elements a, b, c), such reference is
intended to include any one of the listed elements by itself, any
combination of less than all of the listed elements, and/or a
combination of all of the listed elements. Section divisions in the
specification are for ease of review only and do not limit any
combination of elements discussed.
[0056] As used herein, "substantially" means sufficient to work for
the intended purpose. As used herein, "substantially" means
sufficient to work for the intended purpose. The term
"substantially" thus allows for minor, insignificant variations
from an absolute or perfect state, dimension, measurement, result,
or the like such as would be expected by a person of ordinary skill
in the field but that do not appreciably affect overall
performance. When used with respect to numerical values or
parameters or characteristics that can be expressed as numerical
values, "substantially" means within ten percent.
[0057] The term "ones" means more than one. As used herein, the
term "plurality" can be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.
[0058] As used herein, "air" refers to the composition of gases
predominating in the atmosphere of the earth. The four most
plentiful gases are nitrogen (typically present at a concentration
of about 78% by volume, e.g., in a range from about 70-80%), oxygen
(typically present at about 20.95% by volume at sea level, e.g. in
a range from about 10% to about 25%), argon (typically present at
about 1.0% by volume, e.g. in a range from about 0.1% to about 3%),
and carbon dioxide (typically present at about 0.04%, e.g., in a
range from about 0.01% to about 0.07%). Air may have other trace
gases such as methane, nitrous oxide or ozone, trace pollutants and
organic materials such as pollen, diesel particulates and the like.
Air may include water vapor (typically present at about 0.25%, or
may be present in a range from about 10 ppm to about 5% by volume).
Air may be provided for use in culturing experiments as a filtered,
controlled composition and may be conditioned as described
herein.
[0059] As used herein, the term "disposed" encompasses within its
meaning "located."
[0060] As used herein, a "microfluidic device" or "microfluidic
apparatus" is a device that includes one or more discrete
microfluidic circuits configured to hold a fluid, each microfluidic
circuit comprised of fluidically interconnected circuit elements,
including but not limited to region(s), flow path(s), channel(s),
chamber(s), and/or pen(s), and at least two ports configured to
allow the fluid (and, optionally, micro-objects suspended in the
fluid) to flow into and/or out of the microfluidic device.
Typically, a microfluidic circuit of a microfluidic device will
include at least one microfluidic channel and at least one chamber,
and will hold a volume of fluid of less than about 1 mL, e.g., less
than about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9,
8, 7, 6, 5, 4, 3, or 2 .mu.L. In certain embodiments, the
microfluidic circuit holds about 1-2, 1-3, 1-4, 1-5, 2-5, 2-8,
2-10, 2-12, 2-15, 2-20, 5-20, 5-30, 5-40, 5-50, 10-50, 10-75,
10-100, 20-100, 20-150, 20-200, 50-200, 50-250, or 50-300
.mu.L.
[0061] As used herein, a "nanofluidic device" or "nanofluidic
apparatus" is a type of microfluidic device having a microfluidic
circuit that contains at least one circuit element configured to
hold a volume of fluid of less than about 1 .mu.L, e.g., less than
about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8,
7, 6, 5, 4, 3, 2, 1 nL or less. Typically, a nanofluidic device
will comprise a plurality of circuit elements (e.g., at least 2, 3,
4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300,
400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500,
4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000, or more). In
certain embodiments, one or more (e.g., all) of the at least one
circuit elements are configured to hold a volume of fluid of about
100 pL to 1 nL, 100 pL to 2 nL, 100 pL to 5 nL, 250 pL to 2 nL, 250
pL to 5 nL, 250 pL to 10 nL, 500 pL to 5 nL, 500 pL to 10 nL, 500
pL to 15 nL, 750 pL to 10 nL, 750 pL to 15 nL, 750 pL to 20 nL, 1
to 10 nL, 1 to 15 nL, 1 to 20 nL, 1 to 25 nL, or 1 to 50 nL. In
other embodiments, one or more (e.g., all) of the at least one
circuit elements are configured to hold a volume of fluid of about
100 to 200 nL, 100 to 300 nL, 100 to 400 nL, 100 to 500 nL, 200 to
300 nL, 200 to 400 nL, 200 to 500 nL, 200 to 600 nL, 200 to 700 nL,
250 to 400 nL, 250 to 500 nL, 250 to 600 nL, or 250 to 750 nL.
[0062] A "microfluidic channel" or "flow channel" as used herein
refers to flow region of a microfluidic device having a length that
is significantly longer than both the horizontal and vertical
dimensions. For example, the flow channel can be at least 5 times
the length of either the horizontal or vertical dimension, e.g., at
least 10 times the length, at least 25 times the length, at least
100 times the length, at least 200 times the length, at least 300
times the length, at least 400 times the length, at least 500 times
the length, or longer. In some embodiments, the length of a flow
channel is in the range of from about 20,000 microns to about
100,000 microns, including any range therebetween. In some
embodiments, the horizontal dimension is in the range of from about
100 microns to about 1000 microns (e.g., about 150 to about 500
microns) and the vertical dimension is in the range of from about
25 microns to about 200 microns, e.g., from about 40 to about 150
microns. It is noted that a flow channel may have a variety of
different spatial configurations in a microfluidic device, and thus
is not restricted to a perfectly linear element. For example, a
flow channel may be, or include one or more sections having, the
following configurations: curve, bend, spiral, incline, decline,
fork (e.g., multiple different flow paths), and any combination
thereof. In addition, a flow channel may have different
cross-sectional areas along its path, widening and constricting to
provide a desired fluid flow therein.
[0063] As used herein, the term "obstruction" refers generally to a
bump or similar type of structure that is sufficiently large so as
to partially (but not completely) impede movement of target
micro-objects between two different regions or circuit elements in
a microfluidic device. The two different regions/circuit elements
can be, for example, a microfluidic incubation chamber and a
microfluidic channel, or a connection region and an isolation
region of a microfluidic incubation chamber.
[0064] As used herein, the term "constriction" refers generally to
a narrowing of a width of a circuit element (or an interface
between two circuit elements) in a microfluidic device. The
constriction can be located, for example, at the interface between
a microfluidic incubation chamber and a microfluidic channel, or at
the interface between an isolation region and a connection region
of a microfluidic incubation chamber.
[0065] As used herein, the term "transparent" refers to a material
which allows visible light to pass through without substantially
altering the light as is passes through.
[0066] As used herein, the term "micro-object" can encompass one or
more of the following: inanimate micro-objects such as
microparticles, microbeads (e.g., polystyrene beads, Luminex.TM.
beads, or the like), magnetic beads, paramagnetic beads, microrods,
microwires, quantum dots, and the like; biological micro-objects
such as cells (e.g., embryos, oocytes, sperms, cells dissociated
from a tissue, blood cells, immunological cells, including T cells,
B cells, macrophages, NK cells, dendritic cells (DCs), and the
like, hybridomas, cultured cells, cells dissociated from a tissue,
cells from a cell line, such as CHO cells, which may be transfected
and/or transformed, cancer cells, including circulating tumor cells
(CTCs), infected cells, reporter cells, and the like), liposomes
(e.g., synthetic or derived from membrane preparations), lipid
nanorafts, and the like; or a combination of inanimate
micro-objects and biological micro-objects (e.g., microbeads
attached to cells, liposome-coated micro-beads, liposome-coated
magnetic beads, or the like). Beads may further have other
moieties/molecules covalently or non-covalently attached, such as
fluorescent labels, proteins, small molecule signaling moieties,
antigens, or chemical/biological species capable of use in an
assay.
[0067] As used herein, the term "cell" refers to a biological cell,
which can be a plant cell, an animal cell (e.g., a mammalian cell),
a bacterial cell, a fungal cell, or the like. A mammalian cell can
be, for example, from a human, a mouse, a rat, a horse, a goat, a
sheep, a cow, a primate, or the like.
[0068] As used herein, the term "maintaining (a) cell(s)" refers to
providing an environment comprising both fluidic and gaseous
components and, optionally a surface, that provides the conditions
necessary to keep the cells viable and/or expanding.
[0069] A "component" of a fluidic medium is any chemical or
biochemical molecule present in the medium, including solvent
molecules, ions, small molecules, antibiotics, nucleotides and
nucleosides, nucleic acids, amino acids, peptides, proteins,
sugars, carbohydrates, lipids, fatty acids, cholesterol,
metabolites, or the like.
[0070] As used herein in reference to a fluidic medium, "diffuse"
and "diffusion" refer to thermodynamic movement of a component of
the fluidic medium down a concentration gradient.
[0071] The phrase "flow of a medium" means bulk movement of a
fluidic medium primarily due to any mechanism other than diffusion.
For example, flow of a medium can involve movement of the fluidic
medium from one point to another point due to a pressure
differential between the points. Such flow can include a
continuous, pulsed, periodic, random, intermittent, or
reciprocating flow of the liquid, or any combination thereof. When
one fluidic medium flows into another fluidic medium, turbulence
and mixing of the media can result.
[0072] The phrase "substantially no flow" refers to a rate of flow
of a fluidic medium that is less than the rate of diffusion of
components of a material (e.g., an analyte of interest) into or
within the fluidic medium. The rate of diffusion of components of
such a material can depend on, for example, temperature, the size
of the components, and the strength of interactions between the
components and the fluidic medium.
[0073] As used herein in reference to different regions within a
microfluidic device, the phrase "fluidically connected" means that,
when the different regions are substantially filled with fluid,
such as fluidic media, the fluid in each of the regions is
connected so as to form a single body of fluid. This does not mean
that the fluids (or fluidic media) in the different regions are
necessarily identical in composition. Rather, the fluids in
different fluidically connected regions of a microfluidic device
can have different compositions (e.g., different concentrations of
solutes, such as proteins, carbohydrates, ions, or other molecules)
which are in flux as solutes move down their respective
concentration gradients and/or fluids flow through the device.
[0074] A microfluidic (or nanofluidic) device can comprise "swept"
regions and "unswept" regions. As used herein, a "swept" region is
comprised of one or more fluidically interconnected circuit
elements of a microfluidic circuit, each of which experiences a
flow of medium when fluid is flowing through the microfluidic
circuit. The circuit elements of a swept region can include, for
example, regions, channels, and all or parts of chambers. As used
herein, an "unswept" region is comprised of one or more fluidically
interconnected circuit element of a microfluidic circuit, each of
which experiences substantially no flux of fluid when fluid is
flowing through the microfluidic circuit. An unswept region can be
fluidically connected to a swept region, provided the fluidic
connections are structured to enable diffusion but substantially no
flow of media between the swept region and the unswept region. The
microfluidic device can thus be structured to substantially isolate
an unswept region from a flow of medium in a swept region, while
enabling substantially only diffusive fluidic communication between
the swept region and the unswept region. For example, a flow
channel of a micro-fluidic device is an example of a swept region
while an isolation region (described in further detail below) of a
microfluidic device is an example of an unswept region.
[0075] As used herein, a "non-sweeping" rate of fluidic medium flow
means a rate of flow sufficient to permit components of a second
fluidic medium in an isolation region of the incubation chamber to
diffuse into the first fluidic medium in the flow region and/or
components of the first fluidic medium to diffuse into the second
fluidic medium in the isolation region; and further wherein the
first medium does not substantially flow into the isolation
region.
[0076] As used herein, a "flow path" refers to one or more
fluidically connected circuit elements (e.g. channel(s), region(s),
chamber(s) and the like) that define, and are subject to, the
trajectory of a flow of medium. A flow path is thus an example of a
swept region of a microfluidic device. Other circuit elements
(e.g., unswept regions) may be fluidically connected with the
circuit elements that comprise the flow path without being subject
to the flow of medium in the flow path.
[0077] The capability of biological micro-objects (e.g., biological
cells) to produce specific biological materials can be assayed in
such a microfluidic device. For example, sample material comprising
biological micro-objects to be assayed for production of an analyte
of interest can be loaded into a swept region of the microfluidic
device. Ones of the biological micro-objects can be selected for
particular characteristics and disposed in unswept regions. The
remaining sample material can then be flowed out of the swept
region and an assay material flowed into the swept region. Because
the selected biological micro-objects are in unswept regions, the
selected biological micro-objects are not substantially affected by
the flowing out of the remaining sample material or the flowing in
of the assay material. The selected biological micro-objects can be
allowed to produce the analyte of interest, which can diffuse from
the unswept regions into the swept region, where the analyte of
interest can react with the assay material to produce localized
detectable reactions, each of which can be correlated to a
particular unswept region. Any unswept region associated with a
detected reaction can be analyzed to determine which, if any, of
the biological micro-objects in the unswept region are sufficient
producers of the analyte of interest.
[0078] Microfluidic Devices and Systems for Operating and Observing
Such Devices.
[0079] FIG. 1 illustrates an example of a microfluidic device 100
and a system 150 which can be used in the practice of the present
invention. A perspective view of the microfluidic device 100 is
shown having a partial cut-away of its cover 110 to provide a
partial view into the microfluidic device 100. The microfluidic
device 100 generally comprises a microfluidic circuit 120
comprising a flow path 106 through which a fluidic medium 180 can
flow, optionally carrying one or more micro-objects (not shown)
into and/or through the microfluidic circuit 120. Although a single
microfluidic circuit 120 is illustrated in FIG. 1, suitable
microfluidic devices can include a plurality (e.g., 2 or 3) of such
microfluidic circuits. Regardless, the microfluidic device 100 can
be configured to be a nanofluidic device. In the embodiment
illustrated in FIG. 1, the microfluidic circuit 120 comprises a
plurality of microfluidic incubation chambers 124, 126, 128, and
130, each having one or more openings in fluidic communication with
flow path 106. As discussed further below, the microfluidic
incubation chambers comprise various features and structures that
have been optimized for retaining micro-objects in the microfluidic
device, such as microfluidic device 100, even when a medium 180 is
flowing through the flow path 106. Before turning to the foregoing,
however, a brief description of microfluidic device 100 and system
150 is provided.
[0080] As generally illustrated in FIG. 1, the microfluidic circuit
120 is defined by an enclosure 102. Although the enclosure 102 can
be physically structured in different configurations, in the
example shown in FIG. 1 the enclosure 102 is depicted as comprising
a support structure 104 (e.g., a base), a microfluidic circuit
structure 108, and a cover 110. The support structure 104,
microfluidic circuit structure 108, and cover 110 can be attached
to each other. For example, the microfluidic circuit structure 108
can be disposed on an inner surface 109 of the support structure
104, and the cover 110 can be disposed over the microfluidic
circuit structure 108. Together with the support structure 104 and
cover 110, the microfluidic circuit structure 108 can define the
elements of the microfluidic circuit 120.
[0081] The support structure 104 can be at the bottom and the cover
110 at the top of the microfluidic circuit 120 as illustrated in
FIG. 1. Alternatively, the support structure 104 and the cover 110
can be configured in other orientations. For example, the support
structure 104 can be at the top and the cover 110 at the bottom of
the microfluidic circuit 120. Regardless, there can be one or more
ports 107 each comprising a passage into or out of the enclosure
102. Examples of a passage include a valve, a gate, a pass-through
hole, or the like. As illustrated, port 107 is a pass-through hole
created by a gap in the microfluidic circuit structure 108.
However, the port 107 can be situated in other components of the
enclosure 102, such as the cover 110. Only one port 107 is
illustrated in FIG. 1 but the microfluidic circuit 120 can have two
or more ports 107. For example, there can be a first port 107 that
functions as an inlet for fluid entering the microfluidic circuit
120, and there can be a second port 107 that functions as an outlet
for fluid exiting the microfluidic circuit 120. Whether a port 107
function as an inlet or an outlet can depend upon the direction
that fluid flows through flow path 106.
[0082] The support structure 104 can comprise one or more
electrodes (not shown) and a substrate or a plurality of
interconnected substrates. For example, the support structure 104
can comprise one or more semiconductor substrates, each of which is
electrically connected to an electrode (e.g., all or a subset of
the semiconductor substrates can be electrically connected to a
single electrode). The support structure 104 can further comprise a
printed circuit board assembly ("PCBA"). For example, the
semiconductor substrate(s) can be mounted on a PCBA.
[0083] The microfluidic circuit structure 108 can define circuit
elements of the microfluidic circuit 120. Such circuit elements can
comprise spaces or regions that can be fluidly interconnected when
microfluidic circuit 120 is filled with fluid, such as flow
channels, chambers, pens, traps, and the like. In the microfluidic
circuit 120 illustrated in FIG. 1, the microfluidic circuit
structure 108 comprises a frame 114 and a microfluidic circuit
material 116. The frame 114 can partially or completely enclose the
microfluidic circuit material 116. The frame 114 can be, for
example, a relatively rigid structure substantially surrounding the
microfluidic circuit material 116. For example, the frame 114 can
comprise a metal material.
[0084] The microfluidic circuit material 116 can be patterned with
cavities or the like to define circuit elements and
interconnections of the microfluidic circuit 120. The microfluidic
circuit material 116 can comprise a flexible material, such as a
flexible polymer (e.g. rubber, plastic, elastomer, silicone,
polydimethylsiloxane ("PDMS"), or the like), which can be gas
permeable. Other examples of materials that can compose
microfluidic circuit material 116 include molded glass, an etchable
material such as silicone (e.g. photo-patternable silicone or
"PPS"), photo-resist (e.g., SU8), or the like. In some embodiments,
such materials--and thus the microfluidic circuit material 116--can
be rigid and/or substantially impermeable to gas. Regardless,
microfluidic circuit material 116 can be disposed on the support
structure 104 and inside the frame 114.
[0085] The cover 110 can be an integral part of the frame 114
and/or the microfluidic circuit material 116. Alternatively, the
cover 110 can be a structurally distinct element, as illustrated in
FIG. 1. The cover 110 can comprise the same or different materials
than the frame 114 and/or the microfluidic circuit material 116.
Similarly, the support structure 104 can be a separate structure
from the frame 114 or microfluidic circuit material 116 as
illustrated, or an integral part of the frame 114 or microfluidic
circuit material 116. Likewise, the frame 114 and microfluidic
circuit material 116 can be separate structures as shown in FIG. 1
or integral portions of the same structure.
[0086] In some embodiments, the cover 110 can comprise a rigid
material. The rigid material may be glass or a material with
similar properties. In some embodiments, the cover 110 can comprise
a deformable material. The deformable material can be a polymer,
such as PDMS. In some embodiments, the cover 110 can comprise both
rigid and deformable materials. For example, one or more portions
of cover 110 (e.g., one or more portions positioned over incubation
chambers 124, 126, 128, 130) can comprise a deformable material
that interfaces with rigid materials of the cover 110. In some
embodiments, the cover 110 can further include one or more
electrodes. The one or more electrodes can comprise a conductive
oxide, such as indium-tin-oxide (ITO), which may be coated on glass
or a similarly insulating material. Alternatively, the one or more
electrodes can be flexible electrodes, such as single-walled
nanotubes, multi-walled nanotubes, nanowires, clusters of
electrically conductive nanoparticles, or combinations thereof,
embedded in a deformable material, such as a polymer (e.g., PDMS).
Flexible electrodes that can be used in microfluidic devices have
been described, for example, in U.S. 2012/0325665 (Chiou et al.),
the contents of which are incorporated herein by reference. In some
embodiments, the cover 110 can be modified (e.g., by conditioning
all or part of a surface that faces inward toward the microfluidic
circuit 120) to support cell adhesion, viability and/or growth. The
modification may include a coating of a synthetic or natural
polymer. In some embodiments, the cover 110 and/or the support
structure 104 can be transparent to light. The cover 110 may also
include at least one material that is gas permeable (e.g., PDMS or
PPS).
[0087] FIG. 1 also shows a system 150 for operating and controlling
microfluidic devices, such as microfluidic device 100. System 150,
as illustrated, includes an electrical power source 192, an imaging
device 194, and a tilting device 190.
[0088] The electrical power source 192 can provide electric power
to the microfluidic device 100 and/or tilting device 190, providing
biasing voltages or currents as needed. The electrical power source
192 can, for example, comprise one or more alternating current (AC)
and/or direct current (DC) voltage or current sources. The imaging
device 194 can comprise a device, such as a digital camera, for
capturing images inside microfluidic circuit 120. In some
instances, the imaging device 194 further comprises a detector
having a fast frame rate and/or high sensitivity (e.g. for low
light applications). The imaging device 194 can also include a
mechanism for directing stimulating radiation and/or light beams
into the microfluidic circuit 120 and collecting radiation and/or
light beams reflected or emitted from the microfluidic circuit 120
(or micro-objects contained therein). The emitted light beams may
be in the visible spectrum and may, e.g., include fluorescent
emissions. The reflected light beams may include reflected
emissions originating from an LED or a wide spectrum lamp, such as
a mercury lamp (e.g. a high pressure mercury lamp) or a Xenon arc
lamp. As discussed with respect to FIG. 3, the imaging device 194
may further include a microscope (or an optical train), which may
or may not include an eyepiece.
[0089] System 150 can further comprise a tilting device 190
configured to rotate a microfluidic device 100 about one or more
axes of rotation. In some embodiments, the tilting device 190 is
configured to support and/or hold the enclosure 102 comprising the
microfluidic circuit 120 about at least one axis such that the
microfluidic device 100 (and thus the microfluidic circuit 120) can
be held in a level orientation (i.e. at 0.degree. relative to x-
and y-axes), a vertical orientation (i.e. at 90.degree. relative to
the x-axis and/or the y-axis), or any orientation therebetween. The
orientation of the microfluidic device 100 (and the microfluidic
circuit 120) relative to an axis is referred to herein as the
"tilt" of the microfluidic device 100 (and the microfluidic circuit
120). For example, the tilting device 190 can tilt the microfluidic
device 100 at 0.1.degree., 0.2.degree., 0.3.degree., 0.4.degree.,
0.5.degree., 0.6.degree., 0.7.degree., 0.8.degree., 0.9.degree.,
1.degree., 2.degree., 3.degree., 4.degree., 5.degree., 10.degree.,
15.degree., 20.degree., 25.degree., 30.degree., 35.degree.,
40.degree., 45.degree., 50.degree., 55.degree., 60.degree.,
65.degree., 70.degree., 75.degree., 80.degree., 90.degree. relative
to the x-axis or any degree therebetween. The level orientation
(and thus the x- and y-axes) is defined as normal to a vertical
axis defined by the force of gravity. The tilting device can also
tilt the microfluidic device 100 (and the microfluidic circuit 120)
to any degree greater than 90.degree. relative to the x-axis and/or
y-axis, or tilt the microfluidic device 100 (and the microfluidic
circuit 120) 180.degree. relative to the x-axis or the y-axis in
order to fully invert the microfluidic device 100 (and the
microfluidic circuit 120). Similarly, in some embodiments, the
tilting device 190 tilts the microfluidic device 100 (and the
microfluidic circuit 120) about an axis of rotation defined by flow
path 106 or some other portion of microfluidic circuit 120.
[0090] In some instances, the microfluidic device 100 is tilted
into a vertical orientation such that the flow path 106 is
positioned above or below one or more incubation chambers. The term
"above" as used herein denotes that the flow path 106 is positioned
higher than the one or more incubation chambers on a vertical axis
defined by the force of gravity (i.e. an object in an incubation
chamber above a flow path 106 would have a higher gravitational
potential energy than an object in the flow path). The term "below"
as used herein denotes that the flow path 106 is positioned lower
than the one or more incubation chambers on a vertical axis defined
by the force of gravity (i.e. an object in an incubation chamber
below a flow path 106 would have a lower gravitational potential
energy than an object in the flow path).
[0091] In some instances, the tilting device 190 tilts the
microfluidic device 100 about an axis that is parallel to the flow
path 106. Moreover, the microfluidic device 100 can be tilted to an
angle of less than 90.degree. such that the flow path 106 is
located above or below one or more incubation chambers without
being located directly above or below the incubation chambers. In
other instances, the tilting device 190 tilts the microfluidic
device 100 about an axis perpendicular to the flow path 106. In
still other instances, the tilting device 190 tilts the
microfluidic device 100 about an axis that is neither parallel nor
perpendicular to the flow path 106.
[0092] System 150 can further include a media source 178. The media
source 178 (e.g., a container, reservoir, or the like) can comprise
multiple sections or containers, each for holding a different
fluidic medium 180. Thus, the media source 178 can be a device that
is outside of and separate from the microfluidic device 100, as
illustrated in FIG. 1. Alternatively, the media source 178 can be
located in whole or in part inside the enclosure 102 of the
microfluidic device 100. For example, the media source 178 can
comprise reservoirs that are part of the microfluidic device
100.
[0093] FIG. 1 also illustrates simplified block diagram depictions
of examples of control and monitoring equipment 152 that constitute
part of system 150 and can be utilized in conjunction with a
microfluidic device 100. As shown, examples of such control and
monitoring equipment 152 include a master controller 154 comprising
a media module 160 for controlling the media source 178, a motive
module 162 for controlling movement and/or selection of
micro-objects (not shown) and/or medium (e.g., droplets of medium)
in the microfluidic circuit 120, an imaging module 164 for
controlling an imaging device 194 (e.g., a camera, microscope,
light source or any combination thereof) for capturing images
(e.g., digital images), and a tilting module 166 for controlling a
tilting device 190. The control equipment 152 can also include
other modules 168 for controlling, monitoring, or performing other
functions with respect to the microfluidic device 100. As shown,
the equipment 152 can further include a display device 170 and an
input/output device 172.
[0094] The master controller 154 can comprise a control module 156
and a digital memory 158. The control module 156 can comprise, for
example, a digital processor configured to operate in accordance
with machine executable instructions (e.g., software, firmware,
source code, or the like) stored as non-transitory data or signals
in the memory 158. Alternatively, or in addition, the control
module 156 can comprise hardwired digital circuitry and/or analog
circuitry. The media module 160, motive module 162, imaging module
164, tilting module 166, and/or other modules 168 can be similarly
configured. Thus, functions, processes acts, actions, or steps of a
process discussed herein as being performed with respect to the
microfluidic device 100 or any other microfluidic apparatus can be
performed by any one or more of the master controller 154, media
module 160, motive module 162, imaging module 164, tilting module
166, and/or other modules 168 configured as discussed above.
Similarly, the master controller 154, media module 160, motive
module 162, imaging module 164, tilting module 166, and/or other
modules 168 may be communicatively coupled to transmit and receive
data used in any function, process, act, action or step discussed
herein.
[0095] The media module 160 controls the media source 178. For
example, the media module 160 can control the media source 178 to
input a selected fluidic medium 180 into the enclosure 102 (e.g.,
through an inlet port 107). The media module 160 can also control
removal of media from the enclosure 102 (e.g., through an outlet
port (not shown)). One or more media can thus be selectively input
into and removed from the microfluidic circuit 120. The media
module 160 can also control the flow of fluidic medium 180 in the
flow path 106 inside the microfluidic circuit 120. For example, in
some embodiments media module 160 stops the flow of media 180 in
the flow path 106 and through the enclosure 102 prior to the
tilting module 166 causing the tilting device 190 to tilt the
microfluidic device 100 to a desired angle of incline.
[0096] The motive module 162 can be configured to control
selection, trapping, and movement of micro-objects (not shown) in
the microfluidic circuit 120. As discussed below with respect to
FIGS. 2A and 2B, the enclosure 102 can comprise a dielectrophoresis
(DEP), optoelectronic tweezers (OET) and/or opto-electrowetting
(OEW) configuration (not shown in FIG. 1), and the motive module
162 can control the activation of electrodes and/or transistors
(e.g., phototransistors) to select and move micro-objects (not
shown) and/or droplets of medium (not shown) in the flow path 106
and/or incubation chambers 124, 126, 128, 130.
[0097] The imaging module 164 can control the imaging device 194.
For example, the imaging module 164 can receive and process image
data from the imaging device 194. Image data from the imaging
device 194 can comprise any type of information captured by the
imaging device 194 (e.g., the presence or absence of micro-objects,
droplets of medium, accumulation of label, such as fluorescent
label, etc.). Using the information captured by the imaging device
194, the imaging module 164 can further calculate the position of
objects (e.g., micro-objects, droplets of medium) and/or the rate
of motion of such objects within the microfluidic device 100.
[0098] The tilting module 166 can control the tilting motions of
tilting device 190. Alternatively, or in addition, the tilting
module 166 can control the tilting rate and timing to optimize
transfer of micro-objects to the one or more incubation chambers
via gravitational forces. The tilting module 166 is communicatively
coupled with the imaging module 164 to receive data describing the
motion of micro-objects and/or droplets of medium in the
microfluidic circuit 120. Using this data, the tilting module 166
may adjust the tilt of the microfluidic circuit 120 in order to
adjust the rate at which micro-objects and/or droplets of medium
move in the microfluidic circuit 120. The tilting module 166 may
also use this data to iteratively adjust the position of a
micro-object and/or droplet of medium in the microfluidic circuit
120.
[0099] In the example shown in FIG. 1, the microfluidic circuit 120
is illustrated as comprising a microfluidic channel 122 and
incubation chambers 124, 126, 128, 130. Each chamber comprises an
opening to channel 122, but otherwise is enclosed such that the
chambers can substantially isolate micro-objects inside the chamber
from fluidic medium 180 and/or micro-objects in the flow path 106
of channel 122 or in other chambers. In some instances, chambers
124, 126, 128, 130 are configured to physically corral one or more
micro-objects within the microfluidic circuit 120. Incubation
chambers in accordance with the present invention can comprise
various shapes, surfaces and features that are optimized for use
with DEP, OET, OEW, and/or gravitational forces, as will be
discussed and shown in detail below.
[0100] The microfluidic circuit 120 may comprise any number of
microfluidic incubation chambers. Although five incubation chambers
are shown, microfluidic circuit 120 may have fewer or more
incubation chambers. In some embodiments, the microfluidic circuit
120 comprises a plurality of microfluidic incubation chambers,
wherein two or more of the incubation chambers comprise differing
structures and/or features.
[0101] In the embodiment illustrated in FIG. 1, a single channel
122 and flow path 106 is shown. However, other embodiments may
contain multiple channels 122, each configured to comprise a flow
path 106. The microfluidic circuit 120 further comprises an inlet
valve or port 107 in fluid communication with the flow path 106 and
fluidic medium 180, whereby fluidic medium 180 can access channel
122 via the inlet port 107. In some instances, the flow path 106
comprises a single path. In some instances, the single path is
arranged in a zigzag pattern whereby the flow path 106 travels
across the microfluidic device 100 two or more times in alternating
directions.
[0102] In some instances, microfluidic circuit 120 comprises a
plurality of parallel channels 122 and flow paths 106, wherein the
fluidic medium 180 within each flow path 106 flows in the same
direction. In some instances, the fluidic medium within each flow
path 106 flows in at least one of a forward or reverse direction.
In some instances, a plurality of incubation chambers is configured
(e.g., relative to a channel 122) such that they can be loaded with
target micro-objects in parallel.
[0103] In some embodiments, microfluidic circuit 120 further
comprises one or more micro-object traps 132. The traps 132 are
generally formed in a wall forming the boundary of a channel 122,
and may be positioned opposite an opening of one or more of the
microfluidic incubation chambers 124, 126, 128, 130. In some
embodiments, the traps 132 are configured to receive or capture a
single micro-object from the flow path 106. In some embodiments,
the traps 132 are configured to receive or capture a plurality of
micro-objects from the flow path 106. In some instances, the traps
132 comprise a volume approximately equal to the volume of a single
target micro-object.
[0104] The traps 132 may further comprise an opening which is
configured to assist the flow of targeted micro-objects into the
traps 132. In some instances, the traps 132 comprise an opening
having a height and width that is approximately equal to the
dimensions of a single target micro-object, whereby larger
micro-objects are prevented from entering into the micro-object
trap. The traps 132 may further comprise other features configured
to assist in retention of targeted micro-objects within the trap
132. In some instances, the trap 132 is aligned with and situated
on the opposite side of a channel 122 relative to the opening of a
microfluidic incubation chamber, such that upon tilting the
microfluidic device 100 about an axis parallel to the channel 122,
the trapped micro-object exits the trap 132 at a trajectory that
causes the micro-object to fall into the opening of the incubation
chamber. In some instances, the trap 132 comprises a side passage
134 that is smaller than the target micro-object in order to
facilitate flow through the trap 132 and thereby increase the
likelihood of capturing a micro-object in the trap 132.
[0105] In some embodiments, dielectrophoretic (DEP) forces are
applied across the fluidic medium 180 (e.g., in the flow path
and/or in the incubation chambers) via one or more electrodes (not
shown) to manipulate, transport, separate and sort micro-objects
located therein. For example, in some embodiments, DEP forces are
applied to one or more portions of microfluidic circuit 120 in
order to transfer a single micro-object from the flow path 106 into
a desired microfluidic incubation chamber. In some embodiments, DEP
forces are used to prevent a micro-object within an incubation
chamber (e.g., incubation chamber 124, 126, 128, or 130) from being
displaced therefrom. Further, in some embodiments, DEP forces are
used to selectively remove a micro-object from an incubation
chamber that was previously collected in accordance with the
teachings of the instant invention. In some embodiments, the DEP
forces comprise optoelectronic tweezer (OET) forces.
[0106] In other embodiments, optoelectrowetting (OEW) forces are
applied to one or more positions in the support structure 104
(and/or the cover 110) of the microfluidic device 100 (e.g.,
positions helping to define the flow path and/or the incubation
chambers) via one or more electrodes (not shown) to manipulate,
transport, separate and sort droplets located in the microfluidic
circuit 120. For example, in some embodiments, OEW forces are
applied to one or more positions in the support structure 104
(and/or the cover 110) in order to transfer a single droplet from
the flow path 106 into a desired microfluidic incubation chamber.
In some embodiments, OEW forces are used to prevent a droplet
within an incubation chamber (e.g., incubation chamber 124, 126,
128, or 130) from being displaced therefrom. Further, in some
embodiments, OEW forces are used to selectively remove a droplet
from an incubation chamber that was previously collected in
accordance with the teachings of the instant invention.
[0107] In some embodiments, DEP and/or OEW forces are combined with
other forces, such as flow and/or gravitational force, so as to
manipulate, transport, separate and sort micro-objects and/or
droplets within the microfluidic circuit 120. For example, the
enclosure 102 can be tilted (e.g., by tilting device 190) to
position the flow path 106 and micro-objects located therein above
the microfluidic incubation chambers, and the force of gravity can
transport the micro-objects and/or droplets into the chambers. In
some embodiments, the DEP and/or OEW forces can be applied prior to
the other forces. In other embodiments, the DEP and/or OEW forces
can be applied after the other forces. In still other instances,
the DEP and/or OEW forces can be applied at the same time as the
other forces or in an alternating manner with the other forces.
[0108] FIGS. 2A-2F illustrates various embodiments of microfluidic
devices that can be used in the practice of the present invention.
FIG. 2A depicts an embodiment in which the microfluidic device 200
is configured as an optically-actuated electrokinetic device. A
variety of optically-actuated electrokinetic devices are known in
the art, including devices having an optoelectronic tweezer (OET)
configuration and devices having an opto-electrowetting (OEW)
configuration. Examples of suitable OET configurations are
illustrated in the following U.S. patent documents, each of which
is incorporated herein by reference in its entirety: U.S. Patent
No. RE 44,711 (Wu et al.) (originally issued as U.S. Pat. No.
7,612,355); and U.S. Pat. No. 7,956,339 (Ohta et al.). Examples of
OEW configurations are illustrated in U.S. Pat. No. 6,958,132
(Chiou et al.) and U.S. Patent Application Publication No.
2012/0024708 (Chiou et al.), both of which are incorporated by
reference herein in their entirety. Yet another example of an
optically-actuated electrokinetic device includes a combined
OET/OEW configuration, examples of which are shown in U.S. Patent
Publication Nos. 20150306598 (Khandros et al.) and 20150306599
(Khandros et al.) and their corresponding PCT Publications
WO2015/164846 and WO2015/164847, all of which are incorporated
herein by reference in their entirety.
[0109] Motive microfluidic device configurations. As described
above, the control and monitoring equipment of the system can
comprise a motive module for selecting and moving objects, such as
micro-objects or droplets, in the microfluidic circuit of a
microfluidic device. The microfluidic device can have a variety of
motive configurations, depending upon the type of object being
moved and other considerations. For example, a dielectrophoresis
(DEP) configuration can be utilized to select and move
micro-objects in the microfluidic circuit. Thus, the support
structure 104 and/or cover 110 of the microfluidic device 100 can
comprise a DEP configuration for selectively inducing DEP forces on
micro-objects in a fluidic medium 180 in the microfluidic circuit
120 and thereby select, capture, and/or move individual
micro-objects or groups of micro-objects. Alternatively, the
support structure 104 and/or cover 110 of the microfluidic device
100 can comprise an electrowetting (EW) configuration for
selectively inducing EW forces on droplets in a fluidic medium 180
in the microfluidic circuit 120 and thereby select, capture, and/or
move individual droplets or groups of droplets.
[0110] One example of a microfluidic device 200 comprising a DEP
configuration is illustrated in FIGS. 2A and 2B. While for purposes
of simplicity FIGS. 2A and 2B show a side cross-sectional view and
a top cross-sectional view, respectively, of a portion of an
enclosure 102 of the microfluidic device 200 having an open
region/chamber 202, it should be understood that the region/chamber
202 may be part of a fluidic circuit element having a more detailed
structure, such as a growth chamber, an incubation chamber, a flow
region, or a flow channel. Furthermore, the microfluidic device 200
may include other fluidic circuit elements. For example, the
microfluidic device 200 can include a plurality of growth chambers
or incubation chambers and/or one or more flow regions or flow
channels, such as those described herein with respect to
microfluidic device 100. A DEP configuration may be incorporated
into any such fluidic circuit elements of the microfluidic device
200, or select portions thereof. It should be further appreciated
that any of the above or below described microfluidic device
components and system components may be incorporated in and/or used
in combination with the microfluidic device 200. For example,
system 150 including control and monitoring equipment 152,
described above, may be used with microfluidic device 200,
including one or more of the media module 160, motive module 162,
imaging module 164, tilting module 166, and other modules 168.
[0111] As seen in FIG. 2A, the microfluidic device 200 includes a
support structure 104 having a bottom electrode 204 and an
electrode activation substrate 206 overlying the bottom electrode
204, and a cover 110 having a top electrode 210, with the top
electrode 210 spaced apart from the bottom electrode 204. The top
electrode 210 and the electrode activation substrate 206 define
opposing surfaces of the region/chamber 202. A medium 180 contained
in the region/chamber 202 thus provides a resistive connection
between the top electrode 210 and the electrode activation
substrate 206. A power source 212 configured to be connected to the
bottom electrode 204 and the top electrode 210 and create a biasing
voltage between the electrodes, as required for the generation of
DEP forces in the region/chamber 202, is also shown. The power
source 212 can be, for example, an alternating current (AC) power
source.
[0112] In certain embodiments, the microfluidic device 200
illustrated in FIGS. 2A and 2B can have an optically-actuated DEP
configuration. Accordingly, changing patterns of light 222 from the
light source 220, which may be controlled by the motive module 162,
can selectively activate and deactivate changing patterns of DEP
electrodes at regions 214 of the inner surface 208 of the electrode
activation substrate 206. (Hereinafter the regions 214 of a
microfluidic device having a DEP configuration are referred to as
"DEP electrode regions.") As illustrated in FIG. 2B, a light
pattern 222 directed onto the inner surface 208 of the electrode
activation substrate 206 can illuminate select DEP electrode
regions 214a (shown in white) in a pattern, such as a square. The
non-illuminated DEP electrode regions 214 (cross-hatched) are
hereinafter referred to as "dark" DEP electrode regions 214. The
relative electrical impedance through the DEP electrode activation
substrate 206 (i.e., from the bottom electrode 204 up to the inner
surface 208 of the electrode activation substrate 206 which
interfaces with the medium 180 in the flow region 106) is greater
than the relative electrical impedance through the medium 180 in
the region/chamber 202 (i.e., from the inner surface 208 of the
electrode activation substrate 206 to the top electrode 210 of the
cover 110) at each dark DEP electrode region 214. An illuminated
DEP electrode region 214a, however, exhibits a reduced relative
impedance through the electrode activation substrate 206 that is
less than the relative impedance through the medium 180 in the
region/chamber 202 at each illuminated DEP electrode region
214a.
[0113] With the power source 212 activated, the foregoing DEP
configuration creates an electric field gradient in the fluidic
medium 180 between illuminated DEP electrode regions 214a and
adjacent dark DEP electrode regions 214, which in turn creates
local DEP forces that attract or repel nearby micro-objects (not
shown) in the fluidic medium 180. DEP electrodes that attract or
repel micro-objects in the fluidic medium 180 can thus be
selectively activated and deactivated at many different such DEP
electrode regions 214 at the inner surface 208 of the
region/chamber 202 by changing light patterns 222 projected from a
light source 220 into the microfluidic device 200. Whether the DEP
forces attract or repel nearby micro-objects can depend on such
parameters as the frequency of the power source 212 and the
dielectric properties of the medium 180 and/or micro-objects (not
shown).
[0114] The square pattern 224 of illuminated DEP electrode regions
214a illustrated in FIG. 2B is an example only. Any pattern of the
DEP electrode regions 214 can be illuminated (and thereby
activated) by the pattern of light 222 projected into the device
200, and the pattern of illuminated/activated DEP electrode regions
214 can be repeatedly changed by changing or moving the light
pattern 222.
[0115] In some embodiments, the electrode activation substrate 206
can comprise or consist of a photoconductive material. In such
embodiments, the inner surface 208 of the electrode activation
substrate 206 can be featureless. For example, the electrode
activation substrate 206 can comprise or consist of a layer of
hydrogenated amorphous silicon (a-Si:H). The a-Si:H can comprise,
for example, about 8% to 40% hydrogen (calculated as 100 * the
number of hydrogen atoms/the total number of hydrogen and silicon
atoms). The layer of a-Si:H can have a thickness of about 500 nm to
about 2.0 microns. In such embodiments, the DEP electrode regions
214 can be created anywhere and in any pattern on the inner surface
208 of the electrode activation substrate 208, in accordance with
the light pattern 222. The number and pattern of the DEP electrode
regions 214 thus need not be fixed, but can correspond to the light
pattern 222. Examples of microfluidic devices having a DEP
configuration comprising a photoconductive layer such as discussed
above have been described, for example, in U.S. Patent No. RE
44,711 (Wu et al.) (originally issued as U.S. Pat. No. 7,612,355),
the entire contents of which are incorporated herein by
reference.
[0116] In other embodiments, the electrode activation substrate 206
can comprise a substrate comprising a plurality of doped layers,
electrically insulating layers (or regions), and electrically
conductive layers that form semiconductor integrated circuits, such
as is known in semiconductor fields. For example, the electrode
activation substrate 206 can comprise a plurality of
phototransistors, including, for example, lateral bipolar
phototransistors, each phototransistor corresponding to a DEP
electrode region 214. Alternatively, the electrode activation
substrate 206 can comprise electrodes (e.g., conductive metal
electrodes) controlled by phototransistor switches, with each such
electrode corresponding to a DEP electrode region 214. The
electrode activation substrate 206 can include a pattern of such
phototransistors or phototransistor-controlled electrodes. The
pattern, for example, can be an array of substantially square
phototransistors or phototransistor-controlled electrodes arranged
in rows and columns, such as shown in FIG. 2B. Alternatively, the
pattern can be an array of substantially hexagonal phototransistors
or phototransistor-controlled electrodes that form a hexagonal
lattice. Regardless of the pattern, electric circuit elements can
form electrical connections between the DEP electrode regions 214
at the inner surface 208 of the electrode activation substrate 206
and the bottom electrode 210, and those electrical connections
(i.e., phototransistors or electrodes) can be selectively activated
and deactivated by the light pattern 222. When not activated, each
electrical connection can have high impedance such that the
relative impedance through the electrode activation substrate 206
(i.e., from the bottom electrode 204 to the inner surface 208 of
the electrode activation substrate 206 which interfaces with the
medium 180 in the region/chamber 202) is greater than the relative
impedance through the medium 180 (i.e., from the inner surface 208
of the electrode activation substrate 206 to the top electrode 210
of the cover 110) at the corresponding DEP electrode region 214.
When activated by light in the light pattern 222, however, the
relative impedance through the electrode activation substrate 206
is less than the relative impedance through the medium 180 at each
illuminated DEP electrode region 214, thereby activating the DEP
electrode at the corresponding DEP electrode region 214 as
discussed above. DEP electrodes that attract or repel micro-objects
(not shown) in the medium 180 can thus be selectively activated and
deactivated at many different DEP electrode regions 214 at the
inner surface 208 of the electrode activation substrate 206 in the
region/chamber 202 in a manner determined by the light pattern
222.
[0117] Examples of microfluidic devices having electrode activation
substrates that comprise phototransistors have been described, for
example, in U.S. Pat. No. 7,956,339 (Ohta et al.) (see, e.g.,
device 300 illustrated in FIGS. 21 and 22, and descriptions
thereof), the entire contents of which are incorporated herein by
reference. Examples of microfluidic devices having electrode
activation substrates that comprise electrodes controlled by
phototransistor switches have been described, for example, in U.S.
Patent Publication No. 2014/0124370 (Short et al.) (see, e.g.,
devices 200, 400, 500, 600, and 900 illustrated throughout the
drawings, and descriptions thereof), the entire contents of which
are incorporated herein by reference.
[0118] In some embodiments of a DEP configured microfluidic device,
the top electrode 210 is part of a first wall (or cover 110) of the
enclosure 102, and the electrode activation substrate 206 and
bottom electrode 204 are part of a second wall (or support
structure 104) of the enclosure 102. The region/chamber 202 can be
between the first wall and the second wall. In other embodiments,
the electrode 210 is part of the second wall (or support structure
104) and one or both of the electrode activation substrate 206
and/or the electrode 210 are part of the first wall (or cover 110).
Moreover, the light source 220 can alternatively be used to
illuminate the enclosure 102 from below.
[0119] With the microfluidic device 200 of FIGS. 2A-2B having a DEP
configuration, the motive module 162 can select a micro-object (not
shown) in the medium 180 in the region/chamber 202 by projecting a
light pattern 222 into the device 200 to activate a first set of
one or more DEP electrodes at DEP electrode regions 214a of the
inner surface 208 of the electrode activation substrate 206 in a
pattern (e.g., square pattern 224) that surrounds and captures the
micro-object. The motive module 162 can then move the captured
micro-object by moving the light pattern 222 relative to the device
200 to activate a second set of one or more DEP electrodes at DEP
electrode regions 214. Alternatively, the device 200 can be moved
relative to the light pattern 222.
[0120] In other embodiments, the microfluidic device 200 can have a
DEP configuration that does not rely upon light activation of DEP
electrodes at the inner surface 208 of the electrode activation
substrate 206. For example, the electrode activation substrate 206
can comprise selectively addressable and energizable electrodes
positioned opposite to a surface including at least one electrode
(e.g., cover 110). Switches (e.g., transistor switches in a
semiconductor substrate) may be selectively opened and closed to
activate or inactivate DEP electrodes at DEP electrode regions 214,
thereby creating a net DEP force on a micro-object (not shown) in
region/chamber 202 in the vicinity of the activated DEP electrodes.
Depending on such characteristics as the frequency of the power
source 212 and the dielectric properties of the medium (not shown)
and/or micro-objects in the region/chamber 202, the DEP force can
attract or repel a nearby micro-object. By selectively activating
and deactivating a set of DEP electrodes (e.g., at a set of DEP
electrodes regions 214 that forms a square pattern 224), one or
more micro-objects in region/chamber 202 can be trapped and moved
within the region/chamber 202. The motive module 162 in FIG. 1 can
control such switches and thus activate and deactivate individual
ones of the DEP electrodes to select, trap, and move particular
micro-objects (not shown) around the region/chamber 202.
Microfluidic devices having a DEP configuration that includes
selectively addressable and energizable electrodes are known in the
art and have been described, for example, in U.S. Pat. No.
6,294,063 (Becker et al.) and Pat. No. 6,942,776 (Medoro), the
entire contents of which are incorporated herein by reference.
[0121] As yet another example, the microfluidic device 200 can have
an electrowetting (EW) configuration, which can be in place of the
DEP configuration or can be located in a portion of the
microfluidic device 200 that is separate from the portion which has
the DEP configuration. The EW configuration can be an
opto-electrowetting configuration or an electrowetting on
dielectric (EWOD) configuration, both of which are known in the
art. In some EW configurations, the support structure 104 has an
electrode activation substrate 206 sandwiched between a dielectric
layer (not shown) and the bottom electrode 204. The dielectric
layer can comprise a hydrophobic material and/or can be coated with
a hydrophobic material. For microfluidic devices 200 that have an
EW configuration, the inner surface 208 of the support structure
104 is the inner surface of the dielectric layer or its hydrophobic
coating.
[0122] The dielectric layer (not shown) can comprise one or more
oxide layers, and can have a thickness of about 50 nm to about 250
nm (e.g., about 125 nm to about 175 nm). In certain embodiments,
the dielectric layer may comprise a layer of oxide, such as a metal
oxide (e.g., aluminum oxide or hafnium oxide). In certain
embodiments, the dielectric layer can comprise a dielectric
material other than a metal oxide, such as silicon oxide or a
nitride. Regardless of the exact composition and thickness, the
dielectric layer can have an impedance of about 10 kOhms to about
50 kOhms.
[0123] In some embodiments, the surface of the dielectric layer
that faces inward toward region/chamber 202 is coated with a
hydrophobic material. The hydrophobic material can comprise, for
example, fluorinated carbon molecules. Examples of fluorinated
carbon molecules include perfluoro-polymers such as
polytetrafluoroethylene (e.g., TEFLON.RTM.) or
poly(2,3-difluoromethylenyl-perfluorotetrahydrofuran) (e.g.,
CYTOP.TM.). Molecules that make up the hydrophobic material can be
covalently bonded to the surface of the dielectric layer. For
example, molecules of the hydrophobic material can be covalently
bound to the surface of the dielectric layer by means of a linker
such as a siloxane group, a phosphonic acid group, or a thiol
group. Thus, in some embodiments, the hydrophobic material can
comprise alkyl-terminated siloxane, alkyl-termination phosphonic
acid, or alkyl-terminated thiol. The alkyl group can be long-chain
hydrocarbons (e.g., having a chain of at least 10 carbons, or at
least 16, 18, 20, 22, or more carbons). Alternatively, fluorinated
(or perfluorinated) carbon chains can be used in place of the alkyl
groups. Thus, for example, the hydrophobic material can comprise
fluoroalkyl-terminated siloxane, fluoroalkyl-terminated phosphonic
acid, or fluoroalkyl-terminated thiol. In some embodiments, the
hydrophobic coating has a thickness of about 10 nm to about 50 nm.
In other embodiments, the hydrophobic coating has a thickness of
less than 10 nm (e.g., less than 5 nm, or about 1.5 nm to 3.0
nm).
[0124] In some embodiments, the cover 110 of a microfluidic device
200 having an electrowetting configuration is coated with a
hydrophobic material (not shown) as well. The hydrophobic material
can be the same hydrophobic material used to coat the dielectric
layer of the support structure 104, and the hydrophobic coating can
have a thickness that is substantially the same as the thickness of
the hydrophobic coating on the dielectric layer of the support
structure 104. Moreover, the cover 110 can comprise an electrode
activation substrate 206 sandwiched between a dielectric layer and
the top electrode 210, in the manner of the support structure 104.
The electrode activation substrate 206 and the dielectric layer of
the cover 110 can have the same composition and/or dimensions as
the electrode activation substrate 206 and the dielectric layer of
the support structure 104. Thus, the microfluidic device 200 can
have two electrowetting surfaces.
[0125] In some embodiments, the electrode activation substrate 206
can comprise a photoconductive material, such as described above.
Accordingly, in certain embodiments, the electrode activation
substrate 206 can comprise or consist of a layer of hydrogenated
amorphous silicon (a-Si:H). The a-Si:H can comprise, for example,
about 8% to 40% hydrogen (calculated as 100 * the number of
hydrogen atoms/the total number of hydrogen and silicon atoms). The
layer of a-Si:H can have a thickness of about 500 nm to about 2.0
microns. Alternatively, the electrode activation substrate 206 can
comprise electrodes (e.g., conductive metal electrodes) controlled
by phototransistor switches, as described above. Microfluidic
devices having an opto-electrowetting configuration are known in
the art and/or can be constructed with electrode activation
substrates known in the art. For example, U.S. Pat. No. 6,958,132
(Chiou et al.), the entire contents of which are incorporated
herein by reference, discloses opto-electrowetting configurations
having a photoconductive material such as a-Si:H, while U.S. Patent
Publication No. 2014/0124370 (Short et al.), referenced above,
discloses electrode activation substrates having electrodes
controlled by phototransistor switches.
[0126] The microfluidic device 200 thus can have an
opto-electrowetting configuration, and light patterns 222 can be
used to activate photoconductive EW regions or photoresponsive EW
electrodes in the electrode activation substrate 206. Such
activated EW regions or EW electrodes of the electrode activation
substrate 206 can generate an electrowetting force at the inner
surface 208 of the support structure 104 (i.e., the inner surface
of the overlaying dielectric layer or its hydrophobic coating). By
changing the light patterns 222 (or moving microfluidic device 200
relative to the light source 220) incident on the electrode
activation substrate 206, droplets (e.g., containing an aqueous
medium, solution, or solvent) contacting the inner surface 208 of
the support structure 104 can be moved through an immiscible fluid
(e.g., an oil medium) present in the region/chamber 202.
[0127] In other embodiments, microfluidic devices 200 can have an
EWOD configuration, and the electrode activation substrate 206 can
comprise selectively addressable and energizable electrodes that do
not rely upon light for activation. The electrode activation
substrate 206 thus can include a pattern of such electrowetting
(EW) electrodes. The pattern, for example, can be an array of
substantially square EW electrodes arranged in rows and columns,
such as shown in FIG. 2B. Alternatively, the pattern can be an
array of substantially hexagonal EW electrodes that form a
hexagonal lattice. Regardless of the pattern, the EW electrodes can
be selectively activated (or deactivated) by electrical switches
(e.g., transistor switches in a semiconductor substrate). By
selectively activating and deactivating EW electrodes in the
electrode activation substrate 206, droplets (not shown) contacting
the inner surface 208 of the overlaying dielectric layer or its
hydrophobic coating can be moved within the region/chamber 202. The
motive module 162 in FIG. 1 can control such switches and thus
activate and deactivate individual EW electrodes to select and move
particular droplets around region/chamber 202. Microfluidic devices
having a EWOD configuration with selectively addressable and
energizable electrodes are known in the art and have been
described, for example, in U.S. Pat. No. 8,685,344 (Sundarsan et
al.), the entire contents of which are incorporated herein by
reference.
[0128] Regardless of the configuration of the microfluidic device
200, a power source 212 can be used to provide a potential (e.g.,
an AC voltage potential) that powers the electrical circuits of the
microfluidic device 200. The power source 212 can be the same as,
or a component of, the power source 192 referenced in FIG. 1. Power
source 212 can be configured to provide an AC voltage and/or
current to the top electrode 210 and the bottom electrode 204. For
an AC voltage, the power source 212 can provide a frequency range
and an average or peak power (e.g., voltage or current) range
sufficient to generate net DEP forces (or electrowetting forces)
strong enough to trap and move individual micro-objects (not shown)
in the region/chamber 202, as discussed above, and/or to change the
wetting properties of the inner surface 208 of the support
structure 104 (i.e., the dielectric layer and/or the hydrophobic
coating on the dielectric layer) in the region/chamber 202, as also
discussed above. Such frequency ranges and average or peak power
ranges are known in the art. See, e.g., U.S. Pat. No. 6,958,132
(Chiou et al.), US Patent No. RE44,711 (Wu et al.) (originally
issued as U.S. Pat. No. 7,612,355), and US Patent Application
Publication Nos. US2014/0124370 (Short et al.), US2015/0306598
(Khandros et al.), and US2015/0306599 (Khandros et al.).
[0129] Incubation chambers. Non-limiting examples of generic
incubation chambers 244, 246, and 248 are shown within the
microfluidic device 240 depicted in FIGS. 2C and 2D. Each
incubation chamber 244, 246, and 248 can comprise an isolation
structure 250 defining an isolation region 258 and a connection
region 254 fluidically connecting the isolation region 258 to a
channel 122. The connection region 254 can comprise a proximal
opening 252 to the channel 122 and a distal opening 256 to the
isolation region 258. The connection region 254 can be configured
so that the maximum penetration depth of a flow of a fluidic medium
(not shown) flowing from the channel 122 into the incubation
chamber 244, 246, 248 does not extend into the isolation region
258. Thus, due to the connection region 254, a micro-object (not
shown) or other material (not shown) disposed in an isolation
region 258 of an incubation chamber 244, 246, 248 can thus be
isolated from, and not substantially affected by, a flow of medium
180 in the channel 122.
[0130] The channel 122 can thus be an example of a swept region,
and the isolation regions 258 of the incubation chambers 244, 246,
248 can be examples of unswept regions. As noted, the channel 122
and incubation chambers 244, 246, 248 can be configured to contain
one or more fluidic media 180. In the example shown in FIGS. 2C-2D,
the ports 242 are connected to the channel 122 and allow a fluidic
medium 180 to be introduced into or removed from the microfluidic
device 240. Prior to introduction of the fluidic medium 180, the
microfluidic device may be primed with a gas such as carbon dioxide
gas. Once the microfluidic device 240 contains the fluidic medium
180, the flow 260 of fluidic medium 180 in the channel 122 can be
selectively generated and stopped. For example, as shown, the ports
242 can be disposed at different locations (e.g., opposite ends) of
the channel 122, and a flow 260 of medium can be created from one
port 242 functioning as an inlet to another port 242 functioning as
an outlet.
[0131] FIG. 2E illustrates a detailed view of an example of an
incubation chamber 244 according to the present invention. Examples
of micro-objects 270 are also shown.
[0132] As is known, a flow 260 of fluidic medium 180 in a
microfluidic channel 122 past a proximal opening 252 of incubation
chamber 244 can cause a secondary flow 262 of the medium 180 into
and/or out of the incubation chamber 244. To isolate micro-objects
270 in the isolation region 258 of an incubation chamber 244 from
the secondary flow 262, the length L.sub.con of the connection
region 254 of the incubation chamber 244 (i.e., from the proximal
opening 252 to the distal opening 256) should be greater than the
penetration depth D.sub.p of the secondary flow 262 into the
connection region 254. The penetration depth D.sub.p of the
secondary flow 262 depends upon the velocity of the fluidic medium
180 flowing in the channel 122 and various parameters relating to
the configuration of the channel 122 and the proximal opening 252
of the connection region 254 to the channel 122. For a given
microfluidic device, the configurations of the channel 122 and the
opening 252 will be fixed, whereas the rate of flow 260 of fluidic
medium 180 in the channel 122 will be variable. Accordingly, for
each incubation chamber 244, a maximal velocity Vmax for the flow
260 of fluidic medium 180 in channel 122 can be identified that
ensures that the penetration depth D.sub.p of the secondary flow
262 does not exceed the length L.sub.con of the connection region
254. As long as the rate of the flow 260 of fluidic medium 180 in
the channel 122 does not exceed the maximum velocity Vmax, the
resulting secondary flow 262 can be limited to the channel 122 and
the connection region 254 and kept out of the isolation region 258.
The flow 260 of medium 180 in the channel 122 will thus not draw
micro-objects 270 out of the isolation region 258. Rather,
micro-objects 270 located in the isolation region 258 will stay in
the isolation region 258 regardless of the flow 260 of fluidic
medium 180 in the channel 122.
[0133] Moreover, as long as the rate of flow 260 of medium 180 in
the channel 122 does not exceed Vmax, the flow 260 of fluidic
medium 180 in the channel 122 will not move miscellaneous particles
(e.g., microparticles and/or nanoparticles) from the channel 122
into the isolation region 258 of an incubation chamber 244. Having
the length L.sub.con of the connection region 254 be greater than
the maximum penetration depth D.sub.p of the secondary flow 262 can
thus prevent contamination of one incubation chamber 244 with
miscellaneous particles from the channel 122 or another incubation
chamber (e.g., incubation chambers 246, 248 in FIG. 2D).
[0134] Because the channel 122 and the connection regions 254 of
the incubation chambers 244, 246, 248 can be affected by the flow
260 of medium 180 in the channel 122, the channel 122 and
connection regions 254 can be deemed swept (or flow) regions of the
microfluidic device 240. The isolation regions 258 of the
incubation chambers 244, 246, 248, on the other hand, can be deemed
unswept (or non-flow) regions. For example, components (not shown)
in a first fluidic medium 180 in the channel 122 can mix with a
second fluidic medium 280 in the isolation region 258 substantially
only by diffusion of components of the first medium 180 from the
channel 122 through the connection region 254 and into the second
fluidic medium 280 in the isolation region 258. Similarly,
components (not shown) of the second medium 280 in the isolation
region 258 can mix with the first medium 180 in the channel 122
substantially only by diffusion of components of the second medium
280 from the isolation region 258 through the connection region 254
and into the first medium 180 in the channel 122. The first medium
180 can be the same medium or a different medium than the second
medium 280. Moreover, the first medium 180 and the second medium
280 can start out being the same, then become different (e.g.,
through conditioning of the second medium 280 by one or more cells
in the isolation region 258, or by changing the medium 180 flowing
through the channel 122).
[0135] The maximum penetration depth D.sub.p of the secondary flow
262 caused by the flow 260 of fluidic medium 180 in the channel 122
can depend on a number of parameters, as mentioned above. Examples
of such parameters include: the shape of the channel 122 (e.g., the
channel can direct medium into the connection region 254, divert
medium away from the connection region 254, or direct medium in a
direction substantially perpendicular to the proximal opening 252
of the connection region 254 to the channel 122); a width W.sub.ch
(or cross-sectional area) of the channel 122 at the proximal
opening 252; and a width W.sub.con (or cross-sectional area) of the
connection region 254 at the proximal opening 252; the velocity V
of the flow 260 of fluidic medium 180 in the channel 122; the
viscosity of the first medium 180 and/or the second medium 280, or
the like.
[0136] In some embodiments, the dimensions of the channel 122 and
incubation chambers 244, 246, 248 can be oriented as follows with
respect to the vector of the flow 260 of fluidic medium 180 in the
channel 122: the channel width W.sub.ch (or cross-sectional area of
the channel 122) can be substantially perpendicular to the flow 260
of medium 180; the width W.sub.con (or cross-sectional area) of the
connection region 254 at opening 252 can be substantially parallel
to the flow 260 of medium 180 in the channel 122; and/or the length
L.sub.con of the connection region can be substantially
perpendicular to the flow 260 of medium 180 in the channel 122. The
foregoing are examples only, and the relative position of the
channel 122 and incubation chambers 244, 246, 248 can be in other
orientations with respect to each other.
[0137] As illustrated in FIG. 2E, the width W.sub.con of the
connection region 254 can be uniform from the proximal opening 252
to the distal opening 256. The width W.sub.con of the connection
region 254 at the distal opening 256 can thus be in any of the
ranges identified herein for the width W.sub.con of the connection
region 254 at the proximal opening 252. Alternatively, the width
W.sub.con of the connection region 254 at the distal opening 256
can be larger than the width W.sub.con of the connection region 254
at the proximal opening 252.
[0138] As illustrated in FIG. 2E, the width of the isolation region
258 at the distal opening 256 can be substantially the same as the
width W.sub.con of the connection region 254 at the proximal
opening 252. The width of the isolation region 258 at the distal
opening 256 can thus be in any of the ranges identified herein for
the width W.sub.con of the connection region 254 at the proximal
opening 252. Alternatively, the width of the isolation region 258
at the distal opening 256 can be larger or smaller than the width
W.sub.con of the connection region 254 at the proximal opening 252.
Moreover, the distal opening 256 may be smaller than the proximal
opening 252 and the width W.sub.con of the connection region 254
may be narrowed between the proximal opening 252 and distal opening
256. For example, the connection region 254 may be narrowed between
the proximal opening and the distal opening, using a variety of
different geometries (e.g. chamfering the connection region,
beveling the connection region). Further, any part or subpart of
the connection region 254 may be narrowed (e.g. a portion of the
connection region adjacent to the proximal opening 252).
[0139] FIGS. 4A-C depict another exemplary embodiment of a
microfluidic device 400 containing a microfluidic circuit 432 and
flow channels 434, which are variations of the respective
microfluidic device 100, circuit 132 and channel 134 of FIG. 1. The
microfluidic device 400 also has a plurality of incubation chambers
436 that are additional variations of the above-described
incubation chambers 124, 126, 128, 130, 244, 246 or 248. In
particular, it should be appreciated that the incubation chambers
436 of device 400 shown in FIGS. 4A-C can replace any of the
above-described incubation chambers 124, 126, 128, 130, 244, 246 or
248 in devices 100, 200, 240 and 290. Likewise, the microfluidic
device 400 is another variant of the microfluidic device 100, and
may also have the same or a different DEP configuration as the
above-described microfluidic device 100, 200, 240, 290, as well as
any of the other microfluidic system components described
herein.
[0140] The microfluidic device 400 of FIGS. 4A-C comprises a
support structure (not visible in FIGS. 4A-C, but can be the same
or generally similar to the support structure 104 of device 100
depicted in FIG. 1), a microfluidic circuit structure 412, and a
cover (not visible in FIGS. 4A-C, but can be the same or generally
similar to the cover 122 of device 100 depicted in FIG. 1). The
microfluidic circuit structure 412 includes a frame 414 and
microfluidic circuit material 416, which can be the same as or
generally similar to the frame 114 and microfluidic circuit
material 116 of device 100 shown in FIG. 1. As shown in FIG. 4A,
the microfluidic circuit 432 defined by the microfluidic circuit
material 416 can comprise multiple channels 434 (two are shown but
there can be more) to which multiple incubation chambers 436 are
fluidically connected.
[0141] Each incubation chamber 436 can comprise an isolation
structure 446, an isolation region 444 within the isolation
structure 446, and a connection region 442. From a proximal opening
472 at the channel 434 to a distal opening 474 at the isolation
structure 436, the connection region 442 fluidically connects the
channel 434 to the isolation region 444. Generally in accordance
with the above discussion of FIGS. 2D and 2E, a flow 482 of a first
fluidic medium 402 in a channel 434 can create secondary flows 484
of the first medium 402 from the channel 434 into and/or out of the
respective connection regions 442 of the incubation chambers
436.
[0142] As illustrated in FIG. 4B, the connection region 442 of each
incubation chamber 436 generally includes the area extending
between the proximal opening 472 to a channel 434 and the distal
opening 474 to an isolation structure 446. The length L.sub.con of
the connection region 442 can be greater than the maximum
penetration depth D.sub.p of secondary flow 484, in which case the
secondary flow 484 will extend into the connection region 442
without being redirected toward the isolation region 444 (as shown
in FIG. 4A). Alternatively, at illustrated in FIG. 4C, the
connection region 442 can have a length L.sub.con that is less than
the maximum penetration depth D.sub.p, in which case the secondary
flow 484 will extend through the connection region 442 and be
redirected toward the isolation region 444. In this latter
situation, the sum of lengths L.sub.c1 and L.sub.c2 of connection
region 442 is greater than the maximum penetration depth D.sub.p,
so that secondary flow 484 will not extend into isolation region
444. Whether length L.sub.con of connection region 442 is greater
than the penetration depth D.sub.p, or the sum of lengths L.sub.c1
and L.sub.c2 of connection region 442 is greater than the
penetration depth D.sub.p, a flow 482 of a first medium 402 in
channel 434 that does not exceed a maximum velocity V.sub.max will
produce a secondary flow having a penetration depth D.sub.p, and
micro-objects (not shown but can be the same or generally similar
to the micro-objects 270 shown in FIG. 2E) in the isolation region
444 of a incubation chamber 436 will not be drawn out of the
isolation region 444 by a flow 482 of first medium 402 in channel
434. Nor will the flow 482 in channel 434 draw miscellaneous
materials (not shown) from channel 434 into the isolation region
444 of an incubation chamber 436. As such, diffusion is the only
mechanism by which components in a first medium 402 in the channel
434 can move from the channel 434 into a second medium 404 in an
isolation region 444 of an incubation chamber 436. Likewise,
diffusion is the only mechanism by which components in a second
medium 404 in an isolation region 444 of an incubation chamber 436
can move from the isolation region 444 to a first medium 402 in the
channel 434. The first medium 402 can be the same medium as the
second medium 404, or the first medium 402 can be a different
medium than the second medium 404. Alternatively, the first medium
402 and the second medium 404 can start out being the same, then
become different, e.g., through conditioning of the second medium
by one or more cells in the isolation region 444, or by changing
the medium flowing through the channel 434.
[0143] As illustrated in FIG. 4B, the width W.sub.ch of the
channels 434 (i.e., taken transverse to the direction of a fluid
medium flow through the channel indicated by arrows 482 in FIG. 4A)
in the channel 434 can be substantially perpendicular to a width
W.sub.con1 of the proximal opening 472 and thus substantially
parallel to a width W.sub.con2 of the distal opening 474. The width
W.sub.con1 of the proximal opening 472 and the width W.sub.con2 of
the distal opening 474, however, need not be substantially
perpendicular to each other. For example, an angle between an axis
(not shown) on which the width W.sub.con1 of the proximal opening
472 is oriented and another axis on which the width W.sub.con2 of
the distal opening 474 is oriented can be other than perpendicular
and thus other than 90.degree.. Examples of alternatively oriented
angles include angles in any of the following ranges: from about
30.degree. to about 90.degree., from about 45.degree. to about
90.degree., from about 60.degree. to about 90.degree., or the
like.
[0144] In various embodiments of incubation chambers (e.g. 124,
126, 128, 130, 244, 246 , 248, or 436), the isolation region (e.g.
258 or 444) is configured to contain a plurality of micro-objects.
In other embodiments, the isolation region can be configured to
contain only one, two, three, four, five, or a similar relatively
small number of micro-objects. Accordingly, the volume of an
isolation region can be, for example, at least 3.times.10.sup.3,
6.times.10.sup.3, 9.times.10.sup.3, 1.times.10.sup.4,
2.times.10.sup.4, 4.times.10.sup.4, 8.times.10.sup.4,
1.times.10.sup.5, 2.times.10.sup.5, 4.times.10.sup.5,
8.times.10.sup.5, 1.times.10.sup.6, 2.times.10.sup.6,
4.times.10.sup.6, 6.times.10.sup.6, 1.times.10.sup.7,
2.times.10.sup.7, 4.times.10.sup.7, 6.times.10.sup.7,
1.times.10.sup.8, cubic microns, or more.
[0145] In various embodiments of incubation chambers, the width
W.sub.ch of the channel 122, 434 at a proximal opening (e.g. 252,
472) can be within any of the following ranges: 50-1000 microns,
50-500 microns, 50-400 microns, 50-300 microns, 50-250 microns,
50-200 microns, 50-150 microns, 50-100 microns, 70-500 microns,
70-400 microns, 70-300 microns, 70-250 microns, 70-200 microns,
70-150 microns, 90-400 microns, 90-300 microns, 90-250 microns,
90-200 microns, 90-150 microns, 100-300 microns, 100-250 microns,
100-200 microns, 100-150 microns, and 100-120 microns. The
foregoing are examples only, and the width W.sub.ch of the channel
122, 434 can be in other ranges (e.g., a range defined by any of
the endpoints listed above). Moreover, the W.sub.ch of the channel
122, 434 can be selected to be in any of these ranges in regions of
the channel other than at a proximal opening of an incubation
chamber.
[0146] In some embodiments, an incubation chamber has a
cross-sectional height of about 30 to about 200 microns, or about
50 to about 150 microns. In some embodiments, the incubation
chamber has a cross-sectional area of about 100,000 to about
2,500,000 square microns, or about 200,000 to about 2,000,000
square microns. In some embodiments, a connection region has a
cross-sectional height that matches the cross-sectional height of
the corresponding incubation chamber. In some embodiments, the
connection region has a cross-sectional width of about 50 to about
500 microns, or about 100 to about 300 microns.
[0147] In various embodiments of incubation chambers the height
H.sub.ch of the channel 122, 434 at a proximal opening 252, 472 can
be within any of the following ranges: 20-100 microns, 20-90
microns, 20-80 microns, 20-70 microns, 20-60 microns, 20-50
microns, 30-100 microns, 30-90 microns, 30-80 microns, 30-70
microns, 30-60 microns, 30-50 microns, 40-100 microns, 40-90
microns, 40-80 microns, 40-70 microns, 40-60 microns, or 40-50
microns. The foregoing are examples only, and the height H.sub.ch
of the channel 122, 434 can be in other ranges (e.g., a range
defined by any of the endpoints listed above). The height H.sub.ch
of the channel 122, 434 can be selected to be in any of these
ranges in regions of the channel other than at a proximal opening
of an incubation chamber.
[0148] In various embodiments of incubation chambers a
cross-sectional area of the channel 122, 434 at a proximal opening
252, 472 can be within any of the following ranges: 500-50,000
square microns, 500-40,000 square microns, 500-30,000 square
microns, 500-25,000 square microns, 500-20,000 square microns,
500-15,000 square microns, 500-10,000 square microns, 500-7,500
square microns, 500-5,000 square microns, 1,000-25,000 square
microns, 1,000-20,000 square microns, 1,000-15,000 square microns,
1,000-10,000 square microns, 1,000-7,500 square microns,
1,000-5,000 square microns, 2,000-20,000 square microns,
2,000-15,000 square microns, 2,000-10,000 square microns,
2,000-7,500 square microns, 2,000-6,000 square microns,
3,000-20,000 square microns, 3,000-15,000 square microns,
3,000-10,000 square microns, 3,000-7,500 square microns, or 3,000
to 6,000 square microns. The foregoing are examples only, and the
cross-sectional area of the channel 122 at a proximal opening 252,
472 can be in other ranges (e.g., a range defined by any of the
endpoints listed above).
[0149] In various embodiments of incubation chambers, the length
L.sub.con of the connection region 254, 442 can be in any of the
following ranges: 1-200 microns, 5-150 microns, 10-100 microns,
15-80 microns, 20-60 microns, 20-500 microns, 40-400 microns,
60-300 microns, 80-200 microns, and 100-150 microns. The foregoing
are examples only, and length L.sub.con of a connection region 254,
442 can be in a different range than the foregoing examples (e.g.,
a range defined by any of the endpoints listed above).
[0150] In various embodiments of incubation chambers the width
W.sub.con of a connection region 254, 442 at a proximal opening 252
can be in any of the following ranges: 20-500 microns, 20-400
microns, 20-300 microns, 20-200 microns, 20-150 microns, 20-100
microns, 20-80 microns, 20-60 microns, 30-400 microns, 30-300
microns, 30-200 microns, 30-150 microns, 30-100 microns, 30-80
microns, 30-60 microns, 40-300 microns, 40-200 microns, 40-150
microns, 40-100 microns, 40-80 microns, 40-60 microns, 50-250
microns, 50-200 microns, 50-150 microns, 50-100 microns, 50-80
microns, 60-200 microns, 60-150 microns, 60-100 microns, 60-80
microns, 70-150 microns, 70-100 microns, and 80-100 microns. The
foregoing are examples only, and the width W.sub.con of a
connection region 254, 442 at a proximal opening 252 can be
different than the foregoing examples (e.g., a range defined by any
of the endpoints listed above).
[0151] In various embodiments of incubation chambers the width
W.sub.con of a connection region 254, 442 at a proximal opening
252, 472 can be in any of the following ranges: 2-35 microns, 2-25
microns, 2-20 microns, 2-15 microns, 2-10 microns, 2-7 microns, 2-5
microns, 2-3 microns, 3-25 microns, 3-20 microns, 3-15 microns,
3-10 microns, 3-7 microns, 3-5 microns, 3-4 microns, 4-20 microns,
4-15 microns, 4-10 microns, 4-7 microns, 4-5 microns, 5-15 microns,
5-10 microns, 5-7 microns, 6-15 microns, 6-10 microns, 6-7 microns,
7-15 microns, 7-10 microns, 8-15 microns, and 8-10 microns. The
foregoing are examples only, and the width W.sub.con of a
connection region 254, 442 at a proximal opening 252, 472 can be
different than the foregoing examples (e.g., a range defined by any
of the endpoints listed above).
[0152] In various embodiments of incubation chambers, a ratio of
the length L.sub.con of a connection region 254, 442 to a width
W.sub.con of the connection region 254, 442 at the proximal opening
252, 472 can be greater than or equal to any of the following
ratios: 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0,
8.0, 9.0, 10.0, or more. The foregoing are examples only, and the
ratio of the length L.sub.con of a connection region 254 to a width
W.sub.con of the connection region 254 442 at the proximal opening
252 472 can be different than the foregoing examples.
[0153] In various embodiments of microfluidic devices 100, 200,
240, 290, 400, V.sub.max can be set around 0.2, 0.3, 0.4, 0.5, 0.6,
0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 microliters/sec. In
some other embodiments, V.sub.max can be set at about 0.2, 0.3,
0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,
1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 microliters/sec. In
yet other embodiments, V.sub.max can be set at or about 2.0, 2.2,
2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8,
5.0, 6.0, 7.0, 8.0 or about 9.0 microliters/sec.
[0154] In various embodiments of microfluidic devices having
incubation chambers, the volume of an isolation region 258, 444 of
an incubation chamber can be, for example, at least
3.times.10.sup.3, 6.times.10.sup.3, 9.times.10.sup.3,
1.times.10.sup.4, 2.times.10.sup.4, 4.times.10.sup.4,
8.times.10.sup.4, 1.times.10.sup.5, 2.times.10.sup.5,
4.times.10.sup.5, 8.times.10.sup.51.times.10.sup.6,
2.times.10.sup.6, 4.times.10.sup.6, 6.times.10.sup.6 cubic microns,
or more. In various embodiments of microfluidic devices having
incubation chambers, the volume of an incubation chamber may be
about 5.times.10.sup.3, 7.times.10.sup.3, 1.times.10.sup.4,
3.times.10.sup.4, 5.times.10.sup.4, 8.times.10.sup.4,
1.times.10.sup.5, 2.times.10.sup.5, 4.times.10.sup.5,
6.times.10.sup.5, 8.times.10.sup.5, 1.times.10.sup.6,
2.times.10.sup.6, 4.times.10.sup.6, 8.times.10.sup.6,
1.times.10.sup.7, 3.times.10.sup.7, 5.times.10.sup.7, or about
8.times.10.sup.7 cubic microns, or more. In some embodiments, the
microfluidic device has incubation chambers wherein no more than
1.times.10.sup.2 biological cells may be maintained, and the volume
of an incubation chamber may be no more than 2.times.10.sup.6 cubic
microns. In some embodiments, the microfluidic device has
incubation chambers wherein no more than 1.times.10.sup.2
biological cells may be maintained, and an incubation chamber may
be no more than 4.times.10.sup.5 cubic microns. In yet other
embodiments, the microfluidic device has incubation chambers
wherein no more than 50 biological cells may be maintained, an
incubation chamber may be no more than 4.times.10.sup.5 cubic
microns.
[0155] In various embodiment, the microfluidic device has
incubation chambers configured as in any of the embodiments
discussed herein where the microfluidic device has about 100 to
about 500 incubation chambers; about 200 to about 1000 incubation
chambers, about 500 to about 1500 incubation chambers, about 1000
to about 2000 incubation chambers, or about 1000 to about 3500
incubation chambers.
[0156] In some other embodiments, the microfluidic device has
incubation chambers configured as in any of the embodiments
discussed herein where the microfluidic device has about 1500 to
about 3000 incubation chambers, about 2000 to about 3500 incubation
chambers, about 2500 to about 4000 incubation chambers, about 3000
to about 4500 incubation chambers, about 3500 to about 5000
incubation chambers, about 4000 to about 5500 incubation chambers,
about 4500 to about 6000 incubation chambers, about 5000 to about
6500 incubation chambers, about 5500 to about 7000 incubation
chambers, about 6000 to about 7500 incubation chambers, about 6500
to about 8000 incubation chambers, about 7000 to about 8500
incubation chambers, about 7500 to about 9000 incubation chambers,
about 8000 to about 9500 incubation chambers, about 8500 to about
10,000 incubation chambers, about 9000 to about 10,500 incubation
chambers, about 9500 to about 11,000 incubation chambers, about
10,000 to about 11,500 incubation chambers, about 10,500 to about
12,000 incubation chambers, about 11,000 to about 12,500 incubation
chambers, about 11,500 to about 13,000 incubation chambers, about
12,000 to about 13,500 incubation chambers, about 12,500 to about
14,000 incubation chambers, about 13,000 to about 14,500 incubation
chambers, about 13,500 to about 15,000 incubation chambers, about
14,000 to about 15,500 incubation chambers, about 14,500 to about
16,000 incubation chambers, about 15,000 to about 16,500 incubation
chambers, about 15,500 to about 17,000 incubation chambers, about
16,000 to about 17,500 incubation chambers, about 16,500 to about
18,000 incubation chambers, about 17,000 to about 18,500 incubation
chambers, about 17,500 to about 19,000 incubation chambers, about
18,000 to about 19,500 incubation chambers, about 18,500 to about
20,000 incubation chambers, about 19,000 to about 20,500 incubation
chambers, about 19,500 to about 21,000 incubation chambers, or
about 20,000 to about 21,500 incubation chambers.
[0157] FIG. 2F illustrates a microfluidic device 290 according to
one embodiment. The microfluidic device 290 is illustrated in FIG.
2F is a stylized diagram of a microfluidic device 100. In practice
the microfluidic device 290 and its constituent circuit elements
(e.g. channels 122 and incubation chambers 128) would have the
dimensions discussed herein. The microfluidic circuit 120
illustrated in FIG. 2F has two ports 107, four distinct channels
122 and four distinct flow paths 106. The microfluidic device 290
further comprises a plurality of incubation chambers opening off of
each channel 122. In the microfluidic device illustrated in FIG.
2F, the incubation chambers have a geometry similar to the pens
illustrated in FIG. 2E and thus, have both connection regions and
isolation regions. Accordingly, the microfluidic circuit 120
includes both swept regions (e.g. channels 122 and portions of the
connection regions 254 within the maximum penetration depth D.sub.p
of the secondary flow 262) and non-swept regions (e.g. isolation
regions 258 and portions of the connection regions 254 not within
the maximum penetration depth D.sub.p of the secondary flow
262).
[0158] FIGS. 3A through 3B shows various embodiments of system 150
which can be used to operate and observe microfluidic devices (e.g.
100, 200, 240, 290) according to the present invention. As
illustrated in FIG. 3A, the system 150 can include a structure
("nest") 300 configured to hold a microfluidic device 100 (not
shown), or any other microfluidic device described herein. The nest
300 can include a socket 302 capable of interfacing with the
microfluidic device 360 (e.g., an optically-actuated electrokinetic
device 100) and providing electrical connections from power source
192 to microfluidic device 360. The nest 300 can further include an
integrated electrical signal generation subsystem 304. The
electrical signal generation subsystem 304 can be configured to
supply a biasing voltage to socket 302 such that the biasing
voltage is applied across a pair of electrodes in the microfluidic
device 360 when it is being held by socket 302. Thus, the
electrical signal generation subsystem 304 can be part of power
source 192. The ability to apply a biasing voltage to microfluidic
device 360 does not mean that a biasing voltage will be applied at
all times when the microfluidic device 360 is held by the socket
302. Rather, in most cases, the biasing voltage will be applied
intermittently, e.g., only as needed to facilitate the generation
of electrokinetic forces, such as dielectrophoresis or
electrowetting, in the microfluidic device 360.
[0159] As illustrated in FIG. 3A, the nest 300 can include a
printed circuit board assembly (PCBA) 320. The electrical signal
generation subsystem 304 can be mounted on and electrically
integrated into the PCBA 320. The exemplary nest 300 includes
socket 302 mounted on PCBA 320, as well.
[0160] Typically, the electrical signal generation subsystem 304
will include a waveform generator (not shown). The electrical
signal generation subsystem 304 can further include an oscilloscope
(not shown) and/or a waveform amplification circuit (not shown)
configured to amplify a waveform received from the waveform
generator. The oscilloscope, if present, can be configured to
measure the waveform supplied to the microfluidic device 360 held
by the socket 302. In certain embodiments, the oscilloscope
measures the waveform at a location proximal to the microfluidic
device 360 (and distal to the waveform generator), thus ensuring
greater accuracy in measuring the waveform actually applied to the
device. Data obtained from the oscilloscope measurement can be, for
example, provided as feedback to the waveform generator, and the
waveform generator can be configured to adjust its output based on
such feedback. An example of a suitable combined waveform generator
and oscilloscope is the Red Pitaya.TM..
[0161] In certain embodiments, the nest 300 further comprises a
controller 308, such as a microprocessor used to sense and/or
control the electrical signal generation subsystem 304. Examples of
suitable microprocessors include the Arduino.TM. microprocessors,
such as the Arduino Nano.TM.. The controller 308 may be used to
perform functions and analysis or may communicate with an external
master controller 154 (shown in FIG. 1) to perform functions and
analysis. In the embodiment illustrated in FIG. 3A the controller
308 communicates with a master controller 154 through an interface
310 (e.g., a plug or connector).
[0162] In some embodiments, the nest 300 can comprise an electrical
signal generation subsystem 304 comprising a Red Pitaya.TM.
waveform generator/oscilloscope unit ("Red Pitaya unit") and a
waveform amplification circuit that amplifies the waveform
generated by the Red Pitaya unit and passes the amplified voltage
to the microfluidic device 100. In some embodiments, the Red Pitaya
unit is configured to measure the amplified voltage at the
microfluidic device 360 and then adjust its own output voltage as
needed such that the measured voltage at the microfluidic device
360 is the desired value. In some embodiments, the waveform
amplification circuit can have a +6.5V to -6.5V power supply
generated by a pair of DC-DC converters mounted on the PCBA 320,
resulting in a signal of up to 13 Vpp at the microfluidic device
100.
[0163] As illustrated in FIG. 3A, the nest 300 can further include
a thermal control subsystem 306. The thermal control subsystem 306
can be configured to regulate the temperature of microfluidic
device 360 held by the nest 300. For example, the thermal control
subsystem 306 can include a Peltier thermoelectric device (not
shown) and a cooling unit (not shown). The Peltier thermoelectric
device can have a first surface configured to interface with at
least one surface of the microfluidic device 360. The cooling unit
can be, for example, a cooling block (not shown), such as a
liquid-cooled aluminum block. A second surface of the Peltier
thermoelectric device (e.g., a surface opposite the first surface)
can be configured to interface with a surface of such a cooling
block. The cooling block can be connected to a fluidic path 330
configured to circulate cooled fluid through the cooling block. In
the embodiment illustrated in FIG. 3A, the nest 300 comprises an
inlet 332 and an outlet 334 to receive cooled fluid from an
external reservoir (not shown), introduce the cooled fluid into the
fluidic path 330 and through the cooling block, and then return the
cooled fluid to the external reservoir. In some embodiments, the
Peltier thermoelectric device, the cooling unit, and/or the fluidic
path 330 can be mounted on a casing 340 of the nest 300. In some
embodiments, the thermal control subsystem 306 is configured to
regulate the temperature of the Peltier thermoelectric device so as
to achieve a target temperature for the microfluidic device 360.
Temperature regulation of the Peltier thermoelectric device can be
achieved, for example, by a thermoelectric power supply, such as a
Pololu.TM. thermoelectric power supply (Pololu Robotics and
Electronics Corp.). The thermal control subsystem 306 can include a
feedback circuit, such as a temperature value provided by an analog
circuit. Alternatively, the feedback circuit can be provided by a
digital circuit.
[0164] In some embodiments, the nest 300 can include a thermal
control subsystem 306 with a feedback circuit that is an analog
voltage divider circuit (not shown) which includes a resistor
(e.g., with resistance 1 kOhm+/-0.1%, temperature coefficient
+/-0.02 ppm/CO) and a NTC thermistor (e.g., with nominal resistance
1 kOhm+/-0.01%). In some instances, the thermal control subsystem
306 measures the voltage from the feedback circuit and then uses
the calculated temperature value as input to an on-board PID
control loop algorithm. Output from the PID control loop algorithm
can drive, for example, both a directional and a
pulse-width-modulated signal pin on a Pololu.TM. motor drive (not
shown) to actuate the thermoelectric power supply, thereby
controlling the Peltier thermoelectric device.
[0165] The nest 300 can include a serial port 350 which allows the
microprocessor of the controller 308 to communicate with an
external master controller 154 via the interface 310. In addition,
the microprocessor of the controller 308 can communicate (e.g., via
a Plink tool (not shown)) with the electrical signal generation
subsystem 304 and thermal control subsystem 306. Thus, via the
combination of the controller 308, the interface 310, and the
serial port 350, the electrical signal generation subsystem 308 and
the thermal control subsystem 306 can communicate with the external
master controller 154. In this manner, the master controller 154
can, among other things, assist the electrical signal generation
subsystem 308 by performing scaling calculations for output voltage
adjustments. A Graphical User Interface (GUI) (not shown), provided
via a display device 170 coupled to the external master controller
154, can be configured to plot temperature and waveform data
obtained from the thermal control subsystem 306 and the electrical
signal generation subsystem 308, respectively. Alternatively, or in
addition, the GUI can allow for updates to the controller 308, the
thermal control subsystem 306, and the electrical signal generation
subsystem 304.
[0166] As discussed above, system 150 can include an imaging device
194. In some embodiments, the imaging device 194 comprises a light
modulating subsystem 422. The light modulating subsystem 422 can
include a digital mirror device (DMD) or a microshutter array
system (MSA), either of which can be configured to receive light
from a light source 420 and transmits a subset of the received
light into an optical train of microscope 450. Alternatively, the
light modulating subsystem 422 can include a device that produces
its own light (and thus dispenses with the need for a light source
420), such as an organic light emitting diode display (OLED), a
liquid crystal on silicon (LCOS) device, a ferroelectric liquid
crystal on silicon device (FLCOS), or a transmissive liquid crystal
display (LCD). The light modulating subsystem 422 can be, for
example, a projector. Thus, the light modulating subsystem 422 can
be capable of emitting both structured and unstructured light. One
example of a suitable light modulating subsystem 422 is the
Mosaic.TM. system from Andor Technologies.TM.. In certain
embodiments, imaging module 164 and/or motive module 162 of system
150 can control the light modulating subsystem 422.
[0167] In certain embodiments, the imaging device 194 further
comprises a microscope 450. In such embodiments, the nest 300 and
light modulating subsystem 422 can be individually configured to be
mounted on the microscope 450. The microscope 450 can be, for
example, a standard research-grade light microscope or fluorescence
microscope. Thus, the nest 300 can be configured to be mounted on
the stage 426 of the microscope 450 and/or the light modulating
subsystem 422 can be configured to mount on a port of microscope
450. In other embodiments, the nest 300 and the light modulating
subsystem 422 described herein can be integral components of
microscope 450.
[0168] In certain embodiments, the microscope 450 can further
include one or more detectors 440. In some embodiments, the
detector 440 is controlled by the imaging module 164. The detector
440 can include an eye piece, a charge-coupled device (CCD), a
camera (e.g., a digital camera), or any combination thereof. If at
least two detectors 440 are present, one detector can be, for
example, a fast-frame-rate camera while the other detector can be a
high sensitivity camera. Furthermore, the microscope 450 can
include an optical train configured to receive reflected and/or
emitted light from the microfluidic device 360 and focus at least a
portion of the reflected and/or emitted light on the one or more
detectors 440. The optical train of the microscope can also include
different tube lenses (not shown) for the different detectors, such
that the final magnification on each detector can be different.
[0169] In certain embodiments, imaging device 194 is configured to
use at least two light sources. For example, a first light source
420 can be used to produce structured light (e.g., via the light
modulating subsystem 422) and a second light source 430 can be used
to provide unstructured light. The first light source 420 can
produce structured light for optically-actuated electrokinesis
and/or fluorescent excitation, and the second light source 430 can
be used to provide bright field illumination. In these embodiments,
the motive module 164 can be used to control the first light source
420 and the imaging module 164 can be used to control the second
light source 430. The optical train of the microscope 450 can be
configured to (1) receive structured light from the light
modulating subsystem 422 and focus the structured light on at least
a first region in a microfluidic device, such as an
optically-actuated electrokinetic device, when the device is being
held by the nest 300, and (2) receive reflected and/or emitted
light from the microfluidic device and focus at least a portion of
such reflected and/or emitted light onto detector 440. The optical
train can be further configured to receive unstructured light from
a second light source and focus the unstructured light on at least
a second region of the microfluidic device, when the device is held
by the nest 300. In certain embodiments, the first and second
regions of the microfluidic device can be overlapping regions. For
example, the first region can be a subset of the second region.
[0170] In FIG. 3B, the first light source 420 is shown supplying
light to a light modulating subsystem 422, which provides
structured light to the optical train of the microscope 450. The
second light source 430 is shown providing unstructured light to
the optical train via a beam splitter 424. Structured light from
the light modulating subsystem 422 and unstructured light from the
second light source 430 travel from the beam splitter 424 through
the optical train together to reach a second beam splitter 424 (or
dichroic filter 448, depending on the light provided by the light
modulating subsystem 422), where the light gets reflected down
through the objective 454 to the sample plane 428. Reflected and/or
emitted light from the sample plane 428 then travels back up
through the objective 454, through the beam splitter and/or
dichroic filter 448, and to a dichroic filter 452. Only a fraction
of the light reaching dichroic filter 452 passes through and
reaches the detector 440.
[0171] In some embodiments, the second light source 430 emits blue
light. With an appropriate dichroic filter 452, blue light
reflected from the sample plane 428 is able to pass through
dichroic filter 452 and reach the detector 440. In contrast,
structured light coming from the light modulating subsystem 422
gets reflected from the sample plane 428, but does not pass through
the dichroic filter 452. In this example, the dichroic filter 452
is filtering out visible light having a wavelength longer than 495
nm. Such filtering out of the light from the light modulating
subsystem 422 would only be complete (as shown) if the light
emitted from the light modulating subsystem did not include any
wavelengths shorter than 495 nm. In practice, if the light coming
from the light modulating subsystem 422 includes wavelengths
shorter than 495 nm (e.g., blue wavelengths), then some of the
light from the light modulating subsystem would pass through filter
452 to reach the detector 440. In such an embodiment, the filter
452 acts to change the balance between the amount of light that
reaches the detector 440 from the first light source 420 and the
second light source 430. This can be beneficial if the first light
source 420 is significantly stronger than the second light source
430. In other embodiments, the second light source 430 can emit red
light, and the dichroic filter 452 can filter out visible light
other than red light (e.g., visible light having a wavelength
shorter than 650 nm).
[0172] Gaseous environment. The system provides a mixture of gases
necessary for cell viability, including but not limited to oxygen
and carbon dioxide. Both gases dissolve into the fluidic medium,
and may be used by the cells, thus altering over time the gas
content of the fluidic medium in an isolation region of an
incubation chamber. In particular, carbon dioxide content can
change over time, which affects the pH of the fluidic media in the
microfluidic device. In some experimental conditions, non-optimal
oxygen partial pressure may be used.
[0173] Flow controller providing perfusion during incubation.
During the incubating step, the second fluidic medium, present
within the isolation region of the incubation chamber may become
depleted of nutrients, growth factors or other growth stimulants.
The second fluidic medium may accumulate cellular waste products.
Additionally, as the at least one biological cell continues to grow
during the period of incubation, it may be desirable to alter the
nutrients, growth factors or other growth stimulants to be
different from those of the first or second media at the start of
the incubation. Culturing in an incubation chamber of a
microfluidic device as described here may afford the specific and
selective ability to introduce and alter chemical gradients sensed
by the at least one biological cell, which may much more closely
approximate in-vivo conditions. Alternatively, altering the
chemical gradients sensed by the at least one biological cell to
purposely non-optimized set of conditions may permit cell expansion
under conditions designed to explore disease or treatment pathways.
The method may therefore include perfusing the first fluidic medium
during the incubating step, wherein the first fluidic medium is
introduced via at least one inlet 124 of the microfluidic device
and wherein the first fluidic medium, optionally comprising
components from the second fluidic medium is exported via at least
one outlet of the microfluidic device.
[0174] Exchange of components of the first fluidic medium, thereby
providing fresh nutrients, soluble growth factors, and the like,
and/or exchange of waste components of the medium surrounding the
cell(s) within the isolation region occurs at the interface of the
swept and unswept regions of the incubation chamber substantially
under conditions of diffusion. Effective exchange has been
surprisingly found to result under substantially no flow
conditions. Accordingly, it has been surprisingly found that
successful incubation does not require constant perfusion. As
result, perfusing may be non-continuous. In some embodiments,
perfusing is periodic, and in some embodiments, perfusing is
irregular. Breaks between periods of perfusion may be of sufficient
duration to permit components of the second fluidic medium in the
isolation region to diffuse into the first fluidic medium in the
flow channel/region and/or components of the first fluidic medium
to diffuse into the second fluidic medium, all without substantial
flow of the first medium into the isolation region.
[0175] In another embodiment, low perfusion rates may also be
employed to obtain effective exchange of the components of fluidic
media within and outside of the unswept region of the incubation
chamber.
[0176] Accordingly, one method of perfusing at least one biological
cell in at least one incubation chamber of a microfluidic device is
shown in FIG. 5 and includes a perfusing step 5002 where the first
fluidic medium is flowed into a flow region fluidically connected
to the incubation chamber at a first perfusion rate R.sub.1 for a
first perfusion time D.sub.1 through a flow region of the
microfluidic device. R.sub.1 may be selected to be a non-sweeping
rate of flow, as described herein. Method 500 further includes the
step S004 of stopping the flow of the fluidic medium for a first
perfusion stop time S.sub.1. Steps S002 and S004 are repeated for W
repetitions, where W may be an integer selected from 1 to about
1000, whereupon the perfusion process 500 is complete. In some
embodiments, W may be an integer of 2 to about 1000.
[0177] Another method 600, of perfusing at least one biological
cell in at least one incubation chamber of a microfluidic device is
shown in FIG. 6, which includes a first perfusion cycle that
includes the step 6002 of flowing the fluidic medium into a flow
region fluidically connected to the incubation chamber at a first
perfusion rate R.sub.1 for a first perfusion time D.sub.1 through a
flow region of the microfluidic device. R.sub.1 may be selected to
be a non-sweeping rate of flow, as described herein. The first
perfusion cycle includes the step 6004 of stopping the flow of the
fluidic medium for a first perfusion stop time S.sub.1. The first
perfusion cycle may be repeated for W repetitions, wherein W is an
integer selected from 1 to about 1000. After the Wth repeat of the
first perfusion cycle is completed, method 600 further includes a
second perfusion cycle, which includes the step 8006 of flowing the
first fluidic medium at a second perfusion rate R.sub.2 for a
second perfusion time D.sub.2, wherein R.sub.2 is selected to be a
non-sweeping rate of flow. The second perfusion cycle of Method 600
further includes the step 6008 of stopping the flow of the fluidic
medium for a second perfusion stop time S.sub.2. Thereafter, the
method returns to step 6002 and 6004 of the first perfusion cycle
and the combined two cycle perfusion process is repeated for V
repeats, wherein V is an integer of 1 to about 5000. The
combination of W and V may be chosen to meet the desired incubation
period endpoint
[0178] In various embodiments of method 500, or 600, perfusing rate
R.sub.1 may be any non-sweeping rate of flow of fluidic medium as
described above for flow controller configurations. In some
embodiments, R.sub.1 may be about 0.009, 0.010, 0.020, 0.030,
0.040, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14,
0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70,
0.80, 0.90, 1.00, 1.10, 1.20, 1.30, 1.40, 1.50, 1.60, 1.70, 1.80,
1.90, 2.00. 2.10, 2.20, 2.40, 2.50, 2.60, 2.70, 2.80, 2.90 or 3.00
microliters/sec.
[0179] In various embodiments of method 600, the second perfusion
rate R.sub.2 may be any non-sweeping rate of flow of fluidic medium
as described as above for flow controller configurations. In some
embodiments, the R.sub.2 may be 0.009, 0.010, 0.020, 0.030, 0.040,
0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15,
0.16, 0.17, 0.18, 0.19, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80,
0.90, 1.00, 1.10, 1.20, 1.30, 1.40, 1.50, 1.60, 1.70, 1.80, 1.90,
2.00. 2.10, 2.20, 2.40, 2.50, 2.60, 2.70, 2.80, 2.90 or 3.00
microliters/sec. The flow rates R.sub.1 and/or R.sub.2 may be
chosen in any combination. Typically, perfusion rate R.sub.2 may be
greater than perfusion rate R.sub.1, and may be about 5.times.,
10.times., 20.times., 30.times., 40.times., 50.times., 60.times.,
70.times., 80.times., 90.times., 100.times., or more than R.sub.1.
In some embodiments, R.sub.2 is at least ten times faster than
R.sub.1. In other embodiments, R.sub.2 is at least twenty times
faster than R.sub.1. In yet another embodiment, R.sub.2 is at least
100.times. the rate of R.sub.1.
[0180] In various embodiments of method 500 or 600, first perfusion
time D.sub.1 may be any suitable duration of perfusion as described
above for flow controller configurations. In various embodiments,
D.sub.1 may be about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,
65, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170 or 180 sec.
In other embodiments, D.sub.1 may be a range of time, e.g., about
10 to about 40 sec, as described above. In some embodiments,
D.sub.1 may be about 30 sec to about 75 sec. In other embodiments,
D.sub.1 may be about 100 sec. In other embodiments, D.sub.1 may be
in a range from about 60 sec to about 150 sec. In yet other
embodiments, D.sub.1 may be about 20 min, 30 min, 40 min, 50 min,
60 min, 80 min, 90 min, 110 min, 120 min, 140 min, 160 min, 180
min, 200 min, 220 min, 240 min, 250 min, 260 min, 270 min, 290 min
or 300 min. In some embodiments, D.sub.1 is about 40 min to about
180 min.
[0181] In various embodiments of method 500 or 600, second
perfusion time D.sub.2 may be any suitable duration of perfusion as
described above for flow controller configurations. In various
embodiments, D.sub.2 may be about 5 sec, 10 sec, 15 sec, 20 sec, 25
sec, 30 sec, 35 sec, 40 sec, 45 sec, 50 sec, 55 sec, 60 sec, 65
sec, 70 sec, 80 sec, 90 sec or about 100 sec. In other embodiments,
D.sub.2 may be a range of time, e.g., about 5 sec to about 20 sec,
as described above. In other embodiments, D.sub.2 may be about 30
sec to about 70 sec. In other embodiments, D.sub.2 may be about 60
sec.
[0182] In various embodiments of method 500 or 600, the first
perfusion time D.sub.1 may be the same or different from the second
perfusion time D.sub.2. D.sub.1 and D.sub.2 may be chosen in any
combination. In some embodiments, the duration of perfusing D.sub.1
and/or D.sub.2 may be selected to be shorter than the stopping
periods S.sub.1 and/or S.sub.2.
[0183] In various embodiments of method 500 or 600, the first
perfusion stop time S.sub.1 may be selected to be any suitable
period of time as described above for an interval of time between
periods of perfusion for flow controller configurations. In some
embodiments, S.sub.1 may be about 0 min, 5 min, about 10 min, about
15 min, about 20 min, about 25 min, about 30 min, about 35 min,
about 40 min, about 45 min, about 60 min, about 65 min, about 80
min, about 90 min, about 100 min, about 120 min, about 150 min,
about 180 min, about 210 min, about 240 min, about 270 min, or
about 300 min. In various embodiments, S.sub.1 may be any
appropriate range of time, as described above for flow controller
configuration intervals between perfusion, e.g. about 20 to about
60 min. In some embodiments, S.sub.1 may be about 10 min to about
30 min. In other embodiments, S.sub.1 may be about 15 min. In yet
other embodiments, S.sub.1 may be about 0 sec, 5 sec, 10 sec, 20
sec, 30 sec, 40 sec, 50 sec, 60 sec, 70 sec, 80 sec, or about 90
sec. In some embodiments, S.sub.1 is about 0 sec.
[0184] In various embodiments of method 500 or 600, the second
perfusion stop time S.sub.2 may be selected to be any suitable
period of time as described above for an interval of time between
periods of perfusion for flow controller configurations. In some
embodiments, S2 may be about 0 min, 5 min, about 6 min, about 7
min, about 8 min, about 9 min, about 10 min, about 20 min, about 30
min, about 45 min, about 50 min, about 60 about 90 min, about 120
min, about 180 min, about 240 min, about 270 min, or about 300 min.
In various embodiments, S.sub.2 may be any appropriate range of
time, as described above for flow controller configuration
intervals between perfusion, e.g. about 15 to about 45 min. In some
embodiments, S.sub.2 may be about 10 min to about 30 min. In other
embodiments, S.sub.2 may be about 8 min or 9 min. In other
embodiments, S.sub.2 is about 0 min.
[0185] In various embodiments of method 500 or 600, the first
perfusion stop time S.sub.1 and the second perfusion stop time
S.sub.2 may be selected independently from any suitable value.
S.sub.1 may be the same or different from S.sub.2.
[0186] In various embodiments of method 500 and 600, the number of
W repetitions may be selected to be the same or different from the
number of V repetitions.
[0187] In various embodiments of methods 500 or 600, W may be about
1, about 4, about 5, about 6, about 8, about 10, about 12, about
15, about 18, about 20, about 24, about 30, about 36, about 40,
about 45, or about 50. In some embodiments, W may be selected to be
about 1 to about 20. In some embodiments, W may be 1.
[0188] In various embodiments of method 600, V may be about 5,
about 10, about 20, about 25, about 30, about 35, about 40, about
50, about 60, about 80, about 100, about 120, about 240, about 300,
about 350, about 400, about 450, about 500, about 600, about 750,
about 900, or about 1000. In some embodiments, V may be selected to
be about 10 to about 120. In other embodiments, V may be about 5 to
about 24. In some embodiments, V may be about 30 to about 50 or may
be about 400 to about 500.
[0189] In various embodiments of method 600, the number of W
repetitions may be selected to be the same or different from the
number of V repetitions.
[0190] In various embodiments of methods 500 or 600 a total time
for the first step of perfusing (represented by steps 002/5004 or
6002/6004) is about 1 h to about 10 h and W is an integer is 1. In
various embodiments, the total time for the first step of perfusing
is about 9 min to about 15 min.
[0191] In various embodiments of method 600, a total time for the
second step of a perfusing cycle (represented by step 6006/6008) is
about 1 min to about 15 min or about 1 min to about 20 min.
[0192] In any of methods 500 or 600, the perfusing method may be
continued for the entire incubation period of the biological cell,
e.g., for about 1, about 2, about 3, about 4, about 5, about 6,
about 7, about 8 about 9, about 10 days or more.
[0193] In another non-limiting embodiment of method 600 of FIG. 6,
the controller may be configured to perfuse the fluidic medium(s)
in the flow region having longer periods of perfusion D.sub.1
during the perfusing step 6002. The controller may perfuse the
fluidic medium at a first rate for a period of about 45 min, about
60 min, about 75 min, about 90 min, about 105 min, about 120 min,
about 2.25 h, about 2.5 h, about 2.45 h, about 3.0 h, about 3.25 h,
about 3.5 h, about 3.75 h, about 4.0 h, about 4.25 h, about 4.5 h,
about 4.75 h, about 5 h, or about 6 h. At the end of the first
perfusion period D.sub.1, the flow of the fluidic medium may be
stopped for a stopping period of time S.sub.1, which may be about 0
sec, 15 sec, 30 sec, about 45 sec, about 1 min, about 1.25 min,
about 1.5 min, about 2.0 min, about 3.0 min, about 4 min, about 5
min or about 6 min. In some embodiments, the first flow rate
R.sub.1 may be selected to be about 0.009, 0.01, 0.02, 0.03, 0.05,
0.1, 0.2, 0.3, 0.4, or about 0.5 microliters/sec. The flow of the
fluidic medium may be stopped for a perfusion stopping period
S.sub.1 of less than about 1 minute or S.sub.1 may be 0 sec.
Alternatively, S.sub.1 may be about 30 sec, about 1.5 min, about
2.0 min, about 2.5 min, or about 3 min. A second perfusion period
D.sub.2 may follow, using a different perfusion rate. In some
embodiments, the second perfusion rate may be higher than the first
perfusion rate. In some embodiments, the second perfusion rate
R.sub.2 may be selected from about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5,
1.7, 1.9, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0,
4.2, 4.4, 4.6, 4.8, 5.0, 6.0, 7.0, 8.0 or about 9.0
microliters/sec. The second perfusion period D.sub.2 may for about
1 sec, about 2 sec, about 3 sec, about 4 sec, about 5 sec, about 6
sec, about 10 sec, about 15 sec, about 30 sec, about 45 sec, about
60 sec, about 65 sec, about 75 sec, about 80 sec, or about 90 sec.
Perfusing may be then stopped for a second perfusion stop period
S.sub.2, which may be about 0 sec, 10 sec, about 20 sec, about 30
sec, about 40 sec, about 50 sec, about 60 sec, about 1.5 min, about
1.75 min, about 2.0 min, about 2.5 min, about 2.75 min, about 3.0
min or about 4.0 min. In some embodiments, D.sub.1 may be about 2
h, about 3 h, or about 4 h. In various embodiments, D.sub.1 may be
about 4 h. In various embodiments, S.sub.1 may be 0 sec or less
than about one minute. The second perfusion period D.sub.2 may be
about 1 sec to about 6 sec. In some embodiments, the second
perfusion stop period S.sub.2 may be about 40 sec to about 1.5
4min.
[0194] Accordingly, a method is provided for perfusing at least one
biological cell in at least one incubation chamber of a
microfluidic device including the steps of: perfusing the at least
one biological cell using a first perfusion step including: flowing
a first fluidic medium at a first perfusion rate R.sub.1 for a
first perfusion time D.sub.1 through a flow region of the
microfluidic device, where the flow region is fluidically connected
to the incubation chamber, wherein R.sub.1 is selected to be a
non-sweeping rate of flow; stopping the flow of the first fluidic
medium for a first perfusion stop time S.sub.1; and repeating the
first perfusion step for W repetitions, where W is an integer
selected from 1 to 1000. The method may further include a step of
perfusing the at least one biological cell using a second perfusion
step comprising: flowing the first fluidic medium at a second
perfusion rate R.sub.2 for a second perfusion time D.sub.2, where
R.sub.2 is selected to be a non-sweeping rate of flow; stopping the
flow of the first fluidic medium for a second perfusion stop time
S.sub.2; and repeating the first perfusion step followed by the
second perfusion step for V repetitions, wherein V is an integer of
1 to 1000.
[0195] The second perfusion rate R.sub.2 may be greater than the
first perfusion rate R.sub.1. The first perfusion time D.sub.1 may
be the same or different from the second perfusion time D.sub.2.
The first perfusion stop time S.sub.1 may be the same or different
from the second perfusion stop time S.sub.2. The number of W
repetitions may be the same or different from the number of V
repetitions, when the second perfusing step is performed. R.sub.2
may be at least ten times faster than R.sub.1. Alternatively,
R.sub.2 may be at least twenty times faster than R.sub.1. R.sub.2
may be at least 100 times as fast as R.sub.1. D.sub.1 may be about
30 sec to about 175 sec. In other embodiments, D.sub.1 may be about
40 min to about 180 min or about 180 min to about 300 min. In some
other embodiments, D.sub.1 may be about 60 sec to about 150 sec.
S.sub.1 may be about 10 min to about 30 min. In other embodiments,
S.sub.1 may be about 5 min to about 10 min. In yet other
embodiments, S.sub.1 may be zero. In some embodiments, D.sub.1 may
be about 40 min to about 180 min, and S.sub.1 may be zero. In other
embodiments, D.sub.1 may be about 60 sec to about 150 sec, and
S.sub.1 may be about 5 min to about 10 min. In yet other
embodiments, D.sub.1 may be about 180 min to about 300 min, and
S.sub.1 may be zero. The total time for the first perfusing step
may be about 1 h to about 10 h. In other embodiments, the total
time for the first perfusing step may be about 2 h to about 4 h. In
some embodiments, W may be an integer greater than 2. In some
embodiments, W may be about 1 to about 20. In some embodiments,
D.sub.2 may be about 10 sec to about 25 sec. In other embodiments,
D.sub.2 may be about 10 sec to about 90 sec. In some embodiments,
S.sub.2 may be about 10 min to about 30 min. In other embodiments,
S.sub.2 may be about 15 min. In some embodiments, V may be about 10
to about 120. In some embodiments, V may be about 30 to about 50 or
may be about 400 to about 500. In some embodiments, D.sub.2 may be
about 1 sec to about 6 sec. and S.sub.2 may be 0 sec. In some
embodiments, D.sub.2 may be about 10 sec to about 90 sec and
S.sub.2 may be about 40 sec to about 1.5 min. In some embodiments,
a total time for one repeat of the second perfusing step may be
about 1 min to about 15 min.
[0196] Temperature Control. In some embodiments, the at least one
conditioned surface of the incubation chamber(s) and/or flow
region(s) is conditioned by controlling the temperature of the at
least one conditioned surface. The system may include a component
that can control and modulate the temperature of the at least one
conditioned surface of the incubation chambers and/or flow regions
of the microfluidic device. The system may include Peltier heating,
resistive heating, or any other suitable method for providing
temperature modulation to the microfluidic device. The system may
also include sensors and/or feedback components to control heat
input to a predetermined range. In some embodiments, the at least
one conditioned surface has a temperature of at least about
25.degree. C., 26.degree. C., 27.degree. C., 28.degree. C.,
29.degree. C., 30.degree. C., 31.degree. C., 32.degree. C.,
33.degree. C., 34.degree. C., 35.degree. C., 36.degree. C.,
37.degree. C., 38.degree. C., 39.degree. C., or about 40.degree. C.
In some embodiments, the at least one surface has a temperature
greater than about 25.degree. C. In other embodiments, the at least
one surface has a temperature in the range from about
30.degree.-40.degree. C.; about 35.degree. C. to about 38.degree.
C.; or about 36.degree. C. to about 37.degree. C. In some
embodiments, the at least one conditioned surface has a temperature
of at least about 30.degree. C.
[0197] Conditioned surface of the microfluidic device. In some
embodiments, at least one surface of the microfluidic device is
conditioned to support cell growth, viability, portability, or any
combination thereof. In some embodiments, substantially all the
inner surfaces are conditioned. A conditioned surface may be one of
the elements facilitating successful cell incubation within the
microfluidic device. Identification of an appropriate conditioned
surface may require balancing a number of operational requirements.
First, the conditioned surface may provide a contacting surface
that acts to shield cells from the types of materials which may be
used in the fabrication of microfluidic devices of this class.
Without being limited by theory, the conditioned surface may be
surrounded by waters of hydration, which provide an aqueous, not a
metallic contact layer with the cells. Second, the conditioned
surface may provide a contacting surface with which the at least
one biological cell may be supported adequately during incubation,
without substantially inhibiting the ability of the cell to be
removed from the incubation chamber after completion of incubation.
For example, many cells require a contacting surface to have some
degree of hydrophilicity in order to adhere sufficiently to be
viable and/or grow. Alternatively, some cells may require a
contacting surface having a degree of hydrophobicity in order to
grow and present desired levels of viability. Additionally, some
cells may require the presence of selected proteins or peptide
motifs within the contacting surface in order to initiate
viability/growth responses. Third, the conditioning of the at least
one surface may permit the motive forces used in the microfluidic
device to function substantially within normal functioning power
range. For example, if light actuated motive forces are employed,
the conditioned surface may substantially permit passage of light
through the conditioned surface such that the light actuated motive
force is not substantially inhibited.
[0198] The at least one conditioned surface may include a surface
of the incubation chamber or a surface of the flow region, or a
combination thereof. In some embodiments, each of a plurality of
incubation chambers has at least one conditioned surface. In other
embodiments each of a plurality of flow regions has at least one
conditioned surface. In some embodiments, at least one surface of
each of a plurality of incubation chambers and each of a plurality
of flow regions are conditioned surfaces.
[0199] Conditioned surface including a polymer. The at least one
conditioned surface may include a polymer. The polymer may be
covalently or non-covalently linked to the at least one surface.
Polymers may have a variety of structural motifs, including block
polymers (and copolymers); star polymers (star copolymers); and
graft or comb polymers (graft copolymers), all of which may be
suitable for use herein.
[0200] The polymer may include a polymer including alkylene ether
moieties. A wide variety of alkylene ether containing polymers may
be suitable for use in the microfluidic device described herein.
One non-limiting exemplary class of alkylene ether containing
polymers are amphiphilic nonionic block copolymers which include
blocks of polyethylene oxide (PEO) and polypropylene oxide (PPO)
subunits in differing ratios and locations within the polymer
chain. Pluronic.RTM. polymers (BASF) are block copolymers of this
type and are known in the art to be suitable for use when in
contact with living cells. The polymers range in average molecular
mass M.sub.w from about 2000 Da to about 20 KDa. In some
embodiments, the PEO-PPO block copolymer can have a
hydrophilic-lipophilic balance (HLB) greater than about 10 (e.g.
12-18). Specific Pluronic.RTM. polymers useful for yielding a
conditioned surface include Pluronic.RTM. L44, L64, P85, and F127
(including F127NF). Another class of alkylene ether containing
polymers is polyethylene glycol (PEG M.sub.w<100,000 Da) or
alternatively polyethylene oxide (PEO, M.sub.w>100,000). In some
embodiments, a PEG may have an M.sub.w of about 1000 Da, 5000 Da,
10,000 Da or 20,000 Da.
[0201] In other embodiments, the polymer conditioned surface may
include a polymer containing carboxylic acid moieties. The
carboxylic acid subunit may be an alkyl, alkenyl or aromatic moiety
containing subunit. One non-limiting example is polylactic acid
(PLA).
[0202] In some other embodiments, the polymer conditioned surface
may include a polymer containing urethane moieties, such as, but
not limited to polyurethane.
[0203] In other embodiments, the polymer conditioned surface may
include a polymer containing sulfonic acid moieties. The sulfonic
acid subunit may be an alkyl, alkenyl or aromatic moiety containing
subunit. One non-limiting example is polystyrene sulfonic acid
(PSSA) or polyanethole sulfonic acid. These latter exemplary
polymers are polyelectrolytes and may alter the characteristics of
the surface to aid/deter adhesion.
[0204] In yet other embodiments, the polymer conditioned surface
may include a polymer containing phosphate moieties, either at a
terminus of the polymer backbone or pendant from the backbone of
the polymer.
[0205] In yet other embodiments, the polymer conditioned surface
may include a polymer containing saccharide moieties. In a
non-limiting example, polysaccharides such as those derived from
algal or fungal polysaccharides such as xanthan gum or dextran may
be suitable to form a polymer conditioned surface which may aid or
prevent cell adhesion. For example, a dextran polymer having a size
about 3 Kda may be used to provide a conditioned surface within a
microfluidic device.
[0206] In yet other embodiments, the polymer conditioned surface
may include a polymer containing nucleotide moieties, i.e. a
nucleic acid, which may have ribonucleotide moieties or
deoxyribonucleotide moieties. The nucleic acid may contain only
natural nucleotide moieties or may contain unnatural nucleotide
moieties which comprise nucleobase, ribose or phosphate moiety
analogs such as 7-deazaadenine, pentose, methyl phosphonate or
phosphorothioate moieties without limitation. A nucleic acid
containing polymer may include a polyelectrolyte which may aid or
prevent adhesion.
[0207] In yet other embodiments, the polymer conditioned surface
may include a polymer containing amino acid moieties. The polymer
containing amino acid moieties may include a natural amino acid
containing polymer or an unnatural amino acid containing polymer,
either of which may include a peptide, a polypeptide or a protein.
In one non-limiting example, the protein may be bovine serum
albumin (BSA). In some embodiments, an extracellular matrix (ECM)
protein may be provided within the conditioned surface for
optimized cell adhesion to foster cell growth. A cell matrix
protein which may be included in a conditioned surface can include,
but is not limited to, a collagen, an elastin, an RGD-containing
peptide (e.g. a fibronectin), or a laminin. In yet other
embodiments, growth factors, cytokines, hormones or other cell
signaling species may be provided within the at least one
conditioned surface of the microfluidic device.
[0208] In further embodiments, the polymer conditioned surface may
include a polymer including amine moieties. The polyamino polymer
may include a natural polyamine polymer or a synthetic polyamine
polymer. Examples of natural polyamines include spermine,
spermidine, and putrescine.
[0209] In some embodiments, the polymer conditioned surface may
include a polymer containing more than one of alkylene oxide
moieties, carboxylic acid moieties, sulfonic acid moieties,
phosphate moieties, saccharide moieties, nucleotide moieties, or
amino acid moieties. In other embodiments, the polymer conditioned
surface may include a mixture of more than one polymer each having
alkylene oxide moieties, carboxylic acid moieties, sulfonic acid
moieties, phosphate moieties, saccharide moieties, nucleotide
moieties, and/or amino acid moieties, which may be independently or
simultaneously incorporated into the conditioned surface.
[0210] Covalently linked conditioned surface. In some embodiments,
the at least one conditioned surface includes a covalently linked
moiety configured to support cell growth, viability, portability,
or any combination thereof of the one or more biological cells
within the microfluidic device. The covalently linked moiety can
include a linking group, wherein the linking group is covalently
linked to a surface of the microfluidic device. The linking group
is also linked to the moiety configured to support cell growth,
viability, portability, or any combination thereof of the one or
more biological cells within the microfluidic device. The surface
to which the linking group links may include a surface of the
substrate of the microfluidic device, which for embodiments in
which the microfluidic device includes a DEP configuration, can
include silicon and/or silicon dioxide. In some embodiments, the
covalently linked conditioned surface includes all of the inner
surfaces of the microfluidic device.
[0211] A schematic representation is shown in FIG. 7 for a
microfluidic device having a conditioned surface. As seen in FIG.
7, a microfluidic device 700 has a first DEP substrate 704 and a
second DEP substrate 706 facing an enclosed region 702 of the
microfluidic device which may include the at least one incubation
chamber and/or the flow region. The device 700 may be otherwise
configured like any of microfluidic devices 100, 200, 240, 290,
400, 500A-E, or 600. The enclosed region 702 may be the region in
which biological cells are either maintained or are imported into
or exported out from. The inner surfaces 710 (of the second DEP
substrate 706) and 712 (of the first DEP substrate 704) are
modified with a conditioned surface 716, which may be any moiety
supporting cell growth, viability, portability, or any combination
thereof. The conditioned surface is covalently linked to oxide
functionalities of the inner surfaces via a siloxy linking group
714 in this embodiment.
[0212] In some embodiments, the covalently linked moiety configured
to support cell growth, viability, portability, or any combination
thereof, may include alkyl or fluoroalkyl (which includes
perfluoroalkyl) moieties; mono- or polysaccharides (which may
include but is not limited to dextran); alcohols (including but not
limited to propargyl alcohol); polyalcohols, including but not
limited to polyvinyl alcohol; alkylene ethers, including but not
limited to polyethylene glycol; polyelectrolytes (including but not
limited to polyacrylic acid or polyvinyl phosphonic acid); amino
groups (including derivatives thereof, such as, but not limited to
alkylated amines, hydroxyalkylated amino group, guanidinium, and
heterocylic groups containing an unaromatized nitrogen ring atom,
such as, but not limited to morpholinyl or piperazinyl); carboxylic
acids including but not limited to propiolic acid (which may
provide a carboxylate anionic surface); phosphonic acids, including
but not limited to ethynyl phosphonic acid (which may provide a
phosphonate anionic surface); sulfonate anions; carboxybetaines;
sulfobetaines; sulfamic acids; or amino acids.
[0213] The covalently linked moiety configured to support cell
growth, viability, portability, or any combination thereof of one
or more biological cells within the microfluidic device may be any
polymer as described herein, and may include one or more polymers
containing alkylene oxide moieties, carboxylic acid moieties,
saccharide moieties, sulfonic acid moieties, phosphate moieties,
amino acid moieties, nucleic acid moieties, or amino moieties.
[0214] In other embodiments, the covalently linked moiety
configured to support cell growth, viability, portability, or any
combination thereof of one or more biological cells may include
non-polymeric moieties such as an alkyl moiety, fluoroalkyl moiety
(including but not limited to perfluoroalkyl), amino acid moiety,
alcohol moiety, amino moiety, carboxylic acid moiety, phosphonic
acid moiety, sulfonic acid moiety, sulfamic acid moiety, or
saccharide moiety.
[0215] In some embodiments, the covalently linked moiety may be an
alkyl group. The alkyl group can comprise carbon atoms that form a
linear chain (e.g., a linear chain of at least 10 carbons, or at
least 14, 16, 18, 20, 22, or more carbons). Thus, the alkyl group
may be an unbranched alkyl. In some embodiments, the alkyl group
may include a substituted alkyl group (e.g., some of the carbons in
the alkyl group can be fluorinated or perfluorinated). The alkyl
group may comprise a linear chain of substituted (e.g., fluorinated
or perfluorinated) carbons joined to a linear chain of
non-substituted carbons. For example, the alkyl group may include a
first segment, which may include a perfluoroalkyl group, joined to
a second segment, which may include a non-substituted alkyl group.
The first and second segments may be joined directly or indirectly
(e.g., by means of an ether linkage). The first segment of the
alkyl group may be located distal to the linking group, and the
second segment of the alkyl group may be located proximal to the
linking group. In other embodiment, the alkyl group may include a
branched alkyl group and may further have one or more arylene group
interrupting the alkyl backbone of the alkyl group. In some
embodiments, a branched or arylene-interrupted portion of the alkyl
or fluorinated alkyl group is located at a point distal to the
covalent linkage to the surface.
[0216] In other embodiments, the covalently linked moiety may
include at least one amino acid, which may include more than one
amino acids. The covalently linked moiety may include a peptide or
a protein. In some embodiments, the covalently linked moiety may
include an amino acid which may provide a zwitterionic surface to
support cell growth, viability, portability, or any combination
thereof.
[0217] The covalently linked moiety may include one or more
saccharides. The covalently linked saccharides may be mono-, di-,
or polysaccharides. The covalently linked saccharides may be
modified to introduce a reactive pairing moiety which permits
coupling or elaboration for attachment to the surface. Exemplary
reactive pairing moieties may include aldehyde, alkyne or halo
moieties. A polysaccharide may be modified in a random fashion,
wherein each of the saccharide monomers may be modified or only a
portion of the saccharide monomers within the polysaccharide are
modified to provide a reactive pairing moiety that may be coupled
directly or indirectly to a surface. One exemplar may include a
dextran polysaccharide which may be coupled indirectly to a surface
via an unbranched linker.
[0218] The covalently linked moiety may include one or more amino
groups. The amino group may be a substituted amine moiety,
guanidine moiety, nitrogen-containing heterocyclic moiety or
heteroaryl moiety. The amino containing moieties may have
structures permitting pH modification of the environment within the
microfluidic device, and optionally, within the incubation
chamber.
[0219] The covalently linked moiety may include one or more
carboxylic acid, phosphonic acid, sulfamic or sulfonic acid
moieties. In some embodiments, the covalently linked moiety may
include one or more nucleic acid moieties, which may have a
sequence of individual nucleotides that is designed to capture
nucleic acids from biological cells within the microfluidic device.
The capture nucleic acids may have a nucleotide sequence that is
complementary to the nucleic acid from the biological cells and may
capture the nucleic acid by hybridization.
[0220] The conditioned surface may be composed of only one kind of
moiety or may include a more than one different kind of moiety. For
example, the fluoroalkyl conditioned surfaces (including
perfluoroalkyl) may have a plurality of covalently linked moieties
which are all the same, e.g. has the same covalent attachment to
the surface and has the same number of fluoromethylene units
comprising the fluoroalkyl moiety supporting growth and/or
viability and/or portability. Alternatively, the conditioned
surface may have more than one kind of moiety attached to the
surface. For example, the conditioned surface may include alkyl or
fluoroalkyl groups having a specified number of methylene or
fluoromethylene units and may further include a further set of
groups attached to the surface having a charged moiety attached to
an alkyl or fluoroalkyl chain that has a greater number of
methylene or fluoromethylene units. In some embodiments, the
conditioned surface having more than one kind of moiety attached
may be designed such that a first set of attached ligands which
have a greater number of backbone atoms and thus having a greater
length from the covalent attachment to the surface, may provide
capacity to present bulkier moieties at the conditioned surface,
while a second set of attached ligands having different, less
sterically demanding termini while having fewer backbone atoms can
help to functionalize the entire substrate surface to prevent
undesired adhesion or contact with a silicon or alumina substrate
itself. In another example, the moieties attached to the surface
may provide a zwitterionic surface presenting alternating charges
in a random fashion on the surface. Methods of preparing these
surfaces may be found in the cross-referenced U.S. patent
application Ser. No. 15/135,707, filed on Apr. 22, 2016.
[0221] Conditioned Surface Properties. In some embodiments, the
covalently linked moieties may form a monolayer when covalently
linked to the surface of the microfluidic device (e.g., a DEP
configured substrate surface). In some embodiments, the conditioned
surface formed by the covalently linked moieties may have a
thickness of less than 10 nm (e.g., less than 5 nm, or about 1.5 to
3.0 nm). In other embodiments, the conditioned surface formed by
the covalently linked moieties may have a thickness of about 10 nm
to about 50 nm. In some embodiments, the conditioned surface does
not require a perfectly formed monolayer to be suitably functional
for operation within a DEP configuration.
[0222] Aside from the composition of the conditioned surface, other
factors such as physical thickness of the hydrophobic material can
impact DEP force. Various factors can alter the physical thickness
of the conditioned surface, such as the manner in which the
conditioned surface is formed on the substrate (e.g. vapor
deposition, liquid phase deposition, spin coating, flooding, and
electrostatic coating). The physical thickness and uniformity of
the conditioned surface can be measured using an ellipsometer.
[0223] In addition to its electrical properties, the conditioned
surface may also have properties that are beneficial in use with
biological molecules. For example, a conditioned surface that
contains fluorinated (or perfluorinated) carbon chains may provide
a benefit relative to alkyl-terminated chains in reducing the
amount of surface fouling. Surface fouling, as used herein, refers
to the amount of indiscriminate material deposition on the surface
of the microfluidic device, which may include permanent or
semi-permanent deposition of biomaterials such as protein and its
degradation products, nucleic acids and respective degradation
products and the like.
[0224] Various properties for conditioned surfaces which may be
used in DEP configurations are included in the table below. As can
be seen, for entries 1 to 7, which were all covalently linked
conditioned surfaces as described herein, the thickness as measured
by ellipsometry were consistently thinner than that of entry 8, a
CYTOP surface which was formed by non-covalent spin coating (N/A
represents data not available throughout the table). Fouling was
found to be more dependent upon the chemical nature of the surface
than upon the mode of formation as the fluorinated surfaces were
typically less fouling than that of alkyl (hydrocarbon) conditioned
surfaces
TABLE-US-00001 TABLE 1 Properties of various conditioned surfaces
prepared by covalently modifying a surface, compared to CYTOP, a
non-covalently formed surface. Surface Formula of surface
modification type modifying reagent Thickness Fouling Alkyl
terminated CH.sub.3--(CH.sub.2).sub.15--Si-- N/A More fouling than
siloxane (OCH.sub.3).sub.3 fluorinated layers. (C.sub.16) Alkyl
terminated CH.sub.3--(CH.sub.2).sub.17--Si-- ~2 nm More fouling
than siloxane (OCH.sub.3).sub.3 fluorinated layers. (C.sub.18)
Alkyl-terminated CH.sub.3--(CH.sub.2).sub.17-- N/A More fouling
than phosphonate ester P.dbd.O(OH).sub.2 fluorinated layers.
C.sub.18P Alkyl terminated CH.sub.3--(CH.sub.2).sub.21--Si-- ~2-2.5
nm More fouling than siloxane (OCH.sub.2CH.sub.3).sub.3 fluorinated
layers. (C.sub.22) Fluoroalkyl- CF.sub.3--(CF.sub.2).sub.7-- ~1 nm
More resistant to terminated (CH.sub.2).sub.2--Si-- fouling than
alkyl- alkyl-siloxane (OCH.sub.3).sub.3 terminated layers C.sub.10F
Fluoroalkyl- CF.sub.3--(CF.sub.2).sub.13-- ~2 nm More resistant to
terminated (CH.sub.2).sub.2--Si-- fouling than alkyl-
alkyl-siloxane (OCH.sub.3).sub.3 terminated layers (C.sub.16F)
Fluoroalkyl- CF.sub.3--(CF.sub.2).sub.5-- ~2 nm N/A terminated
(CH.sub.2).sub.2--O-- alkoxy-alkyl-
(CH.sub.2).sub.11--Si(OCH.sub.3).sub.3 siloxane C.sub.6FC.sub.13
CYTOP ~30 nm More resistant to Fluoropolymer .sup.1,2 fouling than
alkyl- terminated layers 1. CYTOP structure: ##STR00001## 2. Spin
coated, not covalent.
[0225] Linking group to surface. The covalently linked moieties
forming the conditioned surface are attached to the surface via a
linking group. The linking group may be a siloxy linking group
formed by the reaction of a siloxane containing reagent with oxides
of the substrate surface, which may be formed from silicon or
aluminum oxide. In some other embodiments, the linking group may be
a phosphonate ester formed by the reaction of a phosphonic acid
containing reagent with the oxides of the silicon or aluminum
substrate surface.
[0226] Multi-part conditioned surface. The covalently linked
conditioned surface may be formed by reaction of a surface
conditioning reagent which is configured to already contain the
moiety providing the conditioned surface (e.g., an alkyl siloxane
reagent or a fluoro substituted alkyl siloxane reagent, which may
include a perfluoroalkyl siloxane reagent), as is described below.
Alternatively, the conditioned surface may be formed by coupling
the moiety which supports cell growth, viability, portability, or
any combination thereof to a surface modifying ligand that itself
is covalently linked to the surface.
[0227] Structures for a conditioned surface and methods of
preparation. In some embodiments, a conditioned surface covalently
linked to oxides of the surface of the dielectrophoresis substrate
has a structure of Formula 1:
##STR00002##
[0228] The conditioned surface may be linked covalently to oxides
of the surface of the dielectrophoresis substrate. The
dielectrophoresis substrate may be silicon or alumina, and oxides
may be present as part of the native chemical structure of the
substrate or may be introduced as discussed below. The conditioned
surface may be attached to the oxides via a linking group LG which
may be a siloxy or phosphonate ester group, formed from the
reaction of a siloxane or phosphonic acid group with the
oxides.
[0229] The moiety configured to support cell growth, viability,
portability, or any combination thereof, may include alkyl or
fluoroalkyl (which includes perfluoroalkyl) moieties; mono- or
polysaccharides (which may include but is not limited to dextran);
alcohols (including but not limited to propargyl alcohol);
polyalcohols, including but not limited to polyvinyl alcohol;
alkylene ethers, including but not limited to polyethylene glycol;
polyelectrolytes (including but not limited to polyacrylic acid or
polyvinyl phosphonic acid); amino groups (including derivatives
thereof, such as, but not limited to alkylated amines,
hydroxyalkylated amino group, guanidinium, and heterocylic groups
containing an unaromatized nitrogen ring atom, such as, but not
limited to morpholinyl or piperazinyl); carboxylic acids including
but not limited to propiolic acid (which may provide a carboxylate
anionic surface); phosphonic acids, including but not limited to
ethynyl phosphonic acid (which may provide a phosphonate anionic
surface); sulfonate anions; carboxybetaines; sulfobetaines;
sulfamic acids; or amino acids. An alkyl or fluoroalkyl moiety may
have a backbone chain length of equal to or greater than 10
carbons. In some embodiments, the alkyl or fluoroalkyl moiety may
have a backbone chain length of about 10, 12, 14, 16, 18, 20, or 22
carbons.
[0230] The linking group LG may be directly or indirectly connected
to the moiety providing support cell growth, viability,
portability, or any combination thereof within the microfluidic
device. When the linking group LG is directly connected to the
moiety, optional linker L is not present and n is 0. When the
linking group LG is indirectly connected to the moiety, linker L is
present and n is 1. The linker L may have a linear portion where a
backbone of the linear portion may include 1 to 200 non-hydrogen
atoms selected from any combination of silicon, carbon, nitrogen,
oxygen, sulfur and phosphorus atoms, subject to chemical bonding
limitations as is known in the art. It may be interrupted with any
combination of one or more moieties selected from the group
consisting of ether, amino, carbonyl, amido, or phosphonate groups,
in some non-limiting examples. Additionally, the linker L may have
one or more arylene, heteroarylene, or heterocyclic groups
interrupting the backbone of the linker. In some embodiments, the
backbone of the linker L may include 10 to 20 atoms. In other
embodiments, the backbone of the linker L may include about 5 atoms
to about 200 atoms; about 10 atoms to about 80 atoms; about 10
atoms to about 50 atoms; or about 10 atoms to about 40 atoms. In
some embodiments, the backbone atoms are all carbon atoms. In other
embodiments, the backbone atoms are not all carbons, and may
include any possible combination of silicon, carbon, nitrogen,
oxygen, sulfur or phosphorus atoms, subject to chemical bonding
limitations as is known in the art.
[0231] In some embodiments, the moiety supporting cell growth,
viability, portability, or any combination thereof may be added to
the surface of the substrate in a multi-step process. When the
moiety is coupled to the surface in a step wise fashion, the linker
L may further include a coupling group CG, as shown in Formula
2.
##STR00003##
[0232] For Formula 2, the moiety configured to support cell growth,
viability, portability or any combination thereof, linker L, and
linking group LG may be as defined for Formula 1. In some
embodiments, the coupling group CG represents the resultant moiety
from reaction of a reactive moiety R.sub.x and a moiety that it is
configured to react with, a reactive pairing moiety R.sub.px. For
example, one typical CG may include a carboxamidyl group, which is
the result of the reaction of an amino group with a derivative of a
carboxylic acid, such as an activated ester, an acid chloride or
the like. CG may include a triazolylene group, a carboxamidyl,
thioamidyl, an oxime, a mercaptyl, a disulfide, an ether, or
alkenyl group, or any other suitable group that may be formed upon
reaction of a reactive moiety with its respective reactive pairing
moiety. The coupling group CG may be located at the second end of
the linker L, where the moiety is attached. In some other
embodiments, the coupling group CG may interrupt the backbone of
the linker L. In some embodiments, the coupling group CG is
triazolylene, which is the result of a reaction between an alkyne
group and an azide group, either of which may be the reactive
moiety or the reactive pairing moiety, as is known in the art for
use in Click coupling reactions. A triazolylene group may also be
further substituted. For example, a dibenzocylcooctenyl fused
triazolylene moiety may result from the reaction of a conditioning
modification reagent having a dibenzocyclooctynyl reactive pairing
moiety R.sub.px with an azido reactive moiety R.sub.x of the
surface modifying ligand, which are described in more detail in the
following paragraphs. A variety of dibenzocyclooctynyl modified
molecules are known in the art or may be synthesized to incorporate
a moiety configured to support cell growth, viability, portability,
or any combination thereof.
[0233] Conditioned surface containing other components. The
conditioned surface may additionally include other components,
other than or in addition to a polymer or a conditioned surface
formed by a covalently linked moiety, including biologically
compatible metal ions (e.g., calcium, sodium, potassium, or
magnesium), antioxidants, surfactants, and/or essential nutrients.
A non-limiting exemplary list includes vitamins such as B7,
alpha-tocopherol, alpha-tocopherol acetate, vitamin A and its
acetate; proteins such as BSA, Catalase, Insulin, Transferrin,
Superoxide Dismutase; small molecules such as corticosterone,
D-galactaose, ethanolamine hydrochloride, reduced glutathione,
L-carnitine hydrochloride, linoleic acid, linolenic acid,
progesterone, putrescine dihydrochloride, and triiodo--thyronine;
and salts, including but not limited to sodium selenite, sodium
phosphate, potassium phosphate, calcium phosphate, and/or magnesium
phosphate. Antioxidants may include but are not limited to
carotenoids, cinnamic acids and derivatives, ferulic acid,
polyphenols such as flavonoids, quinones and derivatives (including
mitoquinone-Q), N-acetyl cysteine, and antioxidant vitamins such as
ascorbic acid, vitamin E and the like. The conditioned surface may
include a culture medium supplement such as B-27.RTM. Supplement,
which contains antioxidants and many of the other components listed
above. B-27.RTM. Supplement is commercially available (50.times.),
serum free from ThermoFisher Scientific, (Cat# 17504044).
[0234] In some embodiments, the at least one conditioned surface
may include one or more components of mammalian serum. In some
embodiments, the mammalian serum is Fetal Bovine Serum (FBS), or
Fetal Calf Serum (FCS). The conditioned surface may include
specific components of mammalian serum such as specific amounts and
types of proteins usually found in serum, which may be provided in
defined amounts and type from serum free or defined media.
[0235] In other embodiments, the at least one conditioned surface
does not include a mammalian serum. In various embodiments, the at
least one conditioned surface may not include any titanium, nickel,
or iron metal ions. In yet other embodiments, the at least one
conditioned surface may not include any significant concentration
of titanium, nickel, or iron metal ions. In yet another embodiment,
the at least one conditioned surface may not include any gold,
aluminum, or tungsten metal ions.
[0236] Any of the components of the conditioned surface, including
polymers that may be flowed in to condition the surface, may be
used in any combination as a surface replenishment reagent.
[0237] Fluidic medium. With regard to the foregoing discussion
about microfluidic devices having a channel and one or more
incubation chambers, a fluidic medium (e.g., a first medium and/or
a second medium) can be any fluid that is capable of maintaining a
reporter cell and/or biological cell of interest and, optionally a
micro-object in a substantially viable state. The viable state will
depend on the reporter cell, the biological cell of interest, and,
if present, a micro-object, and the nature of the assay(s) being
performed. For example, if the biological cell that is being
evaluated by the methods described herein include analyzing for the
secretion of a protein of interest, the cell would be substantially
analyzable provided that it is viable and capable of expressing and
secreting proteins.
[0238] The fluidic medium may provide both fluidic and dissolved
gaseous components necessary for cell viability, and may also
maintain pH in a desired range, using either buffered fluidic media
or pH monitoring or both.
[0239] If the cell is a mammalian cell, the fluidic medium may
include mammalian serum or a defined serum free medium as is known
in the art, which is capable of providing essential nutrients,
hormones, growth factors or cell growth signals. Similarly to the
conditioned surface above, the fluidic medium may include Fetal
Bovine Serum (FBS), or Fetal Calf Serum (FCS). Alternatively, the
fluidic medium may not include any animal sourced serum but may be
a defined medium which may include any or all of physiologically
relevant metal ions (including but not limited to sodium,
potassium, calcium, magnesium, and/or zinc) antioxidants,
surfactants, and/or essential nutrients. A non-limiting exemplary
list includes vitamins such as B7, alpha-tocopherol,
alpha-tocopherol acetate, vitamin A and its acetate; proteins such
as BSA, Catalase, Insulin, Transferrin, Superoxide Dismutase; small
molecules such as corticosterone, D-galactaose, ethanolamine
hydrochloride, reduced glutathione, L-carnitine hydrochloride,
linoleic acid, linolenic acid, progesterone, putrescine
dihydrochloride, and triiodo--thyronine; and salts, including but
not limited to sodium selenite, sodium phosphate, potassium
phosphate, calcium phosphate, and/or magnesium phosphate. Other
components may be present in the fluidic medium, as is known in the
art, and may be selected for a particular purpose.
[0240] In some embodiments, a suitable culture medium may include
or may be composed entirely of any of Dulbecco's Modified Eagle's
medium (ThermoFisher Scientific, Cat # 11960-051); FreeStyle.TM.
Medium (Invitrogen, ThermoFisher Scientific, Cat. No. 11960-051);
RPMI-1640 (GIBCO.RTM., ThermoFisher Scientific, Cat. No.
11875-127); Hybridoma-SFM (ThermoFisher Scientific, Cat. No.
12045-076); Medium E (Stem Cell, Cat. No. 3805); 1.times. CD CHO
Medium (ThermoFisher Scientific, Cat. No. 10743-011); Iscove's
Modified Dulbecco's Medium (ThermoFisher Scientific, Cat. No.
12440-061); or CD DG44 medium (ThermoFisher Scientific, Cat. No.
10743-011).
[0241] The culture medium may additionally include may include
Fetal Bovine Serum (FBS, available from GIBCO.RTM., ThermoFisher
Scientific), Heat Deactivated Fetal Bovine Serum; or Fetal Calf
Serum (FCS, Sigma-Aldrich Cat Nos. F2442, F6176, F4135 and others).
FBS may be present at a concentration of about 1% to about 20% v/v;
about 1% to about 15%v/v, about 1% to about 10% v/v, or about 1% to
about 5% v/v, or any number within any of the ranges. The culture
medium may additionally include Human AB serum (Sigma-Aldrich, Cat.
No. S2146), and may be present in a concentration of about 1% to
about 20% v/v; about 1% to about 15%v/v, about 1% to about 10% v/v,
or about 1% to about 5% v/v, or any number within any of the
ranges.
[0242] The culture medium may additionally include
penicillin-streptomycin (ThermoFisher Scientific, Cat. No.
15140-163). The pen-strep may be present in a concentration in a
range of about 0.01% to about 10% v/v; about 0.1% to about 10% v/v;
about 0.01% to about 5% v/v; about 0.1% to about 5% v/v; about 0.1%
to about 3% v/v; about 0.1% to about 2% v/v; about 0.1% to about 1%
v/v; or any value within any of the ranges. In other embodiments,
the culture medium may include geneticin (ThermoFisher Scientific,
Cat. No. 101310-035). Geneticin may be present in a concentration
of about 0.5 micrograms/ml; about 1.0 micrograms/ml; about 5.0
micrograms/ml; about 10.0 micrograms/ml; about 15 micrograms/ml;
about 20 micrograms/ml; about 30 micrograms/ml; about 50
micrograms/ml; about 70 micrograms/ml; about 100 micrograms/ml; or
any value in these ranges.
[0243] The culture medium may include a buffer. The buffer may be
one of Good's buffers. The buffer may be, but is not limited to
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
(HEPES)(ThermoFisher Scientific, Cat. No. 15630-080. The buffer may
be present in a concentration of about 1 millimolar; about 3
millimolar; about 5 millimolar; about 7 millimolar; about 9
millimolar; about 10 millimolar; about 12 millimolar; about 15
millimolar; about 20 millimolar; about 40 millimolar; about 60
millimolar; about 100 millimolar; or any values in these
ranges.
[0244] The culture medium may additionally include a dipeptide
substitute for glutamine, GlutaMAX.TM. (GIBCO.RTM. ThermoFisher
Scientfic, Cat No.35050-079). The substitute for glutamine may be
present in a concentration of about 0.2 millimolar; about 0.5
millimolar; about 0.7 millimolar; about 1.0 millimolar; about 1.2
millimolar; about 1.5 millimolar; about 1.7 millimolar; about 2.0
millimolar; about 2.5 millimolar; about 3.0 millimolar; about 4.0
millimolar; about 7.0 millimolar, or about 10.0 millimolar, or any
value in these ranges. The culture medium may include MEM
non-essential Amino Acid (ThermoFisher Scientific, Cat. No.
10370-088). The MEM non-essential Amino Acid may be present in a
concentration of about 0.2 millimolar; about 0.5 millimolar; about
0.7 millimolar; about 1.0 millimolar; about 1.2 millimolar; about
1.5 millimolar; about 1.7 millimolar; about 2.0 millimolar; about
2.5 millimolar; about 3.0 millimolar; about 4.0 millimolar; about
7.0 millimolar, or about 10.0 millimolar, or any value in these
ranges.
[0245] The culture medium may additionally contain glucose
(ThermoFisher Scientific, Cat. No. 15023-021). Glucose may be
present in a concentration of about 0.1 g/L; about 0.1 g/L; about
0.1 g/L; about 0.3 g/L; about 0.5 g/L; about 0.8 g/L; about 1.0
g/L; about 1.5 g/L; about 2.0 g/L; about 2.5 g/L; about 3.0 g/L;
about 3.5 g/L; about 4.0 g/L; about 5.0 g/L; about 7.0 g/L; about
10.0 g/L; or any values in these ranges.
[0246] The culture medium may additionally include mercaptoethanol
(ThermoFisher Scientific, Cat. No. 31350-010). Mercaptoethanol may
be present in a concentration of about about 0.001% to about 1.5%
v/v; about 0.005% to about 1.0% v/v; about 0.01% to about 1.0% v/v;
about 0.15% to about 1.0% v/v; about 0.2% to about 1% v/v; or any
value in these ranges.
[0247] The culture medium may include OPI culture medium additive,
including oxaloacetate, pyruvate, and insulin (Sigma-Aldrich, Cat.
No. O-5003). OPI culture medium additive may be present in a
concentration of about 0.001% to about 1.5% v/v; about 0.005% to
about 1.0% v/v; about 0.01% to about 1.0% v/v; about 0.15% to about
1.0% v/v; about 0.2% to about 1% v/v; or any value in these ranges.
The culture medium may contain B-27 supplement (50.times.), serum
free (ThermoFisher Scientific, Cat. No. 17504-163). B-27 supplement
may be present in a concentration of about 0.01% to about 10.5%
v/v; about 0.05% to about 5.0% v/v; about 0.1% to about 5.0% v/v;
about 0.5% to about 5% v/v; or any value in these ranges.
[0248] As described herein, a culture medium or an additive for a
culture medium may include one or more Pluronic.RTM. polymers
useful for yielding a conditioned surface, and may include
Pluronic.RTM. L44, L64, P85, F68 and F127 (including F127NF). The
Pluronic.RTM. polymer may be present in the culture medium at a
concentration of about 0.001% v/v to about 10% v/v; about 0.01% v/v
to about 5% v/v; about 0.01% v/v to about 1% v/v, or about 0.05% to
about 1% v/v. For a culture medium additive which may be provided
as a kit, the concentration may be 1.times., 5.times., 10.times.,
100.times., or about 100.times. the final culture medium
concentration.
[0249] The culture medium may include IL 6 (Sigma-Aldrich, Cat. No.
SRP3096-20UG). IL 6 may be present in a concentration of about 1
nM; about 5 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM,
about 30 nM, about 40 nM, about 50 nM or any values in these
ranges.
[0250] The culture medium may additionally include sodium pyruvate
(ThermoFisher Scientific, Cat. No. 11360-070). The substitute for
glutamine may be present in a concentration of about 0.1
millimolar; about 0.02 millimolar; about 0.04 millimolar; about
0.06 millimolar; about 0.08 millimolar; about 0.1 millimolar; about
0.5 millimolar; about 0.7 millimolar; about 1.0 millimolar; about
1.2 millimolar; about 1.5 millimolar; about 1.7 millimolar; about
2.0 millimolar; about 2.5 millimolar; about 3.0 millimolar; about
4.0 millimolar; about 7.0 millimolar, or about 10.0 millimolar, or
any value in these ranges.
[0251] The fluidic medium may be sterile filtered through a 0.22
micron filter unit (VWR , Cat. No. 73520-986).
[0252] Summary of the system. A system is provided for assaying at
least one biological cell of interest in a microfluidic device,
including a microfluidic device including a flow region configured
to contain a flow of a first fluidic medium and at least one
incubation chamber, wherein the at least one incubation chamber is
configured to contain at least one reporter cell and the at least
one biological cell of interest; and at least one reporter cell. In
some embodiments, the incubation chamber may be configured to
contain no more than a single biological cell of interest. In other
embodiments, the incubation chamber may contain a plurality of
biological cells. The incubation chamber may contain one, about 3,
about 5, about 7, or fewer than 10 biological cells of interest.
Fewer than ten biological cells may be 2, 3, 4, 5, 6, 7, 8, 9 or 10
cells. The biological cells may be members of a clonal population.
The biological cells may be derived from a nonclonal cell
population. The incubation chamber may include an isolation region.
The conditioned surface of the microfluidic device may include a
polymer. In various embodiments of the system, the at least one
incubation chamber of the microfluidic device may have at least one
surface conditioned to support cell growth, viability, portability,
or any combination thereof. In some embodiments, the at least one
conditioned surface of the incubation chamber may include a
polymer. In some embodiments, the polymer of the at least one
conditioned surface of the microfluidic device may include alkylene
oxide moieties, amino acid moieties or saccharide moieties. In
various embodiments, the at least one conditioned surface of the
microfluidic device may include a covalently linked conditioned
surface. In various embodiments, the covalently linked conditioned
surface may include alkylene ether moieties, alkyl moieties,
fluoroalkyl moieties, amino acid moieties, or saccharide moieties.
In some embodiments, the covalently linked conditioned surface may
be linked to the surface via a siloxy linking group. In various
embodiments, the conditioned surface may be a monolayer. The
microfluidic device of the system may include a plurality of
incubation chambers.
[0253] The incubation chamber may be configured to isolate the at
least one reporter cell and the at least one biological cell of
interest. The incubation chamber may be configured to locate the at
least one reporter cell and the at least one biological cell of
interest at spatially distinct locations within the incubation
chamber. In some embodiments, the reporter cell may be located at
the distal end of the isolation region and the biological cell may
be in the midsection or the proximal end of the isolation region.
Other combinations of reporter cell placement and biological cell
placement are also suitable.
[0254] The microfluidic device further may further include a flow
channel that includes at least a portion of the flow region, and
the incubation chamber may include a connection region that opens
directly into the flow channel. The isolation region of the
incubation chamber may be fluidically connected to the flow channel
via the connection region and may be configured to contain a second
fluidic medium, where when the flow region and the incubation
chamber are substantially filled with the first and second fluidic
media respectively, then components of the second fluidic medium
may diffuse into the first fluidic medium and/or components of the
first fluidic medium may diffuse into the second fluidic medium;
and the first medium may not substantially flow into the isolation
region. In some embodiments, the biological cell(s) are disposed
within the isolation region and the reporter cell is located in the
channel, adjacent to the proximal opening of the isolation
chamber.
[0255] The reporter cell(s) may be configured to provide a
detectable signal. The detectable signal may be produced when the
biological cell(s) of interest include a biological activity of
interest. The reporter cell(s) may be configured to produce a
different detectable signal when the biological cell(s) of interest
do not have the biological activity of interest. The detectable
signal of the reporter cell(s) may be colorimetric, fluorescent, or
bioluminescent.
[0256] The flow region (which may be a flow channel) of the
microfluidic device may further include one or more capture
micro-objects. Each of the capture micro-objects may include a
binding substance configured to specifically bind to a biological
product of the at least one biological cell of interest. The
biological product may be a secreted biological product. In some
embodiments, when the biological product is bound to the binding
substance of the capture micro-object producing a binding product,
each capture micro-object may be a bound capture micro-object. The
bound capture micro-objects may be configured to be detectable. The
bound capture micro-objects may be indirectly or directly
detectable. The detectable signal of bound capture micro-objects
may be fluorescent or chemiluminescent. The capture micro-objects
may include a bead. In some embodiments, the one or more capture
micro-objects may include a magnetic bead.
[0257] The capture micro-objects are in fluid connection with the
one or more biological cells of interest. The one or more capture
micro-objects may be located in the connection region of the
incubation chamber or in the flow region proximal to the incubation
chamber. In some embodiments, the capture micro-objects may be in
the isolation region but may not be in physical contact with the
biological cell(s). In other embodiments, the one or more capture
micro-objects may not be located in the isolation region of the
incubation chamber.
[0258] The detectable signal of the reporter cell(s) and the
detectable signal of the bound capture micro-object(s) are may be
spectrally distinct.
[0259] The microfluidic device of the system may further include at
least one inlet port configured to input the first or second
fluidic medium into the flow region and at least one outlet port
configured to receive the first medium as it exits from the flow
region. The system may be configured to perfuse the first medium
within the microfluidic device to maintain cell viability.
Perfusion may be irregular or periodic.
[0260] In some embodiments, the microfluidic device of the system
may further include a substrate having a plurality of electrodes,
wherein a surface of the substrate may form a surface of the
incubation chamber and the flow region. The plurality of electrodes
may be configured to generate a dielectrophoresis (DEP) force. The
system may further include a selector control module configured to
activate and deactivate each of the electrodes, wherein activation
of an electrode may generate a dielectrophoresis (DEP) force
sufficiently strong to move the biological cell(s) into or out of
the at least one incubation chamber or the isolation region
thereof. Each of the plurality of electrodes may be optically
actuated. The DEP force may be produced by optoelectronic tweezers
(OET).
[0261] In other embodiments, the microfluidic device of the system
may further include a substrate having an electrode connected to a
plurality of transistors, wherein a surface of the substrate may
form a surface of the incubation chamber and the flow region. Each
transistor of the plurality may be configured to generate a
dielectrophoresis (DEP) force. The system may further include a
selector control module configured to activate and deactivate each
of the plurality of transistors, thereby generating a
dielectrophoresis (DEP) force sufficiently strong to move the
biological cell(s) into or out of the incubation chamber or the
isolation region thereof. Each of the transistors may be optically
actuated. In some embodiments, the DEP force may be produced by
optoelectronic tweezers (OET).
[0262] In other embodiments, the microfluidic device of the system
may further include a substrate having an electrode and a layer of
amorphous silicon, wherein a surface of the substrate may form a
surface of the incubation chamber and the flow region. The system
may further include a selector control module configured to
activate and deactivate the virtual electrode in the layer of
amorphous silicon, thereby generating a dielectrophoresis (DEP)
force sufficiently strong to move the biological cell(s) into or
out of the at least one incubation chamber or the isolation region
thereof. The layer of amorphous silicon may be optically activated.
The DEP force may be produced by optoelectronic tweezers (OET).
[0263] Alternatively, the biological cell(s) may be moved into or
out of the incubation chamber by fluid flow and/or gravity. In some
embodiments, no more than one biological cell may be introduced
into any incubation chamber. In some embodiments, a plurality of
biological cells may be introduced into an incubation chamber. When
the microfluidic device includes a plurality of isolation chambers,
a plurality of biological cells may be introduced to one or more of
the plurality of isolation chambers. In other embodiments, a
plurality of biological cells may be introduced to each isolation
chamber of the plurality. In other embodiments, fewer than ten
biological cells may be introduced an incubation chamber. When the
microfluidic device includes a plurality of isolation chambers,
fewer than ten biological cells may be introduced to any of the
plurality, or fewer than ten biological cells may be added to each
isolation chamber of the plurality.
[0264] The system may be configured to move the reporter cell into
or out of the incubation chamber or the isolation region thereof by
fluid flow and/or gravity.
[0265] In various embodiments of the system, the biological cell
may be a mammalian cell. The biological cell may be a hybridoma.
The biological cell may be a lymphocyte or a leukocyte. The
biological cell may be a B cell, T cell, NK cell, dendritic cell,
or macrophage. The biological cell may be an adherent cell.
[0266] The system may further include a light source configured to
provide excitation energy to a moiety configured to be detectable
by fluorescence. The system may further include a detector
configured to capture an image of the at least one incubation
chamber and any biological cells contained therein. The detector
may capture images under visible, infrared, or ultraviolet
wavelengths of light.
[0267] Cells. In the methods described herein, cells from many
different sources may be used. The biological cell may be a
mammalian cell. In some embodiments, the mammalian cell may be
human, murine, porcine, or any other mammal of interest.
Alternatively, the cell may be non-mammalian, e.g., a bacterial
cell, a fungal cell or any kind of suitable cell. The cell may be
derived from a cell culture sample or other bioproduction process.
The cell may be derived from bone marrow, blood, muscle, skin, or
fat. The cell may be derived from a solid tissue, bone, blood,
urine, fecal, tears, sweat, synovial fluid, pleural fluid, or
aqueous humor sample. The biological cell may be derived from a
fine needle aspirate, lung lavage sample, or a biopsy sample.
[0268] The cell may be derived from breast and/or mammary gland,
lymph node, intestinal tissue, liver, lung, neural, bone, blood,
pancreatic sample, or a genitourinary tissue. The cell may be a
lymphocyte or a leukocyte. The cell may be an immune cell. The cell
may be a B or T cell. The cell may be an adherent cell.
[0269] The cell may be a proliferative cell. A proliferative cell
may have uncontrolled growth and demonstrate disregulated growth,
e.g., as in a tumor cell. A proliferative cell may alternatively
have comparatively slow growth, but have high potential for
differentiation such as a cancer stem cell. In some embodiments,
the cell may be a tumor cell. In some embodiments, the cell may be
a stem cell. In some other embodiments, the cell may be a cancer
stem cell.
[0270] Any of the above listed cell types may be used in any of the
methods described herein. Further, the methods are not limited to
reporter cell assays of the classes of cells described specifically
but may be performed with any type of cell which may be found to be
of interest and may be imported into the microfluidic device
described herein.
[0271] Methods of performing reporter cell assays. Reporter cell
assays of various types are commercially available, including
systems providing colorimetric, fluorescent, or bioluminescent
signals when the reporter cell is contacted by an expression
inducing species secreted or excreted by the biological cell of
interest.
[0272] Using the microfluidic devices, systems and methods
described herein to perform reporter cell assays provides the
ability to probe the biological activity of biological cell(s) of
interest. In some embodiments, each biological cell may be placed
in a separate incubation chamber in order to probe its activity
without also probing activity of any other biological cell. The
ability to place reporter cell(s) and biological cell(s)
selectively into an incubation chamber of the microfluidic device
may afford the ability to study an individual biological cell or a
small group of biological cells (e.g., fewer than ten cells)
without interfering signals from other cells or assay components.
In other embodiments, a plurality of biological cells may be placed
in the incubation chamber, and the activity of the plurality of
cells may be examined without interfering signals from other cells
or assay components. In some embodiments, a dielectrophoresis (DEP)
force may be used to provide the selective placement of reporter
cell(s), biological cell(s), and/or micro-object(s) (see below).
The DEP force may be optically actuated. The dielectrophoresis
(DEP) force may be provided by optoelectronic tweezers (OET).
[0273] In particular, reporter cell(s) may be introduced to a
specific location in an isolation region of the incubation chamber.
Reporter cells may be introduced to the distal end of an isolation
region, a particular portion of an isolation region or any
spatially distinct location in the isolation region such that the
reporter cell(s) may not come into physical contact with the
biological cell of interest.
[0274] The biological cell(s) of interest may be introduced to a
specific location in an isolation region of the incubation chamber.
The biological(s) cell may be introduced to a midpoint of an
isolation region, while the reporter cell(s) may be at the distal
end of the isolation region. In other embodiments, the biological
cell(s) may be introduced to a specific location in the isolation
region that is spatially distinct from the location of the reporter
cell(s). Typically, the biological cell may be introduced into the
isolation region such that the biological cell(s) may not come into
physical contact with the reporter cell(s).
[0275] Any useful reporter cell format and signaling species may be
used, where the reporter cell is configured to produce a detectable
signal when the at least one biological cell produces a biological
activity of interest. The detectable signal may be directly or
indirectly observable. The detectable signal may be a colorimetric,
fluorescent or bioluminescent signal. The fluorescent or
bioluminescent signal may be produced by Forster Resonance Energy
Transfer (FRET) or by Bioluminescence Resonance Energy Transfer
(BRET), amongst other mechanisms. A variety of biological
activities of the biological cell(s) may be probed using reporter
cell assays of these classes.
[0276] Some classes of reporter cell assays include reporter genes
based on beta-lactamase, beta-galactosidase, green fluorescent
protein, and/or luciferase, amongst others. In some of the reporter
cell assays, differentiable signals may be produced when the
biological cell has/does not have the biological activity of
interest, e.g., a secreted product of interest. In some embodiments
of these assays, a detectable signal having a first wavelength may
be produced when the biological product of interest is produced,
while a detectable signal having a second wavelength is produced
with none of the biological product is produced. The first and
second signals may be spectrally distinct.
[0277] Additionally, the reporter cell assay may be performed
simultaneously or sequentially with binding assays, where a
biological product of the biological cell(s) (which may be the same
or different from the biological product probed by the reporter
cell assay) binds to a capture micro-object. The ability to perform
more than one type of assay at the same time upon a single
biological cell may be useful for multiplexing assays with limited
materials and/or time. The capture micro-object may be a bead. In
some embodiments, the capture-micro-object may be a magnetic bead.
A capture micro-object may include a binding substance configured
to specifically bind the biological product of the biological cell,
forming a bound capture micro-object which may be detectable. The
binding substance may be covalently or noncovalently attached to
each of the micro-object(s). One non-limiting example of a binding
substance may be an antibody, which may recognize the biological
product. Many other classes of substances may be used as a binding
substance of the capture micro-object, as is known in the art.
[0278] The bound capture micro-object(s) may be configured to be
directly or indirectly detectable. A detectable signal of the bound
capture micro-object(s) may be a colorimetric, fluorescent, or
chemiluminescent signal. In some embodiments, the detectable signal
may be produced by FRET or a separate biological species such as a
pyrophosphatase, which may produce a luminescent signal.
[0279] The capture micro-object(s) may be introduced into various
locations near but not in physical contact with the biological
cell(s). The capture micro-object(s) may be introduced to be
adjacent to a proximal opening of the incubation chamber in the
flow region. In other embodiments, the capture micro-object(s) may
be introduced into the connection region of the incubation region.
In yet other embodiments, introducing the capture micro-object(s)
may not include introducing the at least one capture micro-object
to the isolation region of the incubation chamber.
[0280] Summary of some of the embodiments of the method. A method
is provided for assaying at least one biological cell for a
biological activity in a system comprising a microfluidic device
having at least one incubation chamber and a flow region, the
method including the steps of introducing the biological cell(s)
into the incubation chamber; introducing one or more reporter cells
into the incubation chamber; and analyzing the one or more reporter
cells for an activity stimulated by the presence of a biological
activity of the biological cell.
[0281] In various embodiments, the reporter cell(s) may be
configured to produce a detectable signal when the biological cell
comprises the biological activity. The detectable signal of the
reporter cell(s) may be a colorometric, fluorescent, or
bioluminscent signal.
[0282] The step of introducing the one or more reporter cells into
the incubation chamber may be performed before the step of
introducing the biological cell(s) therein. The one or more
reporter cells may be introduced into an isolation region of the
incubation chamber. The biological(s) cell may be introduced into
an isolation region of the incubation chamber. In some embodiments,
a single biological cell may be introduced into the isolation
region of the incubation chamber. In other embodiments, a plurality
of biological cells may be introduced into the isolation region of
the incubation chamber. The biological cell(s) may be introduced to
a spatially distinct region of the isolation region from the one or
more reporter cells.
[0283] The step of incubating may further include providing the one
or more reporter cell(s) with one or more reagents forming one or
all of the components of the detectable signal of the one or more
reporter cell(s).
[0284] The step of analyzing may include incubating the at least
one biological cell(s) and the reporter cell(s) in the incubation
chamber for a pre-determined period of time, thereby producing the
detectable signal of the reporter cell(s). In some embodiments, the
step of analyzing may further include analyzing the reporter
cell(s) at more than one time point during the incubation period.
The step of analyzing the reporter cell(s) may further include
providing excitation light to excite a fluorophore of the
detectable signal of the reporter cell(s).
[0285] The method(s) may further include detecting the detectable
signal of the reporter cell(s). The methods may further include
quantifying the detectable signal of the reporter cell(s), thereby
quantifying the presence of the biological activity. In various
embodiments, the one or more reporter cells may be configured to
produce a second detectable signal when the at least one biological
cell does not comprise the biological activity. In some
embodiments, the second detectable signal may be different from the
first detectable signal. For example, the first signal may have a
longer wavelength from that of the second signal such that the
first signal is spectrally distinct from the second signal. In
various embodiments, incubating the biological cell(s) and the
reporter cell(s) for the pre-determined period of time may include
producing the second detectable signal of the reporter cell(s),
thereby indicating an absence of the biological activity. In some
embodiments, analyzing the one or more reporter cells may further
include providing excitation light to excite a fluorophore of the
second detectable signal of the reporter cell(s). The method may
further include detecting the second detectable signal of the
reporter cell(s). The method may further include quantifying the
detectable signal of the reporter cell(s), thereby quantifying the
absence of the biological activity.
[0286] The method(s) may further include introducing at least one
capture micro-object into at least the flow region. The capture
micro-object(s) may be introduced magnetically. In various
embodiments, the method further includes introducing the
micro-object(s) to be adjacent to a proximal opening of the
incubation chamber in the flow region. In other embodiments, the
micro-object(s) may be introduced into the connection region of the
incubation region. In yet other embodiments, introducing the
capture micro-object(s) may not include introducing the at least
one capture micro-object to the isolation region of the incubation
chamber. The capture micro-object(s) may include a bead. In some
embodiments, the bead may be a magnetic bead.
[0287] In various embodiments of the method(s), each of the capture
micro-object(s) may include a binding substance configured to
specifically bind a biological product of the at least one
biological cells, thereby forming a bound capture micro-object
configured to be detectable. The binding substance may be
covalently or noncovalently attached to each of the
micro-object(s). The bound capture micro-object(s) may be
configured to be directly or indirectly detectable. A detectable
signal of the bound capture micro-object(s) may be a colorimetric,
fluorescent, bioluminescent or chemiluminescent signal. The
biological product of the biological cell may be a secreted
biological product.
[0288] The method(s) may further include incubating the capture
micro-object(s) during the incubation period, thereby producing the
bound capture micro-object(s). The method may further include
introducing one or more visualization reagents which are configured
to bind to the bound capture micro-object(s) to produce the
detectable signal. The visualization reagent(s) may be present
during the incubation period or may be introduced after completion
of the incubation period. The method may further include providing
excitation light to excite the detectable signal of the bound
capture micro-object(s). The method may further include detecting
the detectable signal of the bound capture micro-object(s). The
method may further include quantifying the detected signal of the
binding substance. The detectable signal of the binding substance
may be same or different from the detectable signal from the
reporter cell(s). If the signals are different, the signal may be
spectrally distinct.
[0289] In the method(s), the microfluidic system used to perform
the assays may include any of the embodiments of the system
described herein, in any combination.
[0290] In various embodiments, introducing the at least one
biological cell into the microfluidic device, incubation chamber,
isolation region or location within the isolation region thereof,
may include using a dielectrophoresis (DEP) force having sufficient
strength to move the biological cell. The DEP force may be produced
by optoelectronic tweezers (OET).
[0291] In various embodiments of the method(s), introducing the
reporter cell(s) into the at least one incubation chamber may
include using fluid flow and/or gravity.
[0292] In various embodiments of the method(s), introducing one or
more capture micro-objects into the flow region may include using
fluid flow and/or gravity.
[0293] The method(s) may further include introducing a first
fluidic medium into a flow channel of the flow region of the
microfluidic device. In various embodiments, the rate of
introducing the first fluidic medium may not sweep the isolation
region of the incubation chamber.
[0294] The method(s) may further comprising perfusing the first
fluidic medium during the incubating step, wherein the first
fluidic medium is introduced via at least one inlet port of the
microfluidic device and wherein the first fluidic medium,
optionally comprising components from the second fluidic medium is
exported via at least one outlet of the microfluidic device. In
some embodiments, perfusing may be non-continuous. In other
embodiments, perfusing may be periodic. In some embodiments, the
first fluidic medium may be perfused at a rate sufficient to permit
components of the second fluidic medium in the isolation region to
diffuse into the first fluidic medium in the flow region and/or
components of the first fluidic medium to diffuse into the second
fluidic medium in the isolation region; and at the rate wherein the
first medium does not substantially flow into the isolation
region.
[0295] In the methods, the biological cell may be a mammalian cell.
In other embodiments, the biological cell may be a hybridoma. The
biological cell may be a lymphocyte or a leukocyte. The biological
cell may be a B cell, NK cell, T cell, dendritic cell, or
macrophage. In some embodiments, the biological cell may be an
adherent cell.
[0296] A composition is provided including a biological cell and
one or more reporter cells in an isolation region of a microfluidic
device, where the one or more reporter cells are configured to
detect a biological activity of the biological cell when contacted
by a first extracellular species produced by the single biological
cell. The microfluidic device of the composition may have at least
one incubation chamber and a flow region, where the at least one
incubation chamber includes an isolation region and a connection
region, wherein the isolation region is fluidically connected to
the connection region and the connection region comprises an
opening directly into the flow region. The microfluidic device may
include at least one conditioned surface configured to support cell
growth, viability, portability or any combination thereof. The at
least one conditioned surface may be covalently linked to at least
one surface of the at least one incubation chamber. The at least
one conditioned surface may include an alkylene ether moiety
configured to support cell growth, viability, portability or any
combination thereof. In other embodiments, the at least one
conditioned surface may include an alkyl or fluoroalkyl (including
perfluoroalkyl) moiety configured to support cell growth,
viability, portability or any combination thereof. In some other
embodiments, the at least one conditioned surface may include a
dextran moiety configured to support cell growth, viability,
portability or any combination thereof. The at least one biological
cell and one or more reporter cells may be in contact with the at
least one conditioned surface.
[0297] The extracellular species produced by the biological cell
may contact the reporter cell(s) without the biological cell
directly contacting any of the reporter cells. When the reporter
cell(s) are contacted by the extracellular species, then the
reporter cell(s) may be configured to produce a first detectable
signal.
[0298] In some embodiments of the composition, when the reporter
cell(s) are not contacted by the extracellular species, then the
reporter cell(s) may be configured to produce a second detectable
signal. The first detectable signal of the one or more reporter
cells may be different from the second detectable signal of the one
or more reporter cells. The first and the second detectable signal
of the one or more reporter cells may be a colorimetric,
fluorescent, or bioluminescent signal.
[0299] The composition may further include at least one capture
micro-object, wherein the capture micro-object may be configured to
bind an extracellular species produced by the biological cell,
without physically contacting the biological cell. The
extracellular species produced by the biological cell that binds to
the at least one capture micro-object may be different from the
extracellular species produced by the single biological cell that
is detected by the one or more reporter cells. The at least one
capture micro-object may not be located within the isolation
region. The capture micro-object(s) may be located within the
connection region. In other embodiments, the capture
micro-object(s) may be located at the proximal opening of the
connection region of the incubation chamber into the flow channel.
In other embodiments the capture micro-object(s) may be located
within the isolation region of the incubation chamber, but may not
physically contact the biological cell. The capture micro-object(s)
may be configured to form at least one detectable bound capture
micro-object when the extracellular species binds to the capture
micro-object(s). The bound capture micro-object(s) may be directly
or indirectly detectable. The detectable signal of the bound
capture micro-object(s) may be fluorescent or chemiluminescent.
[0300] In the composition, the biological cell may be a mammalian
cell. In other embodiments, the biological cell may be a hybridoma.
The biological cell may be a lymphocyte or a leukocyte. The
biological cell may be a B cell, T cell, NK cell, dendritic cell,
or macrophage. In some embodiments, the biological cell may be an
adherent cell.
[0301] Kits. A kit is provided for assaying at least one biological
cell, where the kit includes a microfluidic device having at least
one incubation chamber and a flow region; and one or more reporter
cells configured to test for a biological activity of the
biological cell. In some embodiments, the kit may further include
one or more reagents used to provide a detectable signal from the
reporter cells configured to test for a biological activity of the
biological cell.
[0302] The kit may further include one or more micro-objects
configured to bind a biological product of the biological cell. The
components of the kit may be provided in separate containers.
[0303] The microfluidic device of the kit may further include a
flow channel including at least a portion of the flow region, and
where the incubation chamber includes a connection region that
opens directly into the flow channel. The incubation chamber may
further include an isolation region. The isolation region may be
fluidically connected to the connection region and may be
configured to contain a second fluidic medium, where when the flow
region and the at least one incubation chamber are substantially
filled with the first and second fluidic media respectively, then
components of the second fluidic medium may diffuse into the first
fluidic medium and/or components of the first fluidic medium may
diffuse into the second fluidic medium; and the first medium may
not substantially flow into the isolation region. The microfluidic
device of the kit may further include a plurality of incubation
chambers.
[0304] The microfluidic device of the kit may further include at
least one inlet port configured to input the first or second
fluidic medium into the flow region and at least one outlet
configured to receive the first medium, optionally containing
components of the second fluidic medium, as it exits from the flow
region.
[0305] The microfluidic device of the kit may include a substrate
having a plurality of DEP electrodes, wherein a surface of the
substrate may form a surface of the incubation chamber and the flow
region. The plurality of DEP electrodes may be configured to
generate a dielectrophoresis (DEP) force sufficiently strong to
introduce the cell of interest into or to move one or more cells of
interest out of the incubation chamber or the isolation region
thereof. The plurality of DEP electrodes may be optically actuated.
The DEP force may be produced by optoelectronic tweezers (OET).
[0306] In other embodiments, the microfluidic device of the kit may
include a substrate having a DEP electrode connected to a plurality
of transistors, wherein a surface of the substrate may form a
surface of the incubation chamber and the flow region. The
plurality of transistors may be configured to generate a
dielectrophoresis (DEP) force sufficiently strong to introduce the
biological cell or to move one or more cells of interest out of the
incubation chamber or the isolation region thereof. Each of the
plurality of transistors may be a phototransistor. Each of the
plurality of transistors may be optically actuated. The DEP force
may be produced by optoelectronic tweezers (OET).
[0307] In some other embodiments, the microfluidic device of the
kit may include a substrate having a DEP electrode, wherein a
surface of the substrate may form a surface of the incubation
chamber and the flow region. The electrode may be configured to
generate a dielectrophoresis (DEP) force sufficiently strong to
introduce one or more biological cells into or move the one or more
cells of interest out of the at least one incubation chamber or the
isolation region thereof. The DEP electrode may be optically
activated. The DEP force may be produced by optoelectronic
tweezers.
[0308] In some embodiments of the kit, the microfluidic device may
further include at least one conditioned surface configured to
support cell growth, viability, portability or any combination
thereof. In some embodiments of the kit, the kit may further
include a reagent to replenish the conditioned surface. In some
embodiments, the at least one conditioned surface of the at least
one incubation chamber may include a polymer. In various
embodiments, the polymer of the at least one conditioned surface of
the microfluidic device may include alkylene oxide moieties, amino
acid moieties or saccharide moieties. In other embodiments, the at
least one conditioned surface of the microfluidic device may
include a covalently linked conditioned surface. In various
embodiments, the covalently linked conditioned surface may include
alkylene ether moieties, alkyl moieties, fluoroalkyl moieties,
amino acid moieties, or saccharide moieties. The at least one
conditioned surface may include an alkylene ether moiety configured
to support cell growth, viability, portability or any combination
thereof. In other embodiments, the at least one conditioned surface
may include an alkyl or fluoroalkyl (including perfluoroalkyl)
moiety configured to support cell growth, viability, portability or
any combination thereof. In some other embodiments, the at least
one conditioned surface may include a dextran moiety configured to
support cell growth, viability, portability or any combination
thereof. In some embodiments, the covalently linked conditioned
surface may be linked to the surface via a siloxy linking
group.
[0309] While embodiments have been shown and described, various
modifications may be made without departing from the scope of the
inventive concepts disclosed herein. The invention(s), therefore,
should not be limited, except as defined in the following
claims.
* * * * *