U.S. patent application number 17/176561 was filed with the patent office on 2021-10-14 for flow cells utilizing surface-attached structures, and related systems and methods.
This patent application is currently assigned to Redbud Labs, Inc.. The applicant listed for this patent is Redbud Labs, Inc., THE UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL. Invention is credited to Jay Kenneth Fisher, Richard Chasen Spero, Richard Superfine.
Application Number | 20210316303 17/176561 |
Document ID | / |
Family ID | 1000005678745 |
Filed Date | 2021-10-14 |
United States Patent
Application |
20210316303 |
Kind Code |
A1 |
Spero; Richard Chasen ; et
al. |
October 14, 2021 |
FLOW CELLS UTILIZING SURFACE-ATTACHED STRUCTURES, AND RELATED
SYSTEMS AND METHODS
Abstract
A flow cell is provided that includes surface-attached
structures in a chamber. The structures are movable in response to
a magnetic or electric field. A target extraction or isolation
system includes the flow cell and a driver configured for applying
a magnetic or electric field to the interior of the flow cell to
actuate movement of the structures. The flow cell may be utilized
to extract or isolate a target from a sample flowing through the
flow cell. Further, a microfluidic system is provided that includes
surface-attached structures and a microarray, wherein actuated
motion of the surface-attached structures is used to enhance flow,
circulation, and/or mixing action for analyte capture on the
microarray.
Inventors: |
Spero; Richard Chasen;
(Research Triangle Park, NC) ; Fisher; Jay Kenneth;
(Research Triangle Park, NC) ; Superfine; Richard;
(Chapel Hill, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Redbud Labs, Inc.
THE UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL |
Research Triangle Park
Chapel Hill |
NC
NC |
US
US |
|
|
Assignee: |
Redbud Labs, Inc.
Research Triangle Park
NC
THE UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL
Chapel Hill
NC
|
Family ID: |
1000005678745 |
Appl. No.: |
17/176561 |
Filed: |
February 16, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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15952195 |
Apr 12, 2018 |
10919036 |
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17176561 |
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15761109 |
Mar 18, 2018 |
10900896 |
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PCT/US2016/052463 |
Sep 19, 2016 |
|
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15952195 |
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62347046 |
Jun 7, 2016 |
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62220906 |
Sep 18, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 3/502715 20130101;
B01L 2300/0636 20130101; B01L 2200/0631 20130101; B01L 2200/14
20130101; B01L 2400/0475 20130101; B01L 2400/043 20130101; G01N
33/54366 20130101; B01L 3/502761 20130101; G01N 33/54373 20130101;
B01L 2300/0816 20130101; B01L 3/502746 20130101; B01L 2400/0415
20130101; B01L 2200/0647 20130101 |
International
Class: |
B01L 3/00 20060101
B01L003/00; G01N 33/543 20060101 G01N033/543 |
Claims
1-83. (canceled)
84. A target extraction system, comprising: a flow cell with a
plurality of flow cell units, such that the fluid inlet or the
fluid outlet of each flow cell unit communicates with the fluid
inlet or the fluid outlet of at least one other flow cell unit.
each flow cell unit comprising a chamber and a plurality of
surface-attached structures to the inside surface at a plurality of
respective attachment sites and extending into the interior
therefrom, and a driver configured for applying a magnetic or
electric field to the interior of the flow cell to actuate movement
of the surface-attached structures; wherein application of a
magnetic or electric field actuates the surface-attached structure
into movement relative to the corresponding attachment site.
85. A target extraction system of claim 84, wherein the driver is
configured for varying a parameter of the magnetic or electric
field selected from the group consisting of: magnetic or electric
field strength; magnetic or electric field direction; a frequency
at which the magnetic or electric field is cycled between ON and
OFF states or high-strength and low-strength states; and a
combination of two or more of the foregoing.
86. The target extraction system of claim 84, wherein the driver
comprises electromagnets or electrodes, and the driver is
configured for varying electrical power applied to the
electromagnets or the electrodes.
87. The target extraction system of claim 84, wherein the driver
comprises one or more magnets, and the driver is configured for
moving one or more of the magnets relative to the housing; for
moving one or more of the magnets in a reciprocating manner.
88. The target extraction system of claim 84, wherein the driver is
configured for rotating one or more of the magnets about a
longitudinal axis of the flow cell, or for moving one or more of
the magnets in a direction toward or away from the flow cell, or
both of the foregoing.
89. The target extraction system of any of claim 84, wherein the
driver comprises a motor configured for powering movement of one or
more of the magnets.
90. The target extraction system of any of claim 84, comprising a
fluid supply source configured for flowing a fluid to a fluid input
of the flow cell, the fluid supply source comprising a sample
source configured for flowing a target-containing sample to the
fluid input; wherein the sample source is configured for flowing
the target-containing sample to the fluid inputs of the flow cell
units simultaneously when the plurality of flow cell units are
arranged in parallel; or the sample source is configured for
flowing the target-containing sample to the fluid input of a first
flow cell unit of the plurality of flow cell units arranged in
series.
91. The target extraction system of claim 84, comprising a
processing fluid source configured for flowing a processing fluid
to the fluid input of the flow cell, wherein the processing fluid
source is selected from the group consisting of: a source of
processing fluid comprising a release agent effective for releasing
targets bound to a surface inside the flow cell; a source of
processing fluid comprising a rinsing agent effective for purging
the flow cell of residual components from a previous operation of
the flow cell; and both of the foregoing; wherein the processing
fluid source comprises a source of release agent selected from the
group consisting of: a chemical lysing agent; a pH cell lysing
agent; an enzymatic liquefaction agent; and a solvent.
92. The target extraction system of any of claim 84, further
comprising a photon source configured for directing photons into
the flow cell under conditions effective for releasing targets
bound to the surface-attached structures by photolysis.
93. The target extraction system of any of claim 84, comprising a
receptacle configured for receiving processed fluid from the fluid
output of the flow cell units simultaneously, when the plurality of
flow cell units are arranged in parallel; and the receptacle
configured for receiving processed fluid from the fluid output of a
last flow cell unit when the plurality of flow cell units are
arranged in series; wherein the receptacle is part of or
communicates with an analytical instrument configured for measuring
an attribute of the targets collected from the flow cell units.
94. A method for extracting a target from a sample, the method
comprising: flowing a target-containing sample through a flow cell
and into contact with surface-attached structures disposed in the
flow cell, wherein the surface-attached structures are attached to
an inside surface of the flow cell at a plurality of respective
attachment sites, and the surface-attached structures are movable
in the flow cell relative to the attachment sites in response to
magnetic or electric actuation; and while flowing the sample,
isolating targets of the sample from a remaining portion of the
sample.
95. The method of claim 94, wherein isolating targets comprises
binding the targets to a binding agent disposed in the flow cell;
wherein the binding agent is selected from the group consisting of:
a binding agent disposed on or integrated with an outer surface of
at least some of the surface-attached structures; a binding agent
disposed on or integrated with the inside surface; and both of the
foregoing.
96. The method of claim 94, wherein isolating targets comprises
releasing the targets after binding, such that the released targets
are flowable out from the flow cell; wherein releasing selected
from the group consisting of: flowing a release agent through the
flow cell and into contact with the bound targets; irradiating the
bound targets with photons under conditions effective for inducing
photolysis; applying a shear force to the bound targets at a
magnitude effective for unbinding the bound targets; and a
combination of two or more of the foregoing.
97. The method of claim 94, wherein isolating targets comprises,
releasing the targets after binding, such that the released targets
are flowable out from the flow cell; wherein releasing comprises
flowing a release agent through the flow cell and into contact with
the bound targets, and the release agent is selected from the group
consisting of: a chemical lysing agent; a pH cell lysing agent; an
enzymatic liquefaction agent; and a solvent.
98. The method of claim 94, wherein isolating targets comprises,
releasing the targets after binding, such that the released targets
are flowable out from the flow cell; wherein releasing comprises
applying a shear force to the bound targets at a magnitude
effective for unbinding the bound targets, and applying the shear
force is selected from the group consisting of: flowing a liquid
through the flow cell at a flow rate effective for releasing the
bound targets by shearing; applying a magnetic or electric field to
the flow cell to actuate movement of the surface-attached
structures at a speed effective for releasing the bound targets by
shearing; both of the foregoing.
99. The method of claim 94, wherein a binding agent is disposed on
or integrated with an outer surface of at least some of the
surface-attached structures, and further comprising, while flowing
the sample, applying a magnetic or electric field to the flow cell
to actuate movement of the surface-attached structures in a
reciprocating manner to increase a time-averaged cross-section of
the surface-attached structures.
100. The method of claim 94, wherein isolating targets comprises
trapping the targets by preventing the targets from passing between
neighbouring surface-attached structures; and after trapping,
releasing the targets by applying a magnetic or electric field to
the flow cell to actuate movement of the surface-attached
structures.
101. The method of claim 94, wherein isolating targets comprises
separating the targets from non-targets of the sample by size or
density, such that the targets and the non-targets elute from the
flow cell at different times; wherein: the targets have a different
size than the non-targets; the inside surface is a top inside
surface, and the flow cell further comprises a bottom inside
surface spaced from the top inside surface such that a
structure-free region is between the surface-attached structures
and the bottom inside surface; and the surface-attached structures
are positioned with an inter-structure spacing effective for
forcing either the targets or the non-targets, whichever are
larger, to flow substantially only through the structure-free
region; and wherein: the targets have a different density than the
non-targets; the inside surface is a top inside surface, and the
flow cell further comprises a bottom inside surface spaced from the
top inside surface such that a structure-free region is between the
surface-attached structures and the bottom inside surface and is
below the surface-attached structures; and flowing the sample
through the flow cell is done at a flow rate effective for allowing
a majority of either the targets or the non-targets, whichever are
denser, to diffuse into the structure-free region and toward the
bottom inside surface.
102. The method of claim 94, comprising, while flowing the sample,
applying a magnetic or electric field to the flow cell to actuate
movement of the surface-attached structures; comprising moving the
surface-attached structures at a speed or frequency effective for
causing an effect selected from the group consisting of: adjusting
or varying an inter-structure spacing between the surface-attached
structures; preventing or disrupting clogging of sample material
between the surface-attached structures; preventing or disrupting
non-specific binding of sample material on the surface-attached
structures; and a combination of two or more of the foregoing.
103. The method of any of claim 94, comprising, after isolating the
targets, transferring the targets to an analytical instrument, and
operating the analytical instrument to measure an attribute of the
targets.
104. A method for extracting a target from a sample, the method
comprising: flowing a target-containing sample through a flow cell
and into contact with surface-attached structures disposed in the
flow cell, wherein the surface-attached structures are attached to
an inside surface of the flow cell at a plurality of respective
attachment sites, and the surface-attached structures are movable
in the interior relative to the attachment sites in response to
magnetic or electric actuation; and while flowing the sample,
capturing the targets on the surface-attached structures, or on the
inside surface, or on both the surface-attached structures and the
inside surface, and wherein capturing produces a depleted sample
containing a reduced concentration of the targets.
105. The method of claim 104, comprising outputting the depleted
sample from the flow cell, wherein the captured targets remain
captured in the flow cell, and releasing the captured targets and
outputting the released targets from the flow cell.
106. A method for extracting a target from a sample, the method
comprising: flowing a target-containing sample through a flow cell
and into contact with surface-attached structures disposed in the
flow cell, wherein the surface-attached structures are attached to
an inside surface of the flow cell at a plurality of respective
attachment sites, and the surface-attached structures are movable
in the flow cell relative to the attachment sites in response to
magnetic or electric actuation; and while flowing the sample,
isolating targets of the sample from a remaining portion of the
sample; wherein, applying the magnetic or electric field moves the
surface-attached structures at a speed or frequency effective for
causing an effect selected from the group consisting of: releasing
the targets bound to the surface-attached structures by shearing;
adjusting or varying an inter-structure spacing between the
surface-attached structures; preventing or disrupting clogging of
sample material between the surface-attached structures; preventing
or disrupting non-specific binding of sample material on the
surface-attached structures; and a combination of two or more of
the foregoing.
107. The method for extracting a target from a sample of claim 94,
wherein the target is a non-analyte target comprising an
interferent, suppressant, and/or element contributing only to
background signal, and wherein the non-analyte target is isolated
from the sample to purge the sample of the non-analyte target
and/or to analyze the sample in the absence of the non-analyte
target.
108. A flow cell or system configured for performing the method of
extracting a target from a sample, comprising a plurality of
binding agents, wherein at least some of the binding agents are
disposed on or integrated with outer surfaces of at least some of
the surface-attached structures; wherein the binding agents
disposed on or integrated with the outer surfaces are arranged in a
plurality of groups, and the groups are spaced from each other as a
one-dimensional or two-dimensional array of groups, and wherein
each group is separated by one or more adjacent groups by
surface-attached structures that do not include binding agents;
wherein the groups are spaced from each other as a two-dimensional
array of groups comprising a plurality of rows of groups, and each
row is staggered or offset relative to a row adjacent thereto
109. The flow cell of claim 108, comprising a plurality of binding
agents, wherein at least some of the binding agents are arranged as
a microarray spaced from the plurality of surface-attached
structures by a gap in the interior; wherein the microarray is
arranged as a plurality of capture sites, each capture site
comprising one or more of the binding agents; wherein the
microarray is arranged as a two-dimensional array of capture sites
comprising a plurality of rows of capture sites, and each row is
staggered or offset relative to a row adjacent thereto
110. The flow cell of claim 109, wherein the microarray is selected
from the group consisting of a DNA microarray, an MMChip, a protein
microarray, a peptide microarray, a tissue microarray, a cellular
microarray, a small molecule microarray, a chemical compound
microarray, an antibody microarray, a carbohydrate microarray, a
phenotype microarray, and a reverse phase protein microarray.
111. The flow cell of claim 109, comprising a detector in operative
communication with the microarray, wherein the microarray comprises
a substrate configured for transmitting photons, at an angle from
about 0 degrees to about 45 degrees; and the detector comprises a
florescence-based optical detection mechanism; wherein the
microarray comprises an electrically conductive or semiconductor
substrate, and the detector comprises an electrical signal-based
detection mechanism.
112. The flow cell of claim 109, comprising a detector configured
for measuring a property of a fluid in the interior, a property of
the surface-attached structures, or a property of binding agents in
the interior.
113. The flow cell of claim 109, wherein the surface-attached
structures are oriented substantially along a direction normal to
the inside surface at which are the surface-attached structures
attached, or are oriented at an angle with respect to the direction
normal to the inside surface.
114. The flow cell of claim 109, wherein the surface-attached
structures have a configuration selected from the group consisting
of: the surface-attached structures are configured to move with a
side-to-side two-dimensional motion; the surface-attached
structures are configured to move with a circular motion; the
surface-attached structures are configured to move in a tilted
motion relative to the inside surface to which the surface-attached
structures are attached; a combination or two or more of the
foregoing.
115. A microfluidic device, comprising: a chamber enclosing an
interior configured for containing a fluid, the chamber comprising
an inside surface facing the interior; and a plurality of
surface-attached structures attached to the inside surface at a
plurality of respective attachment sites and extending into the
interior therefrom, each surface-attached structure being movable
in response to an actuation force selected from the group
consisting of: a magnetic force, a thermal force, a sonic force, an
optical force, an electrical force, and a vibrational force.
116. The microfluidic device of claim 115, comprising a fluid port
communicating with the chamber, wherein the fluid port is sealable
or closable, a fluid inlet and a fluid outlet separate from the
fluid inlet, the fluid inlet and the fluid outlet communicating
with the chamber, and a plurality of analyte capture elements
disposed in the interior; wherein the analyte capture elements are
disposed on the inside surface, on a substrate separate from the
inside surface, on one or more of the surface-attached structures,
or a combination of two or more of the foregoing.
117. A method of reducing reaction time of an assay comprising
conducting the assay in the microfluidic device of claim 115
wherein the reaction time is reduced by at least about six times
(6.times.) as compared to microfluidic devices that do not utilize
the motion of surface-attached structures due to actuation forces
to enhance the flow, circulation, and/or mixing action of a fluid
sample.
118. A method of enhancing flow, circulation, and/or mixing action
of a fluid sample comprising: depositing the fluid sample in the
chamber of the microfluidic device of claim 115; and applying the
actuation force to the surface-attached structures; wherein the
flow, circulation, and/or mixing action of the fluid sample is
enhanced as compared to microfluidic devices that do not utilize
the motion of surface-attached structures due to actuation forces.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation application of
U.S. Utility patent application Ser. No. 15/952,195, filed Apr. 12,
2018, which is a continuation-in-part application of U.S. Utility
patent application Ser. No. 15/761,109, filed Mar. 18, 2018, which
is a 35 U.S.C. .sctn. 371 national phase entry of International
Patent Application Number PCT/US2016/052463, filed Sep. 19, 2016,
which claims priority to U.S. Provisional Patent Application No.
62/347,046, filed Jun. 7, 2016, and U.S. Provisional Patent
Application No. 62/220,906, filed Sep. 18, 2015, the disclosures of
which are incorporated herein by reference in their entireties.
TECHNICAL FIELD
[0002] This present invention generally relates to flow cells and
systems and methods utilizing flow cells for processing fluids
containing analytes or other targets of interest. In particular,
the invention relates to flow cells that utilize surface-attached
structures, and related systems and methods.
BACKGROUND
[0003] A wide variety of techniques involve the isolation or
extraction of one or more selected components from a fluid for
analytical or purification purposes, such as immunoassays,
centrifugation, filtering, chromatography, solid phase extraction
(SPE), and others. Conventional techniques include the use of
steric filters and columns of packed beads, both of which are prone
to clogging. Magnetic beads have also been utilized, but generally
cannot achieve the superior surface area-to-volume ratio offered by
packed columns unless a very large number of magnetic beads are
utilized. To reduce clogging, pre-separation techniques may be
utilized such as pre-filters, guard columns, centrifugation,
pipetting, etc., but such measures add to the complexity, cost, and
size of the associated system.
[0004] In addition, there are currently many assays in which a
fluid sample is placed into a microfluidic chamber and then made to
wash over capture sites on a microarray (e.g., dots of reagents,
oligonucleotides, proteins, etc.). In such assays, the fluid sample
sits for a period of time to allow for the reaction of analytes in
the fluid sample with the capture sites on the microarray.
Typically, such assays allow the analytes in the fluid sample to
flow throughout the chamber by diffusion, resulting in very long
reaction times that can last for multiple days. For devices in
which short reaction times are important (e.g., point of care (POC)
devices), the reaction has to be stopped before all of the analyte
has had time to diffuse to corresponding capture sites, resulting
in low analyte utilization and analyte waste.
[0005] Therefore, there is a need for devices, systems, and methods
for isolating or extracting one or more selected components from a
fluid that minimize or avoid clogging. There is also a need for a
device capable of isolating or extracting one or more selected
components from a fluid flowing through the device, and which is
inherently structured to minimize clogging and/or provides a
structure may be actively operated to prevent or disrupt clogging.
Furthermore, there is a need for devices, systems, and methods for
enhancing flow, circulation, and/or mixing action for analyte
capture on a microarray.
SUMMARY
[0006] To address the foregoing problems, in whole or in part,
and/or other problems that may have been observed by persons
skilled in the art, the present disclosure provides methods,
processes, systems, apparatus, instruments, and/or devices, as
described by way of example in implementations set forth below.
[0007] According to one embodiment, a flow cell includes: a chamber
enclosing an interior and comprising a fluid inlet, a fluid outlet,
and an inside surface facing the interior; and a plurality of
surface-attached structures attached to the inside surface at a
plurality of respective attachment sites and extending into the
interior therefrom, each surface-attached structure comprising a
flexible body and a metallic component disposed on or in the body,
wherein application of a magnetic or electric field actuates the
surface-attached structure into movement relative to the
corresponding attachment site.
[0008] According to another embodiment, a flow cell includes: a
plurality of flow cell units, each flow cell unit comprising a
chamber and a plurality of surface-attached structures according to
any of the embodiments disclosed herein, wherein the plurality of
flow cell units has a configuration selected from the group
consisting of: the flow cell units are stacked in parallel; and the
flow cell units are arranged in series such that the fluid inlet or
the fluid outlet of each flow cell unit communicates with the fluid
inlet or the fluid outlet of at least one other flow cell unit.
[0009] According to an embodiment, a flow cell includes: a chamber
enclosing an interior and comprising a fluid inlet, a fluid outlet,
and an inside surface facing the interior; and a plurality of
surface-attached structures attached to the inside surface at a
plurality of respective attachment sites and extending into the
interior therefrom, each surface-attached structure comprising a
flexible body and a metallic component disposed on or in the body,
wherein application of a magnetic or electric field actuates the
surface-attached structure into movement relative to the
corresponding attachment site.
[0010] According to another embodiment, a flow cell includes: a
chamber enclosing an interior and comprising a fluid inlet, a fluid
outlet, and an inside surface facing the interior; and a plurality
of surface-attached structures attached to the inside surface at a
plurality of respective attachment sites and extending into the
interior therefrom, each surface-attached structure being movable
in response to an actuation force selected from the group
consisting of: a magnetic force, a thermal force, a sonic force, an
optical force, an electrical force, and a vibrational force.
[0011] According to another embodiment, the flow cell includes a
binding agent configured for binding to a target present in the
interior. The binding agent may be disposed on or integrated with
an outer surface of at least some of the surface-attached
structures, or disposed on or integrated with the inside surface,
or both of the foregoing.
[0012] According to another embodiment, the flow cell includes a
plurality of binding agents, wherein at least some of the binding
agents are arranged as a microarray spaced from the plurality of
surface-attached structures by a gap in the interior.
[0013] According to another embodiment, a target extraction system
includes: a flow cell according to any of the embodiments disclosed
herein; a driver configured for applying a magnetic force, and
electric force, or other actuation force to the interior of the
flow cell to actuate movement of the surface-attached structures.
In some embodiments, the target extraction system includes a
housing configured for removably receiving the flow cell.
[0014] According to another embodiment, a method for extracting a
target from a sample includes: flowing a target-containing sample
through a flow cell and into contact with surface-attached
structures disposed in the flow cell, wherein the surface-attached
structures are attached to an inside surface of the flow cell at a
plurality of respective attachment sites, and the surface-attached
structures are movable in the flow cell relative to the attachment
sites in response to magnetic or electric actuation; and while
flowing the sample, isolating targets of the sample from a
remaining portion of the sample.
[0015] According to another embodiment, a method for extracting a
target from a sample includes: flowing a target-containing sample
through a flow cell and into contact with surface-attached
structures disposed in the flow cell, wherein the surface-attached
structures are attached to an inside surface of the flow cell at a
plurality of respective attachment sites, and the surface-attached
structures are movable in the interior relative to the attachment
sites in response to magnetic or electric actuation; and while
flowing the sample, capturing the targets on the surface-attached
structures, or on the inside surface, or on both the
surface-attached structures and the inside surface, and wherein
capturing produces a depleted sample containing a reduced
concentration of the targets.
[0016] According to another embodiment, a method for extracting a
target from a sample includes: flowing a target-containing sample
through a flow cell and into contact with surface-attached
structures disposed in the flow cell, wherein the surface-attached
structures are attached to an inside surface of the flow cell at a
plurality of respective attachment sites, and the surface-attached
structures are movable in the flow cell relative to the attachment
sites in response to magnetic or electric actuation; and while
flowing the sample, applying a magnetic or electric field to the
flow cell to actuate movement of the surface-attached
structures.
[0017] According to another embodiment, a flow cell or system is
configured for performing any of the methods disclosed herein.
[0018] Other devices, apparatus, systems, methods, features and
advantages of the invention will be or will become apparent to one
with skill in the art upon examination of the following figures and
detailed description. It is intended that all such additional
systems, methods, features and advantages be included within this
description, be within the scope of the invention, and be protected
by the accompanying claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The invention can be better understood by referring to the
following figures. The components in the figures are not
necessarily to scale, emphasis instead being placed upon
illustrating the principles of the invention. In the figures, like
reference numerals designate corresponding parts throughout the
different views.
[0020] FIG. 1A is a schematic cross-sectional elevation view of an
example of a flow cell according to some embodiments;
[0021] FIG. 1B is a cross-sectional top plan view of the flow cell
illustrated in FIG. 1A with a top layer thereof removed;
[0022] FIG. 2A is a schematic elevation view of an example of a
chamber of a flow cell according to one embodiment;
[0023] FIG. 2B is a schematic elevation view of an example of a
chamber of a flow cell according to another embodiment;
[0024] FIG. 2C is a schematic elevation view of an example of a
chamber of a flow cell according to another embodiment;
[0025] FIG. 3A is a schematic elevation view of an example of a
single surface-attached structure according to some
embodiments;
[0026] FIG. 3B is a schematic elevation view of another example of
the surface-attached structure according to some embodiments;
[0027] FIG. 4 is a scanning electron micrograph (SEM) of an example
of an array of surface-attached structures attached to a substrate
according to one embodiment;
[0028] FIG. 5 is a schematic view of an example of a chamber of a
flow cell, wherein the chamber is configured for extracting or
isolating a target from a fluid sample according to one
embodiment;
[0029] FIG. 6 is a schematic view of an example of a chamber of a
flow cell, wherein the chamber is configured for extracting or
isolating a target according to another embodiment;
[0030] FIG. 7 is a schematic view of an example of a target
extraction system according to some embodiments;
[0031] FIG. 8 is a perspective view of an example of a target
extraction system (or a portion thereof) according to another
embodiment;
[0032] FIG. 9 is a schematic elevation view of an example of a flow
cell according to an embodiment in which the flow cell includes a
plurality of flow cell units arranged in parallel;
[0033] FIG. 10 is a schematic elevation view of an example of a
flow cell according to an embodiment in which the flow cell
includes a plurality of flow cell units arranged in series;
[0034] FIG. 11 is a perspective view of an example of a
microfluidic system that includes a microarray positioned in
relation to a micropost array, according to an embodiment disclosed
herein;
[0035] FIG. 12A and FIG. 12B are a plan view and a cross-sectional
view, respectively, of an example of a flow cell that is based on
the microfluidic system of FIG. 11, according to an embodiment
disclosed herein;
[0036] FIG. 13A and FIG. 13B are side views of examples of
microposts according to an embodiments disclosed herein;
[0037] FIG. 14A through FIG. 14E are plan views of examples of
configurations of the micropost array according to an embodiments
disclosed herein;
[0038] FIG. 15A and FIG. 15B are side views of a micropost and show
examples of actuation motion thereof;
[0039] FIG. 16 shows a close up cross-sectional view of a portion
of an example of a reaction chamber of the flow cell shown in FIG.
12A and FIG. 12B and show the operation thereof, according to an
embodiment disclosed herein;
[0040] FIG. 17A and FIG. 17B show an example of the flow cell shown
in FIG. 12A and FIG. 12B that includes analyte capture elements on
the microposts according to an embodiment disclosed herein;
[0041] FIG. 18A and FIG. 18B show an example of the flow cell shown
in FIG. 12A and FIG. 12B that includes a microarray in combination
with analyte capture elements on the microposts according to an
embodiment disclosed herein;
[0042] FIG. 19 is a flow diagram of an example of a method of using
a micropost array in conjunction with a microarray for rapidly
flowing target analytes through the bulk fluid;
[0043] FIG. 20A and FIG. 20B are block diagrams of examples of
standalone microfluidic systems that can include a micropost array
and a microarray;
[0044] FIG. 21 is a block diagram of an example of a
high-throughput screening system that can include a micropost array
and a microarray;
[0045] FIG. 22 is a schematic view of an example of a liquid assay
system that is configured for mixing a reaction fluid in a
liquid-based assay for analysis of a target analyte; and
[0046] FIG. 23 is a flow diagram of an example of a method of using
a flow cell with surface-attached structures for creating a mixing
action in a liquid phase reaction.
DETAILED DESCRIPTION
[0047] Definitions
[0048] Unless defined otherwise, all technical and scientific terms
used herein have the meaning commonly understood by a person
skilled in the art to which this invention belongs.
[0049] Following long-standing patent law convention, the terms
"a," "an," and "the" refer to "one or more" when used in this
application, including the claims. Thus, for example, reference to
"a subject" includes a plurality of subjects, unless the context
clearly is to the contrary (e.g., a plurality of subjects), and so
forth.
[0050] Throughout this specification and the claims, the terms
"comprise," "comprises," and "comprising" are used in a
non-exclusive sense, except where the context requires otherwise.
Likewise, the term "include" and its grammatical variants are
intended to be non-limiting, such that recitation of items in a
list is not to the exclusion of other like items that can be
substituted or added to the listed items.
[0051] For the purposes of this specification and appended claims,
unless otherwise indicated, all numbers expressing amounts, sizes,
dimensions, proportions, shapes, formulations, parameters,
percentages, quantities, characteristics, and other numerical
values used in the specification and claims, are to be understood
as being modified in all instances by the term "about" even though
the term "about" may not expressly appear with the value, amount or
range. Accordingly, unless indicated to the contrary, the numerical
parameters set forth in the following specification and attached
claims are not and need not be exact, but may be approximate and/or
larger or smaller as desired, reflecting tolerances, conversion
factors, rounding off, measurement error and the like, and other
factors known to those of skill in the art depending on the desired
properties sought to be obtained by the presently disclosed subject
matter. For example, the term "about," when referring to a value
can be meant to encompass variations of, in some embodiments,
.+-.100% in some embodiments .+-.50%, in some embodiments .+-.20%,
in some embodiments .+-.10%, in some embodiments .+-.5%, in some
embodiments .+-.1%, in some embodiments .+-.0.5%, and in some
embodiments .+-.0.1% from the specified amount, as such variations
are appropriate to perform the disclosed methods or employ the
disclosed compositions. Further, the term "about" when used in
connection with one or more numbers or numerical ranges, should be
understood to refer to all such numbers, including all numbers in a
range and modifies that range by extending the boundaries above and
below the numerical values set forth. The recitation of numerical
ranges by endpoints includes all numbers, e.g., whole integers,
including fractions thereof, subsumed within that range (for
example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as
well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the
like) and any range within that range.
[0052] As used herein, a "target" is any particle (or bioparticle)
carried in a fluid (typically a liquid) such as by entrainment,
suspension or colloidal dispersion, for which isolation from the
fluid is desired. Examples of targets include, but are not limited
to, a biological cell; an intracellular component; a microbe; a
pathogen such as a bacterium, a virus, a prion, or a fungus; a
carcinogen; an antigen; a hapten; an antibody (e.g.,
immunoglobulin); an animal or anti-human antibody (e.g.,
antiglobulin); a toxin; a drug; a steroid; a vitamin; a biopolymer
such as a protein, a carbohydrate, or a nucleic acid; a biological
compound such as a peptide; a hormone; an allergen; a pesticide; a
chemical compound; a molecule; a chemical element (e.g., a trace
metal); a fragment, particle, or partial structure of any of the
foregoing; and binding partners of any of the foregoing. In some
cases the target may be a rare particle in the fluid, and isolation
of the rare particle is desired in order to concentrate the rare
particle for subsequent analysis thereof, or to purify the fluid by
removing the rare particle therefrom, etc.
[0053] In some embodiments, the target is an analyte--that is, it
is desired to isolate the target for the purpose of measuring a
property or attribute thereof, or for detecting its presence in the
fluid. In other embodiments, the target is a non-analyte. For
example, the non-analyte target may be isolated from a fluid for
the purpose of purging the fluid of the target, or for analyzing
the fluid (or components thereof) in the absence of the target. As
another example, the non-analyte target may be considered as an
interferent or suppressant, or as contributing only to background
signal, such that it is desired to analyze the fluid without the
target being present, or to subject the fluid to a reaction without
the target being present. In these latter cases the fluid, or a
species of the fluid other than the target to be isolated, may be
an analyte of interest.
[0054] As used herein, the term "fluid sample" generally refers to
any flowable substance, i.e., a substance that can flow passively
or actively (e.g., by pumping) through a fluid conduit such as a
tube, channel, or chamber. The fluid sample may be, for example, a
bodily (human or animal) fluid (e.g., blood, serum, plasma, other
fluids), a solution containing a biological tissue or cell, a
solution derived from the environment (e.g., surface water, or a
solution containing plant or soil components), a solution derived
from food, or a solution derived from a chemical or pharmaceutical
process (e.g., reaction, synthesis, dissolution, etc.). A fluid
sample may be known to contain or suspected of containing one or
more targets, which may be isolated or extracted from the fluid
sample in accordance with methods disclosed herein.
[0055] As used herein, the term "binding agent" or "binding
partner" refers to any molecule capable of binding to another
molecule, i.e., to another binding partner such as a "target" as
described herein. Thus, examples of binding agents include, but are
not limited to, a biological cell; an intracellular component; a
microbe; a pathogen such as a bacterium, a virus, a prion, or a
fungus; a carcinogen; an antigen; a hapten; an antibody (e.g.,
immunoglobulin); an animal or anti-human antibody (e.g.,
antiglobulin); a toxin; a drug; a steroid; a vitamin; a biopolymer
such as a protein, a carbohydrate, or a nucleic acid; a biological
compound such as a peptide; a hormone; an allergen; a pesticide; a
chemical compound; a molecule; a chemical element (e.g., a trace
metal); a fragment, particle, or partial structure of any of the
foregoing; and binding partners of any of the foregoing. Examples
of molecules that are binding partners to each other include, but
are not limited to, antibody-antigen, antibody-hapten,
hormone-hormone receptor, lectin-carbohydrate, enzyme-enzyme
inhibitor (or enzyme cofactor), biotin-avidin (or streptavidin)
(including derivatives of biotin and avidin), ligand-ligand
receptor, protein-immunoglobulin, and nucleic acid-complementary
nucleic acid (e.g., complementary oligonucleotides, DNA or RNA).
Depending on the type of assay being implemented, a binding partner
may be an analyte to be detected, or may be an intermediate binding
partner utilized in various ways in the course of detecting the
analyte.
[0056] In some embodiments, a binding agent is capable of being
surface-immobilized by a suitable technique such as, for example,
surface functionalization, coating, etc., whereby the binding agent
is consequently disposed on or integrated with a surface. Examples
of surface functionalization techniques include, but are not
limited to, physisorption, graft polymerization, electrostatic
interaction, covalent coupling, and biotin-(strept)avidin coupling.
Depending on the type of binding agent utilized, the binding agent
may be bound or attached to the surface with or without the need
for an intermediary binder or linker or cross-linker molecule.
Depending on the type of binding agent utilized, the surface to be
functionalized or coated may need to be prepared or pre-treated
(e.g., silanization, solvent extraction, solvent impregnation), as
appreciated by persons skilled in the art. A surface-immobilized
binding agent may also be referred to as a "receptor" in some
embodiments.
[0057] In some embodiments, a binding agent may be a
"binding-specific" agent. As used herein, a binding-specific agent
is one that has a high affinity for and readily binds to a specific
type of binding partner, and which under normal assaying conditions
does not bind to any other type of molecule. As an example, a
binding-specific agent may be an antibody that will only bind to a
specific type of antigen, antigen analog or hapten. Depending on
the assay format implemented, a binding-specific agent may be an
analyte-specific receptor, i.e., may act as a direct binding
partner for the analyte to be detected in a fluid sample or for a
conjugate of the analyte or a complex containing the analyte.
Alternatively, a binding-specific agent may be a binding partner
for another non-analyte binding partner, and that other non-analyte
binding partner may in turn be a specific binding partner for the
analyte to be detected.
[0058] The occurrence of a binding-specific agent binding to a
specific type of binding partner may be referred to as a "specific
binding" event. On the other hand, a "non-specific binding" event
or "non-specific adsorption" (NSA) event as used herein generally
refers to the occurrence of a component of a fluid sample (e.g., a
non-target or non-analyte) binding to a surface by a mechanism
other than the specific recognition that characterizes a specific
binding event.
[0059] As used herein, the term "(bio)chemical compound"
encompasses chemical compounds and biological compounds. A chemical
compound may, for example, be a small molecule or a high
molecular-weight molecule (e.g., a polymer). A biological compound
may be, for example, a biopolymer.
[0060] As used herein, the term "oligonucleotide" denotes a
biopolymer of nucleotides that may be, for example, 10 to 300 or
greater nucleotides in length. Oligonucleotides may be synthetic or
may be made enzymatically. Oligonucleotides may contain
ribonucleotide monomers (i.e., may be oligoribonucleotides) and/or
deoxyribonucleotide monomers (i.e., may be
oligodeoxyribonucleotides). Oligonucleotides may include modified
nucleobases. Oligonucleotides may be synthesized as part of or in
preparation for methods disclosed herein, or may be pre-synthesized
and provided as a starting material for methods disclosed herein.
For convenience, oligonucleotides are also referred to herein by
the short-hand term "oligos." Oligos utilized to assemble synthons
may be referred to herein as "synthon precursor oligos" to
distinguish them from other types of oligos that may be utilized or
present in the methods and systems, such as the probes of a capture
array and adaptor oligos (AOs).
[0061] The terms "nucleic acid" and "polynucleotide" are used
interchangeably herein to describe a polymer of any length, e.g.,
greater than about 2 bases, greater than about 10 bases, greater
than about 100 bases, greater than about 500 bases, greater than
1000 bases, up to about 10,000 or more bases composed of
nucleotides, e.g., deoxyribonucleotides or ribonucleotides, and may
be produced enzymatically or synthetically (e.g., PNA as described
in U.S. Pat. No. 5,948,902 and the references cited therein) and
which can hybridize with naturally occurring nucleic acids in a
sequence specific manner analogous to that of two naturally
occurring nucleic acids, e.g., can participate in Watson-Crick base
pairing interactions. In addition to deoxyribonucleic acid (DNA)
and ribonucleic acid (RNA), the terms "nucleic acid" and
"polynucleotide" may encompass peptide nucleic acid (PNA), locked
nucleic acid (LNA), and unstructured nucleic acid (UNA). Nucleic
acids or polynucleotides may be synthesized using methods and
systems disclosed herein.
[0062] As used herein, the term "releasing" in the context of
releasing an oligo from the surface of a support structure refers
to breaking or overcoming a bond or cleavage site of the oligo such
that all or part of the oligo is freed (or unbound, liberated,
detached, untethered, de-anchored, etc.) from the surface.
Typically, releasing an oligo entails "cleaving" the oligo such as
by chemical cleaving, enzymatic cleaving, and photocleaving
techniques, as appropriate for the particular embodiment.
[0063] Certain embodiments disclosed herein entail the use of
"surface-attached structures." Generally, a surface-attached
structure has two opposing ends: a fixed end and a free end. The
fixed end may be attached to a substrate by any suitable means,
depending on the fabrication technique and materials employed. The
fixed end may be "attached" by being integrally formed with or
adjoined to the substrate, such as by a microfabrication process.
Alternatively, the fixed end may be "attached" via a bonding,
adhesion, fusion, or welding process. The surface-attached
structure has a length defined from the fixed end to the free end,
and a cross-section lying in a plane orthogonal to the length. For
example, using the Cartesian coordinate system as a frame of
reference, and associating the length of the surface-attached
structure with the z-axis (which may be a curved axis), the
cross-section of the surface-attached structure lies in the x-y
plane. Generally, the cross-section of the surface-attached
structure may have any shape, such as rounded (e.g., circular,
elliptical, etc.), polygonal (or prismatic, rectilinear, etc.),
polygonal with rounded features (e.g., rectilinear with rounded
corners), or irregular. The size of the cross-section of the
surface-attached structure in the x-y plane may be defined by the
"characteristic dimension" of the cross-section, which is
shape-dependent. As examples, the characteristic dimension may be
diameter in the case of a circular cross-section, major axis in the
case of an elliptical cross-section, or maximum length or width in
the case of a polygonal cross-section. The characteristic dimension
of an irregularly shaped cross-section may be taken to be the
dimension characteristic of a regularly shaped cross-section that
the irregularly shaped cross-section most closely approximates
(e.g., diameter of a circle, major axis of an ellipse, length or
width of a polygon, etc.).
[0064] A surface-attached structure as disclosed herein is movable
(flexible, deflectable, bendable, etc.) relative to its fixed end
or point of attachment to the substrate. To facilitate its
movability, the surface-attached structure may include a flexible
body composed of an elastomeric (flexible) material, and may have
an elongated geometry in the sense that the dominant dimension of
the surface-attached structure is its length--that is, the length
is substantially greater than the characteristic dimension.
Examples of the composition of the flexible body include, but are
not limited to, elastomeric materials such as polydimethylsiloxane
(PDMS).
[0065] The overall shape or geometry of the surface-attached
structure may be generally cylindrical, polygonal, or a combination
of cylindrical and polygonal features. Examples include, but are
not limited to, posts, pillars, rods, bars, paddles, blades, and
the like having circular/elliptical or rectilinear cross-sections.
The characteristic dimension of the surface-attached structure may
be generally constant along its length, or may vary gradually or in
a stepped manner. For example, the surface-attached structure may
be conical or pyramidal, with the characteristic dimension tapering
down in the direction either toward the free end or toward the
fixed end. As another example, a selected portion of the
surface-attached structure may have a smaller characteristic
dimension than that of the remaining portion of the
surface-attached structure. This may be done, for example, to
enhance flexure of the surface-attached structure at that selected
portion.
[0066] In some embodiments, the surface-attached structure has at
least one dimension (length or characteristic dimension) on the
order of micrometers (e.g., from about 1 .mu.m to about 1000 .mu.m)
or nanometers (e.g., less than about 1 .mu.m (1000 nm)). In such
micro-scale embodiments, the surface-attached structures may be
fabricated in accordance with techniques practiced in or derived
from various fields of microfabrication such as microfluidics,
microelectronics, microelectromechanical systems (MEMS), and the
like, now known or later developed.
[0067] The surface-attached structure is configured such that the
movement of the surface-attached structure relative to its fixed
end may be actuated or induced in a non-contacting manner,
specifically by an applied magnetic or electric field of a desired
strength, field line orientation, and frequency (which may be zero
in the case of a magnetostatic or electrostatic field). To render
the surface-attached structure movable by an applied magnetic or
electric field, the surface-attached structure may include an
appropriate metallic component disposed on or in the flexible body
of the surface-attached structure. To render the surface-attached
structure responsive to a magnetic field, the metallic component
may be a ferromagnetic material such as, for example, iron, nickel,
cobalt, or magnetic alloys thereof, one non-limiting example being
"alnico" (an iron alloy containing aluminum, nickel, and cobalt).
To render the surface-attached structure responsive to an electric
field, the metallic component may be a metal exhibiting good
electrical conductivity such as, for example, copper, aluminum,
gold, and silver, and well as various other metals and metal
alloys. Depending on the fabrication technique utilized, the
metallic component may be formed as a layer (or coating, film,
etc.) on the outside surface of the flexible body at a selected
region of the flexible body along its length. The layer may be a
continuous layer or a densely grouped arrangement of particles.
Alternatively, the metallic component may be formed as an
arrangement of particles embedded in the flexible body at a
selected region thereof.
[0068] In other embodiments, the actuation force may be a thermal
force, a sonic force, an optical force, or a vibrational force.
[0069] A microfluidic device or system as disclosed herein may be
composed of a material, for example, of the type utilized in
various fields of microfabrication such as microfluidics,
[0070] microelectronics, micro-electromechanical systems (MEMS),
and the like. The composition of the material may be one that is
utilized in these fields as a semiconductor, electrical insulator
or dielectric, vacuum seal, structural layer, or sacrificial layer.
The material may thus be composed of, for example, a metalloid
(e.g., silicon or germanium), a metalloid alloy (e.g.,
silicon-germanium), a carbide such as silicon carbide, an inorganic
oxide or ceramic (e.g., silicon oxide, titanium oxide, or aluminum
oxide), an inorganic nitride or oxynitride (e.g., silicon nitride
or silicon oxynitride), various glasses, or various polymers such
as polycarbonates (PC), polydimethylsiloxane (PDMS), etc. A solid
body of the material may initially be provided in the form of, for
example, a substrate, a layer disposed on an underlying substrate,
a microfluidic chip, a die singulated from a larger wafer of the
material, etc.
[0071] A microfluidic conduit (e.g., channel, chamber, port, etc.)
may be formed in a solid body of material by any technique, now
known or later developed in a field of fabrication, which is
suitable for the material's composition and the size and aspect
ratio (e.g., length:diameter) of the channel. As non-limiting
examples, the conduit may be formed by an etching technique such as
focused ion beam (FIB) etching, deep reactive ion etching (DRIE),
soft lithography, or a micromachining technique such as mechanical
drilling, laser drilling or ultrasonic milling. Depending on the
length and characteristic dimension of the conduit to be formed,
the etching or micromachining may be done in a manner analogous to
forming a vertical or three-dimensional "via" partially into or
entirely through the thickness of the material (e.g., a
"through-wafer" or "through-substrate" via). Alternatively, an
initially open conduit or trench may be formed on the surface of a
substrate, which is then bonded to another substrate to complete
the conduit. The other substrate may present a flat surface, or may
also include an initially open conduit that is aligned with the
open conduit of the first substrate as part of the bonding
process.
[0072] Depending on its composition, the material defining the
conduit may be inherently chemically inert relative to the fluid
flowing through the conduit. Alternatively, the conduit (or at
least the inside surface of the conduit) may be deactivated as part
of the fabrication process, such as by applying a suitable coating
or surface treatment/functionalization so as to render the conduit
chemically inert and/or of low absorptivity to the material.
Moreover, the inside surface of the conduit may be treated or
functionalized so as to impart or enhance a property such as, for
example, hydrophobicity, hydrophilicity, lipophobicity,
lipophilicity, low absorptivity, etc., as needed or desirable for a
particular application. Alternatively or additionally, the outside
of the conduit may also be treated or functionalized similarly
Coatings and surface treatments/functionalizations for all such
purposes are readily appreciated by persons skilled in the art.
[0073] In some embodiments, the material forming the conduit is
optically transparent for a purpose such as performing an
optics-based measurement, performing a sample analysis, detecting
or identifying a substance flowing through the conduit, enabling a
user to observe flows and/or internal components, etc.
[0074] It will be understood that terms such as "communicate" and
"in communication with" (for example, a first component
"communicates with" or "is in communication with" a second
component) are used herein to indicate a structural, functional,
mechanical, electrical, signal, optical, magnetic, electromagnetic,
ionic or fluidic relationship between two or more components or
elements. As such, the fact that one component is said to
communicate with a second component is not intended to exclude the
possibility that additional components may be present between,
and/or operatively associated or engaged with, the first and second
components.
Flow Cells Utilizing Surface-Attached Structures and Related
Systems and Methods
[0075] FIG. 1A is a schematic cross-sectional elevation view of an
example of a flow cell 100 according to some embodiments.
Specifically, FIG. 1A is a view of the flow cell 100 along its
length. For descriptive purposes, FIG. 1A and other figures include
a Cartesian coordinate frame of reference, which has been
arbitrarily oriented such that the length of the flow cell 100 is
in the direction of the x-axis, the width of the flow cell 100 is
in the direction of the y-axis (through the drawing sheet), and the
height of the flow cell 100 is in the direction of the z-axis.
[0076] Generally, the flow cell 100 includes a chamber 104
enclosing a chamber interior and a plurality of surface-attached
structures 108. The chamber 104 includes a fluid inlet 112 and a
fluid outlet 114 communicating with the chamber interior, and an
inside surface 120 (i.e., one or more inside surfaces) facing the
chamber interior and serving as boundaries thereof. The inside
surface 120 may define all or most of the volume of the chamber
interior. In some embodiments, the interior volume may be on the
order of microliters (e.g., less than about 1000 .mu.L). In some
embodiments, the overall dimensions of the flow cell 100 (length,
width, height) may be on the order of millimeters (e.g., from about
1 mm to about 1000 mm) or micrometers. The flow cell 100 may be
coupled to a fluidic circuit by any suitable means, such as by
utilizing appropriate fittings coupled to the fluid inlet 112 and
the fluid outlet 114 as appreciated by persons skilled in the art.
The flow cell 100 may establish a flow of fluid (typically liquid)
from the fluid inlet 112, through the chamber 104, and to the fluid
outlet 114, as indicated by arrows in FIG. 1A.
[0077] Generally, no limitation is placed on the structural
configuration of the chamber 104 or on the manner in which the
chamber 104 is fabricated. In the embodiment illustrated in FIG.
1A, the chamber 104 includes a first layer 124 (or base), a second
layer 126 (or cover or lid), and a third layer 128 (or intermediate
layer, or spacer) between the first layer 124 and the second layer
126. From the perspective of FIG. 1A, the first layer 124 may be
considered as a bottom layer and the second layer 126 may be
considered as a top layer, in the sense that the first layer 124 is
above the second layer 126 and the first layer 124 and second layer
126 are both above an underlying surface that supports the flow
cell 100 (e.g., benchtop, floor, ground, etc.). The first layer 124
includes a first inside surface 130 facing the chamber interior,
and the second layer 126 includes a second inside surface 132
facing the chamber interior opposite to the first inside surface
130. The first layer 124 (and first inside surface 130) is spaced
from the second layer 126 (and second inside surface 132) by the
third layer 128, which in the present embodiment defines the height
of the chamber 104. Also in the present embodiment, inside surfaces
of the third layer 128 define the shape of the chamber 104 in the
plane orthogonal to the drawing sheet of FIG. 1A. The third layer
128 and the configuration of the chamber 104 are best shown in FIG.
1B, which is a cross-sectional top plan view of the flow cell 100
with the second layer 126 removed, and thus is a view of the flow
cell 100 along its width.
[0078] In the illustrated embodiment, the fluid inlet 112 and the
fluid outlet 114 have been arbitrarily located on the same side of
the flow cell 100, specifically the top side. In this case, the
fluid inlet 112 and the fluid outlet 114 are defined in part by
bores (vias) formed (such as by laser drilling) through the second
layer 126 that are aligned with corresponding openings in the third
layer 128. Positioning the fluid inlet 112 and the fluid outlet 114
on the same side may facilitate certain fabrication techniques.
More generally, however, the fluid inlet 112 and the fluid outlet
114 may be located on different sides of the flow cell 100 (e.g.,
top and bottom, opposing ends, etc.).
[0079] The surface-attached structures 108 may be configured
generally as described above. The plurality of surface-attached
structures 108 is attached to the inside surface 120 of the chamber
104 at a plurality of respective attachment sites, such that the
surface-attached structures 108 extend into the chamber interior
from the inside surface 120. In the illustrated embodiment, the
surface-attached structures 108 are attached to a substrate 136
distinct from the first layer 124 of the flow cell 100, and the
substrate 136 is attached to the first layer 124. It may be
advantageous to form the surface-attached structures 108 on a
distinct substrate 136 in one process, and then attach the
surface-attached structures 108 to the inside surface 120 (by way
of the intervening substrate 136) in a separate process. After
being attached to the inside surface 120, the substrate 136 faces
the chamber interior and serves as a boundary of the chamber
interior, and thus the substrate 136 may be considered as being
part of the inside surface 120. Hence, the description of the
surface-attached structures 108 being "attached to the inside
surface" encompasses embodiments in which a distinct substrate 136
supporting the surface-attached structures 108 is utilized.
Alternatively, the surface-attached structures 108 may be directly
attached to a layer of the chamber 104 such as the first layer
124.
[0080] Referring to FIG. 1B, the surface-attached structures 108
may be arranged in a two-dimensional array. Neighboring (adjacent)
surface-attached structures 108 are spaced from each other by an
"inter-structure spacing," i.e., the distance between two
neighboring surface-attached structures 108. The array may be
ordered in a substantially uniform manner, in which case the
inter-structure spacing is substantially constant throughout the
array. Alternatively, the array may be somewhat randomly ordered,
in which case the inter-structure spacing may vary within some
range of distance values. In some embodiments, the plurality of
surface-attached structures 108 has an inter-structure spacing on
the order of micrometers or nanometers. In the case of a randomly
ordered array of surface-attached structures 108, the
inter-structure spacing may be taken to be the average
inter-structure spacing of the array. In some embodiments, the
inter-structure spacing may be effective for performing separation
by size exclusion or filtration on a target-containing sample
flowing through the chamber 104.
[0081] As also shown in FIG. 1B, the surface-attached structures
108 may be positioned in the fluid flow path established by the
flow cell 100, e.g., in the chamber 104 between the fluid inlet 112
and the fluid outlet 114. Hence, a fluid flowing through the flow
cell 100 will come into contact with the surface-attached
structures 108. This configuration enables the surface-attached
structures 108 to interact with the fluid, or with one or more
desired components of the fluid, as described further below. The
size and density (inter-structure spacing) of the surface-attached
structures 108 may be selected to increase the probability of
contact or interaction with a desired component of the fluid. In
some embodiments, the number and size of the surface-attached
structures 108 and their inter-structure spacing may be such as to
increase the surface area in the chamber 104 by about two times
(2.times.) or greater, or in another example from about two times
(2.times.) to about six times (6.times.) greater, as compared to
the chamber 104 without the surface-attached structures 108. In
some embodiments, the number density of the surface-attached
structures 108 may be in a range from 10.sup.3 to 10.sup.6
structures/cm.sup.2. Moreover, as shown in FIG. 1B the array of
surface-attached structures 108 may span substantially the entire
width (in the vertical direction from the perspective of FIG. 1B)
of the chamber 104 to increase the probability of contact or
interaction.
[0082] As described elsewhere in the present disclosure, the
surface-attached structures 108 are movable by an applied magnetic
or electric field. Because the structures 108 are attached to an
underlying surface, they do not aggregate in the presence of the
magnetic or electric field, unlike magnetic beads which do
aggregate in response to a magnetic field.
[0083] As described above, the plurality of surface-attached
structures 108 is attached to the inside surface 120 of the chamber
104 at a plurality of respective attachment sites. Generally, the
surface-attached structures 108 may be attached to any part of the
inside surface 120, and some surface-attached structures 108 may be
attached to one part while other surface-attached structures 108
are attached to a different part. FIGS. 2A, 2B, and 2C illustrate a
few examples of possible arrangements of the surface-attached
structures 108.
[0084] Specifically, FIG. 2A is a schematic elevation view of an
example of a chamber 204 (along either its length or width) of a
flow cell according to one embodiment. The inside surface of the
chamber 204 includes a first (or bottom) inside surface 230 and an
opposing second (or top) inside surface 232, both facing the
chamber interior and spaced from each other such that chamber
interior is between them. The flow cell (specifically, the chamber
204 in the present embodiment) includes an array of
surface-attached structures 108 as described above. In the
embodiment of FIG. 2A (similar to that of FIGS. 1A and 1B), the
surface-attached structures 108 are attached to the first inside
surface 230 at a plurality of respective attachment sites, and thus
extend generally upward and into the chamber interior from the
first inside surface 230. As also shown in FIG. 2A, the length of
the surface-attached structures 108 (or height in the current
perspective) is less than the height of the chamber interior
(between the first inside surface 230 and the second inside surface
232). Consequently, the chamber interior includes a structure-free
region or gap 240 (i.e., a three-dimensional space devoid of
surface-attached structures 108) between the surface-attached
structures 108 and the second inside surface 232.
[0085] Generally, the intended effects of the region containing the
surface-attached structures 108 and the structure-free region 240,
respectively, on the fluid (and/or components thereof) flowing
through the chamber 204 from the fluid inlet to the fluid outlet
may vary depending on the application. Moreover, the relative
lengths (or heights) of the surface-attached structures 108 and the
structure-free region 240 may vary depending on the application.
For example, in some embodiments the length of the structure-free
region 240 may be less than the length of the surface-attached
structures 108, whereby a majority of the flowing fluid encounters
the structure-free region 240. In other embodiments, the length of
the structure-free region 240 may be greater than or about equal to
the length of the surface-attached structures 108. In some
embodiments, the length of the structure-free region 240 may be
minimized to increase the probability of contact or interaction
with a desired component of the fluid, as the fluid may otherwise
preferentially flow through the structure-free region 240 where
flow resistance is lower. As one non-limiting example, the length
of the structure-free region 240 may be about 10 .mu.m, while in
other examples may be more or less than 10 .mu.m.
[0086] FIG. 2B is a schematic elevation view of the chamber 204
according to another embodiment. In this embodiment, the
surface-attached structures 108 are attached to the second inside
surface 232 at a plurality of respective attachment sites, and thus
extend generally downward and into the chamber interior from the
second inside surface 232. The structure-free region 240 is thus
below instead of above the surface-attached structures 108.
[0087] FIG. 2C is a schematic elevation view of the chamber 204
according to another embodiment. In this embodiment, the flow cell
includes an array of first surface-attached structures 108A
attached to the first inside surface 230 at a plurality of
respective attachment sites, and an array of second
surface-attached structures 108B attached to the second inside
surface 232 at a plurality of respective attachment sites.
Consequently, the structure-free region 240 is located between the
first inside surface 230 and the second inside surface 232.
[0088] Generally, the positioning of the surface-attached
structures 108 in the chamber 204 may depend upon the application.
For example, in a given application, gravitational and/or
density-related effects on one or more components of the fluid may
dictate whether to position surface-attached structures 108 on the
first inside surface 230 (FIG. 2A), on the second inside surface
232 (FIG. 2B), or on both inside surfaces 230 and 232 (FIG. 2C). As
another example, the structure-free region 240 when positioned
below the surface-attached structures 108 (FIG. 2B) may serve as a
collection region for sediments or precipitates. As another
example, providing two or more sets of surface-attached structures
108 having different configurations may be desirable in a given
application. For example, in the embodiment shown in FIG. 2C, the
first surface-attached structures 108A may be configured
differently than the second surface-attached structures 108B. As an
example of different configurations, the first surface-attached
structures 108A may have an inter-structure spacing different from
that of the second surface-attached structures 108B, such as to
achieve two different size exclusion or filtering effects on the
fluid flowing through the chamber 204. As another example of
different configurations, the first surface-attached structures
108A may include a binding agent (described below) while the second
surface-attached structures 108B do not (or vice versa).
Alternatively, the first surface-attached structures 108A may
include a binding agent specific for capturing one type of target
in a fluid sample, while the second surface-attached structures
108B include a binding agent specific for capturing a different
type of target in the same fluid sample.
[0089] In embodiments described thus far, the surface-attached
structures 108 are located on the top or bottom inside surface of a
chamber. Additionally or alternatively, however, surface-attached
structures 108 may be located on a laterally oriented
(side-oriented) inside surface of the chamber, such that the
surface-attached structures 108 extend in a direction generally
parallel with the top or bottom inside surface of the chamber.
[0090] FIG. 3A is a schematic elevation view of an example of a
single surface-attached structure 108 according to some
embodiments. As described above, the surface-attached structure 108
includes a fixed end 342 attached to an underlying surface 320 at
an attachment site, and an opposing free end 344. As described
above, the surface 320 may be an inside surface of a flow cell (or
chamber thereof), or may be a substrate that is attachable to
another surface such as that of a flow cell. The surface-attached
structure 108 has a length L from the fixed end 342 to the free end
344. The surface-attached structure 108 also has a characteristic
dimension D defining the size of the cross-section of the
surface-attached structure 108 that lies in the plane orthogonal to
the axis of the length L. The surface-attached structure 108 has a
flexible body 346. For example, a dominant portion of the
surface-attached structure 108 may be composed of a flexible
material. Consequently, the surface-attached structure 108 is
movable through space in generally any direction except at the
fixed end 342. Thus, the surface-attached structure 108 may be
characterized as being movable relative to its attachment site, or
fixed end 342.
[0091] FIG. 3A schematically illustrates an example of movement of
the surface-attached structure 108 by illustrating the
surface-attached structure 108 at three different positions A, B,
and C. Position A corresponds to an upright position of the
surface-attached structure 108. In the upright position, the
surface-attached structure 108 may extend to a maximum height above
the surface 320 equal to the length L. In the present example, the
upright position corresponds to a nominal position at which the
surface-attached structure 108 is in a non-deflected state. In
other embodiments, however, the surface-attached structure 108 may
be fabricated such that it is nominally or inherently bent to some
degree in while in its non-deflected state, i.e., in the absence of
an applied deflecting force. In response to an appropriately
oriented deflecting force, the surface-attached structure 108 may
be moved to various other positions relative to position A, such as
to position B and/or position C. At position B, the
surface-attached structure 108 has generally been rotated clockwise
about an axis passing through the drawing sheet. At position C, the
surface-attached structure 108 has generally been rotated
counterclockwise about the same axis. It will be understood that
positions B and C are but a few examples of deflected positions
attainable by the surface-attached structure 108. Generally, the
surface-attached structure 108 may rotate about any axis relative
to its attachment site or fixed end 342. Moreover, generally no
limitation is placed on the range of movement of the
surface-attached structure 108. In one example, the range of
movement may be defined by the angular positon of the free end 344
relative to the axis of rotation. For example, positions B and C
may correspond to +/-45 degrees of rotation, respectively, in the
plane of the drawing sheet. Depending on the composition, length,
and characteristic dimension (or aspect ratio, i.e., ratio of
length to characteristic dimension, or L:D) of the surface-attached
structure 108, its maximum range of movement in a given plane may
be more or less than 45 degrees.
[0092] As described above, the surface-attached structure 108
includes a metallic component 348 disposed on or in the flexible
body 346, which enables movement of the surface-attached structure
108 to be actuated or induced through application of a magnetic or
electric field. In the illustrated embodiment, the metallic
component 348 is provided in the form of a continuous layer
disposed on a selected region of the flexible body 346. As
illustrated, the region at which the metallic component 348 is
located may be at or near the free end 344 and thus at an
appreciable distance from the fixed end 342. This configuration may
enhance the responsiveness of the surface-attached structure 108 to
an applied magnetic or electric field.
[0093] Generally, when the magnetic or electric field is applied,
the surface-attached structure 108 experiences a torque that works
to align the dominant axis of the surface-attached structure 108
with the magnetic or electric field. The operating parameters of
the magnetic or electric field may be set, varied, or adjusted as
needed to control movement of the surface-attached structure 108 in
a desired manner. As examples, the strength of the magnetic or
electric field may determine the extent to which the
surface-attached structure 108 is deflected from its nominal,
non-deflected state. The strength of the magnetic or electric field
may be adjusted to move the surface-attached structure 108 from,
for example, position B to some other position between position A
and the surface 320. The spatial orientation (as represented by
field lines, for example) or polarity of the magnetic or electric
field may determine the direction in which the surface-attached
structure 108 is deflected, for example to position B or position
C. The ON/OFF state of the magnetic or electric field may control
whether the surface-attached structure 108 is deflected or not. The
magnetic or electric field may be applied once to move the
surface-attached structure 108 to position B, for example, and
maintained in the ON state for a period of time to hold the
surface-attached structure 108 at position B for that period of
time. The magnetic or electric field may then be removed to release
the surface-attached structure 108, whereby due to its elasticity,
the surface-attached structure 108 returns to its non-deflected
state. The magnetic or electric field may be cycled between ON and
OFF states, or between high-strength and low-strength states, to
oscillate the position of the surface-attached structure 108 at a
desired frequency, for example between position A and position B.
The orientation or polarity of the magnetic or electric field may
be cycled to alternate the direction in which the deflecting force
is positively or actively applied, for example to oscillate the
surface-attached structure 108 between position B and position C.
In some embodiments, the magnetic or electric field may be rotated
to change the axis about which the surface-attached structure 108
rotates, or to cause the surface-attached structure 108 to gyrate
relative to its attachment site or fixed end 342, e.g., gyrate
about the axis of position A or other non-deflected position.
[0094] In some embodiments, for a given fluid and fluid flow rate,
the surface-attached structure 108 may be flexible enough to
deflect in response to fluid flow without the assistance of a
magnetic or electric field--that is, movement of the
surface-attached structure 108 may be actuated by the fluid itself.
In such embodiments, a magnetic or electric field may be applied to
hold the surface-attached structure 108 at a desired position
(e.g., position A, B, or C), in resistance to the force imparted by
the flowing fluid, for a desired period of time. The strength of
the magnetic or electric field may be varied as desired to allow
the surface-attached structure 108 to move to a different position
or to oscillate the surface-attached structure 108.
[0095] FIG. 3B is a schematic elevation view of another example of
the surface-attached structure 108 according to some embodiments.
The surface-attached structure 108 includes a binding agent 352
disposed on or integrated with the outer surface of the
surface-attached structure 108 (e.g., the outer surface of the
flexible body 346). Generally, no limitation is placed on the
manner in which the binding agent 352 is disposed on or integrated
with the outer surface of the surface-attached structure 108. As
examples, the binding agent 352 may be surface-immobilized by a
suitable functionalization technique, applied as a coating, etc.
The binding agent 352 may be configured for binding to a target
present in a fluid that is brought into contact with the binding
agent 352, such as by flowing a fluid sample through the interior
of a flow cell as described herein. The binding agent 352 may have
a specific affinity for the target of interest. For example,
schematically illustrates three different components of a fluid
sample, a target 354 and two different "non-targets" 356 and 358.
The target 354 readily binds to the binding agent 352, whereas the
non-targets 356 and 358 do not. Thus, a surface-attached structure
108 according to such embodiments enables a target 354 to be
extracted or isolated from a fluid, specifically by binding or
capturing the target 354 as the comes into contact with or close
proximity to the binding agent 352. Providing an array of such
surface-attached structures 108 in a flow cell as described above
may enhance binding efficiency (or capture yield) and the ability
to process substantial volumes of fluid in this manner. Generally,
the binding agent 352 may be any of the binding agents noted
earlier in this disclosure. As one non-limiting example, the
binding agent 352 may be avidin or streptavidin, which is capable
of conjugation with any biotin-functionalized molecule desired to
be captured.
[0096] In some embodiments, the binding agent 352 may be disposed
on or integrated with one or more inside surfaces of a chamber of a
flow cell, which may be the same chamber in which surface-attached
structures 108 are provided (e.g., the inside surface 120 shown in
FIGS. 1A and 1B). In some embodiments, the binding agent 352 may be
disposed on or integrated with both the surface-attached structures
108 (as shown in FIG. 3B) and the inside surface(s) of a
chamber.
[0097] In some embodiments, the binding agent 352 may be a sequence
of binding agents. As one non-limiting example, the
surface-attached structure 108 may be functionalized with the
following sequence: avidin-biotin-DNA oligo-avidin-biotin-antibody.
In this case a target cell, for example, may be captured by the
antibody. The target cell may thereafter be released by, for
example, a release agent effective for digesting the oligo.
[0098] In some embodiments, the outer surfaces of the
surface-attached structures 108, or the inside surface of an
associated chamber of a flow cell, or both the outer surfaces and
the inside surface, are chemically pacified to suppress
non-specific binding (or non-specific adsorption (NSA)) and/or
contact-activation of clotting. For example, such surfaces may be
pacified with a surfactant such as a Tween or Triton surfactant,
n-Dodecyl-D-maltoside (DDM), etc.
[0099] While the schematic illustration of FIG. 3B might suggest
the use of a direct binding assaying technique, it will be
understood that FIG. 3B illustrates one example of an assaying
technique that may be utilized to capture targets from a fluid
sample. Other techniques may be utilized such as, for example,
competitive assays, inhibition assays, sandwich assays, etc., as
appreciated by persons skilled in the art.
[0100] FIG. 4 is a scanning electron micrograph (SEM) of an example
of an array of surface-attached structures attached to a substrate
according to one embodiment. The outer surface of the flexible body
of each surface-attached structure is a dark shade, and the outer
surface of the metallic component is a lighter shade. In this
example, the surface-attached structures were fabricated as
described by Judith et al., Micro-elastometry on whole blood clots
using actuated surface-attached posts (ASAPs), Lab Chip, Royal
Society of Chemistry (2015), DOI: 10.1039/c41c01478b, the entire
content of which is incorporated by reference herein. The
surface-attached structures each were fabricated as a core-shell
structure in which a nickel shell (metallic component) encapsulates
the upper region of a polydimethylsiloxane (PDMS) core (flexible
body). A polycarbonate track-etched membrane was utilized as a mold
for the core-shell structures. First, the nickel shells were
created by coating one side of the track-etched membrane with a 200
nm thick layer of gold that serves as the cathode for
electrodeposition of nickel into the pores of the track-etched
membrane. Electrodeposition was performed in an electrolytic cell
with an all-sulfate plating bath. Upon completion, the nickel
containing track-etched membrane was rinsed with deionized water
and dried. The cores of the structures were made by filling the
membrane with PDMS at a 10:1 base to cross-linker ratio. Prior to
curing the PDMS, a 22.times.22 mm glass coverslip was pressed into
the uncured PDMS to provide a rigid substrate for the array. After
curing the PDMS, the gold layer was removed using a
nickel-compatible gold etchant, and the surface-attached structures
were released by dissolving the track-etched membrane using
dichloromethane. The released surface-attached structures were then
stored in ethanol until they were dried using a critical point
drier. The PDMS posts had an elastic modulus of approximately 1
megapascal (MPa) and were able to be bent in response to a magnetic
field generated by a soft iron electromagnet. The array of
surface-attached structures shown in FIG. 4 was incorporated into a
flow cell similar to the flow cell 100 described above and
illustrated in FIGS. 1A and 1B. In this example, and referring to
FIGS. 1A and 1B, the glass coverslip corresponds to the first layer
124, a MYLAR.RTM. (biaxially-oriented polyethylene terephthalate,
or BoPET) lid corresponds to the second layer 126, a double-sided
adhesive spacer corresponds to the third layer 128, and an
underlying layer of PDMS formed during fabrication of the
surface-attached structures 108 corresponds to the substrate 136.
The length (or height) of the surface-attached structures 108 was
about 23 .mu.m, and the height of the chamber interior above the
surface-attached structures 108 was about 200 .mu.m.
[0101] It will be understood that the method described above in
conjunction with FIG. 4 for fabricating surface-attached structures
and an associated flow cell is but one example. Other examples of
fabricating surface-attached structures are disclosed in U.S. Pat.
No. 8,586,368, the entire content of which is incorporated by
reference herein. More generally, any method of fabrication that
produces surface-attached structures having structures, geometry,
properties and functionalities as disclosed herein may be
utilized.
[0102] According to an aspect of the present disclosure, a flow
cell with surface-attached structures as described herein may be
utilized in carrying out a method for extracting a target from a
sample. In some embodiments, the method may include flowing a
target-containing sample through the flow cell and into contact
with the surface-attached structures disposed in the flow cell.
While flowing the sample, the flow cell isolates targets of the
sample from a remaining portion of the sample. The flow cell may be
configured to implement various types of isolation mechanisms. One
mode of isolation entails binding the targets to a binding agent
disposed in the flow cell, as described herein.
[0103] FIG. 5 is a schematic view of an example of a chamber 504 of
a flow cell, wherein the chamber 504 is configured for extracting
or isolating a target 554 from a fluid sample according to one
embodiment. FIG. 5 is a lengthwise view of the chamber 504, such
that fluid generally flows from a fluid inlet on the left to a
fluid outlet on the right. The fluid flow is schematically depicted
by curved arrows at different elevations on the left and on the
right. The fluid sample may include any number of other components
in addition to the target 554 of interest. In this embodiment, the
surface-attached structures 108, the inside surface of the chamber
504, or both, include a binding agent specific to the target 554,
as described above. As the fluid sample flows through the chamber
504 and comes into contact with the surface-attached structures 108
(and/or the inside surface), the targets 554 (or at least a
significant fraction of the targets 554) are captured by the
surface-attached structures 108 (as illustrated) and/or by the
inside surface, depending on the location(s) of the binding agent.
As a result, the captured (bound) targets 554 are removed from the
fluid sample as illustrated.
[0104] Subsequently, the captured (bound) targets 554 may be
released from the binding agent, thereby allowing the released
targets 554 to be flowed out from the chamber 504, such as by being
carried by a different fluid flowed into the chamber 504 behind the
fluid sample. Generally, any release mechanism effective for
overcoming the bond or affinity between the targets 554 and the
binding agent may be utilized, and may depend on the types of
target 554 and binding agents involved in a given application. In
some embodiments, a fluid functioning as (or containing) a release
agent may be flowed behind the fluid sample and into contact with
the bound targets 554. Examples of such release agents include, but
are not limited to, a chemical lysing agent, a pH cell lysing
agent, an enzymatic liquefaction agent, and a solvent. In other
embodiments, photolysis may be performed by irradiating the bound
targets 554 with photons under conditions effective for inducing
photolysis (e.g., wavelength, intensity, etc.). In other
embodiments, the release modality may entail applying a shear force
to the bound targets 554 at a magnitude effective for unbinding the
bound targets. As one example of shearing, a shearing liquid may be
flowed through the flow cell behind the fluid sample at a flow rate
effective for releasing the bound targets 554 by shearing. The
shearing liquid may be water or another common solvent, which in
some embodiments may be flowed at a flow rate significantly higher
than the flow rate of the preceding fluid sample. Alternatively,
the liquid may have a viscosity high enough at a given flow rate to
enhance the shearing action imparted to the bound targets 554. As
another example of shearing, a magnetic or electric field may be
applied to the flow cell to actuate movement of the
surface-attached structures (as described above in conjunction with
FIG. 3B) at a speed effective for releasing the bound targets 554
by shearing. Still other embodiments may utilize a combination of
two or more of the foregoing release mechanisms.
[0105] In some embodiments, additional measures may be taken to
enhance the release functionality. One example is electroporation,
which may be implemented by applying a DC or AC voltage of
appropriate magnitude/amplitude across the chamber interior.
[0106] The extraction or isolation of targets 554 from a fluid
sample may be useful in a variety of applications. In some
embodiments, the target 554 is an analyte having an attribute or
property for which measurement is desired, or for which detection
of its presence in the fluid sample is desired. The chamber 504 may
be useful for capturing rare analytes from fluid samples having a
wide range of volumes, as the isolation modality provided by the
chamber 504 may be effective for concentrating rare analytes. After
isolating the analyte (target 554) in the chamber 504, the isolated
analyte may be released and eluted from the chamber 504 to any
desired destination. For example, the analyte may be flowed to a
collection receptacle or other type of collection site. The
receptacle may then be decoupled from the system to enable the
analyte to be transferred to an off-line analytical instrument,
storage site, or other destination. In other embodiments, the
receptacle may be part of an on-line analytical instrument or other
type of sample processing device positioned downstream from the
chamber 504.
[0107] In other embodiments, the target 554 may be an unwanted
component of the fluid sample. For example, the target 554 may be a
toxin, pathogen, carcinogen, or the like, or may interfere with or
suppress a subsequent analysis, detection, or reaction to be
performed on the fluid sample, or may contribute to the background
noise of a measurement signal to be generated from the fluid
sample, etc. Thus, the chamber 504 may be useful for removing the
target 554 for the purpose of sample purification or cleanup, which
may be analogous to techniques that utilize conventional sorbents
and stationary phases such as, for example, solid phase extraction
(SPE), preparative chromatography, and the like. After isolating
the targets 554 from the fluid sample, the purified/cleaned up
fluid sample may be flowed or transported to any desired
destination such as a collection container, analytical instrument,
other type of sample processing device, etc., as described above.
In some embodiments, after a prescribed period of time has elapsed
during which the fluid sample flows through the chamber 504, the
fluid line downstream from the fluid outlet of the chamber 504 may
be switched to a path leading to a collection site for the
purified/cleaned up fluid sample. For a given application, this
period of time may be determined empirically, or by analyzing the
presence or concentration of residual target 554 in the fluid
sample. The latter may be performed off-line at one or more
intervals, or a system associated with the chamber 504 may be
configured for monitoring the fluid sample on-line (intermittently
or continuously) as it elutes from the chamber 504. After removing
the target 554 (or reducing its concentration to a desired level),
the isolated target 554 may be released and eluted from the chamber
504 via a release mechanism such as described herein, and
transferred to waste or another desired destination.
[0108] In some embodiments, while the fluid sample flows through
the chamber 504, a magnetic or electric field may be applied to the
flow cell to actuate movement of the surface-attached structures
108 in an oscillating or reciprocating manner to increase a
time-averaged cross-section of the surface-attached structures 108.
This oscillation or reciprocation may increase the likelihood of
desired binding events occurring.
[0109] In other embodiments, a flow cell as described herein may be
configured to implement other types of isolation mechanisms as an
alternative to, or in addition to, mechanisms based on binding or
affinity.
[0110] FIG. 6 is a schematic view of an example of a chamber 604 of
a flow cell, wherein the chamber 604 is configured for extracting
or isolating a target 654 according to another embodiment. Similar
to FIG. 5, FIG. 6 is a lengthwise view of the chamber 604, such
that fluid generally flows from a fluid inlet on the left to a
fluid outlet on the right. The fluid flow is schematically depicted
by curved arrows at different elevations on the left and on the
right. The fluid sample may include any number of other components
in addition to the target of interest. As an example, FIG. 6
schematically illustrates two different components: a first
component 654 and a second component 656. Depending on the
embodiment, either or both components 654 and 656 may be a target
or analyte of interest. In the illustrated embodiment, the
surface-attached structures 108 are attached to the top inside
surface of the chamber such that a structure-free region 640 is
below the surface-attached structures 108, which may facilitate
certain applications as described below. In other embodiments, the
surface-attached structures 108 may be attached to the bottom
inside surface or to both the top and bottom inside surfaces as
described herein.
[0111] In one embodiment, the chamber 604 is configured for
separating the first component 654 from the fluid sample by
implementing a filtering or size exclusion technique, which may be
analogous to size exclusion chromatography (SEC) but without the
use of porous beads. In this case, the first component 654 may be
considered to be the target or analyte. The inter-structure spacing
of the array of surface-attached structures 108 is set such that as
the fluid sample flows through the chamber 604, the first
components 654 cannot pass through the array of surface-attached
structures 108, i.e., cannot pass between adjacent surface-attached
structures 108. Instead, as illustrated the first components 654
are forced to flow through the structure-free region 640 below the
surface-attached structures 108, while other components such as the
second components 656 are small enough to flow through the array of
surface-attached structures 108. Because the first components 654
travel through a shorter path length and/or fluid volume compared
to the second components 656, the first components 654 and the
second components 656 become separated in time and space.
Consequently, a substantial fraction of the first components 654
elute from the chamber 604 first, i.e., before the second
components 656 elute from the chamber 604, as schematically
depicted at the fluid outlet in FIG. 6. In this manner, the first
components 654 become isolated from the second components 656 (and
possibly one or more other components of the fluid sample) in a
manner analogous to chromatographic bands or peaks.
[0112] Because the first components 654 elute from the chamber 604
separately, they may be collected separately. Fluidics downstream
from the fluid outlet may be configured to route the first
components 654 to a desired receptacle and then, after collection
of the first components 654 is complete, switch the fluid flow to
another receptacle to receive the remaining portion of the fluid
sample. The respective operations of such fluidics and the chamber
604 may be coordinated in a number of ways, as appreciated by
persons skilled in the art. For example, the operation of a
flow-switching device such as a valve may be synchronized with the
operation of the chamber 604. The duration of fluid flow through
chamber 604 required for adequate isolation of the first components
654 may be determined empirically, or through use of an appropriate
detector, etc.
[0113] In another embodiment, the chamber 604 is configured for
separating the first component 654 from the fluid sample by a
technique referred to herein as density separation. In this case,
the first component 654 is a denser particle in comparison to other
components of the fluid sample. The flow rate and the length of the
chamber 604 are selected such that as the fluid sample flows
through the chamber 604, the higher-density first components 654
tend to settle or diffuse toward the bottom inside surface such
that all or a substantial fraction of the first components 654 flow
through the structure-free region 640. To achieve this effect, the
flow rate may be relatively low in comparison to other methods
disclosed herein. Typically, the shorter the length of the chamber
604, the lower the flow rate should be in order to achieve the
density separation effect effectively.
[0114] In some embodiments, the mechanism of isolation may be a
combination of filtering/size exclusion and density separation.
[0115] The isolation techniques described above in conjunction with
FIG. 6 may be useful for separating first components 654 that are
large particles relative to other components of the fluid sample.
For example, the fluid sample may be whole blood and the first
components 654 may be intact red blood cells (erythrocytes). As
more general examples, the fluid sample may be a colloid in which
the first components 654 are the dispersed phase, or a suspension
in which the first components 654 are the suspended solids.
[0116] In some embodiments, the chamber 604 may also include a
binding agent disposed on or integrated with the surface-attached
structures 108, the inside surface of the chamber 604, or both, as
described elsewhere in the present disclosure. The binding agent
may have a specific affinity for a component other than the first
component 654, such as the illustrated second component 656. The
added modality of binding may be useful for enhancing isolation of
the first component 654, providing multi-target isolation, or any
other purpose found to be useful.
[0117] In some embodiments, the first component 654 may be an
unwanted component, in which case isolation of the first component
654 may be done for the purpose of sample purification or
cleanup.
[0118] In another embodiment, a flow cell as described herein may
be configured to isolate a target by trapping the target in the
array of surface-attached structures 108. The target may be trapped
by preventing the target from passing between neighboring
surface-attached structures 108. This may be accomplished by
setting an appropriate inter-structure spacing between the
surface-attached structures 108. The inter-structure spacing may be
adjusted by applying a magnetic or electric field to the flow cell
to actuate movement of the surface-attached structures 108 as
described herein. After trapping the target for a desired period of
time, the target may be released by applying a magnetic or electric
field to the flow cell to actuate movement of the surface-attached
structures 108, specifically to increase the inter-structure
spacing enough for the previously trapped target to be able to pass
through the surface-attached structures 108 and elute from the flow
cell.
[0119] In various embodiments, a magnetic or electric field may be
applied to the flow cell to actuate movement of the
surface-attached structures 108. The surface-attached structures
108 may be moved to achieve various effects. In addition to those
described elsewhere in the present disclosure, examples of effects
achieved by moving the surface-attached structures 108 include, but
are not limited to, adjusting or varying an inter-structure spacing
between the surface-attached structures 108, preventing or
disrupting clogging of sample material between the surface-attached
structures 108, and/or preventing or disrupting non-specific
binding of sample material on the surface-attached structures 108.
The effect achieved by moving the surface-attached structures 108
may be optimized by varying one or more parameters such as, for
example, flow rate, inter-surface spacing (density of the
surface-attached structures 108), and the frequency
oscillation/reciprocation.
[0120] FIG. 7 is a schematic view of an example of a target
extraction system 700 according to some embodiments. The target
extraction system 700 may include a flow cell 100 according to any
of the embodiments disclosed herein, and a driver 760 configured
for applying a magnetic or electric field to the interior of the
flow cell 100 to actuate movement of the surface-attached
structures 108 as described herein. In some embodiments, the target
extraction system 700 may further include a housing 762 configured
for removably receiving the flow cell 100. In such case, the flow
cell 100 may be configured as a cartridge and the housing 762 may
be configured as (or may include) a cartridge support or
receptacle. By this configuration, the flow cell 100 after use may
be replaced with a new or cleaned or sterilized flow cell or with a
differently configured flow cell.
[0121] Generally, devices and methods for generating and
controlling magnetic and electric fields are known to persons
skilled in the art, and thus the driver 760 will be described
herein only briefly as necessary for facilitating an understanding
of the presently disclosed subject matter. The particular
configuration of the driver 760 depends on whether it applies a
magnetic field or an electric field. Generally, the driver 760
includes a field generator 764. The field generator 764 may be
mounted to a suitable field generator support 766. The field
generator support 766 may hold the field generator 764 in a
position relative to the flow cell 100 at which the field generator
764 is able to apply a magnetic or electric field of a desired
strength and orientation to the surface-attached structures 108. In
embodiments applying a magnetic field, the field generator 764 may
include one or more magnets of a suitable size and shape. The
magnets may be permanent magnets, electromagnets, or a combination
of both types. In embodiments applying an electric field, the field
generator 764 may include one or more electrodes of a suitable size
and shape. When the field generator 764 includes only a single
magnet or electrode, the magnetic or electric field may be
established primarily between that magnet or electrode and the
metallic components of the surface-attached structures 108. The
field generator 764 may include additional magnets or electrodes as
needed for generating a magnetic or electric field having a desired
spatial orientation relative to the surface-attached structures
108.
[0122] In embodiments providing electromagnets or electrodes, the
driver 760 may further include a power source 768 in electrical
communication with the electromagnets or electrodes. The power
source 768 may be configured to supply variable electrical power to
the electromagnets or electrodes. In various embodiments, the
driver 760 may be configured for varying a parameter of the
magnetic or electric field, such as magnetic or electric field
strength, magnetic or electric field direction (orientation),
and/or a frequency at which the magnetic or electric field is
cycled between ON and OFF states or high-strength and low-strength
states.
[0123] In some embodiments all or part of the driver 760, such as
the field generator 764 and any associated field generator support
766, may be movable relative to the flow cell 100 (and the housing
762, if provided). By this configuration, the position of the field
generator 764 may be adjusted to adjust the orientation of the
applied magnetic or electric field. Alternatively or additionally,
the field generator 764 (particularly in the case of permanent
magnets) may be oscillated or reciprocated between different
positions to cause the surface-attached structures 108 to oscillate
or reciprocate, as described above in conjunction with FIG. 3A. For
example, the field generator 764 may be rotated about the
longitudinal axis of the flow cell 100. In other embodiments, power
may be supplied at different magnitudes to different electromagnets
or electrodes to adjust and/or oscillate the orientation of the
magnetic or electric field.
[0124] In some embodiments, the driver 760 may include an actuator
coupled (e.g., mechanically) to the magnets or electrodes of the
field generator 764 to actuate movement of the magnets or
electrodes. The actuator may include a motor 770 for powering the
movement, and an appropriate linkage (e.g., shaft) coupled between
the motor 770 and the magnets or electrodes, as appreciated by
persons skilled in the art. The actuator may move the magnets or
electrodes to adjust or oscillate their positions, such as by
rotation about the longitudinal axis of the flow cell 100. In the
case of permanent magnets, the actuator may translate the permanent
magnets toward or away from the flow cell 100 (and the housing 762,
if provided) to adjust the magnetic field strength applied to the
surface-attached structures 108.
[0125] When coupled into the target extraction system 700, the flow
cell 100 may be operated or utilized to process sample fluid in
accordance with any of the methods disclosed herein. While a fluid
sample is flowing through the flow cell 100, the driver 760 may
actuate movement of the surface-attached structures 108 in
accordance with any of the methods disclosed herein.
[0126] In some embodiments, the target extraction system 700 may
include a fluid supply source 772 configured for flowing a fluid to
the fluid input of the flow cell 100. The fluid supply source 772
may include one or more different fluid supply sources
communicating with respective fluid lines (e.g., tubing). For
example, the fluid supply source 772 may include a sample source
774 configured for flowing a target-containing sample to the fluid
input. The fluid supply source 772 may also include one or more
processing fluid sources 776 and 778 configured for flowing other
fluids to the fluid input. The processing fluid sources 776 and 778
may include, for example, a source of a release agent or a shearing
fluid effective for releasing targets bound to a surface inside the
flow cell 100 (or otherwise captured or trapped in the flow cell
100) as described elsewhere herein, and/or a source of rinsing
agent effective for rinsing/washing the flow cell 100 so as to
purge the flow cell 100 of residual components from a previous
operation of the flow cell 100 and prepare the flow cell 100 for
the next operation.
[0127] In some embodiments, the target extraction system 700 may
include a fluid receptacle 782 configured for receiving processed
fluid from the fluid output of the flow cell 100. The fluid
receptacle 782 may include one or more different fluid receptacles
communicating with respective fluid lines (e.g., tubing). For
example, the fluid receptacle 782 may include one or more fluid
receptacles 784 configured for collecting one or more fluids
carrying respective targets isolated by the flow cell 100. In some
embodiments, the fluid receptacle(s) 784 may be part of or
communicate with an analytical instrument or other type of sample
processing device as described herein. The fluid receptacle 782 may
also include one or more fluid receptacles 786 for receiving one or
more different types of processed fluid other than a fluid carrying
targets. For example, one or more of the fluid receptacles 786 may
be configured for receiving purified or cleaned up fluid samples.
As other examples, one or more of the fluid receptacles 786 may
serve generally as waste receptacles for receiving spent process
fluids such as release agents, shearing fluids, and/or rinsing
agents. As another example, one or more of the fluid receptacles
786 may serve as the destination for fluids flowed through the
target extraction system 700 for pre-operation purposes, such as
for purging the fluid lines of bubbles, priming the fluid lines,
etc.
[0128] FIG. 7 schematically depicts different fluid input lines
coupled to a common fluid input line leading to the fluid inlet of
the flow cell 100, and different fluid output lines branching off
from a common fluid output line leading from the fluid outlet of
the flow cell 100. It will be understood that such an arrangement
is but one example of many possible configurations of the fluid
circuitry that may be provided by the target extraction system 700.
In addition, it will be understood that in practice, other fluidic
components and devices may be included as necessary for realizing
different applications, such as valves (proportional valves,
multi-port valves, etc.), flow regulators, pressure regulators,
temperature regulators, flow path switches/selectors, flow
restrictors, sample loops, etc., all as appreciated by persons
skilled in the art. Moreover, depending on the embodiment, fluid
flow through the flow cell 100 may be active (e.g., by employing a
pump upstream of or downstream from the flow cell 100, other means
for actively creating a pressure differential, etc.) or passive
(e.g., by capillary action, wicking, gravity-assist,
electrokinetics, etc.). In some embodiments, the reservoirs or
containers associated with the fluid supply source 772 may be
integral parts of syringes (syringe pumps).
[0129] In some embodiments, the target extraction system 700 may
include a photon source or light source 790 and associated optics
for generating photons and directing the photons to the
surface-attached structures 108 to perform photolysis as described
above. In this case, all or a part of a wall of the flow cell 100
exposed to at least a portion of the interior of the flow cell 100
may be optically transparent to the wavelength(s) of the photons.
The photon source 790 may be positioned, for example, in the
housing 762. Examples of photon sources include, but are not
limited to, broadband light sources (e.g., flash lamps),
light-emitting diodes (LEDs), laser diodes (LDs), and lasers, any
of which may be wavelength-filtered if desired. In some
embodiments, the target extraction system 700 may include a heating
device of any suitable type (not shown) configured to control fluid
temperature in the flow cell 100. The heating device may be
positioned, for example, in the housing 762 proximate to the flow
cell 100. In some embodiments, the photon source 790 or another
photon source may be utilized for infrared (IR) heating of the
fluid flowing through the flow cell 100.
[0130] When coupled into the target extraction system 700, the flow
cell 100 may be operated or utilized to process sample fluid in
accordance with any of the methods disclosed herein. While a fluid
sample is flowing through the flow cell 100, the driver 760 may
actuate movement of the surface-attached structures 108 in
accordance with any of the methods disclosed herein. The fluid
supply source 772 and the receptacle 782 may be utilized as needed
to supply and receive fluids in accordance with any of the methods
disclosed herein.
[0131] FIG. 8 is a perspective view of an example of a target
extraction system 800 (or a portion thereof) according to another
embodiment. The target extraction system 800 may include a stator
802 positioned on an axis, and a rotor 806 that may be rotated
about the axis. The stator 802 may include a housing 862 in which a
flow cell (not shown) may be removably installed for operation in
the target extraction system 800 as described herein. The rotor 806
may serve as the actuator of a driver as described herein. The
rotor 806 may include a field generator support 866 configured for
supporting one or more magnets. For example, the field generator
support 866 may include one or more recesses 810 in which
respective magnets may be mounted. In other embodiments, the field
generator support 866 may be configured for supporting one or more
electrodes. The rotor 806 may also include a shaft support 816
configured for supporting the shaft of a motor (not shown) as
described herein. In the illustrated embodiment, the shaft support
816 includes a square aperture 818 that receives a square motor
shaft. The shaft support 816 may be mechanically coupled to the
field generator support 866, such that the shaft support 816 and
the field generator support 866 rotate together about the axis of
the housing 862 (and the flow cell disposed therein). The position
of the motor may be fixed in space by a suitable mounting
arrangement (not shown). Thus, the rotor 806 (including the shaft
support 816 and the field generator support 866) may be supported
in space by the motor through its fixed mounting arrangement and
the interconnection between the motor and the rotor 806 provided by
the motor shaft. The stator 802 may be supported independently of
the motor and the rotor 806, such that the motion of the rotor 806
is independent of the stator 802 and the mounting arrangement (not
shown) of the stator 802. In some embodiments, the motor may
provide power for actuating oscillatory rotation of the magnet(s)
as described herein. That is, the rotational power generated by the
motor is transferred to the magnet(s) via the motor shaft, shaft
support 816, and field generator support 866.
[0132] Generally, the stator 802 and the rotor 806 may be
fabricated by any suitable technique. In some embodiments, the
stator 802 and the rotor 806 may be fabricated by a
three-dimensional (3D) printing technique as appreciated by persons
skilled in the art. In some embodiments, the stator 802 and the
rotor 806 may be incorporated into the target extraction system 700
described above and illustrated in FIG. 7.
[0133] In some embodiments, a flow cell as described herein may in
effect be partitioned into a plurality of flow cell units, with
each flow cell unit including a chamber and a plurality of
surface-attached structures as described herein. The flow cell
units may be arranged in parallel or in series. FIGS. 9 and 10
illustrate examples of arrangements of flow cell units.
[0134] Specifically, FIG. 9 is a schematic elevation view of an
example of a flow cell 900 according to an embodiment in which the
flow cell 900 includes a plurality of flow cell units 950A, 950B,
and 950C stacked in parallel. FIG. 9 is a lengthwise view of the
flow cell 900, such that fluid generally flows from the left to the
right. By example, FIG. 9 illustrates three flow cell units 950A,
950B, and 950C, with the understanding that more or less than three
flow cell units 950A, 950B, and 950C may be provided. Each flow
cell unit 950A, 950B, and 950C includes a fluid inlet 912A, 912B,
and 912C, a fluid outlet 914A, 914B, and 914C, a chamber 904A,
904B, and 904C between the fluid inlet 912A, 912B, and 912C and the
fluid outlet 914A, 914B, and 914C, and a plurality of
surface-attached structures 108A, 108B, and 108C attached to an
inside surface of the chamber 904A, 904B, and 904C. Thus, the flow
cell units 950A, 950B, and 950C establish separate, parallel fluid
flow paths through the flow cell 900, whereby fluids flowing
simultaneously through the different flow paths encounter separate
sets of surface-attached structures 108A, 108B, and 108C,
respectively.
[0135] In some embodiments, the sets of surface-attached structures
108A, 108B, and 108C have the same configuration, e.g., the same
inter-structure spacing, the same binding agent (if any), etc. In
this case, the multi-unit flow cell 900 may be advantageous for
increasing the volumetric capacity of the flow cell 900 without
affecting any design considerations or constraints relating to the
individual flow cell units 950A, 950B, and 950C. That is, each flow
cell unit 950A, 950B, and 950C may be configured in an optimal
manner that does not require accounting for the total volumetric
capacity desired for the flow cell 900. Any volumetric requirement
of the flow cell 900 may be met by providing a sufficient number of
flow cell units in the parallel stack. Moreover, providing
additional chambers 904A, 904B, and 904C may multiply the surface
area to which a given volume of fluid is exposed. For embodiments
providing binding agents at internal surfaces of the chambers 904A,
904B, and 904C (e.g., at the surface-attached structures 108A,
108B, and 108C and/or at inside surfaces of the chambers 904A,
904B, and 904C), this configuration may enhance the effectiveness
of the binding/capturing functionality of the flow cell 900.
[0136] In other embodiments, at least one set of surface-attached
structures 108A, 108B, and 108C may be configured differently than
the other sets. For example, at least one set may have a different
inter-structure spacing, or a different binding agent, etc. As
another example, at least one set may have a binding agent while
the other sets do not, or at least one set may have no binding
agent while the other sets do have binding agents. As another
example, at least one chamber 904A, 904B, and 904C may include
surface-attached structures while the other chambers 904A, 904B,
and 904C do not, or at least one chamber 904A, 904B, and 904C may
include no surface-attached structures while the other chambers
904A, 904B, and 904C do include surface-attached structures. The
chamber(s) 904A, 904B, and 904C that do not include
surface-attached structures may or may not include binding agents
at the inside surfaces. Embodiments providing differently
configured flow cell units 950A, 950B, and 950C may be useful for
isolating more than one type of target from a fluid sample.
[0137] In some embodiments, one of the flow cell units 950A, 950B,
and 950C may be a reference flow cell unit that is part of a flow
path utilized to generate a reference signal utilized for
calibration or other purposes. The configuration of the reference
flow cell unit may or may not be the same as that of the other flow
cell units. The reference flow cell unit may communicate with a
separate fluid source, a separate fluid receptacle, or with both a
separate fluid source and a separate fluid receptacle.
[0138] In some embodiments, one or more of the individual flow cell
units 950A, 950B, and 950C may communicate with separate fluid
sources and/or separate fluid receptacles. In some embodiments, two
or more of the individual flow cell units 950A, 950B, and 950C may
communicate with a common fluid source and/or a common fluid
receptacle. As illustrated in FIG. 9, in some embodiments all of
the individual flow cell units 950A, 950B, and 950C may communicate
with a common fluid source and/or a common fluid receptacle. In
such embodiments, the flow cell 900 may include a common fluid
input port 934 and/or a common fluid output port 938. The flow cell
900 may also include an input manifold 994 or other type of
transition between the fluid input port 934 and the fluid inlets
912A, 912B, and 912C, and/or an output manifold 998 or other type
of transition between the fluid output port 938 and the fluid
outlets 914A, 914B, and 914C.
[0139] FIG. 10 is a schematic elevation view of an example of a
flow cell 1000 according to an embodiment in which the flow cell
1000 includes a plurality of flow cell units 1050A, 1050B, and
1050C arranged in series. FIG. 10 is a lengthwise view of the flow
cell 1000, such that fluid generally flows from the left to the
right. By example, FIG. 10 illustrates flow cell units 1050A,
1050B, and 1050C, with the understanding that more or less than
three flow cell units 1050A, 1050B, and 1050C may be provided. Each
flow cell unit 1050A, 1050B, and 1050C includes a fluid inlet
1012A, 1012B, and 1012C, a fluid outlet 1014A, 1014B, and 1014C, a
chamber 1004A, 1004B, and 1004C between the fluid inlet 1012A,
1012B, and 1012C and the fluid outlet 1014A, 1014B, and 1014C, and
a plurality of surface-attached structures 108A, 108B, and 108C
attached to an inside surface of the chamber 1004A, 1004B, and
1004C. In the illustrated series arrangement, the fluid inlet or
the fluid outlet of each flow cell unit 1050A, 1050B, and 1050C
communicates with the fluid inlet or the fluid outlet of at least
one other flow cell unit 1050A, 1050B, and 1050C.
[0140] Generally, the series-arranged multi-unit flow cell 1000 may
provide one or more of the same advantages or functions as the
parallel-arranged flow cell 900 described above. As in the case of
the parallel-arranged flow cell 900, the chambers 1004A, 1004B, and
1004C and corresponding surface-attached structures 108A, 108B, and
108C of the series-arranged flow cell 1000 may be configured the
same as or differently from each other.
[0141] As shown in FIG. 10, in some embodiments one or more of the
flow cell units 1050A, 1050B, and 1050C may include an additional
(or auxiliary) fluid inlet (e.g., fluid inlets 1012D and 1012E)
and/or an additional (or auxiliary) fluid outlet (e.g., fluid
outlets 1014D and 1014E). The additional fluid inlets and fluid
outlets may be utilized for various purposes. For example, the
additional fluid inlets and fluid outlets may enable fluid flow
through the flow cell 1000 to bypass one or more selected flow cell
units 1050A, 1050B, and 1050C, thereby imparting a modularity to
the flow cell 1000 and facilitating customization for a desired
application. The bypassing of a selected flow cell unit may be
temporary, such as to enable elution from the additional fluid
outlet of a preceding flow cell unit to occur for a period of time
before resuming fluid flow into the selected flow cell unit.
[0142] As another example, the additional fluid inlets and fluid
outlets may facilitate multi-target isolation as may be implemented
with differently configured flow cell units 1050A, 1050B, and
1050C. For example, the first flow cell unit 1050A may be
configured to capture a first target from a fluid sample containing
multiple different targets. After capturing the first targets, the
first flow cell unit 1050A may release the first targets into a
fluid, and then output the first target-laden fluid through the
additional fluid output 1014D. By this configuration, the first
targets need not flow through the remaining flow cell units 1050B
and 1050C. In a given sample extraction system, this configuration
may facilitate the analysis of the first target separately from
other targets. The configuration may enhance the effectiveness of
the other flow cell units 1050B and 1050C in isolating other
targets. For example, the second flow cell unit 1050B may be
configured to capture a second target from the same fluid sample,
then release the second targets into a fluid, and then output the
second target-laden fluid through the additional fluid output
1014E, and so on.
[0143] In other embodiments, two or more flow cell units 1050A,
1050B, and 1050C may be configured to capture the same type of
target and, after releasing the captured targets, the different
fluid outputs may be utilized to flow the target-laden fluid to two
or more different fluid receptacles, such as to perform different
analyses or reactions on the same target.
[0144] To facilitate the use of additional fluid inlets and fluid
outlets, valves or other flow regulators (not shown) may be
provided in the fluid line between adjacent flow cell units
1050A,
[0145] 1050B, and 1050C, as appreciated by persons skilled in the
art.
[0146] In another embodiment, the flow cell units 1050A, 1050B, and
1050C may be configured for performing multi-stage particle sizing.
In this embodiment, the inter-structure spacing of the
surface-attached structures 108A, 108B, and 108C may be
progressively smaller in each flow cell unit 1050A, 1050B, and
1050C. Hence, the largest particles in a fluid sample may be
isolated in the first flow cell unit 1050A, followed by
intermediate-sized particles in the same fluid sample being
isolated in the second flow cell unit 1050B, followed by even
smaller particles in the same fluid sample being isolated in the
third flow cell unit 1050C.
[0147] In some embodiments, the present invention provides
surface-attached structures (e.g., micropost arrays) for enhancing
flow, circulation, and/or mixing action for analyte capture on a
microarray (or analyte capture array, or probe array), and related
systems and methods. The surface-attached structures are positioned
in relation to the microarray such that actuated motion of the
surface-attached structures can be used to enhance flow,
circulation, and/or mixing action of analytes in a fluid sample,
thereby enhancing analyte capture on the microarray.
[0148] The presently disclosed microfluidic system includes
surface-attached structures (e.g., a micropost array) and a
microarray (e.g., a nucleic acid microarray, a protein array, an
antibody array, a small molecule array, or the like) that are
separated by gap, wherein the gap contains liquid, such as, but not
limited to, fluid sample. Such a gap may at least partially define
a chamber through with the liquid flows. Further, the microfluidic
system includes an actuation mechanism for actuating the
surface-attached structures (e.g., microposts of a micropost
array). For example, microposts are surface-attached posts wherein
each micropost includes a proximal end attached to a substrate and
a distal end that extends into the gap between the micropost array
and the microarray. Accordingly, the distal ends of the microposts
extend into the fluid sample that is in the gap between the
micropost array and the microarray. The actuation mechanism is used
to generate an actuation force in proximity to the micropost array
to actuate the microposts, thereby compelling at least some of the
microposts to exhibit motion.
[0149] In the presently disclosed microfluidic system, the motion
of the surface-attached structures (e.g., microposts) due to the
actuation force serves to enhance flow, circulation, and/or mixing
action of the fluid sample with respect to the full area of the
microarray as compared with the use of diffusion alone for flow
and/or mixing. Accordingly, an aspect of the presently disclosed
microfluidic system and method is that it can be used to
significantly reduce the reaction time (i.e., accelerate reactions)
compared with microarray applications that rely on diffusion alone
for flow and/or mixing. For example, in microarray applications,
the presently disclosed microfluidic system can be used to reduce
the reaction time from hours or days to a few minutes only.
Particularly, the presently disclosed microfluidic system and
methods can be used to reduce the reaction time by at least about
five times (5.times.), at least about six times (6.times.), at
least about seven times (7.times.), at least about eight times
(8.times.), at least about nine times (9.times.), or at least about
ten times (10.times.), as compared to microfluidic systems and
methods that do not utilize the motion of surface-attached
structures (e.g., microposts) due to actuation forces to enhance
the flow, circulation, and/or mixing action of a fluid sample. This
is particularly useful in microarray applications in which the
"time to result" is important (e.g., POC devices).
[0150] Further, because of the enhanced flow, circulation, and/or
mixing action of the fluid sample and accelerated reactions,
another aspect of the presently disclosed microfluidic system and
method is that in microarray applications in which the analyte
concentration is low, such as in liquid biopsy/circulating
cell-free DNA tests, it can be used to increase analyte utilization
and therefore improve sensitivity of the detection operations as
compared with microarray applications that rely on diffusion alone
for flow and/or mixing.
[0151] Yet another aspect of the presently disclosed microfluidic
system and method is that it provides surface-attached structures
(e.g., micropost arrays) in combination with a microarray and is
therefore able to process multiple target analytes with respect to
multiple capture sites in a single reaction chamber.
[0152] Yet another aspect of the presently disclosed microfluidic
system and method is that it provides enhanced flow, circulation,
and/or mixing action of the fluid sample and accelerated reactions
via the surface-attached structures (e.g., a micropost array),
wherein the surface attached structures are a simple and low cost
stirring mechanism compared with microfluidics devices that
include, for example, pumping mechanisms to move the fluid.
[0153] Still another aspect of the presently disclosed microfluidic
system and method is that it includes the surface-attached
structures (e.g., a micropost array) in combination with the
microarray while maintaining compatibility with common detection
methods (e.g., optical and/or electrical detection systems).
[0154] In another embodiment, the presently disclosed microfluidic
system does not include a microarray separate from the
surface-attached structures, and instead includes analyte capture
elements on the surface-attached structures themselves (e.g., a
micropost array). Namely, the surface-attached structures are
functionalized with analyte capture elements. In some embodiments,
such a configuration may be characterized as including a microarray
that is integrated with or provided by the array of
surface-attached structures. In yet another embodiment, the
presently disclosed microfluidic system includes the
[0155] combination of both the microarray and surface-attached
structures (e.g., microposts) that are functionalized with analyte
capture elements. FIG. 11 illustrates a perspective view of an
example of a microfluidic system 1100 that includes a microarray
positioned in relation to a micropost array, wherein the actuated
motion of the microposts is used to enhance flow, circulation,
and/or mixing action for analyte capture on a microarray. In this
example, microfluidic system 1100 includes a microarray 1110 and a
micropost array 1120 that are separated by a gap 1140, wherein gap
1140 can contain liquid, such as, but not limited to, fluid sample.
Microfluidic system 1100 includes an actuator 1160 in proximity to
micropost array 1120. Optionally, microfluidic system 1100 may
include a detector 1165 in proximity to microarray 1110 as often
microarrays are scanned after runtime in a separate instrument.
[0156] Microarray 1110 can be any type of microarray for performing
assays. Examples of microarrays include, but are not limited to,
DNA microarrays (e.g., cDNA microarrays, oligonucleotide
microarrays, bacterial artificial chromosome (BAC) microarrays and
single nucleotide polymorphism (SNP) microarrays), model-based
meta-analysis of chromatin immunoprecipitation (MM-ChIP) arrays,
protein microarrays, peptide microarrays, tissue microarrays,
cellular microarrays, small molecule microarrays, chemical compound
microarrays, antibody microarrays, carbohydrate arrays, phenotype
microarrays, reverse phase protein microarrays, and the like.
[0157] Microarray 1110 includes an arrangement (e.g., an array) of
capture sites 1112 on a microarray substrate 1114. In one example,
the capture sites 1112 are analyte capture elements (or binding
agents, or probes), wherein each capture site 1112 or groups of
capture sites 1112 can be functionalized to capture different
analytes. Microarray substrate 1114 can be, for example, a glass or
silicon substrate. In the case of a glass substrate 1114, detector
1165 can be a florescence-based optical detection mechanism. In the
case of a silicon substrate 1114 in which microarray 1110 is a
semiconductor array, detector 1165 can be an electrical
signal-based detection mechanism.
[0158] Micropost array 1120 includes an arrangement (e.g., an
array) of microposts 1122 on a micropost substrate 1124. Microposts
1122 are surface-attached posts wherein each micropost 1122
includes a proximal end attached to micropost substrate 1124 and a
distal end that extends into gap 1140 between microarray 1110 and
micropost array 1120. Accordingly, the distal ends of micropost
1122 extend into the fluid sample (not shown) that is in gap 1140
between microarray 1110 and micropost array 1120. In one example,
microposts 1122 are chemically inert and will not react with target
analytes in the fluid sample. However, in another example, the
surfaces of the microposts 1122 can be functionalized with analyte
capture elements.
[0159] Microposts 1122 in micropost array 1120 are designed to
exhibit motion when in the presence of an actuation force. As used
herein, the term "actuation force" refers to the force applied to
microposts 1122. Actuator 1160 is used to generate an actuation
force in proximity to micropost array 1120 that compels at least
some of microposts 1122 to exhibit motion. The actuation force may
be, for example, magnetic, thermal, sonic, optical, electrical,
and/or vibrational. Further, the actuation force may be applied as
a function of frequency or amplitude, or as an impulse force (i.e.,
a step function). Similarly, other actuation forces may be used
without departing from the scope of the present invention, such as
fluid flow across micropost array 1120.
[0160] By actuating microposts 1122 and causing motion thereof, the
fluid sample in gap 1140 is in effect stirred or caused to flow or
circulate within gap 1140 and across the surface area of microarray
1110. Micropost array 1120 that includes the arrangement of
microposts 1122 is based on, for example, the microposts described
in the U.S. Patent 9,238,869, entitled "Methods and systems for
using actuated surface-attached posts for assessing biofluid
rheology," issued on January 19, 2016; the entire disclosure of
which is incorporated herein by reference. The '869 patent
describes methods, systems, and computer readable media for using
actuated surface-attached posts for assessing biofluid rheology.
According to one aspect, a method of the '869 patent for testing
properties of a biofluid specimen includes placing the specimen
onto a micropost array having a plurality of microposts extending
outwards from a substrate, wherein each micropost includes a
proximal end attached to the substrate and a distal end opposite
the proximal end, and generating an actuation force in proximity to
the micropost array to actuate the microposts, thereby compelling
at least some of the microposts to exhibit motion. The method of
the '869 patent further includes measuring the motion of at least
one of the microposts in response to the actuation force and
determining a property of the specimen based on the measured motion
of the at least one micropost.
[0161] In one example, according to the '869 patent, microposts
1122 and substrate 1124 of micropost array 1120 can be formed of
polydimethylsiloxane (PDMS). Further, microposts 1122 may include a
flexible body and a metallic component disposed on or in the body,
wherein application of a magnetic or electric field actuates
microposts 1122 into movement relative to the surface to which they
are attached. In this example, the actuation force generated by
actuator 1160 is a magnetic and/or electrical actuation force. More
details of micropost array 1120 and microposts 1122 are shown and
described hereinbelow with reference to FIG. 12A through FIG.
15B.
[0162] FIG. 12A and FIG. 12B illustrate a plan view and a
cross-sectional view, respectively, of an example of a flow cell
1200 that is based on microfluidic system 1100 of FIG. 1 that
includes microarray 1110 positioned in relation to micropost array
1120. Namely, FIG. 12B is a cross-sectional view taken along line
A-A of FIG. 12A.
[0163] In this example, flow cell 1200 includes a first substrate
1210 that includes a reaction chamber 1212 integrated therein,
wherein reaction chamber 1212 is a space or void in substrate 1210.
Substrate 1210 is capped with a second substrate 1214, wherein
substrate 1214 encloses reaction chamber 1212. Substrate 1210 and
substrate 1214 can be formed, for example, of glass or plastic. In
one example, flow cell 1200 includes two loading ports 1216 (e.g.,
one at each end) for suppling liquid (e.g., fluid sample 1218 that
includes target analytes) into or out of reaction chamber 1212.
[0164] Reaction chamber 1212 is sized to receive microarray 1110
such that the capture sites 1112 face into reaction chamber 1212. A
microarray, such as microarray 1110, can support, for example, from
a few dozen capture sites up to many thousands of capture sites.
The size of reaction chamber 1212 can vary according to the size of
microarray 1110. For example, reaction chamber 1212 can hold from
about 1 microliters to about 500 microliters of fluid sample 1218.
Reaction chambers can come in all shapes and sizes. A typical
reaction chamber size might be, for example, about 1 mm.times.1 mm.
In one example, both substrate 1124 of micropost array 1120 and
substrate 1114 of microarray 1110 are 1-inch.times.3-inch glass
slides, wherein the entirety of the 1-inch.times.3-inch substrate
1124 is covered with microposts 1122 and the 1-inch.times.3-inch
substrate 1114 has three 10 mm.times.10 mm microarrays on it.
[0165] Additionally, micropost array 1120 is mounted on substrate
1214 such that microposts 1122 are facing into reaction chamber
1212. Namely, substrate 1124 of micropost array 1120 is mounted on
the inside surface of substrate 1214 and microposts 1122 are facing
capture sites 1112 of microarray 1110. The length of microposts
1122 can vary. The length of microposts 1122 can be from about 1
.mu.m to about 100 .mu.m in one example, or can be from about 10
.mu.m to about 50 .mu.m in another example. Further, the space
between the distal ends of microposts 1122 and microarray 1110 can
vary. The space between the distal ends of microposts 1122 and
microarray 1110 can be from about 0 .mu.m to about 50 .mu.m in one
example, or can be from about 1 .mu.m to about 30 .mu.m in another
example. In another example, the space between the distal ends of
microposts 1122 and microarray 1110 can be about equal to the
length of microposts 1122. In yet another example, the space
between the distal ends of microposts 1122 and microarray 1110 can
be about 10 .mu.m or more or less than 10 .mu.m.
[0166] FIG. 13A and FIG. 13B illustrate side views of an example of
microposts 1122. Again, microposts 1122 and substrate 1124 of
micropost array 1120 can be formed, for example, of PDMS. The
length, diameter, geometry, orientation, and pitch of microposts
1122 in micropost array 1120 can vary. For example, the length of
microposts 1122 can vary from about 1 .mu.m to about 100 .mu.m. The
diameter of microposts 1122 can vary from about 0.1 .mu.m to about
10 .mu.m. The cross-sectional shape of microposts 1122 can vary.
For example, the cross-sectional shape of microposts 1122 can
circular, ovular, square, rectangular, triangular, and so on. The
orientation of microposts 1122 can vary. For example, FIG. 13A
shows microposts 1122 oriented substantially normal to the plane of
substrate 1124, while FIG. 3B shows microposts 1122 oriented at an
angle a with respect to normal of the plane of substrate 1124. In a
neutral positon with no deflection force applied, the angle a can
be, for example, from about 0 degrees to about 45 degrees.
[0167] Further, the pitch of microposts 1122 within micropost array
1120 can vary, for example, from about 0 .mu.m to about 50 .mu.m.
For example, FIG. 14A through FIG. 14D illustrate plan views of
examples of various configurations of micropost array 1120. Namely,
FIG. 14A shows an example of microposts 1122 that are 0.6 .mu.m in
diameter and spaced 1.4 .mu.m apart. FIG. 14B shows an example of
microposts 1122 that are 0.6 .mu.m in diameter and spaced 2.6 .mu.m
apart. FIG. 14C shows an example of microposts 1122 that are 1
.mu.m in diameter and spaced 1.5 .mu.m apart. FIG. 14D shows an
example of microposts 1122 that are 1 .mu.m in diameter and spaced
3 .mu.m apart. It is understood that the size and dimensions
depicted in FIG. 14A through FIG. 14D are exemplary only and not
limiting. FIG. 14E shows a scanning electron microscope (SEM) image
of an example of a micropost array 1120. Further, FIG. 14A through
FIG. 14E show the rows of microposts 1122 staggered or offset,
which is exemplary only. In another configuration, the density of
the microposts 1122 is lower than the density of the capture sites
1112 of microarray 1110.
[0168] FIG. 15A and FIG. 15B illustrate side views of a micropost
1122 and show examples of actuation motion thereof. Namely, FIG.
15A shows an example of a micropost 1122 oriented substantially
normal to the plane of substrate 1124. FIG. 15A shows that the
distal end of the micropost 1122 can move (1) with side-to-side
two-dimensional motion only with respect to the fixed proximal end
or (2) with circular motion with respect to the fixed proximal end,
which is a cone-shaped motion. By contrast, FIG. 15B shows an
example of a micropost 1122 oriented at an angle with respect to
the plane of substrate 1124. FIG. 15B shows that the distal end of
the micropost 1122 can move (1) with tilted side-to-side
two-dimensional motion only with respect to the fixed proximal end
or (2) with tilted circular motion with respect to the fixed
proximal end, which is a tilted cone-shaped motion.
[0169] FIG. 16 shows a close up cross-sectional view of a portion
of reaction chamber 1212 of flow cell 1200 shown in FIG. 12A and
FIG. 12B and shows the operation thereof. Again, the length of
microposts 1122 can vary, for example, from about 1 .mu.m to about
100 .mu.m. Again, the space between the distal ends of microposts
1122 and microarray 1110 can vary, for example, from about 0 .mu.m
to about 50 .mu.m. In another example, the space between the distal
ends of microposts 1122 and microarray 1110 can be about equal to
the length of microposts 1122. In yet another example, the space
between the distal ends of microposts 1122 and microarray 1110 can
be about 10 .mu.m or more or less than 10 .mu.m.
[0170] In operation, actuator 1160 generates an actuation force in
proximity to micropost array 1120 that compels at least some of
microposts 1122 to exhibit motion. In so doing, both regions of
local circulation 1310 and bulk circulation 1315 occurs within
reaction chamber 1212 of flow cell 1200. In the presence of regions
of local circulation 1310 and bulk circulation 1315, target
analytes in fluid sample 1218 can be rapidly flowed through the
bulk fluid sample 1218 to its corresponding capture site 1112 on
microarray 1110. Namely, due to the presence of regions of local
circulation 1310 and bulk circulation 1315 created by the motion of
microposts 1122, in reaction chamber 1212 of flow cell 1200 the
reaction time can be significantly reduced (i.e., accelerated
reactions) compared with microarray applications that rely on
diffusion alone for flow and/or mixing. That is, any given target
analyte can be rapidly flowed through the bulk fluid sample 1218
and to its corresponding capture site 1112 (i.e., its corresponding
analyte capture element). For example, compared with microarray
applications that rely on diffusion alone, flow cell 1200, which is
based on microfluidic system 1100 of FIG. 11, can be used to reduce
the reaction time from hours or days to a few minutes only.
[0171] Microfluidic system 1100 and/or flow cell 1200 are not
limited to the configurations shown in FIG. 11 through FIG. 16.
Other configurations of microfluidic system 1100 and/or flow cell
1200 are possible, examples of which are shown and described
hereinbelow with reference to FIG. 17A, FIG. 17B, FIG. 18A, and
FIG. 18B.
[0172] FIG. 17A and FIG. 17B show an example of flow cell 1200
shown in FIG. 12A and FIG. 12B that does not include the microarray
1110 and instead includes analyte capture elements on the
microposts 1122 themselves. Namely, individual or groups of
microposts 1122 within micropost array 1120 are functionalized with
analyte capture elements. In some embodiments, microposts 1122 of
micropost array 1120 may include one or more capture elements
(binding agents) exhibiting a specificity for one or more target
analytes in a fluid sample. In one example, microposts 1122 can be
functionalized by integration of the binding moiety into the bulk
elastomer. For example, this can be an elastomer made of monomers
that have functional groups as part of the chain, an elastomer made
of multiple different monomers that link together with one (or
more) containing a binding moiety (block co-polymer, for example),
or an elastomer doped with a functionalizing agent that is not
cross-linked into the matrix. In another example, microposts 1122
can be functionalized by surface-functionalization. Namely, the
binding moiety is attached to (e.g., grafted, bonded, etc.) or sits
atop the surface of the micropost 1122. This treatment can be
performed on micropost array 1120 after it is made or can be added
to the mold so that it is integrated into the surface upon
curing.
[0173] Referring now to FIG. 17B, micropost array 1120 can include
certain microposts 1122 that have been functionalized with capture
elements (binding agents) exhibiting a specificity for one or more
target analytes in fluid sample 1218, hereafter called
functionalized microposts 1122A. Micropost array 1120 can also
include certain microposts 1122 that have not been functionalized
(i.e., are chemically inert), hereafter called inert microposts
1122B. In effect, groups of functionalized microposts 1122A, which
are arranged among inert (or non-functionalized, or passive)
microposts 1122B, create capture sites that substantially mimic
capture sites 1112 of microarray 1110. The configuration shown in
FIG. 17A and FIG. 17B is not limited to groups of functionalized
microposts 1122A. In another example, groups and/or individual
functionalized microposts 1122A can be arranged among inert
microposts 1122B.
[0174] In another embodiment, FIG. 18A and FIG. 18B show an example
of flow cell 1200 shown in FIG. 12A and FIG. 12B that includes the
combination of both microarray 1110 that includes capture sites
1112 and microposts 1122 that are functionalized with analyte
capture elements. In effect, individual and/or groups of
functionalized microposts 1122A create capture sites arranged
opposite capture sites 1112 of microarray 1110. In this
configuration, a double array of capture elements is formed, one
facing the other.
[0175] FIG. 19 illustrates a flow diagram of an example of a method
1400 of using a micropost array (e.g., micropost array 1120) in
combination with a microarray (e.g., microarray 1110) for rapidly
flowing target analytes through a bulk fluid. Method 1400 may
include, but is not limited to, the following steps.
[0176] At a step 1410, a micropost array is provided that is
positioned in relation to a microarray in the reaction chamber of
microfluidics device. For example, flow cell 1200 of FIG. 12, which
is based on microfluidic system 1100 of FIG. 11, provides a
micropost array 1120 positioned in relation to microarray 1110
within reaction chamber 1212 and with a space between micropost
array 1120 and microarray 1110.
[0177] At a step 1415, fluid sample is provided within the reaction
chamber. For example, in flow cell 1200, a bulk amount of fluid
sample 1218 is loaded into reaction chamber 1212 and in the space
between micropost array 1120 and microarray 1110.
[0178] At a step 1420, the micropost array is actuated to induce
flow or stirring action of the fluid sample within the reaction
chamber. For example, in flow cell 1200, micropost array 1120 is
actuated using actuator 1160 to induce flow or stirring action in
fluid sample 1218 within reaction chamber 1212. Namely, actuator
1160 is used to generate an actuation force in proximity to
micropost array 1120 to actuate microposts 1122, thereby compelling
at least some of microposts 1122 to exhibit motion. Because the
distal ends of microposts 1122 extend into the bulk fluid sample
1218, the motion thereof creates a flow or stirring action of fluid
sample 1218 within reaction chamber 1212.
[0179] At a step 1425, because of the flow created by the micropost
array, the target analytes rapidly disperse in the bulk fluid and
bind to their corresponding analyte capture locations of the
microarray. For example, in flow cell 1200, because of the flow
created by micropost array 1120, the target analytes rapidly
disperse in the bulk fluid sample 1218 and bind to their
corresponding analyte capture locations (i.e., capture sites 1112)
of microarray 1110. Namely, target analytes rapidly disperse due to
the presence of regions of local circulation 1610 and bulk
circulation 1615 created by the motion of microposts 1122, in
reaction chamber 1212 of flow cell 1200.
[0180] At a step 1430, after a certain amount of time, the
microarray is processed. For example, in flow cell 1200, after a
certain amount of time, microarray 1110 is processed. Namely,
detector 1165 can be used to analyze the absence and/or presence of
certain target analytes captured by microarray 1110.
[0181] FIG. 19 is also representative of a microfluidic system or
flow cell configured to carry out the method just described.
[0182] In summary and referring again to FIG. 11 through FIG. 19,
using the presently disclosed microfluidic system 1100, flow cell
1200, and/or method 1400, the reaction time can be significantly
reduced (i.e., accelerated reactions) compared with microarray
applications that rely on diffusion alone for flow and/or mixing.
For example, compared with microarray applications that rely on
diffusion alone, microfluidic system 1100, flow cell 1200, and/or
method 1400 can be used to reduce the reaction time from hours or
days to a few minutes only. This is particularly useful in
microarray applications in which the "time to result" is important
(e.g., POC devices).
[0183] Further, because of the enhanced flow, circulation, and/or
mixing action of the fluid sample and accelerated reactions, in
microarray applications in which the analyte concentration is low,
such as liquid biopsy/circulating cell-free DNA tests, microfluidic
system 1100, flow cell 1200, and/or method 1400 can be used to
increase analyte utilization and therefore improve sensitivity of
the detection operations as compared with microarray applications
that rely on diffusion alone for flow and/or mixing.
[0184] Further, microfluidic system 1100, flow cell 1200, and/or
method 1400 provide a micropost array (e.g., micropost array 1120)
in combination with a microarray (e.g., microarray 1110) and is
therefore able to process multiple target analytes with respect to
multiple capture locations in a single reaction chamber.
[0185] Further, microfluidic system 1100, flow cell 1200, and/or
method 1400 provide enhanced flow, circulation, and/or mixing
action of the fluid sample and accelerated reactions via micropost
array 1120, wherein micropost array 1120 is a simple and low cost
stirring mechanism compared with, for example, microfluidics
devices that include pumping mechanisms.
[0186] Further, microfluidic system 1100, flow cell 1200, and/or
method 1400 provide a micropost array (e.g., micropost array 1120)
in combination with a microarray (e.g., microarray 1110) while
maintaining compatibility with current detection methods (e.g.,
optical and/or electrical detection systems).
[0187] The presently disclosed micropost array (e.g., micropost
array 1120) positioned in relation to a microarray (e.g.,
microarray 1110), wherein the actuated motion of microposts (e.g.,
microposts 1122) is used to enhance flow, circulation, and/or
mixing action for analyte capture on a microarray, as described
herein above with reference to FIG. 11 through FIG. 19, can be used
in a standalone device, such as, but not limited to, any
microfluidics device (e.g., a disposable microfluidics cartridge, a
digital microfluidics cartridge, a flow cell, a droplet actuator,
or the like). In one example, FIG. 20A shows a block diagram of an
example of a microfluidic system 1500 that includes a microfluidics
cartridge 1510 (e.g., a portable microfluidics cartridge), wherein
microfluidics cartridge 1510 can be based on, for example,
microfluidic system 1100 and/or flow cell 1200 described herein
above with reference to FIG. 11 through FIG. 19. Microfluidic
system 1500 also includes an actuation unit 1514, wherein actuation
unit 1514 may be in close proximity to microfluidics cartridge
1510.
[0188] Microfluidics cartridge 1510 includes a reaction chamber
1512. Processing and/or analysis of a fluid sample may be performed
within reaction chamber 1512. A micropost array (e.g., micropost
array 1120) and a microarray (e.g., microarray 1110) may be
provided inside reaction chamber 1512, wherein the micropost array
1120 may be used to affect the processing and/or analysis of a
fluid sample within reaction chamber 1512. In one example, the
microposts may include a flexible body and a metallic component
disposed on or in the body, wherein application of a magnetic or
electric field actuates the microposts into movement relative to
the surface to which they are attached. In some embodiments, the
microposts may include one or more capture elements (binding
agents) exhibiting a specificity for one or more target analytes in
a fluid sample.
[0189] As used herein, the term "actuation force" refers to the
force applied to the microposts. Actuation unit 1514 is used to
generate an actuation force in proximity to the micropost array
that compels at least some of the microposts to exhibit motion. The
actuation force may be, for example, magnetic, thermal, sonic,
optical, electrical, and/or vibrational. Further, the actuation
force may be applied as a function of frequency or amplitude, or as
an impulse force (i.e., a step function). Similarly, other
actuation forces may be used without departing from the scope of
the present invention, such as fluid flow across the micropost
array.
[0190] In another example, FIG. 20B shows a block diagram of an
example of a microfluidic system 1550 that includes microfluidics
cartridge 1510 that is described in FIG. 20A and a portable device
1515. Portable device 1515 includes actuation unit 1514 that is
described in FIG. 20A. Optionally, portable device 1515 includes a
motion detection unit 1516 and a processing unit (or controller)
1518.
[0191] Optionally, as the microposts exhibit motion in response to
the actuation force from actuation unit 1514, the motion of the
microposts may be measured or detected using motion detection unit
1516. Motion detection unit 1516 may be configured to measure the
motion of individual or specific microposts, groups of microposts,
or all the microposts. In motion detection unit 1516, the means for
detecting and measuring this micropost behavior may include, for
example, an optical (e.g., an imaging system), magnetic (e.g., a
magnetic pickup coil), sonic, and/or electrical tracking
system.
[0192] Optionally, measurement data from motion detection unit 1516
is provided to processing unit 1518 for calculations and analysis.
In another example, processing unit 1518 can be physically separate
from portable device 1515, wherein processing unit 1518 may be in
communication with portable device 1515 via any wired or wireless
means. Measurement data from motion detection unit 1516 can be any
information about the motion of the microposts. Processing unit
1518 processes the measurement data in order to determine at least
one property of the specimen based on the measured motion of the
microposts. The calculations and analysis performed by processing
unit 1518 may include determining a measure of fluid rheology based
on the force applied by actuation unit 1514 and the resulting
motion detected by motion detection unit 1516. In one example, as a
blood specimen begins to clot, the motion of the microposts becomes
restricted, and the resulting measurements may be used to indicate
and determine clotting time.
[0193] The presently disclosed micropost array (e.g., micropost
array 1120) positioned in relation to a microarray (e.g.,
microarray 1110), wherein the actuated motion of microposts (e.g.,
microposts 1122) is used to enhance flow, circulation, and/or
mixing action for analyte capture on a microarray, as described
herein above with reference to FIG. 11 through FIG. 19 can be used
in a high-throughput system. For example, FIG. 21 shows a block
diagram of an example of a high-throughput screening system 1600
that includes mechanisms for receiving and processing microarrays
based on configurations shown and described with reference to
microfluidic system 1100 and/or flow cell 1200 of FIG. 11 through
FIG. 19.
[0194] In one implementation of high-throughput screening system
1600, the actuation and optical system may be similar to that
described in International Patent Pub. No. WO/2008/103430, entitled
"Methods and systems for multiforce high throughput screening,"
published on Oct. 9, 2008, the disclosure of which is incorporated
herein by reference. High-throughput screening system 1600 is
capable of applying a force and measuring micropost responses. In
one example, high-throughput screening system 1600 includes a
control and measurement subsystem 1602, a multiforce generation
subsystem 1604, a multiforce plate subsystem 1606, and an imaging
and tracking optical subsystem 1608.
[0195] Control and measurement subsystem 1602 may be similar in
operation to processing unit 1518 described above in microfluidic
system 1550 of FIG. 20B. Control and measurement subsystem 1602 may
also include a mechanical properties module 1610 that is used to
measure the mechanical properties of the specimen depending on the
measured movement of the microposts.
[0196] Multiforce generation subsystem 1604 is the actuation
portion of high-throughput screening system 1600. Multiforce
generation subsystem 1604 may be similar in operation to actuation
unit 1514 described above in microfluidic system 1500 of FIG. 20A.
In one example, multiforce generation subsystem 1604 comprises a
magnetic drive block, such as exciter assembly. Multiforce
generation subsystem 1604 may also include an appropriate cooling
mechanism (not shown) to dissipate excess heat or to maintain
high-throughput screening system 1600 at a target temperature. In
one example, multiforce generation subsystem 1604 is capable of
producing forces of significant magnitude (e.g., forces greater
than 10 nanoNewtons), in multiple directions over a three
dimensional sphere, and can be varied at frequencies up to more
than three kilohertz.
[0197] Multiforce plate subsystem 1606 of high-throughput screening
system 1600 may comprise a microtiter well plate that includes a
plurality of specimen wells 1612. One or more of the specimen wells
1612 may be configured to include the presently disclosed micropost
array (e.g., micropost array 1120) positioned in relation to a
microarray (e.g., microarray 1110), wherein the actuated motion of
the microposts (e.g., microposts 1122) is used to enhance flow,
circulation, and/or mixing action for analyte capture on a
microarray, as described herein above with reference to FIG. 11
through FIG. 19. The microtiter well plate may also be coupled with
a cover glass sheet that serves as the bottom of the well plate.
Multiforce plate subsystem 1606 may also include a plurality of
field-forming poles that are used to form a magnetic (or electric)
coupling with excitation poles of multiforce generation subsystem
1604.
[0198] Imaging and tracking optical subsystem 1608 is the motion
detection of high-throughput screening system 1600. Imaging and
tracking optical subsystem 1608 may be similar in operation to
motion detection unit 1516 described above in microfluidic system
1550 of FIG. 20B. However, one physical difference between an
actuation system for a high-throughput screening system and a point
of care system is that the actuation system (e.g., multiforce
generation subsystem 1604) may be replicated for each well or small
group of adjacent wells in a multiwell microtiter plate. The motion
detection system for a multiwell microtiter plate may include, but
is not limited to, an optical system (e.g., imaging and tracking
optical subsystem 1608) that measures scattered light to detect
movement of the microposts, an imaging system including a camera
that images each well or group of wells in the microtiter plate, or
a pick up coil that measures amplitude and phase of a current
produced by motion the microposts in each well.
[0199] Imaging and tracking optical subsystem 1608 may also be
employed to perform several kinds of measurements, either
simultaneously with the application of force or after the force
sequence has been applied. For example, imaging and tracking
optical subsystem 1608 may include a single specimen imaging system
with a robotic stage that can systematically position each specimen
well of multiforce plate subsystem 1606 over a microscope
objective. In another example, imaging and tracking optical
subsystem 1608 may include an array based system that is capable of
imaging several specimen wells simultaneously. The recorded images
may be used to track the micropost position or the like.
EXAMPLES
[0200] A few non-limiting Examples of operating a flow cell as
described herein to capture targets will now be described. The
Examples relate to rare analyte extraction from large volumes of
whole blood that, conventionally, has proven to be extremely
challenging. These Examples have been included to provide guidance
to one of ordinary skill in the art for practicing representative
embodiments of the presently disclosed subject matter. In light of
the present disclosure and the general level of skill in the art,
those of skill can appreciate that the following Examples are
intended to be exemplary only and that numerous changes,
modifications, and alterations can be employed without departing
from the scope of the presently disclosed subject matter.
[0201] The prevailing protocols are often modifications of assays
designed for much smaller volumes. The workflows tend to be
operator-intensive, typically involving centrifugation to
concentrate cells into volumes compatible with the available
instruments. Known techniques include the use of steric filters,
magnetic beads, and columns of packed beads that selectively bind
to an analyte. The packing in a column achieves an extremely high
surface-area-to-volume ratio, but its small interstices are prone
to clogging. A clear-bore column that selectively binds to the
analyte would avoid clogging, but the binding efficiency of such
systems is prohibitively low because analyte transport to the walls
is diffusion limited. Pre-fractionating the sample with
conventional techniques (centrifugation, pipetting, etc.) would
also minimize clogging, but this involves complex workflows and
either highly skilled operators or specialized robotic facilities.
Techniques that use magnetic beads can be automated, but their
capture efficiency is typically inferior because it is impractical
and expensive to achieve the same surface area-to-volume ratio
present in the column systems. Also, careful balancing of the bead
population to the target cell population is required. Large volumes
of the bead reagents are also required, because the bead population
must be tuned to the total number of cells, not the number of
target cells. Yet, the target cell population may be a tiny
fraction (e.g., less than 1%) of the total cell population. As a
result, the cost of these assays is very high--typically hundreds
of dollars per unit of blood. Moreover, the kits associated with
known techniques require extensive manual handling.
[0202] A flow cell as described herein may overcome these issues.
In the Examples described below, the flow cell includes an array of
surface-attached structures functionalized with binding agents as
described herein.
Example 1
Progenitor Cell Isolation From Whole Blood
[0203] In treating bone marrow diseases such as leukemia and
multiple myeloma, blood progenitor cells (BPCs) are routinely
collected from peripheral blood for transfusion. However, typical
clinical practice calls for collection of the full range of
monocytes and performs no subsequent purification. The clinical
value of enriched or purified transplantation is an active area of
research. At a minimum, however, there are dramatic practical
concerns about this approach. Protocols often involve freezing and
storing liters of extracted cell product from the donor, especially
for autologous transfusion. Storing this volume of blood product is
expensive and space-intensive. The cell freezing process requires
dimethyl sulfoxide (DMSO), a toxic solvent that triggers unpleasant
side effects in the patient when the transfusion is administered. A
more judicious selection of the cells to be used in transfusion
could minimize the volume to be stored and transfused by a factor
of 20 or more.
[0204] More broadly, curation of the monocyte product extracted
during apheresis is an active area of clinical research. This may
involve depletion (for example, of t-cells) or enrichment (for
example, of CD34 progenitor cells).
[0205] The primary method used to select subpopulations of cells is
a pull-down assay with magnetic beads. In recent years, new
automated systems have improved the throughput of these assays.
However, they are still expensive and limited in the volumes that
they can process. By contrast, the ideal extraction method would
be: 1) scalable and inline, meaning very large volumes (e.g.,
greater than 1 L) of whole blood or monocyte-enriched product could
be processed through a single flow cell; 2) compact and modular,
meaning the extraction system could be integrated into other
systems (for example, placed in-line with an apheresis system), and
that multiple isolation selection steps could be performed
simultaneously by placing additional modules in series; and 3)
rapid, meaning a cartridge could perform isolation on blood moving
as fast (or faster) than the flow rate in a typical apheresis
system. Such a system would dramatically streamline clinical
research studying targeted transplantation techniques. It could
dramatically reduce the storage volumes required for
transplantation and the DMSO exposure of patients. If included as a
modular component in apheresis systems, it could open the door to a
new category of blood product donation, enabling systematic and
pervasive BPC banking.
[0206] A flow cell as described herein may overcome these issues.
Specifically, the surface-attached structures of the flow cell may
include a binding agent exhibiting a specific affinity to CD34
progenitor cells. The flow cell may thus be effective for capturing
CD34 progenitor cells from a large volume of undiluted whole blood.
If needed, the surface-attached structures may be moved under the
influence of a magnetic or electric field as described herein to
prevent or disrupt clogging. Moreover, because the surface-attached
structures are fixed to an underlying substrate, they do not
aggregate in the presence of a magnetic field, in contrast to
magnetic beads typically used for capture assays. After the CD34
progenitor cells have been captured, they may be released via an
appropriate release mechanism as described herein, such as flowing
a buffer containing a release agent that induces oligo cleavage
into the flow cell. This may be done in combination with causing
mechanical disruption by actuated motion of the surface-attached
structures. Other techniques may be utilized to assist in the
release process as well, such as electroporation. The released CD34
progenitor cells may be collected separately from the blood, and
the blood, now depleted of the CD34 progenitor cells, may be
recovered separately.
Example 2
Cell-Free DNA Isolation From Whole Blood
[0207] Liquid biopsy entails the isolation and characterization of
cells and nucleic acids from biological fluids. Liquid biopsy is
among the most exciting areas of new diagnostics development, with
a burst of new tests for fetal screening and cancer genotyping. In
both contexts, circulating nucleic acids (NAs) promise a minimally
invasive method for detecting and monitoring disease. Yet costs and
risks associated with the tests as presently offered are
substantial. Fetal testing is used by patients to make irreversible
decisions, including pregnancy termination; because of poor
specificity, circulating fetal deoxyribonucleic acid (DNA) tests
are recommended only for screening of high-risk patients. Cancer
genotyping may be used to guide the selection of therapies that,
when poorly matched to the cancer, may have very low efficacy; as a
result, the patient suffers needless costs, side effects, and loss
of time that could have been spent with a more effective
therapy.
[0208] A liquid biopsy involves three general steps: sample
collection, analyte purification, and analysis. Sample collection
is a simple blood draw. Analysis is the subject of intense
investment and development. The middle step--purification--has
received relatively little attention. Circulating NAs are present
in very low concentrations. Presently, collection and purification
require either much hands-on processing or expensive robotic
equipment at a specialized Clinical Laboratory Improvement
Amendments (CLIA) lab. In particular, column-based purification is
laborious and time consuming, while automated, bead-based methods
require complex robotic equipment. Special, expensive tubes are
used to preserve samples during storage and transit. The complex,
multi-step protocols--both automated and manual--create
opportunities for sample contamination.
[0209] It would be advantageous to improve the reliability of
liquid biopsy by improving the sample itself. The ideal extraction
method would be: 1) closed, meaning the extraction system is a
low-maintenance, affordable instrument that accepts a sealed
container of raw sample and furnishes a sealed container of the
extracted analyte, with no operator access to the sample in
between; 2) purifying and concentrating, meaning the solvent is
buffer, the solute is predominantly NA, and the eluted volume is
small (.about.100 .mu.L, a typical molecular analyzer capacity);
and 3) rapid and compact, meaning the extraction is performed at
point of care, within 30 minutes of sample collection.
[0210] Such a system could allow sample collection with standard
ethylenediaminetetraacetic acid (EDTA) tubes and on-site
purification. CLIA labs could ship small vials of purified,
stabilized DNA, ready for analysis using their molecular detection
technology of choice. This system could reduce costs for existing
tests by simplifying the workflow. It could reduce the cost of
studying circulating NAs, and therefore accelerate clinical
research and diagnostics development. And it could be a keystone
technology for enabling completely point-of-care cell-free DNA
(cfDNA) diagnostics.
[0211] A flow cell as described herein may be utilized to implement
a method of inline NA extraction from whole blood. The flow cell
may be utilized to isolate cfDNA from a large sample volume (e.g.,
greater than 10 mL) by passing it through a small (e.g., .about.100
.mu.L) chamber of the flow cell. The surface-attached structures of
the flow cell may include a binding agent exhibiting a specific
affinity to the cfDNA, such as histone antibodies as may be
utilized in chromatin immunoprecipitation (ChIP). In a specific
example, the surface-attached structures are impregnated with
biotinylated phospholipid (Bio-DOPE), and then surface-treated with
streptavidin which binds to the biotinylated histone antibody.
Non-specific adsorption (NSA) is suppressed by impregnating
nonionic surfactants into the elastomer (e.g., Brij-35, Tween-20),
surface treating with non-ionic surfactants or lipid treatments
(Egg-PC), and/or adsorbing blocking proteins or polyelectrolytes
(bovine serum albumin (BSA), poly-L-lysine).
[0212] Typical human blood is .about.50% cellular material that is
denser and larger than cfDNA. To take advantage of this, the flow
cell may be operated with the tips of the surface-attached
structures pointing down, so that the gap (e.g., 30 .mu.m) between
the tips and the opposite surface creates a cell settling region.
As the surface-attached structures move, they size-exclude cells
into the region below, which process is assisted by gravity. vAfter
the cfDNA has been captured, it may be released via an appropriate
release mechanism as described herein, such as one that induces
proteolysis (e.g., the protease known as Proteinase K). The flowing
of the protease into the flow cell may be done in combination with
causing mechanical disruption by actuated motion of the
surface-attached structures. Other techniques may be utilized to
assist in the release process as well, such as electroporation.
Subsequent elution into buffer may produce a pure analyte.
[0213] Microfluidic devices have been developed to perform nucleic
acid extraction using other techniques such as electrokinesis, but
these have been demonstrated only with very small volumes (less
than 100 .mu.L of whole blood). By contrast, a flow cell as
described herein may process much larger volumes as indicated
above. Moreover, the surface-attached structures in the flow cell
provide a surface area-to-volume ratio at least as high as that
provided by conventional columns, yet are able to prevent or
disrupt clogging through actuated movement as described herein. The
flow cell is compact and thus may enable superior point-of-care
diagnostics. Also, the flow cell enables a fully closed process,
thereby reducing risk of contamination. Moreover, use of the flow
cell does not destroy the blood that passes through, potentially
enabling apheresis-like diagnostics. Because the binding chemistry
is modular, the flow cell may be modified to process samples such
as urine, bronchoaleveolar lavage, sputum, or spinal fluid. It
could also be modified to capture other analytes such as
circulating tumor cells, stem cells, or pathogens.
Example 3
Pathogen Isolation From Whole Blood
[0214] Hospital acquired infection (HAI) that leads to sepsis is
deadly, expensive, and increasingly common. Severe sepsis affects
more than one million U.S. patients per year; the mortality is
.about.30% and the cost to the health care system is roughly $20
billion. Rapid anti-microbial treatment is key to improving
outcomes, as is identifying the strain of the pathogen. Strain
identification reduces costs, improves care, and avoids overuse of
antimicrobials. Techniques for strain identification are rapidly
improving, but challenges remain. The traditional method relies
exclusively on blood culture (BC). This process takes days. Worse,
blood culture suffers poor specificity due to contamination and
poor sensitivity because pathogens do not always culture
successfully. New molecular techniques from firms like BioFire
shorten the time-to-result, but a culture step is still required.
Novel techniques are under development that are sensitive enough to
sequence pathogens without amplification by culture, but delivering
on their promise will require a cost-effective and automated
solution for extracting the target molecular analyte from large
volumes of blood.
[0215] Blood has antimicrobial activity and patients often have
circulating antibiotics that reduce BC viability; pathogen
purification improves viability, especially when initial pathogen
concentration is low. For molecular assays based on polymerase
chain reaction (PCR), typical protocols lyse all cells (blood and
bacterial), releasing as much as 1000.times. more human DNA than
pathogenic DNA. This results in non-specific amplification,
reducing sensitivity of the technique. Such "molecular background"
often sets the limit of detection in PCR. It also precludes the use
of direct sequencing methods. This background can be eliminated
only by purifying the pathogen from blood prior to lysis.
[0216] At onset of sepsis, bacterial concentration in blood is low
(e.g., 10-100 colony forming units (CFU)/ml in adults, and less
than 10 CFU/ml in neonates). A conventional molecular technique
might process 1 mL of blood and have a limit of detection of 10-50
CFU/mL. However, the same culture-free molecular analysis of
pathogens purified from 10-60 mL would be sensitive enough to
monitor bacteremia from the earliest stages of sepsis--or perhaps,
even earlier, such that optimally targeted therapy could begin
immediately upon diagnosis.
[0217] A flow cell as described herein may be utilized to isolate
bacteria from a large sample volume such as the above-noted range
of 10-60 mL. As in other applications disclosed herein, the
surface-attached structures in the flow cell may be moved to
provide an anti-clogging functionality. The attached structures of
the flow cell may include a binding agent exhibiting a specific
affinity to the bacteria. such as histone antibodies as may be
utilized in chromatin immunoprecipitation (ChIP). In a specific
example, the surface-attached structures are impregnated with
biotinylated phospholipid (Bio-DOPE), and then surface-treated with
streptavidin which binds to the biotinylated mannose-binding lectin
(MBL). Non-specific adsorption (NSA) is suppressed by impregnating
nonionic surfactants into the elastomer (e.g., Brij-35, Tween-20),
surface treating with non-ionic surfactants or lipid treatments
(Egg-PC), and/or adsorbing blocking proteins or polyelectrolytes
(bovine serum albumin (BSA), poly-L-lysine).
[0218] After the bacteria has been captured, a buffer containing an
appropriate lysing agent may be introduced into the flow cell
behind the blood sample to lyse the bacteria and thereby release
its components (including DNA) into a smaller volume of, for
example, 100 .mu.L. As in other applications, the lytic-based
release process may be performed in combination with causing
mechanical disruption by actuated motion of the surface-attached
structures. Other techniques may be utilized to assist in the
release process as well, such as electroporation.
[0219] It will be understood that various aspects or details of the
invention may be changed without departing from the scope of the
invention. Furthermore, the foregoing description is for the
purpose of illustration only, and not for the purpose of
limitation--the invention being defined by the claims.
Liquid Phase Assays
[0220] A variety of assay techniques for qualitatively assessing or
quantitatively measuring the presence, amount, or functional
activity of a target entity (an analyte) in a sample involve
combining reactants in a liquid phase and incubating the reaction
for a period of time sufficient for generation of a product. In
general, the efficiency of a liquid phase reaction (e.g., reaction
rate and yield) is typically greater for a homogeneous mixture
(i.e., the concentration of each reactant is consistent throughout
the reaction volume) than for a heterogeneous mixture.
Heterogeneity in a liquid phase reaction may occur due to the
chemistries used in the assay, such as a relatively high viscosity,
the formation of micro-vesicles, or other phenomenon that limits
the diffusion of the analyte and/or reagents in the reaction
volume. A homogeneous reaction volume can be created and/or
maintained by mixing the reaction liquid to reduce or substantially
eliminate heterogeneity of all the reactants at the microscale,
thereby increasing the efficiency of the reaction.
[0221] According to an aspect of the present disclosure, a flow
cell with surface-attached structures as described herein may be
utilized in carrying out a method of analyzing a target in a
reaction fluid (i.e., a liquid phase assay). In some embodiments,
the method may include flowing a target-containing reaction fluid
into the flow cell and applying a magnetic or electric field to the
flow cell to actuate the movement of the surface-attached
structures. In so doing, a flow or mixing action is created in the
reaction fluid thereby eliminating or substantially reducing
heterogeneity of the reactants at the micro scale level and
effectively increasing the reaction rate and/or improving the
reaction yield. The flow cell may be configured for various types
of assay techniques used to qualitatively assess or quantitatively
measure the presence, amount, or functional activity of a target
entity (the analyte) in a sample.
[0222] FIG. 22 illustrates a schematic view of an example of a
liquid assay system 2200 that is configured for mixing a reaction
fluid in a liquid-based assay for analysis of a target analyte.
Liquid assay system 2200 includes a flow cell 2210 and an actuator
2215 in proximity to flow cell 2210. Optionally, liquid assay
system 2200 may include a detector 2220 in proximity to flow cell
2210 to monitor a reaction and/or detect a reaction event.
[0223] Flow cell 2210 includes a fist substrate 2225 that includes
a reaction chamber 2230 integrated therein, wherein reaction
chamber 2230 is a space or void in substrate 2225. Substrate 2225
is capped with a second substrate 2235, wherein substrate 2235
encloses reaction chamber 2230. Substrate 2225 and substrate 2235
can be formed, for example, of glass or plastic. In this example,
flow cell 2210 includes two loading ports 2240 formed in second
substrate 2235 (e.g., one at each end) for suppling a reaction
fluid 2245 into or out of reaction chamber 2230. Reaction chamber
2230 is sized to receive a volume of reaction fluid 2245. For
example, reaction chamber 2230 can hold from about 1 .mu.L to about
200 .mu.L. A typical reaction chamber size might be, for example,
about 100 .mu.L.
[0224] A plurality (e.g., an array) of surface-attached structures,
such as microposts 1122, is attached to the surface of second
substrate 2235, such that microposts 1122 extend into reaction
chamber 2230. In this example, microposts 1122 are attached at a
plurality of respective attachment sites to a substrate 2255 that
is distinct from second substrate 2235 of flow cell 2210, and
substrate 2255 is attached to second substrate 2235. Each
surface-attached structure 2250 includes a proximal end attached to
substrate 2255 and a distal end that extends into reaction chamber
2230. Accordingly, the distal ends of microposts 1122 extend into
reaction fluid 2245 that is in reaction chamber 2230. Again, the
length of microposts 1122 can vary. The length of microposts 1122
can be from about 1 .mu.m to about 100 .mu.m in one example, or can
be from about 10 .mu.m to about 50 .mu.m in another example. In one
example, microposts 1122 are chemically inert and will not react
with components of reaction fluid 2245.
[0225] Reaction fluid 2245 may include any number of reaction
components 2260 and target analytes 2265. Examples of reaction
components 2260 include, but are not limited to, assay reagents
such as functionalized beads, binding agents (e.g., antibodies or
fragments thereof), salts, buffers, dNTPs, enzymes, and detection
reagents. Examples of target analytes 2265 include, but are not
limited to, nucleic acid molecules (e.g., DNA, RNA), proteins,
enzymes, protein-protein interactions, receptor-ligand
interactions, low-affinity interactions, second messenger levels,
peptides, sugars and small molecules.
[0226] In operation, actuator 2215 generates an actuation force in
proximity to microposts 1122 that compels at least some of
microposts 1122 to exhibit motion. The actuation force may be, for
example, magnetic, thermal, sonic, optical, electrical, and/or
vibrational. Actuator 2215 is substantially the same as actuator
1160 as described hereinabove with reference to FIG. 11 through
FIG. 19. In so doing, both regions of a local circulation 2270 and
a bulk circulation 2275 in reaction fluid 2245 occurs within
reaction chamber 2230 of flow cell 2210. In the presence of regions
of local circulation 2270 and bulk circulation 2275, the
concentration of reaction components 2260 and target analytes 2265
in sample fluid 2245 is consistent or substantially consistent over
the volume of reaction chamber 2230 (i.e., heterogeneity of
components in reaction fluid 2245 is substantially reduced or
eliminated, and areas of reactant depletion are substantially
reduce or eliminated) compared with liquid-based assays that rely
on diffusion alone for flow or mixing. Because the concentration of
reaction components 2260 and target analytes 2265 in sample fluid
2245 is consistent (or substantially consistent) over the volume of
reaction chamber 2230, the reaction time can be significantly
reduced (i.e., accelerated reactions) and/or the reaction yield
significantly improved (i.e., reaction components are used more
completely).
[0227] In some embodiments, an actuation force may be applied
continuously throughout the incubation period of a reaction to
compel at least some of microposts 1122 to exhibit motion and
generate regions of local circulation 2270 and bulk circulation
2275 in reaction fluid 2245.
[0228] In some embodiments, an actuation force may be applied
intermittently during the incubation period of a reaction to
provide more limited motion of microposts 1122 and mixing of
reaction fluid 2245.
[0229] In some embodiments, a reaction may be monitored and/or a
reaction product or event (e.g., generation of a fluorescence
signal) detected. For example, substrate 2225 and substrate 2235 of
flow cell 2210 can be formed of a clear glass or plastic material
and detector 2220 can be an optical detection mechanism (e.g., a
fluorescence-based optical detector).
[0230] Flow cell 2210 or liquid assay system 2200 can be configured
for performing various types of assay techniques used for
qualitatively assessing or quantitatively measuring the presence,
amount, or functional activity of a target entity (the analyte) in
a sample. In one example, flow cell 2210 can be configured for
amplification of a nucleic acid target (e.g., DNA or RNA). Examples
of nucleic acid amplification techniques include thermal cycling
PCR or isothermal amplification reactions (e.g., recombinase
polymerase amplification (RPA), loop mediated isothermal
amplification (LAMP), helicase-dependent amplification (HAD),
rolling circle amplification (RCA), multiple displacement
amplification (MDA), and nucleic acid sequence-based amplification
(NASBA)). In another example, flow cell 2210 can be configured for
a bead-based detection assay. In one example, the bead-based
detection assay can be a fluorescence-based proximity assay for
detecting, for example, a protein, protein-protein interactions, or
protein modifications (e.g., phosphorylation).
[0231] FIG. 23 illustrates a flow diagram of an example of a method
2300 of using a flow cell with surface-attached structures for
creating a flow or mixing action in a liquid phase reaction. Method
2300 may include, but is not limited to, the following steps.
[0232] At a step 2310, a flow cell with surface-attached structures
is provided in a microfluidics device. For example, flow cell 2210
of FIG. 22 provides microposts 1122 that extend into reaction
chamber 2230.
[0233] At a step 2315, a reaction fluid is provided within the
reaction chamber. In one example, a reaction fluid 2245 that is a
"cocktail" comprising a mixture of reaction components 2260 and
target analyte 2265 is loaded into reaction chamber 2230 via a
loading port 2240. In another example, a cocktail fluid comprising
reaction components 2260 and a sample fluid comprising target
analyte 2265 are loaded separately (e.g., sequentially) via a
loading port 2240 into reaction chamber 2230 to generate a reaction
fluid 2245. In yet another example, a plurality of reagent fluids
each comprising at least one reaction component 2260 and a sample
fluid comprising target analyte 2265 are loaded separately (e.g.,
sequentially) via a loading port 2240 into reaction chamber 2230 to
generate a reaction fluid 2245.
[0234] At a step 2320, the surface-attached structures are actuated
to induce a flow or mixing action of the reaction fluid within the
reaction chamber. For example, in flow cell 2210, microposts 1122
are actuated using actuator 2215 to induce flow or mixing action in
reaction fluid 2245. Namely, actuator 2215 is used to generate an
actuation force in proximity of flow cell 2210 to actuate
microposts 1122, thereby compelling at least some of microposts
1122 to exhibit motion. Because the distal ends of microposts 1122
extent into reaction fluid 2245, the motion thereof creates a flow
or mixing action of reaction fluid 2245 within reaction chamber
2230.
[0235] At a step 2325, because of the flow and mixing created by
the surface-attached structures, heterogeneity of all reactants in
the reaction fluid is substantially reduced or eliminated, and the
efficiency of the reaction is increased. For example, in flow cell
2210, because of the flow and mixing action created by microposts
1122, the concentration of reaction components 2260 and target
analytes 2265 in sample fluid 2245 is consistent or substantially
consistent over the volume of reaction chamber 2230 (i.e.,
heterogeneity of components in reaction fluid 2245 is substantially
reduced or eliminated, and areas of reactant depletion are
substantially reduce or eliminated) compared to liquid-based assays
that rely on diffusion alone for flow and/or mixing. Because the
concentration of reaction components 2260 and target analytes 2265
in sample fluid 2245 is consistent (or substantially consistent)
over the volume of reaction chamber 2230, the reaction time can be
significantly reduced (i.e., accelerated reactions) and/or the
reaction yield significantly improved (i.e., reaction components
are used more completely).
[0236] At a step 2330, after a certain amount of time, the reaction
is processed. In one example, in flow cell 2210, after a certain
amount of time, reaction fluid 2245 is processed to assess (e.g.,
qualitatively or quantitatively) a product generated by the
reaction of reaction components 2260 and target analytes 2265.
Namely, detector 2220 can be used to analyze the absence and/or
presence of a certain reaction product generated by the reaction of
reaction components 2260 and target analytes 2265. In another
example, after a certain amount of time sufficient to generate a
reaction product, reaction fluid 2245 with the reaction product
therein can be removed from reaction chamber 2230 of flow cell 2210
via a loading port 2240 for subsequent processing.
* * * * *