U.S. patent application number 16/181050 was filed with the patent office on 2019-03-07 for capturing particles.
The applicant listed for this patent is The General Hospital Corporation. Invention is credited to Chia-Hsien Hsu, Shannon Stott, Mehmet Toner.
Application Number | 20190072465 16/181050 |
Document ID | / |
Family ID | 42060400 |
Filed Date | 2019-03-07 |
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United States Patent
Application |
20190072465 |
Kind Code |
A1 |
Toner; Mehmet ; et
al. |
March 7, 2019 |
CAPTURING PARTICLES
Abstract
Methods and systems capturing particles suspended in a fluid
flowed through a micro-channel, can include flowing the fluid
including the particles to be captured through a micro-channel and
past a groove defined in a surface of a wall of the micro-channel
such that flowing the fluid past the groove forms microvortices in
the fluid; contacting at least some of the particles against an
adherent disposed on one or more of walls of the microchannel after
the microvortices form in the fluid; and capturing at least some of
the particles contacting the adherent.
Inventors: |
Toner; Mehmet; (Charlestown,
MA) ; Stott; Shannon; (Stoneham, MA) ; Hsu;
Chia-Hsien; (Zhunan Town, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The General Hospital Corporation |
Boston |
MA |
US |
|
|
Family ID: |
42060400 |
Appl. No.: |
16/181050 |
Filed: |
November 5, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14843028 |
Sep 2, 2015 |
10126218 |
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16181050 |
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13121130 |
Aug 11, 2011 |
9128091 |
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PCT/US2009/058408 |
Sep 25, 2009 |
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14843028 |
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61100420 |
Sep 26, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 2200/0647 20130101;
B01L 2300/0858 20130101; B01L 2400/086 20130101; G01N 1/405
20130101; B01L 3/502761 20130101; G01N 33/57434 20130101; B01L
2400/0487 20130101; B01L 2300/0816 20130101; B01L 2200/0668
20130101; G01N 33/54366 20130101; G01N 33/56966 20130101 |
International
Class: |
G01N 1/40 20060101
G01N001/40; G01N 33/574 20060101 G01N033/574; G01N 33/543 20060101
G01N033/543; G01N 33/569 20060101 G01N033/569; B01L 3/00 20060101
B01L003/00 |
Claims
1. (canceled)
2. A microfluidic device comprising a micro-channel having an
elongated direction along which the micro-channel extends, wherein
the micro-channel comprises: a first inner wall surface; a first
groove defined in the first inner wall surface of the
micro-channel, wherein the first groove comprises an apex and two
arms connected to the apex, and the first groove is oriented such
that the apex points in the elongated direction of the
micro-channel; and an adherent disposed on a surface of the first
groove, wherein the adherent configured to selectively bind to an
analyte of interest.
3. The device of claim 2, wherein an angle between each arm of the
first groove is between 10.degree. and 170.degree..
4. The device of claim 2, wherein the micro-channel comprises a
second groove defined in the surface of the first wall, the second
groove comprising an apex and two arms connected to the apex, the
apex of the second groove is oriented to point in the elongated
direction of the micro-channel and wherein the apex of the first
groove is laterally offset to a first side of a centerline of the
microfluidic channel and laterally offset from the apex of the
second groove.
5. The device of claim 4, wherein the apex of the second groove is
laterally offset to a second side of the centerline of the
microfluidic channel that is opposite to the first side of the
centerline.
6. The device of claim 2, wherein the first groove comprises a
U-shape.
7. The device of claim 2, wherein the adherent is an antibody.
8. The device of claim 7, wherein the antibody is an anti-CD66
antibody, an anti-CD14 antibody, an anti-CD4 antibody, an anti-CD8
antibody, or an anti-EpCAM antibody.
9. The device of claim 2, wherein the adherent is an aptamer.
10. The device of claim 2, wherein the two arms of the first groove
are symmetric.
11. The device of claim 2, wherein the two arms of the first groove
are asymmetric.
12. The device of claim 2, wherein the groove spans less than a
width of the microchannel.
13. The device of claim 2, wherein the first groove is disposed in
a first column of a first plurality of grooves defined in the first
inner wall surface, wherein each groove within the first plurality
of grooves comprises a respective apex and a respective two arms
connected to the apex.
14. The device of claim 13, comprising a second plurality of
grooves in the first inner wall surface, the second plurality of
grooves defining a second column, wherein each groove within the
second plurality of grooves comprises a respective apex and a
respective two arms connected to the apex, and wherein the second
column is laterally offset from the first column along a plane of
the first inner wall surface.
15. The device of claim 14, wherein the first column and the second
column are interspersed with symmetric grooves and asymmetric
grooves.
16. The device of claim 14, wherein each groove of the second
column extends into a corresponding groove of the first column.
17. The device of claim 14, where an apex of each groove in the
second column is oriented in an opposite direction of the apex of
each groove in the first column.
18. The device of claim 14, comprising a third plurality of grooves
in the first inner wall surface, the third plurality of grooves
defining a third column, wherein each groove within the third
plurality of grooves comprises a respective apex and a respective
two arms connected to the apex, and wherein the third column is
longitudinally offset from the first column in the elongated
direction and along a plane of the first inner wall surface.
19. A microfluidic device comprising a micro-channel having an
elongated direction along which the micro-channel extends, wherein
the micro-channel comprises: a first inner wall surface and a
second inner wall surface directly across from and opposite to the
first inner wall surface; a first groove defined in the first inner
wall surface of the micro-channel, wherein the first groove
comprises an apex and two arms connected to the apex, and the first
groove is oriented such that the apex points in the elongated
direction of the micro-channel; and an adherent disposed on the
second inner wall surface of the first groove, wherein the adherent
configured to selectively bind to an analyte of interest.
20. The device of claim 19, wherein the adherent is an
antibody.
21. The device of claim 19, wherein the adherent is an aptamer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/843,028, filed Sep. 2, 2015, which is a continuation of U.S.
application Ser. No. 13/121,130, filed Aug. 11, 2011, now U.S. Pat.
No. 9,128,091, which is a 371 U.S. National of PCT Application No.
PCT/US2009/058408, filed Sep. 25, 2009, which claims priority to
U.S. Patent Application No. 61/100,420, which was filed on Sep. 26,
2008, the entire contents of which are incorporated herein by
reference in their entirety.
BACKGROUND
[0002] Microfluidic devices find application in micro total
analysis systems (.mu.TAS) or lab-on-a-chip (LOC) systems because
such devices offer the ability to analyze small sample volumes, and
can be developed into highly parallel systems at reduced costs. In
particular, such systems can be used in biological and clinical
applications in which particle manipulation is used to perform
operations, for example, concentrating, detecting, sorting, and
focusing particulate samples, such as cells and colloids. Passive
manipulation of particles flowing through microfluidic devices, by
techniques such as hydrodynamic focusing, size filtration, and
sedimentation, is relatively simple in comparison to active
manipulation using external energy such as optical forces,
magnetism, electro-kinetics, dielectrophoresis, acoustics, and the
like. Passive manipulation does not rely on external sources of
energy, but instead can be accomplished using geometries of
micro-channels in devices, and flow conditions through such
channels. In contrast, active manipulation can employ external
sources of energy and can require the integration of powered
components to the microfluidic devices.
SUMMARY
[0003] This specification describes technologies relating to
affinity-based particle capture in microfluidic devices having
grooves. When we refer to grooves, we include, for example, long
narrow channels (e.g., channels formed extending into and defined
by a wall of a larger channel).
[0004] In one aspect, methods for capturing particles suspended in
a fluid flowed through a micro-channel include: flowing the fluid
including the particles to be captured through a micro-channel and
past a groove defined in a surface of a wall of the micro-channel;
contacting at least some of the particles against an adherent
disposed on one or more of walls of the microchannel; and capturing
at least some of the particles contacting the adherent.
[0005] In one aspect, microfluidic devices include: a micro-channel
including: an inlet, an outlet positioned at a distance from the
inlet, wherein fluid flows from the inlet to the outlet, and a
groove defined into a surface of a wall of the microchannel, the
groove including an apex and two ends, each end connected to the
apex, the groove oriented such that the fluid flows past the ends
towards the apex; and an adherent applied to at least one wall to
selectively attach an analyte of interest.
[0006] Embodiments can include one or more of the following
features alone or in various combinations.
[0007] In some embodiments, the adherent is disposed on the surface
of the wall in which the groove is defined.
[0008] In some embodiments, the groove is defined in a wall of the
micro-channel. In some cases, the groove extends into the wall.
[0009] In some embodiments, the groove and a plurality of
additional grooves are defined in a surface of the wall such that
flowing the fluid past the plurality of additional grooves forms
respective microvortices in the fluid.
[0010] In some embodiments, flowing the fluid past the groove
comprises flowing the fluid past a groove including an apex and two
ends, each end connected to the apex, the groove oriented such that
the fluid flows past the ends towards the apex. In some cases, the
apex and the two ends are defined in the surface in a V shape. A
dimension of the groove can be in a range between 3 .mu.m and 70
.mu.m.
[0011] In some embodiments, flowing the fluid comprises flowing the
fluid at an average flow velocity between 2.4 cm/min and 6.0
cm/min.
[0012] In some embodiments, the particles are cancer cells and the
adherent is an antibody configured to bind the cancer cells. In
some cases, methods also include culturing the captured cancer
cells.
[0013] In some embodiments, flowing the fluid past the groove forms
microvortices in the fluid.
[0014] In some embodiments, the adherent is an antibody.
[0015] In some embodiments, the adherent is an aptamer.
[0016] In some embodiments, the inlet is configured to receive the
fluid that includes the analyte.
[0017] In some embodiments, the apex and the two ends form a
V-shape.
[0018] In some embodiments, the groove spans less than a width of
the micro-channel.
[0019] In some embodiments, each of the two ends are equidistant
from the apex.
[0020] In some embodiments, the groove is formed symmetrically in
the surface of the wall such that the apex is positioned on an axis
passing through a center of the micro-channel and the two ends are
equidistantly positioned from the apex.
[0021] In some embodiments, a first of the two ends is positioned
nearer to the apex than a second of the two ends.
[0022] In some embodiments, the apex is offset from an axis
extending along a center of the micro-channel.
[0023] In some embodiments, the groove is positioned such that a
first end of the groove receives the fluid before a second end.
[0024] In some embodiments, the groove is one of a plurality of
grooves defined in the wall of the micro-channel, each of the
plurality of grooves having an apex and two ends.
[0025] In some embodiments, the plurality of grooves are disposed
in a column of grooves.
[0026] In some embodiments, the device further comprises include an
additional column of grooves formed adjacent the column of
grooves.
[0027] In some embodiments, an apex and two ends of a groove in the
column of grooves are aligned with an apex and two ends of a groove
in the additional column of grooves on corresponding planes that
are perpendicular to an axis passing through the micro-channel.
[0028] In some embodiments, the additional column of grooves is
offset from the column of grooves.
[0029] In some embodiments, a dimension of a groove projecting
outward of the micro-channel is in a range between 3 .mu.m and 70
.mu.m.
[0030] Particular implementations of the subject matter described
in this specification can be implemented to realize one or more of
the following advantages. The techniques described here can
increase a potential for the passive manipulation and capture of
particles suspended in a fluid, for example, cells suspended in a
buffer solution, in a microfluidic environment. The grooves formed
in the micro-channel of the microfluidic device can induce helical
flows that generate microvortices in the fluid flowing through the
channel. The microvortices can be exploited to enhance the
transverse movement of particles flowing axially through the
channel, towards channel walls, causing the particles to more
frequently interact with and bind to the walls. In comparison to
microfluidic devices having micro-channels without the grooves,
cell-substrate interactions can be increased when cells suspended
in a buffer solution are flowed through the micro-channel that
includes the grooves. This, in turn, can increase the capture
efficiency of the device. Further, passive microfluidic fluid
manipulation techniques described here can negate the need for
external sources of energy, and can consequently decrease energy
consumption and cost of manufacture, particularly when the
microfluidic device is scaled up to highly parallel .mu.TAS or LOC
systems or both. The devices can be transparent based on the choice
of materials for manufacturing. The volumes of samples and reagents
consumed can be decreased due to the micrometer-range dimensions
through which the volumes are flowed. Consequently, cost of samples
and reagents can also be decreased. The techniques described are
applicable to capture and culture live cells.
[0031] The details of one or more implementations of the
specification are set forth in the accompanying drawings and the
description below. Other features, aspects, and advantages of the
specification will become apparent from the description, the
drawings, and the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 shows an embodiment of a microfluidic device having
grooves.
[0033] FIGS. 2A-2D illustrate particle flow paths in a
micro-channel having flat walls and another micro-channel having
grooves formed in a wall.
[0034] FIGS. 3A-3C illustrate exemplary grooves.
[0035] FIGS. 4A-4C illustrate an exemplary method of forming the
microfluidic device of FIG. 1.
[0036] FIG. 5 shows capture efficiencies of example microfluidic
devices for different flow rates.
[0037] FIG. 6 shows capture efficiencies of cancer cells spiked in
whole blood.
[0038] FIG. 7 shows an embodiment of a high throughput microfluidic
device having columns of herringbone patterns.
[0039] FIG. 8 shows an embodiment of a microfluidic device for
culturing captured cells.
[0040] FIGS. 9A-9C are micrographs showing the growth of captured
cells on a glass substrate.
[0041] FIG. 10 shows an analysis of EpCAM expression on the cells
captured with the microfluidic device having grooves and control
cells.
[0042] FIGS. 11A-11E show a circulating tumor cell captured from a
prostate cancer patient using the microfluidic device.
[0043] FIG. 12 show healthy donor controls.
[0044] FIG. 13 shows CTC capture from patient samples using the
microfluidic device having grooves.
[0045] FIGS. 14A-14D shows Wright-Giemsa staining of CTCs in the
microfluidic device having grooves.
[0046] FIG. 15 shows a comparison of two microfluidic devices
having different groove dimensions.
[0047] Like reference numbers and designations in the various
drawings indicate like elements.
DETAILED DESCRIPTION
[0048] Methods, apparatuses, and systems for affinity-based
particle capture in microfluidic devices having grooves are
described. A micro-channel formed in a microfluidic device can be
treated to capture particles suspended in a fluid flowing through
the channel. A particle capture efficiency of the microfluidic
device can be defined as a ratio of a number of particles captured
in the channel and a total number of particles flowed through the
channel. As described below, grooves are formed extending into the
walls of the micro-channel to create flow patterns in the fluid
that promote an interaction between the particles suspended in the
fluid and inner surfaces of the walls of the channel. The increased
interaction can lead to an increase in a number of particles
captured in the channel, and consequently, in the particle capture
efficiency of the microfluidic device. The efficiency can further
be increased by tailoring structural features of the microfluidic
device including, for example, device substrate material, channel
and groove dimensions, and the like, as well as fluid flow
parameters such as flow rates based on types of particles and the
types of fluids in which the particles are suspended. An example of
such a microfluidic device manufactured using soft lithography
techniques is described with respect to FIG. 1. As described later,
particles are captured in the micro-channel of the microfluidic
device by forming grooves in a wall of the micro-channel, coating
an adherent on the inner surfaces of the walls of the
micro-channel, and flowing particles suspended in the fluid through
the micro-channel.
[0049] FIG. 1 illustrates a microfluidic device 100 having grooves
135, 140 extending into one of the walls defining a channel 115 of
the device 100. In some embodiments, microfluidic devices include
protrusions extending outward from the wall (e.g., V-shaped
protrusions) rather than grooves extending into a wall of the
channel 115. In some implementations, a microfluidic device 100 can
include an upper substrate 105 bonded to a lower substrate 110,
each of which can be fabricated using an appropriate material. For
example, the upper substrate 105 can be fabricated using an
elastomer such as, for example, polydimethylsiloxane (PDMS), and
the lower substrate can be fabricated using glass, PDMS, or another
elastomer. Alternatively, or in addition, the substrates can be
manufactured using plastics such as, for example,
polymethylmethacrylate (PMMA), polycarbonate, cyclic olefin
copolymer (COC), and the like. In general, the materials selected
to fabricate the upper and lower substrates can be easy to
manufacture, for example, easy to etch, and can offer optical
properties that facilitate ease of testing, for example, can be
optically clear, and can be non-toxic so as to not negatively
affect the cells attached to the substrate. In addition, the
materials are preferred to exhibit no or limited autofluorescence.
Further, the materials can be easy to functionalize so that
analytes can be attached to the substrate. Furthermore, the
materials can be mechanically strong to provide strength to the
microfluidic device 100. The upper substrate 105 can be securely
fastened to the lower substrate 110, with a micro-channel formed
between them, as described below.
[0050] In some implementations, the micro-channel 115 can have a
rectangular cross-section including two side walls 120 and 125, and
an upper wall 130 formed in the upper substrate 105. Terms of
relative location such as, for example, "upper" and "lower" are
used for ease of description and denote location in the figures
rather than necessary relative positions of the features. For
example, the device can be oriented such that the grooves are on a
bottom surface of the channel or such that a central axis of the
channel extends vertically. Alternatively, the cross-section of the
micro-channel 115 can be one of several shapes including but not
limited to triangle, trapezoid, half-moon, and the like. The lower
substrate 110 can form the lower wall of the micro-channel 115 once
bonded to the upper substrate 105. In some implementations, the
micro-channel 115 includes multiple grooves 135 formed in the upper
wall 130 of the micro-channel 115. Alternatively, the grooves 135
can be formed in any of the walls, and/or can be formed in more
than one wall of the micro-channel 115. The grooves 135 can span an
entire length of a wall, or only a portion of the wall.
[0051] FIGS. 2A-2D are schematics illustrating particle suspensions
flowing through a micro-channel having flat walls and another
micro-channel having grooves formed in a wall. FIG. 2A shows a
microfluidic device 200 that includes a micro-channel 205 having a
rectangular cross-section. The walls of the micro-channel 205 do
not include grooves such as those described with respect to the
microfluidic device 100, i.e., surfaces of the walls are flat. A
particle suspension 220 including particles 225 suspended in a
fluid is flowing through the micro-channel 205. In contrast, FIG.
2B shows a similar suspension flowing through the microfluidic
device 100.
[0052] As the fluid flows past a herringbone pattern formed by
arranging grooves 135 in a column in the micro-channel 115, the
grooves 135 in the path of the fluid disrupt fluid flow. In some
embodiments, depending upon flow velocity and the dimensions of the
grooves, specifically, for example, a size of the grooves and an
angle between the two arms of a groove, the disruption in the fluid
flow leads to a generation of microvortices in the fluid. The
microvortices are generated because the grooves induce fluid flow
in a direction that is transverse to a principal direction of fluid
flow, i.e., the axial direction. In some embodiments, although
microvortices are not generated, the grooves 135, 140 induce
sufficient disruption to alter the flow path of portions of the
fluid to increase wall-particle interactions.
[0053] In an absence of the grooves, as shown in FIG. 2C, the
particles 225 suspended in the fluid travel through the flat
micro-channel 205 in a substantially linear fashion such that only
those particles 225 near the edges of the flow field (e.g.,
immediately adjacent to the walls of the micro-channel 205) are
likely to interact with the micro-channel 205 walls. In contrast,
as shown in FIG. 2D, flowpaths of the particles 225 traveling past
the herringbone patterns experience can be disrupted by the
microvortices in the fluid, increasing the number of
particle-micro-channel wall interactions. The microvortices are
affected by the structural features of each groove 445 formed in
the upper wall 130 of the microfluidic device 100. Exemplary
dimensions of a groove 445 are described with reference to FIG.
3.
[0054] FIGS. 3A and 3B illustrate a groove 135 formed on an upper
wall 130 of a micro-channel 115. As shown in FIG. 3A, a symmetric
groove 135 includes two arms, each spanning a length between a
first end 150 and the apex 145 (l.sub.1), and a second end 155 and
the apex 145 (l.sub.2). In the illustrated embodiments, the angle
.alpha. between the two arms is 90.degree.. In some embodiments,
the angle .alpha. between the arms ranges between 10.degree. and
170.degree.. FIG. 3B is a view of the micro-channel 115 including
the groove 135 formed in the upper surface 115. As shown in FIG.
3B, the width of the groove is w, the height of the side walls 120
and 125 of the micro-channel 115 is h.sub.c and the height of the
groove 135 formed on the upper wall 115 is h.sub.g. In some
embodiments, l.sub.1 and l.sub.2, each range between 250 .mu.m-400
.mu.m, h.sub.g ranges between 3 .mu.m and 70 .mu.m, h.sub.c is 100
.mu.m. For example, when h.sub.c is 100 .mu.m, h.sub.g is 25
.mu.m.
[0055] FIG. 3C illustrates an asymmetric groove 140 including two
arms, each spanning a length between a first end 170 and an apex
165 (l.sub.3), and a second end 175 and the apex (l.sub.4),
respectively. In the illustrated embodiment, the angle .beta.
between the two arms is 90.degree., and can range between
10.degree. and 170.degree.. In some implementations, the groove 140
can be manufactured such that a ratio between l.sub.3 and l.sub.4
is 0.5. For example, l.sub.3 is 141 .mu.m and l.sub.4 is 282 .mu.m.
The groove 140 has a thickness of 35 .mu.m. An effect of the height
of the groove, h.sub.g, on particle capture is described with
reference to FIG. 15.
[0056] A herringbone pattern can be created by forming a column of
herringbones in which each groove is positioned adjacent to another
groove. Further, all grooves in the column can face the same
direction. In some embodiments, a distance between each groove is
50 Alternatively, the grooves can be positioned at any distance
from each other. A column can include any number of grooves, for
example, ten grooves. The herringbone pattern can further include
multiple columns of grooves formed serially from an inlet to the
outlet. In some embodiments, two adjacent columns of grooves can be
separated by 100 .mu.m. In other words, a first groove of the
second column can be positioned 100 .mu.m away from a last groove
of the first column. This pattern can be repeated from an inlet to
the micro-channel 115 to the outlet.
[0057] In some embodiments, grooves or groups of grooves in a
column can be laterally offset from each other. For example, as can
be see in FIG. 2B, the column of grooves in microfluidic device 100
includes a first set of grooves with apexes set to the right
(facing downstream) of the channel centerline and a second set of
grooves with apexes set to the left of the channel centerline. Such
offsets are thought to further increase wall-particle
interactions.
[0058] The dimensions shown in FIGS. 3A-3C are exemplary. In
general, the choice of groove heights can depend on factors
including channel dimensions, particle properties including size,
density, and the like, and particle suspension flow rates. Although
deeper grooves offer more disruption, other factors can impose
limits on groove heights. For example, up to a certain limit, the
groove height can be increased in proportion with the channel
height. The channel height, and consequently the groove height, can
depend upon the particle to micro-channel 115 surface contact area.
An increase in channel dimensions can cause a decrease in
particle-micro-channel 115 interactions as surface contact area
available for the particles to interact decreases relative to the
cross-sectional flow area. Also, a lower limit on the channel
height, and consequently the groove height, can be imposed to
prevent clogging. In some implementations, a ratio between groove
height and channel height can be less than one, for example, in a
range between 0.1 to 0.6. In some implementations, the ratio can be
equal to one (e.g., the groove height can be equal to the channel
height), or can be greater than one (e.g., the groove height, for
example, 60 can be greater than the channel height, for example, 50
.mu.m). Further, the shape of the groove can be different from a
"V" shape, for example, "U" shape, "L" shape, and the like.
[0059] The micro-channel 115 can be formed in the upper substrate
105, for example, using soft lithography techniques. In some
implementations, negative photoresist (SU-8, MicroChem, Newton,
Mass., USA) can be photolithographically patterned on silicon
wafers to create masters with two-layer features. The masters thus
formed can include SU-8 features that form the basis for the
features of the micro-channel 115, for example, channel
cross-section, channel size, and the like. The heights of SU-8
features (ranging from 3 .mu.m-100 .mu.m) on the masters can be
measured with a surface profilometer such as a Dektak ST System
Profilometer, commercially available from Veeco Instruments Inc.,
Plainview N.Y. The masters can then be used as molds on which PDMS
pre-polymer can be poured and allowed to cure in a conventional
oven at 65.degree. C. for 24 hours. The upper substrate 105,
including the micro-channel 115, is formed when the poured PDMS
pre-polymer is cured. The cured upper substrate 110 can be removed
from the molds and bonded to the lower substrate 105, for example
using oxygen plasma treatment, to form the microfluidic device 100.
Alternatively, other types of bonding, for example, using a
reversible sealant, using physical clamping and holding under
pressure, and the like, can be used. In some implementations, the
substrates can be securely bonded together through chemical bonds,
and can subsequently be separated by breaking the bonds under the
application of mechanical forces.
[0060] FIGS. 4A-4C illustrate the formation of a microfluidic
device 100 including an upper substrate 405 manufactured using PDMS
and a lower substrate 410 manufactured using glass. The upper
substrate 405 including the upper and side walls of the
microchannel 415 can be formed using previously described
techniques. Alternatively, or in addition, the upper wall can
include multiple grooves 440, each formed in an asymmetric "V"
shape. In some implementations, symmetric grooves 440 and
asymmetric grooves 445 can be interspersed in the herringbone
pattern. Each groove further includes an apex 445 and two ends 450
and 455. In addition, the micro-channel 415 includes two side walls
420 and 425.
[0061] To configure the microfluidic device 400 to capture the
biological analyte of interest, an adherent 460 is disposed on the
inner surfaces of the micro-channel 115. Specifically, surface
modification is performed on the inner surfaces. In some
implementations, as shown in FIG. 4B, the adherent 460 can be mixed
in a solution and flowed through the micro-channel 415. As the
solution flows through the micro-channel 415, the adherent 460
binds to, and is thereby disposed in the inner surfaces of the
channel 415.
[0062] Techniques other than flowing the adherent through the
micro-channel 115 can also be used to dispose the adherent. For
example, in implementations in which plastic substrates are
employed, the adherent can be disposed on the substrate, for
example, by ultra-violet (UV) radiation treatment to alter the
surface properties such that analytes bind to the altered surface
prior to bonding the upper and lower substrates. In implementations
in which the lower substrate is glass, the glass can be
functionalized, for example, by sputtering, by gas phase
deposition, by building up layers of nanoparticle monolayers, and
the like prior to bonding the glass substrate to the upper
substrate.
[0063] As shown in FIG. 4C, the adherent 460 can be disposed
throughout the inner surfaces of the micro-channel 415.
Alternatively, the adherent 460 can be disposed in one or more
walls of the micro-channel 415, for example, in the wall in which
the grooves 445 are formed. In some embodiments, the adherent 460
can be disposed only on a lower substrate 410 manufactured from
glass. In such embodiments, the lower substrate 410 can be bonded
to the upper substrate 405 after the adherent is disposed on the
lower substrate. In such implementations, the flow rate of the
fluid is selected such that the microvortices established by the
grooves 440 drive the cells in the fluid toward the lower substrate
410 increasing a number of cell-lower substrate 410 interactions.
Subsequently, the lower substrate 410 can be separated from the
upper substrate 405 and the captured cells can be cultured.
[0064] In some implementations, the adherent 460 can be selected
such that the micro-channel 415 can be used for affinity-based cell
capture utilizing wet chemistry techniques. In such
implementations, the adherent 460 can be an antibody, for example,
antibody for EpCAM, or an aptamer, for example, aptamer for surface
proteins, with which the inner surfaces of the micro-channel 415
are functionalized. Additional examples of adherent 460 include
avidin coated surfaces to capture amplified target cells that
express biotin through the biotin-avidin linkage. Further examples
of adherents corresponding to cells that can be captured are shown
in Table 1 below.
TABLE-US-00001 TABLE 1 Cell-type Adherent Neutrophil Anti-CD66
Monocyte Anti-CD14 Lymphocyte Anti-CD4; Anti-CD8 Circulating tumor
cells Anti-EpCAM Neutrophils E, P Selectins HIV-specific T cell HAL
A2-SL9 Any disease specific T cell Pentamer
Once functionalized, the inner surfaces function as capture devices
that can bind the analytes of interest. Capture efficiencies of
exemplary microfluidic devices are described with reference to FIG.
5.
Example 1--Capture Efficiency
[0065] FIG. 5 shows capture efficiencies of example microfluidic
devices for different flow rates. As described previously, the
inner surfaces on which the adherent 460 are disposed bind cells
that interact with the surfaces. To study the capture efficiency of
microfluidic devices, a buffer solution spiked with cancer cells
(lung cancer cells--H1650 line) was flowed through a microfluidic
device 400 having herringbone patterns in the upper wall and
microfluidic device 200 having flat wall surfaces. The microfluidic
device 400 used in this example is a small footprint design having
a width of 2 mm and a length of 2 cm. The fluids were flowed
through the micro-channel 415 of the device 400 at flow rates of
0.12 ml/hr, 0.24 ml/hr, 0.36 ml/hr, and 0.48 ml/hr. All fluids that
traveled through the microfluidic devices 200 and 400 were
collected into a specially designed, serpentine waste chamber. Cell
capture efficiency was determined by counting the number of cells
captured in the devices (flat 200 or herringbone 400) and dividing
that number by the total number of cells put through the device
(counting the cells in the waste chamber and adding that to the
number of cells captured in the device).
[0066] For these experiments, three different flow rates were
studied, with four data points taken for each condition. It is
desirable that a device provide a high capture efficiency at high
flow rates. This can reduce the time and sample size required to
capture a desired number of cells of interest. As shown in FIG. 5,
the microfluidic device 400, that included the herringbone pattern,
outperformed the microfluidic device 200, that has only flat
surfaces, in cell capture efficiency for all flow rates. As flow
rates increase, the advantage of the device 400 with the
herringbone pattern increased. Even at very high flow rates, the
capture efficiency for the device 400 with the herringbone patterns
was .about.50%, whereas for the device 200 without the grooves, it
dropped to .about.30%.
Example 2--Capture Efficiency
[0067] FIG. 6 shows capture efficiencies for example microfluidic
devices with and without grooves. Similar to the previously
described experiments, the microfluidic device 200 having flat
surfaces and the device 400 having the herringbone pattern were
compared by determining the capture efficiency of cancer cells
spiked into whole blood (5,000 cells/ml). The microfluidic device
400 used in this example was the small footprint design described
with reference to FIG. 5. Four different flow rates, similar to the
flow rates described with reference to Example 1, were explored. In
addition, control microfluidic devices, one including the
herringbone pattern and the other including the flat surfaces, were
tested. In addition, the control microfluidic devices were also
tested by functionalizing with an irrelevant capture antibody not
configured to capture the cancer cells. For both control
microfluidic devices, zero cell capture was observed. Similar to
the previous results associated with cancer cells in the buffer
solution, the capture efficiency with the microfluidic device 400
having the herringbone pattern was better than the microfluidic
device 200 having the flat surfaces, for all conditions tested.
[0068] Further, a cell line of prostate cancer cells (PC3) was
tested due to the reduced EpCAM express. Cancer cells have less
EpCAM than regular epithelial cells. The new cell line and their
expression level is approximately 40,000 EpCAM molecules/cell. The
number of cells spiked into blood were 1,000 cells/ml so that the
spiking numbers are more relevant to rare cell detection levels.
For the new cell line, experiments were conducted at flow rates of
0.12 ml/hr and 0.24 ml/hr. For the PC3, the EpCAM surface express
was decreased by an order of magnitude relative to the cancer cells
and the spiking concentration was reduced by a factor of five.
Nevertheless, capture efficiencies comparable to the H1650s are
observed using the microfluidic device 400.
Example 3--Cell Viability
[0069] The effect of flow patterns and subsequent higher shear
stress on viability of the captured cells was also studied using
traditional Live/Dead assays. Cancer cells spiked into whole blood
were captured in the micro-channel 415 of a high throughput
microfluidic device 700, as shown in FIG. 7, having columns of
herringbone patterns. The microfluidic device 400 represents a
small footprint version that can be used for initial validation
studies. The microfluidic device 700 is an example of a scaled-up
version of the microfluidic device 400. To scale up the device, the
design of the microfluidic device 400 was repeated and elongated.
In some embodiments, the microfluidic device 700 is 2 cm wide and 4
cm long and includes a header region and a footer region. In this
example, multiple herringbone patterns were formed by forming
columns of herringbone patterns adjacent to each other in an upper
wall of a micro-channel having a larger width than the
micro-channel 415. The volumetric flow rate through the
micro-channel 715 is 2 ml/hr. captured cells were stained on the
substrate to which the cells were bound with Calcein AM and
Ethidium Homodimer. Results indicated that the most (.about.90%) of
the captured cells were viable, demonstrating that the herringbone
pattern had limited negative effects on the captured cells under
these conditions.
[0070] In some embodiments, the cells can be separated from the
substrate and cultured separately. To separate the cells from the
adherents, the linkage can between the adherents and the cells can
be weakened, for example, by dissolving the adherents in a solution
that does not affect the cells.
[0071] The number of columns of herringbone patterns was limited
only by the width of the micro-channel. In some implementations,
the microfluidic device 700 includes eight mini-chambers, i.e.,
eight columns of herringbone patterns. In such implementations, a
header design can be incorporated at an inlet of the micro-channel
to provide stability and uniform fluid volumes to each column of
herringbone patterns. In some implementations, each column of
herringbone patterns is positioned next to an adjacent column of
patterns such that, an apex of a "V" shaped groove in the column is
aligned with an apex of the "V" shaped groove in the adjacent
column. In other words, the apexes of both grooves lie on a line
perpendicular to a principal axis passing through the micro-channel
of the microfluidic device 700. If all grooves in a column are
equidistantly formed in the micro-channel of the device 700, then
all grooves in the device 700 will be aligned with each other. In
some implementations, a column of herringbone patterns can be
offset from an adjacent column. For example, the apex of a
"V"-shaped groove in the column can be offset by 10 .mu.m from the
apex of a "V"-shaped groove in the adjacent column. The offset
column design can further promote mixing. In some implementations,
the multiple columns in the device 700 can include symmetric
grooves 335 and asymmetric grooves 340 randomly interspersed in
each column. The forming of interspersed grooves promotes
transverse movement of the fluid and the particles suspended in
them, thereby increasing the number of cell-micro-channel wall
interactions and consequently increasing cell capture.
Example 4--Cell Culturing
[0072] In another example, a microfluidic device 800 for culturing
captured cells as shown in FIG. 8 was used to capture and culture
cells. The microfluidic device 800, was similar to the high
throughput design described with reference to FIG. 7, and included
a lower substrate manufactured using glass and an upper substrate
810 manufactured using PDMS that included the columns of
herringbone patterns as described previously. The flow rates of
blood containing the cells to be captured were around 2 ml/hr and
were manipulated to cause the cells to contact adhere to the lower
glass substrate 810. In the microfluidic device 800, the both the
lower and the upper substrates were coated with adherent. The lower
and upper substrates were reversibly bonded to each other using
such that, subsequent to cell capture, the upper substrate 810
could be removed from the lower substrate 805, for example, by
applying mechanical forces. In other embodiments, the lower and
upper substrates can be mechanically clamped to form a water-tight
seal or by suitable methods that do not damage the bound cells. In
some embodiments, the adherent 460 can be disposed on either the
lower or the upper substrate. Cells can be captured on the
substrate on which the adherent is disposed.
[0073] FIGS. 9A-9C are micrographs showing the growth of captured
cells on a glass substrate. Following capture of the cancer cells
spiked into blood, the upper substrate 810 of the microfluidic
device 800 was removed and both upper and lower substrates were
placed into a petri dish and incubated at 37.degree. C., 5%
CO.sub.2 with the appropriate cell culture media (FIG. 9A). As
shown in FIG. 9B, the cells were adhering to the substrate and had
started to spread and increase in number within 24 hours. After
more than three weeks of cell culture, the cells continued to
divide, forming a monolayer on both the lower substrate (glass) and
the upper substrate (PDMS). At this point, the cells were removed
from the capture surfaces (via trypsinization) and cultured in
traditional cell culture flasks. In this manner, successful culture
of captured cancer cells for extended periods of time was
demonstrated. Thus, the cells were not only viable but also
functional and can be grown in culture.
Example 5--Phenotype Changes
[0074] FIG. 10 shows fluorescence-activated cell sorting ("FACS")
analysis of EpCAM expression on the cells captured with the
microfluidic device having grooves and control cells. To explore if
the exposure of the cancer cells to the microfluidic device 800 had
any impact on the cell phenotype, one marker, EpCAM, was studied.
Specifically, expression levels between the cells captured on the
device 800 and the control cells (prepared in the same manner, but
never flowed through the device) were compared. Both cell
populations were cultured for 3 weeks post-experiment. Flow
cytometry results indicated that the capture and culture of the
cancer cells did not change their expression levels of EpCAM. These
results indicate that capturing with the microfluidic device 800
does not change the phenotype of the cell.
Example 6--Cell Capture
[0075] FIG. 11A shows a circulating tumor cell (CTC) captured from
a prostate cancer patient using the microfluidic device 100. As
shown in FIG. 11A, a CTC was captured on the grooves of the
microfluidic device 100. FIG. 11B shows that the cell was intact,
demonstrating the intact nucleus and cytoplasm. The cell was
identified as a CTC because it stained positive for PSA
(prostate-specific antigen, green), and a nuclear stain (DAPI,
blue) and negative for CD45 (red), a traditional marker for white
blood cells (see FIGS. 11C and 11D for a gray-scale
representation). Also, there are no contaminating cells. FIG. 11E
shows the intact cell under reflected light.
Example 7--Background CTC Levels
[0076] FIG. 12 show healthy donor controls. To confirm that the CTC
counts observed using patient samples were higher than the
background that would be observed in healthy donor samples, four
distinct donors (3 male, 1 female) were tested using the
microfluidic device 100 and stained with the PSA/CD45 stain. For
all four cases, the healthy donor counts were .ltoreq.5 false
positives/mL, with an average of 2 false positives/mL. Similar
experiments with the silicon chip resulted in a higher number of
false positives, presumably due to the increase in non-specific
binding.
Example 8--Capture Levels
[0077] FIG. 13 shows CTC capture from patient samples using the
microfluidic device 100 having grooves. Initial results indicate
that CTC capture from patient samples when using the microfluidic
device 100 having grooves can be as high as 160 CTCs/mL.
[0078] FIGS. 14A-14D show gray-scale representations of
Wright-Giemsa staining of CTCs in the microfluidic device 100
having grooves. Because the substrates used to manufacture the
microfluidic device 100 are transparent, the patient samples
captured within the microfluidic channel 115 can be stained with
histological stains, for example, Wright-Giemsa. FIGS. 14A-14D show
micrographs taken from a lung cancer patient sample run through the
microfluidic device 100. The cells selected are CTCs.
[0079] FIG. 15 shows a comparison of two microfluidic devices
having different groove dimensions. Two microfluidic devices were
compared to determine an effect of a height of the groove on
capture efficiency. A first microfluidic device had a channel
height of 70 .mu.m and a second microfluidic device had a channel
height of 50 The first microfluidic device had a groove height of
35 .mu.m and the second microfluidic device had a channel height of
25 In comparison to the first microfluidic device, the second
microfluidic device exhibited a three fold increase in capture
efficiency with the low expressor cells, PC3, spiked into whole
blood.
[0080] While this specification contains many specifics, these
should not be construed as limitations on the scope of the
specification or of what may be claimed, but rather as descriptions
of features specific to particular implementations of the
specification. Certain features that are described in this
specification in the context of separate implementations can also
be implemented in combination in a single implementation.
Conversely, various features that are described in the context of a
single implementation can also be implemented in multiple
implementations separately or in any suitable subcombination.
Moreover, although features may be described above as acting in
certain combinations and even initially claimed as such, one or
more features from a claimed combination can in some cases be
excised from the combination, and the claimed combination may be
directed to a subcombination or variation of a subcombination.
[0081] Similarly, while operations are depicted in the drawings in
a particular order, this should not be understood as requiring that
such operations be performed in the particular order shown or in
sequential order, or that all illustrated operations be performed,
to achieve desirable results. In certain circumstances,
multitasking and parallel processing may be advantageous. Moreover,
the separation of various system components in the implementations
described above should not be understood as requiring such
separation in all implementations, and it should be understood that
the described program components and systems can generally be
integrated together in a single software product or packaged into
multiple software products. Thus, particular implementations of the
specification have been described. Other implementations are within
the scope of the following claims. For example, the actions recited
in the claims can be performed in a different order and still
achieve desirable results.
[0082] Cells disposed in blood or in buffer solution can be flowed
through the micro-channel in such an orientation to bind to
adherents disposed in the channel. Techniques such as hot
embossing, injection molding, and the like can be used form the
micro-channel or the grooves or both. In such embodiments, the
master, from which the substrates are manufactured, need not be
silicon. In some embodiments, small molecules such as peptides,
nucleotides, and the like can be used as adherents
[0083] In some embodiments, prior to flowing any blood through a
micro-channel, non-specific binding can be reduced by the addition
of a surfactant to a blocking buffer (typically 1-3% BSA in
1.times.PBS). After adding the blocking buffer (e.g., 0.05% TWEEN20
in 3% BSA in 1.times.PBS), the microfluidic device can be incubated
for a duration, for example, one hour, at a temperature, for
example, room temperature, to provide efficient blocking of the
substrates. Blood flow can be initiated following the blocking
step.
[0084] In some embodiments, decreasing the non-specific binding to
a surface of the micro-channel can be achieved by contacting the
surface comprising the analyte-binding moiety with a nonionic
detergent prior to sample contact with the surface. The nonionic
detergent can be a polysorbate surfactant such as a polyoxyethylene
derivative of sorbitan monolaurate (for example, polysorbate 20,
sold under the tradename TWEEN20). The nonionic detergent can be
contacted with the surface at a concentration lower than the
concentration required to lyse mammalian cells. For example, an
aqueous solution comprising polysorbate 20 at a concentration of up
to about 0.05% can be used to pre-treat a surface before contact
with a biological sample.
[0085] The aqueous solution can further comprise components to
reduce non-specific surface binding from blood components. For
example, the surface can be contacted with a mixture of 0.05%
polysorbate 20, 1% BSA and 1.times. phosphate buffered saline (PBS)
(calcium ion and magnesium ion-free). The volume of the
pre-treatment solution can be selected based on the dimensions of
the channel. For example, about 3 mL of the 0.05% polysorbate 20
solution described above can be passed through a microfluidic
channel at a rate of about 30 ml/hr. The microchannel can be
incubated in the polysorbate 20 solution for about 1 hour before
introducing the biological sample to the channel.
[0086] In some embodiments, the micro-channel having a surface
containing a biotin-binding conjugate is contacted with a solution
comprising 0.05% Tween20 in 1% BSA in 1.times.PBS (Ca2+/Mg2+-free)
(for example, 3 mL of the surfactant solution at a flow rate of
about 30 mL/hr) prior to contact with the biological sample
containing CTCs, a biotinylated EpCAM antibody, biotin and
streptavidin. Pluronics, poloxymer, PEG, and other similar
surfactants can be similarly used instead of or in combination with
polysorbate 20.
* * * * *