U.S. patent application number 15/347709 was filed with the patent office on 2017-05-11 for inertial droplet generation and particle encapsulation.
The applicant listed for this patent is Illumina, Inc.. Invention is credited to Hamed Amini, Arash Jamshidi, Tarun Kumar Khurana, Foad Mashayekhi, Yir-Shyuan Wu.
Application Number | 20170128940 15/347709 |
Document ID | / |
Family ID | 57472026 |
Filed Date | 2017-05-11 |
United States Patent
Application |
20170128940 |
Kind Code |
A1 |
Amini; Hamed ; et
al. |
May 11, 2017 |
INERTIAL DROPLET GENERATION AND PARTICLE ENCAPSULATION
Abstract
Described are microfluidic devices and methods for providing a
predetermined number of microspheres or beads, together with a
cell, within a fluid droplet being processed. The system may
provide each droplet with a single bead and a single cell, and the
bead may contain DNA or other reagents for later identifying the
specific cell associated with that bead.
Inventors: |
Amini; Hamed; (Menlo Park,
CA) ; Jamshidi; Arash; (Menlo Park, CA) ;
Khurana; Tarun Kumar; (Menlo Park, CA) ; Mashayekhi;
Foad; (Menlo Park, CA) ; Wu; Yir-Shyuan;
(Menlo Park, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Illumina, Inc. |
San Diego |
CA |
US |
|
|
Family ID: |
57472026 |
Appl. No.: |
15/347709 |
Filed: |
November 9, 2016 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
62253605 |
Nov 10, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 3/502784 20130101;
B01F 13/0062 20130101; B01L 2300/0867 20130101; B01L 2300/0883
20130101; B01F 3/0807 20130101; B01L 2300/0816 20130101; B01L
3/502761 20130101; B01J 2219/005 20130101; B01L 2300/0858 20130101;
B01J 2219/00468 20130101; B01L 3/502776 20130101; B01L 2200/0647
20130101; B01J 2219/00722 20130101; B01L 2200/0636 20130101; B01L
2200/0652 20130101 |
International
Class: |
B01L 3/00 20060101
B01L003/00 |
Claims
1. A method of generating liquid droplets containing two or more
types of particles, the method comprising: focusing a bead fluid
having beads suspended therein into a first ordered stream of beads
within a first microchannel; focusing a cell fluid having cells
suspended therein into a second ordered stream of cells within a
second microchannel; and merging the first ordered stream with the
second ordered stream to form a plurality of droplets having a
predetermined number of cells and beads within each droplet.
2. The method of claim 1, wherein the first microchannel has a
minimum cross-sectional dimension D and the beads have a
cross-sectional dimension that is at least about 0.1 D.
3. The method of claim 2, wherein the cells have a cross-sectional
dimension that is at least about 0.1 D.
4. The method of claim 2, wherein merging of the first ordered
stream and the second ordered stream comprises contacting the first
ordered stream and the second ordered stream with a third fluid
immiscible in the first fluid and the second fluid.
5. The method of claim 1, wherein focusing the beads comprises
passing the beads through a first inertial focusing portion of the
first microchannel.
6. The method of claim 1, wherein focusing the cells comprises
passing the cells through a second inertial focusing portion of the
second microchannel.
7. The method of claim 1, wherein focusing the beads comprises
passing the beads through a first inertial focusing portion of the
first microchannel, and wherein focusing the cells comprises
passing the cells through a second inertial focusing portion of the
second microchannel.
8. The method of claim 7, wherein at least one of the first
inertial focusing portion of the first microchannel and the second
inertial focusing portion of the second microchannel has a curved
region.
9. The method of claim 8, wherein each curved region is
independently S-shaped, sigmoidal, sinusoidal, or spiral
shaped.
10. The method of claim 1, wherein the beads comprise nucleotide
fragments.
11. The method of claim 10, wherein the nucleotide fragments
comprise a barcode region, an index region, and a capture
region.
12. The method of claim 11, wherein the barcode region of each
nucleotide fragment is at least about six nucleotides in
length.
13. The method of claim 11, wherein the index region of each
nucleotide fragment is at least about four nucleotides in
length.
14. The method of claim 11, wherein the capture region comprises
poly-T nucleotides and is at least about ten nucleotides in
length.
15. The method of claim 1, wherein the predetermined number of
cells is one and the predetermined number of beads is one.
16. The method of claim 15, wherein each bead has a Reynolds number
of at least about 1, wherein the Reynolds number of a bead is
defined as Re = .rho. U m H .mu. ##EQU00009## where .rho. is the
density of the bead fluid, U.sub.m is the maximum flow speed of the
bead fluid, H is the hydraulic diameter of the bead fluid, and .mu.
is the dynamic viscosity of the bead fluid, wherein each cell has a
Reynolds number of at least about 1, and wherein the Reynolds
number of a cell is defined as Re = .rho. U m H .mu. ##EQU00010##
where .rho. is the density of the cell fluid, U.sub.m is the
maximum flow speed of the cell fluid, H is the hydraulic diameter
of the cell fluid, and .mu. is the dynamic viscosity of the cell
fluid.
17. The method of claim 16, wherein the proportion of the plurality
of droplets containing k.sub.1 beads and k.sub.2 cells is greater
than (.lamda..sub.1.sup.k1exp)(-.lamda..sub.1!))
(.lamda..sub.2.sup.k2exp(-.lamda..sub.2)/(k.sub.2!)), where
.lamda..sub.1 is the average number of the beads per droplet and
.lamda..sub.2 is the average number of the cells per droplet.
18. The method of claim 1, wherein the flow rate of the first
ordered stream is at least about 10 .mu.L/min.
19. The method of claim 1, wherein the flow rate of the second
ordered stream is at least about 10 .mu.L/min.
20. A droplet generation system, comprising: a first inlet
connected to a first inertial focusing microchannel disposed in a
substrate; a first flow source configured to drive a bead fluid
containing beads through the first inertial focusing microchannel;
a second inlet connected to a second inertial focusing microchannel
disposed in the substrate, wherein the first inertial focusing
microchannel is connected to the second inertial focusing
microchannel for forming the bead fluid and the cell fluid into a
plurality of droplets; a second flow source configured to drive a
cell fluid containing cells through the second inertial focusing
microchannel.
21. The system of claim 20, wherein the first inertial focusing
microchannel comprises a side wall having an irregular shape.
22. The system of claim 21, wherein the irregular shape comprises a
first irregularity protruding from a baseline surface away from a
longitudinal axis of the inertial focusing microchannel with the
irregular shape.
23. The system of claim 22, wherein each irregular shape is
selected from the group consisting of trapezoidal, triangular,
rounded, and rectangular.
24. The system of claim 20, wherein the second inertial focusing
microchannel comprises a side wall having an irregular shape.
25. The system of claim 20, wherein at least one of the first
inertial focusing microchannel and the second inertial focusing
microchannel has an expansion/contraction region having a side
wall, wherein the side wall has a stepped surface.
26. The system of claim 20, wherein at least one of the first
inertial focusing microchannel and the second inertial focusing
microchannel has an expansion/contraction region having a side
wall, wherein the side wall has a curved surface.
27. The system of claim 20, wherein at least one of the first
inertial focusing microchannel and the second inertial focusing
microchannel has a curved region.
28. The system of claim 27, wherein each curved region is
independently S-shaped, sinusoidal, sigmoidal, or spiral
shaped.
29. The system of claim 27, wherein the curved region has a Dean
number of up to about 30.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 62/253,605, filed Nov. 10, 2015, the content of
which is incorporate by reference in its entirety.
BACKGROUND
[0002] Field
[0003] The invention relates to the fields of microfluidics and
encapsulation of particles such as beads, nucleic acid fragments,
and cells into droplets for performing biological and chemical
reactions.
[0004] Description of Related Art
[0005] Microfluidic devices may be used to move fluids through
narrow channels to perform certain diagnostic or other reactions.
These devices can include inlets for receiving one or more fluids
and outlets for transferring fluids to external devices or
systems.
SUMMARY
[0006] In one aspect, the invention features methods of generating
liquid droplets containing two or more types of particles. The
methods include focusing a bead fluid having beads suspended
therein into a first ordered stream of beads within a first
microchannel; focusing a cell fluid having cells suspended therein
into a second ordered stream of cells within a second microchannel;
and merging the first ordered stream with the second ordered stream
to form a plurality of droplets having a predetermined number of
cells and beads within each droplet. In one example, the first
microchannel has a minimum cross-sectional dimension D and the
beads have a cross-sectional dimension that is at least about 0.1
D. The cells have a cross-sectional dimension that is at least
about 0.1 D. Merging of the first ordered stream and the second
ordered stream includes contacting with a third fluid immiscible in
the first fluid and the second fluid. Focusing the beads includes
passing the beads through a first inertial focusing portion of the
first microchannel. Focusing the cells includes passing the cells
through a second inertial focusing portion of the second
microchannel.
[0007] In certain of these methods, the beads include nucleotide
fragments. The nucleotide fragments include a tag or barcode
region, an index region, and a capture region. The tag or barcode
region of each nucleotide fragment can be at least about six
nucleotides in length. The index region of each nucleotide fragment
can be at least about four nucleotides in length. The capture
region includes poly-T nucleotides and can be at least about ten
nucleotides in length.
[0008] In certain of these methods, the predetermined number of
cells is one and the predetermined number of beads is one. The
Reynolds number (R.sub.e) of each of the beads is at least about 1,
with the Reynolds number of a bead defined as
Re = .rho. U m H .mu. , ##EQU00001##
where .rho. is the density of the bead fluid, U.sub.m is the
maximum flow speed of the bead fluid, H is the hydraulic diameter
of the bead fluid, and .mu. is the dynamic viscosity of the bead
fluid. The Reynolds number of each of the cells is at least about
1, with the Reynolds number of a cell defined as
Re = .rho. U m H .mu. , ##EQU00002##
where .rho. is the density of the cell fluid, U.sub.m is the
maximum flow speed of the cell fluid, H is the hydraulic diameter
of the cell fluid, and .mu. is the dynamic viscosity of the cell
fluid.
[0009] In certain of these methods, the proportion of the plurality
of droplets containing k.sub.1 beads and k.sub.2 cells is greater
than (.lamda..sub.1.sup.k1 exp(-.lamda..sub.1)/( k.sub.1!))
(.lamda..sub.2.sup.k2 exp(-.lamda..sub.2)/(k.sub.2!)), where
.lamda..sub.1 is the average number of the beads per droplet and
.lamda..sub.2 is the average number of the cells per droplet. The
flow rate of the first ordered stream is at least about 10
.mu.L/min, or is about 10 to 100 .mu.L/min, or is about 40 to 70
.mu.L/min, or is about 45 to 65 .mu.L/min, or is about 50 to 60
.mu.L/min, or is about 50 L/min, or is about 60 .mu./min. The flow
rate of the second ordered stream is at least about 10 .mu.L/min,
or is about 10 to 100 .mu.L/min, or is about 40 to 70 .mu.L/min, or
is about 45 to 65 .mu.L/min, or is about 50 to 60 .mu.L/min, or is
about 50 .mu.L/min, or is about 60 .mu.L/min.
[0010] In one embodiment, a droplet generation system includes a
first inlet connected to a first inertial focusing microchannel
disposed in a substrate; a first flow source configured to drive a
bead fluid containing beads through the first inertial focusing
microchannel; a second inlet connected to a second inertial
focusing microchannel disposed in the substrate, where the first
inertial focusing microchannel is connected to the second inertial
focusing microchannel for forming the bead fluid and the cell fluid
into a plurality of droplets; a second flow source configured to
drive a cell fluid containing cells through the second inertial
focusing microchannel.
[0011] In some embodiments, one or more particle channels may have
a curved region to decrease the focusing length required and to
decrease the device foot-print. In some embodiments, one or all
channels for a first particle type A (such as beads) may have a
curved region to decrease the focusing length required and to
decrease the device foot-print. In some embodiments, one or all
channels for a second particle type B (such as cells) may have a
curved region to decrease the focusing length required and to
decrease the device foot-print. The curved regions may be
symmetrically curved. In some embodiments, the curved regions may
be asymmetrically curved, such as S-shaped, sinusoidal, or
sigmoidal shaped, or continuously curved in a spiral pattern. In
some embodiments, the curved regions of some or all of the channels
are sinusoidal. In some embodiments, the curved regions of some or
all of the channels are spiral shaped. In some embodiments, the
bead channels, or the cell channels, or both the bead and cell
channels comprise spiral shaped regions. In some embodiments, the
bead channels, or the cell channels, or both the bead and cell
channels comprise sinusoidal regions. In some embodiments, the bead
channels comprise spiral regions and the cell channels comprise
sinusoidal regions.
[0012] In some embodiments, the bead channel 104 may have an
expansion/contraction region which enables the adjustment of the
spacing between beads inside the channel. In some embodiments, one
or both of the cell channels 108, 110 may have an
expansion/contraction region which enables the adjustment of the
spacing between cells inside the channel.
[0013] Optionally, the first inertial focusing microchannel
includes a side wall having an irregular shape (e.g., a
discontinuity in the linear nature of the side wall). Optionally,
the second inertial focusing microchannel includes a side wall
having an irregular shape. Optionally, the irregular shape includes
a first irregularity protruding from a baseline surface away from a
longitudinal axis of the inertial focusing microchannel with the
irregular shape. In some instances, the irregularity narrows the
microchannel with respect to the longitudinal axis and in other
instances the irregularity expands the microchannel with respect to
the longitudinal axis. Optionally, each irregular shape is
independently selected from the group consisting of trapezoidal,
triangular, rounded, and rectangular. In some embodiments, the
group further includes elliptical or unsymmetrical shapes. In some
embodiments, the microchannel includes a plurality of irregular
shapes along a portion of the microchannel. The irregular shapes
may be of the same shape or different shapes.
[0014] In some embodiments, one or both of the first inertial
focusing microchannel and the second inertial focusing microchannel
have an expansion/contraction region having a side wall, where the
side wall has a stepped surface. In some embodiments, at least one
of the first inertial focusing microchannel and the second inertial
focusing microchannel has an expansion/contraction region having a
side wall, where the side wall has a curved surface. In some
embodiments, at least one of the first inertial focusing
microchannel and the second inertial focusing microchannel has a
curved region having a Dean number of up to about 30. In some
embodiments, one of the first inertial focusing microchannel and
the second inertial focusing microchannel has a side wall with a
stepped surface. In other embodiments, both inertial focusing
microchannels have side walls with stepped surfaces.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1 is a perspective view of one embodiment of a system
for the separation, ordering, and focusing of cells and beads
within microchannels prior to droplet generation.
[0016] FIGS. 2A-D are schematic drawings showing bead focusing
through different sized bead channels. FIG. 2A shows beads flowing
through a square channel. FIG. 2B shows beads flowing through a
rectangular channel having a cross-sectional dimension or flow rate
that allows two beads to flow adjacent one another. FIG. 2C shows
beads flowing through a rectangular channel having a
cross-sectional dimension or flow rate that focuses the beads so
that the beads flow in a single file line within the bead channel
due to presence of bead-present and bead-absent co-flows. FIG. 2D
is a top schematic view of an inertial focusing bead channel having
a dual-inlet co-flow configuration.
[0017] FIG. 3 is a perspective view of an alternate embodiment of a
system using curving channels for the separation, ordering, and
focusing of cells and beads within microchannels.
[0018] FIGS. 4A-B are schematic drawings of embodiments of flow
channels configured to provide an inertial ordering process. FIG.
4A is a schematic drawing of the inertial ordering processing with
an asymmetrical curving channel. FIG. 4B is a schematic drawing
showing the use of an expansion/contraction region within the flow
channels to tune the spacing between ordered beads inside the
channel.
[0019] FIGS. 5A-F show different embodiments of microchannel
configurations for the ordering and focusing of cells and beads
within microchannels.
[0020] FIG. 6 illustrates a microchannel configuration that allows
high efficiency formation of single-cell/single-bead droplets.
[0021] FIG. 7 illustrates a microchannel configuration that allows
high efficiency formation of single-cell/single-bead droplets using
a dual-inlet co-flow system for cells.
[0022] FIG. 8 illustrates the use of an embodiment of the system
for single cell sequencing.
[0023] FIG. 9 is an image of a device according to embodiments
which shows the focusing of 30 .mu.m diameter beads to the four
focusing positions in a square channel within a length of 1.2-3 cm
from the bead fluid inlet.
[0024] FIG. 10 is an image of a device according to embodiments
that shows focusing and ordering of 40 .mu.m diameter polystyrene
beads in a rectangular straight microchannel prior to droplet
formation.
[0025] FIG. 11 is an image of a device according to embodiments
that shows focusing and ordering of 30 to 40 .mu.m PMMA beads in a
rectangular straight microchannel prior to droplet formation.
[0026] FIGS. 12A-12B show the focusing and ordering of 30 to 40
.mu.m sepharose gel beads in a straight rectangular microchannel
prior to droplet formation. FIG. 12A shows the image taken from the
instrument. FIG. 12B depicts the same image as FIG. 12A, except
that the contrast level has been adjusted to allow for easier
visualization of the sepharose gel beads.
[0027] FIGS. 13A-13B show the focusing and ordering of 30 to 40
.mu.m sepharose gel beads in a spiral rectangular microchannel
prior to droplet formation. FIG. 13A shows the image taken from the
instrument. FIG. 13B depicts the same image as FIG. 13A, except the
contrast level has been adjusted to allow for easier visualization
of the sepharose gel beads.
[0028] FIGS. 14A-14B depict two embodiments of systems comprising
spiral channels. FIG. 14A shows a system with two adjacent spiral
channels and one channel comprising a sinusoidal curve. FIG. 14B
shows a system with two spiral channels on opposite ends of the
system, surrounding two concentric channels comprising sinusoidal
regions.
[0029] FIGS. 15A-15B depict the configuration of a microfluidic
system with respect to the width of the channels after the
convergence of two inlet channels. FIG. 15A shows two cell channels
feeding into a bead channel (width b) and resulting in a single
channel of width m. Two oil inlet channels then converge, yielding
a single channel with width d. FIG. 15B shows a variation of the
configuration shown in FIG. 15A in which channel widths m and d are
increased relative to channel width b.
[0030] FIG. 16 depicts an embodiment of a microfluidic system in
which intra-channel structures or constrictions that can de-clump a
pool of clumped beads to yield ordered beads.
DETAILED DESCRIPTION
[0031] In the following detailed description, reference is made to
the accompanying drawings, which form a part hereof. In the
drawings, similar symbols typically identify similar components,
unless context dictates otherwise. The illustrative embodiments
described in the detailed description, drawings, and claims are not
meant to be limiting. Other embodiments may be utilized, and other
changes may be made, without departing from the spirit or scope of
the subject matter presented herein. It will be readily understood
that the aspects of the present disclosure, as generally described
herein, and illustrated in the Figures, can be arranged,
substituted, combined, separated, and designed in a wide variety of
different configurations, all of which are explicitly contemplated
herein.
[0032] Embodiments relate to the fields of microfluidics and
includes devices and methods for encapsulation of particles, such
as beads, nucleic acid fragments, and cells into droplets. Various
embodiments described below use laminar flow of a fluid, such as an
oil, through microfluidic channels to result in the continuous and
accurate self-ordering of particles suspended within the fluid. As
discussed below, embodiments include microfluidic devices having a
variety of specific channel geometries that can be configured to
advantage of the self-ordering liquid flows to create continuous
streams of ordered particles constrained in three spatial
dimensions. Particles order laterally within the y-z plane (or
cross-sectional plane) of a fluidic channel and can also order
longitudinally along the direction of fluid flow (i.e., the x
direction). An additional dimension of rotational ordering can
occur for asymmetrically shaped particles.
[0033] One embodiment includes methods and devices that perform
reactions within droplets flowing in a microchannel device. For
example, in one embodiment a microchannel device is designed to mix
a single cell with a single bead in one droplet. Each bead applied
to the microchannel device bears one or more nucleotide fragments,
and each nucleotide fragment comprises a unique DNA tag. The DNA
tag may be a barcode or other DNA sequence having the same
nucleotide sequence on all fragments bound to a single bead. The
DNA tag may alternatively be an index sequence which has a
different nucleotide sequence for each fragment on a single bead.
The tag may also include a capture region that can be used to
capture the tag by hybridization to other DNA sequences. For
example, the capture region may comprise a poly-T tail in some
embodiments. In this construct, each bead is uniquely tagged in
comparison to all other beads being used in the device. Thus, when
a single cell and a single bead are encapsulated into a droplet and
are exposed to lysis buffer (for example, the lysis buffer is
present in the droplet when encapsulation occurs, or is added to
the droplet after encapsulation), the cell is lysed, and each
polyadenylated mRNA in the cell becomes bound to the poly-T tail of
the capture region on the bead with which it is encapsulated. If
the bead is then subjected to a cDNA reaction using reverse
transcriptase and the appropriate primer, cDNA strands are formed
having the original mRNA sequences along with the unique tag from
the bead that was encapsulated with the cell. This results in all
of the mRNA from a single cell being labeled with a unique tag
sequence from the bead. This procedure allows later sequencing
reactions to be performed in bulk, with cDNA samples from many
cells being sequenced, but each having a unique tag so that they
can be sorted from one another. The index is used to correct for
amplification errors and avoid multiple-counting of a single
molecule. At the end of an experiment, the mRNA expression of
individual cells can be determined by sequencing the cDNA and
determining which mRNA population was present in each cell, and the
expression level of that mRNA.
[0034] In one embodiment, the microchannel device is configured to
separate, order, and focus streams of beads to focusing positions
within a channel flow field that result in the creation of droplets
each with a predetermined number of beads and cells. The focusing
can be based, at least in part, on inertial lift forces. In square
channels, this can lead, for example, to four streams of focused
particles spaced an equal distance apart from a center of each of
the four square faces. For rectangular geometries, this four-fold
symmetry can be reduced to a two-fold symmetry, with streams of
particles spaced apart from each of two opposed faces of the
channel.
[0035] In some embodiments, a dual-inlet co-flow system serves to
create a first focused, ordered stream of particles A and a second
focused, ordered stream of particles B, where particles A and B are
of different types. In some embodiments, the system serves to
create a single focused bead stream and a single focused cell
stream (e.g., the A particles are beads and the B particles are
cells). In some embodiments, the two streams of particles are
merged in the system to create a single stream comprising the
particles A and B, such as beads and cells. The merged stream of
particles is then contacted with an oil or other immiscible fluid
to create a droplet containing the two particle types. Thus, in
some embodiments, a third fluid stream is introduced that serves to
encapsulate the two types of particles. In some embodiments, the
third fluid stream comprises a carrier fluid that is immiscible or
partially immiscible with the first and second stream fluids and/or
the combined first/second stream fluid.
[0036] Embodiments include microchannel devices that encapsulate a
selected number of A and B particles in a droplet. For example, the
device may be configured to encapsulate no more than one A particle
and no more than one B particle in a single droplet, or up to one A
particle and one B particle in a single droplet, or one A particle
and one B particle in a single droplet. Configurations of fewer, or
more, A particles and fewer, or more, B particles in a single
droplet are also contemplated, including but not limited to two A
particles and one B particle, or one A particle and two B
particles.
[0037] Embodiments also relate to microchannel devices that place a
selected number of beads and cells into a droplet. For example, the
device may be configured to encapsulate no more than one bead and
no more than one cell in a single droplet, or up to one bead and
one cell in a single droplet, or one bead and one cell in a single
droplet. Configurations of fewer or more beads and fewer or more
cells in a single droplet are also contemplated, including but not
limited to two beads and one cell, or one bead and two cells. In
one embodiment, the device may be configured to encapsulate one
bead and one cell within a single droplet. Other configurations of
fewer or more beads and cells per droplet are also contemplated,
including but not limited to two beads and one cell, or one bead
and two cells, or one bead and a plurality of cells, or a plurality
of beads and one cell. This process is typically done by merging a
stream of fluid containing beads with a stream of fluid containing
cells. The merged stream of beads and cells is then contacted with
an oil or other immiscible fluid to create a droplet containing the
beads and cells.
[0038] These configurations produce extremely high concentrations
of single droplets with beads and have a .lamda. approaching 1,
where .lamda. is the average of Poisson distribution of beads being
encapsulated into droplets, but avoid having droplets with multiple
bead occupancy--thus creating an underdispersed Poisson
distribution, e.g., a Poisson distribution with average
distribution of .lamda. but variance of .phi. which is smaller than
.lamda., ideally with .phi. approaching 0. This high concentration
of droplets with single bead occupancy allows systems that require
such droplets (such as a high throughput single cell system) to
improve throughput by 10-20 times over systems in which ordered
streams are not used with decreased error rate (e.g., a decreased
number of droplets with an undesired number of beads or cells).
Similarly, the capture efficiency of the cells can be improved to
the same order of magnitude. Thus, embodiments that employ
focusing, such as inertial focusing as described below, for both
beads and cells may overcome both Poisson distributions, one for
beads and one for cells, in double-Poisson statistics, thus
achieving more than 100.times. improvement in throughput.
Embodiments may be operated continuously and at high volumetric
flow rates with cascading outputs yet still produce droplets having
the desired numbers of beads and cells per droplet.
[0039] Systems and methods may relate to inertial microfluidic
technology for high-throughput and precise microscale control of
cell and particle motion. These systems and methods may be suitable
for applications in any type of nucleic acid sequence analysis,
including long-read DNA sequencing, paired-end sequencing, and
single cell sequencing. The generation of droplets each with, for
example, one bead and one cell enable the continuous analysis and
sequencing of single cells.
[0040] While there are many configurations possible in a system for
the self-ordering of particles, such as cells and beads, within
microfluidic channels and encapsulation of particles, one
embodiment of such a microfluidic system 100 is illustrated in FIG.
1. As shown, the microfluidic system 100 generally includes three
inlets: a bead inlet 102 that connects to a bead channel 104, a
cell inlet 106 that connects to two cell channels 108, 110 on the
two sides of the bead channel 104, and an oil inlet 112 that
connects to two oil channels 114, 116 which are the outermost
channels of the system 100 and are next to the cell channels 108,
110 and spaced laterally away from the bead channel 104. The
microfluidic system 100 generally has one system outlet 118. The
microfluidic system 100 can be provided on a microfabricated chip
120 with the various channels formed in the chip 120.
[0041] The bead inlet 102 is configured for introducing beads 122
suspended in a bead fluid 124 into the microfluidic system 100. The
beads 122 can be of any density made up of various materials. The
bead channel 104 formed in the chip 120 can have numerous
configurations which will be described in detail below. In general,
the bead channel 104 can have a specified geometry designed to
separate, order, and focus the beads 122 to pre-determined lateral
positions in the channel when entering a droplet generation
junction 126. These lateral locations correspond to similar flow
velocities in the velocity profile of the bead fluid 124 such that,
once focused, the beads 122 move at more or less similar speeds and
maintain their spacing and generally do not cross each other. The
bead channel 104 may be straight as shown. The bead channels used
in the microfluidic systems can have various geometries and
cross-sections as detailed below for focusing beads of a
predetermined size suspended within a fluid. For example, bead
channel 104 may have a square cross-section.
[0042] In general, the size of the bead channel 104 is related to
the size of the beads 122 intended to be used within the channel.
For example, as mentioned below, 80-125 .mu.m diameter bead
channels were successfully used for separating, ordering, and
focusing beads that were 30-50 .mu.m in size. The closer the size
of the channel was to the bead size, the faster and more efficient
the separating, focusing, and ordering was found to be.
[0043] The cell channels 108, 110 have long serpentine regions 109,
111 respectively. The oil channels 114, 116 also have long
serpentine regions 115, 117 respectively. These long serpentine
regions act as fluidic resistances to ensure equal distribution of
fluid flow on both branches of the corresponding channel.
[0044] In some embodiments, the bead channel 104 may have a curved
region to decrease the focusing length required and to decrease the
device foot-print. In some embodiments, one or both of the cell
channels 108, 110 may have a curved region to decrease the focusing
length required and to decrease the device foot-print. The curved
regions may be symmetrically curved. In some embodiments, the
curved regions may be asymmetrically curved, such as S-shaped,
sinusoidal, or sigmoidal shaped. In some embodiments, the bead
channel 104 may have an expansion/contraction region which enables
the adjustment of the spacing between beads inside the channel. In
some embodiments, one or both of the cell channels 108, 110 may
have an expansion/contraction region which enables the adjustment
of the spacing between cells inside the channel.
[0045] As shown in FIG. 1, the cell inlet 106 is configured for
introducing cells 130 suspended in a cell fluid 132 into the
microfluidic system 100 through the cell channels 108, 109. The oil
inlet 112 is configured for introducing droplet generation oil 134
to the droplet generation junction 126 through the oil channels
114, 116. The two lateral flows of droplet generation oil 134 pull
droplets from the stream of aqueous bead fluid 124 with the same
frequency, or multiple of, that beads reach the droplet generation
junction 126. Similarly, the two lateral flows of droplet
generation oil 134 pull droplets from the stream of aqueous cell
fluid 132 with the same frequency, or multiple of, that cells reach
the droplet generation junction 126. At the device outlet 118,
droplets exit the microfluidic device 100 in an orderly fashion
with every droplet generally encapsulating one bead and/or one cell
in the particular design illustrated in FIG. 1.
[0046] The chip 120 can also include a straight section of channel
at an output region for analysis of focused particles, collection
of focused particles, and/or for recombining stream lines.
Inertial Focusing
[0047] The bead channels used in the microfluidic systems can have
various geometries and cross-sections for focusing beads of a
predetermined size suspended within a fluid. FIG. 2A-FIG. 2D show
dynamic bead self-assembly in a finite-Reynolds number flow. All
views are from above bead channel 204 such that differences in
position along the channel cross-sectional width can be visualized.
Inertial migration focuses beads to transverse equilibrium
positions. Beads migrate to defined equilibrium positions, for
example, four in a square channel (FIG. 2A) and two in a
rectangular channel (FIG. 2B). In one embodiment illustrated in
FIG. 2A, a straight channel is provided having a square
cross-section with an aspect ratio of substantially 1 to 1. Beads
of a predetermined size flowing within such a channel geometry will
be separated, ordered, and focused into four focusing positions
shown in the cross sectional view of FIG. 2A. These four focusing
positions correspond to four equilibrium points, or potential
minimums, at a distance from each face of the four channel
walls.
[0048] By designing a channel with aspect ratio (here defined as
the ratio of the longer side to the shorter side of the
cross-section) larger than 1, the number of focusing positions can
be successfully decreased from four to two. In one embodiment, the
aspect ratio may be greater than about 1.2, although other aspect
ratios of about 1.1, 1.3, 1.4, 1.5 or more are also contemplated.
In one embodiment shown in FIG. 2B, a straight bead channel is
provided having a rectangular cross-section with an aspect ratio of
substantially 2 to 1. Beads of a predetermined size flowing within
such channel geometry can be separated, ordered, and focused into
two focusing positions corresponding to two equilibrium points or
potential minimums along the wider side walls across the width of
the channel. In some embodiments, the wider side of the channel can
be parallel to either y or z direction leading to bead focusing on
either top-and-bottom or left-and-right of the channel
respectively. Through the process of lateral bead focusing, the
beads interact with each other and order themselves longitudinally
as well. Bead may be both laterally focused (in an y-z plane)
and/or longitudinally ordered (in an x direction). The inter-bead
interactions create a repulsive force between bead pairs that
spaces them out along the channel, leading to creation of bead
lattices.
[0049] To obtain a single focusing position shown in FIG. 2C, a
dual-inlet co-flow system 200 with a rectangular inertial focusing
bead channel 204 with a wider side in the z-direction (leading to
left-and-right focusing in the cross-section view) as shown in FIG.
2D can be used. By using two bead inlets 202, 203 connected to the
bead channel 204 and injecting beads in the bead fluid 124 on only
one side of the channel, with the other stream only containing
fluid (and no beads), the focusing position can be further
decreased to 1. This leads to more efficient ordering of the beads
along the channel. Co-flowing with bead-free fluid was found to
confine beads on one side of a microchannel resulting in a single
line of beads with regular and repeatable spacing. A similar
concept can be used for focusing of cells and other types of
particles as well.
[0050] FIG. 2D is a schematic view of a dynamic self-assembling
bead system including a two-inlet bead channel. Randomly
distributed beads are self-assembled through inertial lift forces
and hydrodynamic bead-bead interactions. The dual-inlet co-flow
system 200 reduces the degrees of freedom by focusing beads 222
into a single substantially axially aligned stream at one focusing
position 228 (FIG. 2D). Unprocessed, the beads 222 in a bead fluid
224 are flowed through the "lower" bead inlet 202. A bead-free
fluid 225 is flowed through the "upper" bead inlet 203. As a
result, the bead-free fluid 225 flows through in the "upper" half
of bead channel 204 and the beads 222 are confined to the "lower"
half of the bead channel 204 so that the beads 222 align at the one
focusing position 228. This equilibrium state becomes a
one-dimensional system where inter-bead spacing is a dependent
variable dependent on, for example, flow, fluid and geometric
parameters.
[0051] In general, "focusing" refers to a reduction in the area of
a cross-section of a channel through which a flux of beads passes.
In some embodiments, beads can be localized within an area having a
width of, at most, 1.01, 1.05, 2, 3, 4, or 5 times the width of the
beads. Localization can occur at any location within the channel,
including within an unobstructed portion of the channel. For
example, localization can occur in a portion of the channel having
less than 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1%, or 0.1% reduction in
cross-sectional area. In certain embodiments, localization can
occur in a channel having a substantially constant cross-sectional
area.
[0052] Inertial focusing within microchannels has been described in
Di Carlo et al., Proceedings of the National Academy of Sciences of
the United States of America 104:18892-97 (2007), which is hereby
incorporated by reference. Briefly, self-assembling systems, in
general, require multiple interactions that include positive and
negative feedback, which for bead systems are realized as
attractive and repulsive forces. Viscous reversing wakes, which are
induced by confinement, repel neighboring beads to infinity while
fluid inertia in the form of lift forces act to maintain the beads
at finite distances. This mechanism of dynamic self-assembly of
microscale beads in a finite-Reynolds-number channel flow provides
parameters for controlling bead stream self-assembling and allow
expanded bead control in microchannel systems. Such control is
useful for applications such as low-pass spatial filtering on bead
spacing. Microfluidic devices can be designed and operated to
control bead-bead and bead-wall interactions in order to manipulate
inter-bead spacing and reduce defocusing.
[0053] Although a Stokes flow (e.g., R.sub.e=0) assumption is
widely accepted in analyzing inertial effects in microfluidic
systems, Reynolds numbers in microfluidic channels often reach
.about.1 and even .about.100s in some extreme cases. Reynolds
number, R.sub.e, is determined by
Re = .rho. U m H .mu. , ##EQU00003##
where .rho. is the density of the fluid, U.sub.m is the maximum
flow speed, H is the hydraulic diameter, and .mu. is the dynamic
viscosity of the fluid. Many inertial effects have been observed in
microfluidic devices at such Reynolds numbers. One example is
inertial migration of beads in square and rectangular channels.
Randomly distributed beads migrate across streamlines due to
inertial lift forces, which is a combination of shear gradient lift
that pushes beads towards walls and wall effect lift that pushes
beads towards the center of a channel. These inertial lift forces
focus beads to four (FIG. 2A) or two (FIG. 2B) dynamic "transverse
equilibrium points" that are determined by channel symmetry. The
system is a non-equilibrium system that constantly dissipates
energy and the transverse equilibrium point is where the inertial
lift forces become zero in the cross-section of the channel. As
used herein "focusing position" refers to these transverse
equilibrium points.
[0054] While traveling down the channel, the beads are laterally (y
direction and z direction) focused by inertial lift forces and
simultaneously longitudinally (x direction) self-assembled by
bead-bead interactions. Focusing occurs along the width and height
of a microchannel, and assembling occurs along the longitudinal
axis of the microchannel. In the final organized state, the system
of beads has two degrees of freedom: inter-bead spacing and
focusing position. Inter-bead spacing is determined by fluid and
flow parameters (U.sub.m, .rho., .mu.) and geometric parameters
(bead diameter (a), channel width (w), and height (h)). These
parameters make up a bead Reynolds number
R p = Re ( a H ) 2 , ##EQU00004##
based on the shear rate at the bead scale, and inter-bead spacing
decreases with increasing R.sub.p.
[0055] When beads are aligned at one focusing position, there is a
default inter-bead spacing for any given set of flow and geometric
parameters. However, with more than one focusing position,
different cross-channel spacing and single-stream spacing appear.
Inter-bead spacing does not show a strong dependence on channel
aspect ratio. The selection of a focusing position for beads is
intrinsically a random event, which makes diverse patterns in the
organized structure. However, additional degrees of freedom in the
form of additional focusing positions make the resulting bead
stream more complicated.
Channel Geometry
[0056] The bead channel geometry can have various geometries in
contrast to the straight channel geometry as shown in FIG. 1. FIG.
3 illustrates another embodiment of a microfluidic system 300 with
a curved bead channel 304. As shown, the microfluidic system 300
generally includes three inlets: a bead inlet 302 that connects to
the bead channel 304, a cell inlet 306 that connects to two cell
channels 308, 310 on the two sides of the bead channel 304, and an
oil inlet 312 that connects to two oil channels 314, 316 which are
the outermost channels of the system 300 and are next to the cell
channels 308, 310 away from the bead channel 304. The microfluidic
system 300 generally has one system outlet 318. The microfluidic
system 300 can be provided on a microfabricated chip 320 with the
various channels formed in the chip 320.
[0057] The bead inlet 304 is configured for introducing beads 322
suspended in a bead fluid 324 into the microfluidic system 300. The
beads 322 can be of any density made up of various materials. The
bead channel 304 formed in the chip 320 can have numerous
configurations which will be described in detail below. In general,
the bead channel 304 can have a specified geometry designed to
separate, order, and focus the beads 322 to pre-determined lateral
positions in the channel when entering the droplet generation
junction 326. These lateral locations correspond to similar flow
velocities in the velocity profile of the bead fluid 324 such that,
once focused, the beads 322 move at similar speeds and maintain
their spacing and generally do not cross each other. The bead
channel 304 may be curved as shown. Curving channels can be used to
decrease the focusing length required and to decrease the device
foot-print.
[0058] The cell channels 308, 310 have serpentine regions 309, 311
respectively. The oil channels 314, 316 also have serpentine
regions 315, 317 respectively.
[0059] In one embodiment, symmetrically, asymmetrically, or
continuously curved channels can be provided such as S-shaped,
sinusoidal, or sigmoidal shaped bead channels having a rectangular
cross-section. Beads of a predetermined size flowing within such
channel geometry will be generally focused into two focusing
positions corresponding to one or two equilibrium points or
potential minimums at a distance from left and right side faces of
the channel. An aspect ratio of a sigmoidal channel can be
substantially 1 to 1 and/or can vary along a length thereof. For
example, the aspect ratio of a sigmoidal channel can vary over the
length of the channel between 1 to 1 and 2 to 1 depending on the
configuration chosen.
[0060] In another embodiment as shown in FIG. 4A, the bead channel
404 has a curving region 438. While asymmetrically curved channels
can have various shapes and configurations as needed for a
particular application, in one embodiment an asymmetric bead
channel can generally have the shape of a wave having large and
small turns, where a radius of curvature can change after each
inflection point of the wave. Each large and small turn can have a
specified width of the channel associated with the turn.
Asymmetrically curved channels enable both longitudinal ordering
and lateral focusing.
[0061] In one embodiment, one-half of a wavelength of the channel
wave can have a large curve while one-half of a wavelength of the
channel wave can have a small curve. These curves can then be
repeated as many times as needed, varying after each inflection
point, to provide a specified length of channel with an asymmetric
curve. The asymmetrically curved bead channel 404 can also have a
rectangular cross-section with an aspect ratio that can vary as
needed over the channel length depending on the nature of the
asymmetry in the curves. In one embodiment, the aspect ratio can
vary between 1 to 1 and 2 to 1. In this case, a single focused
stream of beads is created corresponding to a single equilibrium
point or potential minimum within the channel 404.
[0062] In other embodiments, asymmetric curving bead channels, for
example an expanding spiral shaped channel can be provided, having
a rectangular cross-section with an aspect ratio of substantially 2
to 1. This aspect ratio may vary. In this case, beads are focused
into a single stream line a distance away from an inner wall of the
channel corresponding to a single equilibrium point or potential
minimum within the channel. Examples of systems that include spiral
channels are shown in FIGS. 14A and 14B. In some embodiments, the
spiral portion of the channel has an outer diameter of 2 to 10 mm,
or about 3 to 7 mm, or about 5 mm, or about 10 mm.
[0063] In some embodiments, a single chip may have bead channels
with different channel geometries. FIG. 4B shows another embodiment
with the bead channel 405 having varying diameter. An
expansion/contraction region 440 after a bead focusing region
(upstream, not shown) enables the adjustment of the spacing between
beads inside the channel. For example, the expansion/contraction
region 440 after the bead focusing region (upstream, not shown) may
be used to increase the spacing between beads 422A-D inside the
channel (FIG. 4B). Alternatively, an expansion/contraction region
after the bead focusing region may be used to decrease the spacing
between beads inside the channel. In some embodiments, channel
dimensions can decrease over the length of the chip to facilitate
filtering of the sample, or for other reasons specific to an
application, such as creating fluidic resistance. Channel
dimensions can be larger at the input area or at the output area to
enable forks or valve systems to be positioned within the channels,
or to enable multiple stream lines to be separated and directed to
different locations for analysis or collection. In a similar way,
cross-sections of various channels can also be changed as needed
within a single chip to manipulate stream lines of focused beads
for particular applications. In general, any combination of channel
geometries, channel cross-sections, and channel dimensions can be
included on a single chip as needed to sort, separate, order, and
focus beads of a predetermined size or beads of multiple
predetermined sizes. For instance, different channel geometries and
flow rates can be used for the streams of bead fluid and cell fluid
to ensure desired focusing and ordering in each stream prior to
droplet generation.
[0064] In one embodiment, a straight section of bead channel is
formed in the chip near the inlet for transporting and dividing
flow lines as the bead is introduced into the microfluidic system.
The straight section of each channel can transition to any number
of symmetric and/or asymmetric curving channels for focusing beads
of a predetermined size as needed.
[0065] As shown in FIG. 3, the bead channel 304 has a curved region
305 to decrease the focusing length required and to decrease the
device foot-print. In some embodiments, one or both of the cell
channels 308, 310 may have a curved region to decrease the focusing
length required and to decrease the device foot-print as shown in
FIG. 4A. The curved regions may be symmetrically curved. In some
embodiments, the curved regions may be asymmetrically curved, such
as S-shaped, sinusoidal, or sigmoidal shaped.
[0066] In one embodiment, symmetrically, asymmetrically, or
continuously curved channels can be provided such as S-shaped,
sinusoidal, or sigmoidal shaped cell channel having a rectangular
cross-section. Cells of a predetermined size flowing within such
channel geometry will be generally focused into two focusing
positions corresponding to one or two equilibrium points or
potential minimums at a distance from left and right side faces of
the channel. An aspect ratio of a sigmoidal channel can be
substantially 1 to 1 and/or can vary along a length thereof. For
example, the aspect ratio of a sigmoidal channel can vary over the
length of the channel between 1 to 1 and 2 to 1 depending on the
configuration chosen.
[0067] Similar to the bead channel 404 in FIG. 4A having a curving
region 438, cell channels 310, 308 each may have a curving region.
While asymmetrically curved channels can have various shapes and
configurations as needed for a particular application, in one
embodiment an asymmetric cell channel can generally have the shape
of a wave having large and small turns, where a radius of curvature
can change after each inflection point of the wave. Each large and
small turn can have a specified width of the channel associated
with the turn. Asymmetrically curved channels enable both
longitudinal ordering and lateral focusing.
[0068] In one embodiment, one-half of a wavelength of the channel
wave can have a large curve while one-half of a wavelength of the
channel wave can have a small curve. These curves can then be
repeated as many times as needed, varying after each inflection
point, to provide a specified length of channel with an asymmetric
curve. The asymmetrically curved cell channel can also have a
rectangular cross-section with an aspect ratio that can vary as
needed over the channel length depending on the nature of the
asymmetry in the curves. In one embodiment, the aspect ratio can
vary between 1 to 1 and 2 to 1. In this case, a single focused
stream of cells is created corresponding to a single equilibrium
point or potential minimum within the channel.
[0069] In other embodiments, asymmetric curving cell channels, in
particular an expanding spiral shaped channel can be provided,
having a rectangular cross-section with an aspect ratio of
substantially 2 to 1. This aspect ratio may vary. In this case,
cells are focused into a single stream line a distance away from an
inner wall of the channel corresponding to a single equilibrium
point or potential minimum within the channel. Examples of systems
that include spiral channels are shown in FIGS. 14A and 14B. In
some embodiments, the spiral portion of the channel has an outer
diameter of 2 to 10 mm, or about 3 to 7 mm, or about 5 mm, or about
10 mm.
[0070] Microfluidic devices as described herein may be manufactured
using any suitable technology known to one of ordinary skill in the
art. For example, such devices and systems may be manufactured
using master molds combined with soft lithography techniques. As
another example, microfluidic devices or certain components of the
microfluidic devices can be manufactured using three-dimensional
printing technologies.
[0071] In some embodiments, the channels are rectangular in shape.
The rectangular-shaped channels may be formed into a variety of
geometries described herein, such as straight and curved channels.
The channel height to width aspect ratio may be selected to
optimize particle ordering. In some embodiments, the rectangular
channel aspect ratio is 7:1, or 5:1, or 4:1, or 3:1, or 2:1. In
some embodiments, channel height can be in the range of about 0.5
.mu.m to about 200 .mu.m.
[0072] In some embodiments, a single chip may have cell channels
with different channel geometries. Similar to the bead channel 405
in FIG. 4B having varying diameter, one or both of the cell
channels 308, 310 can have varying diameter. An
expansion/contraction region after a cell focusing region enables
the adjustment of the spacing between cells inside the channel. For
example, the expansion/contraction region after the cell focusing
region may be used to increase the spacing between cells inside the
channel (FIG. 4B). Alternatively, an expansion/contraction region
after the cell focusing region may be used to decrease the spacing
between cells inside the channel. In some embodiments, channel
dimensions can decrease over the length of the chip to facilitate
filtering of the sample, or for other reasons specific to an
application, such as creating fluidic resistance. Channel
dimensions can be larger at the input area or at the output area to
enable forks or valve systems to be positioned within the channels,
or to enable multiple stream lines to be separated and directed to
different locations for analysis or collection. In a similar way,
cross-sections of various channels can also be changed as needed
within a single chip to manipulate stream lines of focused cells
for particular applications. In general, any combination of channel
geometries, channel cross-sections, and channel dimensions can be
included on a single chip as needed to sort, separate, order, and
focus cells of a predetermined size or cells of multiple
predetermined sizes. For instance, different channel geometries and
flow rates can be used for the streams of cell flow and cell fluid
to ensure desired focusing and ordering in each stream prior to
droplet generation.
[0073] In one embodiment, a straight section of cell channel is
formed in the chip near the inlet for transporting and dividing
flow lines as the cell is introduced into the microfluidic system.
The straight section of each channel can transition to any number
of symmetric and/or asymmetric curving channels for focusing cells
of a predetermined size as needed.
[0074] In some embodiments, the bead channel 304 may have an
expansion/contraction region as shown in FIG. 4B which enables the
adjustment of the spacing between beads inside the channel. In some
embodiments, one or both of the cell channels 308, 310 may have an
expansion/contraction region which enables the adjustment of the
spacing between beads inside the channel.
[0075] As shown in FIG. 3, the cell inlet 306 is configured for
introducing cells suspended in a cell fluid into the microfluidic
system 300. The oil inlet 312 is configured for introducing droplet
generation oil to the droplet generation junction 326 through oil
channels 314, 316. The two lateral flows of oil pull droplets from
the stream of aqueous bead fluid 324 with the same frequency, or
multiple of, that beads reach the droplet generation junction 326.
Similarly, the two lateral flows of oil pull droplets from the
stream of aqueous cell fluid with the same frequency that cells
reach the droplet generation junction 326. At the device outlet
318, droplets exit the microfluidic device 300 in an orderly
fashion with every droplet generally encapsulating one bead and/or
one cell in the particular design illustrated in FIG. 3. In some
embodiments, every droplet generally encapsulates a predetermined
number of beads greater or equal to zero and a predetermined number
of cells greater or equal to zero. For example, each droplet may
have 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, about 15, 20, 30, 40, 50,
60, 70, 80, 90, or 100 beads, and each droplet may have 0, 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, about 15, 20, 30, 40, 50, 60, 70, 80, 90, or
100 cells. In some embodiments, the statistical distribution of
beads is less than optimal, e.g., less than 1 bead per droplet. In
some embodiments, the statistical distribution of cells is less
than optimal, e.g., less than 1 cell per droplet.
[0076] The chip 320 can also include a straight section of channel
at an output region for analysis of focused particles, collection
of focused particles, and/or for recombining stream lines.
[0077] As will be appreciated by those skilled in the art, any
number of curves or straight sections can be included as needed
within the chip for one or more of the bead channel 304, cell
channels 308, 310. Additional curved sections of channels can serve
as "off-ramps" for focused bead streams to facilitate additional
separation based on labels or tags associated with the beads.
Channel forks or splits can be included at any positions within the
channels to further facilitate manipulation of focused beads as
needed for various applications.
[0078] Aspect ratios of all channels described above and herein,
including straight, symmetric, and asymmetric, can vary as needed
from one application to another and/or as many times as needed over
the course of a channel. In embodiments illustrated in FIGS. 1-4,
aspect ratios are shown as 1 to 1 and 1 to 2; however, a person of
ordinary skill will recognize that a variety of aspect ratios could
be employed. In addition, the choice of width to height as the
standard for determining the aspect ratio is somewhat arbitrary in
that the aspect ratio can be taken to be the ratio of a first
cross-sectional channel dimension to a second cross-sectional
channel dimension, and for rectangular channels this would be
either width to height or height to width. By way of further
example, the aspect ratio of the channel of FIG. 2B could be
expressed as either 2 to 1 or 1 to 2.
[0079] Other channel cross-sections can also be included in each of
the geometries noted above. Channel cross-sections can include, but
are not limited to, circular, triangular, diamond, and
hemispherical. Beads of a predetermined size can be focused in each
of these exemplary cross-sections, and the focusing positions will
be dependent on the geometry of the channel. For example, in a
straight channel having a circular or hemispherical cross-section,
an annulus or arc of focused beads can be formed within the
channel. In a straight channel having a triangular or diamond
cross-section, beads can be focused into streams corresponding to
focusing positions at a distance from the flat faces of each wall
in the geometry. As symmetric and asymmetric curving channels are
included having each of the exemplary cross-sections noted above,
focusing streams and focusing positions can generally correspond to
that described above with respect to the channels having a
rectangular cross-section.
[0080] In general, there are certain parameters within straight,
symmetric, and asymmetric microfluidic channels which lead to
optimal ordering and focusing conditions for beads suspended within
a sample. These parameters can include, for example, channel
geometries, bead size with respect to channel geometries,
properties of fluid flow through microfluidic channels, and forces
associated with beads flowing within microfluidic channels under
laminar flow conditions. Forces acting on the beads may be referred
to as inertial forces, however, it is possible that other forces
contribute to the focusing and ordering behaviors. Exemplary
inertial forces can include, but are not limited to, inertial lift
down shear gradients and away from channel walls, Dean drag
(viscous drag), pressure drag from Dean flow, and centrifugal
forces acting on individual beads.
Multiple Bead Channels in One Chip
[0081] Any number of microfluidic bead channels can be formed in
the chip in any number of ways. In one exemplary embodiment, a
single bead channel is formed on the chip for focusing beads
therein. In other exemplary embodiments, a plurality of bead
channels can be formed in the chip in various configurations of
networks for focusing beads. For example, 2, 4, 6, 8, 10, 12, and
more channels can be formed in the chip. Any number of layers can
also be included within a microfabricated chip of the system, each
layer having multiple bead channels formed therein.
Multiple Cell Channels in One Chip
[0082] Any number of microfluidic cell channels can be formed in
the chip in any number of ways. In one exemplary embodiment, a
single cell channel is formed on the chip for focusing beads
therein. In other exemplary embodiments, a plurality of cell
channels can be formed in the chip in various configurations of
networks for focusing cells. For example, 2, 4, 6, 8, 10, 12, and
more channels can be formed in the chip. Any number of layers can
also be included within a microfabricated chip of the system, each
layer having multiple cell channels formed therein.
Bead Channel Length
[0083] The interplay between different parameters including
channels size, bead size, flow rate, and fluid properties affect
the length required for bead focusing. This interplay in a channel
is determined by the following formula:
L f = .pi. .mu. h 2 .rho. U m a 2 f L ##EQU00005##
where L.sub.f is the length required for bead focusing; .XI. is the
dynamic viscosity of the fluid; h is the size of the bead channel
(or the hydraulic dimeter, or another critical dimension of the
channel); .rho. is the density of the fluid; U.sub.m is the maximum
flow speed; .alpha. is the bead diameter; and f.sub.L is a factor,
which is in the range of 0.02-0.05 for most cases. Other factors
that affect bead channel length include wall features, wall
geometries, wall coatings, fluid types, types and concentrations of
components in fluids other than beads, bead shapes, bead coating,
and bead weight.
[0084] Table 1 shows examples of the lengths for bead separation,
focusing, and ordering for 30-50 .mu.m beads that are relevant to
high throughput single cell experiments in an aqueous liquid with
properties close to that of water. The number of focusing positions
depends on the inlet configuration (single inlet vs. dual inlet)
and their relative flow rates. The first number in that column
corresponds to the standard case of having a single inlet.
TABLE-US-00001 TABLE 1 Lengths required for bead ordering for 30-50
.mu.m beads. Channel # of 50 .mu.L/min 60 .mu.L/min dimension
focusing 50 .mu.m 30 .mu.m 50 .mu.m (.mu.m) Aspect ratio positions
30 .mu.m bead bead bead bead 125 .times. 125 1 4 or 3 or 1 1.4-3.6
cm 0.5-1.3 cm 1.2-3 cm 0.4-1.1 cm 125 .times. 100 1.25 2 or 1
0.7-1.8 cm 0.2-0.7 cm 0.6-1.5 cm 0.2-0.6 cm 125 .times. 80 1.56 2
or 1 0.3-1 cm 0.1-0.3 cm 0.3-0.8 cm 0.1-0.3 cm
[0085] In one embodiment shown in FIG. 5A, a microfluidic system
500A has one straight bead channel that is 125.times.125 .mu.m in
dimension. The microfluidic system 500A includes three inlets: a
bead inlet 502A that connects to a single bead channel 504A, a cell
inlet 506A that connects to two cell channels 508A, 510A on the two
sides of the bead channel 504A, and an oil inlet 512A that connects
to two oil channels 514A, 516A which are the outermost channels of
the system 500A and are next to the cell channels 508A, 510A away
from the bead channel 504A. The microfluidic system 500A generally
has one system outlet 518A.
[0086] The bead inlet 502A is configured for introducing beads
suspended in a bead fluid into the microfluidic system 500A. The
beads can be of any density made up of various materials. The bead
inlet 502A may have bead filters 542A that prevent undesired
particles such as dust from entering and clogging the bead channel
504A. The spacing between the bead filters 542A should be at least
2-3 times the size of the beads so all beads can flow through the
bead filters 542A without the risk of clogging the bead channel
504A. For example, the spacing between the bead filters 542A may be
300 .mu.m, e.g., .about.5-10 times the bead size.
[0087] The cell inlet 506A is configured for introducing cells
suspended in a cell fluid into the microfluidic system 500A. The
cell inlet 506A may have cell filters 544A that prevent undesired
particles such as dust from entering and clogging the cell channels
508A, 510A. The spacing between the cell filters 544A should be at
least 2-3 times the size of the cells so all beads can flow through
the cell filters 544A without the risk of clogging the cell
channels 508A, 510A. For example, the spacing between the cell
filters 544A may be 300 .mu.m, e.g., .about.5-10 times the cell
size.
[0088] The oil inlet 512A is configured for introducing droplet
generation oil to the droplet generation junction 526A through oil
channels 514A, 516A. The oil inlet 512A may have cell filters 546A
that prevent undesired particles such as dust from entering and
clogging the oil channels 514A, 516A. The spacing between the oil
filters 546A depends on the characteristics of the oil used, such
as viscosity, so the oil can flow through the oil filters 546A
without the risk of clogging the oil channels 514A, 516A. For
example, the spacing between the oil filters 546A may be 300
.mu.m.
[0089] The two lateral flows of oil pull droplets from the stream
of aqueous bead fluid 524A with the same frequency, or multiple of,
that beads reach the droplet generation junction 526A because of
inertial focusing. Similarly, the two lateral flows of oil pull
droplets from the stream of aqueous cell fluid with the same
frequency, or multiple of, that cells reach the droplet generation
junction 526A because of inertial focusing. At the device outlet
518A, droplets exit the microfluidic device 500 in an orderly
fashion with every droplet generally encapsulating one bead and/or
one cell in general. The design and input concentrations can be
adjusted such that not all droplets have a single bead or cell if
needed. For example, each droplet may have 0, 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, about 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 beads,
and each droplet may have 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, about
15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 cells. In some
embodiments, the statistical distribution of beads is less than
optimal, e.g., less than 1 bead per droplet. In some embodiments,
the statistical distribution of cells is less than optimal, i.e.
less than 1 cell per droplet.
[0090] In one embodiment as shown in FIG. 5B, a microfluidic system
500B has one straight bead channel that is 125.times.125 .mu.m in
dimension. The microfluidic system 500B includes four inlets: two
bead inlets 502B, 503B that connect to a bead channel 504B (both
inlets have bead solution, but only one of them contains beads
while the other one is bead-free), a cell inlet 506B that connects
to two cell channels 508B, 510B on the two sides of the bead
channel 504B, and an oil inlet 512B that connects to two oil
channels 514B, 516B which are the outermost channels of the system
500B and are next to the cell channels 508B, 510B away from the
bead channel 504B. The dual-inlet co-flow design results in
efficient bead ordering. The two cell channels 508B, 510B in this
dual-inlet co-flow system are longer in length when compared to the
two cell channels 508A, 510A shown in FIG. 5A even though both
microfluidic systems have the same bead channel dimension of
125.times.125 .mu.m. The two oil channels 514B, 516B in this
dual-inlet co-flow system are longer in length when compared to the
two oil channels 514A, 516A shown in FIG. 5A even though both
microfluidic systems have the same bead channel dimension of
125.times.125 .mu.m. The microfluidic system 500B generally has one
system outlet 518B.
[0091] The bead inlet 502B is configured for introducing beads
suspended in a bead fluid into the microfluidic system 500B. The
beads can be of any density made up of various materials. The bead
inlet 503B is configured for introducing fluid not containing any
beads. The bead inlets 502B, 503B may have bead filters 542B, 543B
respectively that prevent undesired particles such as dust from
entering and clogging the bead channels 504B. The spacing between
the bead filters 542B, 543B should be at least 2-3 times the size
of the beads so all beads can flow through the bead filters 542B,
543B without the risk of clogging the bead channel 504B. For
example, the spacing between the bead filters 542B, 543B may be 300
.mu.m, e.g., .about.5-10 times the bead size.
[0092] This dual-inlet co-flow system 500B leads to more efficient
ordering of the beads along the channel. Co-flowing with bead-free
fluid confines beads on one side of a microchannel resulting in a
single line of beads with regular spacing. The cell inlet 506B is
configured for introducing cells suspended in a cell fluid into the
microfluidic system 500B. The cell inlet 506B may have cell filters
544B that prevent undesired particles such as dust from entering
and clogging the cell channels 508B, 510B. The spacing between the
cell filters 544B should be at least 2-3 times the size of the
cells so all beads can flow through the cell filters 544B without
the risk of clogging the cell channels 508B, 510B. For example, the
spacing between the cell filters 544B may be 300 .mu.m e.g.,
.about.5-10 times the cell size.
[0093] The oil inlet 512B is configured for introducing droplet
generation oil to the droplet generation junction 526B through oil
channels 514B, 516B. The oil inlet 512B may have cell filters 546B
that prevent undesired particles such as dust from entering and
clogging the oil channels 514B, 516B. The spacing between the oil
filters 546B depends on the characteristics of the oil used, such
as viscosity, so the oil can flow through the oil filters 546B
without the risk of clogging the oil channels 514B, 516B. For
example, the spacing between the oil filters 546B may be 300
.mu.m.
[0094] The two lateral flows of oil pull droplets from the stream
of aqueous bead fluid 524B at the same frequency, or multiple of,
that beads reach the droplet generation junction 526B due to
inertial focusing. Similarly, the two lateral flows of oil pull
droplets from the stream of aqueous cell fluid with the same
frequency, or multiple of, that cells reach the droplet generation
junction 526B due to inertial focusing. At the device outlet 518B,
droplets were found to exit the microfluidic device 500 in an
orderly fashion with every droplet generally encapsulating one bead
and/or one cell in general.
[0095] With 50 .mu.L/min bead fluid flow rate and 125.times.125
.mu.m, the separation of 30 and 50 .mu.m beads required 1.4-3.6 cm
and 0.5-1.3 cm respectively. With 60 .mu.L/min bead fluid flow
rate, the separation of 30 and 50 .mu.m beads required 1.2-3 cm and
0.4-1.1 cm respectively. The length of the channel can be adjusted
for any different bead solution to accommodate the change in
focusing length due to changes in fluid density or viscosity.
[0096] In one embodiment as shown in FIG. 5C, a microfluidic system
500C has one straight bead channel that is 125.times.100 .mu.m in
dimension. The microfluidic system 500C includes three inlets: a
bead inlet 502C that connects to a bead channel 504C, a cell inlet
506C that connects to two cell channels 508C, 510C on the two sides
of the bead channel 504C, and an oil inlet 512C that connects to
two oil channels 514C, 516C which are the outermost channels of the
system 500C and are next to the cell channels 508C, 510C away from
the bead channel 504C. The microfluidic system 500C generally has
one system outlet 518C.
[0097] The bead inlet 504C is configured for introducing beads
suspended in a bead fluid into the microfluidic system 500C. The
beads can be of any density made up of various materials. The bead
inlet 502C may have bead filters 542C that prevent undesired
particles such as dust from entering and clogging the bead channel
504C. The spacing between the bead filters 542C should be at least
2-3 times the size of the beads so all beads can flow through the
bead filters 542C without the risk of clogging the bead channel
504C. For example, the spacing between the bead filters 542C may be
300 .mu.m, e.g., .about.5-10 times the bead size.
[0098] The cell inlet 506C is configured for introducing cells
suspended in a cell fluid into the microfluidic system 500C. The
cell inlet 506C may have cell filters 544C that prevent undesired
particles such as dust from entering and clogging the cell channels
508C, 510C. The spacing between the cell filters 544C should be at
least 2-3 times the size of the cells so all beads can flow through
the cell filters 544C without the risk of clogging the cell
channels 508C, 510C. For example, the spacing between the cell
filters 544C may be 300 .mu.m e.g., .about.5-10 times the cell
size.
[0099] The oil inlet 512C is configured for introducing droplet
generation oil to the droplet generation junction 526C through oil
channels 514C, 516C. The oil inlet 512C may have cell filters 546C
that prevent undesired particles such as dust from entering and
clogging the oil channels 514C, 516C. The spacing between the oil
filters 546C depends on the characteristics of the oil used, such
as viscosity, so the oil can flow through the oil filters 546C
without the risk of clogging the oil channels 514C, 516C. For
example, the spacing between the oil filters 546C may be 300
.mu.m.
[0100] The two lateral flows of oil pull droplets from the stream
of aqueous bead fluid 524C with the same frequency, or multiple of,
that beads reach the droplet generation junction 526C because of
inertial focusing. Similarly, the two lateral flows of oil pull
droplets from the stream of aqueous cell fluid with the same
frequency, or multiple of, that cells reach the droplet generation
junction 526C because of inertial focusing. At the device outlet
518C, droplets exit the microfluidic device 500C in an orderly
fashion with every droplet generally encapsulating one bead and/or
one cell in general.
[0101] In one embodiment as shown in FIG. 5D, a microfluidic system
500D has one straight bead channel that is 125.times.100 .mu.m in
dimension. The microfluidic system 500D includes four inlets: two
bead inlets 502D, 503D that connect to a bead channel 504D, a cell
inlet 506D that connects to two cell channels 508D, 510D on the two
sides of the bead channel 504D, and an oil inlet 512D that connects
to two oil channels 514D, 516D which are the outermost channels of
the system 500D and are next to the cell channels 508D, 510D away
from the bead channel 504D. The dual-inlet co-flow design results
in efficient bead ordering. The two cell channels 508D, 510D in
this dual-inlet co-flow system are longer in length when compared
to the two cell channels 508C, 510C shown in FIG. 5A even though
both microfluidic systems have the same bead channel dimension of
125.times.100 The two oil channels 514D, 516D in this dual-inlet
co-flow system are longer in length when compared to the two oil
channels 514C, 516C shown in FIG. 5C even though both microfluidic
systems have the same bead channel dimension of 125.times.100
.mu.m. The microfluidic system 500D generally has one system outlet
518D.
[0102] The bead inlet 502D is configured for introducing beads
suspended in a bead fluid into the microfluidic system 500D. The
beads can be of any density made up of various materials. The bead
inlet 503D is configured for introducing fluid not containing any
beads. The bead inlets 502D, 503D may have bead filters 542D, 543D
respectively that prevent undesired particles such as dust from
entering and clogging the bead channels 504D. The spacing between
the bead filters 542D, 543D should be at least 2-3 times the size
of the beads so all beads can flow through the bead filters 542D,
543D without the risk of clogging the bead channel 504D. For
example, the spacing between the bead filters 542D, 543D may be 300
.mu.m, e.g., .about.5-10 times the bead size.
[0103] This dual-inlet co-flow system 500D leads to more efficient
ordering of the beads along the channel. Co-flowing with bead-free
fluid confines beads on one side of a microchannel resulting in a
single line of beads with regular spacing. The cell inlet 506D is
configured for introducing cells suspended in a cell fluid into the
microfluidic system 500D. The cell inlet 506D may have cell filters
544D that prevent undesired particles such as dust from entering
and clogging the cell channels 508D, 510D. The spacing between the
cell filters 544D should be at least 2-3 times the size of the
cells so all beads can flow through the cell filters 544D without
the risk of clogging the cell channels 508D, 510D. For example, the
spacing between the cell filters 544D may be 300 .mu.m, e.g.,
.about.5-10 times the cell size.
[0104] The oil inlet 512D is configured for introducing droplet
generation oil to the droplet generation junction 526D through oil
channels 514D, 516D. The oil inlet 512D may have cell filters 546D
that prevent undesired particles such as dust from entering and
clogging the oil channels 514D, 516D. The spacing between the oil
filters 546D depends on the characteristics of the oil used, such
as viscosity, so the oil can flow through the oil filters 546D
without the risk of clogging the oil channels 514D, 516D. For
example, the spacing between the oil filters 546A may be 300
.mu.m.
[0105] The two lateral flows of oil pull droplets from the stream
of aqueous bead fluid 524D with the same frequency, or multiple of,
that beads reach the droplet generation junction 526D because of
inertial focusing. Similarly, the two lateral flows of oil pull
droplets from the stream of aqueous cell fluid with the same
frequency, or multiple of, that cells reach the droplet generation
junction 526D because of inertial focusing. At the device outlet
518D, droplets exit the microfluidic device 500D in an orderly
fashion with every droplet encapsulating one bead and/or one cell
in general.
[0106] With 50 .mu.L/min bead fluid flow rate and channel dimension
of 125.times.100 .mu.m, the separation of 30 and 50 .mu.m beads
require 0.7-1.8 cm and 0.2-0.7 cm respectively. With 60 .mu.L/min
bead fluid flow rate, the separation of 30 and 50 .mu.m beads
require 0.6-1.5 cm and 0.2-0.6 cm respectively.
[0107] In one embodiment as shown in FIG. 5E, a microfluidic system
500E has one straight bead channel that is 125.times.100 .mu.m in
dimension. The microfluidic system 500E includes three inlets: a
bead inlet 502E that connects to a bead channel 504E, a cell inlet
506E that connects to two cell channels 508E, 510E on the two sides
of the bead channel 504E, and an oil inlet 512E that connects to
two oil channels 514E, 516E which are the outermost channels of the
system 500E and are next to the cell channels 508E, 510E away from
the bead channel 504E. The microfluidic system 500E generally has
one system outlet 518E.
[0108] The bead inlet 504E is configured for introducing beads
suspended in a bead fluid into the microfluidic system 500E. The
beads can be of any density made up of various materials. The bead
inlet 502E may have bead filters 542E that prevent undesired
particles such as dust from entering and clogging the bead channel
504E. The spacing between the bead filters 542E should be at least
2-3 times the size of the beads so all beads can flow through the
bead filters 542E without the risk of clogging the bead channel
504E. For example, the spacing between the bead filters 542E may be
300 .mu.m, e.g., .about.5-10 times the bead size.
[0109] The cell inlet 506E is configured for introducing cells
suspended in a cell fluid into the microfluidic system 500E. The
cell inlet 506E may have cell filters 544E that prevent undesired
particles such as dust from entering and clogging the cell channels
508E, 510E. The spacing between the cell filters 544E should be at
least 2-3 times the size of the cells so all beads can flow through
the cell filters 544E without the risk of clogging the cell
channels 508E, 510E. For example, the spacing between the cell
filters 544E may be 300 .mu.m, e.g., .about.5-10 times the cell
size.
[0110] The oil inlet 512E is configured for introducing droplet
generation oil to the droplet generation junction 526E through oil
channels 514E, 516E. The oil inlet 512E may have cell filters 546E
that prevent undesired particles such as dust from entering and
clogging the oil channels 514E, 516E. The spacing between the oil
filters 546E depends on the characteristics of the oil used, such
as viscosity, so the oil can flow through the oil filters 546E
without the risk of clogging the oil channels 514F, 516F. For
example, the spacing between the oil filters 546E may be 300
.mu.m.
[0111] The two lateral flows of oil pull droplets from the stream
of aqueous bead fluid 524E with the same frequency, or multiple of,
that beads reach the droplet generation junction 526E because of
inertial focusing. Similarly, the two lateral flows of oil pull
droplets from the stream of aqueous cell fluid with the same
frequency, or multiple of, that cells reach the droplet generation
junction 526E because of inertial focusing. At the device outlet
518E, droplets exit the microfluidic device 500E in an orderly
fashion with every droplet generally encapsulating one bead and/or
one cell in general.
[0112] In one embodiment as shown in FIG. 5F, a microfluidic system
500F has one straight bead channel that is 125.times.100 .mu.m in
dimension. The microfluidic system 500F includes four inlets: two
bead inlets 502F, 503F that connect to a bead channel 504F, a cell
inlet 506F that connects to two cell channels 508F, 510F on the two
sides of the bead channel 504F, and an oil inlet 512F that connects
to two oil channels 514F, 516F which are the outermost channels of
the system 500F and are next to the cell channels 508F, 510F away
from the bead channel 504F. The dual-inlet co-flow design results
in efficient bead ordering. The two cell channels 508F, 510F in
this dual-inlet co-flow system are longer in length when compared
to the two cell channels 508E, 510E shown in FIG. 5E even though
both microfluidic systems have the same bead channel dimension of
125.times.80 The two oil channels 514F, 516F in this dual-inlet
co-flow system are longer in length when compared to the two oil
channels 514E, 516E shown in FIG. 5E even though both microfluidic
systems have the same bead channel dimension of 125.times.80 The
microfluidic system 500F generally has one system outlet 518F.
[0113] The bead inlet 502F is configured for introducing beads
suspended in a bead fluid into the microfluidic system 500F. The
beads can be of any density made up of various materials. The bead
inlet 503F is configured for introducing fluid not containing any
beads. The bead inlets 502F, 503F may have bead filters 542F, 543F
respectively that prevent undesired particles such as dust from
entering and clogging the bead channels 504F. The spacing between
the bead filters 542F, 543F should be at least 2-3 times the size
of the beads so all beads can flow through the bead filters 542F,
543F without the risk of clogging the bead channel 504F. For
example, the spacing between the bead filters 542F, 543F may be 300
.mu.m, e.g., .about.5-10 times the bead size.
[0114] This dual-inlet co-flow system 500F leads to more efficient
ordering of the beads along the channel. Co-flowing with bead-free
fluid confines beads on one side of a microchannel resulting in a
single line of beads with regular spacing. The cell inlet 506F is
configured for introducing cells suspended in a cell fluid into the
microfluidic system 500F. The cell inlet 506F may have cell filters
544F that prevent undesired particles such as dust from entering
and clogging the cell channels 508F, 510F. The spacing between the
cell filters 544F should be at least 2-3 times the size of the
cells so all beads can flow through the cell filters 544F without
the risk of clogging the cell channels 508F, 510F. For example, the
spacing between the cell filters 544F may be 300 .mu.m, e.g.,
.about.5-10 times the cell size.
[0115] The oil inlet 512F is configured for introducing droplet
generation oil to the droplet generation junction 526F through oil
channels 514F, 516F. The oil inlet 512F may have cell filters 546F
that prevent undesired particles such as dust from entering and
clogging the oil channels 514F, 516F. The spacing between the oil
filters 546F depends on the characteristics of the oil used, such
as viscosity, so the oil can flow through the oil filters 546F
without the risk of clogging the oil channels 514F, 516F. For
example, the spacing between the oil filters 546F may be 300
.mu.m.
[0116] The two lateral flows of oil pull droplets from the stream
of aqueous bead fluid 524F with the same frequency, or multiple of,
that beads reach the droplet generation junction 526F because of
inertial focusing. Similarly, the two lateral flows of oil pull
droplets from the stream of aqueous cell fluid with the same
frequency, or multiple of, that cells reach the droplet generation
junction 526F because of inertial focusing. At the device outlet
518F, droplets exit the microfluidic device 500F in an orderly
fashion with every droplet encapsulating one bead and/or one cell
in general.
[0117] With 50 .mu.L/min bead fluid flow rate and channel dimension
of 125.times.80 .mu.m, the separation of 30 and 50 .mu.m beads
required 0.7-1.8 cm and 0.2-0.7 cm respectively. With 60 .mu.L/min
bead fluid flow rate, the separation of 30 and 50 .mu.m beads
require 0.6-1.5 cm and 0.2-0.6 cm respectively.
[0118] Cell Channel Length
[0119] The interplay between different parameters including channel
size, cell size, flow rate, and fluid properties affect the length
required for cell focusing. This interplay in a channel is
determined by the following formula:
L f = .pi. .mu. h 2 .rho. U m a 2 f L ##EQU00006##
where L.sub.f is the length required for cell focusing; .mu. is the
dynamic viscosity of the fluid; h is the size of the cell channel
(or the hydraulic dimeter, or another critical dimension of the
channel); .rho. is the density of the fluid; U.sub.m is the maximum
flow speed; a is the cell diameter; and f.sub.L is a factor in the
range of 0.02-0.05 for most cases. Other factors that affect cell
channel length include wall features, wall geometries, wall
coatings, fluid types, types and concentrations of components in
fluids other than cells, cell shape, cell surface coating, and cell
state.
Bead to Volume Ratios
[0120] In another aspect of the system, a bead to volume ratio can
optionally be manipulated or adjusted for conservation of mass
within the channels. In general, separating, ordering, and focusing
of beads is, in part, dependent on inter-bead spacing within
channels as well as the ratio of bead size to hydrodynamic size of
the channel. Various channel geometries described herein may
require a predetermined bead to volume ratio of the bead to be
focused in order to achieve a required inter-bead spacing and
thereby maintain ordering and focusing of that bead. In particular,
the bead to volume ratio of a bead suspended within a fluid can be
calculated and adjusted as needed to achieve focusing within
certain channel geometries. In general, a maximum bead to volume
ratio for a specific bead size and channel geometry can be
determined using the formula, assuming a rectangular channel and
non-overlapping focusing positions:
Max VolumeFraction = N .pi. a 2 6 hw ##EQU00007##
where N is the number of focusing positions in a channel, a is the
focused bead diameter, h is the channel height, and w is the
channel width. Thus, beads can be diluted or concentrated to attain
a predetermined ratio before and/or during introduction of the bead
into the system. Additionally, certain exemplary systems may
require the ratio to be adjusted after the bead is introduced into
the channels.
[0121] Bead to volume ratios of a bead within the channels
described herein can have any value sufficient to enable ordering
and focusing of beads. In general, the bead to volume ratio can be
less than about 50%. In other embodiments, bead to volume ratios
can be less than about 40%, 30%, 20%, 10%, 8%, or 6%. More
particularly, in some embodiments, bead to volume ratios can be in
a range of about 0.001% to about 5%, and can be in a range of about
0.01% to about 4%. Alternatively, the ratio can be in the range of
about 0.1% to about 3%. Alternatively, the ratio can be in the
range of about 0.5% to about 2%. As will be appreciated by those
skilled in the art, the bead to volume ratio of additional or
extraneous beads within the bead, apart from the bead to be
focused, need not necessarily be considered or adjusted. As will be
further appreciated by those skilled in the art, any number of
beads may not require any adjustment to the bead to volume ratio of
the bead to be focused before, during, and/or after introduction
into the system.
[0122] Various commonly used techniques for diluting or
concentrating beads for adjusting a bead to volume ratio can be
used in the embodiments disclosed herein. For example, a bead can
be diluted or concentrated in batches before introduction into the
system such that the bead ultimately introduced into the system has
the required ratio before being introduced through the inlet. In
other embodiments, the system can include two or more inlets for
introducing the bead simultaneously with a diluent or concentrate
to effect dilution or concentration. In this way, the bead to
volume ratio can be adjusted within the system, whether adjustment
occurs within a chamber before the bead and diluent or concentrate
enter the channels or whether adjustment occurs through mixing of
the bead and the diluent or concentrate within the channels. In
another embodiment, the diluent or concentrate can be introduced
into a center portion, fork, or branch of a channel as may be
required by various applications after the unadjusted bead has
traveled within the channel for some distance. A person skilled in
the art will appreciate the variations possible for adjusting the
bead to volume ratio of a bead within the embodiments described
herein.
Cell to Volume Ratios
[0123] In another aspect of the system, a cell to volume ratio can
optionally be manipulated or adjusted for conservation of mass
within the channels. In general, separating, ordering, and focusing
of cells is, in part, dependent on inter-cell spacing within
channels as well as the ratio of cell size to hydrodynamic size of
the channel. Various channel geometries described herein may
require a predetermined cell to volume ratio of the cell to be
focused in order to achieve a required inter-cell spacing and
thereby maintain ordering and focusing of that cell. In particular,
the cell to volume ratio of a cell suspended within a fluid can be
calculated and adjusted as needed to achieve focusing within
certain channel geometries. In general, a maximum cell to volume
ratio for a specific cell size and channel geometry can be
determined using the formula, assuming a rectangular channel and
non-overlapping focusing positions:
Max VolumeFraction = N .pi. a 2 6 hw ##EQU00008##
where N is the number of focusing positions in a channel, a is the
focused cell diameter, h is the channel height, and w is the
channel width. Thus, cells can be diluted or concentrated to attain
a predetermined ratio before and/or during introduction of the cell
into the system. Additionally, certain exemplary systems may
require the ratio to be adjusted after the cell is introduced into
the channels.
[0124] Cell to volume ratios of a cell within the channels
described herein can have any value sufficient to enable ordering
and focusing of cells. In general, the cell to volume ratio can be
less than about 50%. In other embodiments, cell to volume ratios
can be less than about 40%, 30%, 20%, 10%, 8%, or 6%. More
particularly, in some embodiments, cell to volume ratios can be in
a range of about 0.001% to about 5%, and can be in a range of about
0.01% to about 4%. Alternatively, the ratio can be in the range of
about 0.1% to about 3%. Alternatively, the ratio can be in the
range of about 0.5% to about 2%. As will be appreciated by those
skilled in the art, the cell to volume ratio of additional or
extraneous cells within the cell, apart from the cell to be
focused, need not necessarily be considered or adjusted. As will be
further appreciated by those skilled in the art, any number of
cells may not require any adjustment to the cell to volume ratio of
the cell to be focused before, during, and/or after introduction
into the system.
[0125] Various commonly used techniques for diluting or
concentrating cells for adjusting a cell to volume ratio can be
used in the embodiments disclosed herein. For example, a cell can
be diluted or concentrated in batches before introduction into the
system such that the cell ultimately introduced into the system has
the required ratio before being introduced through the inlet. In
other embodiments, the system can include two or more inlets for
introducing the cell simultaneously with a diluent or concentrate
to effect dilution or concentration. In this way, the cell to
volume ratio can be adjusted within the system, whether adjustment
occurs within a chamber before the cell and diluent or concentrate
enter the channels or whether adjustment occurs through mixing of
the cell and the diluent or concentrate within the channels. In
another embodiment, the diluent or concentrate can be introduced
into a center portion, fork, or branch of a channel as may be
required by various applications after the unadjusted cell has
traveled within the channel for some distance. A person skilled in
the art will appreciate the variations possible for adjusting the
cell to volume ratio of a cell within the embodiments described
herein.
Inertial Focusing and Droplet Generation
[0126] In some embodiments, inertial focusing of beads may be
combined with droplet generation to produce extremely high
concentrations of droplets and a bead .lamda. approaching 1, but
avoid having droplets with multiple bead occupancy. .lamda. is the
average of Poisson distribution, the probability of an event
occurring, such as a droplet with one single bead. The effect of
Poisson distribution on single-cell analysis and sorting using
droplet-based microfluidics has been described in Mazutis et al.,
Nature Protocols 8:870-91 (2013), which is hereby incorporated by
reference. This high concentration of droplets with single bead
occupancy allows systems that require such droplets (such as high
throughput single cell systems) to improve throughput, for example
by 2-25 times, or 5-25 times, or 5-10 times, or 10-20 times, as
compared to other encapsulation methods, with decreased error rate
(e.g., decreased proportion of droplets with more than one
bead).
[0127] In some embodiments, inertial focusing of cells may be
combined with droplet generation to produce extremely high
concentrations of droplets and a cell .lamda. approaching 1, but
avoid having droplets with multiple cell occupancy .lamda. for
cells is the probability of a droplet to have only one single cell.
This high concentration of droplets with single cell occupancy
allows systems that require such droplets (such as high throughput
single cell systems) to improve throughput, for example by 2-25
times, or 5-25 times, or 5-10 times, by 10-20 times, as compared to
other encapsulation methods, with decreased error rate (e.g.,
decreased proportion of droplets with more than one cell).
[0128] In some embodiments, focusing, such as inertial focusing, is
employed for both A and B particles, such as beads and cells, to
overcome the two Poisson distributions, for example, one for beads
and one for cells, in double-Poisson statistics. This method
creates a system with double-underdispersed-Poisson statistics and
a further enhanced improvement in throughput (e.g., at least 5, 10,
25, 50, or 100X) over non-ordered systems. Embodiments of the
invention may be operated continuously and at high volumetric flow
rates with cascading outputs. The invention also requires no
interactions with mechanical filters or obstacles and requires very
low maintenance.
[0129] In some embodiments, particles such as beads, nucleic acid
fragments, and cells may have statistical distribution other than
Poisson, such as normal distribution, log-normal distribution,
Pareto distribution, discrete uniform distribution, continuous
uniform distribution, Bernoulli distribution, binomial
distribution, negative binomial distribution, geometric
distribution, hypergeometric distribution, beta-binomial
distribution, categorical distribution, multinomial distribution,
multivariate hypergeometric distribution, log-Poisson distribution,
exponential distribution, Gamma distribution, Rayleigh
distribution, Rice distribution, Chi-squared distribution,
student's t distribution, F-distribution, Beta distribution,
Dirichlet distribution, and Wishart distribution.
[0130] Once the proper channel geometry and flow rate are
determined for a particular bead (30-50 .mu.m for example), the
bead concentration can be adjusted to obtain a large .lamda. (still
smaller than 1). Then bead fluid is injected into the bead inlet
connected to the bead channel at the pre-designated flow rate (for
example, 60 .mu.L/min). Subsequently, cells are injected into the
cell inlet connection to cell channels at the pre-designated flow
rate (for example, 60 .mu.L/min). Finally, the droplet generation
oil is injected into the oil inlet connected to the oil channels at
the appropriate flow rate (for example, 150-250 .mu.L/min). The
third stream (e.g., oil) flow rate may be the same or greater than
the flow rates of the bead and cell fluids.
[0131] Similar principles can be used to focus and order cells in
the cell channel. As a result, one can increase the capture
efficiency of the cells to the same order of magnitude as for the
beads. The net result from combining the two ordered streams and
their improved efficiencies is that both Poisson distributions in
the original double-Poisson statistics are overcome to achieve
greater improvement (e.g., 50.times. or 100.times.) in
throughput.
[0132] FIG. 6 illustrates another embodiment of a microfluidic
system 600. As shown, a bead channel 604 may be curved or straight.
There may be one or two bead inlets connected to the bead channel
604. The microfluidic system 600 generally includes three inlets: a
bead inlet that connects to a bead channel 604, a cell inlet that
connects to two cell channels 608, 610 on the two sides of the bead
channel 604, and an oil inlet that connects to two oil channels
614, 616 which are the outermost channels of the system 600 and are
next to the cell channels 608, 610 away from the bead channel 604.
The microfluidic system 600 generally has one system outlet 618.
The microfluidic system 600 can be provided on a microfabricated
chip with the various channels formed in the chip.
[0133] A bead inlet is configured for introducing beads 622
suspended in a bead fluid 624 into the microfluidic system 600. The
beads 622 can be of any density made up of various materials. In
general, the bead channel 604 can have a specified geometry
designed to separate, order, and focus the beads 622 to
pre-determined lateral positions in the channel when entering the
droplet generation junction 626. These lateral locations correspond
to similar flow velocities in the velocity profile of the bead
fluid 624 such that, once focused, the beads 622 move at similar
speeds and maintain their spacing and do not cross each other. The
bead channels used in the microfluidic systems can have various
geometries and cross-sections for focusing beads of a predetermined
size suspended within a fluid. For example, bead channel 604 may
have a square cross-section.
[0134] The cell inlet is configured for introducing cells 630
suspended in a cell fluid into the microfluidic system 600. The oil
inlet is configured for introducing droplet generation oil 632 to
the droplet generation junction 626 through oil channels 614, 616.
The two lateral flows of oil pull droplets from the stream of
aqueous bead fluid 624 with the same frequency, or multiple of,
that beads reach the droplet generation junction 626. Similarly,
the two lateral flows of oil pull droplets from the stream of
aqueous cell fluid 634 with the same frequency, or multiple of,
that cells reach the droplet generation junction 626. The beads 622
are ordered prior to entering the droplet generation junction 626.
The cells 630 are ordered prior to entering the droplet generation
junction 626. By combining inertial forcing and droplet generation
for both beads 622 and cells 630, droplets 634 are formed with one
bead and one cell each. This embodiment generates more
single-particle droplets (e.g., one cell and one bead) and fewer
empty or multiple-particle droplets (e.g., two beads and one cell)
than would have been possible from stochastic (Poisson)
loading.
[0135] FIG. 7 illustrates another embodiment of a microfluidic
system 700. As shown, a bead channel 704 may be curved or straight.
There may be one or two bead inlets connected to the bead channel
704. The microfluidic system 700 generally includes three inlets: a
bead inlet that connects to a bead channel 704, two cell inlets
that connect to two cell channels 708, 710 on the two sides of the
bead channel 704, and an oil inlet that connects to two oil
channels 714, 716 which are the outermost channels of the system
700 and are next to the cell channels 708, 710 away from the bead
channel 704. The microfluidic system 700 generally has one system
outlet 718. The microfluidic system 700 can be provided on a
microfabricated chip with the various channels formed in the
chip.
[0136] A bead inlet is configured for introducing beads 722
suspended in a bead fluid 724 into the microfluidic system 700. The
beads 722 can be of any density made up of various materials. In
general, the bead channel 704 can have a specified geometry
designed to separate, order, and focus the beads 722 to
pre-determined lateral positions in the channel when entering the
droplet generation junction 726. These lateral locations correspond
to similar flow velocities in the velocity profile of the bead
fluid 724 such that, once focused, the beads 722 move at similar
speeds and maintain their spacing and do not cross each other. The
bead channels used in the microfluidic systems can have various
geometries and cross-sections for focusing beads of a predetermined
size suspended within a fluid. For example, bead channel 704 may
have a square cross-section.
[0137] One cell inlet is configured for introducing cells 730
suspended in a cell fluid into the microfluidic system 700 through
cell channel 708. Another cell inlet is configured for introducing
a cell-free fluid into the microfluidic system 700. The oil inlet
is configured for introducing droplet generation oil 732 to the
droplet generation junction 726 through oil channels 714, 716. The
two lateral flows of oil pull droplets from the stream of aqueous
bead fluid 724 with the same frequency, or multiple of, that beads
reach the droplet generation junction 726. Similarly, the two
lateral flows of oil pull droplets from the stream of aqueous cell
fluid 734 with the same frequency, or multiple of, that cells reach
the droplet generation junction 726. The beads 722 are ordered
prior to entering the droplet generation junction 726. The cells
730 are ordered prior to entering the droplet generation junction
726. By combining inertial forcing and droplet generation for both
beads 722 and cells 730, droplets 734 are formed with one bead and
one cell each. This embodiment generates more single-particle
droplets (e.g., one bead and one cell) and fewer empty or
multiple-particle (e.g., two beads and one cell) droplets than
would have been possible from stochastic (Poisson) loading.
Width of Fluidic Channel at Channel Convergence
[0138] A design parameter to consider is the width of the fluidic
channel after the bead channel and the cell channel meet, and
before the droplet formation junction, e.g., width m in FIG. 15A.
The bead fluid in this region gets squeezed and diluted by the cell
fluid, which increases the distance between the beads and lowers
the occupancy rate of beads in droplets. Therefore, the width m can
be adjusted to compensate for this phenomenon, and in turn increase
the bead's encapsulation efficiency in the droplets.
[0139] To address this, the width of the channel m was increased
proportionally to the ratio of the flow rate of bead and cell
fluids. In one example, the flow rate for the bead fluid was 30
.mu.L/min and the flow rate for cell fluid was 30 .mu.L/min. In
this example, to maintain the same distance between beads after the
bead fluid is combined with the cell fluid, the ratio of channel
width m/b was increased by 200% (since the ratio of total bead and
cell fluids flowrate and bead fluid flow rate=60/30). In addition,
the width of the fluidic channel post droplet generation junction,
e.g., width d, was also wider by .about.200% as shown in FIG. 15B.
The ratio of the channel width m/b can vary from 1 to as high as 3
depending on the flow rates of the bead and cell fluids.
[0140] Channel Modification to Address Bead Clumping
[0141] Clumped beads may adversely affect single bead droplet
rates. Additionally, clumped beads may require a longer channel
length to achieve bead ordering. Therefore, in one embodiment,
non-clumped beads are fed into the bead fluidic channel. Feeding
non-clumped beads can be achieved by introducing structures or
constrictions at the bead inlet or at the beginning of the bead
channel to disrupt the clumps of beads. For example, in FIG. 16,
intra-channel constrictions are shown as wavy structures. In this
example, the channel width at the constriction is greater than the
bead diameter, but less the twice the bead diameter.
Nucleic Acid Sequencing
[0142] The application of inertial forcing and droplet generation
to beads, cells, and nucleic acids is suitable for applications in
any type of DNA sequence analysis, including long-read DNA
sequencing and single cell sequencing. The generation of droplets
each with one bead and one cell enable the continuous high
throughput analysis and sequencing of single cells.
[0143] In one embodiment shown in FIG. 8, a microchannel device is
designed to generate droplets each containing a single cell and a
single bead. Step 1, the microchannel device is configured to
separate, order, and focus streams of barcoded beads to one or more
focusing positions within a channel flow field. Step 2, the
microchannel device is configured to separate, order, and focus
streams of cells to one or more focusing positions within a channel
flow field. Step 3, the microchannel device receives an oil as
another input. Step 4, by combining the ordered barcoded bead, the
ordered cells, and an oil, the microchannel device generates
droplets with a-double-underdispersed-Poisson statistics, where
each droplet contains one bead and one cell. In some embodiments,
the design of the microfluidic device, concentrations of beads,
cells, other components of the bead fluid and cell fluid, the type
of oil, and the flow rates of the bead fluid, cell fluid, and oil
are designed so the microfluidic device generates droplets with any
desired numbers of beads and cells per droplet. In some
embodiments, the statistical distribution of beads is less than
optimal, e.g., less than 1 bead per droplet. In some embodiments,
the statistical distribution of cells is less than optimal, e.g.,
less than 1 cell per droplet. This ratio is beneficial for single
cell sequencing applications, as a high proportion of populated
droplets contain one cell and one bead, and low proportions of
droplets contain one cell and no bead, one bead and no cell, or are
empty. Thus, a high proportion of cells will be sequenced.
[0144] Each barcoded bead shown in FIG. 8 includes numerous
nucleotide fragments, and each nucleotide fragment includes a
unique DNA tag (e.g., a barcode, the same on all fragments on a
single bead), an index (e.g., a unique molecular identifier,
different for each fragment on a single bead), along with a capture
region comprising a poly-T tail. This construct makes each bead
uniquely tagged in comparison to all other beads being used in the
device. For example, each of the four droplets shown in FIG. 8
contains one barcoded bead and one cell. Each of the four barcoded
beads is uniquely tagged in comparison to the other three barcoded
beads. In some embodiments, the poly-T region may be at an internal
region of a nucleotide fragment rather than at the tail region of
the nucleotide fragment.
[0145] In some embodiments, the bead fluid contains a lysis buffer.
Step 5, when a cell and a bead become encapsulated into a droplet
and the droplet contains lysis buffer, the cell is lysed. After
cell lysis at Step 6, each polyadenylated mRNA in each cell becomes
bound to the poly-T tail of a nucleotide fragment on the bead,
e.g., hybridization between the nucleotide fragment on the bead and
the mRNA. Because of the index region, each mRNA from a cell is
uniquely tagged in comparison to other mRNA sequences from the
cell. And because of the unique DNA tag, each mRNA from a cell is
uniquely tagged in comparison to other mRNAs from other cells.
[0146] At Step 7, the emulsion of droplets is broken, releasing
beads with hybridized nucleotide fragments and mRNA into solution.
Resolution of an emulsion may be accomplished by any suitable
means, such as by chemical, physical, or electrolytic means. The
means may be chosen to be compatible with the particles in the
system, or may be chosen to degrade one or both particle types to
allow for subsequent analysis, such as sequencing.
[0147] At Step 8, the hybridized nucleotide fragments and mRNAs are
subject to reverse transcription using a reverse transcriptase to
generate cDNAs.
[0148] At Step 9, the cDNAs are subject to amplification using the
appropriate primers and polymerase. Thus, each cDNA strand formed
has an original mRNA sequence along with the unique DNA tag of the
bead that was encapsulated with the cell and the unique index from
the nucleotide fragment on the bead.
[0149] At Step 10, the amplified cDNAs are subject to library
preparation, such as Nextera library preparation.
[0150] At Step 11, the nucleotides in the library are subject to
sequencing, such as paired-end sequencing. Because each mRNA from a
cell is uniquely tagged in comparison to other mRNAs from the same
cell and mRNAs from other cells, sequencing reactions of the
library can be performed in bulk, with cDNA samples from many cells
being sequenced, but each uniquely tagged so that they can be
sorted from one another. Each library sequence has a unique DNA tag
or barcode, an index, and a capture region comprising a poly-T
region. The index can be used to correct for amplification errors
and avoid multiple-counting of a single molecule. After sequencing,
the mRNA population and expression level of individual cells can be
determined.
[0151] One of ordinary skill in the art will recognize that the
reverse transcription, amplification, and sequencing steps
discussed herein may be accomplished using methods known in the
field.
[0152] In certain of these methods, the beads include nucleotide
fragments. The nucleotide fragments include a barcode region, an
index region, and a capture region comprising a poly-T tail. The
barcode region of each nucleotide fragment is at least about six
nucleotides in length, or is about six to eight nucleotides in
length, or is about six nucleotides in length. The index region of
each nucleotide fragment is at least about four nucleotides in
length, or is about four to ten nucleotides in length, or is about
four nucleotides in length. The capture region includes poly-T
nucleotides and is at least about ten nucleotides in length, or is
about ten to twenty nucleotides in length, or is about ten
nucleotides in length.
Particle Analysis
[0153] In another embodiment, an analysis region is provided in
proximity to the output channel to monitor, sort, count, image, or
otherwise analyze the localized and focused streams of particles.
In one embodiment, a chip can be, or be part of, a particle
enumerating system. In particular, an analysis region, in which the
particles have been focused and ordered, could be subject to
interrogation by a detector for the purpose of counting the
particles. A variety of detectors are discussed below, as are
systems for tagging particles for detection, and these elements can
also be used for enumeration.
Types of Particles
[0154] Any number of different types of particles can be introduced
into the system for particle focusing and should not be limited to
those particle types described herein. Particles can be made of or
derived from various materials, and can have different properties
such as a density higher equal or lower than water.
[0155] Particles suspended within a sample can have any size which
allows them to be ordered and focused within the microfluidic
channels described herein. For example, particles can have a
hydrodynamic size that is in the range of about 100 microns to
about 0.01 microns. Alternatively, particles can have a
hydrodynamic size that is in the range of about 20 microns to about
0.1 microns. Alternatively, particles can have a hydrodynamic size
that is in the range of about 10 microns to about 1 micron. It will
be appreciated that particle size is only limited by channel
geometry, and particles both larger and smaller than the
above-described ranges can be ordered and focused within
predetermined channel geometries having laminar flow
conditions.
[0156] Particles can be cells or nucleic acids. Cells and nucleic
acids can be derived from any biological system, such as animal,
bacteria, virus, fungus, or plant, and any source such as water,
food, soil, or air.
[0157] In some embodiments, a solid sample serves as a source of
particles of interest. If a solid sample is obtained, such as a
tissue sample or soil sample, the solid sample can be liquefied or
solubilized prior to subsequent introduction into the system. If a
gas sample is obtained, it may be liquefied or solubilized as well.
For example, the sample may consist of bubbles of oil or other
kinds of liquids as the particles suspended in an aqueous
solution.
[0158] In some embodiments, a sample can be derived from an animal
such as a mammal. The mammal can be a human. Exemplary fluid
samples containing particles derived from an animal can include,
but are not limited to, whole blood, partitioned blood, blood
components, sweat, tears, ear flow, sputum, bone marrow suspension,
lymph, urine, brain fluid, cerebrospinal fluid, saliva, mucous,
vaginal fluid, semen, ascites, milk, secretions of the respiratory,
intestinal and genitourinary tracts, and amniotic fluid. In other
embodiments, exemplary samples can include fluids that are
introduced into a human body and then removed again for analysis,
including all forms of lavage such as antiseptic, bronchoalveolar,
gastric, peritoneal, cervical, arthroscopic, ductal, nasal, and ear
lavages. Exemplary particles can include any particles contained
within the fluids noted herein and can be both rigid and
deformable. In particular, particles can include, but are not
limited to, cells, alive or fixed, such as adult red blood cells,
fetal red blood cells, trophoblasts, fetal fibroblasts, white blood
cells, epithelial cells, tumor cells, cancer cells, hematopoeitic
stem cells, bacterial cells, mammalian cells, protists, plant
cells, neutrophils, T lymphocytes, CD 4+ cells, B lymphocytes,
monocytes, eosinophils, natural killers, basophils, dendritic
cells, circulating endothelial, antigen specific T-cells, and
fungal cells. In some embodiments, particles may include or be
derived from viruses, organelles, or liposomes.
[0159] Particles can be non-cellular or non-biological items, or
synthetic items, including such as beads, droplets, nanoparticles,
or molecular complexes. Different particle forms include but are
not limited to solid beads, porous solid beads, hydrogel beads,
double- or multi-emulsions, deformable or non-deformable beads,
spherical or complex-shaped beads. In some embodiments, particles
are beads, such as beads suitable for oligonucleotide (DNA or RNA)
sequencing applications. Beads may be synthetic polymer beads, such
as beads of polystyrene, sepharose, agarose, polyacrylamide,
chitosan, gelatin, and the like. Beads may also include magnetic
beads. Beads may be of any diameter, such as 10 to 100 .mu.m, or 10
to 20 .mu.m, or 25 to 50 .mu.m, or 30 .mu.m, or 40 .mu.m.
[0160] Particles may be suspended generally in any suspensions,
liquids, and/or fluids with at least one type of particle, cell,
droplet, or otherwise, disposed therein. Further, focusing can
produce a flux of particles enriched in a first particle based on
size.
[0161] In some embodiments, one or more particles, such as cells,
may stick, group, or clump together within a sample. In such a
configuration, a grouping or clumping of particles can be
considered to be "a particle" for the purposes of systems of the
invention. More particularly, a grouping or clumping of particles
may act and be treated as a single particle within channels of the
invention described herein and can thus be sorted, ordered,
separated, and focused in the same way as a single particle.
[0162] Particles from non-biological samples can include, for
example, any number of various industrial and commercial samples
suitable for particle separating, ordering, and focusing. Exemplary
industrial samples that contain particles that can be introduced
into the system can include, but are not limited to, emulsions,
two-phase chemical solutions (for example, solid-liquid,
liquid-liquid, and gas-liquid chemical process samples), waste
water, bioprocess particulates, and food industry samples such as
juices, pulps, seeds, etc. Similarly, exemplary commercial samples
that contain particles can include, but are not limited to,
bacteria/parasite contaminated water, water with particulates such
as coffee grounds and tea particles, cosmetics, lubricants, and
pigments.
[0163] In some embodiments, particles from a fluid sample obtained
from an animal is directly applied to the system described herein,
while in other embodiments, the sample is pretreated or processed
prior to being delivered to a system of the invention. For example,
a fluid drawn from an animal can be treated with one or more
reagents prior to delivery to the system or it can be collected
into a container that is preloaded with such a reagent. Exemplary
reagents can include, but are not limited to, a stabilizing
reagent, a preservative, a fixant, a lysing reagent, a diluent, an
anti-apoptotic reagent, an anti-coagulation reagent, an
anti-thrombotic reagent, magnetic or electric property regulating
reagents, a size altering reagent, a buffering reagent, an
osmolality regulating reagent, a pH regulating reagent, and/or a
cross-linking agent.
[0164] Suitable carrier fluids for the particle channels include
aqueous solutions, water, buffer solutions, salt-based solutions,
and mixtures thereof. Where the particles are cells, the cell
carrier fluid is compatible with cells, such as an aqueous buffer,
for example, phosphate-buffered saline. Where the particles are
beads, the carrier fluid may be water or an aqueous solution
optionally further comprising a chemical agent that provides the
desired amount of expansion of the polymer bead. In other
embodiments, the bead fluid comprises a cell lysis buffer.
[0165] Suitable oils include organic oils, such as olive oil or
vegetable oil, or mineral oils, or silicone oils (such as
derivatives of octamethyltrisiloxane), or perfluorinated oils (such
as Fluorinert FC-40) or long chain hydrocarbon acids, such as oleic
acid or dioctyl phthalate. Oils used in the third stream may also
comprise stabilizers or surfactants.
[0166] Flow rates for the first and second particle streams may be
the same or different. Flow rates may be in the range of about 10
to 75 .mu.L/min, or about 10 to 50 .mu.L/min, or about 10 to 35
.mu.L/min, or about 40 to 75 .mu.L/min, or about 10, 15, 20, 25,
30, 35, 40, 45, 50, 55, 60, 65, or 75 .mu.L/min. In some
embodiments, the bead stream flow rate is higher than the cell
stream flow rate.
[0167] The particle fluids may be introduced to the system with a
particular particle concentration. For example, particles may be
present in the particle fluids at a concentration of 100 to 3500
per .mu.L, or 100 to 750 per .mu.L, or 100 to 600 per .mu.L, or 100
to 300 per .mu.L, or 500 to 3000 per .mu.L, or 1000 to 3000 per
.mu.L. In some embodiments, the particles are cells, which are
present in the cell fluid at a concentration of 100 to 750 per
.mu.L, or 100 to 300 per .mu.L. In some embodiments, particles are
beads, which are present in the bead fluid at a concentration of
500 to 3000 per .mu.L or 1000 to 3000 per .mu.L.
[0168] In some embodiments, particle A or bead occupancy rates for
droplets produced by the systems described herein are at least 60,
70, 75, 80, 85, or 90%. In some embodiments, particle B or cell
occupancy rates for droplets produced by the systems described
herein are at least 10, 20, 30, 40, 50, 60, 70, 80, or 90%.
EXAMPLES
[0169] Some aspects of the embodiments discussed above are
disclosed in further detail in the following example, which is not
in any way intended to limit the scope of the present
disclosure.
Example 1
Focusing of 30 .mu.m-Diameter Beads to the Four Focusing Positions
in a Straight Square Channel within a Length of 1.2-3 cm from the
Bead Fluid Inlet
[0170] This example demonstrates that the focusing of 30
.mu.m-diameter beads to the four focusing positions in a square
channel was achieved within a length of 1.2-3 cm from the bead
fluid inlet.
[0171] Using the properties of fluid-bead and bead-bead
interactions in the bead channels, a set of microfluidic devices
were designed that allowed for beads of different sizes to focus
and order prior to entering the droplet generation junction.
Referring to FIG. 9, a microfluidic device 900 with a 125.times.125
.mu.m straight bead channel 904 was made that focused 30 .mu.m
diameter beads to their four focusing positions within a length of
1.2-3 cm from the inlet at the flow rate of 60 .mu.L/min. At 20
.mu.L/min of bead fluid 924 flow rate, no particle ordering was
observed and beads were randomly distributed at the droplet
generation junction. At 60 .mu.L/min of bead fluid 924 flow rate,
the beads 922 were ordered prior to entering the droplet generation
junction 926. At the device outlet 918, droplets 934 exited the
microfluidic device 900 in an orderly fashion with every droplet
encapsulating one bead in general. The combination of bead channel
dimension, flow rate generated more single-particle droplets and
fewer empty or multiple-particle droplets than would have been
possible from stochastic (Poisson) loading.
[0172] Altogether, these data indicate that the focusing of 30
.mu.m-diameter beads to the four focusing positions in a square
channel is achievable within a length of 1.2-3 cm from the bead
fluid inlet.
Example 2
Focusing of .ltoreq.40 .mu.m-Diameter Beads to Achieve One Bead Per
Droplet
[0173] This example demonstrates that focusing of .ltoreq.40
.mu.m-diameter beads to the two focusing positions in a rectangular
microchannel was achieved within a length of 1.2-3 cm from the bead
fluid inlet. This configuration of device resulted in the vast
majority of droplets containing one bead per droplet.
[0174] Using the properties of fluid-bead and bead-bead
interactions in the bead channels, a microfluidic device was
designed that allowed for beads of 40 .mu.m or less (for example,
20 to 40 .mu.m, or 30 .mu.m) to focus and order prior to entering a
droplet generation junction of the microfluidic device. The
microfluidic device had a bead channel with a cross-sectional
dimension of 100.times.125 .mu.m that focused the beads to their
two focusing positions within a length of approximately 1.2-3 cm
from the inlet. The flow rate of the bead solution and the cell
solution were set at 50 .mu.L/min. The flow rate of the oil was set
at 300 .mu.L/min, which resulted in approximately 4000 droplets per
second being created. At these flow rates, clear bead ordering was
observed and an ordered stream of beads was observed entering the
droplet generation junction. Only about 0.6% of the resulting
droplets had more than one bead within a droplet and a 90%
reduction compared to Poisson statistics.
[0175] Altogether, these data indicate that the focusing of beads
to the two focusing positions in a rectangular channel is
achievable within a length of 1.2-3 cm from the bead fluid
inlet.
Example 3
Focusing of 40 .mu.m-Diameter Polystyrene Beads to Achieve One Bead
Per Droplet
[0176] This example demonstrates that focusing of 40 .mu.m-diameter
polystyrene beads to the two focusing positions in a rectangular
microchannel was achieved within a length of 1.2-3 cm from the bead
fluid inlet. As shown in FIG. 10, this configuration of device
resulted in the vast majority of droplets containing one bead per
droplet.
[0177] Using the properties of fluid-bead and bead-bead
interactions in the bead channels, a microfluidic device was
designed that allowed for beads of 40 .mu.m or less to focus and
order prior to entering a droplet generation junction of the
microfluidic device. The microfluidic device had a bead channel
with a cross-sectional dimension of 75.times.125 .mu.m that focused
the beads to their two focusing positions within a length of
approximately 1.2-3 cm from the inlet. The flow rate of the bead
solution was set at 50 .mu.L/min and the cell solution were set at
10 .mu.L/min. The input bead concentration was set at 2000
beads/.mu.L. The flow rate of the oil was set at 250 .mu.L/min,
which resulted in approximately .about.2000 droplets per second
being created. At these flow rates, clear bead ordering was
observed and an ordered stream of beads was observed entering the
droplet generation junction (FIG. 10). Only about 2.7% of the
resulting droplets had more than one bead within a droplet and a
83.3% reduction compared to Poisson statistics. Results for the
distribution of beads inside droplets for other flow rate
conditions are shown in Table 2.
TABLE-US-00002 TABLE 2 Percentage of droplets with one polystyrene
bead only ("Desired"), two or more polystyrene beads ("Error"), and
empty droplets ("Empty") for various operating conditions, with a
comparison to Poisson distribution. Bead Fluid Cell Fluid Oil Flow
Desired Error Empty Flow Rate Flow Rate Rate Droplets Droplets
Droplets (.mu.L/min) (.mu.L/min) (.mu.L/min) (%) (%) (%) 40 5 250
73.4 8.4 18.2 40 10 250 64.3 10.8 24.9 50 10 250 72.9 2.7 24.4
Poisson Poisson Poisson 35.0 16.2 48.8
[0178] Altogether, these data indicate that the focusing of 40
.mu.m-diameter polystyrene beads to the two focusing positions in a
rectangular channel is achievable within a length of 1.2-3 cm from
the bead fluid inlet.
Example 4
Focusing of 40 .mu.m-Diameter Polymethylmethacrylate Beads to
Achieve One Bead Per Droplet
[0179] This example demonstrates that focusing of 30 to 40
.mu.m-diameter polymethylmethacrylate (PMMA) beads to the two
focusing positions in a rectangular microchannel was achieved
within a length of 1.2-3 cm from the bead fluid inlet. As shown in
FIG. 11, this configuration of device resulted in the vast majority
of droplets containing one bead per droplet.
[0180] Using the properties of fluid-bead and bead-bead
interactions in the bead channels, a microfluidic device was
designed that allowed for PMMA beads of 40 .mu.m or less to focus
and order prior to entering a droplet generation junction of the
microfluidic device. The microfluidic device had a bead channel
with a cross-sectional dimension of 75.times.125 .mu.m that focused
the beads to their two focusing positions within a length of
approximately 1.2-3 cm from the inlet. The flow rate of the bead
solution was set at 60 .mu.L/min and the cell solution were set at
10 .mu.L/min. The input bead concentration was set at 1500
beads/.mu.L. The flow rate of the oil was set at 260 .mu.L/min,
which resulted in approximately .about.2000 droplets per second
being created. At these flow rates, as shown in FIG. 11, clear bead
ordering was observed and an ordered stream of beads was observed
entering the droplet generation junction. As shown in Table 3, only
about 5.3% of the resulting droplets had more than one bead within
a droplet and a 67.3% reduction compared to Poisson statistics.
TABLE-US-00003 TABLE 3 Distribution of beads inside droplets for
other flow rate conditions. Bead Fluid Cell Fluid Oil Flow Desired
Error Empty Flow Rate Flow Rate Rate Droplets Droplets Droplets
(.mu.L/min) (.mu.L/min) (.mu.L/min) (%) (%) (%) 50 10 250 57.3 13.4
29.3 60 10 260 65.6 5.3 29.1
[0181] Altogether, these data indicate that the focusing of 30 to
40 .mu.m-diameter PMMA beads to the two focusing positions in a
rectangular channel is achievable within a length of 1.2-3 cm from
the bead fluid inlet.
Example 5
Focusing of 40 .mu.m-Diameter Sepharose Gel Beads to Achieve One
Bead Per Droplet
[0182] This example demonstrates that focusing of 30 to 40
.mu.m-diameter sepharose gel beads to the two focusing positions in
a straight rectangular microchannel was achieved within a length of
1.2-3 cm from the bead fluid inlet. As shown in FIGS. 12A and 12B,
this configuration of device resulted in the vast majority of
droplets containing one bead per droplet. The same approach can be
used for other types if porous polymer gel beads, such as
polyacrylamide, agarose, chitosan, gelatin, and the like.
[0183] Using the properties of fluid-bead and bead-bead
interactions in the bead channels, a microfluidic device was
designed that allowed for beads of 40 .mu.m or less to focus and
order prior to entering a droplet generation junction of the
microfluidic device. The microfluidic device had a bead channel
with a cross-sectional dimension of 75.times.125 .mu.m that focused
the beads to their two focusing positions within a length of
approximately 1.2-3 cm from the inlet. The flow rate of the bead
solution was set at 60 .mu.L/min and the cell solution were set at
10 .mu.L/min. The input bead concentration was set at 2100
beads/.mu.L. The flow rate of the oil was set at 270 .mu.L/min,
which resulted in approximately .about.2500 droplets per second
being created. As shown in FIGS. 12A and 12B, at these flow rates,
clear bead ordering was observed and an ordered stream of beads was
observed entering the droplet generation junction. As shown in
Table 4, only about 6.1% of the resulting droplets had more than
one bead within a droplet and a 62.3% reduction compared to Poisson
statistics.
TABLE-US-00004 TABLE 4 Distribution of beads inside droplets for
other flow rate conditions Bead Fluid Cell Fluid Oil Flow Desired
Error Empty Flow Rate Flow Rate Rate Droplets Droplets Droplets
(.mu.L/min) (.mu.L/min) (.mu.L/min) (%) (%) (%) 35 35 270 44.4 3.7
52.0 35 35 280 39.0 4.0 57.1 40 35 280 45.0 3.2 51.8 45 40 250 55.4
5.9 38.7 50 10 240 69.8 9.5 20.6 60 10 270 70.0 6.1 24.0
[0184] Altogether, these data indicate that the focusing of 30 to
40 .mu.m-diameter sepharose gel beads to the two focusing positions
in a rectangular channel is achievable within a length of 1.2-3 cm
from the bead fluid inlet.
Example 6
Focusing of 40 .mu.m-Diameter Sepharose Gel Beads to Achieve One
Bead Per Droplet
[0185] This example demonstrates that focusing of 30 to 40
.mu.m-diameter sepharose gel beads to the two focusing positions in
a spiral rectangular microchannel was achieved within a length of
1.2-3 cm from the bead fluid inlet. As shown in FIGS. 13A and 13B,
this configuration of device resulted in the vast majority of
droplets containing one bead per droplet. The same approach can be
used for other types if porous polymer gel beads, such as
polyacrylamide, agarose, chitosan, gelatin, and the like.
[0186] Using the properties of fluid-bead and bead-bead
interactions in the bead channels, a spiral microfluidic device was
designed that allowed for gel beads of 40 .mu.m or less to focus
and order prior to entering a droplet generation junction of the
microfluidic device. The microfluidic device had a bead channel
with a cross-sectional dimension of 75.times.100 .mu.m that focused
the beads to their two focusing positions within a length of
approximately 1.2-3 cm from the inlet. The flow rate of the bead
solution was set at 50 .mu.L/min and the cell solution were set at
10 .mu.L/min. The input bead concentration was set at 1800
beads/.mu.L. The flow rate of the oil was set at 180 .mu.L/min,
which resulted in approximately .about.2000 droplets per second
being created. At these flow rates, as shown in FIGS. 13A and 13B,
clear bead ordering was observed and an ordered stream of beads was
observed entering the droplet generation junction. As shown in
Table 5, about 5.2% of the resulting droplets had more than one
bead within a droplet and a 67.9% reduction compared to Poisson
statistics.
TABLE-US-00005 TABLE 5 Distribution of beads inside droplets for
other flow rate conditions Bead Fluid Cell Fluid Oil Flow Desired
Error Empty Flow Rate Flow Rate Rate Droplets Droplets Droplets
(.mu.L/min) (.mu.L/min) (.mu.L/min) (%) (%) (%) 40 10 180 42.1 1.9
56.0 40 20 180 39.1 1.8 59.1 50 10 180 52.1 5.2 42.8
[0187] Altogether, these data indicate that the focusing of 30 to
40 .mu.m-diameter 30 to 40 .mu.m-diameter sepharose gel beads to
the two focusing positions in a rectangular channel is achievable
within a length of 1.2-3 cm from the bead fluid inlet.
[0188] In at least some of the previously described embodiments,
one or more elements used in an embodiment can interchangeably be
used in another embodiment unless such a replacement is not
technically feasible. It will be appreciated by those skilled in
the art that various other omissions, additions and modifications
may be made to the methods and structures described above without
departing from the scope of the claimed subject matter. All such
modifications and changes are intended to fall within the scope of
the subject matter, as defined by the appended claims.
[0189] With respect to the use of substantially any plural and/or
singular terms herein, those having skill in the art can translate
from the plural to the singular and/or from the singular to the
plural as is appropriate to the context and/or application. The
various singular/plural permutations may be expressly set forth
herein for sake of clarity.
[0190] It will be understood by those within the art that, in
general, terms used herein, and especially in the appended claims
(e.g., bodies of the appended claims) are generally intended as
"open" terms (e.g., the term "including" should be interpreted as
"including but not limited to," the term "having" should be
interpreted as "having at least," the term "includes" should be
interpreted as "includes but is not limited to," etc.).
[0191] It will be further understood by those within the art that
if a specific number of an introduced claim recitation is intended,
such an intent will be explicitly recited in the claim, and in the
absence of such recitation no such intent is present. For example,
as an aid to understanding, the following appended claims may
contain usage of the introductory phrases "at least one" and "one
or more" to introduce claim recitations. However, the use of such
phrases should not be construed to imply that the introduction of a
claim recitation by the indefinite articles "a" or "an" limits any
particular claim containing such introduced claim recitation to
embodiments containing only one such recitation, even when the same
claim includes the introductory phrases "one or more" or "at least
one" and indefinite articles such as "a" or "an" (e.g., "a" and/or
"an" should be interpreted to mean "at least one" or "one or
more"); the same holds true for the use of definite articles used
to introduce claim recitations. In addition, even if a specific
number of an introduced claim recitation is explicitly recited,
those skilled in the art will recognize that such recitation should
be interpreted to mean at least the recited number (e.g., the bare
recitation of "two recitations," without other modifiers, means at
least two recitations, or two or more recitations). Furthermore, in
those instances where a convention analogous to "at least one of A,
B, and C, etc." is used, in general such a construction is intended
in the sense one having skill in the art would understand the
convention (e.g.,"a system having at least one of A, B, and C"
would include but not be limited to systems that have A alone, B
alone, C alone, A and B together, A and C together, B and C
together, and/or A, B, and C together, etc.). In those instances
where a convention analogous to "at least one of A, B, or C, etc."
is used, in general such a construction is intended in the sense
one having skill in the art would understand the convention
(e.g.,"a system having at least one of A, B, or C" would include
but not be limited to systems that have A alone, B alone, C alone,
A and B together, A and C together, B and C together, and/or A, B,
and C together, etc.). It will be further understood by those
within the art that virtually any disjunctive word and/or phrase
presenting two or more alternative terms, whether in the
description, claims, or drawings, should be understood to
contemplate the possibilities of including one of the terms, either
of the terms, or both terms. For example, the phrase "A or B" will
be understood to include the possibilities of "A" or "B" or "A and
B."
[0192] In addition, where features or aspects of the disclosure are
described in terms of Markush groups, those skilled in the art will
recognize that the disclosure is also thereby described in terms of
any individual member or subgroup of members of the Markush
group.
[0193] As will be understood by one skilled in the art, for any and
all purposes, such as in terms of providing a written description,
all ranges disclosed herein also encompass any and all possible
sub-ranges and combinations of sub-ranges thereof. Any listed range
can be easily recognized as sufficiently describing and enabling
the same range being broken down into at least equal halves,
thirds, quarters, fifths, tenths, etc. As a non-limiting example,
each range discussed herein can be readily broken down into a lower
third, middle third and upper third, etc. As will also be
understood by one skilled in the art all language such as "up to,"
"at least," "greater than," "less than," and the like include the
number recited and refer to ranges which can be subsequently broken
down into sub-ranges as discussed above. Finally, as will be
understood by one skilled in the art, a range includes each
individual member. Thus, for example, a group having 1-3 articles
refers to groups having 1, 2, or 3 articles. Similarly, a group
having 1-5 articles refers to groups having 1, 2, 3, 4, or 5
articles, and so forth.
[0194] While various aspects and embodiments have been disclosed
herein, other aspects and embodiments will be apparent to those
skilled in the art. The various aspects and embodiments disclosed
herein are for purposes of illustration and are not intended to be
limiting, with the true scope and spirit being indicated by the
following claims.
* * * * *