U.S. patent application number 14/167393 was filed with the patent office on 2015-06-18 for devices for separation of particulates, associated methods and systems.
This patent application is currently assigned to General Electric Company. The applicant listed for this patent is General Electric Company. Invention is credited to Jason Louis Davis, Craig Patrick Galligan, Gregory Andrew Grossmann, Erik Leeming Kvam, Jason Michael Nichols, Christopher Michael Puleo, Xuefeng Wang.
Application Number | 20150166956 14/167393 |
Document ID | / |
Family ID | 53367219 |
Filed Date | 2015-06-18 |
United States Patent
Application |
20150166956 |
Kind Code |
A1 |
Puleo; Christopher Michael ;
et al. |
June 18, 2015 |
DEVICES FOR SEPARATION OF PARTICULATES, ASSOCIATED METHODS AND
SYSTEMS
Abstract
A device is configured for separation of particulates dispersed
within a base fluid, wherein the particulates have a relative
density difference compared to the base fluid. The device comprises
a microchannel of length l and height h comprising an inlet and an
outlet; a microporous surface on one or more walls of the
microchannel; a collection chamber on an opposing side of the
microporous surface; and an applied force field across the height h
of the microchannel to sediment the particles through the
microporous surface into the collection chamber. The microporous
body operationally generates a fluid flow regime comprising a first
fluid flow having a first flow rate through the microchannel and a
second fluid flow having a second flow rate through the collection
chamber and the second flow rate is a fraction of the first flow
rate.
Inventors: |
Puleo; Christopher Michael;
(Niskayuna, NY) ; Kvam; Erik Leeming; (Niskayuna,
NY) ; Grossmann; Gregory Andrew; (Halfmoon, NY)
; Galligan; Craig Patrick; (Niskayuna, NY) ;
Nichols; Jason Michael; (Schenectady, NY) ; Wang;
Xuefeng; (Niskayuna, NY) ; Davis; Jason Louis;
(Albany, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
General Electric Company |
Schenectady |
NY |
US |
|
|
Assignee: |
General Electric Company
Schenectady
NY
|
Family ID: |
53367219 |
Appl. No.: |
14/167393 |
Filed: |
January 29, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61916379 |
Dec 16, 2013 |
|
|
|
Current U.S.
Class: |
435/2 ;
435/309.1 |
Current CPC
Class: |
B01D 21/2444 20130101;
C02F 2001/007 20130101; B01L 2400/0469 20130101; B01D 21/0006
20130101; B01L 2200/0668 20130101; C02F 1/00 20130101; C02F 1/444
20130101; C02F 2101/32 20130101; C02F 2103/10 20130101; C02F 1/48
20130101; B01L 2200/0631 20130101; B01D 21/0042 20130101; B01L
2200/0652 20130101; B01L 2400/0421 20130101; C02F 1/24 20130101;
B01L 2400/0457 20130101; B01D 21/02 20130101; B01D 21/0087
20130101; B01D 21/10 20130101; B01D 17/02 20130101; B01L 2400/043
20130101; B01D 21/2433 20130101; B01L 3/502753 20130101; B01L
2300/0681 20130101; B01L 2300/0851 20130101; C12M 47/02 20130101;
C02F 2103/08 20130101; C12N 5/0641 20130101; B01D 21/245 20130101;
C02F 2201/002 20130101; B01D 2221/04 20130101; B01L 3/502761
20130101 |
International
Class: |
C12N 5/078 20060101
C12N005/078 |
Claims
1. A device for separating particulates dispersed within a base
fluid and having a relative density difference compared to the base
fluid, comprising: a microchannel of length l and height h disposed
between a fluid inlet and a fluid outlet; a microporous body
defining at least a portion of the microchannel; and a collection
chamber on an opposing side of the microporous body; wherein, the
particulates and a portion of the base fluid traverse the
microporous body under the influence of an external force field,
and are entered and collected in the collection chamber; and
wherein the microporous body operationally generates a fluid flow
regime comprising a first fluid flow having a first flow rate
through the microchannel and a second fluid flow having a second
flow rate through the collection chamber and the second flow rate
is a fraction of the first flow rate.
2. The device of claim 1, wherein the external force field is a
gravitational field.
3. The device of claim 1, wherein the external force field is an
applied force field selected from among an applied magnetic field
and an applied electric field.
4. The device of claim 1, wherein the microchannel has a length l
between about 10 millimeters and about 100 millimeters (mm)
5. The device of claim 1, wherein the microchannel has a height h
between about 10 micron and about 1000 microns (.mu.m).
6. The device of claim 1, wherein the particulates have an average
largest dimension between about 1 micron and about 250 microns.
7. The device of claim 1, wherein the microporous body comprises
pores with an average diameter between about 10 microns and about
500 microns.
8. The device of claim 1, wherein the microporous body has porosity
between about 10 percent and about 75 percent.
9. The device of claim 1, further comprising one or more of a
collection chamber fluid inlet and a collection chamber fluid
outlet.
10. The device of claim 1, further comprising one or more
controllers for controlling the applied external force field.
11. The device of claim 1, further comprising a fluid driver to
induce a flow of particulates dispersed within a base fluid through
the microchannel and to drive out a processed fluid enriched in the
base fluid and depleted in particulates.
12. The device of claim 1, further comprising a fluid driver
configured to facilitate recovery of particulates from the
collection chamber.
13. The device of claim 1, further comprising one or more
controllers to control the first fluid flow.
14. The device of claim 1, wherein the device is fully automated or
partially automated.
15. The device of claim 1, wherein one or more of the fluid inlet,
the fluid outlet, the microchannel, the microporous body, and the
collection chamber is configured to integrate with an analytical
device.
16. The device of claim 1 is configured to separate particulates
from one or more of whole blood, petroleum, water, a cell extract,
or a tissue extract.
17. The device of claim 1 is configured to separate particulates
from whole blood.
18. The device of claim 1 is configured to separate red blood cells
from whole blood.
19. The device of claim 1, wherein the particulates comprise one or
more of red blood cells, white blood cells, blood platelets,
non-hematic biological cells, tissue fragments, metals, minerals,
and non-cellular biological solids.
20. A device for separating one or more cells dispersed within a
base fluid and having a relative density difference compared to the
base fluid, the device comprising: a microchannel of length l and
height h disposed between a fluid inlet and a fluid outlet; a
microporous body defining at least a portion of the microchannel;
and a collection chamber on an opposing side of the microporous
body; wherein the cells and a portion of the base fluid traverse
the microporous body under the influence of an external force
field, and are entered and collected in the collection chamber; and
wherein the microporous body operationally generates a fluid flow
regime comprising a first fluid flow having a first flow rate
through the microchannel and a second fluid flow having a second
flow rate through the collection chamber and the second flow rate
is a fraction of the first flow rate.
21. The device of claim 20, wherein the cells have an average cell
diameter (d) between about 1 micron and about 100 microns.
22. The device of claim 20, wherein the microchannel has a height h
between about 10 microns and about 1000 microns.
23. The device of claim 20, wherein the microporous body has an
average pore diameter (p) between about 10 microns and about 500
microns.
24. The device of claim 20, wherein the microporous body has an
average porosity (q) between about 10 percent and about 75
percent.
25. A method for separating particulates dispersed within a base
fluid and having a relative density difference compared to the base
fluid, comprising: providing a separation device comprising: a
microchannel of length l and height h disposed between a fluid
inlet and a fluid outlet; a microporous body defining at least a
portion of the microchannel; and a collection chamber on an
opposing side of the microporous body; wherein the particulates and
a portion of the base fluid traverse the microporous body under the
influence of an external force field, and are entered and collected
in the collection chamber; introducing a sample of unprocessed
fluid comprising particulates dispersed within a base fluid into
the microchannel via the fluid inlet; separating at least a portion
of the particulates from the unprocessed fluid to provide a stream
of processed fluid at the fluid outlet; and recovering at least a
portion of the particulates initially present in the unprocessed
fluid in the collection chamber; wherein the particulates and a
portion of the base fluid traverse the microporous body under the
influence of an external force field, and are entered and collected
in the collection chamber; and wherein the microporous body
operationally generates a fluid flow regime comprising a first
fluid flow having a first flow rate through the microchannel and a
second fluid flow having a second flow rate through the collection
chamber and the second flow rate is a fraction of the first flow
rate.
26. The method of claim 25, further comprising a step of priming
the device prior to introducing the unprocessed fluid into the
microchannel.
27. The method of claim 25, further comprising re-traversing the
fluid through the microporous body and re-entering the
microchannel.
28. The method of claim 25, wherein the unprocessed fluid is a
biological sample.
29. The method of claim 28, wherein the unprocessed fluid comprises
one or more of whole blood, a cell extract, or a tissue
extract.
30. The method of claim 28, wherein the unprocessed fluid comprises
whole blood.
31. The method of claim 28, wherein the particulates are blood
cells.
32. The method of claim 28, wherein the processed fluid comprises
blood plasma.
33. A method for separating cells dispersed within a base fluid of
whole blood sample, comprising: providing a separation device
comprising: a microchannel of length l and height h disposed
between a fluid inlet and a fluid outlet; a microporous body
defining at least a portion of the microchannel; and a collection
chamber on an opposing side of the microporous body; wherein the
particulates and a portion of the base fluid traverse the
microporous body under the influence of an external force field,
and are entered and collected in the collection chamber;
introducing the whole blood sample of unprocessed fluid comprising
cells dispersed within a base fluid into the microchannel via the
fluid inlet; separating at least a portion of the cells from the
unprocessed fluid to provide a stream of processed fluid at the
fluid outlet; and recovering at least a portion of the cells
initially present in the unprocessed fluid in the collection
chamber; wherein the particulates and a portion of the base fluid
traverse the microporous body under the influence of an external
force field, and are entered and collected in the collection
chamber; and wherein the microporous body operationally generates a
fluid flow regime comprising a first fluid flow having a first flow
rate through the microchannel and a second fluid flow having a
second flow rate through the collection chamber and the second flow
rate is a fraction of the first flow rate.
34. The method of claim 33, wherein the processed fluid comprises
blood plasma which is substantially free of blood cells.
35. The method of claim 33, wherein the cells recovered in the
collection chamber is substantially free of blood plasma.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/916379, entitled "System for separation of
particulates and associated methods and devices", filed on Dec. 16,
2013, which is herein incorporated by reference.
FIELD
[0002] The invention relates to microfluidic systems, devices and
methods useful for separating particulate materials from fluids. In
a particular aspect, the invention relates to a method for
separating cells from biological samples using the systems, devices
and methods provided herein.
BACKGROUND
[0003] Preparation and manipulation of high quality cells or
biomolecules are primary requirements for a variety of diagnostic
or therapeutic applications. Though filtration techniques are
commonly used for capturing cells, they pose various challenges
including the inability to obtain high separation efficiencies for
heterogeneous cell populations, clogging of pores or harsh
filtration conditions causing cellular damage. The challenges are
exacerbated when filtering larger volumes of crude biological
samples. In addition to filtration, a number of microfluidic
separation techniques exist that rely on the application of a
force, which acts to push or pull cells in a direction
perpendicular to the direction of flow of the sample fluid being
processed. Several of these continuous flow techniques have
recently proven useful in efficiently separating out cells from
blood, including hydrodynamic filtration, inertial and
deterministic lateral flow separation. However, most of the
techniques require pre-dilution of the blood sample and are limited
to relatively low volumetric flow rates.
[0004] Recent developments on "filter-free" mechanisms for
biological sample preparation offer portable purification devices
for limited volumes. Primary applications have been in
point-of-care diagnostics, where relatively small samples are
collected and directly processed within the collection vessel.
However, there remains a need to expand these simple separation
methods to larger volumetric flow rates to provide alternatives to
large scale centrifugation and filtration techniques.
[0005] A well-known continuous flow separation technique is field
flow fractionation (FFF) in which differential retention of
particles being eluted through a microchannel results in separation
of particles having different characteristics. However, the
relevance of field flow fractionation for whole blood separation
remains uncertain as most studies suggest the need for relatively
dilute starting blood samples. Recent combinations of newer
separation techniques, such as hydrodynamic filtration and inertial
focusing, have increased the traditional throughput limits
associated with continuous flow separations. However, each of these
"high throughput" implementations requires carefully controlled
flow rates and/or upstream sample pre-filtration or dilution.
[0006] Sedimentation-based devices may provide simpler methods of
cell and/or particle separation from fluids containing them without
the need for careful fluid flow control and/or excessive sample
dilution. However, there exists a need to provide devices and
methods that enable high speed separation of particulates such as
cells and/or dispersed particles from fluids containing them
without the need to augment sedimentation rates via capital
intensive equipment, such as centrifuges. A fast and efficient
separation and collection of particles or cells from a large sample
volume without complex equipment is an unmet need. Therefore,
inexpensive devices that can accelerate particle separation via
sedimentation, and enable use of a large sample volume with minimal
human intervention are highly desirable.
BRIEF DESCRIPTION
[0007] In one embodiment, the present invention provides a device
for separating particulates dispersed within a base fluid and
having a relative density difference compared to the base fluid,
comprises a microchannel of length l and height h disposed between
a fluid inlet and a fluid outlet; a microporous body defining at
least a portion of the microchannel; and a collection chamber on an
opposing side of the microporous body; wherein, the particulates
and a portion of the base fluid traverse the microporous body under
the influence of an external force field, and are entered and
collected in the collection chamber; and wherein the microporous
body operationally generates a fluid flow regime comprising a first
fluid flow having a first flow rate through the microchannel and a
second fluid flow having a second flow rate through the collection
chamber and the second flow rate is a fraction of the first flow
rate.
[0008] One embodiment of a device for separating one or more cells
dispersed within a base fluid and having a relative density
difference compared to the base fluid, the device comprises a
microchannel of length l and height h disposed between a fluid
inlet and a fluid outlet; a microporous body defining at least a
portion of the microchannel; and a collection chamber on an
opposing side of the microporous body; wherein the cells and a
portion of the base fluid traverse the microporous body under the
influence of an external force field, and are entered and collected
in the collection chamber; and wherein the microporous body
operationally generates a fluid flow regime comprising a first
fluid flow having a first flow rate through the microchannel and a
second fluid flow having a second flow rate through the collection
chamber and the second flow rate is a fraction of the first flow
rate.
[0009] In another embodiment, a method for separating particulates
dispersed within a base fluid and having a relative density
difference compared to the base fluid, comprises: providing a
separation device comprising: a microchannel of length l and height
h disposed between a fluid inlet and a fluid outlet; a microporous
body defining at least a portion of the microchannel; and a
collection chamber on an opposing side of the microporous body;
wherein the particulates and a portion of the base fluid traverse
the microporous body under the influence of an external force
field, and are entered and collected in the collection chamber;
introducing a sample of unprocessed fluid comprising particulates
dispersed within a base fluid into the microchannel via the fluid
inlet; separating at least a portion of the particulates from the
unprocessed fluid to provide a stream of processed fluid at the
fluid outlet; and recovering at least a portion of the particulates
initially present in the unprocessed fluid in the collection
chamber; wherein the particulates and a portion of the base fluid
traverse the microporous body under the influence of an external
force field, and are entered and collected in the collection
chamber; and wherein the microporous body operationally generates a
fluid flow regime comprising a first fluid flow having a first flow
rate through the microchannel and a second fluid flow having a
second flow rate through the collection chamber and the second flow
rate is a fraction of the first flow rate.
[0010] One embodiment of a method for separating cells dispersed
within a base fluid of whole blood sample, comprises providing a
separation device comprising: a microchannel of length l and height
h disposed between a fluid inlet and a fluid outlet; a microporous
body defining at least a portion of the microchannel; and a
collection chamber on an opposing side of the microporous body;
wherein the particulates and a portion of the base fluid traverse
the microporous body under the influence of an external force
field, and are entered and collected in the collection chamber;
introducing the whole blood sample of unprocessed fluid comprising
cells dispersed within a base fluid into the microchannel via the
fluid inlet; separating at least a portion of the cells from the
unprocessed fluid to provide a stream of processed fluid at the
fluid outlet; and recovering at least a portion of the cells
initially present in the unprocessed fluid in the collection
chamber; wherein the particulates and a portion of the base fluid
traverse the microporous body under the influence of an external
force field, and are entered and collected in the collection
chamber; and wherein the microporous body operationally generates a
fluid flow regime comprising a first fluid flow having a first flow
rate through the microchannel and a second fluid flow having a
second flow rate through the collection chamber and the second flow
rate is a fraction of the first flow rate.
DRAWINGS
[0011] These and other features, aspects, and advantages of the
present invention will become better understood when the following
detailed description is read with reference to the accompanying
drawings in which like characters represent like parts throughout
the drawings, wherein:
[0012] FIG. 1A is a schematic drawing of front view of a device
suitable for use in one embodiment of the devices.
[0013] FIG. 1B is a schematic drawing of side view of a device
suitable for use in one embodiment of the devices.
[0014] FIG. 1C is a schematic drawing of top view of a device
suitable for use in one embodiment of the devices.
[0015] FIG. 2 is a schematic view of method steps for separating
cells from whole blood using one embodiment of the devices.
[0016] FIG. 3 illustrates a method of separating particles based on
density from a fluid using one embodiment of the devices.
[0017] FIG. 4 illustrates a method of separating particles based on
density from a fluid using another embodiment of the devices.
[0018] FIG. 5 illustrates a method of separating particles from a
fluid using one embodiment of the devices.
[0019] FIG. 6 illustrates a method of separating particles from a
fluid using one embodiment of the devices.
[0020] FIGS. 7A and 7B are images taken from computational fluid
dynamic (CFD) models showing fluid flow pattern through the device
without microporous surface and with microporous surface,
respectively.
[0021] FIGS. 8A and 8B illustrate the performance characteristics
of a cell separation device showing loss of leukocytes with
increasing fluid flow rate using a device without a microporous
surface and with a microporous surface, respectively.
[0022] FIGS. 8C and 8D illustrate the performance characteristics
of a cell separation device, showing blood sample loaded through
inlet and processed fluid samples driven out from the outlet of a
device without a microporous surface and a device with a
microporous surface, respectively.
[0023] FIG. 9 illustrates the performance characteristics of a cell
separation device showing a percentage of cells captured in a
collection chamber from a blood sample loaded into one embodiment
of the device (I) compared with a commercial benchmark (II).
[0024] FIG. 10 illustrates the performance characteristics of a
cell separation device showing percentage recovery of captured
cells from one embodiment of the device (I) relative to a
commercial benchmark (II).
[0025] FIG. 11A illustrates the performance characteristics of a
cell separation device showing a percentage of captured and
recovered cells from a blood sample with different flow rates using
one embodiment of the device.
[0026] FIG. 11B illustrates the performance characteristics of a
cell separation device showing length for varying flow rate using
one embodiment of the device.
[0027] FIG. 12 illustrates one embodiment of a microfluidic
separation device.
[0028] FIG. 13 illustrates one embodiment of a portion of the
microfluidic separation device of FIG. 12 in an exploded
perspective view.
[0029] FIG. 14 illustrates one embodiment of a portion of the
microfluidic separation device of FIG. 12 in an exploded
perspective view.
DETAILED DESCRIPTION
[0030] Separation of particulates dispersed within a base fluid
(collectively "the unprocessed fluid"), for example separation of
blood cells dispersed within blood plasma, or separation of
particulate impurities dispersed in water, may be effected using
various embodiments of systems, devices and methods provided by the
present invention. Embodiments of the device and its device
components (e.g. FIGS. 1A-1C, FIGS. 12-13) comprise a fluid inlet
for introducing the unprocessed fluid into the device, a fluid
outlet for removing processed fluid from the device, and a
separation region comprising a microchannel disposed between the
fluid inlet and the fluid outlet, a microporous body defining at
least a portion of the microchannel; and a collection chamber. The
particles are separated from the base fluid of the fluidic sample
through the microporous body using fluidic flow and sedimentation,
which is unlike a filtration device that relies entirely on
physical barriers to filter particles.
[0031] To more clearly and concisely describe the subject matter of
the disclosed invention, the following definitions are provided for
specific terms, which are used in the following description and the
appended embodiments. Throughout the specification, exemplification
of specific terms should be considered as non-limiting
examples.
[0032] The terms "particulate" and "particle", and their plural
referents "particulates" and "particles", are used interchangeably
herein and are intended to have the same meaning, particle being
treated as a synonym for particulate. As used herein, the term
"particles" refers to a portion of the fluidic sample loaded into
the device which excludes the base fluid. The term "particles"
includes without limitation cells, inorganic colloids, polymers,
biopolymers, immiscible liquids, heterogenous solids, and nominally
gaseous/liquid materials in a solid phase. For example, blood
cells, grains of sand, oil droplets, nucleic acids, or ice crystals
are all particles.
[0033] The terms "microporous body" and "microporous surface" may
be used interchangeably herein and are intended to have the same
meaning.
[0034] As used herein, the term "operationally generates" refers to
a function of generating one or more fluid flow regimes by
microporous surface during operation of the device. For example,
when a fluid sample loaded into the device and flows through the
microchannel for separation of the particles from the fluid, the
microporous surface generates a field of flow with two fluid flow
regimes under the operating conditions of the device.
[0035] As used herein, the term "fluidic sample" refers to a
mixture that comprises a non-fluid component and a fluid component.
The mixture may be heterogeneous or homogenous in nature. The
loaded sample is interchangeably used herein with a "sample",
"fluidic sample" or "fluid loaded into the device". The sample may
comprise without limitation, a fluid comprising one or more
particles, a fluid comprising one or more cells, water with
particles, water-oil emulsion, or a fluid with impurities. For
example, a sample comprises a slurry of sand and water, or a
fluidic sample comprises cells and plasma. The term "fluidic
sample" is used interchangeably and without limitation with the
term "dispersion", "particle dispersion", or "cellular dispersion"
when identifying a generic class of fluidic sample. In some
embodiments, a cellular dispersion refers to a sample of cells
dispersed in a fluid, for example blood cells dispersed in plasma,
cells dispersed in growth media, or cells dispersed in
stabilization media.
[0036] As used herein, the term "base fluid" refers to a portion of
the fluidic sample which excludes the particles. For example, a
whole blood sample comprises blood cells in plasma, wherein the
plasma is a base fluid. For another example, an aqueous sand
dispersion comprises sand in water, wherein water is a base
fluid.
[0037] As used herein, the term "relative density difference"
refers to a difference between the density of the particulates
present in the base fluid and the density of the base fluid.
[0038] As used herein, the term "sediment" refers to particle
motion induced by an applied force field. Motion or movement of
particulates in a fluid in response to a force may be active or
passive, and the movement is referred to herein as sedimentation.
For example, in cases where the force of gravity augments the
action of an externally applied electric field in inducing
particulate movement. Sedimentation usually provides a simple means
of separating particulates from a base fluid. In one embodiment, in
an aqueous sand dispersion, the sand particles have a density
greater than that of the base fluid and sediment in the direction
of the gravitational field. In another embodiment, in an
oil-in-water emulsion, the oil droplets have a density less than
that of the base fluid and transit in the direction opposing the
gravitational field. In another embodiment, in a dispersion of
magnetic particles, the magnetic particles are sediment in the
direction of an applied magnetic field. In another embodiment, in a
dispersion of charged particles, the charged particles
differentially sediment based on their polarity within an applied
electric field.
[0039] The singular forms "a", "an" and "the" include plural
referents unless the context clearly dictates otherwise.
[0040] Approximating language, as used herein throughout the
specification and claims, may be applied to modify any quantitative
representation that could permissibly vary without resulting in a
change in the basic function to which it is related. Accordingly, a
value modified by a term such as "about" is not to be limited to
the precise value specified. In some instances, the approximating
language may correspond to the precision of an instrument for
measuring the value. Where necessary, ranges have been supplied,
and those ranges are inclusive of all sub-ranges there between.
[0041] In one or more embodiments, a device is configured to
separate particulates present in a fluidic sample. The sample may
comprise particulates dispersed in a base fluid and the
particulates have a relative density difference compared to the
base fluid. In these embodiments, the device for separating
particulates from the base fluid comprises a microchannel of length
l and height h disposed between a fluid inlet and a fluid outlet; a
microporous body defining at least a portion of the microchannel;
and a collection chamber on an opposing side of the microporous
body. The particulates and a portion of the base fluid traverse the
microporous body under the influence of an external force field,
and are entered and collected in the collection chamber. The
microporous body operationally generates a fluid flow regime
comprising a first fluid flow having a first flow rate through the
microchannel and a second fluid flow having a second flow rate
through the collection chamber and the second flow rate is a
fraction of the first flow rate. Non-limiting examples of an
embodiment of the device are shown in FIGS. 1A, 1B and 1C and the
related methods steps are shown in FIG. 2.
[0042] The term microchannel is used to describe the channel into
which the particulates dispersed in a base fluid are introduced. As
noted, the microchannel comprises an inlet and an outlet; and one
or more walls, wherein the one or more walls comprise a microporous
body, such as a microporous surface. The inlet of the microchannel
may be configured to receive a sample, such as a biological sample,
a water sample or an oil sample. The inlet and the outlet may be
present, without limitation at the ends of the channel. The
microchannel may comprise one or more walls, and a microporous
surface constitutes the one or more walls of the microchannel.
[0043] The microchannel is further described as microfluidic
channel since at least one of the dimensions of the microchannel is
appropriately measured in microns. In various embodiments of the
device, a microchannel has a length l and a height h. In some
embodiments, the microchannel is a horizontal channel configured to
permit the flow of a fluid at a predetermined flow rate. The device
may be set for a specific flow rate as desired before operating the
device. Typically, the microchannel has a length appropriately
measured in units larger than microns, for example millimeters
(mm), centimeters (cm) or meters (m). In some embodiments of the
device, the microchannel has a length l between 5 mm and 25 cm. In
another embodiment, the microchannel has a length l between 10 mm
and 10 cm. In yet another embodiment, the microchannel has a length
l between 10 mm and 25 mm
[0044] The microchannel may be of a regular shape (for example
cylindrical) and be of uniform height h. In one embodiment, the
average height of the microchannel is between about l and about
1000 microns (.mu.m). In an alternate embodiment, the average
height of the microchannel is between about 10 and about 500
microns. In yet another embodiment, the average height of the
microchannel is between about 20 and about 250 microns. The
microchannel may be of an irregular shape (for example a channel
defined in part by an undulating wall) and be characterized by a
plurality of heights h. Typically, however, the microchannel is
rectangular in shape and is defined on three sides by walls
enclosing the microchannel and on a fourth side by the microporous
body.
[0045] As noted, the microchannel comprises a microporous body, in
some embodiments, the microporous body is a microporous surface. In
one embodiment, the microporous body constitutes one or more walls
defining the microchannel. The microporous body may be a membrane
or a solid body through which holes have been created. In one or
more embodiments, the microporous body comprises pores originating
at a first surface of the microporous body and terminating at a
second surface of the microporous body. For example, pores
traversing a film may be created by chemical etching techniques
and/or laser ablative techniques.
[0046] The term "microporous" is used herein because the pores have
dimensions appropriately measured in microns. In one embodiment,
the pores have an average diameter between about 1 micron and about
500 microns. In an alternate embodiment, the pores have an average
diameter between about 10 microns and about 250 microns. In yet
another embodiment, the pores have an average diameter between
about 20 microns and about 100 microns. In one embodiment, the
porosity of the microporous body is between about 10 and about 75
percent. In an alternate embodiment, the porosity of the
microporous body is between about 20 and about 65 percent. In yet
another embodiment, the porosity of the microporous body is between
about 30 and about 60 percent.
[0047] As noted, in one embodiment, the microporous body may be a
microporous film such as a monofilament screen or mesh made from,
for example, polyester, nylon, polypropylene. Alternatively, the
microporous body may be a chemically-etched KAPTON, titanium, or
NiTinol film. In one embodiment, the microporous body is a laser
etched organic film made from an organic polymeric material such as
KAPTON.
[0048] As noted, the microchannel is disposed between a fluid inlet
and a fluid outlet. Fluid enters the microfluidic separation device
via the fluid inlet as unprocessed fluid, travels the length of the
microchannel to the fluid outlet. During the passage of fluid from
the inlet to the outlet, particulates migrate out of the
microchannel and enter into the collection chamber under the
influence of the fluid flow through the microchannel and one or
more additional forces such as the ambient gravitational field, an
applied force field, or buoyancy forces. The action within the
microchannel converts the unprocessed fluid introduced at the fluid
inlet into processed fluid merging at the fluid outlet. In one
embodiment, the fluid inlet is configured to receive and hold a
fluid sample comprising particulates dispersed within a base fluid,
and then to deliver the fluid sample to the microchannel under the
influence of a device component, for example a vacuum line applied
to the fluid outlet.
[0049] As noted, the device comprises a collection chamber in fluid
communication with the microchannel configured such that the
collection chamber is situated on an opposing side of the
microporous body. Typically, the collection chamber is configured
such that every pore of the microporous body enables direct fluid
communication between the microchannel and the collection chamber.
The portion of the microchannel-microporous body interface is
referred to herein as the separation zone, wherein the condition of
direct fluid communication between the microchannel and the
collection chamber through each of the pores is met. The collection
chamber is configured to collect particles, which sediment from the
fluid sample during processing. For example, the collection chamber
collects the cells from a blood sample that is loaded to the
microchannel of the device. In the collection chamber, the fluid
may be in a quasi-static flow during operation. In some
embodiments, the particles sediment and pass through the pores of
the microporous surface and are entrapped within the collection
chamber. The collection chamber may comprise one or more outlets
for collecting the particles which sediment through the microporous
surface. The outlet may be connected to a conduit or a syringe to
retrieve the particles from the collection chamber of the device.
For example, in the case of a blood sample, the separated cells are
recovered from the collection chamber using a tube or syringe for
downstream applications. In one or more embodiments, the collection
chamber is coupled to a pump to recover the particles from the
collection chamber during operation.
[0050] In various embodiments, the collection chamber is primed,
may be partially or completely filled with a priming fluid (e.g.
buffer solution) prior to the introduction of the unprocessed fluid
into the microchannel. In some embodiments, the collection chamber
is filled with a priming fluid prior to initiate a flow through the
microchannel. As fluid flows through the device, a first flow
regime is established in the microchannel and a second flow regime
is established in the collection chamber, the second flow regime
being characterized by a lower overall flow rate than that of the
first flow regime. In various embodiments, an average time required
for the particles to sediment through the microporous body is less
than the time required for the particles to transit across the
length l of the microchannel.
[0051] In one or more embodiments, the device further comprises an
applied force field across one or more dimensions of the
microchannel, for example across the height h of the microchannel
to cause the particles to pass through the microporous surface and
into the collection chamber. The applied force field may function
across the height h of the microchannel under operating conditions
of the device. The applied force field may decrease the time
required for the particles being impelled through the microchannel
by the moving fluid at relatively high flow rate to sediment
through the microchannel and be trapped within the collection
chamber, wherein the fluid flow rate is relatively low.
[0052] In one or more embodiments, an external force causes the
particulates to migrate through the microporous body. For example,
the external force may be ambient gravity at times herein referred
to as ambient gravitational forces. In some embodiments, the
applied force field is selected from a magnetic field, an electric
field, an electrophoretic field or combinations thereof. In an
alternate embodiment, the external force may be a combination of
the ambient gravitational forces present together with an applied
force field, such as an applied electric field or magnetic field.
In an alternate embodiment, the forces causing the particulates to
migrate across the microporous body are exerted by the fluid being
processed. For example, buoyancy forces may dominate gravitational
forces in the separation of oil in water emulsions. In some
embodiments, the device further comprises one or more controllers
for controlling the applied force field.
[0053] In one embodiment, the microfluidic separation device is
configured such that the ambient gravitational force acts across
the height h of the microchannel and causes particulates dispersed
within the base fluid to sediment through the microporous body and
into the collection chamber.
[0054] The particulates dispersed in the base fluid and traverse
together with a portion of the base fluid itself under the
influence of passive and/or active forces through the microporous
body and are separated from the base fluid. The particulates and a
portion of the base fluid are collected in a collection chamber
disposed on an opposing side of the microporous body. The fluid
sample comprises a plurality of particles in a fluid base, wherein
the particles may have a relative density difference compared to
the fluid base.
[0055] In various embodiments, the separation of particulates from
the base fluid occurs as the fluid is flowing through the
microchannel for processing. Having traversed the microporous body,
the particulates continue to migrate away from the microporous body
under the influence of the passive and/or active forces. The base
fluid is typically much less susceptible to the influence of the
passive and/or active forces as compared to the particulates, and
in various embodiments the base fluid entering the collection
chamber may remain in relatively close proximity to the microporous
body, and is subject to return to the microchannel by re-traversing
the microporous surface. This dynamic of particulates and base
fluid traversing and base fluid re-traversing the microporous
surface creates a flow regime within the collection chamber which
has a lower flow rate relative to the flow rate of the fluid being
processed through the microchannel. In some embodiments, the
microporous surface operationally generates a fluid flow regime
comprising a fluid with high flow rate flowing through the
microchannel and a fluid with low flow rate flowing through the
collection chamber.
[0056] As noted, the device for separating particles from a fluid
sample comprises a plurality of particulates having a relative
density difference when compared to the fluid base. The relative
density difference, at least in part, enables the particles to
sediment through the microporous surface. The separation of
particles using the device and the associated methods, at least in
part, is based on sedimentation of the particles through a
microporous body (surface).
[0057] Sedimentation is one of the simplest methods of cell or
particle separation, wherein the separation is based on the density
and size of the particle itself. However, sedimentation is
typically only thought of as a useful method of high speed cell
separation if coupled to a centrifuge and/or density gradient
medium (DGM). In the device, as noted, the force which causes the
particulates to traverse the microporous body may be an active
force such as an applied electric field, a passive force such as
the ambient gravitational field, or a combination thereof. For
example, in cases where the force of gravity augments the action of
an externally applied electric field in inducing particulate
movement or sedimentation. In some cases, sedimentation rates are
too low to be useful for applications, such as blood cell
separation from plasma, or separation of particulate impurities
from water. Thus, separation of particulates using the ambient
gravitational field or natural particle buoyancy at useful rates
has remained an objective.
[0058] In the device of FIGS. 1A to 1C, the sedimentation rate of
the particles in suspension is proportional to the centrifugal
force applied to the particles, where (equation 1):
v = d 2 ( .rho. p - .rho. l ) 18 .eta. .times. g c ##EQU00001##
v is the sedimentation rate, d is the diameter of the particle,
.rho..sub.p is the density of the particle, .rho..sub.1 is the
density of the DGM, .eta. is the viscosity of the DGM, and g.sub.c
is the centrifugal force. In the case where the centrifugal force
is simply equal to gravity, sedimentation of blood components is
slow with erythrocytes settling at .about.1 micron/s. Previously,
this has limited the utility of the earth's gravitational field (g)
for high speed, sedimentation-based cell separation, and
necessitated the equipment intensive centrifugation approach. As an
example, centrifugal forces of 10-800.times.g are typically applied
to blood to separate out specific cell types, which are stratified
across the DGM. With the application of the excess centrifugal
force, separation can take place on the order of minutes. A simpler
and automated separation is enabled by the claimed device that can
use only the earth's gravitational field to achieve similar
separation rates. It should be noted that other forces would follow
a similar relationship where centrifugal, or gravitational force
fields could be substituted without limitation for magnetic,
electric, or dielectric fields.
[0059] As noted, the sample loaded to the device may be processed
in the microchannel, as the particles or cells from a fluidic
sample or oil from a water sample may be separated and sediment
through the microporous surface and enter into the collection
chamber. The particles, cells or other materials present in the
fluidic sample may be of interest, which are collected or captured
in the collection chamber and described herein as "captured
particles" or "captured cells". The captured particles or captured
cells may be recovered from the collection chamber of the device,
which are described at times herein as "recovered particles" or
"recovered cells". In some embodiments, the recovery of particles
or cells is less than 100%, wherein the number of captured
particles or captured cells in the collection chamber is different
than the number of recovered particles or recovered cells.
[0060] The fluid that flows through the microchannel and emerges at
the fluid outlet after removal of at least a portion of the
particulates in the unprocessed fluid (at times herein referred to
as the "loaded sample") may be recovered from the device's fluid
outlet. The fluid recovered from the fluid outlet may be referred
to herein as "processed sample" and/or "processed fluid". In some
embodiments, the processed sample may be a sample of interest. For
example, the processed water sample recovered after removal of the
particles is purified water, which is a sample of interest. In some
other embodiments, the processed sample may be a waste product. For
example, in case of a purification of cells from a whole blood
sample, the plasma generated as "processed sample" is collected to
a waste chamber and the sample of interest may be the recovered
blood cells. In some other examples, the processed plasma may be a
sample of interest, depending on the user requirement. In one or
more embodiments, the processed sample is collected from the device
outlet, wherein the outlet is coupled to a pump to drive out the
processed sample.
[0061] As noted, the device is configured such that the microporous
surface operationally generates a fluid flow regime comprising a
high flow rate portion flowing through the microchannel and a low
flow rate portion flowing through the collection chamber. The
microporous surface effectively generates a resultant force derived
from a fluid drag force in the direction of fluid flow across the
length l of the microchannel and a force for particle sedimentation
across the height h of the microchannel. In some embodiments, the
resultant force favors sedimentation and captures the particles in
the collection chamber. The fluid flow rate above the microporous
surface may be high, which allows processing of larger sample
volume using the device compared to the sample volume typically
used for known devices.
[0062] The device may be configured, such that the time required
for the particles to sediment through the microporous surface is
less than the time required for the particles to transit across the
length l of the microchannel. The particle capture efficiency
and/or volumetric throughput may be improved by either increasing
the length l or decreasing the height h of the microchannel. An
increase in length l of the microchannel increases the time
available for the particles to interact with the microporous
surface. As noted, "capture efficiency" refers to a probability
function, dependent on the average number of particles interacting
with the pores during transit through the device and the
probability of particle passage through a pore for each such
interaction. The probability of particle passage through the pore
for each interaction may depend on the ratio of particle/pore
diameters.
[0063] In addition to particle collection, the device may be
configured to distinguish particles with respect to size,
sedimentation velocity, or density. The particles with different
size may be separated using the device. The particles having
different sedimentation velocity may also be separated using the
device. In some embodiments, the particle has an average diameter
between 1 and 250 .mu.m. As the microporous surface comprises pores
with an average diameter in a range of 1 to 500 .mu.m, the
particles that are smaller than the pore size pass through the
microporous surface. The particles, which are passed through the
pores of the microporous surface, are captured in the collection
chamber.
[0064] One embodiment of a device for separating one or more cells
of average diameter (d) from a blood sample, comprises a
microchannel of length l and height h comprising an inlet and an
outlet; a microporous surface with an average pore diameter (p) and
porosity (q), on one or more walls of the microchannel; a
collection chamber on the opposing side of the microporous surface;
and an applied gravitational field across the height h of the
microchannel to sediment the cells through the microporous surface
into the collection chamber. In this embodiment, as noted before,
the microporous surface operationally generates a fluid flow regime
comprising a high flow rate portion that flows through the
microchannel and a low flow rate portion that flows through the
collection chamber. In this embodiment, an average time required
for the cells to sediment through the microporous surface is less
than the average time required for the cells to transit across the
length l of the microchannel.
[0065] As illustrated in FIG. 1A, the device (front view) 10
comprises a fluid inlet 12, a fluid outlet 14 and a microchannel
16. In some embodiments, the term "microchannel" is used
interchangeably with the term "separation channel". The fluid inlet
12 may at times function as and be referred to as an inlet well
which connects to the microchannel. In the embodiment shown, fluid
inlet 12 is configured as an inlet well in fluid communication with
microchannel 16 via conduit 18. The microchannel 16 is bounded on
its lower side by microporous body 32. The device 10 also comprises
a collection chamber 22 on an opposing side of the microporous body
32.
[0066] As shown in FIG. 1B, in one exemplary embodiment, a side
view of the device 10 comprises a collection chamber 22. In some
embodiments, a vacuum or a syringe 24 is coupled to the collection
chamber of the device to pull a sample loaded to the device. In
another embodiment, the device 10 comprises a vacuum 20 coupled to
the device outlet 14 to pull the sample loaded to the device to a
waste-tub or waste-chamber and drive the sample fluid through the
device 10. In other embodiments, the fluid-flow across the device
is accomplished using a positive pressure applied to the device
inlet 12 for sample load. In some embodiments, a gravity-driven
flow provided through the device-inlet.
[0067] In some embodiments, a top view of the device 10 comprises a
microporous surface 32, as shown in FIG. 1C. The pore size of the
microporous surface is small enough to provide distinct flow
regimes above and below the microporous surface, whereas the pore
size is large enough to allow sedimentation of the particles
through the pores and capture within the collection chamber.
[0068] The device enables high speed separation without using a
centrifuge or additional equipment. In an exemplary embodiment of
the device 10, a high velocity flow-stream flows across a wide
sedimentation area of the microchannel 16 over the microporous
surface 32. For example, the microchannel comprises a sedimentation
area of 10 mm.times.400 mm over the microporous surface. In one
embodiment, the flow-stream within the microchannel 16 extends
across the microporous surface 32, which covers the collection
chamber 22. Unlike a filtration device, a pressure drop across the
microporous surface 32 is significantly minimized as the fluid
stream enters into the microchannel 16 through the opening 18 and
spreads over the wide area of the microporous surface 32. The
majority of the fluid-flow occurs over the microporous surface 32.
The device is configured such that the fluid sample is entered into
the microchannel 16, spread over the microporous surface 32. The
particles or cells of the fluid sample are sediment through the
pores of the microporous surface, and are trapped into the
collection chamber 22 underneath the microporous surface 32.
[0069] In an example of a method for separating one or more
particles from a fluidic sample, the method comprises loading the
sample to a device, wherein the device comprises a microchannel of
length l and height h, comprising an inlet and an outlet; a
microporous surface constitutes one or more walls of the
microchannel; a collection chamber on an opposing side of the
microporous surface, and an applied force field across the height h
of the microchannel to sediment the particles through the
microporous surface and capture into the collection chamber. The
method further comprises contacting the sample with the microporous
surface; generating a fluid flow regime comprising a high velocity
portion flowing through the microchannel and a low velocity portion
flowing within the collection chamber; sedimenting the particles
through the microporous surface into the collection chamber;
collecting the particles in the collection chamber under the
applied force field and retaining a fluid in the microchannel; and
driving out the retained fluid through the outlet of the
microchannel. An average time required for the particles to
sediment through the microporous surface is less than the average
time required for the particles to transit across the length l of
the microchannel. The fluid recovered after sedimentation of the
particles or cells from the sample comprises a reduced number of
particles or cells compared to the number of particles or cells
initially present during loading of the sample.
[0070] In one embodiment, the sample is a whole blood sample,
wherein the method is employed to separate one or more cell types
from the blood sample. In this embodiment, the method comprises
loading the blood sample to the device comprising a microchannel
with a microporous surface and a collection chamber, contacting the
blood sample to the microporous surface; generating a fluid flow
regime comprising a high velocity portion flowing through the
microchannel and a low velocity portion flowing through the
collection chamber; sedimenting the cells through the microporous
surface and capturing into the collection chamber under the applied
force field and retaining a plasma fluid in the microchannel. The
retained fluid, here plasma, is driven out through the outlet of
the microchannel, wherein a time required for the cells to sediment
through the microporous surface is less than the time required for
the cells to transit across the length l of the microchannel.
[0071] Examples of methods for separation of cells from a whole
blood sample are illustrated in FIG. 2. FIG. 2 illustrates
separation of cells and plasma from a 0.5 mL sample of whole blood.
The method resolves the challenge of using gravity to separate
cells in a high throughput manner. As shown in FIG. 2, the method
40 encompasses various steps of an exemplary embodiment. In step
one 42, 0.5 mL sample of whole blood is loaded to the device 10
through the inlet 12. In step two, 44, a vacuum is applied to the
outlet 14 of the device to drive the sample through the device. The
sample enters to the device from the inlet 12, passing through the
microchannel 16 and exits from the device through the outlet 14. In
step three, 46, the device runs until the collection chamber is
filled with the captured cells from the whole blood sample. Step
four, 48, includes the sedimentation of the cells to the collection
chamber and driving out the plasma from the device outlet 14 to a
waste chamber or to a second collection chamber. In step five, 50,
the cells that entered to the collection chamber 22 due to
sedimentation through the pores, are visible in the collection
chamber from the bottom. In step six, 52, the collection chamber 22
is opened through an outlet to drain the collected cells out form
the device 10.
[0072] The spreading of the fluid flow stream across the wide
separation surface provides a significant opportunity for
interaction of the particles with the surface. The addition of a
static collection chamber 22 allowed sedimentation of the particles
into the collection chamber, without traversing the particles
across entire length l of the microchannel, unlike standard
centrifuge tubes or large sedimentation tanks. The pressure drop
across the microporous surface is minimized enough such that
re-entry of the particles into the high speed flow-stream and loss
of particles into the waste chamber is minimized, which is
demonstrated by showing clear plasma in FIG. 2, step four, 48. The
efficiency of the separation and the relative performance of the
device compared to the filtration technology are significantly
high.
[0073] FIG. 3 illustrates one embodiment of the device 60 under
operating condition, wherein the unprocessed fluid (sample) 62
containing particles 66 enters into the device through the device
inlet 12 and the fluid flow 64 exits from the device through the
device outlet 14. The particles 66 are sediment through the porous
surface 32 of the microchannel and trapped into the collection
chamber 22. In this embodiment, the percentage of particles 66 is
significantly reduced in the processed fluid 64, as the particles
66 are sediment through the microporous surface and trapped within
the collection chamber 22. The length and height of the
microchannel is l and h, respectively.
[0074] FIG. 4 illustrates an additional embodiment of the device 70
under operating condition, wherein the unprocessed fluid (sample)
72 comprising smaller particles 76 and larger particles 78. The
sample 72 enters into the device through the inlet 12 and the
processed fluid 74 exits from the device through the outlet 14. The
larger particles 78 are larger than the diameter of the pores
within the microporous surface 32. The particles 78 cannot pass
through the pores to trap into the collection chamber 22, hence
particles 78 are retained within the microchannel in the processed
fluid 74. In this embodiment, the smaller particles 76 sediment
through the microporous surface 32, collect to the collection
chamber 22, and are separated from the larger particles 78. The
length and height of the microchannel is l and h, respectively. In
this device, there is a limited fluid flow across the microporous
surface 32 and particles enter into the collection chamber 22
through sedimentation, unlike a tangential flow filtration
process.
[0075] FIG. 5 shows another embodiment of the device 80 under
operating condition. In this embodiment, the unprocessed fluid
(loaded sample) 82 comprises a plurality of particles 86 and 88,
wherein the particles 86 and 88 have different sedimentation rates.
The particles 86 and 88 are captured in segmented portions of the
collection chamber 22A and 22B respectively. In this embodiment,
the similar or same size particles may be separately sediment in
two different segments of the collection chambers 22A and 22B based
on the particle's sedimentation rate. The particles 86 having
higher sedimentation rate sediment faster and collect to the
segment 22A of the collection chamber closer to the inlet. The
particles 88 having lower sedimentation rate sediment later and
collect into the segment 22B of the collection chamber closer to
the outlet. The processed sample 84 is driven out from the device
outlet. The length and height of the microchannel is l and h,
respectively.
[0076] Another embodiment of the device 90 is shown in FIG. 6,
wherein a fluid sample 92 is loaded to the device and a processed
fluid 94 is recovered from the device outlet. In this embodiment,
an additional force, such as a magnetic force field 98 is applied
to sediment the particles 96 through the microporous surface 32 and
are trapped into the collection chamber 22. The magnetic field is
applied across the microporous surface to allow only certain
particle types to enter into the collection chamber 22. For
example, the particles 96 pass through the micropores have a
magnetic property. A population of cells having magnetic property
may also be separated from a fluid base using this embodiment of
the device.
[0077] FIG. 7A is an image from a computational fluid dynamic (CFD)
model showing fluid flow pattern through a representative device
without a microporous surface. The representative device is a
channel with one inlet and an outlet, without any microporous
surface in it. The representative device without a microporous
surface has the same dimension of inlet, outlet, channel length,
channel height as of the present device. A significant portion of
the fluid flows through the device enters into the collection
chamber through the microchannel. FIG. 7B is computational fluid
dynamic (CFD) model showing fluid flow pattern through the device
with a microporous surface 32, wherein distinct flow regimes are
generated above the microporous surface 32 versus below the surface
32. The presence of microporous surface provides a fluid
resistance, which may limit the fluid to flow into the collection
chamber.
[0078] FIG. 8A is a graph showing loss of white blood cells (or
leukocytes) to the fluid that recovered from the device outlet with
increasing fluid flow rate using a representative device without a
microporous surface, as described above. A significant cell loss
occurs when the microporous surface is absent. FIG. 8B is a graph
showing minimum loss of leukocytes to the fluid that recovered from
the device outlet with increasing fluid flow rate using a device
with a microporous surface, wherein nearly 100% of the leukocytes
of the blood are captured in the collection chamber.
[0079] FIG. 8C is an image of a blood sample 110 loaded through a
device inlet and a fluid sample 112 recovered from the device
outlet using a representative device without a microporous surface,
as described above. The image (8C) shows the sample 112 is plasma
contaminated with red blood cells. FIG. 8D is an image of a blood
sample 110 loaded through a device inlet at a flow rate of 1000
.mu.l/min and a fluid sample 114 recovered from the device outlet
using a device with a microporous surface. The image of 8D clearly
shows the recovered fluid 114 is clear plasma, collected from the
device comprising a microporous surface.
[0080] FIG. 9 shows higher cell separation efficiency of the
microfluidic separation device (I) compared to the cell separation
efficiency using a commercial filtration device designed
specifically for capturing white blood cells (II). The cell
separation efficiency is measured in terms of capture of the white
blood cells in the collection chamber. In addition, a common
problem with currently available white blood cell filters is loss
of cells due to retention within the filter membrane. In contrast,
in embodiment of the present device, the loss of cells is addressed
by collection of cells within the liquid filled collection chamber
below the microporous surface. FIG. 9 shows the ability of the
device that competes with traditional filtration techniques for
capturing cells. FIG. 10 shows much higher (.about.80%) actual
white blood cells recovery from the present microfluidic device
comprising a microporous surface (I) compared to a commercially
available cell filter membrane (II) (.about.60%). The difficulty in
recovering captured cells from the surface of filtration membranes
is addressed.
[0081] FIGS. 11A and 11B are graphs showing the device is
operational with higher flow rates compared to a flow rate used by
commercially available microfluidic separation devices. The
recovery of white blood cells from the processed fluid was
consistent for the flow rate range of 50 .mu.l/min to 1 mL/min The
recovery of cells was not decreased even at 1 mL/min flow rate
(FIG. 11A). The percent of cells captured in the collection chamber
slightly decreases with increasing flow rate. In the present
embodiment of the device, the microporous surface is positioned
perpendicular to the fluid flow path. The cells sediment and
separate from the fluid flow stream after a certain length (l) of
the microchannel. With increase in flow rate, the length of the
microchannel that is necessary to achieve separation of the cells
is also increased, as shown in FIG. 11B. The separation distance is
estimated by visual observation of red cell front within the device
during operation.
[0082] As noted, the sample loaded to the device is a fluidic
sample. In some embodiments, the fluid may include a biological
sample, water sample, aqueous slurry, oil slurry, oil-water
emulsion or combinations thereof. As noted, the biological
materials used in the embodiments may comprise a physiological body
fluid, a pathological body fluid, a cell extract, a tissue sample,
a cell suspension, a forensic sample and combinations thereof. In
some embodiments, the biological material is a physiological body
fluid or a pathological body fluid, such as the fluid generated
from secretions, excretions, exudates, and transudates, or cell
suspensions such as, blood, lymph, synovial fluid, semen, saliva
containing buccal swab or sputum, skin scrapings or hair root
cells, cell extracts or cell suspensions of humans or animals. In
some embodiments, the physiological/pathological liquids or cell
suspensions may be extracted from plants. In one or more
embodiments, the extracts or suspensions of parasites, bacteria,
fungi, plasmids, or viruses, human or animal body tissues such as
bone, liver or kidney. In some embodiments, the sample fluid is a
biological sample selected from whole blood, cell extract, tissue
extract or combinations thereof. In one embodiment, the sample
fluid comprises whole blood.
[0083] In one or more embodiments, the particle comprises red blood
cells, white blood cells, platelets, biological cells, tissue
fragments, metals, minerals, polymers or combinations thereof.
[0084] In some embodiments, the device 10 (FIG. 1A, B, or C) may be
a portable or field-able device, so that the biological materials
can be collected at any location and loaded into the device for
cell separation. In some examples, the device may run using a pump.
In one embodiment, the device is packaged with a power source,
wherein the entire assembly may be self-contained. In such
embodiments, the device is portable, simple, and user friendly
compared to existing devices in the market.
[0085] The applications for the device 10 (FIG. 1A, B, or C)
include, but are not limited to, therapeutic application,
biochemical analysis, proteomics, healthcare related applications,
pharmaceutical or biotech research applications, environmental
monitoring, in vitro diagnostic and point-of-care applications, or
medical devices.
[0086] In one or more embodiments, the device 10 (FIG. 1A, B, or C)
is fully automated or partially automated. The automation of the
device is required to reduce human intervention during collection
of cells. The use of an automated device further helps in
minimizing contamination during purification of biological samples,
aqueous samples, and oil samples. Fully automatic devices are
desirable for various applications, wherein the objective is to
purify blood cells, blood serum or water or oil from a sample. An
externally located controller may be operationally coupled to the
device to drive the device, excluding any manual intervention after
application of the biological sample, water or oil to the
device-inlet.
[0087] In some embodiments, the device is configured to integrate
with another device or system, more specifically with an analytical
device. As noted, the device may have one or more coupling means
through which the device may integrate with another device
depending on the requirement. The coupling means may include but is
not limited to, an adapter, or a connector. In some embodiments,
the device itself is configured to have one or more holders,
connecting ports or combination thereof, which mechanically couples
the device to another device. The device may be electronically or
mechanically coupled to another device for downstream
applications.
[0088] In one or more embodiments, the device further comprises one
or more containers for collecting waste or fluid after separation
of the particles or cells. In embodiments, where blood is a sample,
the plasma generated after separation of the cells may be collected
to a waste chamber. In some other embodiments, when the sample is
water or oil, after separation of the particles, the purified fluid
is collected to a collection chamber coupled to the outlet. This
collection chamber is different than the collection chamber for
particles present opposing side of the microporous surface. In one
or more embodiments, the non-limiting examples of containers are
bag, chamber and vessels. The containers may be disposable or
reusable. Various components of the device may be operationally
connected to each other using conduits, holder, adapter, or valves.
The device may further comprise one or more sensors, such as
temperature sensor, pressure sensor, flow sensor or pH sensor,
depending on the requirement.
Experimental Part
Device Fabrication
[0089] A microfluidic separation device housing was created using a
commercially available rapid prototyping instrument and an ABS-like
photopolymer (DSM Somos WaterShed XC 11122). The microfluidic
separation device was assembled from three parts, created on the
rapid prototyping instrument together with a porous KAPTON film
which served as the microporous body or microporous surface, and a
set of pressure sensitive adhesive films which joined the parts
together and served to create the microchannel. Useful reference
may be made to FIGS. 12-14 to better understand the fabrication of
the microfluidic separation device.
[0090] The first part 101 (FIG. 14) comprised a fluid inlet 12 and
fluid outlet 14 with slots 18 linking each to microchannel 16 (FIG.
12). The second part 102 (FIG. 14) defined the collection chamber
22 (FIG. 12). A third part 103 (FIG. 13) formed a wall of the
collection chamber. A 50 micron (.mu.m) thick pressure sensitive
adhesive 104 (FIG. 14) was used to define the microchannel having
dimensions 50 millimeters by 10 millimeters by 50 microns and
comprised features cut out using a cutter/plotter (Graphtec Craft
Robo ProS). The adhesive film 104 also served to fix the
microporous body 105 (FIGS. 12-14) (the porous KAPTON film) to the
first part 101. Additional cut adhesive films 106 and 107 were used
to fix the second part 102 to the microporous body 105 and the
third part 103 respectively. In the embodiment shown microporous
body 105 comprises pores 134.
[0091] Two different types of microporous bodies were used in the
devices. As mentioned, the first type of microporous body was
formed from a KAPTON sheet with laser-machined pore arrays having
average pore diameter of about 21.7 microns with a 50 micron
center-to-center pore spacing. The second type of microporous body
employed was a medical grade polyamide woven mesh having 40 micron
pores and 40% porosity (SEFAR MEDIFAB, 07-40/40).
[0092] The collection chamber 22 defined by second part 102 had
dimensions of 40 millimeters by 10 millimeters by 2 millimeters,
resulting in a 750 microliter (.mu.L) holding volume. As
configured, the microfluidic separation device could process a
total volume of about 0.5 milliliters of blood before the
collection chamber reached its maximum cell holding capacity.
Device Operation
[0093] The microfluidic separation device was equipped with two
ports 123 and 124 (FIGS. 13-14) which enabled the device to be
primed easily before use. Typically, the device was primed by
introducing deionized water through one of the two ports 123 and
124 and completely filing both the collection chamber 22 and the
microchannel 16 before use. Alternatively, the device could be
primed by flowing deionized water from the fluid inlet and into the
microchannel and collection chamber. Typically, the priming liquid
could be introduced into the microfluidic separation device without
introducing air bubbles.
[0094] Once primed, a sample fluid comprising particulates
dispersed with a base fluid (such as whole blood comprising blood
cells are the particulates) dispersed within blood plasma (the base
fluid) was introduced into fluid inlet 12 and was made to flow
through the microchannel 16 contact the upper surface of the
microporous body by the application of a vacuum on the fluid outlet
14 side of the device. Owing to the gravitational forces present,
particulates within the sample fluid flowing within the
microchannel tended to sediment downwardly through the pores of the
microporous body and into the underlying water-filled collection
chamber, wherein the downward motion of the particulates continued.
Operation of the microfluidic separation device typically effected
at least a substantial separation of particulates and base fluid.
The processed fluid, a mixture of the base fluid and water
exchanged with the collection chamber was collected in the fluid
outlet.
EXAMPLES
[0095] Cell Separation-Whole blood or cell-suspensions were used to
test collection efficiencies of mammalian cells using the
microfluidic separation device. Typical white blood cell dimensions
are 10 to 12 microns, and thus the microporous body having 21.7
micron pore diameters provided an about a 2 to 1 ratio of pore
diameter to cell dimension. A syringe pump was attached to the
fluid outlet used to create a flow of the cell-containing sample in
the fluid inlet through the microchannel and into the fluid outlet
at defined rates (PicoPlus syringe pump, Harvard Apparatus) from 50
to 1000 microliters per minute (.mu.L/min). Phosphate buffered
saline (PBS), deionized water or cell culture medium were used as
priming fluids. Separated base fluid was collected by pipetting
from the fluid outlet. Separated cells were recovered from the
collection chamber using a syringe. In all cell separation examples
disclosed herein, the external force field which caused the cells
to traverse the microporous body from the microchannel to the
collection chamber was gravity and the microfluidic separation
device was oriented such that the passage of cells from the
microchannel to the collection chamber was in a downward
direction.
[0096] Cell collection efficiency was assessed on a SysMex XE2100
Hematology Analyzer and provided red and white blood cell counts
from whole blood samples. White blood cell viability and collection
efficiency were assessed on a NucleoCounter.RTM. *(chemometec)
Live/Dead Analyzer. Collection efficiencies were recorded as the
ratio of the number of cells introduced through the device fluid
inlet 12 (FIG. 12) to the number of cells collected in the
collection chamber 22 (FIG. 12). The number of cells actually
recovered from the collection chamber was also recorded as there
was some additional loss of cells during the transfer of the
contents of the collection chamber. Total cell loss was
significantly less than losses occurring in standard filtration
protocols used for white blood cell capture. Viability of the cells
was not affected by passage through the microfluidic separation
device over the range of flow rates tested. Cell collection
efficiency was assessed at sample flow rates of from 50 microliters
per minute to 1000 microliters per minute (.mu.L/min) In addition,
a total surface area of the microporous body necessary to enable
efficient cell separation was estimated for each flow rate.
Example 1
Analysis of Cell-Separation Using Computational Fluid Dynamics
(CFD)
[0097] Computational Fluid Dynamics: A finite element method
analysis solution to the full Navier-Stokes equation was also
performed using Comsol.RTM. multiphysics. Velocity fields were
extracted for scaled devices (large enough to model ten pores
across the microporous surface) with and without the microporous
surface. This allowed visualization of the effect of the
microporous surface on flow conditions within the collection
chamber. The output boundary condition was set to pressure at 0 Pa,
inflow velocities were set to match the 50 uL/min experimental
conditions. Collection efficiencies over a range of particle/fluid
density ratios were estimated using the particle tracing function
in Comsol.RTM., and counting the number of particles that entered
the pore array. Additional simulations were then run to investigate
the effect of changing the particle/pore size ratio for densities
that match those reported for white blood cells and plasma in the
literature. FIGS. 7A and 7B show CFD model analysis of blood sample
passing through a representative device without a microporous
surface and a device with a microporous surface, respectively.
[0098] Whole blood (0.5 mL) was introduced into to the fluid inlet
of a primed microfluidic separation device and caused to flow
through the device at a flow rate of 1000 .mu.l/min A microfluidic
separation device without a microporous body that separate the
collection chamber from a microchannel was used as a control. This
device had an idealized configuration and fluid dynamics is shown
in FIG. 7A in which the microchannel 16 is absent in the separation
zone 32. FIG. 7B provides a useful point of reference and shows
fluid dynamics using an idealized microfluidic separation device
with the microporous body in place and the microchannel extending
fully across the separation zone 33, which includes the portion of
the microchannel in contact with the microporous body. Fluid
flowing into the device shown in FIG. 7A enters the collection
chamber without being constrained by the presence of a microporous
body. As a result a complex flow regime is created in the
collection chamber. In the absence of the microporous body, cells
are both captured within and pass through the collection chamber.
In the experiments carried out in the absence of the microporous
body the efficiency of cell separation was strongly dependent of
sample flow rate through the device.
Example 2
Recovery of White Blood Cells from Whole Blood Using Both of the
Device and a Representative Device Without a Microporous Body
[0099] Whole blood (0.5 mL) was introduced into to the fluid inlet
of a primed microfluidic separation device and caused to flow
through the device at a flow rate of 50, 250, 500 or 1000 .mu.l/min
The microfluidic separation device identical to that used in
present Example with the exception that no microporous body to
separate the collection chamber from a microchannel was used as a
control. Data are gathered in FIG. 8A for cell separation using a
device without a microporous body, which shows a steady decline in
cell separation efficiency as the loss of cells increases to the
waste with increasing flow rate. In contrast, FIG. 8B shows high
cell separation efficiency and minimum loss of cells to the waste
with increasing flow rate while using a device with a microporous
body. The loss of white blood cells (or leukocytes) to the fluid
that recovered from the device outlet was increased with increasing
fluid flow rate (FIG. 8A). While only a minimal loss of leukocytes
to the fluid that recovered from the device outlet was observed,
even with increasing fluid flow rates (FIG. 8B). A significant
number of leukocytes of the blood was captured and recovered in the
collection chamber of the device.
Example 3
Recovery of Red Blood Cells From Whole Blood Using the Microfluidic
Device and a Representative Device Without a Microporous Body
[0100] Red blood cell separation was carried out with a flow rate
of 1000 .mu.l/min on a sample consisting of 0.5 mL of whole blood
using a primed microfluidic separation device disclosed herein.
Separation of red blood cells from the processed fluid collected in
the fluid outlet was essentially quantitative. The relative
performance of the device was compared with a representative device
without a microporous body. Whole blood (0.5 mL) was introduced
into to the fluid inlet of a primed microfluidic separation device.
The microfluidic separation device identical to that used in
present Example with the exception that no microporous body to
separate the collection chamber from a microchannel was used as a
control. The plasma recovered from the outlet of the device is
transparent and clear fluid without contamination of red blood
cells, as shown in FIG. 8D, compared to the plasma collected from
the outlet of the representative device, which is turbid and
contaminated with red blood cells, as shown in FIG. 8C.
[0101] The role of the microporous body is evident in FIG. 8C
(microporous body absent) and FIG. 8D (microporous body present)
wherein in each case element 110 is the starting whole blood sample
and elements 112 and 114 are the processed fluid collected in the
fluid outlet in the presence and absence of the microporous body
respectively. FIG. 8C shows clearly the presence of red blood cells
which escaped capture in the separation zone in the processed fluid
collected in the fluid outlet. FIG. 8D show the processed fluid 114
as essentially free of red blood cells.
Example 4
Separation of Blood Cells From a Whole Blood
[0102] A whole blood sample (0.5 mL) was introduced into the primed
microfluidic separation device configured as in FIG. 12 and was
made to flow through the microchannel at a flow rate of 250
.mu.l/min The processed fluid was analyzed and shown to be free of
white blood cells (FIG. 9, left column). The results obtained were
compared to the performance of a commercial filter designed for
capturing white blood cells. FIG. 9 illustrates the effectiveness
of the present invention in overcoming a common problem associated
with filtration techniques wherein cell separation efficiency is
limited by a tendency of the filter to bind cells.
[0103] FIG. 10 illustrates that the actual recovery of blood cells
from the collection chamber and microporous body of the
microfluidic separation device provided by the present invention is
enhanced relative to the commercial filter. The recovery of cells
from the blood sample was about 80% using the microfluidic
separation device of the present invention, whereas cell recovery
using the benchmark filter was only about 60%.
[0104] Additional experiments were carried out at higher and lower
flow rates using the microfluidic separation device configured as
in FIG. 12. Results are gathered in FIG. 11A, which show that the
processed fluid collected from the fluid outlet is substantially
free of blood cells and that a significant percentage of the blood
cells are recoverable from the device following processing. Thus,
even at a flow rate of 1000 .mu.l/min (FIG. 11A) the efficiency at
which white blood cells were removed from the blood plasma was not
decreased relative to the results obtained at a 50 .mu.l/min flow
rate. A commercial flow filter was unable to fully separate out
cells from 0.5 mL of blood, as the filter stalled at 5 PSI running
pressure due to occlusion of the filter pores with captured cells
(data not shown). The commercial benchmark also required a stack of
5 filters having a 15 mm diameter each (883 5 mm.sup.2 area) in
order to achieve separation efficiencies comparable to those
observed for the microfluidic separation device provided by the
present invention.
[0105] While only certain features of the invention have been
illustrated and described herein, many modifications and changes
will occur to those skilled in the art. It is, therefore, to be
understood that the appended embodiments are intended to cover all
such modifications and changes as fall within the scope of the
invention.
* * * * *