DEVICES FOR SEPARATION OF PARTICULATES,
ASSOCIATED METHODS AND SYSTEMS
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 December 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 prefiltration
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 1 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 1and 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 1and
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 1and 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. IB 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 11 A 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. 11 B 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. FIG.s 1A-1C,
FIG.s 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 1and height h disposed between a fiuid 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 fiuid 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, IB 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 fiuid 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 1and 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 1between 5 mm and 25 cm. In another embodiment, the
microchannel has a length 1between 10 mm and 10 cm. In yet another embodiment,
the microchannel has a length 1between 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 1 and about 1000 microns (m i) . 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 chemicallyetched
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 1of 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 FIG. 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 is the sedimentation rate, d is the diameter of the particle, pp is the density of the
particle, pi is the density of the DGM, h is the viscosity of the DGM, and gc 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 ~ 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 x 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 fiuidic 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 fiuidic
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 1of 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 1 of the microchannel. The particle capture
efficiency and/or volumetric throughput may be improved by either increasing the
length 1or decreasing the height h of the microchannel. An increase in length 1of 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 mih. As the
microporous surface comprises pores with an average diameter in a range of 1 to 500
mih, 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 1and 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 1 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. 1 B, 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 wastechamber
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. 1 C. 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 x 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 1and 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 1of 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 1of 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 0.5mL
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 1of 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 1and
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 1 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 22 A and 22 B respectively. In this embodiment, the similar
or same size particles may be separately sediment in two different segments of the
collection chambers 22 A and 22 B based on the particle's sedimentation rate. The
particles 86 having higher sedimentation rate sediment faster and collect to the
segment 22 A of the collection chamber closer to the inlet. The particles 88 having
lower sedimentation rate sediment later and collect into the segment 22 B 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 1and 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. 7 A 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. 7 B 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 mΐ/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 (-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) (-60%). The
difficulty in recovering captured cells from the surface of filtration membranes is
addressed.
[0081] FIGs. 11 A and 11 B 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 mΐ/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 (1) 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. 11 B. 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 (FIGs. 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 (FIGs. 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 (FIGs. 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 micro fluidic separation device housing was created using a commercially
available rapid prototyping instrument and an ABS-like photopolymer (DSM Somos
Watershed XC 11122). The micro fluidic 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 FIG.s 12-14 to better understand
the fabrication of the micro fluidic 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 (m h) 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 (FIG.s 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 (m )
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 (FIG.s 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 (m /h h) . 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® *(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 (m /h h) . 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® 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®, 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 mΐ/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. A 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 m /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 micro fluidic
device and a representative device without a microporous body
[0100] Red blood cell separation was carried out with a flow rate of 1000 mΐ/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
microfiuidic separation device configured as in FIG. 12 and was made to flow through
the microchannel at a flow rate of 250 mΐ/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 microfiuidic 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 microfiuidic 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 mΐ/min (FIG. 11 A) the efficiency at which white blood cells were removed
from the blood plasma was not decreased relative to the results obtained at a 50
mΐ/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 mm2 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.
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 1and 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 1 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 (m h) .
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 1and 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 fiuid, comprising:
providing a separation device comprising:
a microchannel of length 1and height h disposed between a fluid inlet
and a fiuid 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 fiuid 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 fiuid comprising particulates
dispersed within a base fiuid into the microchannel via the fiuid inlet;
separating at least a portion of the particulates from the unprocessed fluid
to provide a stream of processed fluid at the fiuid 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.
A method for separating cells dispersed within a base fluid of whole blood
e, comprising:
providing a separation device comprising:
a microchannel of length 1and 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.