U.S. patent application number 17/323868 was filed with the patent office on 2021-09-09 for separation using angled acoustic waves.
The applicant listed for this patent is FloDesign Sonics, Inc.. Invention is credited to Kedar C. Chitale, Jason Dionne, Bart Lipkens, Walter M. Presz, JR., Benjamin Ross-Johnsrud.
Application Number | 20210277381 17/323868 |
Document ID | / |
Family ID | 1000005594450 |
Filed Date | 2021-09-09 |
United States Patent
Application |
20210277381 |
Kind Code |
A1 |
Lipkens; Bart ; et
al. |
September 9, 2021 |
SEPARATION USING ANGLED ACOUSTIC WAVES
Abstract
Methods and systems for processing material in a host fluid use
an acoustophoresis device. These methods and systems can deflect
material (e.g., a second fluid, cells, beads or other particles,
exosomes, viruses, oil droplets) in host fluid streams at high flow
rates.
Inventors: |
Lipkens; Bart; (Bloomfield,
CT) ; Dionne; Jason; (Simsbury, CT) ; Presz,
JR.; Walter M.; (Wilbraham, MA) ; Chitale; Kedar
C.; (Little Rock, AR) ; Ross-Johnsrud; Benjamin;
(Northampton, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
FloDesign Sonics, Inc. |
Wilbraham |
MA |
US |
|
|
Family ID: |
1000005594450 |
Appl. No.: |
17/323868 |
Filed: |
May 18, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15942316 |
Mar 30, 2018 |
11021699 |
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17323868 |
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15613790 |
Jun 5, 2017 |
10550382 |
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15942316 |
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15143481 |
Apr 29, 2016 |
9670477 |
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15613790 |
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62316933 |
Apr 1, 2016 |
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62154690 |
Apr 29, 2015 |
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62485229 |
Apr 13, 2017 |
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62479309 |
Mar 30, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61M 1/3678 20140204;
B01L 2400/0436 20130101; B01L 2300/0864 20130101; B01L 3/502761
20130101; C12M 47/04 20130101; G01N 2015/0288 20130101; G01N
2015/1006 20130101; G01N 15/1484 20130101; C12M 47/02 20130101;
G01N 2015/149 20130101; B01L 2300/0867 20130101; B01D 21/283
20130101; G01N 15/0255 20130101; G01N 2015/0294 20130101; C12N 1/02
20130101; G01N 2015/0053 20130101; B01L 2400/0439 20130101; C12N
13/00 20130101 |
International
Class: |
C12N 13/00 20060101
C12N013/00; B01L 3/00 20060101 B01L003/00; G01N 15/14 20060101
G01N015/14; B01D 21/28 20060101 B01D021/28; C12M 1/00 20060101
C12M001/00; C12N 1/02 20060101 C12N001/02 |
Claims
1. A system for processing fluid, comprising: a fluid chamber for
containing the fluid; an ultrasonic transducer coupled to the fluid
chamber for generating an acoustic wave in the fluid chamber at an
acute angle to a direction of mean flow through the fluid chamber;
at least one inlet in fluid communication with the fluid chamber;
at least one outlet in fluid communication with the fluid chamber;
and at least one pump engaged with one or more of the at least one
inlet or the at least one outlet and configured to control one or
more of the fluid entering the fluid chamber via the at least one
inlet or the fluid exiting the fluid chamber via the at least one
outlet, such that the at least one pump can influence a fluid
stream that passes through the acoustic wave in the fluid
chamber.
2. The system of claim 1, wherein the at least one pump further
comprises a first pump and a second pump.
3. The system of claim 2, wherein the at least one inlet further
comprises a first inlet and a second inlet, the first pump being
engaged with the first inlet and the second pump being engaged with
the second inlet, whereby a flow rate in the first inlet and a flow
rate in the second inlet can be controlled independently with the
first pump and with the second pump, respectively.
4. The system of claim 3, wherein the first pump and the second
pump are configured to control the flow rate in one or more of the
first inlet or the second inlet to contribute to separation of
material entrained in the fluid stream in the fluid chamber.
5. The system of claim 2, wherein the at least one outlet further
comprises a first outlet and a second outlet, the first pump being
engaged with the first outlet and the second pump being engaged
with the second outlet, whereby a flow rate in the first outlet and
a flow rate in the second outlet can be controlled independently
with the first pump and with the second pump, respectively.
6. The system of claim 5, wherein the first pump and the second
pump are configured to control the flow rate in one or more of the
first outlet or the second outlet to contribute to separation of
material entrained in a fluid stream in the fluid chamber.
7. The system of claim 1, further comprising the at least one pump
being configured to control flow velocity of the fluid in one or
more of the at least one inlet or the at least one outlet.
8. The system of claim 7, further comprising a first control for
controlling the ultrasonic transducer and a second control for
controlling the at least one pump.
9. The system of claim 8, wherein the first control and the second
control are configured to influence a ratio of acoustic radiation
force produced by the acoustic wave to viscous drag force, such
that material entrained in a fluid stream in the fluid chamber is
deflected at a predetermined angle.
10. The system of claim 1, wherein the fluid chamber comprises an
internal dimension that is at least 10 times the wavelength of the
acoustic wave.
11. A method for processing fluid, comprising: controlling, with at
least one pump, one or more of a fluid provided to at least one
inlet to a fluid chamber or a fluid exiting at least one outlet of
the fluid chamber; generating an acoustic wave in the fluid chamber
with an ultrasonic transducer, such that the acoustic wave
propagates in a direction that is at an acute angle to a direction
of mean fluid flow through the fluid chamber; and controlling the
at least one pump to influence a fluid stream that passes through
the acoustic wave in the fluid chamber.
12. The method of claim 11, wherein the at least one pump further
comprises a first pump and a second pump.
13. The method of claim 12, wherein the at least one inlet further
comprises a first inlet and a second inlet, the first pump being
engaged with the first inlet and the second pump being engaged with
the second inlet; and controlling a flow rate in the first inlet
and a flow rate in the second inlet independently with the first
pump and with the second pump, respectively.
14. The method of claim 13, further comprising controlling the
first pump and the second pump to control the flow rate in one or
more of the first inlet or the second inlet to contribute to
separation of material entrained in a fluid stream in the fluid
chamber.
15. The method of claim 12, wherein the at least one outlet further
comprises a first outlet and a second outlet, the first pump being
engaged with the first outlet and the second pump being engaged
with the second outlet; and controlling a flow rate in the first
outlet and a flow rate in the second outlet independently with the
first pump and with the second pump, respectively.
16. The method of claim 15, further comprising controlling the
first pump and the second pump to control the flow rate in one or
more of the first outlet or the second outlet to contribute to
separation of material entrained in a fluid stream in the fluid
chamber.
17. The method of claim 11, further comprising controlling the at
least one pump to control flow velocity of the fluid in one or more
of the at least one inlet or the at least one outlet.
18. The method of claim 17, further comprising controlling the
ultrasonic transducer and the at least one pump to influence a
ratio of acoustic radiation force produced by the acoustic wave to
viscous drag force, such that material entrained in a fluid stream
in the fluid chamber is deflected at a predetermined angle.
19. The method of claim 11, wherein the fluid chamber comprises an
internal dimension that is at least 10 times the wavelength of the
acoustic wave.
20. A system for processing fluid, comprising: a fluid chamber for
containing the fluid; an ultrasonic transducer coupled to the fluid
chamber for generating an acoustic wave in the fluid chamber at an
acute angle to a direction of mean flow through the fluid chamber;
at least two inlets in fluid communication with the fluid chamber;
at least two outlets in fluid communication with the fluid chamber;
and a first pump engaged with one or more of the at least two
inlets to control fluid entering the fluid chamber via the one or
more inlets to which the first pump is engaged; and a second pump
engaged with one or more of the at least two outlets to control
fluid exiting the fluid chamber via the one or more outlets to
which the second pump is engaged; wherein the first pump and the
second pump are configured to influence a fluid stream that passes
through the acoustic wave in the fluid chamber.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of Ser. No.
15/942,316, filed Mar. 30, 2018, which is a continuation-in-part
application of Ser. No. 15/613,790, filed Jun. 5, 2017, now U.S.
Pat. No. 10,550,382, which is a continuation application of Ser.
No. 15/143,481, filed Apr. 29, 2016, now U.S. Pat. No. 9,670,477,
which claims priority to U.S. Provisional Patent Application Ser.
No. 62/316,933, filed on Apr. 1, 2016; and to U.S. Provisional
Patent Application Ser. No. 62/154,690, filed on Apr. 29, 2015.
Application Ser. No. 15/942,316 also claims priority to U.S.
Provisional Patent Application Ser. No. 62/479,309, filed on Mar.
30, 2017; and to U.S. Provisional Patent Application Ser. No.
62/485,229, filed on Apr. 13, 2017. The entire disclosures of all
of these applications are hereby incorporated herein by
reference.
BACKGROUND
[0002] In the medical field, it often is desirable to separate low
concentration cells from a fluid mixture with no harm to the cells,
wash cells, concentrate cells in a fluid mixture, differentiate
cells based on key parameters, or even fractionate many different
types of cells. Such processes are key in the development of
possible cures to many common diseases. It may also be desirable to
separate particles or cells different in size, density and or
acoustic contrast factor using an acoustic field where the
particles may be separated from each other as well. Examples
include the separation of live from dead cells and the separation
of differentiated from undifferentiated cells. The methods
described herein provide for such a separation or fractionation
method that is label-free.
[0003] In the food and beverage industry, filter cartridges and
filter membranes have conventionally been used to filter particles
from liquids. Such filters are expensive and become clogged and
non-functional as material is processed. In contrast,
acoustophoresis provides, among other possible advantages, a
solid-state, low-cost alternative to filter cartridges and filter
membranes that is capable of processing large quantities of a host
medium, for example water or beer, that is laden with yeast or
other suspended particles.
[0004] In the food and beverage industry, host fluid is flowed
through filters at flow rates up to ten times greater than those
through conventional acoustophoresis devices. At these higher flow
rates, trapping of the particles in the host fluid is decreased,
thereby leading to decreased separation efficiency. It would
therefore be desirable to provide systems and methods capable of
separating a second fluid or a particulate from a host fluid at
much higher flowrates, or at much lower concentrations, than
conventional macro-scale acoustic separators.
[0005] In the oil and water industry, efficiently and economically
separating oil and other contaminants from water has become an
important process. The rise of fracking techniques has led to many
settling ponds and large costs for transportation of contaminated
water. These settling ponds are a challenge to the environment and
better means are needed to clarify fracking water more effectively.
Acoustophoresis provides, among other possible advantages, a
solid-state, effective means of clarifying fracking, but the flow
rates associated with such macro-scale acoustophoresis devices is
still too low to be feasible. It would therefore be desirable to
provide systems and methods capable of separating a second fluid,
cell, or particulate from a host fluid at much higher
flowrates.
SUMMARY
[0006] This disclosure describes various embodiments of mini to
macro-scale systems, devices, and methods for acoustophoresis to
separate, fractionate, isolate, concentrate, wash, detect, or even
differentiate cells or particles in fluid suspension. The devices
and methods include a flow chamber and an ultrasonic transducer and
reflector that set up an angled acoustic standing wave oriented at
an acute angle relative to the direction of mean flow through the
flow chamber, which includes the particle path through the angled
acoustic standing wave. At higher flow rates, acoustic standing
waves may be used to deflect the particles in a desired direction,
without causing the particles to become trapped in the standing
wave. By applying the acoustic standing wave to the host fluid at
an angle thereto, desired deflection of the particles can be
achieved.
[0007] These systems and methods can separate, sort, and
differentiate various particles using bulk ultrasonic standing
waves oriented at an angle .gamma. relative to the fluid velocity.
This approach offers a sensitive separation capability with respect
to size and acoustic contrast of particles.
[0008] In one aspect, systems for separating material from a host
fluid include: a flow chamber defining a direction of mean flow; an
ultrasonic transducer including a piezoelectric material configured
to be excited to generate an angled bulk acoustic standing wave
with a wavelength and an acoustic radiation force in the flow
chamber oriented at an acute angle relative to the direction of
mean flow through the flow chamber, wherein the flow chamber has a
minimum internal dimension that is at least 10 times the wavelength
of the angled acoustic standing wave; a reflector opposite the at
least one ultrasonic transducer; a first inlet fluidly connected to
the flow chamber; a second inlet fluidly connected to the flow
chamber; a first outlet fluidly connected to the flow chamber; and
a second outlet fluidly connected to the flow chamber. Embodiments
of these systems can include one or more of the following
features.
[0009] In some embodiments, the first inlet is at least 0.1 inches
(e.g., 0.2, 0.3, 0.4, 0.5, or 1 inch) from the angled bulk acoustic
standing wave.
[0010] In some embodiments, systems also include a first channel
ending at the first inlet, wherein the first channel has a
substantially straight section extending at least 0.1 inches (e.g.,
0.25, 0.5, 0.75, or 1 inch) from the first inlet.
[0011] In some embodiments, a space between the ultrasonic
transducer and the reflector comprises a first portion within the
flow chamber and a second portion outside the flow chamber. In some
cases, systems also include an acoustically transparent material
separating the first portion from the second portion. In some
cases, systems also include a cooling water system in fluid
connection with the second portion. In some cases, the second
portion is filled with solid material having an acoustic impedance
equal to an acoustic impedance of the host fluid.
[0012] In some embodiments, the system comprises a plurality of
ultrasonic transducers.
[0013] In some embodiments, the first inlet and the second inlet
are coaxial. In some cases, the first outlet and the second outlet
are coaxial. In some cases, the first inlet has a rectangular
cross-section. In some cases, an area of the rectangular
cross-section of the first inlet is at least 0.05 square inches
(e.g., 0.1, 0.25, 0.5, 0.75, or 1 inch).
[0014] In some embodiments, the first inlet has an aspect ratio of
at least 5 (e.g., 10, 15, 20, 25, or 50).
[0015] In some embodiments, systems also include a third outlet,
wherein second outlet is disposed between the first outlet and the
third outlet and a cross-sectional area of the third outlet is
smaller than a cross-sectional area of the second outlet. In some
cases, the second outlet has a rectangular cross-section and third
outlet has a rectangular cross-section. In some cases, a width of
the second outlet is the same as a width of the third outlet. In
some cases, a height of the second outlet is at least 2 times a
height of the third outlet.
[0016] In some embodiments, systems also include a plurality of
third outlets, each of the plurality of third outlets offset from
an axis of the second outlet in a direction of deflection of the
angled acoustic wave.
[0017] In some embodiments, systems also include a first channel
ending at the first inlet, wherein the first channel has a
substantially straight section extending at least 0.1 inches (e.g.,
0.25, 0.5, 0.75, or 1 inch) from the first inlet at a first acute
angle relative to a plane perpendicular to the angled acoustic
standing wave. In some cases, a second channel ending at the second
inlet, wherein the second channel has a substantially straight
section extending at least 0.1 inches (e.g., 0.25, 0.5, 0.75, or 1
inch) from the second inlet at a second acute angle relative to the
plane perpendicular to the angled acoustic standing wave. In some
cases, the first acute and the second acute angle are equal. In
some cases, system also include a third channel ending at the first
outlet, wherein the third channel has a substantially straight
section extending from the first outlet at a third acute angle
relative to the plane perpendicular to the angled acoustic standing
wave. In some cases, the first acute and the third acute angle are
equal. In some cases, systems also include a fourth channel ending
at the second outlet, wherein the first outlet located in a
direction of deflection of the angled acoustic wave relative to the
second outlet, wherein the fourth channel has a first
cross-sectional area, the third channel has a first section with
the first cross-sectional area and a second section with a second
cross-sectional area that is smaller than the first cross-sectional
area, and the second section of the third channel is located
between the first outlet and the first section of the third
channel. In some cases, the third channel has a substantially
straight section extending from the first outlet at a third acute
angle. In some cases, the first acute angle is between 80 degrees
and 90 degrees.
[0018] In some embodiments, a wall of the flow chamber adjacent to
the first outlet in a direction of deflection of the angled
acoustic wave extends at an acute angle relative to a plane
perpendicular to the angled acoustic standing wave. In some cases,
the acute angle is between 1 and 20 degrees (e.g., more than 2
degrees, more than 3 degrees, more than 5 degrees, more than 10
degrees, less than 15 degrees, less than 10 degrees, less than 7.5
degrees, less than 5 degrees).
[0019] In one aspect, systems for separating material from a host
fluid include: a flow chamber extending between a first end and a
second end; an inlet located at the first end of the flow chamber;
a first outlet located at between the first end of the flow chamber
and the second end of the flow chamber, the inlet and the first
outlet defining a direction of mean flow through the flow chamber;
an ultrasonic transducer including a piezoelectric material
configured to be excited to generate an angled acoustic standing
wave between the inlet and the first outlet, the angled acoustic
standing wave with a wavelength and an acoustic radiation force in
the flow chamber oriented at an acute angle relative to the
direction of mean flow through the flow chamber; and a reflector
opposite the at least one ultrasonic transducer; wherein the first
outlet is spaced apart from the second end of the flow chamber.
[0020] In some embodiments, the flow chamber has a minimum internal
dimension that is at least 10 times the wavelength of the angled
acoustic standing wave.
[0021] In some embodiments, the first outlet is located at least
0.5 inches from the second end of the flow chamber.
[0022] In some embodiments, the flow chamber has a distance between
the first end and the second end and the first outlet is located at
away from the second end by at least 30% of the distance. In some
cases, the first outlet is located at away from the second end by
at most 70% of the distance.
[0023] In some embodiments, systems also include a second outlet
located at the second end of the chamber.
[0024] In one aspect, methods of separating material from a host
fluid include: flowing an initial mixture of the host fluid and the
material via an inlet into an acoustophoretic device at a flow
rate, the acoustophoretic device including: an acoustic chamber
communicating with the inlet; an ultrasonic transducer coupled to
the chamber and arranged to be excited to produce an acoustic wave
at an angle with a mean direction of flow of the initial mixture;
controlling a ratio of acoustic radiation force produced by the
ultrasonic transducer and a viscous drag force of the initial
mixture to cause a first subgroup of the material passing through
the acoustic wave to deflect at an angle that is different than
that of a second subgroup of the material, to thereby permit the
first and second subgroups to be separated. Embodiments of these
methods can include one or more of the following features.
[0025] In some embodiments, methods also include controlling the
ratio by controlling one or more of the angle, the flow rate, a
frequency of excitation of the ultrasonic transducer or power
supplied to the ultrasonic transducer.
[0026] In some embodiments, methods also include controlling the
ratio based on characteristics of one or more subgroups. In some
cases, methods also include controlling the ratio based on one or
more of material size, density, compressibility or acoustic
contrast factor.
[0027] In some embodiments, methods also include controlling the
ratio to deflect at least some of the material at the angle of the
acoustic wave.
[0028] In some embodiments, the material further includes a third
subgroup that is different from the first subgroup and the second
subgroup, and controlling the ratio further comprises causing the
third subgroup to deflect at an angle that is different than that
of the first subgroup or the second subgroup.
[0029] In some embodiments, methods also include controlling the
ratio in a range that is determined by characteristics of subgroups
of materials in the mixture to be separated. In some cases, the
range is determined by the relative sizes of the material in the
subgroups to be separated. In some cases, the range spans at least
an order of magnitude.
[0030] In some embodiments, methods also include collecting the
first subgroup or the second subgroup in a collection duct
communicating with the acoustic chamber.
[0031] In some embodiments, the material comprises particulates,
cells, or fluids, that include at least two subgroups possessing
different characteristics.
[0032] These systems and methods can separate, sort, and
differentiate various particles using bulk ultrasonic standing
waves oriented at an angle .gamma. relative to the fluid velocity.
This approach offers a sensitive separation capability with respect
to size and acoustic contrast of particles.
[0033] "Bulk acoustic standing waves" indicate acoustic waves that
propagate through volume of a medium such as water with little
attenuation. In contrast, "surface acoustic standing waves" are
acoustic waves that travel along the surface of a material
exhibiting elasticity, with an amplitude that typically decays
exponentially with depth into the substrate. Surface acoustic waves
do not penetrate very far into a volume of a medium such as water,
e.g. several millimeters from a substrate into the water volume at
most.
[0034] The singular forms "a," "an," and "the" include plural
referents unless the context clearly dictates otherwise.
[0035] Numerical values should be understood to include numerical
values which are the same when reduced to the same number of
significant figures and numerical values which differ from the
stated value by less than the experimental error of conventional
measurement technique of the type described in the present
application to determine the value.
[0036] All ranges disclosed herein are inclusive of the recited
endpoint and independently combinable (for example, the range of
"from 2 grams to 10 grams" is inclusive of the endpoints, 2 grams
and 10 grams, and all the intermediate values). The endpoints of
the ranges and any values disclosed herein are not limited to the
precise range or value; they are sufficiently imprecise to include
values approximating these ranges and/or values.
[0037] The modifier "about" used in connection with a quantity is
inclusive of the stated value and has the meaning dictated by the
context. When used in the context of a range, the modifier "about"
should also be considered as disclosing the range defined by the
absolute values of the two endpoints. For example, the range of
"from about 2 to about 10" also discloses the range "from 2 to 10."
The term "about" may refer to plus or minus 10% of the indicated
number. For example, "about 10%" may indicate a range of 9% to 11%,
and "about 1" may mean from 0.9-1.1.
[0038] It should be noted that some of the terms used herein may be
relative terms. For example, the terms "upper" and "lower" are
relative to each other in location, i.e. an upper component is
located at a higher elevation than a lower component in a given
orientation, but these terms can change if the device is flipped.
The terms "inlet" and "outlet" are relative to a fluid flowing
through them with respect to a given structure, e.g. a fluid flows
through the inlet into the structure and flows through the outlet
out of the structure. The terms "upstream" and "downstream" are
relative to the direction in which a fluid flows through various
components, i.e. the flow fluids through an upstream component
prior to flowing through the downstream component. It should be
noted that in a loop, a first component can be described as being
both upstream of and downstream of a second component.
[0039] The terms "horizontal" and "vertical" are used to indicate
direction relative to an absolute reference, i.e. ground level.
However, these terms should not be construed to require structures
to be absolutely parallel or absolutely perpendicular to each
other. For example, a first vertical structure and a second
vertical structure are not necessarily parallel to each other. The
terms "top" and "bottom" or "base" are used to refer to surfaces
where the top is always higher than the bottom/base relative to an
absolute reference, i.e. the surface of the earth. The terms
"upwards" and "downwards" are also relative to an absolute
reference; upwards is always against the gravity of the earth. It
is to be understood that gravity, or the effects of gravity, are
negligible in the angled wave deflection process described herein,
because the process works on individual particles, not much larger
particle clusters as used in other systems.
[0040] The term "parallel" should be construed in its lay sense of
two surfaces that maintain a generally constant distance between
them, and not in the strict mathematical sense that such surfaces
will never intersect when extended to infinity.
[0041] Two numbers are of the same order of magnitude if the
quotient of the larger number divided by the smaller number is a
value of at least 1 and less than 10.
[0042] The details of one or more embodiments of these systems,
devices, and methods are set forth in the accompanying drawings and
the description below. Other features, objects, and advantages will
be apparent from the description and drawings, and from the
claims.
DESCRIPTION OF DRAWINGS
[0043] FIG. 1 is a schematic of particle deflection by the acoustic
radiation force of an angled acoustic standing wave oriented at an
angle .gamma. relative to the flow velocity V.
[0044] FIGS. 2A and 2B are schematics of the normal and tangential
velocity components for a left running acoustic standing wave (FIG.
2A), and a right running acoustic standing wave (FIG. 2B).
[0045] FIG. 3 is a schematic of the Galilean transformation
decomposing the angled acoustic standing wave system into a system
of two equations, i.e., normal to the wavefront and tangential to
the wavefront.
[0046] FIG. 4 is a schematic illustration of net particle
deflection by the tangential velocity component after a one-half
wavelength propagation in the normal direction to the
wavefront.
[0047] FIG. 5 is a plot of particle deflection angle
.DELTA..theta..sub.M versus wave angle .gamma. for M parameter
values of 0 to 1.
[0048] FIG. 6 is a plot of particle deflection angle MM versus M
parameter for acoustic standing wave angles of 30.degree.,
45.degree., and 60.degree..
[0049] FIG. 7A is a plot of particle deflection angle
.DELTA..theta..sub.M versus M parameter curve highlighting two
possible regions of particle deflection. FIG. 7B is a schematic of
a particle deflection angle .DELTA..theta..sub.M at an angle less
than the wave angle .gamma. and FIG. 7C is a schematic of a
particle deflection angle MM that equals the wave angle
.gamma..
[0050] FIGS. 8A and 8B show the numerical particle deflection
trajectory for a CHO cell for (a) M/sin .lamda. value less than one
and (b) M/sin .lamda. value greater than one for a frequency of 2
MHz, an acoustic pressure amplitude of 1 MPa, a particle diameter
of 18 .mu.m, and an acoustic contrast factor of 0.03.
[0051] FIG. 9A shows the numerical particle deflection trajectory
for CHO cells of diameter 16, 18, and 20 .mu.m and acoustic
contrast factor of 0.03. FIG. 9B shows the numerical particle
deflection trajectory for CHO cells of diameter 20 .mu.m and
acoustic contrast factors of 0.03, 0.035, 0.04, 0.045, and 0.05.
Frequency is 2 MHz, acoustic pressure amplitude is 1 MPa and
velocity amplitude is 6 cm/min.
[0052] FIG. 10 shows numerical particle trajectories for a CHO cell
as a function of velocity magnitude of the fluid through the
channel.
[0053] FIG. 11A is a chart comparing the universal analytical
predictions for particle deflection with numerical particle
trajectories over a wide range of M values. FIGS. 11B and 11C are
charts comparing the universal analytical predictions for particle
deflection, with numerical particle trajectories over a wide range
of M values
[0054] FIG. 12A-12G show an angled wave device (AWD) system with a
45.degree. angled standing wave. FIG. 12A is a photograph of the
AWD system with multiple flow inlets on the right and multiple flow
outlets on the left. FIG. 12B is a schematic of the setup showing
locations of the transducer, reflector and flow channels. FIG. 12C
is a schematic illustrating one possible mode of operation of the
AWD with the dash lines representing the nodal plane locations of
the standing wave. FIG. 12D is a schematic of the flow profiles
within the AWD. FIG. 12E is a cross-section of the AWD and FIGS.
12F and 12G are cross-sections of alternate duct arrangements for
the AWD.
[0055] FIG. 13 is a particle size distribution of the polystyrene
beads used in experiments.
[0056] FIGS. 14A-14F are photos of polystyrene bead deflection as a
function of electrical power to the 1 MHz transducer setting up an
acoustic standing wave at a 45.degree..
[0057] FIGS. 15A and 15B illustrate an AWD system configured for
concentrating particles or cells by lowering the mixture duct and
constricting the lower buffer stream.
[0058] FIG. 16 is a schematic of an AWD system configured for
particle fractionation.
[0059] FIGS. 17A, 17B, and 17C are schematics illustrating aspects
of an angled fluid device (AFD) system. FIG. 17A shows system
geometry and flow characteristics. FIG. 17B shows particle transfer
between fluids. FIG. 17C schematically depicts the fluid flow
direction of the system.
[0060] FIG. 18A is a photograph of the AFD and FIG. 18B is a
schematic of the setup showing fluid streamlines from a CFD
prediction.
[0061] FIGS. 19A and 19B are photographs of the acoustic chamber
window of the AFD system showing particle movement through the AFD
system without acoustics (FIG. 19A) and with acoustics (FIG.
19B).
[0062] FIGS. 20A and 20B are, respectively, a cross-section and a
schematic of a system in which a flow construction is used to
increase the concentration of the particle mixture separated using
the system.
[0063] FIG. 21 is a schematic of an AFD system designed for
particle fractionation.
[0064] FIGS. 22A, 22B, and 22C are, respectively, a schematic, a
plot of modeled flow velocities, and a cross-section of an AFD
system designed for particle collection.
[0065] FIGS. 23A and 23B are, respectively, a cross-section and a
schematic of a low angle AFD system.
[0066] FIGS. 24A-24 C present the results of using an AWD system to
fractionate T-cells from 35 um beads. FIGS. 24A and 24 B are
schematics illustrating the anticipated separation of T-cells from
beads. FIG. 24C is a chart of the results.
[0067] FIGS. 25A-25C present the results of using an AWD system to
fractionate a mixed population of beads. FIG. 25A is a schematic
illustrating the anticipated separation of larger beads from
smaller beads. FIGS. 25B and 25C are charts of the results.
[0068] FIGS. 26A-26C present the results of using an AWD system to
fractionate a population of PMMA beads. FIGS. 26A, 26B, and 26C
show the distribution of beads between the center outlet and the
buffer outlet without acoustics, with 1 W of power applied and with
1.2 W of power applied.
[0069] FIG. 27 shows a 10-degree AWD system with a center channel
and a buffer channel around it.
[0070] FIG. 28 shows an AWD system that has one small inlet on a
side, buffer flow on top of it, and 5 outlets where different
fractions from a mixture population will end up.
[0071] FIG. 29 shows an AWD system in which viewing of the flow is
made possible by 2 glass windows.
[0072] Like reference symbols in the various drawings indicate like
elements.
DETAILED DESCRIPTION
[0073] The present disclosure relates to acoustophoretic devices
that employ multi-dimensional ultrasonic acoustic standing waves,
planar acoustic standing waves or combinations of planar and
multidimensional acoustic standing waves (collectively referred to
herein as angled acoustic standing waves) oriented at an angle
relative to the direction of mean flow through the device. The
direction of mean flow through the chamber is to be understood to
include the path traveled by a second fluid, cell, or particulate
that is flowed through an angled acoustic standing wave generated
in the device. These angled acoustic standing waves deflect
particles in a host fluid stream, rather than trapping the
particles for agglomeration. This is an important distinction from
many current acoustophoresis devices. These devices described can
operate at high flowrates and can be used to replace costly and
clog-prone filter cartridges and filter membranes in various
industries. The devices and methods of the present disclosure rely
primarily on the axial force component to deflect the particles out
of the acoustic field, rather than relying on trapping,
agglomeration, and gravitational and buoyancy forces. The devices
and methods presented herein are capable of being operated
independent of gravity (i.e., in any orientation), and do not rely
on gravitational settling. In this way, the axial force of an
angled acoustic standing wave oriented at an angle relative to the
flow direction is capable of advantageously deflecting material
(e.g., a second fluid, cells, beads or other particles, exosomes,
viruses, oil droplets) in host fluid streams at high flow rates of
up to about 400 mL/min, and more preferably up to about 600 mL/min
or about 700 mL/min in devices with a cross section of 1 inch by 1
inch. Devices have also been produced with a 0.5 inch.times.0.5
inch total flow channel, with the center inlet being 0.1
inch.times.0.1 inch. For these devices, volumetric flow rates on
the order of 0 to 100 ml/min with typical buffer flow rate of 20 to
100 ml/min and center flow rate of 1 to 10 ml/min. This corresponds
to linear velocities on the order of 1 to 100 mm/sec regardless of
the size of the device.
[0074] Thus, bulk acoustic standing waves angled relative to a
direction of flow through a device can be used to deflect, collect,
differentiate, or fractionate particles or cells from a fluid
flowing through the device. The angled acoustic standing waves can
be used to separate or fractionate particles in the fluid by size,
density, speed of sound, or shape. The angled acoustic standing
wave can be a three-dimensional acoustic standing wave. The
acoustic standing wave may also be a planar wave where the
piezoelectric material is excited in a piston fashion or the
acoustic standing waves may be a combination of the planar acoustic
standing waves and the multidimensional acoustic standing waves.
For the purposes of this disclosure, a standing wave where the
lateral force is at least an order of magnitude less than the
magnitude of the axial force is considered a "planar acoustic
standing wave." However, standing waves that are not planar
acoustic standing waves may be used with the approaches described
in this disclosure as well. This can be used to separate live cells
from dead cells, damaged cells from healthy cells, or
differentiated from undifferentiated cells. The deflection of the
particles by the standing wave can also be controlled or amplified
by the strength of the acoustic field, the angle of the acoustic
field, the properties of the fluid, the three dimensionality of the
standing wave, the frequency of the standing wave, the acoustic
chamber shape, and the mixture flow velocity.
[0075] When acoustic standing waves propagate in liquids, the fast
oscillations may generate a non-oscillating force on particles
suspended in the liquid or on an interface between liquids. This
force is known as the acoustic radiation force. The force
originates from the non-linearity of the propagating wave. As a
result of the non-linearity, the wave is distorted as it propagates
and the time-averages are nonzero. By serial expansion (according
to perturbation theory), the first non-zero term will be the
second-order term, which accounts for the acoustic radiation force.
The acoustic radiation force on a particle, or a cell, in a fluid
suspension is a function of the difference in radiation pressure on
either side of the particle or cell. The physical description of
the radiation force is a superposition of the incident wave and a
scattered wave, in addition to the effect of the non-rigid particle
oscillating with a different speed compared to the surrounding
medium thereby radiating a wave. The following equation presents an
analytical expression for the acoustic radiation force F.sub.R on a
particle, or cell, in a fluid suspension in a standing wave.
F R = 3 .times. .pi. .times. P 0 2 .times. V P .times. .beta. f 2
.times. .lamda. .times. X .times. sin .function. ( 2 .times. k
.times. x ) ( 1 ) ##EQU00001##
where .beta..sub.m is the speed of sound in the fluid medium, .rho.
is density, X is acoustic contrast factor, V.sub.p is particle
volume, .lamda. is wavelength, k is 2.pi./.lamda., P.sub.0 is
acoustic pressure amplitude, x is the axial distance along the
standing wave (i.e., perpendicular to the wave front), and
X = 1 3 .function. [ 5 .times. .rho. .rho. - 2 .times. .rho. f 2
.times. .rho. .rho. + .rho. f - .beta. .rho. .beta. f ] ( 2 )
##EQU00002##
where .rho..sub..rho. is the particle density, .rho..sub.f is the
fluid medium density, .beta..sub..rho. is the compressibility of
the particle, and .beta..sub.f is the compressibility of the fluid
medium.
[0076] The acoustic radiation force on a particle is seen to be a
symmetric function having a period that is one half the acoustic
wavelength. This means the radiation force distribution repeats
every half wavelength. This also means a particle will be
accelerated and decelerated by the radiation force presented by Eq.
(1).
[0077] FIG. 1 schematically shows the particle deflection that such
a force variation will generate when a mixture flows through a
standing wave at an angle, .gamma.. V is the velocity of the
mixture of fluid and particles. The pluses and minuses in the
figure represent the direction of the radiation force. Plus sign
means the radiation force is in the flow direction and increases
particle velocity, and minus sign means the radiation force slows
down the particle. The particles will always be deflected toward
the wave front, or away from the wave axial direction as shown.
FIG. 1 is a left running wave, or the wave slants to the left when
looking in the direction of the fluid mixture flow.
[0078] FIGS. 2A and 2B are schematics of the normal and tangential
velocity components for a left running acoustic standing wave (FIG.
2A), and a right running acoustic standing wave (FIG. 2B). As shown
in FIGS. 2A and 2B, the fluid velocity (V) in FIG. 1 can be
decomposed into a velocity component perpendicular to the running
wave (V.sub.N), and one parallel to the wave (V.sub.T). The
particles will always be deflected in the direction of the
tangential velocity component. It is the fluid motion in the
tangential direction that carries, or drags the particle at a
constant velocity, as the normal velocity component is slowed down
or sped up by the axial radiation force. In this case, any particle
in suspension will again be deflected in the V.sub.T direction.
[0079] An angled flow problem, as presented in FIG. 2 can often be
analyzed more simply by using a Galilean transformation, as shown
in FIG. 3. This transformation amounts to looking at the same
problem while running along the wave at a velocity V.sub.T.
Theoretically, the physics of the problem do not change with such a
transformation. As seen in FIG. 3, this amounts to solving the flow
through a standing wave with the flow direction perpendicular to
the wave front, or in the axial direction of the wave. In this
direction, the acoustic radiation force variation, as presented in
Eq. (1), will result in a symmetrical series of velocity increases
and decreases in the normal flow direction. Using v as the particle
perturbation velocity resulting from the acoustic radiation forces
on a particle as the mixture flows through a normal acoustic
standing wave, the following governing equation can be generated to
describe the particle trajectory (i.e. from Newton's second law,
Eq. (1) and Stokes' drag), where r.sub.p is particle radius:
.rho. .rho. .times. V P .function. ( d .times. v d .times. t ) + 6
.times. .mu. .times. r p .times. v = 3 .times. .times. P 0 2
.times. V P .times. .beta. f 2 .times. .lamda. .times. X .function.
( .beta. , .rho. ) .times. sin .function. ( 2 .times. k .times. x )
( 3 ) ##EQU00003##
As such, v is actually .DELTA.V.sub.N, or the change in particle
velocity normal to the standing wave resulting from the effects of
the acoustic radiation forces on the particles as generated by the
standing wave relative to the normal fluid flow velocity. The
viscosity effects always oppose the perturbation velocity, and act
in a direction toward the mean velocity. As a result, the viscosity
always drives the particle perturbation velocity to fluctuate about
the mean flow velocity with an amplitude of .DELTA.V.sub.N. The
particles in suspension are assumed small enough to instantly react
to the viscous and radiation forces. With this assumption, the
first term on the left side drops out, and Equation 3 can be
reduced to:
v=C sin(2kx) (4)
where
C = .pi. 3 .times. r p 2 .times. .beta. m .times. .phi. .mu.
.times. .lamda. .times. P 0 2 , ##EQU00004##
C is the maximum perturbation velocity in the normal direction and
is seen to be a function of the acoustic pressure amplitude,
particle radius, acoustic contrast factor, fluid viscosity and the
acoustic wavelength. With this assumption, the particle velocity
instantly adjusts to the Stokes velocity generated by the radiation
force.
[0080] FIG. 4 schematically presents the particle deflection effect
caused by the decrease and increase of the velocity component
normal to the acoustic standing wave, when the standing wave is at
an angle .gamma. to the flow. As inferred by the Galilean
transformation, the tangential velocity component has to remain
constant as the velocity component normal to the acoustic standing
wave varies symmetrically about the mean normal velocity.
[0081] The trajectory of the fluid compared to the average
trajectory of a particle are also shown in FIG. 4. P.sub.1P.sub.2
is the fluid trajectory during a time period .DELTA.t.sub.0.
P.sub.1P.sub.3 is the average particle trajectory. V.sub.N is the
component of velocity perpendicular to the wave, V.sub.T is the
tangential component of the velocity along the wave front, V is the
incoming mixture velocity, t is time, and .DELTA..theta..sub.M is
particle deflection from the fluid direction. P.sub.1 is where the
mixture enters the half wavelength of a standing wave. A planar
standing wave is assumed. The fluid is not deflected by the
radiation forces and leaves the half wavelength at P.sub.2 which is
horizontally aligned with P.sub.1, in the direction of fluid
velocity. On the other hand, the particle is carried down the wave
front by the tangential component of the fluid velocity, and is
deflected to P.sub.3 as seen in the figure. The phrase "a direction
of deflection of the angled acoustic wave" is used to indicate the
direction of this deflection.
[0082] The problem of interest is to determine the particle
deflection with acoustic wave angle under different flow and
acoustic conditions. .DELTA.V.sub.N is the maximum normal velocity
perturbation, C, associated with a sinusoidal acoustic radiation
force acting on a particle as shown in Eq. (4).
[0083] Particle or cell deflection can be characterized
.DELTA.V_N/V which is a non-dimensional parameter that will be
defined as M in the following analytical equation development:
M = .DELTA. .times. V N V ( 5 ) ##EQU00005##
which can be expanded to
M = C V = .pi. 3 .times. r p 2 .times. .beta. f .times. P 0 2
.times. X .mu. .times. .lamda. .times. V ( 6 ) ##EQU00006##
where C is the maximum normal velocity perturbation
(.DELTA.V.sub.N) from Eq. (4), and V is the fluid free stream
velocity. This non-dimensional parameter, M, is important since it
represents the ratio of acoustic radiation force on a particle, to
the viscous drag force on the particle. M is the key parameter for
particle deflection by an angled standing wave. Both acoustic
pressure and particle size are squared in the expression. This
means they are the most dominant factors for determining particle
deflection. An accurate expression for particle deflection in an
angled wave, in terms of M, can be obtained by solving particle
movement with the normal wave, and then transforming the results to
the angled wave flow field (i.e., using the Galilean transformation
presented in FIG. 3). The Galilean transformation has no effect on
time. Therefore, the time of transit between half wavelengths
(which repeat) will be the same in the normal wave plane, and the
transformed angled wave plane.
[0084] Equation 7 presents an expression for .DELTA.t.sub.M which
is the time it takes a particle in suspension to travel through one
half wavelength of the normal standing wave (i.e. process repeats
every half wavelength) as it is being accelerated and decelerated
by the acoustic axial radiation force. Equation 8 is the expression
for .DELTA.t.sub.o which is the time it takes the fluid to pass
through one half wavelength of the normal wave. These two time
values are independent of the Galilean transformation, and combined
with FIG. 4, can be used to obtain particle deflection from the
fluid flow direction.
.DELTA. .times. t M = .intg. 0 .lamda. 2 .times. d .times. x V N +
.DELTA. .times. V N .times. sin .times. 2 .times. k .times. x = 1 V
N .times. .intg. 0 .lamda. 2 .times. d .times. x 1 + M sin .times.
.lamda. .times. sin .times. 2 .times. k .times. x ( 7 ) .DELTA.
.times. t o = .lamda./2 V N ( 8 ) ##EQU00007##
The ratio of these times is defined as
M = .DELTA. .times. t M .DELTA. .times. t o ( 9 ) ##EQU00008##
Equations 10 and 11 use M combined with the wave angle .gamma. to
generate an expression for particle deflection in the angled wave
field.
tan .times. .theta. M = .DELTA. .times. t M .times. V T .DELTA.
.times. t o .times. V N = M tan .times. .gamma. ( 10 ) .DELTA.
.times. .theta. M = tan - 1 .function. ( M tan .times. .gamma. ) -
.pi. 2 + .gamma. ( 11 ) ##EQU00009##
[0085] FIG. 4 helps interpret Eq. (10) and (11). The angled waves
in FIG. 4 represent the results of transforming the normal wave by
adding V.sub.T to all the velocities. P.sub.1 is the point that the
flow mixture enters the standing wave. The standing wave is at an
angle .gamma. with respect to the flow direction. The dash wave
lines represent regions in the standing wave where the radiation
forces on the particles are zero. The direction of the radiation
forces reverse when crossing the dash lines shown in FIG. 4.
P.sub.2 and P.sub.3 are points on the zero force line that is
.lamda./2 distance away from P.sub.1. The particle in suspension
when flowing through P.sub.1 will be deflected by the acoustic wave
and will pass through P.sub.3 as shown in FIG. 4. P.sub.2 is the
point it would have passed through with no acoustic radiation
forces, and it represents the fluid flow direction. The dashed line
connecting P.sub.1 and P.sub.3 represents the average trajectory of
the particle through one cycle of the acoustic radiation force.
.theta..sub.M is the total angle that the same line makes with the
normal direction of the wave. Therefore, .DELTA..theta..sub.M is
the particle deflection angle generated by the acoustic wave as
measured from the flow direction (i.e. dash line connecting P.sub.1
and P.sub.2). The particle transit times calculated from the normal
wave analysis are used with the tangential velocity transformation
to get particle displacements in the wave front direction. The
particle wave front distance generated by the transformation with
no radiation forces is .DELTA.t.sub.oV.sub.T and the particle wave
front distance generated by both the Galilean transformation and
the integrated effects of acoustic radiation forces on the movement
is .DELTA.t.sub.MV.sub.T. The difference
(.DELTA.t.sub.oV.sub.T-.DELTA.t.sub.MV.sub.T) is the particle
deflection along the wave front direction generated by the
sinusoidal acoustic radiation forces acting on the particle. For
.DELTA..theta..sub.M, or particle deflection angles, to be
calculated at different wave angles and different deflection
parameters, M, the integral expression for epsilon has to be solved
in Eq. (11).
[0086] An analytical solution for particle deflection as a function
of wave angle and the non-dimensional parameter, M, defined by the
ratio of acoustic radiation forces and viscous forces on a mixture
flowing through an acoustic standing wave, was developed using
substitution of variables. This analytical solution which allows
particle deflection angle to be predicted for all values of M and
.gamma., is shown in Eq. (12).
.DELTA. .times. .theta. M = { tan - 1 .function. ( 1 tan .times.
.gamma. .times. .times. 1 - ( M sin .times. .gamma. ) 2 ) - .pi. 2
+ .gamma. .times. if .times. .times. M sin .times. .gamma. < 1
.gamma. .times. if .times. .times. M sin .times. .gamma. .gtoreq. 1
( 12 ) ##EQU00010##
[0087] FIG. 5 presents calculated particle deflection angles from
Eq. (12) as a function of the wave angle .gamma., and the
non-dimensional deflection parameter M. The different M curves in
FIG. 5 can represent the effects of power on particle deflection
versus wave angle while particle size, fluid compressibility
factor, acoustic wavelength, fluid viscosity and fluid velocity are
all held constant at a baseline condition. The wave angle variation
is from zero to ninety degrees. The particle deflection, at any
constant M value, starts at zero where the wave angle is zero and
moves up along the forty-five.degree. line until a maximum is
reached. Increasing wave angle, with M fixed, increases the
component of the radiation force slowing down the particles. At
some wave angle condition, the particles are stopped from moving
through the waves by the normal radiation force, and are forced by
the fluid to move along the wave front direction. At this point,
the particle deflection reaches a maximum for that M value (i.e.
M=0.667, at a wave angle of 42 degrees is an example).
[0088] The triangular solution region under the 45.degree. line
shown in FIG. 5 represents all particle deflections possible with a
mixture flowing at an angle relative to the bulk acoustic standing
waves. It can be applied to any fluid, standing wave, particle or
acoustic pressure. It presents particle deflection at all wave
angles as a function of a non-dimensional parameter, M, which is
the ratio of acoustic radiation force to viscous drag force on the
particle. Deflection angles are seen to either fall on, or lie
below the 45.degree. line as shown in FIG. 5. The forty-five degree
line represents the case where the deflection angle
.DELTA..theta..sub.M and acoustic wave angle .gamma. are equal.
This is the maximum particle deflection for any angled acoustic
wave and occurs when M/sin .gamma..gtoreq.1, i.e., the acoustic
radiation force equals or exceeds the viscous drag force. This
analytical solution enables angled wave systems to designed and
controlled to provide the M values necessary to obtain desired
results as discussed in more detail later in this disclosure.
[0089] Each M curve in FIG. 5 is seen to have a steep gradient near
the maximum deflection value where the particle deflection shifts
from the difference between the up and down deflection regions
shown in FIG. 1 for a left running wave, to the up deflection only.
This steep gradient represents a change in the physical mode of the
deflection process and is reflected in the experimental results
presented later in this disclosure. This occurs when the radiation
force in the upward deflection region reaches a value large enough
to stop the particle motion through the wave. The results show that
particles flowing in a fluid suspension can be deflected along an
acoustic standing wave of any strength, if the wave angle is small
enough. The different M curves in FIG. 5 can represent the effects
of acoustic pressure on particle deflection versus wave angle while
particle size, fluid compressibility factor, acoustic wavelength,
fluid viscosity and fluid velocity are all held constant at the
baseline condition.
[0090] For example, the M=0.8 curve in the figure can represent
many different applications. One exemplary application with M=0.8
has a fluid mixture velocity, V=7.75.times.10.sup.-4 m/sec, an
acoustic standing wave wavelength, .lamda.=7.4.times.10.sup.-4 m, a
mixture viscosity, .mu.=1.0.times.10.sup.-3 Pas, a contrast factor,
X=0.12, a mixture compressibility, .beta..sub.f=4.06.times.10-10
m2/N, particle radius, r.sub.p=3.times.10.sup.-6 m, and acoustic
pressure amplitude, P.sub.0=1.0 MPa as a discussion point. The
particle deflection curve presented in FIG. 5 for various M
parameters is for all wave angles. Looking at this curve as wave
angles are varied from zero to ninety degrees helps interpret the
physics. The particle deflection initially moves up the 45.degree.
line. Along this line, the particle is stopped between waves, and
moves tangentially along the wave front. This effect continues with
increasing wave angles until the axial radiation force can no
longer stop the normal velocity component of the particle. At this
point, the particle moves through multiple waves and is deflected
by each wave it passes through. The particle deflection is a
maximum of 53.degree., for M=0.8, at a wave angle of 53.degree.. At
a wave angle of 55.degree. with M=0.8, the particle deflection
angle drops to 38.degree. and at a wave angle of 60.degree. with
M=0.8, the particle deflection is 26.5.degree..
[0091] FIG. 6 presents the particle deflection variation with M
that occurs through waves angled at 30.degree., 45.degree., and
60.degree.. M is varied from 0 to 1 in FIG. 6. The particle
deflection angle .DELTA..theta..sub.M increases with increasing
values of M. The rate of increase of particle deflection angle also
grows with increasing values of M. A steep gradient in the
deflection curve is observed near the maximum deflection angle for
all curves. The magnitude of the gradient is seen to increase with
increasing wave angle .gamma.. This steep gradient provides a
mechanism for the separation of particles with only slight
differences in acoustic properties.
[0092] FIGS. 7A, 7B, and 7C present the particle deflection curve
versus M for an acoustic wave angle of 45.degree. only. In region
1, the particles pass through all the waves, and get deflected down
(for the right running wave shown) at a constant angle,
.DELTA..theta..sub.M smaller than .gamma. as shown in FIG. 7B. The
particle net deflection in region 1 is the difference between
downward deflection (particle slowed down by the radiation force)
and upward deflection (particle accelerated by the radiation
force). The curve in FIG. 7A shows the large gradient that occurs
when region 1 transitions into region 2. In the vicinity of this
transition, a small change in M generates a large change in
particle deflection angle .DELTA..theta..sub.M. The separation of
particles with minute size, stiffness or density differences may be
accomplished in this transition region. Region 2 presents the
operating parameter space where the acoustic radiation force is
large enough to stop the particles from moving through the waves.
The particles move parallel to the wave front and
.DELTA..theta..sub.M=.gamma. in region 2. Theoretically, in region
2, all the particles will be deflected down the wave front in the
first wave as shown in FIG. 7C.
[0093] The analytical model results, as presented in FIG. 5,
predict that particles in suspension can be deflected down an
acoustic standing wave of any strength, if the wave angle is small
enough. As the wave angle .gamma. is decreased, the fluid and
particle velocity normal to the wave decreases. At some point, the
acoustic radiation force will overcome the oncoming particle normal
velocity component, and as a result, the particle will stop moving
through the wave and will travel along the wave front. This process
occurs when the wave angle is low enough to cause the resultant
particle velocity component normal to the wave to reach zero. The
forty-five degree line in FIG. 5 represents a locus of such points.
The analysis predicts the maximum deflection for any value of M
always falls on this forty-five degree locus line. Since the
acoustic power parameter M is equal to C/V where C represents the
maximum particle normal velocity perturbation generated by the
acoustic radiation forces, it also can be interpreted as the
.DELTA.VN/V where V is the oncoming fluid and particle velocity.
When .DELTA.VN=V sin(.gamma.), the acoustic perturbation velocity
is equal to the fluid normal velocity component to the wave.
Therefore, at any power, or acoustic pressure of the acoustic
standing wave, there will be an angle of the standing wave where
the radiation force can stop the particle velocity normal to the
wave. The following equation defines this point which represents
the maximum particle deflection as well as where the deflection
curve for a given M value intersects the forty five degree line in
FIG. 5:
.DELTA..theta..sub.max=sin.sup.-1(M)=.gamma. (13)
Equation 13 defines the maximum deflection angle possible, and the
wave angle .gamma. needed for maximum particle deflection using
angled acoustic standing waves as a function of the non-dimensional
parameter M.
[0094] The M parameter can also be used to determine the desired
operation characteristics, for example, to be used in deflecting
extremely small particles in suspension. The smaller the particle
size, the lower the M factor. Assuming flow velocity is reduced as
low as possible for system feasibility, and power is increased as
large as possible, then the M operating curves specify that the
system should be operated at as low a wave angle as possible as
particle deflection peaks at lower wave angles for low M values.
This indicates that systems used with small particles, or
nanoparticles, should be operated at extremely small angles (e.g.,
<5.degree., <4.degree., <3.degree., <2.degree.,
<1.degree.).
[0095] The predictions presented above are based on analytical
procedures for ideal standing waves and fluid velocity fields, and
were used as guidance for more accurate numerical particle
trajectory studies and experimental verification tests showing the
benefits of using acoustic standing waves to deflect, collect,
differentiate, separate, purify, or fractionate one population of
particles or cells from a mixture that may contain multiple
different types of particles, i.e., different in size and/or
material properties such as density or compressibility.
[0096] The particle trajectory can be solved by numerically
integrating the equation of motion of the particle, i.e., Eq. (3)
given some initial conditions of the particle. The equation is
solved by a fourth order Runge Kutta method with automatic time
stepping. In the following results, a uniform velocity profile of
fluid for a flow channel of one inch width is used. Typical
conditions used in the computations are an acoustic standing wave
with a frequency of 2 MHz and an acoustic pressure amplitude of 1
MPa. The acoustic standing wave has a width of one inch and has an
angle of 45.degree..
[0097] FIGS. 8A and 8B presents deflection results for a particle
with properties that are similar to a Chinese Hamster Ovary (CHO)
cell. CHO cells are of interest since they are widely used in the
production of recombinant proteins and monoclonal antibodies. A
typical CHO cell has a diameter of 18 .mu.m and an acoustic
contrast factor of 0.03.
[0098] FIGS. 8A and 8B show the numerical particle deflection
trajectory for a CHO cell for M/sin .gamma. value less than one and
for M/sin .gamma. value greater than one, respectively. The
simulation used: a frequency of 2 MHz, an acoustic pressure
amplitude of 1 MPa, a CHO cell diameter of 18 .mu.m, and a CHO cell
acoustic contrast factor of 0.03. The numerical particle trajectory
results further verify the physics of angled standing waves and the
analytical predictions presented for two cases, M/sin .gamma.<1
and M/sin .gamma..gtoreq.1. These results include inertial effects.
Viscosity modifies inertial effects to generate a symmetrical
perturbation velocity about the mean normal velocity component
which generates the net constant deflection as shown in FIGS. 8A
and 8B. Therefore, the particle deflection in the first half
wavelengths can vary depending on the exact location of the
particle relative to the standing wave, as is shown in FIG. 8 where
the initial particle location of the two particles differs by a
quarter wavelength in the y direction. The viscosity damps this
initial length effect out quickly. The results verify the constant
angle of deflection as the particle passes through each half
wavelength of the standing wave. When M/sin .gamma..gtoreq.1 (i.e.,
the condition in FIG. 8B), the particle deflection angle equals the
standing wave angle. After the initial transient of the particle
motion, the particle deflection is along the wave angle.
[0099] FIG. 9A shows numerical particle trajectory for CHO cells of
diameter 16, 18, and 20 .mu.m and acoustic contrast factor of 0.03.
FIG. 9B shows the numerical particle trajectory for CHO cells of
diameter 20 .mu.m and acoustic contrast factors of 0.03, 0.035,
0.04, 0.045, and 0.05. The simulation used a frequency of 2 MHz, an
acoustic pressure amplitude of 1 MPa, and a velocity amplitude is 6
cm/min.
[0100] FIG. 9A shows CHO particle deflections for three slightly
different sizes, 16, 18, and 20 .mu.m, representing a size
variation of about .+-.10%. The smallest particle deflection is
that of a particle with an M/sin .gamma. value less than one. The
18 .mu.m particle deflects according to an M/sin .gamma. value of
less than one but greater than that of the 16 .mu.m particle,
resulting in a larger deflection. The 20 .mu.m particles deflection
is that of an M/sin .gamma..gtoreq.1 type trajectory. These small
size differences lead to large differences in particle
trajectories. FIG. 9B shows similar results but as a function of
small changes in acoustic contrast factor, i.e., values of 0.03,
0.035, 0.04, 0.045, and 0.05. These results indicate that angled
standing waves can be used to separate, or fractionate particles in
suspension by size, acoustic contrast factor, i.e., density and
compressibility, and shape. This technique may allow live cells to
be separated from dead cells, or even damaged cells from healthy
cells. For example, Table 1 presents the acoustic contrast factors
for several types of cells.
TABLE-US-00001 TABLE 1 Speed of Acoustic Cell Density Sound
Contrast Type (g/cc) (m/s) Factor Jurkat T-cell 1.06 1615 0.079
Primary T-cell 1.04 1560 0.049 Yeast 1.1 1700 0.12 CHO 1.03 1550
0.03
[0101] FIG. 10 shows numerical particle trajectories for a CHO cell
as a function of velocity magnitude of the fluid through the
channel. These particle trajectories verify the effects of normal
velocity variation on the particle deflection resulting from a
mixture flowing into an acoustic standing wave at an angle of
45.degree.. As the flow velocity increases, .DELTA.V.sub.N/V
decreases and particle deflection angles. This effect provides a
means to increase the ability to detect minor differences in
particle properties by manipulating the fluid velocity. The
deflection of the particle by the standing wave can also be
controlled and/or amplified by the strength of the acoustic field,
the angle of the acoustic field, the properties of the fluid, the
three dimensionality of the standing wave, the frequency of the
standing wave, the acoustic chamber shape and the mixture flow
velocity.
[0102] FIG. 11A compares the universal analytical predictions for
particle deflection, with numerical particle trajectories over a
wide range of M values. The different lines in the figure represent
analytical predictions from FIG. 5. The symbols represent numerical
data from CFD. Each line or symbol type represents a different M
value in FIG. 11. The agreement between analytical predictions and
numerical results are good. The errors seen in narrow regions near
wave angles of 0.degree., and 90.degree. are believed to be a
result of singularities that occur at these two extremes. The
results verify the importance of the deflection parameter M, the
location of the maximum deflection, and the existence of a steep
gradient region near the maximum deflection point.
[0103] FIGS. 11B and 11C present the results of an experiment with
an angled wave device that has two outer channels adjacent to a
central channel and a wave angle of 45 degrees. The device was
operated at a frequency of 2.1 MHz and a flow rate of 2 ml/min in
the central channel and 40 ml/min in the outer channels. The outer
channels contained clear or buffer fluid, and the central channel
was provided with a fluid containing beads of a given size. The
power was varied and the deflection angle of the beads versus power
was measured and plotted for each of four different groups of beads
in FIG. 11B. In addition, as the power was varied, the deflection
angle of the beads versus the M factor was measured and plotted for
each of four different groups of beads in FIG. 11C. Each of the
four different groups of beads had bead sizes falling in a range of
sizes that was different for each group of beads. The group denoted
with circle shapes in the graph is sized in the range of 10-20
micrometers. The group denoted with triangle shapes in the graph is
sized in the range of 27-32 micrometers. The group denoted with
diamond shapes in the graph is sized in the range of 32-38
micrometers. The group denoted with square shapes in the graph is
sized in the range of 45-53 micrometers. As shown in FIG. 11B, the
deflection angle for the beads changed with the change in power. As
shown in in FIG. 11C, the M factor for all the beads agrees fairly
well with the analytical result shown in solid black squares.
[0104] The numerical particle trajectory model can easily be
modified to take into account more realistic acoustic and flow
fields. Computational Fluid Dynamics simulations can be done to
determine the fluid velocities in a realistic fluid channel
geometry. Similarly, numerical solvers for acoustic fields
generated by piezoelectric transducers can be used to predict more
accurate solutions for the acoustic field. The particle trajectory
model can then make use of the numerically predicted acoustic and
fluid velocity fields to obtain more realistic predictions. Another
extension is the inclusion of gravitational and buoyancy forces
acting on the particles.
[0105] Two macroscale, ultrasonic, angled wave separator
configurations were fabricated and tested. Two different approaches
were used to generate the desired fluid/acoustic interactions. The
first concept is that of an Angled Wave Device (AWD) where an
angled acoustic standing wave propagates through one or more
parallel fluid streams flowing in a straight duct. The second is an
Angled Fluid Device (AFD) where narrow fluid streams are injected
and controlled to flow through an acoustic standing wave chamber at
an angle to the standing wave. These macro scale, ultrasonic
separators were shown to have the potential to operate effectively
at much higher flow rates and/or at much lower particle
concentrations, than conventional acoustic separators. For example,
while earlier acoustic separators typically operate a linear
velocity of less than 1 mm/s, the systems described in this
disclosure can operate at linear velocities of up to 100 mm/s. The
test results verified the analytical predictions, and demonstrated
the potential to separate, or fractionate particles in suspension
by size, density, and speed of sound using angled acoustic standing
waves.
[0106] FIGS. 12A, 12B, and 12C show an AWD system 100 with a
45.degree. angled standing wave. FIG. 12A is a photograph of the
AWD system 100 with multiple flow inlets 110, 112 on the right and
multiple flow outlets 114, 116 on the left. FIG. 12B is a schematic
of the system 100 showing locations of the transducer 118,
reflector 120, and flow channels. FIG. 12C is a schematic
illustrating one possible mode of operation of the AWD system 100
with the dash lines representing the nodal plane locations of the
standing wave. FIG. 12D is a schematic of the flow profiles within
the AWD system 100. FIG. 12E is a cross-section of the AWD system
100 and FIGS. 12F and 12G are cross-sections of alternate duct
arrangements for the AWD system.
[0107] The AWD system 100 can be operated in horizontal and
vertical orientations. Multiple inlets 110, 112 are shown on the
right and multiple outlets 114, 116 on the left. The inlet 110 and
the inlet 112 are coaxial rectangular ducts with an axis 107. In
the orientation shown, the flow travels horizontally from right to
left through a flow chamber 109, in this case a rectangular duct.
In general, AWD systems include a piezoelectric material configured
to be excited to generate an angled acoustic standing wave with a
wavelength and an acoustic radiation force in the flow chamber
oriented at an acute angle relative to the direction of mean flow
through the flow chamber and the flow chamber has a minimum
internal dimension that is at least 10 times (e.g., at least 50
times, at least 100 times, or at least 1000 times) the wavelength
of the angled acoustic standing wave. In the AWD system 100, an
angled standing wave is generated at 45.degree. to the flow
direction by a PZT-8, 1 MHz, 1 inch by 1 inch transducer and a
stainless steel reflector. Optionally, some systems include
multiple transducer/reflector pairs. The minimum internal dimension
of the flow chamber 109 of this system is the height 108 of the
flow chamber which is about 0.75 inches. In a test described in
more detail below, the AWD system 100 was operated vertically with
flow downwards to eliminate gravity effects on particle
deflections. A mixture of polystyrene beads and water was pumped
downward through the 0.2-inch middle inlet channel at a velocity of
155 cm/min. In the AWD system 100, the middle inlet channel (inlet
110) has a cross-sectional area of about 0.15 square inches. In
general, AWD systems have mixture inlets with cross-sectional areas
of between 0.01 and 2 square inches (e.g., 0.05, 0.1, 0.25, 0.5,
0.75, or 1 square inches).
[0108] The space between the ultrasonic transducer and the
reflector has a first portion within the flow chamber and a second
portion outside the flow chamber. In the acoustic chamber of the
AWD system 100, thin acoustically transparent membranes 121 are
used to separate the mixture flow from the prismatic void regions
(i.e., the second portion outside the flow chamber) set up by the
angular transducer and reflector set up. Optionally, the system can
include cooling water system in fluid connection with the prismatic
void regions. For example, pumps can circulate water through these
regions to maintain a constant fluid temperature. In some systems,
these prismatic void regions are filled with solid material having
an acoustic impedance equivalent to the host fluid. This approach
has been found to eliminate flow problems associated with the
triangular regions while allowing the angled wave to pass with
minimal reflections.
[0109] As shown in FIG. 12E, the straight, rectangular duct
includes an inner duct (inlet 110) which flows a mixture of
particles and a host fluid, and an outer duct (inlet 112) which
flows a buffer flow. The buffer flow duct (inlet 110) completely
surrounds the mixture flow duct (inlet 112). The mixture flow duct
stops before the acoustic region where the acoustic standing wave
passes through the system at an angle to the flow direction. The
mixture flow duct (outlet 114) is then continued in the rectangular
duct system after the acoustic standing wave. As a result, the
angled acoustic standing wave passes through both the mixture flow
stream and the buffer stream as shown in FIG. 12C. The system
amounts to two inlet flows entering the acoustic standing wave and
two exit flows leaving the standing wave. The inlet and exit ducts
are aligned. The acoustic standing wave is at an angle to the flow
direction in the duct.
[0110] The flow rates are set to generate laminar flow in the
chamber and operate below a Reynolds number of 200 based on
equivalent duct diameter. The low Reynolds number results in shear
dominated flow, with no turbulence. The flow rate is set in three
of the four streams. The two inlet flow rates are set to push the
flow, and either outlet flow duct can be set to pull the flow. This
push and pull operation assures the flow streams stay laminar and
straight, and also provide a means to modify flow profiles for
desired particle separation. The average buffer flow velocity can
be set above or below the average flow velocity of the mixture
flow. As the mixture flow passes through the angled, acoustic
standing wave, the particles in suspension will be deflected
downward along the wave front as shown. The particle deflection
from the horizontal direction can vary from zero up to the wave
direction. The deflection is a factor of the M factor. If the M
factor is large enough to stop the flow through the waves, the
particles will travel along the wave angle. The particles will be
carried by the fluid velocity component parallel to the wave. The
host fluid direction will be unaffected by the acoustics, and will
travel horizontally to the mixture exit duct shown.
[0111] Typical velocity profiles through the acoustic section of
the AWD system 100 are shown in FIG. 12D. The flow is at an
extremely low Reynolds number, which provides shear flow with rapid
development in a duct. The flow wants to flow in layers, or laminar
flow. This is why at low Reynolds numbers, cylinders have lower
drag coefficients than spheres. Three-dimensional regions in the
duct shape should be avoided. The inner mixed flow duct should have
a high aspect ratio (e.g., of at least 5:1, 10:1, 15:1, 20:1, 25:1,
50:1, 100:1)) such that the mixture duct provides approximately
two-dimensional flow for stability. In the AWD system 100, the
aspect ratio is about 7:1.
[0112] Buffer flow around the duct at the side edges as shown in
FIG. 12E is anticipated to limit wall boundary flow effects. No
eddies will exist due to viscous dissipation at low Reynolds
number. As a result, fully developed, two dimensional laminar flow
profiles will develop quickly in the ducts and will enter near the
acoustic region in both the mixture duct and the buffer duct as
shown. The buffer flow rate is set to allow rapid energizing of the
shear layer between the streams thereby providing near constant
velocity in the mixture stream flowing through the angled standing
wave. The mixture flow duct is terminated well before the acoustic
region to allow elimination of the shear layers between the streams
as shown in FIG. 12D. In general, the distance di between the
inlets and the space between the ultrasonic transducer and the
reflector 120 where the angled wave is formed is between 0.025 and
2 (0.5 e.g., 0.05, 0.25, 0.5, 0.75, or 1 inches). In the AWD system
100, the distance di between the inlets and the space between the
ultrasonic transducer 118 and the reflector 120 where the angled
wave is formed is approximately 0.5 inches.
[0113] FIG. 13 is a particle size distribution of polystyrene beads
used to test the AWD system 100. The beads used have a mean
diameter of about 150 .mu.m and sizes as small as 20 .mu.m and as
large as 220 .mu.m. The mixture contained two grams of beads per
liter of water. This allowed for visual observation of the mixture
flow. A water buffer flow was pumped around and parallel to the
mixture at a velocity of 23 cm/min. Electrical power to the
transducer was varied from zero to 3.2 Watt (W) and particle
deflection was recorded. The wide range of particle size resulted
in an even bigger variation of the M parameter. The expectation was
that for certain power and fluid velocity, the larger particles
will deflect at the 45.degree. wave angle, while the smaller
particles would not deflect at all, or at a small angle.
[0114] FIGS. 14A-14F are photos of polystyrene beads 122 flowing
through the AWD system 100 during the test to show bead deflection
as a function of electrical power to the 1 MHz transducer setting
up an acoustic standing wave at a 45.degree.. These figures show
that bead deflection increased as the power (0 W in FIG. 14A, 0.8 W
in FIG. 14B, 1.5 W in FIG. 14C, 1.8 W in FIG. 14D, 2.4 W in FIG.
14E, and 3.2 W in FIG. 14F) was increased. In these photos, the
mixture flow is from right to left, gravity forces are right to
left, and the acoustic standing wave axial direction is from upper
left to lower right in the model window orientation shown. FIG. 14A
shows the mixture flow with no acoustics. Without acoustic forces,
the beads 122 flow horizontally with the fluid and no particle
deflection is Observed with the all of the beads flowing to an exit
region 123.
[0115] The M factor and particle deflection increases directly with
electric power supplied to the transducer. The mixture stream was
seen to deflect down at an angle less than the wave angle, as it
moved through the angled wave from right to left for power up to
1.5 W (see FIG. 14B). At 1.5 W, larger beads started to deflect
along the angled wave front while smaller particles traveled
straight through the acoustic field, thereby exhibiting
fractionation (FIG. 14C). As the power was increased above 1.5 W,
medium-sized and smaller-sized beads deflected at the 45.degree.
wave angle (FIG. 14D and FIG. 14E), until at 3.2 W, all the visible
beads were deflected along the standing wave (FIG. 14F). In
addition, the exit region 123 of the smaller beads that were not
deflected along the standing wave began to exhibit a gradually
increasing deflection as the power increased.
[0116] The bead diameter variation was calculated using the M
factor based on power variation measured from first noticeable bead
deflection to all bead deflection along the wave front. At a power
of 1.5 W, the analytical calculations indicated that large
particles of 200 .mu.m were deflected along the wave front and a
majority of all particles larger than 130 .mu.m were deflected
along the wave front at a power of 3.2 W. The analytical prediction
is that identical values of the product of the square of particle
diameter and acoustic energy, which is proportional to power, yield
identical particle deflections. The results agree well with the
documented size distribution for the beads and the observed bead
behaviour. These test results verify the analytical model, and
demonstrate the ability to select and differentiate by size or
material property using angled wave technology.
[0117] Some AWD systems have a third outlet configured to
concentrate material being deflected. For example, these systems
can have the second outlet disposed between the first outlet and
the third outlet where a cross-sectional area of the third outlet
is smaller than a cross-sectional area of the second outlet.
[0118] FIGS. 15A and 15B illustrate an AWD system 200 with such a
duct configuration. The AWD system 200 is configured for
concentrating particles or cells by lowering the mixture duct 110
and constricting the lower buffer stream. The outlet mixture duct
114 is attached at the side walls after the flow passes through the
acoustic wave to provide particle collection with higher
concentrations. This mixture duct attachment is made after the flow
passes the acoustic field, allowing buffer flow around the inlet
mixture duct 110 before the acoustic wave and thus providing good
flow profiles and particle concentration. The duct flow rates can
be varied with push/pull mechanisms as described above to help
obtain the desired separation and concentration. The mixture duct
wall attachment isolates an exit duct 124 from the buffer flow duct
(outlet 116). D is the height of the outlet mixture duct 114. h is
the height of the buffer flow duct 116 above the mixer duct 114. d
is much smaller and is the height of the third outlet duct 124. In
the AWD system 200, the width of the ducts is the same. The
concentration rate should ideally be D/d if the velocities in both
ducts were the same. D/d could be varied accordingly. In AWD
systems with this configuration, height D of the outlet mixture
duct 114 is generally have between 2 and 100 times the height d of
the third outlet wall buffer duct 124 (e.g., 3, 5, 10, 25, 50, 75
times the height d of the third outlet wall buffer duct 124). The
mixture to lower buffer flow rate ratio should ideally be D/d if
the velocities in both ducts were the same. In the AWD system 200,
the height D of the mixture duct 114 is about 3 times the height d
of the third outlet 124. The height h of the buffer inlet 112 and
buffer outlet 116 can be much smaller than shown in the figure and
will be chosen with CFD for specific applications.
[0119] Some AWD systems have a plurality of third outlets, each of
the plurality of third outlets offset from an axis of the second
outlet in a direction of deflection of the angled acoustic
wave.
[0120] FIG. 16 is a schematic of an AWD system 300 configured for
particle fractionation. In the orientation of FIG. 16, the
direction of deflection of the angled acoustic wave is downward and
the multiple collection ducts 124a-124e are provided below the
mixing duct (outlet 114) to collect different size particles. The
AWD system 300 has five collection ducts 124a-124e but some AWD
systems configured for fractionation have more collection ducts
(e.g., 10 collection ducts 15, collection ducts, or 20 collection
ducts) or fewer collection ducts (e.g., 4 collection ducts 3,
collection ducts, or 2 collection ducts). The M factor is set in
Region 1 along with push/pull flow rate settings to provide
operation where different deflections occur with different particle
sizes. One example configuration has a total system height of one
inch with the five collection channels spanning a typical distance
of 0.4 inches in total. The system is scalable up or down as needed
to accommodate smaller or larger flow rates. Fractionation systems
can be used, for example, for cell enrichment from a leukopack
(e.g., to fractionate the different cells such as red blood cells,
monocytes, granulocytes, and lymphocytes); to fractionate a
starting population of T cells according to size; to fractionate an
affinity bead/cell complex from unbound free cells; or to
fractionate a population of free cells, affinity bead/cell complex
A, and affinity bead/cell complex B. Use of the M-factor can aid in
design and operation of acoustic separation systems
[0121] In one example, a mixed population of two particles of the
same material, one of size 5 micron and one of size 10 micron need
to be separated in a 45.degree. angled wave device. Operating
parameters such as flowrate, power, and frequency are selected such
that the M-factor for the bigger particle is M.sub.10=0.8. For a
45.degree. angled wave device, this M-factor that results in a
particle deflection of 45 degree for the ten-micron particle. Since
M scales with the square of the particle radius, the M-factor for
the smaller particle is M.sub.5=0.8/4=0.2. The deflection angle for
this particle is about two degrees. Therefore, a proper angled wave
setup with a wave angle of 45.degree. is able to fractionate these
two populations.
[0122] In a second example, the goal is to fractionate three
different cells, lymphocytes, monocytes, and neutrophils, cells
found within the white blood cell population. Lymphocytes have a
typical size of 6 micron. Monocytes and neutrophils are about ten
microns. In addition, the acoustic contrast factor of the
lymphocytes is smaller than that of the monocytes. A 45.degree.
degree angled wave device can be tuned such that the monocytes have
an M-factor of 0.75. The neutrophils being of the same size and
slightly smaller acoustic contrast factor have a slightly smaller
M-factor of about 0.725. The smaller lymphocytes M-factor scales as
( 6/10).sup.2=0.36, or 36% that of the monocytes, resulting in an
M-factor of 0.27. The deflection curve for a 45.degree. wave angle
indicates that the monocytes and neutrophils deflect at 45.degree.,
whereas the lymphocytes deflect at about 5.degree.. A system with
properly designed outlets will be able to harvest separately the
monocytes and neutrophils in one channel, and the lymphocytes in a
separate outlet, thereby separating and enriching the
lymphocytes.
[0123] In a third example, the goal is to fractionate the output of
an affinity cell selection process. An 25 micron affinity bead is
used for a TCR+ T-cell negative cell selection process. The TCR+
T-cells are bound to the affinity bead and form a complex of
affinity bead with multiple TCR+ cells attached to the bead. The
TCR- T-cells are not bound, remaining in solution as free unbound
cells. An angled wave system is then used to fractionate these two
populations, free unbound TCR- cells from the affinity bead/TCR+
cell complexes. The radius of the T-cell is about 6 micron.
Therefore, the ratio of the M-factor is (25/6).sup.2=17. Choosing
system parameters such that the affinity/cell complex has an
M-factor of 1 results in the deflection of the complex at the wave
angle. The unbound free cell then has an M-factor of 1/17=0.06
which means the free cells deflect at an angle of less than
1.degree., thereby effectuating a fractionation process of the
affinity bead/cell complex from the free cells.
[0124] In a fourth example, the goal is to fractionate a mixed cell
populations consisting of similar sized cells but with a difference
in acoustic contrast factor, with cell A having a contrast factor
of 0.03 and cell B having a contrast factor of 0.06. An 45.degree.
angled wave system is used to separate cells A from cells B. The
system is tuned such that the M-factor of cell B is 0.75, resulting
in a deflection of cells B at an angle of 45.degree.. Since the
M-factor scales with the contrast factor, the M-factor for cell A
is 0.75/2=0.375, resulting in a deflection of about 5 degrees for
cell A. A properly designed system should allow for the separation
of cells A at 5 degrees from cells B at 45 degrees.
[0125] FIGS. 17A, 17B, and 17C are schematics illustrating aspects
of an AFD system 400. FIG. 17A shows system geometry and flow
characteristics. FIG. 17B shows particle transfer between
fluids.
[0126] FIG. 17A shows the AFD system 400 with an acoustic chamber
which has an ultrasonic transducer 118 on one side, a reflector 120
on the opposite side of the chamber, and multiple flow inlets 110,
112 and outlets 114, 116. The transducer 118 and chamber are
designed to generate a bulk, ultrasonic acoustic standing wave
traveling horizontally in the chamber as shown n FIG. 17A. The
vertical dash lines shown in the figure represent the nodal plane
locations of the standing wave. Two inlets 110, 112 are shown in
the right top of the chamber, and two outlets 114, 116 are shown at
the left bottom of the chamber. A first channel 110' ends at the
first inlet 110 and a second channel 112' ends at the first inlet
112. The channels 110', 112' are at an angle alpha (.alpha.) with a
plane perpendicular to the angled acoustic standing wave (in the
case the horizontal direction) of 60.degree.. In AFD system 400,
the first channel 110' and the second channel 112' both have a
substantially straight section extending at least 0.5 inches from
their respective inlet 110, 112.
[0127] The two lower exit ducts are at an angle beta (.beta.) with
the horizontal of 70.degree.. In some systems, the angles alpha and
beta are the same. In some systems, the four ducts all enter the
acoustic chamber at different angles. These angles though, will
vary between zero and ninety degrees. In some systems, the angle
alpha is between 30.degree. and 88.degree. (e.g., more than
35.degree., more than 40.degree., more than 45.degree., more than
50.degree., more than 55.degree., more than 60.degree., less than
80.degree., less than 75.degree., less than 70.degree., less than
65.degree., less than 60.degree., less than 55.degree., less than
50.degree.). In some systems, the angle beta is between 30.degree.
and 88.degree. (e.g., more than 35.degree., more than 40.degree.,
more than 45.degree., more than 50.degree., more than 55.degree.,
more than 60.degree., less than 80.degree., less than 75.degree.,
less than 70.degree., less than 65.degree., less than 60.degree.,
less than 55.degree., less than 50.degree.).
[0128] Fluid enters the acoustic chamber through the inlet ducts,
and exits the chamber through the exit ducts. Typical duct
dimensions are channels depths of 0.5 to 1 inch and channel widths
of 0.1 to 0.4 inches. The flow rates are set to generate laminar
flow in the chamber and operate below a Reynolds number of 200
based on duct diameter. The low Reynolds number results in shear
dominated flow, with no turbulence. The flow rate is set in three
of the four ducts attached to the acoustic chamber in FIG. 17A. The
flow is both pushed and pulled. The two inlet flow rates are set to
push the flow, and the outlet flow carrying the particles is set to
pull the flow. This push and pull operation assures the flow
streams go where desired. The outlet mixture flow can be set above
or below the flowrate entering one of the inlet ducts. Typical flow
profiles are shown in the chamber in FIG. 17A. Fully developed
laminar flow profiles enter the acoustic chamber as shown. The wall
shear layer between at the chamber inlet quickly mix out. A fairly
uniform flow develops for a while near the interface of the two
injected streams as shown in FIG. 17A. The flow shear forces on the
fluid in the corners causes flow rotation, and will generate large
scale eddies as shown. This flow rotation will be slow and solid
body like rotation because of the low Reynolds number. The flow
rates are controlled at both the inlets and the exits. In most of
the operations, both inlet duct flow rates will be specified and
one of the exit flow ducts will be specified. This type of
operation is called push/pull. The acoustic standing wave can be
planar, or three dimensional. Planar standing waves are preferred.
The wall of the flow chamber adjacent to the first outlet in a
direction of deflection of the angled acoustic wave can extend at
an acute angle relative to a plane perpendicular to the angled
acoustic standing wave. In the AFD system 400, the lower wall of
the chamber is tilted down an angle gamma (.gamma.) as shown. This
wall slope is designed to help collect particles deflected by
acoustic radiation forces. Some AFD systems have wall slopes
between 1 and 20 degrees (e.g., more than 2 degrees, more than 3
degrees, more than .kappa. degrees, more than 10 degrees, less than
15 degrees, less than 10 degrees, less than 7.5 degrees, less than
5 degrees).
[0129] FIG. 17B presents the AFD system 400 operating with a fluid
mixture with particles in suspension entering through the inlet 110
and clear fluid entering the chamber in the inlet 112. The
particles are assumed to have a positive acoustic contrast factor,
which means they will deflect towards the nodal plane surfaces as
shown. In this manner, all the particles get deflected in the down
direction. The angled wall at the bottom of the chamber allows
particles to drop out of the acoustic field without getting trapped
in the wall shear layer, or held by the acoustic edge effect.
[0130] FIG. 17C schematically depicts the fluid flow direction of
the AWD system 400. The fluid velocity is decomposed into
components normal and tangent to the acoustic standing wave nodal
planes. The normal direction represents the axial direction of the
standing wave. For a planar wave, this is the direction of the
radiation forces on the particles in the mixture. As a result, the
radiation forces slow and speed up the normal velocity component of
the particles with respect to the fluid normal velocity. The
tangential velocity component of the particles remains the same as
the fluid. As a result of this effect, the particles are deflected
at an angle to the fluid towards the downward direction. If the
radiation forces are large enough, the normal velocity of the
particle will approach zero, and the particles will move vertically
downward while the fluid continues to flow across the chamber
towards the exit duct. It is important to realize that these
particle deflections use fluid velocity, and more specifically the
component of fluid velocity in the downward direction. The fluid
carries the particles down. This effect is completely separate from
gravity. The process is gravity independent.
[0131] FIG. 18A is a photograph of a prototype of the AFD system
400 tested and FIG. 18B is a schematic of the setup showing fluid
streamlines from a CFD prediction. In FIG. 18B, the red represents
a mixture stream and blue represent a buffer stream. The CFD
results show that the flow is regular and uniform without any
mixing between the streams. There are two flow inlets 110, 112 and
two flow outlets 114, 116 in the AFD system 400. The top inlets are
at an angle of 60.degree. with respect to the horizontal direction.
The outlets are at 70.degree.. Pumps are used to control both the
amount of flow entering the acoustic chamber through the inlets and
the flow exiting the outlets (push-pull control). The AFD device
was tested with a 1 MHz acoustic standing wave operating at 1 W.
The two flow streams make about a 30.degree. angle with the
standing wave in the acoustic chamber.
[0132] FIGS. 19A and 19B are photographs of the acoustic chamber
window of the AFD system showing particle movement through the AFD
system without acoustics (FIG. 19A) and with acoustics (FIG. 19B).
The test was conducted with 200 ml/min flowing through all inlets
and outlets. This results in mixture flow velocities of about 160
cm/min. The mixture stream of polystyrene beads 122 and water is
readily visible. The mixture was 2 grams of beads 122 per liter of
water. The beads 122 are those described with reference to FIG.
13.
[0133] In FIG. 19A (acoustics off), the mixture stream is seen to
flow directly from the upper left inlet 110 to the lower left
outlet 116 as predicted with CFD. The second stream is water, and
it is not visible in the photograph. FIG. 19B (acoustics on) shows
the effect of the angled standing waves on the motion of the beads
122. The M/sin(.gamma.) parameter for the test was greater than
1.0. The beads 122 were deflected along the angled wave front
almost immediately as they enter the acoustic chamber. This
resulted in the vertical motion of all the visible beads 122 from
the mixture stream to the buffer stream and down to the bottom of
the chamber and into the lower right outlet 114. This result occurs
at all buffer flow rates and shows the ability of this system to be
used for particle washing, or for particle separation and/or
collection at high flow rates when compared to conventional
ultrasonic separator systems. The AFD system 400 is not limited to
two streams and can be modified to include many different angle
variations. The AFD system 400 has the potential to work with a
variety of fluid/particle mixtures where the substance in
suspension could be beads, cells, exosomes, viruses, oil droplets,
or any material that has a different density, compressibility, or
contrast factor than the host fluid. The system can work with
nanoparticles since the acoustic radiation force effect is
amplified by the angle that the flow makes with the acoustic
wave.
[0134] Some systems are configured to provide fractionation by
providing a constriction in the outlet channel positioned to
receive deflected material. For example, the fourth channel ending
at the second outlet can have a first cross-sectional area. The
third channel ending at the first outlet can have a first section
with the first cross-sectional area and a second section with a
second cross-sectional area that is smaller than the first
cross-sectional area with the second section of the third channel
located between the first outlet and the first section of the third
channel.
[0135] FIGS. 20A and 20B illustrates this approach to increasing
the concentration of the particle mixture drawn off with AFD
systems. In AFD system 500, the lower outlet duct 114 is
constricted near the acoustic chamber. This constriction is shown
in the particle outlet that is pulled, for example, by a pump to a
desired flowrate. Since the flowrate is set by the pull rate, any
area constriction results in a velocity increase. The outlet duct
constriction d/D increases the flow velocity by D/d for the two
dimensional duct shown in FIGS. 20A and 20B. FIG. 20B presents
approximate flow profiles for a moderate constriction with a pull
flow rate such that the peak velocity in the acoustic chamber
occurs in the area near the exit duct constriction region. The
higher velocities occurring near the entrance of the exit ducts
114, 116, and near the separation streamline dividing the two flows
will provide better separation with fewer particles moving back to
the mixture stream. The higher velocities mean higher tangential
velocity components to carry the particles (e.g., beads 122) down.
As an example, if the flowrates in all four ducts are set the same
by the push-pull mode and the constriction is 90% of the duct area,
then the velocity near Q4 exit duct entrance will increase ten fold
when compared to the second outlet duct flow, Q3, or with respect
to the inlet flow of both ducts (Q1 and Q2). This effect is
reflected flow profile shown in FIG. 20B. The length of the
constriction region shown in the schematic of the velocity profiles
provides velocity directions toward the constriction channel. This
means less chance of particles re-entering the original mixture
stream, and better separation efficiency. In the same
configuration, and still assuming a 90% constriction, if the
flowrate is set so the Q4 exit duct has one tenth the flow rate of
the incoming inlet ducts, the throttled velocity is nearly or
exactly the same as the un-throttled exit duct velocity in Q3 and
therefore still provides the downward velocity component desired
for separating the particles, while flowing much less fluid in exit
duct flowrate Q4. This arrangement results in a possible ten times
concentration of the mixture with each pass through the AFD
separator.
[0136] FIG. 21 presents an AFD system 600 designed for particle
fractionation. Four inlet ducts 110, 111, 112, 113 and four outlet
ducts 114, 115, 116, 117 are shown. The different shadings
represent CFD predictions showing the ability to maintain the four
angled streams through the acoustic chamber. Again, push/pull
operation allows such unique definition in the acoustic chamber.
Some AWD systems include many more streams. All four flows pass
though the chamber at an angle to the acoustic standing wave. If
the top, or blue stream was a mixture of fluid mixture with
multiple particle sizes in suspension, the particles could be
fractionated into the lower three collection ducts shown in FIG. 21
using the angled wave deflection process. The system may be
operated at an M factor which allows different particles to be
deflected for the collection configuration shown. The same AFD
system shown in FIG. 21 could be expanded to have five or more
inlet ducts and five or more outlet ducts. The incoming mixture and
buffer streams could then pass through many different adjacent duct
pairs. Operated with push/pull technology, this would allow
particle separation with many different wave angles. In the same
manner, the different duct streams could be used to set different
velocity profiles for different particle separation
requirements.
[0137] FIGS. 22A, 22B, and 22C are, respectively, a schematic, a
plot of modeled flow velocities, and a cross-section of an AFD
system 700 designed for particle collection. FIG. 22A shows the
flow through the system. The system contains one inlet duct 110 and
one outlet duct 116 attached to the acoustic chamber. A collection
region 130 is shown below the flow stream passing through the
acoustic chamber at an angle to the standing wave as shown. This
collection is enhanced by a large scale collection vortex shown in
FIG. 22A. This collection vortex is driven by the flow stream
passing through the chamber and provides tangential velocity
components parallel to the nodal planes which can carry the
particles downward out of the mixture stream into the collection
regions. The collection vortex can be furthered enhanced by drawing
off fluid through the collector outlet 114 at the bottom of system.
Turning the acoustics off and on in an appropriate manner to allow
particles to fall down to the collection bottom could also enhance
performance.
[0138] FIGS. 23A and 23B are, respectively, a cross-section and a
schematic of a low angle AFD system 700. The M parameter can be
used to determine the desired operation characteristics for
deflecting extremely small particles (e.g., nanoparticles of the
range of 100 nm to 1000 nm or 10 nm to 100 nm, or 1 to 10 nm,
bacteria, viruses such as lenti or retro viruses, adeno associated
viruses, exosomes, microvesicles, and other nano-sized particles)
in suspension. The smaller the particle size, the lower the M
factor. In a system with flow velocity reduced as low as possible
for system feasibility, and power is increased as large as
possible, then the M operating curves specify that the system
should be operated at as low a wave angle as possible. Typical
operating parameters for these systems are angles between
nearly-zero and 15 degrees, frequencies between 2 and 50 MHz,
acoustic pressure amplitude between 1 and 20 MPa), and linear
velocities are on the order of l0 mm/s, 1 mm/s or 0.1 mm/s. For low
M values, deflection peaks at lower wave angles. The AFD system 700
is configured for use with nanoparticles. Two inlet flow ducts 110,
112 feed the acoustic chamber and two exit flow ducts 114, 116 are
used to exit the flow streams. Both the inlet angle .phi..sub.i and
outlet angle .phi..sub.o are approximately 5.degree.. In other ASWD
system, the inlet angle .phi..sub.i and outlet angle .phi..sub.o
are different angles (e.g., between 2.degree. and 10.degree., more
than 3.degree., more than 4.degree., less than 9.degree., less than
8.degree., less than 7.degree., less than 6.degree.). Again,
push/pull techniques can be used to control the flow streams to
generate the particle fractionation desired.
[0139] The systems and methods described in this disclosure can
provide macro-scale, ultrasonic separators that use bulk, acoustic
standing waves angled to the direction of a fluid mixture flow
field to generate particle deflection that can be used to collect,
differentiate, separate, purify, or fractionate one population of
particles or cells from a mixture that may contain multiple
different types of particles. Particle trajectory equations provide
the key physics. The universal prediction curves developed for
particle deflection at all wave angles as a function of the
non-dimensional parameter M defined by the ratio of acoustic
radiation force to viscous drag force on the particle can be used
in system design and operation. Particle deflection, measured from
the fluid flow direction, varies continuously from zero to a
maximum value equal to the wave angle .gamma., which is the angle
that the standing wave makes with the flow direction. The
analytical results agreed well with both numerical trajectory
computations and model test results. The acoustic pressure
amplitude, particle diameter and wave angle were shown to have the
largest effect on particle deflection.
[0140] Results also showed that for any acoustic pressure amplitude
of a standing wave, there is a wave angle of the standing wave
where the radiation force stops the particle velocity normal to the
wave, and as a result, the particles start to move along the wave
front. This point is defined by the non-dimensional parameter M,
and angle of the wave .gamma. and operating near this point
generates large particle deflection with small changes in
controllable parameters such as acoustic power or flow velocity.
This operating point is quite useful, since it could allow the
separation of particles with minute size, stiffness or density
differences.
[0141] Some of these systems and methods use standing waves angled
to a flow channel or narrow flow streams injected at an angle
through a fixed acoustic chamber. Both systems were shown to
effectively separate polystyrene beads from a flowing mixture at
high speeds when compared to conventional ultrasonic separators.
Such macro scale, ultrasonic separators were also shown to
effectively operate at much higher flow rates, or at much lower
particle concentrations, than conventional acoustic separators.
Model test results agreed very well with theory, and verified the
prediction system developed. The angled wave system could work with
a fluid/substance mixture where the substance in suspension could
be micro carrier beads, cells, exosomes, virus, oil, or any
material that has a different density, compressibility, or contrast
factor than the host fluid. The analytical model predicts that the
system can theoretically work even with nanoparticles since the
acoustic radiation force effect is amplified by the angle the flow
makes with the acoustic wave.
[0142] FIGS. 24A-24 C present the results of using an AWD system
similar to that shown in FIGS. 12A-12E to fractionate T-cells from
35 um beads. The system had a 30.degree. wave angle and was
operated with a frequency of 2.1 MHz and flow rates of 5 ml/min of
a T-cell/bead mixture through the center inlet and of 30 ml/min of
a buffer through the buffer inlet. FIGS. 24A and 24 B are
schematics illustrating the anticipated separation of T-cells from
beads. FIG. 24C presents the results. When the system was operated
without acoustics, 98% of the T-cells and 95% of the beads flowed
through the center outlet. When 2.3 W of power were applied, 92% of
the T-cells flowed through the center outlet and 100% of the beads
were deflected into the buffer outlet.
[0143] FIGS. 25A-25C present the results of using the same AWD
system to fractionate a mixed population of beads with sizes mainly
10 um-29 um and 32 um-42 um. The system had a 30.degree. wave angle
and was operated with a frequency of 2.1 MHz and flow rates of 2
ml/min of a bead mixture through the center inlet and of 40 ml/min
of a buffer through the buffer inlet. The resulting linear flow
velocity was 48 cm/min. FIG. 25A is a schematic illustrating the
anticipated separation of larger beads from smaller beads. FIGS.
25B and 25C presents the results. When the system was operated
without acoustics, most of the beads of both sizes flowed through
the center outlet. When 1.5 W of power were applied, most of the
smaller beads still flowed through the center outlet but most of
the larger beads were deflected into the buffer outlet.
[0144] FIGS. 26A-26C present the results of using the same AWD
system to fractionate a population of PMMA beads with sizes 5 um-20
um. The system had a 30.degree. wave angle and was operated with a
frequency of 2.1 MHz and flow rates of 2 ml/min of a bead mixture
through the center inlet and of 40 ml/min of a buffer through the
buffer inlet. The resulting linear flow velocity was 48 cm/min.
FIGS. 26A, 26B, and 26C show the distribution of beads between the
center outlet and the buffer outlet without acoustics, with 1 W of
power applied and with 1.2 W of power applied. When the system was
operated without acoustics, most of the beads flowed through the
center outlet. When 1 W of power was applied, the larger beads
started preferentially being diverted into the buffer outlet. When
1.2 W of power were applied, most of the beads larger than 12 um
were deflected into the buffer outlet. These results demonstrate
the ability to selectively fractionate material with very small
differences.
[0145] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention.
[0146] For example, FIG. 27 shows a 10-degree AWD system 900 with a
center channel and a buffer channel around it. The center channel
cross section can be circular or rectangular. A typical
cross-section of the system can be from 0.1''.times.0.1'' to a
1''.times.1'' channel with a center channel width ranging from 1/2
to 1/10th of the channel cross-section. Applications include cell
fractionation, bead-cell fractionation.
[0147] In another example, FIG. 28 shows an AWD system 930 that has
one small inlet on a side and buffer flow on top of it and has 5
outlets where different fractions from a mixture population will
end up. A typical size of the channel could be from
0.25''.times.0.25'' to 1''.times.1'' and relative side inlet width
could vary from 1/2 of the channel width to 1/10th of the channel
width. Applications include, for example, leukopack fractionation,
T-cell.
[0148] In another example, FIG. 29 shows an AWD system 960 in which
viewing of the flow is made possible by 2 glass windows.
Attachments are included so that the system can be suspended
vertically with the help of a metal rod. The angled wave system 960
is the same as the 10-degree AWD system 900. This system can be
configured with wave angles between 5 to 85 degrees.
[0149] Accordingly, other embodiments are within the scope of the
following claims.
* * * * *