Note: Descriptions are shown in the official language in which they were submitted.
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APPARATUSES AND METHODS FOR FIELD FLOW FRACTIONATION OF
PARTICLES USING ACOUSTIC AND OTHER FORCES
Technical Field
This invention relates generally to the field of field-flow-fractionation. In
particular, the invention provides apparatuses and methods for the
discrimination of matters
utilizing acoustic force, or utilizing acoustic force with electrophoretic or
dielectrophoretic
force, in field flow fractionation.
Background Art
Electrical-field-flow-fractionation (E-FFF) and dielectrophoretic-field-flow-
fractionation (DEP-FFF) are known in the art. For example, U.S. Patent No.
5,240,618
discloses an electrical field-flow-fractionation method, and U.S. Patent Nos.
5,888,370,
5,993,630 and 5,993,632 disclose methods and apparatuses for fractionation
using
conventional and generalized dielectrophoresis and field flow fractionation.
In E-FFF
(Caldwell and Gao, 1993), electrophoretic forces are used to balance
sedimentation forces
(for large particle application, particle size being ~ several micron or
larger) and/or
diffusion forces (for small particle application) and to control particle
equilibrium positions
(or equilibrium distribution profile) in a fluid flow velocity profile.
Particles of different
charges or sizes or densities exhibit different equilibrium positions (or
different distribution
profiles), and are caused to move through the chamber at different velocities,
and can thus
be separated into different fractionations. In DEP-FFF (Huang et al, 1997,
Markx et al,
1997, Wang et al, 1998), DEP force components in the vertical direction are
used to
balance sedimentation forces and control particle equilibrium positions in a
fluid flow
profile. Particles of different dielectric properties are positioned at
different heights in the
flow profile and are thereby transported at different velocities. A particle
mixture
introduced into an E-FFF or DEP-FFF chamber can be fractionated into sub-
populations
according to the time they exit the chamber. E-FFF separation has been
demonstrated on
colloidal adsorption complexes. DEP-FFF separation has been demonstrated on
synthetic
polystyrene beads and biological cells.
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However, the currently available apparatuses and methods used in field-flow-
fractionation suffer from the following limitations. For E-FFF, electrode
polarization
presents a significant problem since majority of the applied voltage is
dropped across the
electrode/medium interface. Furthermore, electrical charge may be used as a
separation or
fractionation parameter for only certain cases. Similarly, for DEP-FFF,
dielectric
properties may be used as separation bases for only certain problems. Positive
DEP force
has not been exploited for particle DEP-FFF separation. The separation
efficiency using
current field-flow-fractionation methods is still not satisfactory for many
application
problems. Thus, there is a need to further improve field-flow-fractionation
methods so that
the methods have improved applicability and separation efficiency to many
separation
problems. The present invention addresses these and other related needs in the
art.
Disclosure of the Invention
This invention provides apparatuses and methods for particle characterization,
manipulation and separation using acoustic radiation force (or acoustic
force),
electrophoretic (E) force, dielectrophoretic (DEP) force, gravitational force,
hydrodynamic
force and a fluid flow profile. A new force component, i.e., acoustic-
radiation-force, is
introduced to the techniques of field-flow-fractionation (FFF), electrical-
field-flow-
fractionation (E-FFF), dielectrophoretic-field-flow-fractionation (DEP-FFF),
and thus the
present invention has a number of advantages over current FFF or E-FFF or DEP-
FFF
apparatuses and methods:
~ Particles, e.g., cells, can be separated according to their properties such
as size, density,
dielectric parameters, electrical charges, as well as their acoustic impedance
- a new
parameter for particle discrimination and separation.
~ Positive DEP forces may also be exploited for particle separation - a new
dimension to
the DEP-FFF method where only negative DEP forces are used.
~ Better particle separation efficiency can be achieved.
Since an acoustic radiation force (or for simplicity, acoustic force) is used
as an
additional force component to influence particle positions including particle
equilibrium
positions, or particle distribution profiles including particle equilibrium
distribution
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profiles, in FFF or E-FFF or DEP-FFF operation, the methods are termed
acoustic-FFF or
acoustic-E-FFF or acoustic-DEP-FFF. Panicles refer to any matter, particular
matter, or
solubilized matter, or any combination thereof. The acoustic force is produced
by
establishing an acoustic wave, e.g., an acoustic standing wave, in the chamber
in the
direction normal to that of the fluid flow and parallel to the active DEP
force components
or electrophoretic force components. The acoustic standing waves can be
generated using
any methods known in the an, e.g., using piezoelectric transducers.
The present invention provides methods and apparatuses for the discrimination
of
particulate matter and solubilized matter of different types. This
discrimination may
include, for example, separation, characterization, differentiation and
manipulation of the
particulate matter. According to the present invention, the particulate matter
may be placed
in liquid suspension before input into the apparatus. The discrimination
occurs in the
apparatus, which may be a thin, enclosed chamber. Particles may be
distinguished, for
example, by differences in their density, size, dielectric permitivity,
electrical conductivity,
surface charge, surface configuration, and/or acoustic impedance. The
apparatuses and
methods of the present invention may be used to discriminate different types
of matter
simultaneously.
The apparatuses and methods are applicable to the characterization,
manipulation
and separation of many types of particles - solid particles such as glass
beads, latex
particles, liquid panicles such as liquid droplets, or gaseous particles such
as gas bubble.
Particles can be organic ones, e.g., mammalian cells, bacteria, virus, or
other
microorganisms, or inorganic ones, e.g., metal particles. Particles can be of
different
shapes, e.g., sphere, elliptical sphere, cubic, discoid, needle-type, and can
be of different
sizes, e.g., nano-meter-size gold sphere, to micrometer-size cells, to
millimeter-size
particle-aggregate. Examples of particles include, but not limited to,
biomolecules such as
DNA, RNA, chromosomes, protein molecules, e.g., antibodies, cells, colloid
particles, e.g.,
polystyrene beads.
The apparatuses and methods are applicable to any particle separation
problems, in
particular cell separations in biomedical setting. Examples of particle
separation include,
but not limited to, separation of cancer cells from normal cells, metastatic
cancer cells from
blood, fetal nucleated cells from maternal erythrocytes/nucleated cells, virus-
infected cells
from normal counterpart cells, red blood cells from white blood cells,
bacteria from blood
or urine or other body fluid, etc. For biological applications using living
cells, the present
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invention allows cells to be separated without the need to alter them with
ligands, stains,
antibodies or other means. Non-biological applications similarly require no
such alteration.
It is recognized however, that the apparatuses and methods according to the
present
invention are equally suitable for separating such biological matter even if
they have been
so altered. The separation process in the present invention introduces little
or non-stress on
the matter to be separated. Living cells remain undamaged, unaltered and
viable during
and following separation using the present invention. Or, the stress on the
cells is small
enough so that the separated cells are still applicable for further
characterization, assay or
analysis, or growth following separation using the present invention.
A. Apparatuses using acoustic forces (acoustic-FFF apparatuses)
In one aspect, the present invention provides an apparatus fox the
discrimination of
a matter utilizing acoustic forces in field flow fractionation, which
apparatus comprises: a)
a chamber having at least one inlet port and at least one outlet port, said
chamber having
such structural characteristics that when a carried medium is caused to travel
through said
chamber, the traveling velocity of said carried medium at various positions
within said
chamber is different; (b) at least one piezoelectric transducer adapted along
a portion of
said chamber, wherein said piezoelectric transducer can be energized via at
least one
electrical signal provided by an electrical signal generator to create an
acoustic wave,
thereby causing at least one acoustic force having components normal to the
traveling
direction of said carrier medium on a matter in said Garner medium.
The apparatus can have a single inlet port and a single outlet port.
Alternatively, the
apparatus can have a plurality of inlet and/or outlet ports. Preferably, the
outlet port is
connected to a collection device or a characterization device. The outlet port
of the
chamber according to the present invention may take many forms. Specifically,
the outlet
port may be a single port, or a plurality of ports, or an array of ports. The
outlet port, for
example, may be located along the entire width or a part of the width of the
chamber. The
outlet port may be adapted to receive matter of various shapes and sizes. For
example, the
size of the outlet port may vary from approximately twice the size of the
matter to be
discriminated to the entire width of the chamber. In one embodiment, the
outlet port may
be constructed of one or more tubing elements, such as TEFLON tubing. The
tubing
elements may be combined to provide an outlet port. Further, for example, the
outlet port
may be connected to fraction collectors or collection wells that are used to
collect separated
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matter. Other components that may be connected to the apparatus of the present
invention
are, for example, measurement or diagnostic equipment, such as cytometers,
particle
counters and spectrometers. Other devices or apparatus used for further assay
or analyses
on the separated matters may also be connected to the apparatus of the present
invention.
The chamber of the apparatus should be designed to have such structural
characteristics that when a fluid (liquid or gas) is caused to travel through
the chamber, the
velocity of the fluid (liquid or gas) at various positions within said chamber
is different and
the fluid (liquid or gas) travels through the chamber according to a velocity
profile. For
example, the chamber may be rectangular in shape and may include, for example,
a top
wall, a bottom wall and two side walls. The top wall and bottom wall may be
parallel to
each other, or substantially parallel to each other, and the distance betweem
the top wall and
the bottom is referred to as chamber height. The distance between the inlet
port and outlet
port is referred to as chamber length when the chamber comprises one inlet
port and one
outlet port. The two side walls may be parallel to each other, or
substantially parallel to
each other, and the distance between the two side walls of the chamber that
are parallel to
each other is referred to as chamber width. The two side walls may be parts of
a gasket or
a spacer between the top wall and bottom wall. The gasket or spacer may be cut
in the
middle to form a rectangular thin channel with taper ends. Alternatively, the
gasket or
spacer may be cut in the middle to form thin channels of other shapes such as
ellipse,
circle, or any other shape. In certain embodiments, the chamber may be
constructed so that
the top wall and bottom wall are of a much greater magnitude than the side
walls (e.g., both
chamber length and chamber width are substantially greater than the chamber
height for a
chamber with a rectangular shape), thereby creating a thin chamber. For such a
thin
chamber having a rectangular channel in the middle, when a carrier medium is
caused to
?5 travel through the thin rectangular channel (or called "travel through the
chamber"), the
velocity of the carrier medium in the chamber may follow a parabolic or a near-
parabolic
profile. The velocity of the carrier medium at the top and bottom walls is
zero, and with
increasing the distances from the top wall or from the bottom wall, the
velocity of the
carrier medium increases to a maximum value at the middle position between the
top and
.0 bottom walls. In other embodiments, the chamber may be constructed so that
the top wall
and bottom wall are of a much smaller magnitude than the side walls (e.g.,
both chamber
length and chamber height are substantially greater than the chamber width for
a chamber
with a rectangular shape), again creating a thin chamber. In addition to the
rectangular
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shape of the chamber, the chamber may be of circular construction, elliptical,
triangular,
hexadecagonal, or of other geometrical shapes. The chamber may be constructed
having a
top wall, a bottom wall, and a gasket or a spacer between the top and bottom
wall. The
gasket or spacer may be cut in the middle to form rectangular thin channel
with taper ends.
Alternatively, the gasket or spacer may be cut in the middle to form thin
channels of other
shapes such as ellipse, circle, or any other shape. In the case of a thin
rectangular channel
where the top and bottom walls on two different planes are parallel to each
other, the
velocity of the carrier medium in the chamber may follow a parabolic or a near-
parabolic
velocity profile. The velocity of the carrier medium at the top and bottom
walls is zero,
and with increasing the distances from the top wall or from the bottom wall,
the velocity of
the carrier medium increases to a maximum value at the middle position between
the top
and bottom walls. Preferably, for a rectangular channel, the width of the
channel is from
about 1 mm to about 20 cm, and the thickness of the channel is from about 20
micron to
about 10 mm, and the length of the channel is from about 1 cm to about 200 cm,
and
preferably from about 10 cm to about 50 cm. As such, the present invention is
not intended
to be limited to a particular geometric shape and the chamber may be
constructed of many
different materials, for example, glass, polymeric material, plastics, quartz,
coated metal,
or the like, provided that the chamber has such structural characteristics
that when a carrier
medium is caused to travel through the chamber, the velocity of the medium at
different
positions in the chamber is different.
The apparatus can comprise a single piezoelectric transducer or comprise a
plurality
of piezoelectric transducers. The plurality of piezoelectric transducers may
be energized
via common electrical signals or via different electrical signals. The
plurality of
piezoelectric transducers can be adapted along the interior or exterior
surface of the
chamber. The plurality of piezoelectric transducers can also be configured on
a plane
substantially parallel to traveling direction of the carrier medium that is
caused to travel
througTi the chamber.
Preferably, the electrical signal generator for energizing the piezoelectric
transducer
to create the acoustic force is capable of varying magnitude, and frequency of
said
electrical signals.
In a preferred embodiment, the chamber of the apparatus comprises a tube. The
piezoelectric transducer, or a plurality thereof, can be adapted along the
interior surface of
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the tube. Alternatively, the piezoelectric transducer, or a plurality thereof,
can be adapted
along the exterior surface of the tube.
In another preferred embodiment, the chamber comprises a top wall, a bottom
wall,
and two side walls. The piezoelectric transducer, or a plurality thereof, can
be configured
on the top wall of the chamber. Alternatively, the piezoelectric transducer,
or a plurality
thereof, can be configured on the bottom wall of the chamber. In another
configuration, the
piezoelectric transducer, or a plurality thereof, can be adapted on opposing
surfaces of the
chamber. Preferably, the chamber height between the top and bottom walls is
about half
wavelength of the standing acoustic wave.
An apparatus for the discrimination of a matter utilizing acoustic forces in
field
flow fractionation is also provided, which apparatus consists essentially of,
or consists of,
a) a chamber having at least one inlet port and at least one outlet port, said
chamber having
such structural characteristics that when a carried medium is caused to travel
through said
chamber, the traveling velocity of said carried medium at various positions
within said
chamber is different; b) at least one piezoelectric transducer adapted along a
portion of
said chamber, wherein said piezoelectric transducer can be energized via at
least one
electrical signal provided by an electrical signal generator to create an
acoustic wave,
thereby causing at least one acoustic force having components normal to the
traveling
direction of said carrier medium on a matter in said carrier medium.
B. Apparatuses using electrophoretic (E) and acoustic forces (acoustic-E-FFF
apparatuses)
In another aspect, the present invention provides an apparatus for the
discrimination
of a matter utilizing electrophoretic and acoustic forces in field flow
fractionation, which
apparatus comprises: a) a chamber having at least one inlet port and at least
one outlet port,
said chamber having such structural characteristics that when a carried medium
is caused to
travel through said chamber, the traveling velocity of said carried medium at
various
positions within said chamber is different; b) at least two electrode elements
adapted along
a portion of said chamber, wherein said electrode elements can be energized
via at least one
electrical signal provided by an electrical signal generator to create an
electrical field,
thereby causing at least one electrophoretic force having components normal to
the
traveling direction of said Garner medium on a matter in said Garner medium;
and c) at least
one piezoelectric transducer adapted along a portion of said chamber, wherein
said
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piezoelectric transducer can be energized via at least one electrical signal
provided by an
electrical signal generator to create an acoustic wave, thereby causing at
least one acoustic
force having components normal to the traveling direction of said carrier
medium on a
matter in said carrier medium.
The apparatus can have a single inlet port and a single outlet port.
Alternatively,
the apparatus can have a plurality of inlet and/or outlet ports. Preferably,
the outlet port is
connected to a collection device or a characterization device. The outlet port
of the
chamber according to the present invention may take many forms. Specifically,
the outlet
port may be a single port, or a plurality of ports, or an array of ports. The
outlet port, for
example, may be located along the entire width or a part of the width of the
chamber. The
outlet port may be adapted to receive matter of various shapes and sizes. For
example, the
size of the outlet port may vary from approximately twice the size of the
matter to be
discriminated to the entire width of the chamber. In one embodiment, the
outlet port may
be constructed of one or more tubing elements, such as TEFLON tubing. The
tubing
elements may be combined to provide an outlet port. Further, for example, the
outlet port
may be connected to fraction collectors or collection wells that are used to
collect separated
matter. Other components that may be connected to the apparatus of the present
invention
are, for example, measurement or diagnostic equipment, such as cytometers,
particle
counters and spectrometers. Other devices or apparatus used for further assay
or analyses
on the separated matters may also be connected to the apparatus of the present
invention.
The chamber of the apparatus should be designed to have such structural
characteristics that when a fluid (liquid or gas) is caused to travel through
the chamber, the
velocity of the fluid (liquid or gas) at various positions within said chamber
is different and
the fluid (liquid or gas) travels through the chamber according to a velocity
profile. For
example, the chamber may be rectangular in shape and may include, for example,
a top
wall, a bottom wall and two side walls. The top wall and bottom wall may be
parallel to
each other, or substantially parallel to each other, and the distance between
the top wall and
the bottom is referred to as chamber height. The distance between the inlet
port and outlet
is referred to as chamber length when the chamber comprises one inlet port and
one outlet
port. The two side walls may be parallel to each other, or substantially
parallel to each
other, and the distance between the two side walls of the chamber is referred
to as chamber
width. The two side walls may be parts of a gasket or a spacer between the top
wall and
bottom wall. The gasket or spacer may be cut in the middle to form a
rectangular thin
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channel with taper ends. Alternatively, the gasket or spacer may be cut in the
middle to
form thin channels of other shapes such as ellipse, circle, or any other
shape. In certain
embodiments, the chamber may be constructed so that the top wall and bottom
wall are of a
much greater magnitude than the side walls (e.g., both chamber length and
chamber width
axe substantially greater than the chamber height for a chamber with a
rectangular shape),
thereby creating a thin chamber. For such a thin chamber having a rectangular
channel in
the middle, when a Garner medium is caused to travel through the thin
rectangular channel
(or called "travel through the chamber"), the velocity of the carrier medium
in the chamber
may follow a parabolic or a near-parabolic profile. The velocity of the
carrier medium at
the top and bottom walls is zero, and with increasing the distances from the
top wall or
from the bottom wall, the velocity of the carrier medium increases to a
maximum value at
the middle position between the top and bottom walls. In other embodiments,
the chamber
may be constructed so that the top wall and bottom wall are of a much smaller
magnitude
than the side walls (e.g., both chamber length and chamber height are
substantially greater
than the chamber width for a chamber with a rectangular shape), again creating
a thin
chamber. In addition to the rectangular shape of the chamber, the chamber may
be of
circular construction, elliptical, triangular, hexadecagonal, or of other
geometrical shapes.
The chamber may be constructed having a top wall, a bottom wall, and a gasket
or a spacer
between the top and bottom wall. The gasket or spacer may be cut in the middle
to form
rectangular thin channel with taper ends. Alternatively, the gasket or spacer
may be cut in
the middle to form thin channels of other shapes such as ellipse, circle, or
any other shape:
In the case of a thin rectangular channel where the top and bottom walls on
two different
planes are parallel to each other, the velocity of the carrier medium in the
medium may
follow a parabolic or a near-parabolic velocity profile. The velocity of the
carrier medium
at the top and bottom walls is zero, and with increasing the distances from
the top wall or
from the bottom wall, the velocity of the carrier medium increases to a
maximum value at
the middle position between the top and bottom walls. Preferably, for a
rectangular
channel, the width of the channel is from about 1 mm to about 20 cm, and the
thickness of
the channel is from about 20 micron to about 10 mm, and the length of the
channel is from
about 1 cm to about 200 cm, preferably from about 10 cm to about 50 cm. As
such, the
present invention is. not intended to be limited to a particular geometric
shape and the
chamber may be constructed of many different materials, for example, glass,
polymeric
material, plastics, quartz, coated metal, or the like, provided that the
chamber has such
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structural characteristics that when a carrier medium is caused to travel
through the
chamber, the velocity of the medium at different positions in the chamber is
different.
The apparatus can comprise two, or more than two electrode elements. Each of
the
electrode elements can be individually connected to one of a plurality of
electrical
conductor buses electrically connected to the electrical signal generator. The
electrode
elements can be adapted substantially longitudinally or latitudinally along a
portion of the
chamber. The electrode elements can be adapted along the interior or exterior
surface of
the chamber. The electrode elements can be configured on a plane substantially
parallel to
traveling direction of the carrier medium caused to travel through said
chamber.
Preferably, the electrode elements configured on a plane form an electrode
array. The
electrode array may be an interdigitated electrode array, interdigitated
castellated electrode
array, interdigitated electrode array with arc-shape tip extensions.
Preferably, the electrode
element is a metal layer, e.g., a gold layer, coated on a surface of the
chamber. Other
metals such as platinum, aluminum, chromium, titanium, copper and silver may
also be
used.
The electrical signal generator for energizing the electrode element to create
the
electrophoretic force may be a DC signal source capable of varying magnitude
of DC
voltage, or may be an AC signal source capable of varying magnitude and
frequency, of
electrical signals. Preferably, the electrical signal for energizing the
electrode element to
create the electrophoretic force is a direct current (DC) electrical signal or
a low-frequency-
alternating current (AC) signal.
The apparatus can comprise a single piezoelectric transducer or comprise a
plurality of piezoelectric transducers. The plurality of piezoelectric
transducers may be
energized via common electrical signals or via different electrical signals.
The plurality of
piezoelectric transducers can be adapted along the interior or exterior
surface of the
chamber. The plurality of piezoelectric transducers can also be configured on
a plane
substantially parallel to traveling direction of the carrier medium that is
caused to travel
through the chamber.
Preferably, the electrical signal generator for energizing the piezoelectric
transducer
to create the acoustic force is capable of varying magnitude and frequency of
said electrical
signals.
Common electrical conductor buses may be used to connect a plurality of
electrode
elements to the signal generator. The common electrical conductor buses may be
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fabricated by the same process as the fabricated electrode elements in the
apparatus, or may
be one or more conducting assemblies, such as a ribbon conductor, metallized
ribbon or
metallized plastic. For an interdigitated electrode array, alternating
electrode elements
along the array may be connected together so as to receive electrical signals
from the signal
generator. The electrical generator may be a DC voltage supply capable of
generating
voltages of varying magnitude. Voltage signals desired for the apparatuses and
methods of
the present invention are in the range of about 0 to about 15 volts, and more
preferably
between about 0 to about 2 volts. The signal generator may be a signal
generator capable
of generating signals of varying voltage and frequency and may be, for
example, a function
generator, such as a Hewlett Packard generator Model No. 8116A. Signals
desired for the
apparatuses and methods of the present invention are in the range of about 0
to about 15
volts, and about 0.1 Hz to about 100 kHz, and more preferably between about 0
to about 2
volts, and about 0.1 Hz to 1 kHz.
In a preferred embodiment, the chamber of the apparatus comprises a tube. The
electrode element and/or the piezoelectric transducer, or a plurality thereof,
can be adapted
along the interior surface of the tube. Alternatively, the electrode element
and/or the
piezoelectric transducer, or a plurality thereof, can be adapted along the
exterior surface of
the tube.
In another preferred embodiment, the chamber comprises a top wall, a bottom
wall,
and two side walls. The electrode element andlor the piezoelectric transducer,
or a plurality
thereof, can be configured on the top wall of the chamber. Alternatively, the
electrode
element and/or the piezoelectric transducer, or a plurality thereof, can be
configured on the
bottom wall of the chamber. In another configuration, the electrode element
and/or the
piezoelectric transducer, or a plurality thereof, can be adapted on opposing
surfaces of the
chamber. Preferably, the chamber height between the top and bottom walls is
about half
wavelength of the standing acoustic wave.
In still another preferred embodiment, the apparatus consists essentially of,
or
consists of: a) a chamber having at least one inlet port and at least one
outlet port, said
chamber having such structural characteristics that when a carned medium is
caused to
travel through said chamber, the traveling velocity of said carried medium at
various
positions within said chamber is different; b)at least two electrode elements
adapted along
a portion of said chamber, wherein said electrode elements can be energized
via at least one
electrical signal provided by an electrical signal generator to create an
electrical field,
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thereby causing at least one electrophoretic force having components normal to
the
traveling direction of said Garner medium on a matter in said carrier medium;
and c) at
least one piezoelectric transducer adapted along a portion of said chamber,
wherein said
piezoelectric transducer can be energized via at least one electrical signal
provided by an
electrical signal generator to create an acoustic wave, thereby causing at
least one acoustic
force having components normal to the traveling direction of said carrier
medium on a
matter in said carrier medium.
C. Apparatuses using dielectrophoretic (DEP) and acoustic forces
(acoustic-DEP-FFF apparatuses)
In another aspect, the present invention provides an apparatus for the
discrimination
of a matter utilizing dielectrophoretic and acoustic forces in field flow
fractionation, which
apparatus comprises: a) a chamber having at least one inlet port and at least
one outlet port,
said chamber having such structural characteristics that when a carried medium
is caused to
travel through said chamber, the traveling velocity of said carried medium at
various
positions within said chamber is different; b) at least two electrode elements
adapted along
a portion of said chamber, wherein said electrode elements can be energized
via at least one
electrical signal provided by an electrical signal generator to create a non-
uniform electrical
field, thereby causing at least one dielectrophoretic force having components
normal to the
traveling direction of said carrier medium on a matter in said Garner medium;
and c) at least
one piezoelectric transducer adapted along a portion of said chamber, wherein
said
piezoelectric transducer can be energized via at least one electrical signal
provided by an
electrical signal generator to create an acoustic wave, thereby causing at
least one acoustic
force having components normal to the traveling direction of said Garner
medium on a
matter in said carrier medium.
The apparatus can have a single inlet port and a single outlet port.
Alternatively,
the apparatus can have a plurality of inlet andlor outlet ports. Preferably,
the outlet port is
connected to a collection device or a characterization device. The outlet port
of the
chamber according to the present invention may take many forms. Specifically,
the outlet
port may be a single port, or a plurality of ports, or an array of ports. The
outlet port, for
example, may be located along the entire width or a part of the width of the
chamber. The
outlet port may be adapted to receive matter of various shapes and sizes. For
example, the
size of the outlet port may vary from approximately twice the size of the
matter to be
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discriminated to the entire width of the chamber. In one embodiment, the
outlet port may
be constructed of one or more tubing elements, such as TEFLON tubing. The
tubing
elements may be combined to provide an outlet port. Further, for example, the
outlet port
may be connected to fraction collectors or collection wells that are used to
collect separated
matter. Other components that may be connected to the apparatus of the present
invention
are, for example, measurement or diagnostic equipment, such as cytometers,
particle
counters and spectrometers. Other devices or apparatus used for further assay
or analyses
on the separated matters may also be connected to the apparatus of the present
invention.
The chamber of the apparatus should be designed to have such structural
characteristics that when a fluid (liquid or gas) is caused to travel through
the chamber, the
velocity of the fluid (liquid or gas) at various positions within said chamber
is different and
the fluid (liquid or gas) travels through the chamber according to a velocity
profile. For
example, the chamber may be rectangular in shape and may include, for example,
a top
wall, bottom wall and two side walls. The top wall and a bottom wall may be
parallel to
each other, or substantially parallel to each other, and the distance between
the top wall and
the bottom is referred to as chamber height. The distance between the inlet
port and outlet
is referred to as chamber length when the chamber comprises one inlet port and
one outlet
port. The two side walls may be parallel to each other, or substantially
parallel to each
other, and the distance between the two side walls of the chamber is referred
to as chamber
width. The two side walls may be parts of a gasket or a spacer between the top
wall and
bottom wall. The gasket or spacer may be cut in the middle to form a
rectangular thin
channel with taper ends. Alternatively, the gasket or spacer may be cut in the
middle to
form thin channels of other shapes such as ellipse, circle, or any other
shape. In certain
embodiments, the chamber may be constructed so that the top wall and bottom
wall are of a
much greater magnitude than the side walls (e.g., both chamber length and
chamber width
are substantially greater than the chamber height for a chamber with a
rectangular shape),
thereby creating a thin chamber. For such a thin chamber having a rectangular
channel in
the middle, when a carrier medium is caused to travel through the thin
rectangular channel
(or called "travel through the chamber"), the velocity of the carrier medium
in the chamber
may follow a parabolic or a near-parabolic profile. The velocity of the
carrier medium at
the top and bottom walls is zero, and with increasing the distances from the
top wall or
from the bottom wall, the velocity of the carrier medium increases to a
maximum value at
the middle position between the top and bottom walls. In other embodiments,
the chamber
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may be constructed so that the top wall and bottom wall are of a much smaller
magnitude
than the side walls (e.g., both chamber length and chamber height are
substantially greater
than the chamber width for a chamber with a rectangular shape), again creating
a thin
chamber. In addition to the rectangular shape of the chamber, the chamber may
be of
circular construction, elliptical, triangular, hexadecagonal, or of other
geometrical shapes.
The chamber may be constructed having a top wall, a bottom wall, and a gasket
or a spacer
between the top and bottom wall. The gasket or spacer may be cut in the middle
to form
rectangular thin channel with taper ends. Alternatively, the gasket or spacer
may be cut in
the middle to form thin channels of other shapes such as ellipse, circle, or
any other shape.
In the case of a thin rectangular channel where the top and bottom walls on
two different
planes are parallel to each other, the velocity of the carrier medium in the
medium may
follow a parabolic or a near-parabolic velocity profile. The velocity of the
Garner medium
at the top and bottom walls is zero, and with the increasing distances from
the top wall or
from the bottom wall, the velocity of the carrier medium increases to a
maximum value at
the middle position between the top and bottom walls. Preferably, for a
rectangular
channel, the width of the channel is from about 1 mm to about 20 cm, and the
height of the
channel is from about 20 micron to about 10 mm, and the length of the channel
is from
about 1 cm to about 200 cm, preferably, from about 10 cm to about 50 cm. As
such, the
present invention is not intended to be limited to a particular geometric
shape and the
chamber may be constructed of many different materials, for example, glass,
polymeric
material, plastics, quaxtz, coated metal, or the like, provided that the
chamber has such
structural characteristics that when a carrier medium is caused to travel
through the
chamber, the velocity of the medium at different positions in the chamber is
different.
The apparatus can comprise two, or more than two electrode elements. Each of
the
electrode elements can be individually connected to one of a plurality of
electrical
conductor buses electrically connected to the electrical signal generator. The
electrode
elements can be adapted substantially longitudinally or latitudinally along a
portion of the
chamber. The electrode elements can be adapted along the interior or exterior
surface of
the chamber. The electrode elements can be configured on a plane substantially
parallel to
traveling direction of the carrier medium caused to travel through said
chamber.
Preferably, the electrode elements configured in a plane form an electrode
array. The
electrode array may be an interdigitated electrode axray, interdigitated
castellated electrode
array, interdigitated electrode array with arc-shape tip extensions.
Preferably, the electrode
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element is a metal layer, e.g., a gold layer, coated on a surface of the
chamber. Other
metals such as platinum, aluminum, chromium, titanium, copper and silver may
also be
used.
The electrical signal generator for energizing the electrode element to create
the
dielectrophoretic force may be an AC signal source capable of varying
magnitude and
frequency of electrical signals.
The apparatus can comprise a single piezoelectric transducer or comprise a
plurality of piezoelectric transducers. The plurality of piezoelectric
transducers may be
energized via common electrical signals or via different electrical signals.
The plurality of
piezoelectric transducers can be adapted along the interior or exterior
surface of the
chamber. The plurality of piezoelectric transducers can also be configured on
a plane
substantially parallel to traveling direction of the Garner medium that is
caused to travel
through the chamber.
Preferably, the electrical signal generator for energizing the piezoelectric
transducer
to create the acoustic force is capable of varying magnitude and frequency of
said electrical
signals.
Common electrical conductor buses may be used to connect a plurality of
electrode
elements to the signal generator. The common electrical conductor buses may be
fabricated by the same process as the fabricated electrode elements in the
apparatus, or may
~0 be one or more conducting assemblies, such as a ribbon conductor,
metallized ribbon or
metallized plastic. For an interdigitated electrode array, alternating
electrode elements
along the array may be connected together so as to receive electrical signals
from the signal
generator. The electrical generator may be a DC voltage supply capable of
generating
voltages of varying magnitude. Voltage signals desired for the apparatuses and
methods of
2,5 the present invention are in the range of about 0 to about 15 volts, and
more preferably
between about 0 to about 10 volts. The signal generator may be a signal
generator capable
of generating signals of varying voltage and frequency and may be, for
example, a function
generator, such as a Hewlett Packard generator Model No. 8116A. Signals
desired for the
apparatuses and methods of the present invention are in the range of about 0
to about 15
30 volts, and about 0.1 Hz to about 500 MHz, and more preferably between about
0 to about
10 volts, and about 0.1 kHz to 10 MHz. These frequencies are exemplary only,
as the
frequency required for matter discrimination using dielectrophoresis forces is
dependent
upon the conductivity of, for example, the cell suspension medium. Further,
the desired
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frequency is dependent upon the characteristics of the matter to be
discriminated. The
discrimination obtained depends on the shape, size and configuration of the
electrode
elements, for example. In an exemplary embodiment, the signals are sinusoidal,
however it
is possible to use signals of any periodic or aperiodic waveform. The
electrical signals may
be developed in one or more electrical signal generators that are capable of
varying voltage
and frequency of electrical signals.
In a preferred embodiment, the chamber of the apparatus comprises a tube. The
electrode element and/or the piezoelectric transducer, or a plurality thereof,
can be adapted
along the interior surface of the tube. Alternatively, the electrode element
andlor the
piezoelectric transducer, or a plurality thereof, can be adapted along the
exterior surface of
the tube.
In another preferred embodiment, the chamber comprises a top wall, a bottom
wall,
and two side walls. The electrode element and/or the piezoelectric transducer,
or a plurality
thereof, can be configured on the top wall of the chamber. Alternatively, the
electrode
element and/or the piezoelectric transducer, or a plurality thereof, can be
configured on the
bottom wall of the chamber. In another configuration, the electrode element
and/or the
piezoelectric transducer, or a plurality thereof, can be adapted on opposing
surfaces of the
chamber. Preferably, the chamber height between the top and bottom walls is
about half
wavelength of the standing acoustic wave.
In still another preferred embodiment, the apparatus consists essentially of,
or
consists of a) a chamber having at least one inlet port and at least one
outlet port, said
chamber having such structural characteristics that when a carried medium is
caused to
travel through said chamber, the traveling velocity of said carried medium at
various
positions within said chamber is different; b) at least two electrode elements
adapted along
a portion of said chamber, wherein said electrode elements can be energized
via at least one
electrical signal provided by an electrical signal generator to create an
electrical field,
thereby causing at least one dielectrophoretic force having components normal
to the
traveling direction of said earner medium on a matter in said carrier medium;
and c) at least
one piezoelectric transducer adapted along a portion of said chamber, wherein
said
piezoelectric transducer can be energized via at least one electrical signal
provided by an
electrical signal generator to create an acoustic wave, thereby causing at
least one acoustic
force having components normal to the traveling direction of said earner
medium on a
matter in said carrier medium.
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D. Methods of discriminating a matter using acoustic force, or acoustic force
with electrophoretic or dielectrophoretic force
In still another aspect, the present invention provides a "continuous-mode"
method
of discriminating a matter using acoustic forces in field flow fractionation,
which method
comprises: a) obtaining an apparatus described in Section A; b) introducing a
carrier
medium containing a matter to be discriminated into the chamber of the
apparatus via its
inlet port, wherein said introducing causes the carrier medium to travel
through the
chamber according to a velocity profile; c) applying at least one electrical
signal provided
by an electrical signal generator to the piezoelectric transducer, wherein
said energized
piezoelectric transducer creates an acoustic wave, thereby causing at least
one acoustic
force on said matter having components normal to the traveling direction of
said carnet
medium travelling through said chamber; whereby said matter is displaced to a
position
within said carrier medium along a direction normal to the traveling direction
of said carrier
medium travelling through said chamber and discriminated according to its
position within
said carrier medium along the direction normal to the traveling direction of
said carnet
medium travelling through said chamber. This is a continuous mode of acoustic-
field-
flow-fractionation (acoustic-FFF). It can be used with any apparatus described
in Section
A.
In still another aspect, the present invention provides a "batch-mode" method
of
discriminating a matter using acoustic forces in field flow fractionation,
which method
comprises: a) obtaining an apparatus described in Section A; b) loading a
carnet medium
into the chamber of apparatus via its inlet port until the chamber is filled
with the carrier
medium; c) delivering, e.g., injecting, a sample that contains a matter to be
discriminated
into the carrier medium in the chamber; d) applying at least one electrical
signal provided
by an electrical signal generator to the piezoelectric transducer, wherein
said energized
piezoelectric transducer creates an acoustic wave, thereby causing at least
one acoustic
force on said matter; e) introducing the carrier medium into the chamber of
the apparatus
via its inlet port, wherein said introducing causes the carrier medium to
travel through the
chamber according to a velocity profile; whereby said matter is displaced to a
position
within said carrier medium along a direction normal to the traveling direction
of said Garner
medium travelling through said chamber and discriminated according to its
position within
said carrier medium along the direction normal to the traveling direction of
said carrier
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medium travelling through said chamber. This "batch-mode" of acoustic-FFF can
be used
with any apparatus described in Section A.
In the above "batch-mode" method of acoustic-FFF, preferably, prior to the
introducing of Garner medium into the chamber that causes the carrier medium
to travel
through the chamber according to a velocity profile (step e), applying
electrical signal to
the piezoelectric transducer to cause acoustic force on said matter results in
the matter
being displaced into an equilibrium position along a direction normal to the
traveling
direction of the carrier medium traveling through the chamber.
In still another aspect, the present invention provides a "continuous-mode"
method
of discriminating a matter using electrophoretic and acoustic forces in field
flow
fractionation, which method comprises: a) obtaining an apparatus described in
above
Section B; b) introducing a carrier medium containing a matter to be
discriminated into the
apparatus via its inlet port, wherein said introducing causes the carrier
medium to travel
through the chamber according to a velocity profile; c) applying at least one
electrical
signal provided by an electrical signal generator to the electrode elements,
wherein said
energized electrode elements creates an electrical field, thereby causing at
least one
electrophoxetic force on said matter having components normal to the traveling
direction of
said carrier medium travelling through said chamber; and d) applying at least
another
electrical signal provided by an electrical signal generator to the
piezoelectric transducer,
wherein said energized piezoelectric transducer creates an acoustic wave,
thereby causing
at least one acoustic force on said matter having components normal to the
traveling
direction of said carrier medium travelling through said chamber; whereby said
matter is
displaced to a position within said carrier medium along a direction normal to
the traveling
direction of said caxrier medium travelling through said chamber and
discriminated
according to its position within said Garner medium along the direction normal
to the
traveling direction of said carrier medium travelling through said chamber.
Any of the
apparatuses described in Section B can be used in the present method of the
continuous-
mode of acoustic-electrophoretic-field-flow-fractionation (acoustic-E-FFF).
In yet another aspect, the present invention provides a "batch-mode" method of
discriminating a matter using electrophoretic and acoustic forces in field
flow fractionation,
which method comprises: a) obtaining an apparatus described in above Section
B; b)
loading a carrier medium into the chamber of apparatus via its inlet port
until the chamber
is filled with the carrier medium; c) delivering, e.g., injecting, a sample
that contains a
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matter to be discriminated into the Garner medium in the chamber; d) applying
at least one
electrical signal provided by an electrical signal generator to the electrode
element, wherein
said energized electrode element creates an electrical field, thereby causing
at least one
electrophoretic force on said matter; e) applying at least another electrical
signal provided
by an electrical signal generator to the piezoelectric transducer, wherein
said energized
piezoelectric transducer creates an acoustic wave, thereby causing at least
one acoustic
force on said matter; f) introducing the carrier medium into the chamber of
the apparatus
via its inlet port, wherein said introducing causes the carrier medium to
travel through the
chamber according to a velocity profile; whereby said matter is displaced to a
position
within said carrier medium along a direction normal to the traveling direction
of said carrier
medium travelling through said chamber and discriminated according to its
position within
said carrier medium along the direction normal to the traveling direction of
said carrier
medium travelling through said chamber. Any of the apparatuses described in
Section B
can be used in the present method of the "batch-mode" of acoustic-E-FFF.
In the above described "batch-mode" of acoustic-E-FFF method, preferably,
prior to
the introducing of Garner medium into the chamber that causes the carrier
medium to travel
through the chamber according to a velocity profile (step fj, applying
electrical signal to
the electrode element to cause electrophoretic force on said matter and
applying electrical
signal to the piezoelectric transducer to cause acoustic force on said matter
result in the
matter being displaced into an equilibrium position along a direction normal
to the
traveling direction of the carrier medium traveling through the chamber.
In yet another aspect, the present invention provides a "continuous-mode"
method
of discriminating a matter using dielectrophoretic and acoustic forces in
field flow
fractionation, which method comprises: a) obtaining an apparatus described in
the above
.Section C ; b) introducing a carrier medium containing a matter to be
discriminated into the
apparatus via its inlet port, wherein said introducing causes the Garner
medium to travel
through the chamber of the apparatus according to a velocity profile; c)
applying at least
one electrical signal provided by an electrical signal generator to the
electrode element,
wherein said energized electrode element creates a non-uniform electrical
field, thereby
causing at least one dielectrophoretic force on said matter having components
normal to the
traveling direction of said carrier medium travelling through said chamber;
and d) applying
at least another electrical signal provided by an electrical signal generator
to the
piezoelectric transducer, wherein said energized piezoelectric transducer
creates an acoustic
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wave, thereby causing at least one acoustic force on said matter having
components normal
to the traveling direction of said Garner medium travelling through said
chamber; whereby
said matter is displaced to a position within said carrier medium along a
direction normal to
the traveling direction of the carrier medium traveling through the chamber
and
discriminated according to its position within said carrier medium along the
direction
normal to the traveling direction of the carrier medium traveling through the
chamber. Any
apparatus described in Section C can be used in the present method of the
"continuous-
mode" of acoustic-dielectrophoretic-field-flow-fractionation (acoustic-DEP-
FFF).
In yet another aspect, the present invention provides a "batch-mode" method of
discriminating a matter using dielectrophoretic and acoustic forces in field
flow
fractionation, which method comprises: a) obtaining an apparatus described in
above
Section C; b) loading a carrier medium into the chamber of apparatus via its
inlet port until
the chamber is filled with the carrier medium; c) delivering, e.g., injecting
a sample that
contains a matter to be discriminated into the carrier medium in the chamber;
d) applying at
1 S least one electrical signal provided by an electrical signal generator to
the electrode
element, wherein said energized electrode element creates a non-uniform
electrical field,
thereby causing at least one dielectrophoretic force on said matter; e)
applying at least
another electrical signal provided by an electrical signal generator to the
piezoelectric
transducer, wherein said energized piezoelectric transducer creates an
acoustic wave,
thereby causing at least one acoustic force on said matter; f) introducing the
carrier medium
into the chamber of the apparatus via its inlet port, wherein said introducing
causes the
carrier medium to travel through the chamber according to a velocity profile;
whereby said
matter is displaced to a position within said carrier medium along a direction
normal to the
traveling direction of said carrier medium travelling through said chamber and
discriminated according to its position within said Garner medium along the
direction
normal to the traveling direction of said carrier medium travelling through
said chamber.
Any of the apparatuses described in Section C can be used in the present
method of "batch-
mode" of acoustic-DEP-FFF.
In the above described "batch-mode" of acoustic-DEP-FFF method, preferably,
prior to the introducing of carrier medium into the chamber that causes the
carrier medium
to travel through the chamber according to a velocity profile (step f),
applying electrical
signal to the electrode element to cause dielectrophoretic force on said
matter and applying
electrical signal to the piezoelectric transducer to cause acoustic force on
said matter result
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in the matter being displaced into an equilibrium position along a direction
normal to the
traveling direction of the carrier medium traveling through the chamber
In the above acoustic-E-FFF or acoustic-DEP-FFF methods, identical, but
preferably, different electric signals can be used to generate the acoustic
force and the
electrophoretic or the dielectrophoretic force.
In the above acoustic-E-FFF or acoustic-DEP-FFF method, the acoustic force and
the electrophoretic or the dielectrophoretic force can be generated
simultaneously or'
sequentially.
The above acoustic-FFF, acoustic-E-FFF and acoustic-DEP-FFF methods can
fiuther comprise a step of discriminating the matter according to the velocity
profile of
carrier medium travelling through the chamber and the matter moves within the
chamber at
velocities dependent on its displacement within the velocity profile.
The above acoustic-FFF, acoustic-E-FFF and acoustic-DEP-FFF methods can
further comprise a step of displacing the discriminated matter from the
apparatus, and
preferably results in the separation of the discriminated matter from each
other. After
being displaced within the carrier medium travelling through the chamber of
the present
invention, the displaced matter may exit from the outlet port or ports at a
time dependent on
the displacement of the matter within the velocity profile of the carrier
medium traveling
through the chamber. Specifically, matter at different levels of displacement
within the
velocity profile travels at different speeds. Therefore, the displaced matter
is discriminated
by its displacement within the velocity profile and by its traveling speed.
Matter that is
displaced to different positions within the velocity profile travels at
different velocities and
exit from the outlet port or ports of the chamber at different times.
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This velocity profile may be, for example, a hydrodynamic fluid profile such
as a
parabolic flow profile. For a chamber of rectangular shape, the chamber
structural
characteristics is defined by the chamber length, chamber width and chamber
height. The
velocity profile may be determined by knowing the flow rate of the fluid, and
the chamber
width, height and length. Fox example, for a rectangular chamber with the
chamber length
and width being substantially greater than the chamber height, a laminar flow
may be
established in the chamber. The velocity of the carrier medium at different
positions is
mainly determined by its distance from the chamber bottom walls, and the
velocity profile
is an approximately parabolic flow profile (or a near parabolic velocity
profile), given by,
Y",=6~T~m~H~1-H~,
where ~Y", ~ is the average velocity of the carrier medium, H is the chamber
height, V", is
the velocity of the carrier medium located at a distance z from the chamber
bottom wall.
We would like to point out that the above parabolic velocity profile is only
an
approximation of the velocity profile under the condition that the chamber
length and width
in a rectangular chamber is much greater than the chamber height. That is why
we use the
term "near parabolic" profile or "near parabolic" profile for describing such
velocity
profile. Along the chamber width direction, the parabolic profile in the above
equation is
more accurate for the positions in the middle part across the chamber width
than for the
positions at the end regions. Similarly, along the chamber length direction,
the parabolic
profile in the above equation is more accurate for the positions in the middle
part across the
chamber length than for the positions at the end regions. The average velocity
may be
calculated according to the equation:
Average Velocity ~Ym ~ =(flow rate)/(chamber width X chamber height (or
thickness)).
Thus, the structural characteristics of the chamber that influence the
velocity profile of the
fluid flow in a rectangular chamber include: chamber width, chamber height (or
chamber
thickness) and chamber length. Chamber of different size and different
geometrical shape
will result in different velocity profile when a fluid is caused to travel
through the chamber.
Parameters that determine the velocity profile of the fluid flow include, but
are not limited
to, chamber geometrical dimensions; constrictions or expansions of the fluid
flow path
which may include, for example, those arising for a non-parallel disposition
of opposing
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chamber walls, or from the presence of suitably-placed obstructions or vanes;
surface
roughness of the chamber walls; structural features of the chamber walls that
give rise to
periodic or aperiodic modifications of the thickness of the fluid stream,
including the
electrode elements and other surface structural configurations; and the
geometrical form of
the chamber which may be, for example, rectangular, circular, wedge-shaped,
stepped, or
the like.
In the above methods, the displaced matter may exit from one of a plurality of
the
outlet ports of the chamber dependent on its displacement within the velocity
profile. . In a
preferred embodiment, the gravitational force acting on the matter acts in a
direction
normal to the traveling direction of the carrier medium in the chamber.
The present acoustic-FFF, acoustic-E-FFF and acoustic-DEP-FFF methods can be
used to discriminate any matters. In specific embodiments, matters to be
discriminated are
cells, cellular organelles, viruses, molecules or an aggregate or complex
thereof. Non-
limiting examples of discriminatable cells include animal, plant, fungus,
bacterium cultured
or recombinant cells. Non-limiting examples of discriminatable cellular
organelles include
nucleus, mitochondria, chloroplasts, ribosomes, ERs, Golgi apparatuses,
lysosomes,
proteasomes, secretory vesicles,. vacuoles or microsomes. Discriminatable
molecules can
be inorganic molecules such as ions, organic molecules or a complex thereof.
Non-limiting
examples of discriminatable ions include sodium, potassium, magnesium,
calcium,
chlorine, iron, copper, zinc, manganese, cobalt, iodine, molybdenum, vanadium,
nickel,
chromium, fluorine, silicon, tin, boron or arsenic ions. Non-limiting examples
of
discriminatable organic molecules include amino acids, peptides, proteins,
nucleosides,
nucleotides, oligonucleotides, nucleic acids, vitamins, monosaccharides,
oligosaccharides,
carbohydrates, lipids or a complex thereof. The matters to be discriminated
can be of any
size. Preferably, the dimension of the matter to be discriminated is from
about 0.01 micron
to about 1000 micron.
Brief Description of the Drawings
Figure 1. Schematic diagram of an acoustic-FFF chamber with a rectangular
channel cut in the middle. .Also shown is the operation principle of the
acoustic-FFF.
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Figure 2. Schematic diagram of an acoustic-FFF chamber with an ellipse-shaped
channel cut in the middle.
Figure 3. Schematic diagram of an ellipse-shaped acoustic-FFF chamber with a
channel cut in the middle.
Figure 4. Schematic diagram of an acoustic-FFF chamber with multiple outlet
ports
at the outlet end of the chamber.
Figure 5. Schematic diagram of an acoustic-E-FFF chamber with a rectangular
channel cut in the middle. Also shown is the operation principle of the
acoustic-E-FFF.
Figure 6. Schematic diagrams for electrode arrays that may be used for
acoustic-E-
FFF apparatus. (A) The interdigitated electrode array. (B) The interdigitated
castellated
electrode array.
Figure 7. Schematic diagram of a acoustic-DEP-FFF chamber with a rectangular
channel cut in the middle. Also shown is the operation principle of the
acoustic-DEP-FFF.
Figure 8. Schematic diagrams for electrode arrays that may be used for
acoustic
DEP-FFF apparatus. (A) The interdigitated electrode array with periodic
triangular-shaped
tips on the electrode elements. (B) The interdigitated electrode array with
periodic arc-
shaped tips on the electrode elements.
Figure 9. Schematic diagram showing the "batch mode operation principle" for
using acoustic-FFF, acoustic-E-FFF and acoustic-DEP-FFF apparatuses. (A)
Different
types of particles are displaced to different equilibrium height positions
under the influence
of the applied forces during the "relaxation" process. (B) The particles that
have been
displaced to different equilibrium positions move along the chamber at
different velocities
under influence of an established fluid flow in the chamber.
Figure 10. Schematic diagram showing the "continuous mode operation principle"
for using acoustic-FFF, acoustic-E-FFF and acoustic-DEP-FFF apparatuses.
Particles in a
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carrier medium are continuously fed into the chamber, are continuously
displaced to
different height positions in the carrier medium and exit the chamber at
different outlet
ports.
Modes of Carryin~ Out the Invention
A. Definitions
Unless defined otherwise, all technical and scientific terms used herein have
the
same meaning as is commonly understood by one of ordinary skill in the art to
which this
invention belongs. All patents, applications, published applications and other
publications
and sequences from GenBank and other data bases referred to herein in any
section of this
application are incorporated by reference in their entirety. If a definition
set forth in this
section is contrary to or otherwise inconsistent with a definition set forth
in applications,
published applications and other publications and sequences from GenBank and
other data
bases that are herein incorporated by reference, the definition set forth in
this section
prevails over the definition that is incorporated herein by reference.
As used herein, "a" or "an" means "at least one" or "one or more."
As used herein, "matter" refers to particulate matter, solubilized matter, or
any
combination thereof.
. As used herein, "electrode element (or electrode)" refers to a structure of
highly
electrically-conductive material over which an applied electrical voltage is
constant or
nearly-constant. Nearly-constant means that the voltage drop across such
electrically-
conductive structure is so small that sufficiently strong electrical field at
the regions around
the electrode elements can be produced when the electrical signals are applied
to the
electrode elements. Typically, the highly electrically-conductive materials
include metal
films (e.g., gold, platinum, titanium, chromium, etc) and semiconductor
materials such as
silicon doped with impurities (e.g., silicon doped with phosphorus, or
arsenic, or antimony,
or aluminum, or gallium, or indium), and other materials whose electrical
conductivity is
high. For this invention, highly electrically-conductive material refers to
the material
whose electrical conductivity is substantially larger than that of carrier
medium used for the
fractionation (e.g., the conductivity of the conductive material is twice or
more than twice
of that of the carrier medium). It is to be understood that these terms
include all of the
electrode configurations described in the present specification and claims.
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As used herein, "electrode array" refers to a collection of more than one
electrode
elements. In an electrode array, each individual element may be displaced in a
well-
defmed geometrical relationship with respect to one another. This array may
be, for
example, an interdigitated electrode array, an interdigitated castellated
array, a polynomial
array, an interdigitated electrode array having periodic triangular-shaped
tips on the
electrode elements (or arc-like tips, or rectangular tips), or the like.
"Electrode array" may
also include multiple electrode elements that have same or different
geometrical shapes.
As used herein, "normal to the traveling direction" of the carrier medium
travelling
through the chamber refers to a direction which is substantially non-opposing
and
substantially nonlinear to the flow direction of the carrier medium traveling
through the
chamber. For example, when a carrier medium is caused to flow along the
chamber length
direction of a rectangular chamber, the "normal to the traveling direction"
may be a
direction across the chamber width, or across chamber height, or any direction
in a plane
parallel to the plane defined by the chamber width and chamber height, or any
other
direction that is non-opposing to the flow direction of the carrier medium.
Ordinarily, the
angle between the travelling direction and the "normal to the traveling
direction" is from
about 45 degree to about 135 degree. Preferably, the angle between the
travelling direction
and the "normal to the traveling direction" is from about 80 degree to about
100 degree.
More preferably, the angle between the travelling direction and the "normal to
the traveling
direction" is from about 85 degree to about 95 degree.
As used herein, "fraction collectors (or collection wells or collection
devices)"
refers to storage and collection devices for discretely retaining the
separated and/or
discriminated and/or displaced matter.
As used herein, "characterization device" refers to any device that is capable
of
characterizing the separated andlor displaced and/or discriminated matter.
Characterization
device may be a particle counter that counts the particles and record the
particle number
and arrival time as they exit the chamber. Characterization device could be an
assay device
that is capable of performing further assay or analysis on separated matter.
As used herein, "piezoelectric transducer" refers a structure of
"piezoelectric
material" that can produce an electrical field when exposed to a change in
dimension
caused by an imposed mechanical force, and that can be energized by an applied
electrical
signal to produce mechanical stress in the materials. In the present
invention, we apply AC
electrical signals to the piezoelectric transducer and produce alternating
mechanical stress
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in the material, that is coupled as an acoustic wave into the carrier medium
used for
fractionation and discrimination of matter.
As used herein, "structural characteristics of the chamber" refers to the
structural
properties of the chamber, including, but not limited to, the chamber
geometrical shape,
chamber dimensions, structure and composition of each of chamber components
(e.g.; top
wall, bottom wall, side walls).
As used herein, "the traveling velocity of said carned medium at various
positions
within said chamber is different" means that the structural characteristics of
the chamber is
designed or chosen so that the traveling velocities of said carried medium at
least two
positions within said chamber are different. It is not necessary that the
traveling velocities
of said carned medium at all positions within said chamber are different. In
many cases, it
is sufficient that the traveling velocities of the carned medium at a certain
height (or
width), or within a certain plane normal to the travelling direction, is
identical, but is
different from the traveling velocities of the carried medium at another
height (or width), or
within another plane normal to the travelling direction
As used herein, "matter is displaced to a position along a direction" means
that the
matter is caused to move to a position along a direction of interest under the
influence of
forces exerting on matter. Here the positions axe identified as locations or
points along the
direction. For example, in a rectangular chamber comprising a top wall and a
bottom wall
that are separated by a thin gasket that is cut in the middle to form a thin,
rectangular
channel, a carrier medium containing the matter to be discriminated is caused
to move
along the channel length direction. Electrode elements and piezoelectric
transducers are
adapted on the chamber top and/or bottom walls. When electrical signals are
applied to the
electrode elements and piezoelectric transducers, acoustic forces and
dielectrophoretic
forces are produced on the matter that is placed in the carrier medium. These
forces have
components along the vertical direction, which are normal to the traveling
direction of the
chamber. These force components will cause the matter to move to various
positions along
the vertical direction. ~ For example, the matter may be iutially located
close to the chamber
bottom wall and may be caused to move to certain heights from the chamber
bottom wall
when the electrical signals are applied to energize electrode elements and
piezoelectric
transducers. In the present invention, the forces that influence the positions
of "matter"
include acoustic forces, electrophoretic forces, dielectrophoretic forces,
gravitational
forces, hydrodynamic lifting forces, thermal diffusion forces. For the matter
whose
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positions are influenced by the thermal diffusion effects, the positions of
"matter" along a
direction refer to a distribution profile or a concentration profile of the
matter along the ~ .
direction. For the matter whose positions are not influenced by the thermal
diffusion
effects, the positions of "matter" along a direction refer to the locations of
"matter" along
the direction.
As used herein; "displacement within the velocity profile" refers to the
displacement of matter within the velocity profile of the carrier medium
traveling through
the chamber. Here the displacement of the matter is identified within the
frame of the
velocity profile. The matter displaced to the fast-moving part of the velocity
profile may be
caused to move faster than the matter displaced to the slow-moving part of the
velocity
profile. For the matter whose positions are influenced by the thermal
diffusion of the
matter, the displacement of "matter" within the velocity profile refers to a
re-distribution
profile of the matter within the reference frame of the velocity profile. For
the matter
whose positions are not influenced by the thermal diffusion of the matter, the
displacement
of "matter" within the velocity profile refers to the displacement of "matter"
within the
reference frame of the velocity profile.
As used herein, "the matter being displaced into an equilibrium position along
a
direction" refers to the matter being caused to move to an equilibrium
position along a
direction of interest under the influence of forces exerting on matter. Here
the equilibrium
positions are identified as locations or points along the direction. The
equilibrium positions
refer to the positions at which the net force on the matter is zero, or almost
zero so that the
matter will remain on such equilibrium positions. For the matter whose
positions are
influenced by the thermal diffusion of the matter, the equilibrium position of
"matter"
along a direction refers to an equilibrium distribution profile of the matter
along the
direction. For the matter whose positions are not influenced by the thermal
diffusion of the
matter, the equilibrium position of "matter" along a direction refers to the
location of
"matter" along the direction when the matter is at force equilibrium.
As used herein, "the chamber's length is substantially greater than its width
or
height" means that, regardless of the actual chamber shape, the characteristic
length of the
chamber is at least twice as long as the characteristic width or height of the
chamber.
Preferably, the characteristic length of the chamber is at least three-times
as long as the
characteristic width or height of the chamber. More preferably, the
characteristic length of
the chamber is at least five-times as long as the characteristic width or
height of the
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chamber. Although the width and height of the chamber can be identical or
roughly
identical, it is preferable that one it substantially longer than the other,
e.g., the width being
substantially longer than the height, or vice vef sa. For example, the
characteristic width of
the chamber can be at least twice, three-times or five-times, as long as the
characteristic
height, or vice versa.
For clarity of disclosure, and not by way of limitation, the detailed
description of
the invention is divided into the subsections that follow.
B. Exemplary apparatuses
B.1. Acoustic-FFF chamber.
Figure 1 shows an embodiment of acoustic-FFF chamber and the operational
principle of acoustic-FFF. The chamber has a top wall 10 and a bottom wall 20.
The top
wall and bottom wall are separated by a gasket or spacer 30 that has a
rectangular channel
40 cut in it. The channel 40 has tapered ends. For clarity, the top wall 10,
the gasket 30
and the bottom wall 20 are shown separated from each other. In use, these
components are
bound to each other to form an acoustic-FFF chamber. An inlet port 50 and an
outlet port
60 are located on the top wall and bottom wall, at the inlet end and outlet
end of the
chamber, respectively. The inlet port 50 is connected with an infusion device
70 that can
introduce carrier medium and introduce the matter to be discriminated into the
chamber.
The infusion device may be a syringe pump coupled with an injection valve
(Wang et al.,
1998; Huang et al., 1999; Yang et al., 1999). The outlet port 60 is connected
with a
collection or characterization device 80 that is capable of characterizing the
matter that has
been separated and discriminated after the acoustic-FFF process. The
collection or
characterization device may be a particle counter, a flow cytometer or a
fractionation
collector.
In the exemplary figure, the whole bottom wall 20 is a piezoelectric
transducer.
The top surface 90 and bottom surface 100 of the bottom wall 20 has been
coated with
metal films or other electrically conductive material. AC electrical signals
from a signal
generator 110 can be applied to the top surface 90 and bottom surface 100 of
the
piezoelectric transducer 20 to energize the piezoelectric transducer to
produce an acoustic
wave in the chamber in the direction normal to surfaces 90 and 100. The
acoustic wave
transmitted from the piezoelectric transducer is reflected back by the top
wall 10. The
superimposition of the transmitted wave from the piezoelectric transducer and
the reflected
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wave from the top wall form the total acoustic wave field in the chamber. The
total
acoustic wave field may have two components, i.e., standing-wave component and
traveling wave component. The ratio of the magnitude of the standing-wave
component to
the magnitude of the traveling-wave component is determined by the chamber
height (i.e.,
the distance between the top wall and bottom wall), the wavelength of the
acoustic wave,
the acoustic properties of the top wall 10 and the bottom wall 20, the
decaying factor for
the acoustic wave in the carrier medium. In one embodiment, the chamber height
is half
wavelength of the standing acoustic wave, and a standing acoustic wave is
established in
the chamber. An acoustic pressure node exists at the center plane of the
chamber. In
another embodiment, the chamber height is larger or smaller than half
wavelength of the
standing acoustic wave.
In the example of Figure l, the bottom wall 20 of the chamber corresponds to a
piezoelectric transducer. There may be many variations in adapting one or more
piezoelectric transducers along the portions of the chamber. The piezoelectric
transducers
may be adapted on the top and/or the bottom walls. Fox adapting the
piezoelectric
transducer on the bottom wall, the transducer may be bound to a solid plate
from the
bottom side so that the solid plate forms the bottom surface of the chamber.
The acoustic
wave may be generated from the piezoelectric transducer and be coupled into
the carrier
medium placed in the chamber through the solid plate. Similarly, for adapting
the
piezoelectric transducer on the top wall, the transducer may be bound to a
solid plate from
the top side so that the solid plate forms the top surface of the chamber. The
acoustic wave
may be generated from the piezoelectric transducer and be coupled into the
carrier medium
placed in the chamber through the solid plate. The acoustic-FFF chamber shown
in Figure
1 comprises one piezoelectric transducer in the chamber. Multiple
piezoelectric
transducers may be employed in one chamber. These transducers may be adapted
on the
top wall in series, or on bottom wall in series, or on both top and bottom
walls to form a
piezoelectric transducer array. The multiple piezoelectric transducers may be
energized by
same or different electrical signals to produce acoustic waves in the chamber.
The matter being introduced into the chamber will experience different forces
in the
chamber. We consider the case that the matter introduced is microscopic
particles and the
chamber is disposed horizontally. These forces are:
Acoustic radiation force Facousti~ 120 in the vertical direction pointing
towards or
away from the top (or bottom) wall, depending on a factor which relates to the
densities of
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the particles and the suspending medium, and to the acoustic impedance of the
of the
particles and the medium. The force Fa~oust« 120 may be a component of total
acoustic
force acting on the particle.
Gravitational force F~ 130 levitating or sedimenting the particles, depending
on the
relative magnitude of the densities of the particles and the suspending
medium.
Hdynamic liftin force FLT 140 that tends to drive the particles away from the
chamber walls. Various theoretical and experimental studies have been
conducted on such
hydrodynamic forces yet its nature remains in question (Williams et al., 1992;
1994; 1996;
1997). However, it is generally accepted that this force plays an important
role only when
the particles are very close to the chamber walls (e.g., < 5 micrometer in a
chamber of 200
micrometer thick). Some recent work in DEP-FFF (Huang et al., 1997; Wang et
al., 1998)
shows that this force plays little role in DEP-FFF operation.
These three forces are acted on the particles, driving the particles towards
equilibrium positions at which these forces balance so that the net force
acting on
individual particles in the vertical direction is zero, i.e.,
Facoustic ~Z~ ~' FLijt ~Z) - FG = ~
Particles of different properties (e.g., size, geometrical shape, density,
acoustic
impedance) equilibrate to different height positions. For example, particles
150 and 160
are displaced to different heights from the chamber bottom wall. When a fluid
flow is
introduced by infusing the carrier medium through inlet port 50 into the
chamber, a flow
velocity profile 170 is generated. The traveling direction in this case is
parallel to the
chamber top and bottom walls, and points from the chamber inlet end towards
the chamber
outlet end. The carrier medium at various positions of the chamber exhibit
different
velocities. For the example shown in Figure 1, when the chamber length (i.e.,
the length of
the channel cut in the middle) and chamber width (i.e., the width of the
channel) is
substantially greater than the chamber height (i.e., the distance between the
top wall and
bottom wall), the velocity of the carrier medium at the positions not close to
the channel
walls defined by the gasket follows an approximate parabolic velocity profile
in the vertical
direction,
Vin= = 6wm ~ ~ Cl FI
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where Y", is the velocity of the medium at a height z from the chamber bottom
wall, ~Y",
is the average velocity of the medium, H is the chamber height. Thus, a near-
parabolic
velocity profile is established along the vertical direction for the carrier
medium in such a
chamber: Thus, particles 150 and 160 may be discriminated according to the
height
positions la, and Ia2 along a vertical direction that is normal to the
traveling direction of the
carrier medium. Furthermore, because of the velocity profile, the particles
150 and 160
may be further discriminated according to the vertical positions within the
velocity profile.
Even furthermore, the particles 150 and 160 are caused to travel across the
chamber at
different velocities Y, and YZ . If the particles 150 and 160 are introduced
into the chamber
at similar time, the particles 150 and 160 will exit the chamber at different
times because
they are transported through the chamber at different velocities. The
particles of different
properties (e.g.: size, density, geometry, acoustic impedance) may be
displaced to different
positions along the vertical direction, may be discriminated according to
their
displacement positions along the vertical direction or within the velocity
profile, maybe.
discriminated according to the velocities at which the particles travel
through the chamber
or according to the exit times of the particles leaving the chamber. Particles
of different
properties may be fractionated into subpopulations. Alternatively, particles
displaced to
different heights may be fractionated into sub-populations as they exit the
chamber through
different outlet ports if the different outlet ports are arranged vertically
along the outlet end
of the chamber.
In the above discussion, the chamber has been considered as being disposed
horizontally. However, the chamber may be disposed along any direction or
having any
angle with respect to the horizontal plane. In these cases, we would still
consider forces
acting on the matter to be discriminated primarily along the direction normal
to the
traveling direction of the carnet medium. The difference between these cases
and the
above case where the chamber is disposed horizontally is that the
gravitational force may
be different. In the above case, the gravitational force acts in a direction
perpendicular to
the traveling direction of the carnet medium. In the cases where the chamber
is not
disposed horizontally, the gravitational force may act in a direction not
perpendicular to the
traveling direction of the carrier medium. Thus, only a component of the
gravitational
force should be considered for analyzing the forces exerting on the matter to
be
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discriminated along a direction perpendicular to the traveling direction of
the Garner
medium.
The velocity profile in the rectangular chamber shown in Figure 1 depends on
the
structural characteristics of the chamber. When the chamber length and width
is
substantially larger than the chamber height, a parabolic or a near-parabolic
velocity profile
along the vertical direction exits in the chamber. The reason for being "near-
parabolic" is
that the velocity profile at the positions close to the gasket walls does not
follow the
"parabolic profile". When the chamber width and the chamber height are of
similar sizes,
the velocity of the Garner medium in the chamber will follow other velocity
profile than the
"parabolic velocity profile" discussed above. Furthermore, the top and the
bottom walls
have been considered as flat and parallel to each other during the above
discussions. When
the top wall and/or the bottom wall are not flat, or when the top wall and the
bottom wall
are not parallel to each other, or when the top wall or the bottom wall is
modified with
structures elements of various thickness, the velocity profile of the carrier
medium will be
different from the "near-parabolic velocity profile" described above.
To produce different velocity profile of the carrier medium, the gasket 30
between
the top wall 10 and the bottom wall 20 may be cut in the middle to form
channels of other
shapes. For example, the channel 40 in the acoustic-FFF chamber shown in
Figure 2 has an
ellipse shape. When a carrier medium is caused to travel through such a
chamber, the
velocity profile of the carrier medium will be different from that for the
chamber shown in
Figure 1. Similarly, the channel for the chamber shown in Figure 3 will result
in a unique
velocity profile for the carrier medium when it is caused to travel through
the channel.
The above discussion of the acoustic-FFF chamber has focused on the
discrimination of particles where we ignored the influence of thermal
diffusion effects. For
matter of small sizes to be discriminated in an acoustic-FFF chamber, it may
be necessary
to take into account the thermal diffusion forces. In such cases, the position
of the matter
being displaced along a direction or within a velocity profile by the applied
forces refers to
the distribution of the matter that has been influenced by the applied forces.
Such
distributions of the matter along a direction or within a velocity profile
refer to the
concentration profile or the distribution profile of the matter along the
direction or within
the velocity profile.
The acoustic-FFF chamber may have one or more inlet ports through which the
matter to be discriminated and the carrier medium are introduced. The chamber
may have
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one or more outlet ports through which the discriminated matter and the Garner
medium
may exit the chamber. The inlet and outlet ports may be located on the top
or/and bottom
walls of the chamber. The inlet and outlet ports may be holes (as small as
from about
several microns or as large as about several mm in diameter) drilled on the
chamber top
and/or bottom walls. PEEK or plastic, or metal tubing may be inserted into the
holes and
serve as the fluid connection between the chamber and the external fluid-
circuits such as
infusion devices or collection devices. Alternatively, the inlet and outlet
port may be a slot
(from about microns) to about mm in width) drilled across the chamber outlet
end.
Multiple tubing, arranged in a ribbon form, can be interfaced with such slots.
In the
exemplary chamber shown in Figure 4, a single inlet port - a hole - is located
at the bottom
wall of the chamber. The two outlet ports 180 and 190, positioned at both the
top and the
bottom walls, are the thin slots cut at the walls. A plurality of tubing
arranged in a ribbon
form is used to connect to the thin slots as the outletports. The two outlet
ports arranged at
the bottom and top walls correspond to the split-configuration employed in
many field-
flow-fractionation devices (Springston et al, 1987; Lee et al, 1989; Levin and
Giddings,
1991).
B.2. Acoustic-Electrical-FFF chamber.
Figure 5 shows an embodiment of acoustic-E-FFF chamber and the operational
principle of acoustic-E-FFF. The chamber has a top wall 210 and a bottom wall
220. The
top wall and bottom wall are separated by a gasket or spacer 230 that has a
rectangular
channel 240 cut in it. The channel 240 has tapered ends. For clarity, the top
wall 210, the
gasket 230 and the bottom wall 220 are shown separated from each other. In
use, these
components are bound to each other to form an acoustic-E-FFF chamber. An inlet
port 250
and an outlet port 260 are located on the top wall and bottom wall, at the
inlet end and
outlet end of the chamber, respectively. The inlet port 250 is connected with
an infusion
device 270 that can introduce carrier medium and introduce the matter to be
discriminated
into the chamber. The infusion device may be a syringe pump coupled with an
injection
valve (Wang et al., 1998; Huang et al., 1999; Yang et al., 1999). The outlet
port 260 is
connected with a collection or characterization device 280 that is capable of
characterizing
the matter that has been separated and discriminated after the acoustic-E-FFF
process. The
collection or characterization device may be a particle counter, a flow
cytometer or a
fractionation collector.
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In the exemplary figure 5, the whole bottom wall 220 is a piezoelectric
transducer.
The top surface 290 and bottom surface 300 of the bottom wall 220 has been
coated with
metal films or other electrically conductive material. AC electrical signals
from a signal
generator 310 can be applied to the top surface 290 and bottom surface 300 of
the
piezoelectric transducer 220 to energize the piezoelectric transducer to
produce an acoustic
wave in the chamber in the direction that is normal to the surfaces 290 and
300. The
acoustic wave transmitted from the piezoelectric transducer is reflected back
by the tap
wall 210. The superimposition of the transmitted wave from the piezoelectric
transducer
and the reflected wave from the top wall form the total acoustic wave field in
the chamber.
The total acoustic wave field may have two components, i.e., standing-wave
component
and traveling wave component. The ratio of the magnitude of the standing-wave
component to the magnitude of the traveling-wave component is determined by
the
chamber height (i. e., the distance between the top wall and bottom wall), the
wavelength of
the acoustic wave, the acoustic properties of the top wall 210 and the bottom
wall 220, the
decaying factor for the acoustic wave in the carrier medium. In one
embodiment, the
chamber height is half wavelength of the standing acoustic wave, and a
standing acoustic
wave is established in the chamber. An acoustic pressure node exists at the
center plane of
the chamber. In another embodiment, the chamber height is larger or smaller
than half
wavelength of the standing acoustic wave.
In the example of Figure 5, the bottom wall 220 of the chamber corresponds to
a
piezoelectric transducer. There may be many variations in adapting one or more
piezoelectric transducers along the portions of the chamber. The piezoelectric
transducers
may be adapted on the top andlor the bottom walls. For adapting the
piezoelectric
transducer on the bottom wall, the transducer may be bound to a solid plate
from the
bottom side so that the solid plate forms the bottom surface of the chamber.
The acoustic
wave may be generated from the piezoelectric transducer and be coupled into
the carrier
medium placed in the chamber through the solid plate. Similarly, for adapting
the
piezoelectric transducer on the top wall, the transducer may be bound to a
solid plate from
the top side so that the solid plate forms the top surface of the chamber. The
acoustic wave
may be generated from the piezoelectric transducer and be coupled into the
carrier medium
placed in the chamber through the solid plate. The acoustic-E-FFF chamber
shown in
Figure 5 comprises one piezoelectric transducer in the chamber. Multiple
piezoelectric
transducers may be employed in one chamber. These transducers may be adapted
on the
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top wall in series, or on bottom wall in series, or on both top and bottom
walls to form a
piezoelectric transducer array. The multiple piezoelectric transducers may be
energized by
same or different electrical signals to produce acoustic waves in the chamber.
In the exemplary figure 5, electrode elements employed for generating an
electric
field in the chamber correspond to the top surface 290 of the bottom wall 220,
and the
bottom surface 295 of the top wall 210. The top surface 290 of the bottom wall
220 and the
bottom surface 295 of the top wall 210 have been coated with metal films or
other
electrically conductive material. Thus, DC electrical signals or low-frequency-
AC signals
from a signal generator 315 may be applied across the surface 290 and 295 to
produce an
electric field that in the direction normal to the top wall and the bottom
wall. The matter
being introduced into such an electric field will experience electrophoretic
forces that
depend on the field strength and the effective charge of the matter.
In the example shown in figure 5, the top surface 290 of the bottom wall 220
is used
as one electrode element for generating electric field and also used as one
electrode for
energizing the piezoelectric transducer. In general, the electrode elements
for generating
the electrical field and for energizing the piezoelectric transducer may be
different.
Furthermore, the electrode elements for generating the electric field may be
covering only
portions of the bottom surface of the top wall, or the portions of the top
surface of the
bottom wall. Electrode arrays of different configurations may be utilized on
these surfaces
(i.e., the bottom surface of the top wall or the top surface of the bottom
wall). Figure 6
shows an interdigitated electrode array 400 and an interdigitated castellated
electrode array
410. The electrode elements may be adapted substantially latitudinally (as
shown in Figure
6A or Figure 6B) or longitudinally (i.e., the electrode elements in Figure 6A
are turned by
90 degree) along a portion of the chamber. Individual electrode elements in
these electrode
arrays axe connected to one of two common electrical conductor buses 405 and
408.
Electrode elements are energized to produce electric fields when electrical
signals from
signal sources are connected to such electrical conductor buses. For these
cases where
electrode arrays are used to produce electric fields, various electrical
signal connection
modes for producing electrical field and for energizing the piezoelectric
transducers may be
utilized. For example, the bottom surface of the top wall forms a conductive
plane as one
electrode element used to produce electrical field in the chamber, the bottom
wall forms a
piezoelectric transducer whose bottom surface is covered with conductive thin
film and top
surface is covered with an interdigitated electrode array such as that shown
in Figure 6A.
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The signal for producing electrical field in the chamber may be applied across
the bottom
surface of the-top wall and either one or both electrical conductor buses (405
and 408 in
Figure 6A) in the interdigitated array. The signal for producing acoustic
waves in the
chamber may be applied across the bottom surface of the bottom wall and either
one or
both electrode buses (405 and 408 in Figure 6A) in the interdigitated array.
The matter being introduced into an acoustic-E-FFF chamber will experience
different forces in the chamber. We consider the case that the matter
introduced is
microscopic particles and the chamber is disposed horizontally. Referring to
Figure 5,
these forces are:
Acoustic radiation force FQCOUStrc 320 in the vertical direction pointing
towards or
away from the top (or bottom) wall, depending on a factor which relates to the
densities of
the particles and the suspending medium, and to the acoustic impedance of the
of the
particles and the medium. The acoustic force F~~oustic 320 may be a component
of the total
acoustic radiation force acting on particles.
Electrophoretic force FE 325 in the vertical direction on the charged
particles.
Depending on whether the particles are positively or negatively charged and
depending on
the direction of the DC electrical field, this force points towards or away
from the chamber
bottom wall. The electrophoretic force FE 325 may be a component of the total
electrophoretic force acting on particles.
Gravitational force FG 330 levitating or sedimenting the particles, depending
on the
relative magnitude of the densities of the particles and the suspending
medium.
Hydrodynamic liftin force F~,ft 340 that tends to drive the particles away
from the
chamber walls. Various theoretical and experimental studies have been
conducted on such
hydrodynamic forces yet its nature remains in question (Williams et al., 1992;
1994; 1996;
1997). However, it is generally accepted that this force plays an important
role only when
the particles are very close to the chamber walls (e.g.: < 5 micrometer in a
chamber of 200
micrometer thick). Some recent work in DEP-FFF (Huang et al., 1997; Wang et
al., 1998)
shows that this force plays little role in DEP-FFF operation.
These forces are acted on the particles, driving the particles towards
equilibrium
positions at which these forces balance so that the net force acting on
individual particles in
the vertical direction is zero, i.e.,
Facousrfc (~') + FLiJt ~Z) + Fg (Z) - FG = ~
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Particles of different properties (e.g.: size, geometrical shape, density,
charge,
acoustic impedance) equilibrate to different height positions. For example,
particles 350
and 360 are displaced to different heights la, and hz from the chamber bottom
wall. When
a fluid flow is introduced by infusing the carrier medium through inlet port
250 into the
chamber, a flow velocity profile 370 is generated. The traveling direction in
this case is
parallel to the chamber top and bottom walls, and points from the chamber
inlet end
towards the chamber outlet end. The carrier medium at various positions of the
chamber
exhibit different velocities. For the example shown in Figure 5, when the
chamber length
(i.e., the length of the channel cut in the middle) and chamber width (i.e.,
the width of the
channel) is substantially greater than the chamber height (i.e., the distance
between the top
wall and bottom wall), the velocity of the Garner medium at the positions not
close to the
channel walls defined by the gasket follows an approximate parabolic velocity
profile in
the vertical direction,
Ym -6~Ym~H~1_H~
where Y", is the velocity of the medium at a height z from the chamber bottom
wall, ~Y",
is the average velocity of the medium, H is the chamber height. Thus, a near-
parabolic
velocity profile is established along the vertical direction for the carrier
medium in such a
chamber. Thus, particles 350 and 360 may be discriminated according to the
height
positions ( h, versus h2 ) along a vertical direction that is normal to the
traveling direction
of the Garner medium. Furthermore, because of the velocity profile, the
particles 350 and
360 may be further discriminated according to the vertical positions within
the velocity
profile. Even furthermore, the particles 350 and 360 are caused to travel
across the
chamber at different velocities Vi and YZ . If the particles 350 and 360 are
introduced into
the chamber at similar time, the particles 350 and 360 will exit the chamber
at different
times because they are transported through the chamber at different
velocities. The
particles of different properties (e.g.: size, density, geometry, charge,
acoustic impedance)
may be displaced to different positions along the vertical direction ( la,
versus laa ); may be
discriminated according to their displacement positions along the vertical
direction or
within the velocity profile; maybe discriminated according to the velocities
at which the
particles travel through the chamber ( Y, versus YZ ) or according to the exit
times of the
particles leaving the chamber. Particles of different properties may be
fractionated into
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subpopulations. Alternatively, particles displaced to different heights may be
fractionated
into sub-populations as they exit the chamber through different outlet ports
if the different
outlet ports are arranged vertically along the outlet end of the chamber.
In the above discussion, the chamber has been considered as being disposed
horizontally. However, the chamber may be disposed along any direction or
having any
angle with respect to the horizontal plane. In these cases, we would still
consider forces
acting on the matter to be discriminated primarily along the direction normal
to the
traveling direction of the carrier medium. The difference between these cases
and the
above case where the chamber is disposed horizontally is that the effect of
the gravitational
force may be different. In the above case, the gravitational force acts in a
direction
perpendicular to the traveling direction of the carrier medium. In the cases
where the
chamber is not disposed horizontally, the gravitational force may act in a
direction not
perpendicular to the traveling direction of the Garner medium. Thus, only a
component of
the gravitational force should be considered for analyzing the forces exerting
on the matter
to be discriminated along a direction perpendicular to the traveling direction
of the carrier
medium.
The velocity profile in the rectangular chamber shown in figure 5 depends on
the
structural characteristics of the chamber. When the chamber length and width
is
substantially larger than the chamber height, a parabolic or near-parabolic
velocity profile
along the vertical direction exits in the chamber. The reason for being "near-
parabolic" is
that the velocity profile at the positions close to the gasket walls does not
follow the
"parabolic profile". When the chamber width and the chamber height are of
similar sizes,
the velocity of the Garner medium in the chamber will follow other velocity
profile than the
"parabolic velocity profile" discussed above. Furthermore, the top and the
bottom walls
have been considered as flat and parallel to each other during the above
discussions. When
the top wall andlor the bottom wall are not flat, or when the top wall and the
bottom wall
are not paxallel to each other, or when the top wall or the bottom wall is
modified with
structures elements of various thickness, the velocity profile of the carrier
medium will be
different from the "near-parabolic velocity profile" described above.
To produce different velocity profile of the carrier medium, the gasket 230
between
the top wall 210 and the bottom wall 220 may be cut in the middle to form
channels of
other shapes. For example, the channel in the acoustic-E-FFF chamber may have
an ellipse
shape, similar to that shown in Figure 2 for an acoustic-FFF chamber. When a
carrier
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medium is caused to travel through such a chamber, the velocity profile of the
carrier
medium will be different from that for the chamber shown in Figure 5.
Similarly, the
channel for the acoustic-FFF chamber shown in Figure 3 may be used for an
acoustic-E-
FFF chamber. Such a chamber will result in a unique velocity profile for the
carrier
medium when it is caused to travel through the channel.
The above discussion of the acoustic-E-FFF chamber has focused on the
discrimination of particles where we ignored the influence of thermal
diffusion effects. For
matter of small sizes to be discriminated in an acoustic-E-FFF chamber, it may
be
necessary to take into account the thermal diffusion forces. In such cases,
the position of
the matter being displaced along a direction or within a velocity profile by
the applied
forces refers to the distribution of the matter that has been influenced by
the applied forces.
Such distributions of the matter along a direction or within a velocity
profile refer to the
concentration profile or distribution profile of the matter along the
direction or within the
velocity profile.
The acoustic-E-FFF chamber may have one or more inlet ports through which the
matter to be discriminated and the carrier medium are introduced. The chamber
may have
one or more outlet ports through which the discriminated matter and the
carrier medium
may exit the chamber. The inlet and outlet ports may be located on the top
or/and bottom
walls of the chamber. The inlet and outlet ports may be holes (as small as
from about
several microns or as large as about several mm in diameter) drilled on the
chamber top
and/or bottom walls. PEEK or plastic, or metal tubing may be inserted into the
holes and
serve as the fluid connection between the chamber and the external fluid-
circuits such as
infusion devices or collection devices. Alternatively, the inlet and outlet
port may be a slot
(from about microns) to about mm in width) drilled across the chamber outlet
end.
Multiple tubing, arranged in a ribbon form, can be interfaced with such slots.
The
exemplary acoustic-FFF chamber shown in Figure 4 could be used as an example
for an
acoustic-E-FFF chamber. In this case, a single inlet port - a hole - is
located at the bottom
wall of the chamber. The two outlet ports 180 and 190, positioned at both the
top and the
bottom walls, are the thin slots cut at the walls. A plurality of tubing
arranged in a ribbon
form is used to connect to the thin slots as the outlet ports. The two outlet
ports arranged at
the bottom and top walls correspond to the split-configuration employed in
many field-
flow-fractionation devices (Springston et al, 1987; Lee et al, 1989; Levin and
Giddings,
1991).
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8.3. Acoustic-DEP-FFF chamber.
Figure 7 shows an embodiment of acoustic-DEP-FFF chamber and the operational
principle of acoustic-DEP-FFF. The chamber has a top wall 510 and a bottom
wall 520.
The top wall and bottom wall are separated by a gasket or spacer 530 that has
a rectangular
channel 540 cut in it. The channel 540 has tapered ends. Under the bottom
wall, there is a
piezoelectric transducer 525. For clarity, the top wall 510, the gasket 530,
the bottom wall
520, and the piezoelectric transducer 525 are shown separated from each other.
In use,
these components are bound to each other to form an acoustic-DEP-FFF chamber.
An inlet
port 550 and an outlet port 560 are located on the top wall and bottom wall,
at the inlet end
and outlet end of the chamber, respectively. The inlet port 550 is connected
with an
infusion device 570 that can introduce carrier medium and introduce the matter
to be
discriminated into the chamber. The infusion device may be a syringe pump
coupled with
an injection valve (Wang et al., 1998; Huang et al., 1999; Yang et al., 1999).
The outlet
port 560 is connected with a collection or characterization device 580 that is
capable of
characterizing the matter that has been separated and discriminated after the
acoustic-DEP-
FFF process. The collection or characterization device may be a particle
counter, a flow
cytometer or a fractionation collector.
In the exemplary figure 7, the piezoelectric transducer 525 has the same size
as the
entire area of the chamber bottom wall. The top surface 590 and bottom surface
600 of the
piezoelectric transducer 525 has been coated with metal films or other
electrically
conductive material. AC electrical signals from a signal generator 610 can be
applied to
the top surface 590 and bottom surface 600 of the piezoelectric transducer 525
to energize
the piezoelectric transducer so to produce an acoustic wave in the chamber in
the direction
normal to the surfaces 590 and 600. The acoustic wave transmitted from the
piezoelectric
transducer is coupled through the bottom wall 520 into the chamber and is
reflected back
by the top wall 510. The superimposition of the transmitted wave from the
piezoelectric
transducer and the reflected wave from the top wall form the total acoustic
wave field in the
chamber. The total acoustic wave field may have two components, i.e., standing-
wave
component and traveling wave component. The ratio of the magnitude of the
standing-
wave component to the magnitude of the traveling-wave component is determined
by the
chamber height (i.e. the distance between the top wall and bottom wall), the
wavelength of
the acoustic wave, the acoustic properties of the top wall 510 and the bottom
wall 520 and
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the piezoelectric transducer 525, the decaying factor for the acoustic wave in
the Garner
medium. In one embodiment, the chamber height between the top wall and bottom
wall is
half wavelength of the standing acoustic wave, and a standing acoustic wave is
established
in the chamber. An acoustic pressure node exists at the center plane of the
chamber. In
another embodiment, the chamber height is larger or smaller than half
wavelength of the
standing acoustic wave.
In the example of Figure 7, a piezoelectric transducer 525 is bound to the
chamber
bottom wall 520. There may be many variations in adapting one or more
piezoelectric
transducers along the portions of the chamber. The piezoelectric transducers
may be
adapted on the top and/or the bottom walls. For adapting the piezoelectric
transducer on
the top wall, the transducer may be bound to a solid plate from the top side
so that the solid
plate forms the top wall of the chamber. The acoustic wave may be generated
from the
piezoelectric transducer and be coupled into the carrier medium placed in the
chamber
through the solid plate. Alternatively, the piezoelectric transducer may be
used directly as
the top wall. For adapting the piezoelectric transducer on the bottom wall,
the transducer
may be used directly as the chamber bottom wall. The microelectrode elements
or arrays
may be fabricated directly on the top surface of such piezoelectric
transducers. The
acoustic-DEP-FFF chamber shown in Figure 7 comprises one piezoelectric
transducer in
the chamber. Multiple piezoelectric transducers may be employed in one
chamber. These
transducers may be adapted on the top wall in series, or on bottom wall in
series, or on both
top and bottom walls to form a piezoelectric transducer array. The multiple
piezoelectric
transducers may be energized by same or different electrical signals to
produce acoustic
waves in the chamber.
In the exemplary figure 7, electrode elements employed for generating an
electric
field in the chamber correspond to the electrode array 545 on the top surface
of the bottom
wall 520. AC signals may be applied from signal generator 615 across the
electrode array
545 to produce a non-uniform electric field in the chamber. The matter being
introduced
into such a non-uniform electric field will experience dielectrophoretic
forces that depend
on the dielectric properties of the matter and the medium surrounding the
matter and
depend on the field non-uniform distributions.
In the example shown in figure 7, the electrode array 545 is supported on the
top
surface of the bottom wall 520 for generating non-uniform electric field. In
general, the
electrode elements for generating the electric field may be covering only
portions of the
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bottom surface of the top wall, or the portions of the top surface of the
bottom wall, or
both. Electrode arrays of different configurations may be utilized on these
surfaces (i.e.,
the bottom surface of the top wall or the top surface of the bottom wall). The
interdigitated
electrode array 400 and an interdigitated castellated electrode array 410 in
Figures 6A and
6B may be used. The electrode elements may be adapted substantially
latitudinally (as
shown in Figure 6A or 6B, or as shown in Figure 7 for the electrode elements
in the array
545) or longitudinally (i.e., the electrode elements in Figure 6A are turned
by 90 degree, or
the electrode elements in the array 545 of Figure 7 are turned by 90 degree)
along a portion
of the chamber. Similarly, the interdigitated electrode arrays with periodic
triangular (700)
or arc-like electrode tips (710) shown in Figures 8 may also be used. The
electrode
elements may be adapted substantially latitudinally (as shown in Figure 8A or
8B) or
longitudinally (i.e., the electrode elements in Figure 8A and 8B are turned by
90 degree)
along a portion of the chamber. Individual electrode elements in these
electrode arrays
shown Figure 8A and Figure 8B are connected to one of two common electrical
conductor
buses 705 and 708. Electrode elements are energized to produce electric fields
when
electrical signals from signal sources are connected to such electrical
conductor buses.
The example shown in Figure 7 has the configuration that the electrode array
545 is
supported on the chamber bottom wall 520 and the piezoelectric transducer 525
is bound to
the bottom wall 520. In other embodiments, electrode arrays of various
geometrical types
may also be fabricated dzrectly on piezoelectric transducers. For example, PZT
is a type of
piezoelectric material and could be used as a piezoelectric transducer. After
its surface
being polished to sufficient smoothness, microfabrication methods could be
used to
fabricate microelectrodes on such piezoelectric substrate. In constructing an
acoustic-DEP-
FFF chamber operation, the piezoelectric transducer with microelectrodes on
the top
surface and a planner electrode on the bottom surface may be used as bottom
wall of the
acoustic-DEP-FFF chamber. The electrical signals could be applied to
microelectrode
array on the top surface of the piezoelectric transducer to produce
dielectrophoresis forces.
Simultaneously, electrical signals could be applied to the top array (e.g.
through orie of the
common electrical conductor buses 705 or 708) and the bottom planer electrodes
for
producing acoustic field and forces. The advantage of this approach is that
the electrode
array for producing dielectrophoresis forces is integrated onto the surface of
the
piezoelectric transducer. Such an integration of the electrode array with the
piezoelectric
transducer is similar to that of the acoustic-E-FFF chamber shown in Figure 5,
where the
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electrode element for producing electrophoresis force is on the top surface of
the
piezoelectric transducer.
The matter being introduced into an acoustic-DEP-FFF chamber will experience
different forces in the chamber. We consider the case that the matter
introduced is
microscopic particles and the chamber is disposed horizontally. Referring to
Figure 7 these
forces are:
Acoustic radiation force Facoustic 620 in the vertical direction pointing
towards or
away from the top (or bottom) wall, depending on a factor which relates to the
densities of
the particles and the suspending medium, and to the acoustic impedance of the
of the
particles and the medium. The force Fa~oustic 620 may be a component of total
acoustic
force acting on the particle.
Dielectrophoretic force FDEP 625 in the vertical direction on the polarized
particles.
Depending on whether the particles are more or less polarizable than the
surrounding
medium, this force points downwards to the electrode elements or upwards away
from the
electrode elements. The dielectrophoretic force FDEp 625 may be a component of
the total
dielectrophoretic force acting on particles.
Gravitational force F~ 630 levitating or sedimenting the particles, depending
on the
relative magnitude of the densities of the particles and the suspending
medium.
Hdynamic lifting force FLIP 640 that tends to drive the particles away from
the
chamber walls. Various theoretical and experimental studies have been
conducted on such
hydrodynamic forces yet its nature remains in question (Williams et al., 1992;
1994; 1996;
1997). However, it is generally accepted that this force plays an important
role only when
the particles are very close to the chamber walls (e.g.: < 5 micrometer in a
chamber of 200
micrometer thick). Some recent work in DEP-FFF (Huang et al., 1997; Wang et
al., 1995)
shows that this force plays little role in DEP-FFF operation.
These forces are acted on the particles, driving the particles towards
equilibrium
positions at which these forces balance so that the net force acting on
individual particles in
the vertical direction is zero, i.e.,
Facoustic z ~O + FLift ~Z~ + FDEP W FG -
Particles of different properties (e.g.: size, geometrical shape, density,
dielectric
properties, acoustic impedance) equilibrate to different height positions. For
example,
particles 650 and 660 are displaced to different heights ( la, and h2 ) from
the chamber
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bottom wall. When a fluid flow is introduced by infusing the carrier medium
through inlet
port 550 into the chamber, a flow velocity profile 670 is generated. The
traveling direction
in this case is parallel to the chamber top and bottom walls, and points from
the chamber
inlet end towards the chamber outlet end. The Garner medium at various
positions of the
chamber exhibit different velocities. For the example shown in Figure 7, when
the
chamber length (i.e., the length of the channel cut in the middle) and chamber
width (i.e.,
the width of the channel) is substantially greater than the chamber height
(i.e., the distance
between the top wall and bottom wall), the velocity of the Garner medium at
the positions
not close to the channel walls defined by the gasket follows an approximate
parabolic
velocity profile in the vertical direction,
6wm ~ H Cl
where Ym is the velocity of the medium at a height z from the chamber bottom
wall, ~Y"t
is the average velocity of the medium, H is the chamber height. Thus, a near-
parabolic
velocity profile is established along the vertical direction for the carrier
medium in such a
chamber. Thus, particles 550 and 560 may be discriminated according to the
height
positions ( la, versus la2 ) along a vertical direction that is normal to the
traveling direction
of the carrier medium. Furthermore, because of the velocity profile, the
particles 550 and
560 may be further discriminated according to the vertical positions within
the velocity
profile. Even furthermore, the particles 550 and 560 are caused to travel
across the
chamber at different velocities V, and Yz . If the particles 550 and 560 are
introduced into
the chamber at similar time, the particles 550 and 560 will exit the chamber
at different
times because they are transported through the chamber at different velocities
( Y, versus
Ya ). The particles of different properties (e.g.: size, density, geometry,
charge, acoustic
impedance) may be displaced to different positions along the vertical
direction; may be
discriminated according to their displacement positions along the vertical
direction or
within the velocity profile ( la, versus h2 ); maybe discriminated according
to the velocities
at which the particles travel through the chamber ( Y, versus YZ ) or
according to the exit
times of the particles leaving the chamber. Particles of different properties
may be
fractionated into subpopulations. Alternatively, particles displaced to
different heights may
be fractionated into sub-populations as they exit the chamber through
different outlet ports
if the different outlet ports are arranged vertically along the outlet end of
the chamber.
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In the above discussion, the chamber has been considered as being disposed
horizontally. However, the chamber may be disposed along any direction or
having any
angle with respect to the horizontal plane. In these cases, we would still
consider forces
acting on the matter to be discriminated primarily along the direction normal
to the
traveling direction of the carrier medium. The difference between these cases
and the
above case where the chamber is disposed horizontally is that the effect of
the gravitational
force may be different. In the above case, the gravitational force acts in a
direction
perpendicular to the traveling direction of the Garner medium. In the cases
where the
chamber is not disposed horizontally, the gravitational force may act in a
direction not
perpendicular to the traveling direction of the carrier medium. Thus, only a
component of
the gravitational force should be considered for analyzing the forces exerting
on the matter
to be discriminated along a direction perpendicular to the traveling direction
of the carrier
medium.
The velocity profile in the rectangular chamber shown in figure 7 depends on
the
structural characteristics of the chamber. When the chamber length and width
is
substantially larger than the chamber height, a parabolic or near-parabolic
velocity profile
along the vertical direction exits in the chamber. The reason for being "near-
parabolic" is
that the velocity profile at the positions close to the gasket walls does not
follow the
"parabolic profile". When the chamber width and the chamber height are of
similar sizes,
the velocity of the carrier medium in the chamber will follow other velocity
profile than the
"parabolic velocity profile" discussed above. Furthermore, the top and the
bottom walls
have been considered as flat and parallel to each other during the above
discussions. When
the top wall and/or the bottom wall are not flat, or when the top wall and the
bottom wall
are not parallel to each other, or when the top wall or the bottom wall is
modified with
structures elements of various thickness, the velocity profile of the carrier
medium will be
different from the "near-parabolic velocity profile" described above.
To produce different velocity profile of the Garner medium, the gasket 530
between
the top wall 510 and the bottom wall 520 may be cut in the middle to form
channels of
other shapes. For example, the channel in the acoustic-DEF-FFF chamber may
have an
ellipse shape, similar to that shown in Figure 2 for an acoustic-FFF chamber.
When a
Garner medium is caused to travel through such a chamber, the velocity profile
of the
carrier medium will be different from that for the chamber shown in Figure 7.
.Similarly,
the channel for the acoustic-FFF chamber shown in Figure 3 may be used for an
acoustic-
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DEP-FFF chamber. Such a chamber will result in a unique velocity profile for
the carrier
medium when it is caused to travel through the channel.
The above discussion of the acoustic-DEP-FFF chamber has focused on the
discrimination of particles where we ignored the influence of thermal
diffusion effects. For
matter of small sizes to be discriminated in an acoustic-DEP-FFF chamber, it
may be
necessary to take into account the thermal diffusion forces. In such cases,
the position of
the matter being displaced along a direction or within a velocity profile by
the applied
forces refers to the distribution of the matter that has been influenced by
the applied forces.
Such distributions of the matter along a direction or within a velocity
profile refer to the
concentration profile or the distribution profile of the matter along the
direction or within
the velocity profile.
The acoustic-DEP-FFF chamber may have one or more inlet ports through which
the matter to be discriminated and the carrier medium are introduced. The
chamber may
have one or more outlet ports through which the discriminated matter and the
carrier
medium may exit the chamber. The inlet and outlet ports may be located on the
top orland
bottom walls of the chamber. The inlet and outlet ports may be holes (as small
as from .
about several microns or as large as about several mm in diameter) drilled on
the chamber
top and/or bottom walls. PEEK or plastic, or metal tubing may be inserted into
the holes
and serve as the fluid connection between the chamber and the external fluid-
circuits such
as infusion devices or collection devices. Alternatively, the inlet and outlet
port may be a
slot (from about microns) to about mm in width) drilled across the chamber
outlet end.
Multiple tubing, arranged in a ribbon form, can be interfaced with such slots.
The
exemplary acoustic-FFF chamber shown in Figure 4 could be used as an example
for an
acoustic-DEP-FFF chamber. In this case, a single inlet port - a hole - is
located at the
bottom wall of the chamber. The two outlet ports 180 and 190 in Figure 4,
positioned at
both the top and the bottom walls, are the thin slots cut at the walls. A
plurality of tubing
arranged in a ribbon form is used to connect to the thin slots as the outlet
ports. The two
outlet ports arranged at the bottom and top walls correspond to the split-
configuration
employed in many field-flow-fractionation devices (Springston et al, 1957; Lee
et al, 1959;
Levin and Giddings, 1991).
C. Exemplary methods
C.1. Batch-mode operation for acoustic-FFF, acoustic-E-FFF and acoustic-DEP-
FFF.
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In the batch-mode operation, an acoustic-FFF (or acoustic-E-FFF or acoustic-
DEP-
FFF) chamber is preloaded with a Garner medium through the inlet port.
Particle-mixture
samples (e.g. 100 pL) are then delivered, e.g., injected or otherwise
introduced into the
medium in the chamber through the inlet port. For acoustic-FFF, appropriate
acoustic field
condition is applied so that particles are given a specified time tb reach
their equilibrium
positions under the influence of the acoustic force and other forces (e.g.,
gravitational
force, hydrodynamic lifting force). The acoustic field conditions in the
chambers) are
applied through energizing the piezoelectric transducers) with appropriate
electrical
signals. For acoustic-E-FFF, appropriate electric field and acoustic field
conditions are
applied so that particles are given a specified time to reach their
equilibrium positions
under the influence of the acoustic force, electrophoretic force and other
forces (e.g.,
gravitational force, hydrodynamic lifting force). For acoustic-DEP-FFF,
appropriate
dielectrophoretic field and acoustic field conditions are applied so that
particles are given a
period of time to reach their equilibrium positions under the influence of the
acoustic force,
dielectrophoretic force and other forces (e.g., gravitational force,
hydrodynamic lifting
force). This step is called the "relaxation" in typical field-flow-
fractionation (Giddings,
1981, Giddings, 1993). During the relaxation step (Figure 9A), particles of
different
properties are displaced to different equilibrium positions within the chamber
under the
influence of applied forces. For particles (or matter) of small sizes where
the thermal
diffusion effect plays role in particle equilibrium positions, the equilibrium
positions of
different particle types correspond to the equilibrium concentration profile
for different
particle types. Following the relaxation process, fluid flow is then
established and
particles at different heights axe driven to move through the chamber at
different velocities.
In this process, the externally applied field conditions are maintained.
Particles of different
properties are separated into fractions according to the time they exit the
chamber (Figure .
9B). Figure 9A and 9B show such a batch-mode process in a cross-sectional view
of a
rectangular,.acoustic-FFF (or acoustic-E-FFF, or acoustic-DEP-FFF) chamber.
Figure 9A
shows that during the relaxation step, particle types 800 and 850 have been
displaced to
different heights under the influence of the applied forces. Figure 9B shows
that after the
fluid flow profile (i.e., a velocity profile 780) is established in the
chamber following the
relaxation step, particle type 800 has moved ahead of particle type 850 and
will exit the
chamber earlier at the outlet port 795 The fluid flow is established by
infusing a carrier
medium into the chamber inlet port 790.
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Batch-mode is the typical mode of operation for most field-flow-fractionation.
Particles can exit the chamber from a single outlet port or multiple outlet
ports (e.g. one on
the top and one on the bottom wall). The two-outlet-ports located at the top
and bottom
walls may be used to collect two separated fractions directly. The
"relaxation" step may
not be necessary in some applications. Depending on the type of particle
mixtures, it is
possible that particles will attain their equilibrium height positions during
the fluid flow
shortly after being introduced into the chamber. In this case, particles can
be separated in a
batch mode without the need of "relaxation step".
Thus, the "batch-mode" operation of discriminating a matter using acoustic
force in
field flow fractionation comprises the following steps: a) obtaining an
acoustic-FFF
apparatus described in this invention; b) loading a carrier medium into the
chamber of
apparatus via its inlet port until the chamber is filled with the Garner
medium; c) delivering
a sample that contains a matter to be discriminated into the carrier medium in
the chamber;
d) applying at least one electrical signal provided by an electrical signal
generator to the
piezoelectric transducer, wherein said energized piezoelectric transducer
creates an acoustic
wave, thereby causing at least one acoustic force on said matter; e)
introducing the carrier
medium into the chamber of the apparatus via its inlet port, wherein said
introducing causes
the carrier medium to travel through the chamber according to a velocity
profile; whereby
said matter is displaced to a position within said carrier medium along a
direction normal to
the traveling direction of said carrier medium travelling through said chamber
and
discriminated according to its position within said.carrier medium along the
direction
normal to the traveling direction of said carrier medium travelling through
said chamber.
This "batch-mode" acoustic-FFF can be used with any acoustic-FFF apparatus
described in
this invention.
In the above "batch-mode" method of acoustic-FFF, preferably, prior to the
introducing of Garner medium into the chamber that causes the Garner medium to
travel
through the chamber according to a velocity profile (i.e., prior to step e),
the matter to be
discriminated is displaced into equilibrium position along a direction normal
to the
traveling direction of the carrier medium traveling through the chamber by
applying
electrical signal to the piezoelectric transducer to cause acoustic force on
said matter.
The "batch-mode" operation of discriminating a matter using electrophoretic
and
acoustic forces in field flow fractionation comprises the following steps: a)
obtaining an
acoustic-E-FFF apparatus described in the present invention; b) loading a
carrier medium
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into the chamber of apparatus via its inlet port until the chamber is filled
with the carrier
medium; c) delivering a sample that contains a matter to be discriminated into
the carrier
medium in the chamber; d) applying at least one electrical signal provided by
an electrical
signal generator to the electrode element, wherein said energized electrode
element creates
an electrical field, thereby causing at least one electrophoretic force on
said matter; e)
applying at least another electrical signal provided by an electrical signal
generator to the
piezoelectric transducer, wherein said energized piezoelectric transducer
creates an acoustic
wave, thereby causing at least one acoustic force on said matter; f)
introducing the carrier
medium into the chamber of the apparatus via its inlet port, wherein said
introducing causes
the carrier medium to travel through the chamber according to a velocity
profile; whereby
said matter is displaced to a position within said Garner medium along a
direction normal to
the traveling direction of said Garner medium travelling through said chamber
and
discriminated according to its position within said carrier medium along the
direction
normal to the traveling direction of said Garner medium travelling through
said chamber.
Any of the acoustic-E-FFF apparatus described in the present invention can be
used in the
"batch-mode" operation of acoustic-E-FFF.
In the above described "batch-mode" of acoustic-E-FFF method, preferably,
prior to
the introducing of carrier medium into the chamber that causes the carrier
medium to travel
through the chamber according to a velocity profile (i.e., prior to step f),
the matter to be
discriminated is displaced into equilibrium position along a direction normal
to the
traveling direction of the Garner medium traveling through the chamber
applying by
applying electrical signal to the electrode element to cause electrophoretic
force on said
matter and applying electrical signal to the piezoelectric transducer to cause
acoustic force
on said matter.
The "batch-mode" operation of discriminating a matter using dielectrophoretic
and
acoustic forces in field flow fractionation comprises the following steps: a)
obtaining an
acoustic-DEP-FFF apparatus described in the present invention; b) loading a
carrier
medium into the chamber of apparatus via its inlet port until the chamber is
filled with the
carrier medium; c) delivering a sample that contains a matter to be
discriminated into the
Garner medium in the chamber; d) applying at least one electrical signal
provided by an
electrical signal generator to the electrode element, wherein said energized
electrode
element creates a non-uniform electrical field, thereby causing at least one
dielectrophoretic
force on said matter; e) applying at least another electrical signal provided
by an electrical
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signal generator to the piezoelectric transducer, wherein said energized
piezoelectric
transducer creates an acoustic wave, thereby causing at least one acoustic
force on said
matter; f) introducing the carrier medium into the chamber of the apparatus
via its inlet
port, wherein said introducing causes the carrier medium to travel through the
chamber
according to a velocity profile; whereby said matter is displaced to a
position within said
carrier medium along a direction normal to the traveling direction of said
carrier medium
travelling through said chamber and discriminated according to its position
within said
carrier medium along the direction normal to the traveling direction of said
carrier medium
travelling through said chamber. Any of the acoustic-DEP-FFF apparatuses
described in .
the present~invention can be used in the "batch-mode" operation of acoustic-
DEP-FFF.
In the above described "batch-mode" of acoustic-DEP-FFF method, preferably,
prior to the introducing of carrier medium into the chamber that causes the
carrier medium
to travel through the chamber according to a velocity profile (step f), the
matter to be.
discriminated is displaced into equilibrium position along a direction normal
to the
traveling direction of the Garner medium traveling through the chamber by
applying
electrical signal to the electrode element to cause dielectrophoretic force on
said matter and
applying electrical signal to the piezoelectric transducer to cause acoustic
force on said
matter.
In the above acoustic-E-FFF or acoustic-DEP-FFF methods, identical, but
preferably, different electric signals are used to generate the acoustic force
and the
electrophoretic or the dielectrophoretic force.
In the above acoustic-E-FFF or acoustic-DEP-FFF method, the acoustic force and
the electrophoretic or the dielectrophoretic force can be generated
simultaneously or
sequentially.
C.2. Continuous mode operation for acoustic-FFF, acoustic-E-FFF and acoustic-
DEP-FFF.
In the continuous-mode operation, particle-mixture samples are continuously
fed
into the acoustic-FFF (or acoustic-E-FFF or acoustic-DEP-FFF) chamber through
the inlet
port. For acoustic-FFF, appropriate acoustic field condition is applied so
that particles that
are continuously fed into the chamber are continuously being driven towards
their
equilibrium positions under the influence of the acoustic force and other
forces (e.g.
gravity, hydrodynamic lift force). The acoustic field conditions in the
chambers) are
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applied through energizing the piezoelectric transducers) with appropriate
electrical
signals. For acoustic-E-FFF, appropriate electric field and acoustic wave
conditions are
applied so particles that are continuously fed into the chamber are
continuously being
driven towards their equilibrium positions under the influence of the acoustic
force,
electrophoretic force and other forces (e.g. gravity, hydrodynamic lift
force). For acoustic-
DEP-FFF, appropriate dielectrophoretic field and acoustic wave conditions are
applied so
that particles that are continuously fed into the chamber are continuously
being driven
towards their equilibrium positions under the influence of the acoustic force,
dielectrophoretic force and other forces (e.g. gravity, hydrodynamic lift
force). Take a
particle-mixture consisting of two subpopulations as an example to be
separated or
analyzed in an acoustic-FFF (or acoustic-E-FFF, or acoustic-DEP-FFF) chamber.
The
subpopulation having higher equilibrium positions in the acoustic-FFF (or
acoustic-E-FFF,
or acoustic-DEP-FFF) chamber may exit the chamber from an outlet port that is
located at
higher positions at the outlet end. The subpopulations having lower
equilibrium positions.
may exit the chamber from an outlet port that is located at lower positions at
the outlet end.
Thus particle mixtures can be continuously separated into two fractions.
Clearly, split-flow
at the chamber outlet end is needed (Springston et al, 1987; Lee et al, 1989;
Levin and
Giddings, 1991).
Multiple fractions can be collected if multiple outlet ports are located at
either the
top or the bottom or both walls for a chamber having top and bottom walls.
Figure 10
shows an example where the chamber has one outlet port 870 on the top wall and
two
outlet ports 880 and 890 on the bottom wall. Such a chamber can be used to
separate
particle mixture having three (or more than three) subpopulations. The chamber
may be an
acoustic-FFF chamber, or acoustic-E-FFF chamber, or acoustic-DEP-FFF chamber.
A
fluid velocity profile (i.e. fluid flow profile) 860 is established in the
chamber. The .
subpopulation 900 that is displaced to the highest positions by the applied
forces during the
transit time through the chamber may exit the outlet port 870 at the top wall.
The
subpopulation 910 that is displaced to the lowest positions by the applied
forces during the
transit time through the chamber may exit the chamber from the first outlet
port 880 on the
bottom wall. The subpopulation 920 that is displaced to the middle heights may
exit the
chamber from the second outlet port 890 on the bottom wall. Such continuous
operation
corresponds to the procedure for the FFF systems with split-configurations
(Springeston et
al, 1987; Lee et al, 1989; Levin and Giddings, 1991).
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Thus, "continuous-mode" operation of discriminating a matter using acoustic
forces
in field flow fractionation comprises the following steps: a) obtaining an
acoustic-FFF
apparatus described in the present invention; b) introducing a carrier medium
containing a
matter to be discriminated into the chamber of the apparatus via ifs inlet
port, wherein said
introducing causes the tamer medium to travel through the chamber according to
a
velocity profile; c) applying at least one electrical signal provided by an
electrical signal
generator to the piezoelectric transducer, wherein said energized
piezoelectric transducer
creates an acoustic wave, thereby causing at least one acoustic force on said
matter having
components normal to the traveling direction of said carrier medium travelling
through said
chamber; whereby said matter is displaced to a position within said carrier
medium along a
direction normal to the traveling direction of said carrier medium travelling
through said
chamber and discriminated according to its position within said carrier medium
along the
direction normal to the traveling direction of said carrier medium travelling
through said
chamber. The continuous mode of acoustic-FFF can be used with any acoustic-FFF
apparatus described in this invention.
The "continuous-mode" operation of discriminating a matter using
electrophoretic
and acoustic forces in field flow fractionation comprises the following steps:
a) obtaining
an acoustic-E-FFF apparatus described in the present invention; b) introducing
a carrier
medium containing a matter to be discriminated into the apparatus via its
inlet port,
wherein said introducing causes the tamer medium to travel through the chamber
according to a velocity profile; c) applying at least one electrical signal
provided by an
electrical signal generator to the electrode elements, wherein said energized
electrode
elements creates an electrical field, thereby causing at least one
electrophoretic force on
said matter having components normal to the traveling direction of said tamer
medium
travelling through said chamber; and d) applying at least another electrical
signal provided
by an electrical signal generator to the piezoelectric transducer, wherein
said energized
piezoelectric transducer creates an acoustic wave, thereby causing at least
one acoustic
force on said matter having components normal to the traveling direction of
said carrier
medium travelling through said chamber; whereby said matter is displaced to a
position
within said carrier medium along a direction normal to the traveling direction
of said carrier
medium travelling through said chamber and discriminated according to its
position within
said tamer medium along the direction normal to the traveling direction of
said carrier
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medium travelling through said chamber. Any of the acoustic-FFF apparatuses
described
in the present invention can be used the "continuous-mode" of acoustic-E-FFF.
The "continuous-mode" operation of discriminating a matter using
dielectrophoretic
and acoustic forces in field flow fractionation comprises the following steps:
a) obtaining
an acoustic-DEP-FFF apparatus described in the present invention; b)
introducing a carrier
medium containing a matter to be discriminated into the apparatus via its
inlet port,
wherein said introducing causes the carrier medium to travel through the
chamber of the
apparatus according to a velocity profile; c) applying at least one electrical
signal provided
by an electrical signal generator to the electrode element, wherein said
energized electrode
element creates a non-uniform electrical field, thereby causing at least one
dielectrophoretic
force on said matter having components normal to the traveling direction of
said carrier
medium travelling through said chamber; and d) applying, at least another
electrical signal
provided by an electrical signal generator to the piezoelectric transducer,
wherein said
energized piezoelectric transducer creates an acoustic wave, thereby causing
at least one
acoustic force on said matter having components normal to the traveling
direction of said
Garner medium travelling through said chamber; whereby said matter is
displaced to a
position within said carrier medium along a direction normal to the traveling
direction of
the carrier medium traveling through the chamber and discriminated according
to its
position within said carrier medium along the direction normal to the
traveling direction of
the Garner medium traveling through the chamber. Any acoustic-DEP-FFF
apparatus
described in the present invention can be used in the "continuous-mode"
operation of
acoustic-DEP-FFF.
D. Forces
Although not wish to be bound by any theories and mechanisms described herein,
the following illustrates the various forces through which the present
apparatuses and
methods are used or operated.
D.1. Acoustic radiation forces
Acoustic radiation force is a non-contact force that can be used for trapping,
handling, moving particles in fluid. The use of the acoustic radiation force
in a standing
ultrasound wave for particle manipulation has been demonstrated for
concentrating
erythrocytes (Yasuda et al, 1997), focusing micron-size polystyrene beads (0.3
to 10
micron in diameter, Yasuda and Kamakura, 1997), concentrating DNA molecules
(Yasuda
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et al, 1996C), batch and semicontinuous aggregation and sedimentation of cells
(Pui, et al,
1995). By competing electrostatic and acoustic radiation forces, separation of
polystyrene
beads of different size and charges have been reported (Yasuda et al, 1996A,
B).
Furthermore, in terms of ion leakage (for erythrocytes, Yasuda et al, 1997) or
antibody
production (for hybridoma cells, Pui. et al, 1995), little or no damage or
harming effect was
observed when acoustic radiation force was used to manipulate mammalian cells.
A standing plane, acoustic wave can be established in an acoustic-FFF chamber,
or
acoustic-E-FFF or acoustic-DEP-FFF chamber by applying AC signals to the
piezoelectric
transducers. Alternatively, an acoustic wave that has a standing-wave
component can be
established in an acoustic-FFF chamber, or acoustic-E-FFF or acoustic-DEP-FFF
chamber
by applying AC signals to the piezoelectric transducers. We will now examine
the acoustic
radiation force exerting a particle in s,standing acoustic wave field. Assume
that the
standing wave is established along a particular direction (e.g., z-axis
direction) in a fluid.
The standing wave spatially varying along the z axis in the fluid can be
expressed as:
Op(z) = p0 sin(kz) cos(wt)
where ~p is acoustic pressure at z, po is the acoustic pressure amplitude, k
is the
wave number ( 2~c / a , where ~, is the wavelength), z is the distance from
the pressure node,
w is the angular frequency, and t is the time. According to the theory
developed by
Yoshioka and Kawashima (1955), the radiation force Fa~ousn~ acting on a
spherical particle
in the stationary standing wave field is given by (see "Acoustic radiation
pressure on a
compressible sphere" by K. Yoshioka and Y. Kawashima in Acustica, 1955, Vol.
5, pages
167-173),
4~ 3 ( )
F'a~ousr~~ _ - 3 ~' k Ea~ousr~~ A sm 2kz
where r is the particle radius, Ea~ouSt;~ is the average acoustic energy
density, A is a
constant given by
A= SPp -2pm _ Yp
2 pp + pm Ym
where p", and pp are the density of the particle and the medium, y", and y p
are the
acoustic impedance of the particle and medium, respectively. A is termed
herein as the
acoustic-polarization-factor. The acoustic impedance ( y", and yp for the
medium and the
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particle) of a material is defined as the product between the density ( p",
and pp for the
medium and the particle) of the material and the acoustic velocity ( C", or Cp
for the
medium and the particle) in the material ( y", = p", ~ C", and y p = pp ~ Cp
).
When A>0, the particle moves towards the pressure node (z=0) of'the standing
wave.
When A<0, the particle moves away from the pressure node.
Clearly, particles having different density and acoustic impedance will
experience different
acoustic-radiation-forces when they are placed into the same standing acoustic
wave field:
For example, the acoustic radiation force acting on a particle of 10 micron in
diameter can
vary somewhere between 0.01 and 1000 pN, depending on the established acoustic
energy
density distribution.
The paper of "Acoustic radiation pressure on a compressible sphere" by K.
Yoshioka and Y. Kawashima published in Acustica, 1955, Vol. 5, pages 167-173
also
described a theory for the acoustic radiation forces exerting on a particle in
a traveling
wave acoustic field. Using such theories, those skilled in the art of acoustic
manipulation
of particles may readily analyze and calculate the acoustic radiation forces
on a particle in a
given acoustic field.
The piezoelectric transducers are made from "piezoelectric materials" that
produce
an electric field when exposed to a change in dimension caused by an imposed
mechanical
force (piezoelectric or generator effect). Conversely, an applied electric
field will produce
a mechanical stress (electrostrictive or motor effect) in the materials. They
transform
energy from mechanical to electrical and vice-versa. The piezoelectric effect
was
discovered by Pierre Curie and his brother Jacques in 1880. It is explained by
the
displacement of ions, causing the electric polarization of the materials'
structural units.
When an electric field is applied, the ions are displaced by electrostatic
forces, resulting in
the mechanical deformation of the whole material. Thus, in an acoustic-FFF or
acoustic-E-
~FFF or acoustic-DEP-FFF apparatus, when AC voltages are applied to the
piezoelectric
transducers, the vibration occurred to the transducers will be coupled into
the fluid in the
chamber and result in an acoustic wave in the chamber. Such an acoustic wave
may have
standing wave and traveling wave components.
Separation of particles in a medium using acoustic wave has been reported
previously. For example, U.S. Patent No. 4,523,682, which is hereby
incorporated by
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reference in its entirety, discloses a method for separating particles of
different sizes,
densities and other properties in an acoustic chamber. U.S. Patent No.
4,523,682 describes
the spatial separation of particles of different properties in an acoustic
wave. The aspect of
acoustic-FFF separation in the present invention provides the apparatus and
methods for
separating particles from a mixture. The purified particle populations may be
obtained
using the present invention, while the US 4523682 can separate particles only
according to
the positions the particles occupy in an acoustic chamber.
D.2. Electrophoretic forces
The electrostatic force or electrophoretic force FE on a particle in an
applied
electrical field EZaz is given by
FE = QpEzaz
where Qp is the effective electric charge on the particle. The direction of
the electrostatic
force (or electrophoretic force) on the charged particle depends on the
polarity of the
particle charge as well as the applied-field direction.
D.3. Dielectrouhoretic forces
The dielectrophoretic force FDEPz acting on a particle of radius r subjected
to a non-
uniform electrical field is given by
FDEPz - 2~~m~3xDEPvErms az
where Erms is the RMS value of the field strength, sm is the dielectric
permitivity of the
medium. ,'~DEP 1S the particle polarization factor, given by
*_
Ep Em
~DEP = Re
~p + 2s",
"Re" refers to the real part of the "complex number". The symbol ~x = sx - j ~
is the
2
complex permitivity (of the particle x=p, and the medium x=gin). The
parameters sp and
a-p are the effective permitivity and conductivity of the particle,
respectively. These
parameters may be frequency dependent. For example, a typical biological cell
will have
frequency dependent, effective conductivity and permitivity, at least, because
of cytoplasm
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membrane polarization. In the above equation, FDEPz is the component of total
dielectrophoretic force along the direction aZ . In the present application,
the
dielectrophoretic (DEP) force refers to the force acting on particles due to
electric fields of
non-uniform strength. In the literature, such forces were sometimes referred
as
conventional dielectrophoretic forces. However, in the present application,
for the sake of
simplicity, we call the forces acting on particles due to electrical fields of
non-uniform
strength "dielectrophoretic forces" or "DEP forces".
The above equation for the dielectrophoretic force can be also be written as
_ 3 2
FDEPz - 2?LfimY ,'~DEP V p(Z) az 1
where p(z) is the squaxe-field distribution for a unit-voltage excitation (V
=1 V) on the
electrodes, V is the applied voltage.
A non-uniform electrical field can be established in an acoustic-DEP-FFF
chamber
by applying AC signals to the microelectrodes incorporated on the chamber
surfaces. For
1 S an interdigitated electrode array, the dielectrophoretic forces will
follow an approximately
exponential decay with the distance from the electrode plane, as shown by
Huang et al,
1997.
Particles or cells having different dielectric property (as defined by
permitivity and
conductivity) will experience different dielectrophoretic forces in the
vertical direction in
an acoustic-DEP-FFF chamber. For DEP manipulation of particles (including
biological
cells), DEP forces acting on a particle of 10 micron in diameter can vary
somewhere
between about 0.01 and about 1000 pN.
In previously reported DEP-FFF technology (Huang et al, Biophys. J. Vol. 73,
p1118-1129, 1997; Wang et al., Biophys. J. Vol. 74, p2689-2701, 1998; Yang, J.
et al.
Anal. Chem. Vol. 71, p911-918, 1999), only negative DEP forces can be used to
influencelcontrol/manipulate particle positions in fluid flow velocity profile
because the use
of positive DEP may result in the particles being directed and trapped on the
electrode
elements. However, in the acoustic-DEP-FFF apparatus and acoustic-DEP-FFF
method in
the present invention, both positive and negative DEP forces can be used since
the use of
spatially-varying acoustic radiation forces provides additional control for
displacing
particles in a fluid stream. Positive DEP forces may be balanced by the
spatially-varying
acoustic radiation forces that are in an opposing direction.
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D.4. Hydrodynamic lifting forces
A fluid flow profile may be established in an acoustic-FFF chamber, or
acoustic-E-
FFF or acoustic-DEP-FFF chamber for particle separation and analysis. For a
rectangular
chamber with the chamber length and chamber width substantially greater than
the chamber
height, a laminar, parabolic flow profile may be generated. Such a velocity
profile may be
described as,
Yn~ - 6~V~n ~ H Cl _ H
where V"~ is the fluid velocity at the height z from the chamber bottom. H is
the chamber
height and ~V", ~ is the average fluid-velocity in the chamber. When a
particle is carried
with the fluid in such a profile, there is a hydrodynamic lifting force ( Ftft
) acting on the
particle in the vertical direction if the particle is placed close to the
chamber bottom (or top)
wall and if the chamber is disposed horizontally or nearly-horizontally. If
the distance
between the particle and the chamber bottom wall is very small (e.g. < 1
micron for a 10
micron particle in a 200 micron height chamber), the hydrodynamic lifting
force will direct
the particle away from the chamber wall (Williams et al., 1992; 1994; 1996;
1997). This
force has been used in the classical, hyperlayer-FFF operation in which the
particles are
positioned at different height from the chamber wall by balancing the
hydrodynamic lifting
force and sedimentation force (e.g., Ratanathanawongs S. K. and Giddings,
1992,
Williams et al, 1996). It is known that this hydrodynamic lifting force decay
rapidly to
zero with the distance from the chamber wall, yet its origin remains to be
debated.
D.5. Force Balance in Acoustic-FFF, Acoustic-E-FFF and Acoustic-DEP-FFF
chambers
In an acoustic- FFF chamber that is disposed horizontally or nearly
horizontally
with a fluid flow along the horizontal direction being established in the
chamber, three
types of forces are exerted on a particle in the vertical direction, i. e.,
F~~oust,~ , Frn , and
sedimentation forces F~ _ - 3 ~-3 (pp - pm ) . When these forces are balanced,
FHCOtlStlC (Z) + FI~ (~) + F~ = 0
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the particle experience no-force and would cease to move up or down. With the
position-
dependent acoustic and hydrodynamic lifting forces, particles may equilibrate
at such zero-
force height positions ( heq ). Such a position is dependent on the applied
acoustic wave
energy density, and more importantly, particle density and acoustic impedance,
and particle
size. Particles of different properties (density, acoustic impedance, size)
will equilibrate at
different heights, and will move at different velocities (VP , dependent on
hey ) under the
influence of the fluid-flow, and will transverse through the chamber at
different time
(tP = L l VP ). Similar analysis can be performed for an acoustic-FFF chamber
that is not
disposed horizontally. In such a case, the effect of the sedimentation force
on the
displacement of the particles within the flow velocity profile is different
from that shown
above. Only a component of the sedimentation force, which is within the
velocity profile
and is perpendicular to the traveling direction of the carrier medium,
contributes to the
displacement of the particles in the velocity profile.
In an acoustic-E-FFF chamber that is disposed horizontally or nearly
horizontally
with a fluid flow along the horizontal direction being established in the
chamber, four types
of forces are exerted on a particle in the vertical direction, i.e., Fa~ous~;~
, F'E = Flijt , and
sedimentation forces F~ _ - 3 ~r3 ( p p - p", ) . When these forces are
balanced,
Fa~ousra (z) + FE + Fr fr (z) + F~ = 0
the particle experience no-force and would cease to move up or down. With the
position-
dependent acoustic and hydrodynamic lifting forces, particles may equilibrate
at such zero-
force height positions ( he9 ). Such a position is dependent on the applied
acoustic wave
energy density, the applied electrical field, and more importantly, particle
density and
acoustic impeance, particle effective charge and size. Particles of different
properties
(density, acoustic impedance, effective charge, size) will equilibrate at
different heights,
and will move at different velocities ( VP , dependent on heq ) under the
influence of the
fluid-flow, and will transverse through the chamber at different time ( tP = L
l Vp ).
Compared with electrical-FFF, the acoustic-radiation-force has been added into
the force
equation. Similar force-balance analysis can be performed for an acoustic-E-
FFF chamber
that is not disposed horizontally. In such a case, the effect of the
sedimentation force on
the displacement of the particles within the flow velocity profile is
different from that
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shown above. Only a component of the sedimentation force, which is within the
velocity
profile and is perpendicular to the traveling direction of the carrier medium,
contributes to
the displacement of the particles in the velocity profile.
By competing the acoustic radiation force with electrostatic (electrophoretic)
forces,
Yasuda et al. (1996A, B) demonstrated the spatial separation of more than 20
micron for
polystyrene particles of different sizes and different charges in a chamber of
about 750
micron thick.
Similarly, in an acoustic-DEP-FFF chamber that is disposed horizontally or
nearly
horizontally with a fluid flow along the horizontal direction being
established in the
chamber, the force balance equation for a particle in the acoustic-DEP-FFF
chamber is
given by
Focoustic ~Z~ + FDEp (z) + F,;ft (z) + F~ = O .
Again, the zero-net-force position may correspond to an equilibrium height 1e9
. Such
position is .dependent on the applied acoustic wave energy density, the
applied non-uniform
electrical field, and more importantly, particle density, and dielectric
property and size.
Particles of different properties (density, acoustic impedance, dielectric
property, size) will
equilibrate at different heights, and will move at different velocities ( hp ,
dependent on heq )
under the influence of the fluid-flow, and will transverse through the chamber
at different
time ( t p = L l Vp ). Compared with DEP-FFF, the acoustic-radiation-force has
been added
into the force equation. Similar force-balance analysis can be performed for
an acoustic-
DEP-FFF chamber that is not disposed horizontally. In such a case, the effect
of the
sedimentation force on the displacement of the particles within the flow
velocity profile is
different from that shown above. Only a component of the sedimentation force,
which is
within the velocity profile and is perpendicular to the traveling direction of
the carrier
medium, contributes to the displacement of the particles in the velocity
profile.
The above analysis is based on single (large) particles, and particle
diffusion effect
is essentially ignored. Acoustic-FFF, acoustic-E-FFF and acoustic-DEP-FFF
characterization and separation can also be applied to small particles (down
to molecule
levels). The above analyses need to be modified to take into account the
diffusion effects.
For example, Yasuda et al. (1996A) described particle concentration profile in
a 1-D
ultrasound standing wave and electrical field for particles whose diffusion
effects were
brought into consideration. For these cases where particle diffusion effects
are taken into
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account, particle equilibrium concentration (or distribution) profile in the
vertical direction
determines the elution-times of particular particle types. Theoretical
analyses for FFF
effects of small particles having concentration profiles in the direction
normal to the fluid
flow were given in many FFF literatures (e.g., Caldwell and Gao, 1993; and
Giddings
1993). Those who are skilled in the art, e.g., FFF separation and analysis of
molecules and
small particles and acoustic/electrophoretic/ dielectrophoretic effects of
particles, may
readily perform theoretical analyses of applying the acoustic-FFF, or acoustic-
E-FFF or
acoustic-DEP-FFF methods for separating and analyzing molecules and small
particles.
References
Barmatz MB et al., US patent 4523682, June 1985.
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Giddings, Araal. Chem. Vol. 53, p1170A-1178A, 1981.
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Huang Y et al., Biophys. J. Vol. 73, p1118-1129, 1997.
Huang Y, et al., J. Hematotherapy and Stem Cell Research Vol. 8, 481-490, 1999
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Wang X-B. et al., Biophys. J. Vol. 74, p2689-2701, 1998.
Williams et al., Chem. Eng. Comm. Vol . 111, p121-147, 1992.
Williams et al., Chem. Eng. Comm. Vol. 130, p143-166, 1994.
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Yang, J. et al. Anal. Chena. Vol. 71, p911-918, 1999.
Yasuda K. et al, J. Acoust. Soc. Am. Vol. 99(4), p1965-1970, April, 1996 A.
Yasuda K. et al., Jpn. J. Appl. Phys. Vol. 35(1), No. 5B, p3295-3299, 1996 B.
Yasuda K. et al, J. Acoust. Soc. Arn., Vol. 99 (2), p1248-1251, 1996 C.
Yasuda K. et al, J. Acoust. Soc. Am. Vol. 102 (1), p642-645, July, 1997.
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Yasuda K. and Kamakura T. Appl. Phys. Lett, Vol. 71 (13), p1771-1773, Sep.
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The above examples are included for illustrative purposes only and is not
intended
to limit the scope of the invention. Since modifications will be apparent to
those of skill in
this art, it is intended that this invention be limited only by the scope of
the appended
claims.
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