U.S. patent application number 10/436647 was filed with the patent office on 2004-01-22 for method and apparatus for fractionation using conventional dielectrophoresis and field flow fractionation.
This patent application is currently assigned to Board of Regents, The University of Texas System. Invention is credited to Becker, Frederick, Gascoyne, Peter R.C., Huang, Ying, Wang, Xiaobo, Yang, Jun.
Application Number | 20040011651 10/436647 |
Document ID | / |
Family ID | 23494900 |
Filed Date | 2004-01-22 |
United States Patent
Application |
20040011651 |
Kind Code |
A1 |
Becker, Frederick ; et
al. |
January 22, 2004 |
Method and apparatus for fractionation using conventional
dielectrophoresis and field flow fractionation
Abstract
Methods and apparatus for discriminating matter in a chamber
having an inlet port and an outlet port utilizing dielectrophoresis
and field flow fractionation. A carrier medium is introduced into
the inlet port and is directed from the inlet port to the outlet
port according to a velocity profile. A programmed voltage signal
is applied to an electrode element coupled to the chamber to form a
dielectrophoretic force on the matter. The dielectrophoretic force
is balanced with a gravitational force to displace the matter to
positions within said velocity profile in the carrier medium to
discriminate the matter. A chamber having a top and bottom outlet
port may be utilized to withdraw a first portion of a carrier
medium from the top outlet port at a first, controllable fluid flow
rate and to withdraw a second portion of the carrier medium from
the bottom outlet port at a second, controllable fluid flow
rate.
Inventors: |
Becker, Frederick; (Houston,
TX) ; Gascoyne, Peter R.C.; (Belaire, TX) ;
Huang, Ying; (San Diego, CA) ; Wang, Xiaobo;
(San Diego, CA) ; Yang, Jun; (Houston,
TX) |
Correspondence
Address: |
Michael C. Barrett, Esq.
FULBRIGHT & JAWORSKI L.L.P.
Suite 2400
600 Congress Avenue
Austin
TX
78701
US
|
Assignee: |
Board of Regents, The University of
Texas System
|
Family ID: |
23494900 |
Appl. No.: |
10/436647 |
Filed: |
May 13, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10436647 |
May 13, 2003 |
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09378874 |
Aug 23, 1999 |
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6641708 |
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09378874 |
Aug 23, 1999 |
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08604779 |
Jan 31, 1996 |
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5993630 |
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60010904 |
Jan 31, 1996 |
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Current U.S.
Class: |
204/547 ;
204/643 |
Current CPC
Class: |
G01N 30/0005 20130101;
B03C 5/028 20130101; G01N 2030/0035 20130101; G01N 2030/007
20130101; B03C 5/026 20130101; G01N 2030/0065 20130101 |
Class at
Publication: |
204/547 ;
204/643 |
International
Class: |
G01N 027/27 |
Claims
What is claimed is:
1. A method of discriminating matter in a chamber having an inlet
port and an outlet port, said chamber defined by a pair of side
walls, a top wall and a bottom wall, and an electrode element
adapted to said chamber, said chamber having a substantially
greater width than thickness, utilizing dielectrophoresis and field
flow fractionation, comprising: introducing a carrier medium
including said matter into said inlet port and directing said
carrier medium from said inlet port to said outlet port, such that
said carrier medium travels through said chamber according to a
velocity profile; applying a programmed voltage signal to said
electrode element to create a spatially inhomogeneous electric
field which causes a dielectrophoretic force on said matter having
components normal to the direction of said carrier medium traveling
through said chamber; and controlling said spatially inhomogeneous
electric field to balance said dielectrophoretic force with a
gravitational force on said matter to displace said matter to
positions within said velocity profile in said carrier medium to
discriminate said matter.
2. The method of claim 1, wherein said programmed voltage signal
comprises a time dependent amplitude or frequency.
3. The method of claim 1, wherein said programmed voltage signal
comprises frequency modulation.
4. The method of claim 1, wherein said programmed voltage signal
comprises amplitude modulation.
5. The method of claim 1, wherein said programmed voltage signal
comprises a sweeping frequency.
6. The method of claim 1, wherein said programmed voltage signal
comprises a series of voltage signals, said voltage signals having
different waveforms.
7. The method of claim 6, wherein said different waveforms differ
in signal frequency, signal amplitude, frequency modulation, or
amplitude modulation.
8. The method of claim 1, wherein said matter travels through said
chamber at a velocity proportionate to its displacement within said
velocity profile.
9. The method of claim 1, wherein said matter exits from said
outlet port at time intervals proportionate to its displacement
within said velocity profile.
10. The method of claim 1, wherein said matter exits from said
outlet port at positions laterally displaced from said inlet
port.
11. The method of claim 1, wherein said method of discriminating
matter is in continuous mode.
12. The method of claim 1, wherein said method of discriminating
matter is in batch mode.
13. A method of discriminating matter, utilizing dielectrophoresis
and field flow fractionation, in a chamber having an inlet port and
an outlet port, said chamber defined by a pair of side walls, a top
wall and a bottom wall, and an electrode element adapted to said
chamber, said chamber having a substantially greater width than
thickness, comprising: introducing a carrier medium from said inlet
port into said chamber; introducing said matter into said inlet
port; introducing a transport fluid into said inlet port and
directing said transport fluid from said inlet port to said outlet
port, such that said transport fluid travels through said chamber
according to a velocity profile; applying a programmed voltage
signal to said electrode element to create a spatially
inhomogeneous electric field which causes a dielectrophoretic force
on said matter having components normal to the direction of said
transport fluid traveling through said chamber; and controlling
said spatially inhomogeneous electric field to balance said
dielectrophoretic force with a gravitational force on said matter
to displace said matter to positions within said velocity profile
in said transport fluid to discriminate said matter.
14. The method of claim 13, wherein said matter travels through
said chamber at a velocity proportionate to its displacement within
said velocity profile.
15. The method of claim 13, wherein said matter exits from said
outlet port at time intervals proportionate to its displacement
within said velocity profile.
16. A method of discriminating matter utilizing dielectrophoresis
and field flow fractionation in a chamber defined by a pair of side
walls, a top wall, and a bottom wall, said method comprising:
introducing a carrier medium including said matter into an inlet
port of said chamber and directing said carrier medium from said
inlet port toward top and bottom outlet ports coupled to said top
and bottom walls, respectively; applying an electrical signal to an
electrode element coupled to said chamber to create a spatially
inhomogeneous electric field to generate a dielectrophoretic force
on said matter having components normal to the direction of said
carrier medium traveling through said chamber; controlling said
spatially inhomogeneous electric field to balance said
dielectrophoretic force with a gravitational force on said matter
to displace said matter to different heights within said chamber to
discriminate said matter; withdrawing a first portion of said
carrier medium from said top outlet port; and withdrawing a second
portion of said carrier medium from said bottom outlet port.
17. The method of claim 16, wherein at least a portion of said
matter is withdrawn from said top outlet port.
18. The method of claim 16, wherein at least a portion of said
matter is withdrawn from said bottom outlet port.
19. The method of claim 16, wherein said carrier medium is
introduced into said inlet port at a first fluid flow rate, and
wherein said first portion is withdrawn at a second fluid flow
rate, said second fluid flow rate being less than or equal to said
first fluid flow rate.
20. The method of claim 16, wherein said carrier medium is
introduced into said inlet port at a first fluid flow rate, and
wherein said first portion is withdrawn at a second fluid flow rate
equal to about one half of said first fluid flow rate.
21. The method of claim 16, wherein said first portion is withdrawn
at a first fluid flow rate and wherein said second portion is
withdrawn at a second fluid flow rate.
22. The method of claim 21, further comprising varying said first
and second fluid flow rates to reduce fluid pressure of at least
one of said top or bottom outlet ports.
23. The method of claim 21, further comprising varying said first
and second fluid flow rates to further discriminate said
matter.
24. The method of claim 16, wherein said first portion or said
second portion is withdrawn with a syringe pump.
25. The method of claim 16, wherein said method of discriminating
is in continuous mode.
26. The method of claim 16, wherein said method of discriminating
is in batch mode.
27. The method of claim 16, wherein said carrier medium travels
through said chamber according to a velocity profile such that said
carrier medium moves more rapidly at the center of said
chamber.
28. The method of claim 27, wherein said velocity profile is
parabolic.
29. The method of claim 16, wherein said carrier medium travels
through said chamber according to a plug-like profile.
30. The method of claim 16, wherein said electrical signal
comprises a programmed voltage signal.
31. The method of claim 16, wherein said chamber has a
substantially greater width than thickness.
32. A method of discriminating matter utilizing dielectrophoresis
and field flow fractionation in a chamber defined by a pair of side
walls, a top wall, and a bottom wall, said method comprising:
introducing a carrier medium including said matter into an inlet
port of said chamber and directing said carrier medium from said
inlet port toward top and bottom outlet ports coupled to said top
and bottom walls, respectively; applying an electrical signal to an
electrode element coupled to said chamber to create a spatially
inhomogeneous electric field to generate a dielectrophoretic force
on said matter having components normal to the direction of said
carrier medium; controlling said spatially inhomogeneous electric
field to balance said dielectrophoretic force with a gravitational
force on said matter to displace said matter to different heights
within said chamber to discriminate said matter; withdrawing a
first portion of said carrier medium from said top outlet port at a
first fluid flow rate; and withdrawing a second portion of said
carrier medium from said bottom outlet port at a second fluid flow
rate.
33. The method of claim 32, further comprising controlling said
first and second fluid flow rates to further discriminate said
matter.
34. The method of claim 32, further comprising detecting
discriminated matter with a detector coupled to said chamber.
35. The method of claim 32, wherein said carrier medium is
introduced into said inlet port at a third fluid flow rate, and
wherein said first fluid flow rate is less than or equal to said
third fluid flow rate.
36. The method of claim 32, wherein said carrier medium is
introduced into said inlet port at a third fluid flow rate, and
wherein said first fluid flow rate is about one half of said third
fluid flow rate.
37. The method of claim 32, wherein said method of discriminating
is in continuous mode.
38. The method of claim 32, wherein said method of discriminating
is in batch mode.
39. The method of claim 32, wherein said electrical signal
comprises a programmed voltage signal.
40. A method for treating a condition in a patient indicated by
presence of an identified matter, in a chamber having an inlet port
and an outlet port, a center and a pair of side walls, and an
electrode element, said chamber having a substantially greater
width than thickness, utilizing dielectrophoresis and field flow
fractionation, comprising: introducing a sample of said patient
including said identified matter into said inlet port; introducing
a transport fluid into a duct, causing a fluid flow according to a
velocity profile within said chamber; applying at least one
electrical signal at a holding frequency of said identified matter
to said electrode element to create a spatially inhomogeneous
electric field to cause a dielectrophoretic force on said
identified matter having components normal to the direction of said
fluid flow; holding said identified matter in close proximity to
said electrode element; transporting said sample without said
identified matter by said transport fluid at a velocity according
to said velocity profile; and collecting said sample without said
identified matter at said outlet port, thus treating the
condition.
41. The method of claim 40, further comprising collecting said
identified matter at said outlet port.
42. The method of claim 40, wherein said electrical signal
comprises a programmed voltage signal.
43. A method for diagnosing a condition by determining a presence
of unidentified matter within a patient sample, in a chamber having
an inlet port and an outlet port, a center and a pair of side
walls, and an electrode element, said chamber having a
substantially greater width than thickness, utilizing
dielectrophoresis and field flow fractionation, comprising:
introducing a carrier medium including said unidentified matter
into said inlet port, causing said carrier medium to travel through
said chamber according to a velocity profile; applying at least one
electrical signal to said electrode element at a holding frequency
of a known matter to create a spatially inhomogeneous electric
field to cause a dielectrophoretic force on said matter having
components normal to the direction of said carrier medium traveling
through said chamber; and determining whether said unidentified
matter is held in close proximity to said electrode element,
indicating said condition.
44. The method of claim 43, wherein said electrical signal
comprises a programmed voltage signal.
45. An apparatus for discriminating matter, comprising: a chamber
defined by a top and bottom wall; a top outlet port coupled to said
top wall; a bottom outlet port coupled to said bottom wall; an
inlet port coupled to said chamber and in spaced relation with said
top and bottom outlet ports; an electrode element coupled to said
chamber; and a detector in operative relation with at least one of
said top or bottom outlet ports;
46. The apparatus of claim 45, further comprising an injection
valve in operative relation with said inlet port.
47. The apparatus of claim 45, further comprising a spacer
positioned between said top and bottom walls, said spacer defining
an open channel within said chamber.
48. The apparatus of claim 45, wherein said detector comprises a
flow cytometer.
49. The apparatus of claim 45, wherein said electrode elements
comprises an array of microelectrodes.
50. An apparatus for discriminating matter, comprising: a chamber
defined by a top and bottom wall; an outlet port coupled to said
chamber; an inlet port coupled to said chamber and in spaced
relation with outlet port; an electrode element coupled to said
chamber and configured to create a spatially inhomogeneous electric
field to generate a dielectrophoretic force on said matter; and two
or more sensing electrode elements coupled to said chamber and
defining a detector integral with said chamber.
Description
[0001] The present application is a continuation-in-part of U.S.
patent application Ser. No. 08/604,779 filed Feb. 23, 1996, which
claims priority to U.S. Provisional Application Serial No.
60/010,904 filed Jan. 31, 1996. The entire text of each of the
above-referenced disclosures is specifically incorporated by
reference herein without disclaimer.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to the fields of
molecular separation and particle discrimination. More
particularly, it concerns the fractionation of particulate matter
utilizing a combination of electrical, hydrodynamic or
gravitational forces.
[0004] 2. Description of the Related Art
[0005] The ability to identify, characterize and purify cell
subpopulations is fundamental to numerous biological and medical
applications, often forming the starting point for research
protocols and the basis for current and emerging clinical
protocols. Cell separation has numerous applications in areas such
as medicine, biotechnology, biomedical research, environmental
monitoring and bio/chemical warfare defense. For example, cell
separation can make possible life-saving procedures such as
autologous bone marrow transplantation for the remediation of
advanced cancers where the removal of cancer-causing metastatic
cells from a patient's marrow is necessitated (Fischer, 1993). In
other applications, such as the study of signaling between blood
cells (Stout, 1993; Cantrell et al., 1992), highly purified cell
subpopulations permit studies that would otherwise be impossible.
Current approaches to cell sorting most frequently exploit
differences in cell density (Boyum, 1974), specific immunologic
targets (Smeland et al., 1992), or receptor-ligand interactions
(Chess et al., 1976) to isolate particular cells.
[0006] These techniques are often inadequate and sorting devices
capable of identifying and selectively manipulating cells through
novel physical properties are therefore desirable. The application
of the principles of AC electrokinetics has been used for the
dielectric characterization of mammalian cells through the method
of electrorotation (ROT) (Arnold and Zimmermann, 1982; Fuhr, 1985;
Holzel and Lamprecht, 1992; Wang et al., 1994) and for cell
discrimination and sorting (Hagedom et al., 1992; Huang et al.,
1993; Gascoyne et al., 1992; Gascoyne et al., 1994; Huang et al.,
1992). In these techniques, cells become electrically polarized
when they are subjected to an AC electric field. In ROT, a
rotational electrical field is applied and the interaction between
the cells' polarization and the applied field results in cell
rotation. If that field is inhomogeneous, then the cells experience
a lateral dielectrophoretic (DEP) force, the frequency response of
which is a function of their intrinsic electrical properties
(Gascoyne et al., 1992). In turn, these properties depend strongly
on cell composition and organization, features that reflect cell
morphology and phenotype. Cells differing in their electrical
polarizabilities can thus experience differential forces in the
inhomogeneous electric field (Becker et al., 1994; Becker et al.,
1995). Analysis of the dielectrophoretic motion of mammalian cells
as a function of applied frequency permits cell membrane
biophysical parameters, such as capacitance and surface
conductance, to be probed. Because DEP effectively maps biophysical
properties into a translational force whose direction and magnitude
reflects cellular properties, DEP force may induce separation
between particles of different characteristics. For example, DEP
has been used on a microscopic scale to separate bacteria from
erythrocytes (Markx et al., 1994), viable from nonviable yeast
cells (Wang et al., 1993), and erythroleukemia cells from
erythrocytes (Huang et al., 1992). However, the differences in the
electrical polarizabilities of the cell types in those various
mixtures were greater than those to be expected in many typical
cell sorting applications.
[0007] Field flow fractionation (FFF) has also been generally
employed for separation of matter, utilizing particle density,
size, volume, diffusivity, and surface charge as parameters
(Giddings, 1993). The technique can be used to separate many
different types of matter, from a size of about 1 mm to more than
about 100 micrometers, which may include, for example, biological
and non-biological matter. Separation according to field flow
fractionation occurs by differential retention in a stream of
liquid flowing through a thin channel. The FFF technique combines
elements of chromatography, electrophoresis, and
ultracentrifugation, and it utilizes a flow velocity profile
established in the thin channel when the fluid is caused to flow
through the chamber. Such velocity profile may be, for example,
linear or parabolic. A field is then applied at right angles to the
flow and serves to drive the matter into different displacements
within the flow velocity profile. The matter being displaced at
different positions within the velocity profile will be carried
with the fluid flow through the chamber at differing velocities.
Fields may be based on sedimentation, crossflow, temperature
gradient, centrifugal forces, and the like. The technique suffers,
however, from producing insufficiently pure cell populations, being
too slow, or being too limited in the spectrum of target cells or
other matter.
[0008] Thus, there exists a need in the art for highly discriminate
separation of particulate matter, especially biological matter.
Furthermore, such a technique should operate without physically
modifying the structure of the matter to be separated. In addition,
it should allow for the sensitive manipulation of such particles,
which may include characterization and purification of desired
matter from extraneous or undesired matter.
SUMMARY OF THE INVENTION
[0009] The present invention seeks to overcome these drawbacks
inherent in the prior art by combining the use of
frequency-dependent dielectric and conductive properties of
particles with the properties of the suspending and transporting
medium. As used herein, the term "matter" is intended to include
particulate matter, solubilized matter, or any combination thereof.
The invention provides a novel apparatus and novel methods by which
different particulate matter and solubilized matter may be
identified and selectively manipulated. These particles may also be
fractionated or collected separately by changing the DEP force or
the fluid flow characteristics. Utilizing the invention in this
manner, particulate matter and solubilized matter may be
discriminated and separated. The apparatus and methods of the
present invention may discriminate many different types of matter
simultaneously.
[0010] The present invention provides a method and apparatus 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 permittivity, electrical conductivity, surface charge,
and/or surface configuration. In the case of the biological cells,
they may be discriminated according to differences in their size,
density, membrane electrical capacitance and conductance, interior
conductivity and permittivity, and/or surface charges.
[0011] The methods according to the present invention may be used
to discriminate particulate matter, including inorganic matter,
such as minerals, crystals, colloidal, conductive, semiconductive
or insulating particles and gas bubbles. The methods of the present
invention may also be used to discriminate biological matter, such
as cells, cell organelles, cell aggregates, nucleic acids,
bacterium, protozoans, or viruses. Further, the particulate matter
may be, for example, a mixture of cell types, such as fetal
nucleated red blood cells in a mixture of maternal blood, cancer
cells such as breast cancer cells in a mixture with normal cells,
or red blood cells infested with malarial parasites. Additionally,
the methods of the present invention may be used to discriminate
solubilized matter such as a molecule, or molecular aggregate, for
example, proteins, or nucleic acids.
[0012] Particles to be discriminated may be any size. However, the
present invention is generally practical for particles between
.about.10 nm and .about.1 mm, and may include, for example,
chemical or biological molecules (including proteins, DNA and RNA),
assemblages of molecules, viruses, plasmids, bacteria, cells or
cell aggregates, protozoans, embryos or other small organisms, as
well as non-biological molecules, assemblages thereof, minerals,
crystals, colloidal, conductive, semiconductive or insulating
particles and gas bubbles. For biological applications using living
cells, the present invention allows cells to be separated without
the need to alter them with ligands, stains, antibodies or other
means. Cells remain undamaged, unaltered and viable during and
following separation. Non-biological applications similarly require
no such alteration. It is recognized however, that the apparatus
and methods according to the present invention are equally suitable
for separating such biological matter even if they have been so
altered.
[0013] The apparatus may include, for example, a chamber. The
chamber may have at least one inlet and one outlet port, an
interior surface and an exterior surface. The chamber may further
be designed to have structural characteristics so that a desired
flow velocity profile is generated when a fluid is caused to flow
through the chamber. The flow velocity profile refers to the fact
that the fluid at different positions travels at differing
velocities. The chamber may be rectangular in shape and may
include, for example, a top wall, bottom wall and two side walls.
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, thereby creating a thin chamber capable of creating a
velocity profile. For example, for a rectangular chamber having a
width W, a height H and a length L where the condition W>>H
is held, the velocity profile for a fluid flow in the chamber in
the direction along its length can be described as follows
(Pazourek and Chmelik, 1992), 1 V ( x , h ) = 6 V m h H ( 1 - h H )
( 1 - cosh ( 2 3 ( x - W / 2 ) / H ) cosh ( 3 W / H ) ) ( 1 )
[0014] where x is the distance from the chamber side wall
(measuring horizontally) and h is the distance from the bottom wall
(measuring vertically). The factor (V.sub.m) is the average
velocity of the fluid traveling through the chamber. Equation (1)
indicates that the fluid velocity follows a parabolic dependency on
the vertical position in the chamber. Since W>>H, the fluid
velocity expression in Equation (1) can be approximated as a
parabolic dependency, as 2 V ( h ) = 6 V m h H ( 1 - h H ) , ( 2
)
[0015] where the edge-effect along the chamber width is ignored. In
such a flow profile, the fluid velocity increases with increasing
distance from the chamber top or bottom walls. Fluid close to the
top and bottom walls travels at near zero velocity; and that at the
mid point between the top and bottom walls travels at the highest
velocity.
[0016] 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, again creating a thin chamber capable of creating a
velocity profile. In that case, the parameter h in equation (2)
describes the horizontal distance in the chamber from a side wall
and H is the width of the chamber. Alternately, the chamber may be
of circular construction, triangular, rectangular, hexadecagonal,
or of other geometrical shapes. In such cases, modified versions of
equation (2) will apply as is known in the art of hydrodynamics. As
such, the present invention is not intended to be limited to a
particular geometric shape. The chamber according to the present
invention may be constructed of many different materials, for
example, glass, polymeric material, plastics, quartz, coated metal,
or the like.
[0017] The chamber includes at least one electrode element adapted
along a portion or all of the chamber walls. Each of these one or
more electrode elements may be electrically connected to an
electrical conductor, which in turn are connected to an electrical
signal source. In the discussion which follows, the terms
"electrode element" or "electrodes" will be used. As used herein,
"electrode element" is a structure of highly
electrically-conductive material over which an applied electrical
signal voltage is constant. It is to be understood that these terms
include all of the electrode configurations described below. An
electrical signal generator, which may be capable of varying
voltage, frequency, phase or all the three may provide at least one
electrical signal to the electrode elements. The electrode elements
of the present invention may include, for example, a plurality of
electrode elements which may be connected to a plurality of
electrical conductors, which in turn are connected to the electric
signal generator.
[0018] The chamber according to the present invention may include a
plurality of electrode elements which comprise an electrode array.
As used herein, an "electrode array" is a collection of more than
one electrode element in which each individual element may be
displaced in a well-defined geometrical relationship with respect
to one another. This array may be, for example, an interdigitated
(or parallel) array, interdigitated castellated array, a polynomial
array, plane electrode, or the like. Further, the array may be
comprised of microelectrodes of a given size and shape, such as an
interdigitated array. The electrode array may be adapted along any
interior or exterior surface of the chamber. Alternately, it is
envisioned that the electrode array may be incorporated into the
material which comprises the chamber walls. In certain embodiments,
the electrode array may be a multilayer array in which conducting
layers may be interspersed between insulating layers. Further, the
present invention may have a plurality of electrode arrays which
may be adapted, for example, on opposing surfaces of the chamber.
However, it may be possible to place the plurality of electrode
arrays on adjacent surfaces or on all surfaces of the chamber.
Fabrication of such an electrode array, depending on electrode
dimensions, may use any of the standard techniques known in the art
for patterning and manufacturing microscale structures.
[0019] For an interdigitated (parallel) array, the parallel
electrode elements may be adapted to be substantially
longitudinally or latitudinally along a portion of the chamber.
Other configurations of electrode elements are contemplated by the
present invention, such as electrode elements adapted at angles to
the chamber. It is also possible to use a three-dimensional
electrode element that may or may not be attached to the surface of
the chamber. For example, electrode elements may be fabricated from
silicon wafers, using the semiconductor microfabrication techniques
known in the art. If the electrodes are adapted along the exterior
surface of the chamber, it is envisioned that a means of
transmitting energy into the chamber, such as a microwave
transmitter may be present. The electrode elements may be
configured to be on a plane substantially normal or parallel to a
flow of fluid travelling through said chamber. However, it is to be
understood that the electrode elements may be configured at many
different planes and angles to achieve the benefits of the present
invention.
[0020] When the electrode elements are energized by at least one
electrical signal from the electrical signal generator, the
electrode elements thereby create spatially inhomogeneous
alternating electric field, which causes a DEP force on the
particulate matter and solubilized matter. This DEP force may be a
conventional DEP force (cDEP), or it may be a travelling wave DEP
force (twDEP), or the combination of the both. The cDEP force may
have components acting in a direction substantially normal to the
electrode element plane, that is, the cDEP force may cause matter
to move towards or away from this plane. In reference to the fluid
traveling through the chamber, the DEP force may act substantially
in a direction normal to the fluid flow. As used herein, "a
direction normal to the fluid flow" means in a direction which is
substantially non-opposing and substantially nonlinear to the flow
of a fluid traveling through the chamber. This direction may be for
example, vertically, sideways, or in another non-opposing
direction. By effect of this DEP force, particulate matter and
solubilized matter is displaced to different positions within the
fluid, in particular within the flow velocity profile established
in the chamber. This displacement may be relative to the electrode
elements, or may relate to other references, such as the chamber
walls.
[0021] It is noted that by altering phase of the alternating
electrical signal, a second DEP force, known as traveling wave DEP
(twDEP), is created. The cDEP force is dependent on the spatial
inhomogeneity of the electric field and causes matter to move
towards or away from regions of high electrical field strength. The
twDEP force is dependent upon the phase distribution of the applied
electric field, and caused matter to move towards or away from the
direction of increasing phase values.
[0022] In the present invention, the cDEP force is dependent on the
magnitude of the spatial inhomogeneity of the electric field and
the in-phase (real) part of the electrical polarization induced in
matter by the field. It is to be understood that the term
"electrical polarization" is related to the well-known
Clausius-Mossotti factor, described below. This field-induced
electrical polarization is dependent on the differences between the
dielectric properties between the matter and the suspending medium.
These dielectric properties include dielectric permittivity and
electrical conductivity. Together, these two properties are known
as complex permittivity. The cDEP force causes the matter to move
towards or away from regions of high electrical field strength,
which in an exemplary embodiment, may be towards or away from the
electrode plane.
[0023] The equation for the time-averaged dielectrophoretic force
in a non-uniform electrical field is (Wang et al., 1995): 3 F DEP =
2 m r 3 Re ( f CM ) E rms 2 + 2 m r 3 Im ( f CM ) ( E x0 2 x + E y0
2 y + E z0 2 z ) ( 3 )
[0024] where E.sub.rms is the rms value of the electric field
distribution, E.sub..alpha.0 and .phi..sub..alpha.(.alpha.=x,y,z)
are the magnitudes and phases of each field component. The
parameters E.sub.rms, E.sub..alpha.0and .phi..sub..alpha. are, in
general, functions of spatial coordinates (x,y,z) and dependent on
positions. Nevertheless for the sake of simplicity, the explicit
spatial coordinates (x,y,z) have be omitted. The factor f.sub.CM is
the well-known Clausius-Mossotti factor, defined as 4 f CM = ( p *
- m * ) ( p * + 2 m * ) ,
[0025] where .di-elect cons..sub.p* and .di-elect cons..sub.m* are
the complex permittivities of the matter and its suspending medium,
respectively. Each complex permittivity is defined as .di-elect
cons.*=.di-elect cons.i.sigma./.sub.(2.pi.f), .di-elect cons. and
.sigma. are the permittivity and conductivity, respectively. The
parameters is the frequency of the applied field, r is the radius
of the matter on which the DEP force is acting. Equation (3)
indicates that dielectrophoretic force in general consists of two
components. The first component, cDEP (conventional
dielectrophoretic) force component, is dependent on the real part
Re(f.sub.CM) (in-phase component) of the Clausius-Mossotti factor
f.sub.CM and the magnitude non-uniformity factor 5 E rms 2
[0026] of the applied electric field. If the in-phase part of the
Clausius-Mossotti factor is greater than zero, then the cDEP force
component will move the matter towards the location of the strong
field. If the in-phase part of the Clausius-Mossotti factor is less
than zero, the cDEP force component will move the matter towards
the location of the weak field. The second component, twDEP
(traveling-wave dielectrophoretic) force component, is dependent on
the imaginary part Im(f.sub.CM) (out-of-phase component) of the
Clausius-Mossotti factor f.sub.CM and the phase non-uniformity
factor (.gradient..phi..sub.x, .gradient..phi..sub.y and
.gradient..phi..sub.z) of the applied electric field. Depending on
the polarity of Im(f.sub.CM), the twDEP force component will tend
to move the matter towards the direction of increasing or
decreasing phase values (.phi..sub.x, .phi..sub.y, .phi..sub.z) of
the field components. Whether the matter experiences a cDEP force
component, or a twDEP force component, or both, will depend upon
the electrode geometry and the manner in which the electrical
signals are applied.
[0027] In the present invention, the purpose of applying the DEP
force is to cause particulate matter and solubilized matter to be
displaced to different positions within the fluid flow velocity
profile established in the chamber. Specifically, the DEP force is
applied so that it acts in conjunction with other forces so that
different types of particulate matter is equilibrated at different,
characteristic positions within the flow profile (or solubilized
matter will attain equilibrium concentration distribution within
the flow profile). Examples of other forces that may be used in
conjunction with the applied DEP force include gravitational
forces, electrical forces and hydrodynamic lifting forces.
Gravitational forces arise because of the density difference
between the matter and its suspending medium. If the density of the
matter is larger than that of the medium, the matter will
experience a gravitational force pointing downwards. If the density
of the matter is smaller than that of the medium, the matter will
experience a gravitational force pointing upwards. An electrical
force may be produced on the charged matter when a DC electrical
field is established in the chamber. Hydrodynamic lifting forces
refer to the forces acting on matter when it is close to a chamber
wall and there is a fluid-velocity profile in the chamber (Williams
et al., 1992). Such lifting forces tend to push the matter away
from the chamber walls. The magnitude of the hydrodynamic lifting
forces may depend on the size, density, shape of the matter, the
density and viscosity of the medium, and the fluid-flow profile in
the chamber. In some cases, the hydrodynamic lifting forces may be
significant and may be of comparable magnitude to the gravitational
forces and dielectrophoretic forces. In other cases, the
hydrodynamic lifting forces may be much smaller than the
dielectrophoretic forces and may play a negligible role in
positioning matters in the hydrodynamic flow profile.
[0028] A feature of the present invention is that DEP forces are
applied to the matter in combination with at least one other force
so that matter having different properties (dielectric/electrical
property, density property, charge property) will be positioned
differently in the flow-velocity profile established in the
chamber. In one embodiment, DEP forces may be balanced with
gravitational forces so that different matter attains different
equilibrium positions. In another embodiment, DEP forces may be
balanced with gravitational forces plus the hydrodynamic lifting
forces so as to influence the equilibrium positions of the matter.
In another embodiment, DEP forces may be balanced by electrical
forces to control the equilibrium positions of different matter.
DEP and other forces depend on the properties of the matter (e.g.
dielectric property, density, size, electrical charge etc),
therefore, the balance of these forces and the resulting matter
equilibrium positions (or displacement) are also dependent on the
properties of the matter. The matter of different properties will
be displaced to different positions within the chamber or within
the flow-velocity profile. These equilibrium positions may also be
referred as "levitation" or "levitation height". As used herein,
"levitate" or "levitation height" means that matter is displaced at
different levels with respect to the electrode elements, in any
direction, or matter is equilibrated at different positions with
respect to the electrode elements under the balance of DEP forces
and other forces.
[0029] In one embodiment of the present invention, an
interdigitated (or parallel) electrode array may be adopted on the
bottom wall of the separation chamber. The geometry of the
interdigitated electrode array is characterized by the electrode
element width to electrode element spacing. In one embodiment, the
electrode element width and spacing are the same and an electrical
voltage is applied to the neighboring electrode elements. Under
this condition, only the cDEP force component is present in DEP
forces exerting on the matter in the chamber. This cDEP force
mainly lies in the vertical direction, especially for positions
some distances away from the chamber bottom wall. This force acting
on a matter of the radius r can be approximated as (Huang et al.,
1997; Wang et al., 1998)
F.sub.DEP=2.pi..di-elect
cons..sub.mr.sup.3Re(f.sub.CM)U.sup.2Aexp(-2.pi.h- /d) (4)
[0030] where U is the applied root-mean-squared (RMS) voltage,
.di-elect cons..sub.m is the dielectric permittivity of the medium.
The DEP forces fall approximately exponentially with height h above
the electrode plane, with a decay constant that is characterized by
the periodic distance d
(=2*electrode-element-width+2*electrode-element-spacing) of the
electrode array and a unit voltage force coefficient A. Thus,
changing electrode element width and/or spacing may modify DEP
forces acting on the matter. The DEP forces shown Equation (4) may
be used to balance the gravitational forces acting on the matter to
achieve positioning particulate matter at different heights from
the electrode plane. The gravitation forces are given by 6 - 4 3 r
3 ( p - m ) .
[0031] Here .rho..sub.p and .rho..sub.m are the densities of the
matter and its suspending medium, respectively, satisfying the
relationship .rho..sub.p>.rho..sub.m. The balance of
gravitational and DEP levitation forces positions the matter at a
stable equilibrium height, given by 7 h eq = d 4 ln ( 3 m U 2 g A
Re ( f CM ) ( p - m ) ) . ( 5 )
[0032] Thus, equilibrium levitation heights are dependent on the
dielectric property (as characterized by the dielectric
polarization factor, Re(f.sub.CM)) and density (.rho..sub.p) of the
matter, of the electrode dimensions (A and d), of the applied DEP
field strength (U). The factor Re(f.sub.CM) is also dependent on
the frequency of the applied field. In this embodiment, the DEP
force acts in combination with the gravitational forces, and the
levitation height of the matter is in the vertical direction with
respect to the electrode plane. In other embodiments, the DEP force
may act in combination with other forces and the levitation height
may not be along the vertical direction.
[0033] In another embodiment, the interdigitated electrode array
may have different electrode width from electrode spacing. The DEP
force may take different form from those shown in Equation (4).
Thus, the ratio of electrode width to electrode spacing may be
modified to change the particulate matter and solubilized matter
levitation height. Specifically, by changing this ratio, the
electric field which is created is thereby altered. When the
electric field is thereby altered, in magnitude and/or
inhomogeneity, the levitation height of the matter similarly
change. This levitation need not be in a vertical direction, and
may include displacement in a horizontal direction, for
example.
[0034] Common electrical conductors may be used to connect the one
or more sets of electrode elements to the signal generator. The
common electrical conductors may be fabricated by the same process
as the electrodes, or may be one or more conducting assemblies,
such as a ribbon conductor, metallized ribbon or metallized
plastic. A microwave assembly may also be used to transmit signals
to the electrode elements from the signal generator. All of the
electrode elements may be connected so as to receive the same
signal from the generator. It is envisioned that such a
configuration may require presence of a ground plane. More
typically, alternating electrodes along an array may be connected
so as to receive different signals from the generator. The
electrical generator may be capable of generating signals of
varying voltage, frequency and phase and may be, for example, a
function generator, such as a Hewlett Packard generator Model No.
8116A. Signals desired for the methods of the present invention are
in the range of about 0 to about 50 volts, and about 0.1 kHz to
about 180 MHz, and more preferably between about 0 to about 15
volts, and about 10 kHz to 10 MHz. These frequencies are exemplary
only, as the frequency required for matter discrimination is
dependent upon the conductivity of for example, the cell suspension
medium. Further, the desired frequency is dependent upon the
characteristics of the matter to be discriminated. The variation of
the frequency will generally alter the polarization factor (the
Clausius-Mossotti factor) of the matter and change the DEP forces
exerted on the matter. Thus to enhance the discrimination of
matters using the present invention, the operational frequency may
be chosen so as to maximize the difference in the DEP forces
exerting on the matter or maximize the difference in the DEP-force
induced levitation height between different matter. In one
embodiment of the invention using the interdigitated (parallel)
electrode array, the levitation height of the matter may be
expressed in Equation (5). As an example, the operation frequency
for discriminating two different matters (A and B) with such an
embodiment of the invention may be chosen to maximize the
levitation height difference .vertline.h.sub.eqA-h.sub.eqB.v-
ertline.: 8 Max frequency { h eqA - h eqB or ( 6 ) Max frequency d
4 ln ( ( pB - m ) Re ( f CMA ) ( pA - m ) Re ( f CMB ) ) . ( 7
)
[0035] Here the polarization factors f.sub.CMA and f.sub.CMB depend
on the applied field frequency, the maximum discrimination may be
found by scanning the frequency empirically. Alternatively, if the
dielectric property of the matter A and B can be obtained from some
other methods, then the discrimination frequency may then be
predicted through theoretical calculation. For example, the
dielectric properties of mammalian cells may be readily determined
using the technique of electrorotation in which individual cells
are subjected to a rotating electrical field and cells are caused
to rotate as a result of the interaction between the rotating field
and the field-induced polarization. The frequency spectra of cell
rotations are obtained by measuring cell rotational rate as a
function of the frequency and can be analyzed in terms of
dielectric shell-models to obtain cell dielectric parameters. The
use of electrorational method for cell dielectric characterization
is known to those skilled in the art (Wang, X.-B. et al. 1995;
Huang, Y. et al, 1996; Fuhr & Hagedorn, 1996). The dielectric
parameters from electrorotational measurements may then be used to
calculate the frequency dependency of cell polarization factor
f.sub.CM and to determine the frequency using Formula (7) at which
the discrimination between two cell types is maximized.
[0036] The discrimination between matters depends also on the
shape, size and configuration of the electrode elements. The change
in these variables may significantly alter electrical field
distribution and affect DEP forces acting on matters. Thus, it may
be necessary to design different geometries of electrode array for
different applications of the present invention. Electrode array
may be, for example, an interdigitated (or parallel) array,
interdigitated castellated array, a polynomial array, plane
electrode, or the like. Further, the array may be comprised of
microelectrodes of a given size and shape, such as an
interdigitated array.
[0037] 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 which may be capable of varying
voltage, frequency and phase. Furthermore, DEP forces acting on
matters may be programmed and varied by electrical signals applied
to electrode arrays so that the signal amplitude, frequency,
waveforms, and/or phases are a function of the time. For example,
the applied sinusoidal signal may have a frequency (f.sub.1) for
certain-length of time and may then be changed to a frequency
(f.sub.2). Alternatively, electrical signals with
frequency-modulation (frequency continuously changes with time) and
amplitude-modulation (amplitude continuously changes with time) may
be applied. The signals applied to electrode arrays can therefore
be programmed according to the specific separation goals and the
specific separation problems. By employing such programmed signals,
the DEP force may be varied with time for enhancing separation
performance and the discrimination of DEP-FFF separator may be
tailed to specific applications.
[0038] A chamber according to the present invention may have at
least one inlet and outlet port. These ports may be the same port,
or the chamber may be constructed to have different ports. The
inlet port may take the form of drilled holes on the major walls of
the chamber at the positions close to the chamber inlet end. The
inlet port would allow the introduction of the matter to be
discriminated into the chamber. The matter may be suspended or
solubilized in a liquid medium, and may be introduced into the
chamber through an injection valve equipped with an injection loop.
The use of such injection valve for introducing the matter to be
discriminated is known to those skilled in the art, as typically
employed in chromatography. The inlet port may also be used for the
introduction of the medium into the chamber so to establish a flow
velocity profile. The reference by Wang et al (1998) provided a
detailed description of using an injection valve for introducing
the matter to be discriminated and for introducing the medium
through an inlet port.
[0039] The outlet port may be arranged to be vertically lower than
the at least one inlet port. Such an arrangement thereby permits
sedimentation of the particulate matter and solubilized matter as
it travels throughout the chamber. In addition to the at least one
inlet port and one outlet port, the chamber may also include one or
more input ducts which allow the fluid to flow through the
apparatus.
[0040] 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. In
one embodiment, the outlet port may be two ports and may be located
on the two major facing walls at positions close to the outlet end
of a rectangular chamber. Because the matters to be discriminated
attain equilibrium levitation heights in the chamber and are
transported through the chamber under the influence of the
flow-velocity profile, the matters may exit the chamber from one of
the two outlet ports and the carrier medium may exit the chamber
from the other outlet port. The advantage of this approach would
increase the concentration of the matters at the port where they
exit the chamber so that the matters may be collected and analyzed.
In another embodiment, the matters to be discriminated may exit the
two outlet ports, i.e. one population of the matter from one outlet
port and all the others from the second outlet port. This
embodiment may further allow the continuous operation of the
discrimination and separation of the matter using the present
invention. The matters may continuously be introduced into the
chamber through the inlet port, and upon their introduction into
the chamber, the matters would experience dielectrophoretic forces
and other operational forces (such as gravity and hydrodynamic
lifting forces) and would be directed towards to different
levitation heights within the chamber. At these heights, all the
forces acting on the matters would balance each other and the net
force would be zero. During this process of moving the matters to
their equilibrium positions, the flow velocity profile would carry
the matter through the chamber. Depending on their levitation
positions in the profile, the matter would exit the chamber at one
of the two outlet ports. Which of the two ports the matter may exit
from would depend on DEP and other forces acting on the matter and
thus depend on the properties of the matter, allowing the
discrimination of the matter.
[0041] In another embodiment, the outlet port may be located along
the entire width or a part of the width of the chamber. The outlet
port may be adapted to receive the matters of various shapes and
sizes. For example, the size of the outlet port may vary from
approximately twice the size of the matter desired 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 having a cross section comprised
of individual tubing elements. Further, for example, the outlet
port may be connected to fraction collectors or collection wells
which are used to collect separated matter. As used herein,
"fraction collectors" and "collection wells" include storage and
collection devices for discretely retaining the discriminated
particulate matter and solubilized matter. Other components that
may be included in the apparatus of the present invention are, for
example, measurement or diagnostic equipment, such as flow
cytometers, lasers, particle counters, particle impedance sensors,
impedance analyzers, and spectrometers. These analytical
instruments connected directly to the outlet port of the chamber
may serve not only detection step for measuring and recording the
time of the arrival of the particulate or solubilized matter but
also analyzing step for characterizing the properties of the
matter. For example, an AC impedance sensor may be connected to the
outlet port of the chamber, and coupled with AC impedance sensing
electronics, may serve an analytical step for determining the AC
impedance of individual particles when they exit the separation
chamber.
[0042] The matter being discriminated using the chamber of the
present invention attains equilibrium positions within the chamber
at which DEP and other forces (e.g. gravity or hydrodynamic lifting
forces) balance each other. A fluid flow may be established in the
chamber so to establish a flow velocity profile. After being
displaced within such a fluid flow profile , the displaced matter
may exit from the outlet port or ports at a time proportionate to
the displacement of the matter within the fluid. Specifically, the
matter equilibrated at different positions within the flow profile
is carried by the fluid flow at different speeds or matter at
different levels of displacement within the fluid travels at
different speeds. Therefore, the matter is discriminated by its
displacement within the fluid flow. Matters of different properties
attain different equilibrium positions or are displaced at
different levels within the fluid flow profile. Particulate matter
and solubilized matter within the fluid flow velocity profile will
travel through the chamber at velocities according to their
positions within the velocity profile.
[0043] This velocity profile may be, for example, a hydrodynamic
fluid profile such as a parabolic flow profile. For a chamber of a
rectangular shape, the velocity profile may be determined by
knowing the average fluid velocity, and the chamber width and
thickness, as shown in Equations (1) and (2). The average fluid
velocity may be calculated based on the flow rate of the fluid, and
the chamber width and thickness, according to the equation:
average fluid velocity=(flow rate)/(chamber width.times.chamber
thickness) (8)
[0044] Parameters that determine the velocity profile of the fluid
flow include (but are not limited to): the chamber width or
thickness, which in a rectangular embodiment may be the distance
between opposing walls; constrictions or expansions of the fluid
flow path which may include, for example, those arising for a
non-parallel disposition of opposing 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.
[0045] In one embodiment of the present invention, an
interdigitated (parallel) electrode array may be adopted on the
bottom wall of a rectangular separation chamber. The matter to be
discriminated is introduced into the chamber with appropriate
electrical signals applied and is positioned at equilibrium heights
with respect to the electrode elements under the influence of the
DEP forces and the gravitational forces. Matters of different
properties (e.g.: dielectric, density, size) are displaced to
different heights, allowing the discrimination of the matter.
Further, when a fluid flow profile such as those described in
Equations (1) and (2) is generated in the chamber, the matter being
displaced at different heights from the electrode plane is
transported at different velocities under the influence of the
fluid flow. For example, when a parabolic flow profile (along the
vertical direction as in Equation (2)) is established, the matter
being displaced to positions close to the half-height of the
chamber travels at higher speeds than the matter being displaced to
positions close to the chamber top and bottom walls. Thus the
matter may be discriminated by such differing velocities. As an
example, two particulate matters A and B are positioned at
equilibrium heights h.sub.eqA and h.sub.eqB within a parabolic flow
profile (along the vertical direction as in Equation (2)). Their
velocities caused by the fluid flow are 9 V A = 6 K V m h eqA H ( 1
- h eqA H ) , ( 9 )
[0046] and 10 V B = 6 K V m h eqB H ( 1 - h eqB H ) . ( 10 )
[0047] Here K.sub..alpha. is a factor which lies between 0 and 1,
and it reflects the retardation effects due to the chamber wall
(Williams et al, 1992). If the matter A is positioned higher than
the matter B but less than the half chamber height H,
h.sub.eqB<h.sub.eqA<H, then A would travel at larger velocity
than B, V.sub.B<V.sub.A. Further, different matter, when
introduced into the chamber at a fixed time, would take differing
time to travel through the chamber and to exit the chamber outlet
port. The matter having larger velocities would exit the chamber
ahead of the matter having smaller velocities. For the matter A and
B described above, the time they take to travel through the chamber
of length L would be 11 t A = L V A ( 11 )
[0048] and 12 t B = L V B . ( 12 )
[0049] Thus, the matter may be further discriminated and separated
by such differential exit-time (t.sub.B>t.sub.A since
V.sub.B<V.sub.A). For The matter exiting the chamber earlier may
be collected separately from those exiting the chamber later,
allowing the matter separation and discrimination. In another
embodiment of an apparatus according to the present invention, a
chamber may have two facing electrode arrays adapted on opposing
surfaces. The chamber may be oriented so that the electrode planes
stand substantially vertical and the thin sides of the chamber are
vertically arranged. It is understood, however, that the electrode
planes need not be only vertical, and the present invention
contemplates adapting the apparatus at varying angles. Different
electrical signals (frequency and magnitude) may be applied to the
facing electrodes from the signal generator so that particles
experience different cDEP forces. Further, within each electrode
array, each alternate element may receive different electrical
signals to create an inhomogeneous alternating electric field.
[0050] This further embodiment may have, for example, one inlet
port adapted to receive the particulate matter to be discriminated.
The inlet port may be located, for example, close to the top of one
end of the chamber. This apparatus may also include one or more
ducts to introduce a fluid that travels through the chamber. The
ducts, which may be arranged substantially along the entire width
of the input end of the chamber, serve to introduce a sheet of
fluid that travels throughout the chamber in a substantially linear
direction. As used herein, a "sheet" of fluid may be a flow of
fluid or gas entering the chamber at a substantially uniform fluid
velocity. The uniform distribution in the fluid velocity here
refers to that the fluid velocity does not vary with positions
along the entire width of the input end of the chamber. However,
the fluid velocity may be a function of the distance from electrode
planes located at two major, facing walls. The introduced "sheet"
fluid carries the particulate matter through the chamber. Following
transit through the chamber, fluid leaves at the opposite end. This
exit end of the chamber may include, for example, one or more exit
ports, which may be arranged in one or more arrays of exit ports.
The outlet port may be constructed so that matter having different
lateral positions at one vertical level may be separately
discriminated. For example, it may be possible to utilize a laser
as a tool to determine characteristics of matter exiting at
selected lateral positions.
[0051] Different electrical signals (frequency or magnitude or
both) are applied to electrode elements located on each of the side
walls. There is a synergistic interaction between these different
electrical signals which creates an inhomogeneous electric field.
Particulate matter to be discriminated is subjected to DEP forces
(F.sub.DEP1 and F.sub.DEP2) from electrical fields induced from
electrode elements located on both the side walls,
F.sub.total=F.sub.DEP1+F.sub.DEP2. (13)
[0052] Under the combined influence of these forces, the matter is
directed equilibrium positions with respect to the side-walls. The
equilibrium position, defined as the distances from the two
side-walls, is therefore determined by DEP forces on the
particulate matter. Since both DEP force components are dependent
on the dielectric and conductive properties of the matter, this
equilibration position depends on these properties of the matter.
Other factors influencing the equilibrium positions include the
magnitude and frequency of the electrical fields applied to the
electrodes on the opposing chamber walls, the fluid density,
viscosity, and flow rate. Different matter, because of their
different properties, equilibrates at different characteristic
distances from the side-walls of the chamber and attains different
equilibrium positions based on this synergistic interaction of the
DEP forces induced by the differing electrical signals. When a
fluid flow is established in the chamber, the velocity of the
different matter within the fluid is controlled by the velocity
profile of the fluid and the equilibrium position of the matter
with in the flow-velocity profile. This velocity profile has a
maximum velocity towards the center of the chamber, with this
velocity proportionately diminishing as distance from the
side-walls decreases. Because of this velocity profile, matter that
has equilibrated at different equilibrium distances from the
chamber walls will be carried at different velocities and therefore
take varying amounts of time to traverse the chamber. Those skilled
in the art would appreciate that the equations describing the
velocity and the transit time of matter through the chamber under
the influence of the fluid flow are similar to Equations (9, 10,
11, 12).
[0053] The distance that matter sediments during its passage across
the chamber will depend upon its transit time, as gravitational
forces act on the matter during its transit through the chamber,
and this is known as a "sedimentation effect." For a spherical
particle having a radius r and density .rho..sub.p, the
sedimentation velocity V.sub.sed in a medium having a density
.rho..sub.m and viscosity .eta..sub.m can be written as 13 V sed =
2 r 2 ( p - m ) 9 m . ( 14 )
[0054] The sedimentation velocity is a function of the particle
size and density, medium viscosity and density. Consequently,
different particles will sediment to different depths (D.sub.sed)
based upon the sedimentation velocity (V.sub.sed) and the transit
time (t.sub.transit) of matter through the chamber,
D.sub.sed=V.sub.sed.times.t.sub.transit. (15)
[0055] Thus, particle sedimentation depends on matter
characteristics, such as size, mass, and volume, for example. As
described above, the time (t.sub.transit) required for particles to
travel across the entire length of the chamber is controlled by the
fluid flow profile and the positions of particles within the
flow-velocity profile. The placement of particles within the fluid
flow profile is in turn determined by the synergism of the
differing electrical signals. Thus, particles with different
characteristics (e.g.: dielectric property, size) may be placed at
different positions in the flow profile and therefore exhibit
different transit times. The combination of differences in transit
time and in sedimentation velocity between particles of different
properties (e.g. dielectric property, density, size) may lead to
different sedimentation depths for these particles. They may exit
the chamber through different outlet ports which may be placed at
different heights with respect to the inlet ports. Discrimination
may be accomplished either in "batch mode" or in "continuous mode."
In batch mode, an aliquot of particles is injected and collected
with respect to the time of transit (t.sub.transit) for the
particles and the height of exit (D.sub.sed) at the outlet ports.
In continuous mode, a constant stream of particles is injected into
the inlet port, and matter emerging at different heights
(D.sub.sed) are continuously collected.
[0056] The methods and apparatus of the present invention introduce
for the first time the use of the frequency-dependent dielectric
and conductive properties of particles as well as those of the
suspending medium. These new criteria for particle fractionation
allow sensitive manipulation of particles because the
dielectrophoretic force is large and strongly dependent on particle
properties. Appropriate choices of the suspending medium and
applied field conditions allow for high levels of
discrimination.
[0057] Previously reported field flow fractionation techniques have
limitations for biological samples because of the narrow range of
cell densities, demanding complex centrifuges and centrifugation
techniques for good discrimination. The cDEP affinity method
demands large differences in the dielectric characteristics of the
particles to be separated so that selected particulate matter and
solubilized matter can be completely immobilized while others are
swept away by fluid flow forces. Since, for biological cells,
damage can occur at high electric field strengths, there is a
practical limitation to the maximum cDEP force that can be applied
and this in turn limits the maximum fluid flow rate in the cDEP
affinity approach. This may result in a slow cell sorting rate. In
the methods of the present invention, these limitations are
substantially reduced. Furthermore, the cDEP affinity method of the
prior art utilizes the dielectrophoretic force component that
generally immobilizes particles on electrode elements. The cDEP/FFF
approach of the present invention utilizes the DEP forces and other
forces to determine the positions of particles or other matter in a
flow-velocity profile and exploits the flow-velocity profile.
[0058] Also, in the present invention, the flow profile is an
active mechanism for the separation and discrimination of
particles, and the dielectrophoretic force (mainly the force
component in the direction normal to the fluid flow direction), in
conjunction with other forces (e.g. gravity, hydrodynamic lifting
force, or another dielectrophoretic force), is the primary means by
which the heights or positions of particles in the fluid flow
profile are controlled. As discussed above, the fluid profile may
be controlled by apparatus design, fluid rate, density and the
like. By combining FFF and dielectrophoretic forces, the present
invention takes advantage of particle volume and density in
synergism with the frequency-dependent particle dielectric and
conductive properties as well as surface configuration. The
operation of an apparatus according to the present invention may be
controlled by varying experimental conditions including, but not
limited to, the particle suspending medium conductivity and
permittivity, the fluid flow rate, viscosity and density, the
applied electrical field strength, the applied frequency and the
applied electrical signal waveform. This utilization of many
parameters in setting the operational conditions for fractionation
greatly increases the ability to discriminate between different
particulate matter and solubilized matter. In the methods according
to the present invention, particles emerging from the outlet ports
of the apparatus may be collected, for example, by one or more
fraction collectors, or may be fed directly into analytical
apparatus such as flow cytometer or impedance sensors to
characterize separated particles. Furthermore, when necessary or
desired, particles may be transferred to collection wells
containing appropriate solutions or media, such as neutral salt
buffers, tissue culture media, sucrose solutions, lysing buffers,
solvents, fixatives and the like. In the case of biological cells,
the collected, separated cells may be further cultured and analyzed
for their molecular characteristics. Alternatively, the separated
cells may be subjected to other molecular, biochemical studies.
[0059] In an illustrative embodiment, the chamber may be
constructed in a rectangular shape using, for example, two glass
slides as chamber walls. These chamber walls may be spaced apart by
spacers to create the rectangular design. These spacers may be made
of, for example, glass, polymeric material such as TEFLON, or any
other suitable material. The size of the chamber and spacing
between chamber walls is dependent on the size of the particles
which are to be discriminated. To practice the methods of the
present invention, an apparatus may have spacing between about 100
nm and about 10 mm, and more preferably between about 20 microns
and about 600 microns in an illustrative embodiment for the purpose
of discriminating mammalian cells. Further, a longer chamber may be
desired to permit greater discrimination throughput. An apparatus
according to the present invention can discriminate cells at a rate
between about 100 and about 3 million cells per second. Factors
that determine discrimination rate include, for example, the
dielectric properties of the particles to be discriminated, the
electrode design, length of the chamber, fluid flow rate, frequency
and voltage of the electrical signals, and the signal waveforms.
The chamber dimensions may be chosen to be appropriate for the
input matter type, characteristics, and degree of discrimination
desired or required.
[0060] In other embodiments, one or more surfaces of the chamber
may support an electrode array. The electrode array may be a
microelectrode array of, for example, parallel electrode
(interdigitated) elements. In certain embodiments, the parallel
electrode elements may be spaced about 20 microns apart. The
apparatus may accommodate electrode element widths of between about
0.1 microns and about 1000 microns, and more preferably between
about 1 micron and about 100 microns for embodiments for the
discrimination of cellular matter. Further, electrode element
spacing may be between about 0.1 microns and about 1000 microns,
and for cellular discrimination more preferably between about 1
micron and about 100 microns. Alteration of the ratio of electrode
width to electrode spacing in the parallel electrode design changes
the magnitude of the dielectrophoretic force and thereby changes
the particle levitation characteristics of the design. The
electrode elements may be connected to a common electrical
conductor, which may be a single electrode bus carrying an
electrical signal from the signal generator to the electrode
elements. Alternately, electrical signals may be applied by more
than one bus which provides the same or different electrical
signals. In certain embodiments, alternate electrode elements may
be connected to different electrode buses along the two opposite
long edges of the electrode array. In this configuration, alternate
electrode elements are capable of delivering signals of different
characteristics. As used herein, "alternate electrode elements" may
include every other element of an array, or another such repeating
selection of elements. The electrode elements may be fabricated
using standard microlithography techniques that are well known in
the art. For example, the electrode array may be fabricated by ion
beam lithography, ion beam etching, laser ablation, printing, or
electrodeposition. The array may be comprised of for example, a 100
nm gold layer over a seed layer of 10 nm chromium or titanium. An
apparatus according to the present invention may be used with
various methods of the present invention. For example, an apparatus
according to the present invention may be used in a method of
discriminating particulate matter and solubilized matter utilizing
dielectrophoresis and field flow fractionation. This method
includes the following steps.
[0061] First, the chamber is preloaded through one inlet port with
a carrier medium, such as a cell suspension medium, tissue culture
medium, a sucrose solution, or the like. Cautions should be taken
during the loading to ensure that no bubble (or only few small
bubbles) is introduced into the chamber.
[0062] Secondly, the matter to be discriminated suspended or
solubilized in a medium may then be introduced into one or more
inlet ports of the chamber. During this introduction, certain
electrical signals may be applied so that the matter to be
discriminated is subjected to dielectrophoretic forces which may
prevent the matter to be in contact with the walls containing the
electrode elements. Alternatively, in some applications, no
electrical signals are applied during the introduction of the
matter.
[0063] Thirdly, after the introduction of the matter into the inlet
region of the chamber, certain electrical signals may be applied
for some time prior to the commencement of the fluid flow in the
chamber. During this period, the matter may move to their
appropriate positions (or equilibrium positions) under the
influence of dielectrophoretic forces generated by the application
of electrical signals and other forces such as gravity. At these
positions, all the forces acting on the matter balance each other
and the net force is zero or close to zero. The matters of
different characteristics may attain different equilibrium
positions within the chamber and are therefore discriminated
according to their equilibrium positions. Alternatively, in some
applications, no electrical signals are applied so that the matter
may move to appropriate positions (equilibrium positions) under the
influence of forces such as gravity.
[0064] Finally, a fluid flow is established in the chamber by, for
example, pumping the carrier medium into the chamber using a
syringe pump. This causes the carrier medium to travel through the
chamber according to a velocity profile so that the velocity of the
medium at different positions with respect to the chamber walls may
be different. At least one alternating electrical signal may be
applied to the one or more electrode elements, which creates an
inhomogeneous alternating electric field within the chamber. This
field causes dielectrophoretic forces to act on the matter within
the chamber. Dielectrophoretic forces, together with other forces
such gravity and hydrodynamic lifting forces, cause the matter to
be displaced to equilibrium positions in the flow velocity profile
within the carrier medium. At these equilibrium positions, the
dielectrophoretic forces are balanced by other forces acting on the
matter. The matters are discriminated according to their positions
within the carrier medium. In addition, the matters at different
positions are caused to travel at different velocities under the
influence of the flow-velocity profile of the carrier medium. Thus,
the matters are further discriminated according to their
velocities. To further discriminate matter, the frequency, or
magnitude or both of the electrical signal may be varied with time.
Such change thereby causes a change in the inhomogeneous
alternating electric field which, in turn, changes the
dielectrophoretic forces acting on the matter and alters the
displacement of the matter with respect to the electrode elements.
These changes further influence the equilibrium positions of the
matter within the flow velocity profile and the velocity of the
matter. The matters are further discriminated according to their
exit time from the chamber. The matter having larger velocities
will exit the chamber ahead of others having small velocities. The
matter after exiting the chamber may be collected and/or
analyzed.
[0065] Another method according to the present invention for
discriminating particulate matter and solubilized matter using
dielectrophoresis and field flow fractionation includes the
following steps. First, the chamber is preloaded through one inlet
port with a carrier medium, such as a cell suspension medium,
tissue culture medium, a sucrose solution, or the like. Secondly,
the matter to be discriminated suspended or solubilized in a medium
may then be flown into one or more inlet ports of the chamber. This
introduction causes the carrier medium to travel through the
chamber according to a velocity profile. The carrier medium at
different positions of the chamber may travel at different
velocities. At least one alternating electrical signal may be
applied to the one or more electrode elements, which creates an
inhomogeneous alternating electric field within the chamber. This
field generates dielectrophoretic forces acting on the matter and
causes the matter within the chamber to be displaced to a position
in the flow velocity profile within the carrier medium. At such an
equilibrium position, dielectrophoretic force acting on the matter
is balanced by other forces such as gravity, or hydrodynamic
lifting forces, or another dielectrophoretic force in the chamber.
Thus, the matter is discriminated according to its position within
the carrier medium. Furthermore, the matter may be discriminated,
for example, according to its velocity. The matter at different
positions of the flow velocity profile travels at different
velocities under the influence of the fluid flow. To further
discriminate matter, the electrical signal may be varied
(frequency, or magnitude, or both). Such a change thereby causes a
change in the inhomogeneous alternating electric field which, in
turn, changes the dielectrophoretic force acting on the matter and
changes the displacement of the matter with respect to the
electrode elements. These changes further influence the equilibrium
positions of the matter within the flow velocity profile and the
velocity of the matter. The matters are further discriminated
according to their exit time from the chamber. The matter having
larger velocity will exit the chamber earlier than other having
smaller velocities. The matter after exiting the chamber may be
collected and/or analyzed.
[0066] Another method according to the present invention includes
discriminating particulate matter and solubilized matter utilizing
dielectrophoresis and field flow fractionation according to the
following steps. First, the chamber is preloaded through one inlet
port with a carrier medium, such as a cell suspension medium,
tissue culture medium, a sucrose solution, or the like. Secondly,
the matter to be discriminated is introduced into the chamber
through one inlet port of a chamber. Next, a transport fluid, which
may be, for example, a tissue culture medium or a gas, is flown
into at least one duct. This causes a fluid flow in the chamber
according to a velocity profile. The fluid at different positions
of the chamber may travel at different speeds. At least one
electrical signal is applied to at least one electrode element.
These one or more electrical signals thereby create an
inhomogeneous electric field within the chamber. The field causes a
DEP force on the matter causing the matter to be displaced to a
position within the transport fluid. At such an equilibrium
position, DEP force is balanced by other forces (such as gravity,
hydrodynamic lifting forces or another DEP force) acting on the
matter. The matter is discriminated according to its position in
the flow velocity profile within the transport fluid. As this
transport fluid is subjected to a velocity profile, the matter
moving at different velocities is thereby partitioned according to
its position in the direction of the fluid flow. The matter is
discriminated according to its velocity and its position within the
fluid flow. To further discriminate matter, the electrical signal
may be varied (frequency, or magnitude, or both). Such a change
thereby causes a change in the inhomogeneous alternating electric
field which, in turn, changes the dielectrophoretic force acting on
the matter and changes the displacement of the matter with respect
to the electrode elements. These changes further influence the
equilibrium positions of the matter within the flow profile and the
velocity of the matter. Furthermore, the separated particulate or
solubilized matters may be collected at times dependent upon their
velocities. It is further possible to collect the matter at one or
more outlet ports for further analysis and characterization.
[0067] Another method according to the present invention for
discriminating particulate matter and solubilized matter utilizing
dielectrophoresis and field flow fractionation includes the
following steps. In this case, a continuous discrimination and
separation of particulate matter and solubilized matter is
achieved. The chamber according to the present invention has two
outlet ports located on the two major facing walls. At least one of
the two major walls supports an electrode array. First, the chamber
is preloaded through one inlet port with a carrier medium, such as
a cell suspension medium, tissue culture medium, a sucrose
solution, or the like. Secondly, the matter to be discriminated
suspended or solubilized in a medium is continuously introduced
into the chamber. This causes a fluid flow in the chamber according
to a velocity profile. At least one electrical signal is applied to
at least one electrode element. These one or more electrical
signals thereby create an inhomogeneous electric field within the
chamber. The field causes a DEP force on the matter causing the
matter to move towards equilibrium position within the transport
fluid. Thus, the matter is not only carried with the fluid flow but
also driven by combined influences of DEP and other forces such as
gravity, hydrodynamic lifting forces or another DEP forces. When
the matter reaches the outlet end, it will exit the chamber at one
of the two outlet ports, depending on its position along the
direction normal to the two major walls of the chamber. The matter
is discriminated according to the outlet port it exits the chamber.
For example, if the matter consists of two subpopulations having
different dielectric properties, the two subpopulation of the
matter may attain different positions in the fluid flow when they
reach the chamber outlet end. The separation of the two populations
is achieved since the two populations exit the chamber at the two
different outlet ports. Clearly the discrimination of the matter
using this approach can be operated continuously. The separated
matter from the two outlet ports may be further collected and
analyzed.
[0068] There are further steps possible to more precisely
discriminate matter. These steps include the following. First, the
alternating electrical signal or signals may be selected at a
frequency and voltage combination which causes the matter to be
either attracted towards or repelled from the electrode elements.
By doing so, the matter is more clearly displaced within the
transport fluid. By application of such a voltage and frequency
combination, it is possible to hold the matter in close proximity
to the electrode elements.
[0069] It is possible to select a frequency to attract desired or
nondesired matter. As used herein, desired matter may be any matter
which is desired to be discriminated and collected for further use.
For example, the separation of normal blood cells from a sample
containing "contaminated" cancer cells may be desired for use in
returning these normal cells into a patient's bloodstream. So
normal cells may be called "desired matter" in this case.
Nondesired matter may be matter which is desired to be
discriminated for other purposes. For example, cancer cells from a
patient's blood or bone marrow may be discriminated so that a
sample of blood not containing the cancer cells may be returned to
the patient. In this case, the cancer cells may be called
"nondesired matter".
[0070] A method for discriminating such a combination of matter may
include the following. A frequency is selected so that the
nondesired matter is held in close proximity to the electrode
elements while simultaneously the desired matter is carried with
the fluid flow and is separated from the nondesired matter. This
frequency may be known as a holding frequency. The fluid flow then
carries the desired matter to the outlet port or ports of the
chamber, where it may be collected. During this process, the
desired matter may also be subjected to further discrimination and
separation so that the subpopulations of the desired matter are
separated under the cDEP/FFF operation. After collection, the
desired matter may, for example, be returned to a patient's
bloodstream or bones, or it may be used for diagnosis or other
molecular or biochemical analysis. Then, to clear the chamber, the
frequency may be changed, or the voltage itself may be turned off.
This will cause the nondesired matter to be released from close
proximity to the electrode element and will be partitioned by the
fluid flow. This nondesired matter may then flow through the
chamber in the fluid, and may be collected, if required. After
collection, the nondesired matter may be used, for example, for
diagnosis or other purposes.
[0071] In an alternate embodiment, it may be possible to hold
desired matter in close proximity to the electrode elements, and
first partition the nondesired matter by the fluid flow, following
the same steps outlined above.
[0072] The apparatus and methods of the present invention may be
used for a number of different useful manners. For example, the
methods according to the present invention may be used to determine
characteristics of an unknown particulate matter and unknown
solubilized matter in a sample of matter. These characteristics can
then be compared to known matter. Additionally, the methods of the
present invention may be used to diagnose a condition by
determining a presence of unidentified particulate matter and
unidentified solubilized matter in a patient sample. This
unidentified matter may be, for example, the presence of a cancer,
a virus, parasite, or the like. After determining the presence of a
condition, the methods of the present invention may be used to
treat the condition by using an apparatus according to the present
invention to discriminate the cancer, virus, parasite or the like
from normal blood or bone marrow cells.
[0073] "Manipulation or discrimination" as used in relation to the
present invention may include, for example, characterization,
separation, fractionation, concentration and/or isolation.
[0074] Typical biological applications for the device useful for
specific products and services include the manipulation or
discrimination of tumor cells, such as epithelial tumor cells or
leukemia cells, from blood and hemopoietic stem cells, purging of
tumor cells from bone marrow and hemopoietic stem cells and
mixtures with other normal cells, purging of residual T-lymphocytes
from stem cells, and enrichment of specific target cell types
including tumor cells, stem cells, etc. Also included is the
manipulation or discrimination of leukocyte cell subpopulations,
removal and concentration of parasitized erythrocytes from normal
erythrocytes in malaria and of other parasitized cells from their
normal counterparts, manipulation of cells at different phases of
the cell cycle, manipulation of viable and non-viable cells,
manipulation of free cell nuclei, and manipulation of nucleated
fetal erythrocytes and trophoblast cells from maternal blood for
further analysis including genetic testing. Moreover, the invention
contemplates the manipulation of bacteria, viruses, plasmids and
other primitive organisms from water, blood, urine, cell mixtures
and other suspensions, manipulation and identification of tumor
cells in biopsies, plaques and scrape tests including Pap smears,
and the manipulation and identification of metastatic tumor cells
from cell mixtures.
[0075] With different and smaller electrode geometries, it is
contemplated that the technology can be used for molecular
applications including manipulation of DNA or RNA molecules and/or
DNA or RNA fragments according to their molecular weight, folding
characteristics and dielectric properties, manipulation of
chromosomes, manipulation of specific protein/DNA and protein/RNA
aggregates, manipulation of individual proteins from a mixture, and
manipulation of specific subcellular molecular complexes and
structures.
[0076] The chamber used for cDEP/FFF application may vary
significantly in size to fit the need of different sample sizes.
For example, the large size chamber may be implemented for
separating many millions of the cells at each operation. On the
other extreme, the chamber may be miniaturized so to form a
microfluidic cell separation step in an integrated bioanalytical
system. Such miniaturized chamber may be integrated with other
microfluidic devices or components. In order to optimize particle
discrimination in different applications it is understood that the
present invention may encompass use of specifically-targeted
electrodes and chamber designs. These designs should provide a
sensitive dependency of the height of particle levitation on the
particle dielectric properties. For example, alteration of the
ratio of electrode width to electrode spacing in the parallel
electrode design changes the vertical component of the
dielectrophoretic force and thereby changes the particle levitation
characteristics of the design. Other strategies for providing
improved particle discrimination include, for example, using more
than two sets of electrode elements with different frequencies
and/or voltages applied to them and the exploitation of synergism
between electrical signals applied to electrode arrays on both the
chamber bottom and top walls. In addition, dielectric (i.e.
non-conducting) elements can be placed within the chamber to modify
both the electrical field distribution and the hydrodynamic flow
profile. The electrode element size and shape may be designed to
optimize discrimination. Furthermore, several electrode geometries
(energized with the same or different electrical signals) can be
connected serially so as to provide for stepwise discrimination
between different particulate matter and solubilized matter.
Different chamber configurations can also be used in series.
Finally, cells that have been separated by an upstream cDEP/FFF
configuration can be collected and held downstream by cDEP trapping
for characterization.
BRIEF DESCRIPTION OF THE DRAWINGS
[0077] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0078] FIG. 1A is a block diagram of an apparatus according to the
present invention.
[0079] FIG. 1B is a diagram a parallel (interdigitated) electrode
array is positioned normal to a fluid flow.
[0080] FIG. 1C is a force diagram of the apparatus of FIG. 1A.
[0081] FIG. 2A is a side view of an apparatus according to the
present invention which describes a typical trajectory of matter
introduced into the apparatus.
[0082] FIG. 2B is a top view of the apparatus of FIG. 2A which
describes a typical trajectory of matter introduced into the
apparatus.
[0083] FIG. 2C is an end view of the apparatus of FIG. 2A.
[0084] FIG. 2D is a three-dimensional view of the apparatus
described in FIGS. 2A-2C.
[0085] FIG. 2E is a force diagram of the apparatus of FIG. 2A.
[0086] FIG. 3A is a graphical representation of the number of HL-60
cells exiting an apparatus according to the present invention under
the influence of field flow fractionation, as a function of time
(flow rate=200 ul/min).
[0087] FIG. 3B is a graphical representation of the number of HL-60
cells exiting an apparatus according to the present invention under
the influence of field flow fractionation, as a function of time
(flow rate=100 ul/min).
[0088] FIG. 3C is a graphical representation of the number of HL-60
and human-blood cells exiting an apparatus according to the present
invention under the influence of field flow fractionation, as a
function of time. The first and second peak corresponds to HL-60
cells and human blood cells, respectively, (flow rate=100
ul/min).
[0089] FIG. 4A is a graphical representation of DS19 cell
levitation height under the influence of cDEP and gravitational
forces as a function of frequency.
[0090] FIG. 4B is a graphical representation of DS 19 cell
levitation height under the influence of cDEP and gravitational
forces as a function of voltage.
[0091] FIG. 5A is a graphical representation of velocity of HL-60
cells travelling through a cDEP-FFF apparatus according to the
present invention as a function of frequency, (flow rate=10
ul/min).
[0092] FIG. 5B is a graphical representation of velocity of MDA 468
cells travelling through a cDEP-FFF apparatus according to the
present invention as a function of frequency. (flow rate=40
ul/min).
[0093] FIG. 5C is a graphical representation of velocity of MDA 435
cells travelling through a cDEP-FFF apparatus according to the
present invention as a function of frequency. (flow rate=40
ul/min).
[0094] FIG. 5D is a graphical representation of velocity of MDA 435
cells travelling through a cDEP-FFF apparatus according to the
present invention as a function of voltage. (flow rate=40
ul/min).
[0095] FIGS. 6A-6F illustrate programmable electrical signals that
can be applied to electrodes under DEP-FFF operation according to
embodiments of the present invention.
[0096] FIG. 7 shows a chamber having a single output port according
to one embodiment of the present invention. Particles may be loaded
into the chamber and attain equilibrium height positions with
respect to the electrode plane on the bottom surface of the
chamber. Under the balance of dielectrophoretic and gravitational
forces, the smaller particles may be levitated higher than larger
particles.
[0097] FIG. 8 shows a chamber having a single output port according
to one embodiment of the present invention. Particles move under
the influence of a fluid flow velocity profile at velocities
corresponding to their height positions within the profile. Smaller
particles, because of their higher levitation, move faster than
larger particles.
[0098] FIG. 9 shows a chamber having a top outlet port and a bottom
output port according to one embodiment of the present invention.
Particles may be loaded into the chamber and may attain equilibrium
height positions with respect to the electrode plane on the bottom
surface of the chamber. Under the balance of dielectrophoretic and
gravitational forces, small particles may be levitated higher than
larger particles.
[0099] FIG. 10 shows a chamber having a top and a bottom output
port according to one embodiment of the present invention.
Particles move under the influence of a fluid flow velocity profile
at velocities corresponding to their height positions within the
profile. Smaller particles, because of their higher levitation,
move faster than larger particles. The particles may exit the
chamber from the bottom outlet port while a particle-free buffer
may be eluted from the top outlet port.
[0100] FIG. 11 shows a chamber having a top outlet port and a
bottom output port according to one embodiment of the present
invention. The chamber may be operated at a continuous separation
mode where particle mixtures are continuously introduced into the
chamber.
[0101] FIG. 12 shows continuous mode operation of a chamber having
a top outlet port and a bottom output port according to one
embodiment of the present invention. The particle mixtures may be
continuously introduced into the chamber and may be transported
through the chamber under the influence of the fluid flow.
Simultaneously, particles may be subjected to dielectrophoretic
forces generated by electrodes on the chamber bottom-surface and to
gravitational forces and may move in the vertical direction. As
particles reach the end of the chamber, smaller particles attain
higher positions and exit the chamber from the top outlet port
while larger particles attain lower positions and exit the chamber
from the bottom outlet port.
[0102] FIG. 13 is a schematic representation of a DEP/G-FFF system
showing a chamber in exploded view. The microfabricated,
interdigitated electrode array on the bottom surface of the chamber
may be energized with voltage signals from the power amplifier to
generate DEP levitation forces. Cell mixtures may be introduced
into the chamber via the injection valve. The syringe pump may be
operated to establish a fluid-flow profile in the chamber. Cell
kinetic responses may be monitored through video microscopy. Cells
exiting the chamber may be detected by a UV detector or collected
by a fraction collector.
[0103] FIG. 14 is a three-dimensional representation of DEP/G-FFF
fractionation of NF-PS (10.57.+-.1.03 .mu.m) and COOH-PS
(9.44.+-.0.95 .mu.m) beads. Particle counts were plotted as a
function of time at eight inspection locations having distances of
36, 64, 92, 132, 165, 225, 272, 360 mm from the chamber inlet. To
show the progress of separation, the time after the emergence of
the first bead at each position was used to define zero time for
that location.
[0104] FIG. 15A is a DEP/G-FFF Fractogram showing the separation of
NF-PS (10.57.+-.1.03 .mu.m, the second peak) and COOH-PS
(9.44.+-.0.95 .mu.m, the first peak) beads.
[0105] FIG. 15B is a DEP/G-FFF Fractogram showing the separation of
NF-PS beads of different sizes (6.14.+-.0.45, 10.57.+-.1.03 and
15.5.+-.1.84 .mu.m in diameter).
[0106] FIGS. 16A-16B (A) Dependency of elution peak times for NF-PS
(10.57.+-.1.03 .mu.m, solid curve) and COOH-PS (9.44.+-.0.95 .mu.m,
broken curve ) beads on the fluid flow rate in a DEP/G-FFF
separation. (B) Plot of elution peak times for NF-PS (upper line)
and COOH-PS (lower line) beads versus the reciprocal of the fluid
flow rate. The solid line is a linear fit to the experimental
data.
[0107] FIGS. 17A-17B (A) Dependency of elution peak times for NF-PS
(10.57.+-.1.03 .mu.m, solid curve) and COOH-PS (9.44.+-.0.95 .mu.m,
broken curve) beads on the voltage (50 kHz) applied to the
microelectrodes. (B) Plot of the ratio of the two elution-peak
times for NF-PS and COOH-PS beads versus the applied voltage.
Particles were allowed to relax in the chamber for 10 minutes after
injection and prior to the application of fluid flow. Sucrose
buffer of electrical conductivity 10 mS/m was pumped through the
chamber at a rate of 800 .mu.l/min.
[0108] FIG. 18 Dependency of elution peak times for NF-PS beads of
6 (triangle), 10 (circle) and 15 (square) .mu.m diameter on the
voltage (50 kHz) applied to the microelectrode array. Particles
were allowed to relax to equilibrium height positions in the
chamber for 25 minutes after injection and prior to the application
of fluid flow. Sucrose buffer of electrical conductivity 2.2 mS/m
was pumped through the chamber at a rate of 800 .mu.l/min.
[0109] FIG. 19 Dependency of elution peak times for PS beads of 6
(triangle), 10 (circle) and 15 (square) .mu.m diameter on the
applied field frequency. The voltage of the applied electrical
signals was 0.53 V RMS. Particles were allowed to relax to their
equilibrium height positions in the chamber for 25 minutes prior to
the application of fluid flow. Sucrose buffer of electrical
conductivity 2.2 mS/m was pumped through the chamber at a rate of
800 .mu.l/min.
[0110] FIG. 20 Dependencies of vertical (levitation) DEP forces on
the particle position relative to electrode edges for a parallel
electrode array of 50 .mu.m width and spacing. The bold line on the
X-axis represents an electrode element. The electrical field
simulation was performed using the Green's theorem-based analytical
method (Wang et al., 1996). For the force calculation, a particle
of radius r=5 .mu.m and Re[f.sub.CM]=-0.5 was subjected to an
applied field of 1 V RMS. DEP levitation forces were calculated for
particle heights between 5 (largest force) and 95 .mu.m (smallest
force) at 10 .mu.m increments above the electrode plane.
[0111] FIG. 21 Schematic representation of the instantaneous forces
acting on a particle in a DEP/G-FFF chamber according to the
present disclosure.
[0112] FIG. 22 Schematic drawing of dielectrophoretic/gravitational
field-flow-fractionation principle. Cell equilibrium height in the
fluid-flow profile may be determined by the balance of DEP
levitation forces (F.sub.DEPz) generated by the microelectrodes and
the sedimentation force (F.sub.grav). Cells that are farthest from
the bottom electrode plane are carried faster by the fluid
(V.sub.FFF2>V.sub.FFF1- ) and exit the chamber earlier than
those at lower positions.
[0113] FIG. 23 Time dependency of the numbers of MDA-435 cells
(first peak, square) and erythrocytes (second peak, circle) passing
by an inspection widow at the chamber outlet port for a DEP field
of 1.4 V RMS at a frequency of 5 kHz. MDA-435 cells moved ahead of
erythrocytes, and the two populations were well separated. The
cells were suspended in an isotonic sucrose/dextrose buffer that
had an electrical conductivity of 56 mS/m. The flow rate was 0.5
mL/min, corresponding to a mean fluid velocity of 780
.mu.m/sec.
[0114] FIG. 24A Frequency dependency of the elution-time for
MDA-435 cells (square) and erythrocytes (circle), with the error
bars representing the elution-peak width. The two cell populations
were well separated at frequencies below 20 kHz. The elution-time
and elution-peak width were derived from the cell fractograms like
those shown in FIG. 23 according to the method described in the
Experimental Section. Cell suspension, DEP field, and fluid-flow
conditions are the same as in FIG. 23.
[0115] FIG. 24B Frequency dependencies of equilibrium levitation
height for MDA-435 cells (square) and erythrocytes (circle) under a
fluid flow of mean velocity 16 .mu.m/sec. MDA-435 cells were
levitated higher than erythrocytes with height difference up to 15
.mu.m. The heights are averages for at least 10 cells. Error bars
indicate the standard deviations of the measured heights. Cell
suspension and DEP field conditions are the same as for FIG.
23.
[0116] FIG. 24C Frequency dependencies of the polarization factor
.alpha..sub.DEP for the MDA-435 cells (square) and erythrocytes
(circle) derived from levitation height curves in (B) using
Equation (19) with the following parameters:
A=-2.2072.times.10.sup.13/m.sup.3; U=1.41 V; d=200 .mu.m; .di-elect
cons..sub.m=78.di-elect cons..sub.0 (8.854.times.10.sup.-12 F/m);
.rho..sub.c=1.072 (MDA-435), 1.095 (erythrocytes) kg/dm.sup.3;
.rho..sub.m=1.033 kg/dm.sup.3. Continuous curves (-MDA-435; - - -
erythrocytes) are polarization factors calculated from dielectric
modeling (equations 21-24) using parameters derived from cell
electrorotation measurements. For MDA-435 cells, r=7.5 .mu.m,
C.sub.mem (.di-elect cons..sub.mem/d)=24 mF/m.sup.2; G.sub.mem
(.sigma..sub.mem/d)=200 S/m.sup.2; .di-elect cons..sub.int=60; and
.sigma..sub.int=0.5 S/m. For erythrocytes, a=b=3.5 .mu.m, ellipsoid
factor e=4; C.sub.mem (.di-elect cons..sub.mem/d)=8.7 mF/M.sup.2;
G.sub.mem (.sigma..sub.mem/d)=200 S/m.sup.2; .di-elect
cons..sub.int=60; and .sigma..sub.int=0.5 S/m.
[0117] FIG. 25A Voltage dependency of the elution-time for MDA-435
(square ) and erythrocytes (circle), with the error bars
representing the elution-peak width. Large applied voltages
increased DEP levitation forces so that cells were positioned
higher above the chamber bottom wall, leading to larger FFF
velocity and shorter elution time. Cells were suspended in an
isotonic sucrose/dextrose medium of 56 mS/m. The applied DEP field
frequency was 20 kHz and flow rate=1 mL/min.
[0118] FIG. 25B Flow-rate dependency of the elution-time for the
MDA-435 (square) and erythrocytes (circle), with the error bars
representing the elution-peak width. Elution-time was found to be
inversely proportional to the flow rate, indicating that cell
positions in the flow profile were not influenced by the change in
the flow rate. Experimental conditions are the same as in FIG.
25(A), except the applied voltage U=1.41 V.
[0119] FIG. 26 Schematic representation of a DEP-FFF chamber of
dimension 200 .mu.m (H).times.25 mm (L).times.17 mm (W). The cell
sample is preloaded into the inlet regions of the chamber from the
cell-sample-port. After cells settle onto the bottom wall, eluate
buffer is pumped through the chamber via the eluate-buffer-port so
as to establish a hydrodynamic laminar flow profile in the chamber.
Appropriate voltage signals are then applied to the electrode
elements to cause cells to equilibrate at different heights and
travel at correspondingly different velocities in the flow profile.
In this study, parallel electrodes with equal electrode width and
spacing of 20 (or 50) .mu.m were used with alternate elements
connected to electrode buses running along the two long edges of
the chamber.
[0120] FIG. 27 The frequency dependency of levitation height
(diamond symbol) of a typical HL-60 cell (radius=6.6 .mu.m)
suspended in a sucrose/dextrose medium of conductivity 56 mS/m
under an applied voltage of 1.06 V (RMS) on a parallel electrode
array (20 .mu.m width and spacing). No fluid flow is present. The
continuous curve represents the best fit of the experimental data
using DEP levitation theory (Equations 18-19, Huang et al, 1997).
The factor dielectric polarization Re(f.sub.CM) is based on a
single-shell dielectric model (Irimajiri et al., 1979; Huang et
al., 1992), for which, the cell interior relative permittivity and
conductivity are assumed to be 75 an 0.75 S/m, respectively. The
best fit yielded values for the membrane capacitance and
conductance of 16.3 mF/m.sup.2 and <50 S/M.sup.2,
respectively.
[0121] FIG. 28 The voltage dependency of the levitation height
(diamond symbol) of an HL-60 cell (radius=6.3 .mu.m) suspended in a
medium of conductivity 56 mS/m for an applied filed of frequency
17.8 kHz field. No fluid flow is present. The continuous curve
represents the best fit to the experimental data using
dielectrophoretic levitation theory (Huang et al, 1997). The
dielectric polarization factor Re(f.sub.CM) is derived as -0.43. In
the simulation, the value for the electrode polarization factor
p(f) at 17.8 kHz was 0.67 (Huang et al., 1997).
[0122] FIG. 29 The frequency dependency of the mean velocity for
HL-60 cells suspended in a sucrose/dextrose medium of conductivity
44 mS/m in a thin chamber containing parallel microelectrode arrays
(20 .mu.m width and spacing) at flow rates of 20 ((square, lower
curve <.nu.>=98); 40 (diamond, middle curve,
<.nu.>=196); and 80 .mu.l/min (circle, upper curve,
<.nu.>=392 .mu.m/s). The parameter <.nu.> is the
average fluid flow velocity in the chamber. The applied voltage was
1.06 V (RMS). Chamber dimensions were 200 .mu.m (H).times.25 mm
(L).times.17 mm (W). Each symbol represents the mean velocity of
about 20 cells. The continuous lines show the best fit of
dielectrophoretic field-flow-fractionation theory (Huang et al,
1997), the broken lines (20: - - - ; 40: --- - ; 80 .mu.l/min: ---
) the averaged cell velocity when the electrical field was turned
off. In the single shell model (Huang et al, 1992; 1997), the cell
radius, interior relative permittivity and conductivity are taken
to be 5.8 .mu.m (measured by microscopy), 75 and 0.75 S/m,
respectively. The best fits for the three flow rates gave values
for cell membrane specific capacitance of 15.6 (.+-.0.95)
mF/m.sup.2 and conductance of 220 (.+-.76) S/m.sup.2.
[0123] FIG. 30 The voltage dependency of the mean velocity of HL-60
cells suspended in a sucrose/dextrose medium of conductivity 44
mS/m in a thin chamber containing parallel microelectrode arrays
(20 .mu.m width and spacing) at flow rates of 20 (square, lower
curve, <.nu.>=98), 40 (diamond, middle curve,
<.nu.>=196), and 80 .mu.l/min (circle, upper curve,
<.nu.>=392 .mu.m/s). The parameter <.nu.> is the
average fluid flow velocity in the chamber. The applied field
frequency was 31.6 kHz. Chamber dimension were H (200
.mu.m).times.L (25 mm).times.W (17 mm). The continuous curve
represents a best fit of dielectrophoretic field-flow-fractionation
(DEP-FFF) theory to the experimental data for which the factor
Re(f.sub.CM) was derived as -0.46 (.+-.0.065).
[0124] FIG. 31 DEP-FFF experimental setup. Microfabricated
electrodes on the bottom wall of the separation chamber were
energized with electrical signals and provided DEP levitation
forces. After being introduced to the chamber through the injection
valve, the cells of different types in a mixture were levitated to
different equilibrium heights under the balance of DEP and
sedimentation forces. A fluid flow profile was produced in the
chamber from the injection syringe pump. The cells were transported
through the chamber at different velocities corresponding to their
heights, exited the chamber from the bottom outlet port and were
detected by the flow cytometer.
[0125] FIG. 32 Frequency dependency of DEP-FFF elution fractograms
for CD34.sub.+ stem cells obtained by the flow cytometer. Cells
were suspended at 1.5.times.10.sup.6/mL in the sucrose buffer
having an electrical conductivity of 10 mS/m. The applied voltage
was 4 V p-p. The injection and withdrawal syringe pumps were
operated at 2 and 1.8 mL/min, respectively.
[0126] FIG. 33 Frequency dependency of DEP-FFF elution fractograms
for MDA-435 cells obtained by the flow cytometer. Cells were
suspended at 1.5.times.10.sup.6/mL in the sucrose buffer having an
electrical conductivity of 10 mS/m. The applied voltage was 4 V
p-p. The injection and withdrawal syringe pumps were operated at 2
and 1.8 mL/min, respectively.
[0127] FIG. 34 Frequency dependency of elution-time for MDA-435
cells (square) and CD34.sup.+ stem cells (circle). Error bars stand
for the elution peak width for each DEP-FFF fractogram.
[0128] FIG. 35 DEP-FFF fractograms for separating MDA-435 cells
from CD34.sup.+ cells using the trap-and-release protocol. DEP
field was operated at 40 kHz for 7 min and switched to 5 kHz for 7
min. CD34.sup.+ cells were pre-labeled with PE-conjugated CD34
antibodies and were identified by flow cytometer to elute the
chamber earlier than MDA-435 cells. DEP signal voltage and
fluid-flow conditions were the same as those used for FIG. 33.
[0129] FIG. 36 DEP-FFF fractogram for separating MDA-435 cells from
CD34.sup.+ cells by the swept-frequency protocol. The DEP field was
swept between 15 and 35 kHz for 7 min and then switched to 5 kHz
for 7 min. DEP signal voltage and fluid-flow conditions were the
same as those used in FIG. 33.
[0130] FIG. 37 Contour plot for fluorescence level vs elution time
for cells that exited the DEP-FFF chamber for the separation shown
in FIG. 36.
[0131] FIG. 38A-38B
[0132] (A) Typical electrorotation spectra for CD34.sup.+ (circle)
and MDA-435 (square) cells in the sucrose buffer having a
conductivity of 56 mS/m. Continuous curves show best fit of the
single-shell dielectric model (Irimajiri et al., 1979; Huang et al,
1992).
[0133] (B) The frequency spectra of .alpha..sub.DEP (normalized DEP
response) for CD34.sup.+ (--) and MDA-435 ( - - - ) cells under
separation conditions (conductivity 10 mS/m) calculated using the
dielectric parameters (Table 1) derived from ROT measurements.
[0134] FIG. 39. Schematic showing DEP-FFF set-up and operating
principles according to the present disclosure. A thin, rectangular
chamber may be constructed with microfabricated, interdigitated
electrodes on its bottom wall. Different cell types may be
levitated to different equilibrium heights under the influence of
the opposing DEP (F.sub.DEPz) and sedimentation (F.sub.sed) forces.
With a flow velocity profile established in the chamber from an
injector, cells at different heights (h.sub.2>h.sub.1) may be
carried through the chamber at different velocities
(V.sub.2>V.sub.1) and thereby separated. Cells may exit the
chamber from the bottom outlet port and may be detected and counted
by a detector such as an on-line flow cytometer.
[0135] FIGS. 40A-40B. (A) Frequency dependency of DEP-FFF elution
fractograms for T-lymphocytes obtained by the on-line flow
cytometer.
[0136] (B) Frequency dependency of DEP-FFF elution fractograms for
human breast cancer MDA-435 cells obtained by the on-line flow
cytometer. Compared with T-lymphocytes, MDA-435 cells exhibited
rapidly-broadening elution fractograms as frequencies increased
above 10 kHz. Note the frequency scale difference. Cells were
suspended at 1.2.times.10.sup.6/ml in an isotonic sucrose/dextrose
buffer having an electrical conductivity of 10 mS/m. The applied
voltage was 4 V p-p. The injection and withdrawal syringe pumps
were operated at 2 and 1.6 mL/min, respectively.
[0137] FIGS. 41A-41D. (A) DEP-FFF fractograms showing the
separation of human breast cancer MDA-435 cells from T-lymphocytes
by a DEP field at 40 kHz followed by a 5 kHz field (see Method and
Materials for details).
[0138] (B) DEP-FFF fractograms showing the separation of human
breast cancer MDA-435 cells from T-lymphocytes by a DEP field swept
between 15 and 35 kHz followed by a 5 kHz field.
[0139] (C) contour plots show fluorescence level vs elution time
for cells exiting the DEP-FFF chamber for the separation in FIG.
41A.
[0140] (D) contour plots show fluorescence level vs elution time
for cells exiting the DEP-FFF chamber for the separation in FIG.
41B. To allow identification, the T-lymphocytes were fluorescently
labeled with PE-conjugated CD3 antibodies. Cell suspension, DEP
signal voltage and fluid-flow conditions were the same as for FIG.
40A-40B.
[0141] FIG. 42. DEP-FFF fractogram showing the separation of
monocytes from B-lymphocytes. The injection and withdrawal syringe
pumps were operated at 2 and 1.9 mL/min, respectively.
Identification of monocytes and B-lymphocytes by flow cytometry was
made possible by pre-labeling them with PE-CD14 and FITC-CD19
antibodies, respectively. The cell suspension and DEP field
conditions were the same as FIG. 40A-40B, except that the DEP field
was swept between 20 and 40 kHz.
[0142] FIG. 43. Flow cytometric cell count for leukocytes
(CD45.sup.+, solid line) and erythrocytes (CD45.sup.-, dashed line)
as a function of elution time during DEP-FFF enrichment of
leukocytes from blood. More than 95% of leukocytes eluted between
15 and 17.5 min. The leukocyte: erythrocyte ratio increased from
1:700 to 1:19, a 35-fold enrichment. The DEP-FFF was operated under
a 10 kHz DEP field with the injection and withdrawal syringe pumps
operated at 0.5 and 0.4 mL/min, respectively.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0143] DEP/FFF may employ a dielectrophoretic force that is
balanced against gravitational forces to position cells at
different equilibrium heights within a fluid-flow profile. Cells or
other particles at different heights in the flow profile may be
transported at different velocities, and may therefore be separated
via velocity and/or via height differences In embodiments described
herein, the DEP force in DEP-FFF may be generated by applying
electrical signals to microelectrode arrays configured on the
bottom surface of a separation chamber. In one embodiment, the
signal used for separation is a sinusoidal signal with fixed
frequency and voltage. Using such an approach, it has been shown
that polystyrene beads of different sizes and with different
surface modification may be separated. Additionally, DEP-FFF
separations of model cell mixtures (cultured breast cancer cells
and cultured HL-60 leukemia cells) from normal blood cells has been
demonstrated.
[0144] Turning now to FIGS. 6A-6F, there is shown an embodiment
that utilizes a dielectrophoretic force field that is programmed
and varied by adjusting voltage signals applied to microelectrodes
so that the signal amplitude, frequency, waveforms, and/or phases
are a function of the time. Specifically, FIGS. 6A-6F show several
examples of such "programmed" voltage signals. FIG. 6A shows a
signal with a fixed amplitude/frequency. FIGS. 6B-6F depict various
embodiments using "programmed" voltage signals, and more
particularly, embodiments using time-dependent amplitude/frequency.
FIG. 6E and FIG. 6F show signals with frequency-modulation
(frequency continuously changes with time) and amplitude-modulation
(amplitude continuously changes with time). Applying such signals
shown in FIGS. 6A-6F to electrodes described herein, DEP-FFF may be
programmed according to specific problems one is facing and
according to specific separation goals. By employing such
programmed signals, DEP forces may be varied in time for better
separation performance and the discrimination of a DEP-FFF
separator may be tailored to specific applications.
[0145] Certain DEP-FFF chamber embodiments described herein contain
one or two inlet ports and one outlet port. The chamber may be
preloaded with a buffer solution. A sample (which, for convenience
may be assumed to be made of a cell mixture) may be introduced into
the chamber with voltage signals applied to microelectrodes. With
electrical signals applied, cells are allowed to settle for some
time (e.g., on the order of minutes) to reach equilibrium heights,
resulting from a balancing of forces (including dielectrophoretic
forces and gravitational forces). A flow profile may then be
established in the chamber by driving fluid through at least one
inlet port. Fluid exits the chamber at an outlet port. Cells at
different heights within the flow profile may exit the chamber at
different times because they are transported at different
velocities. Exiting cells may be detected using a UV detector, or a
flow cytometer, or are collected by a fraction collector.
[0146] The speed of DEP-FFF operation may be increased by using a
higher fluid flow rate. However, the maximum flow rate may be
limited by the cell (or particle) detector one uses because high
flow rates lead to excessively high fluid pressure at the cell
detector. For example, a HS BRYTE flow cytometer (Bio-Rad,
Microsciences Ltd) may operate at up to 50-100 microliters per
minute, whilst the flow rate for DEP/FFF operation may range up to
several mL per minute. It follows that a separation that may be
achieved in the DEP/FFF separator with a flow rate of 5 mL/min
would have to be slowed at least 50 fold if an HS BRYTE flow
cytometer were used for detection. Such a slow-down is not only
inefficient but also means that cells may be subjected to
electrical fields and suspended in a non-physiological buffer for
50-times longer, which may lead to undesired effects.
[0147] To reduce the fluid pressure at the cell detector downstream
from the DEP/FFF separator, a chamber according to the present
invention may use two outlet ports, one adjacent the top of the
chamber, and one adjacent the bottom of the chamber. Because, in
many DEP/FFF applications, cells are levitated to heights less than
half the chamber height, the top half of the fluid may not contain
any, or may contain only few, cells. Thus, when fluid exiting the
top outlet port is withdrawn by an extracting agent, such as a
syringe pump, running at half the fluid-flow rate as the chamber
inlet port, the great majority of cells or all the cells may exit
the chamber at the bottom outlet port. In this embodiment, the
fluid flow rate and fluid pressure at the detector may be reduced
by half.
[0148] In one embodiment, such a two outlet port system may be
operated with a fluid-flow rate through the top outlet port set to
more than half the inlet fluid flow rate because cells may occupy
much less than half the chamber height. Thus, fluid pressure at the
cell detector may be reduced even further. Furthermore, cells
exiting the chamber may be at higher concentrations because less
fluid exits with them. In the operation of the two outlet port
system, the fluid-flow rates at the top and bottom outlet ports may
vary with time, provided that the sum of the two equals to the
inlet flow rate. Thus, the fluid flow rates at the inlet and outlet
ports may be programmed and varied with time during the operation.
Such variation in the flow rates with time may speed up the
separation process. For example, in the case of the separation of
the mixtures of two cell populations, after the complete elution of
the first population of cells, the second population may be quickly
removed from the chamber by increasing the inlet flow rate and,
correspondingly the two outlet-flow rates.
[0149] In FIGS. 7, 8, 9 and 10, there are shown operating
principles for a DEP/FFF chamber in accordance with this embodiment
of the present invention n. FIG. 7 and FIG. 8 illustrate DEP/FFF
operation for a single outlet port where the particles, which for
convenience may be assumed to be a particle mixture, are first
introduced into the chamber and equilibrated for some time to
attain equilibrium heights (FIG. 7). The particles may then be
subjected to a fluid flow profile that separates them as a result
of differential velocities at different equilibrium heights. The
particles and all the DEP/FFF buffer exits the chamber from the
outlet port (FIG. 8).
[0150] FIGS. 9 and 10 show DEP/FFF operation for an embodiment
having a chamber including two outlet ports positioned on the top
and the bottom walls of the chamber. FIG. 9 shows that the
particles may be introduced into the chamber and allowed to reach
equilibrium positions. Particles may then be subjected to a fluid
flow profile and are separated as before. However, in this
embodiment there are two outlet ports. The particles may exit the
chamber from the bottom outlet port while most of the buffer fluid
may exit the chamber from the top outlet port (FIG. 10). Depending
on the ratio of flow rates at the two outlet ports, the fluid
pressure at the particle detector (which may be coupled to the
bottom outlet port) may be significantly reduced.
[0151] While the description above and many examples given below
discuss the DEP/FFF chambers with outlet port(s) from which
particles (e.g. cells) are eluted, the inventors envisage
embodiments of the present invention in which the particle
detectors are integrated with the DEP/FFF separation chamber. In
one embodiment, particle detection may be based on the electrical
impedance change between two sensing electrode elements located at
the two opposite sides of a channel when particles flow through the
channel. Such electrode elements may be fabricated using the same
methods as those for making DEP separation microelectrodes, and the
channel may be on the same substrate (e.g. glass or silicon) as
that for the separation electrodes. In such cases, particle
separation and detection are accomplished in the same integrated
device.
[0152] Continuous DEP/FFF Particle Separation Using DEP-FFF
Chambers With Multiple Outlet Ports
[0153] By increasing the fluid flow rate from the upper fluid
outlet port of FIG. 10, particles may be withdrawn from the top.
Indeed, by adjusting the ratio of flow rates through the top and
bottom outlet ports appropriately, desired fractions of cells may
be caused to exit through those respective ports. In this
embodiment, the particle mixture need not be injected as a single
batch and separated in time, but instead may be continuously
introduced in the chamber (FIGS. 11 and 12) Particles are carried
with the fluid flow. Simultaneously, they move in the vertical
direction to reach equilibrium heights determined by their
properties and the balance of forces such as dielectrophoretic
levitation and gravitational forces (FIG. 12). If the fluid flow
rate is controlled appropriately, particles may reach their
equilibrium height positions before they arrive at chamber outlets.
As fluid exits the chamber from both the top and the bottom outlet
ports, the fluid from the bottom surface up to a certain height (a
threshold height) in the chamber will exit the chamber through the
bottom outlet port while fluid above the threshold height will exit
through the top outlet port. Particle having equilibrium heights
lower than the threshold height exit the chamber through the bottom
outlet port (FIG. 12). On the other hand, particles having
equilibrium heights above the threshold height exit the chamber
through the top outlet port (FIG. 12). Separation may thus be
achieved according to particle heights as particles reach the
chamber outlet end. Such separation may be operated in a batch, or
alternatively, a continuous mode as the height-differential between
different particles are exploited for separation directly, without
need for exploiting differential velocities created by the fluid
flow profile.
[0154] Again, the height-differential between different particle
types may be the basis of the differential velocity and exit time
for batch-mode DEP-FFF operation, as described herein. Using the
two port system of FIGS. 11-12 eliminates the need for a flow
profile, and it enables continuous separation.
[0155] In the embodiment of FIGS. 11-12, particles do not have to
reach their equilibrium positions as they arrive at the chamber
outlet end. As long as there are sufficient differential heights
between sub-populations of particles to be separated, the
continuous DEP-FFF operation may separate particles. Because flow
profile is not actively exploited in this embodiment, separation
may be performed even for a "plug-like" flow profile--this opens
the possibility that fluid flow may be generated by effects such as
electro-osmosis or surface acoustic wave, rather than by a typical
syringe pump. Nevertheless, flow profile does influence the time
cells take to travel through the chamber--the time that is
available for particles to "move" in the vertical direction under
the combined action of the dielectrophoretic levitation and
sedimentation forces--so it may influence, in turn, the
distribution of particle vertical positions as they exit the
chamber.
[0156] FIGS. 11 and 12 show embodiments of the DEP/FFF principle of
continuous separation with two outlet ports. In this embodiment,
particle may be continuously fed into the DEP-FFF chamber and
transported by fluid flow to the chamber outlet ports. As the
particles are transported, they experience vertical DEP and
gravitational forces that drive them up or down. Different particle
types, possessing different dielectric properties and therefore
experiencing different dielectrophoretic forces, attain different
heights as they reach the end of the chamber. Because there are two
outlet ports, some particles, depending on their height positions,
exit the chamber at the bottom outlet port and others at the top
outlet port. Although a typical parabolic profile is drawn in the
figure for the fluid flow, those having skill in the art, with the
benefit of the present disclosure, will recognize that the
illustrated flow profile is not necessary.
[0157] In one embodiment, multiple outlet ports may be placed on
the bottom or bottom plates of the chamber. Particles being
separated may exit from such outlet ports, again, depending on
their relative position in the chamber.
[0158] While chambers used for continuous separation may be similar
to those used batch mode, important differences lie in the fact
that flow rates through the top and bottom ports may be chosen in
continuous mode so that particles of different types may exit the
chamber through the top and bottom outlet ports. Thus the fluid
flow rate, chamber thickness, the particle levitation properties
may all have to be taken into account when designing separation
protocol to ensure that a separation would work as desired.
[0159] In one embodiment of continuous DEP/FFF separation may
include the use of a splitter at the outlet end of the chamber.
Such a splitter may be several to tens of micrometers, and may be
used to divide the fluid at the outlet end of the chamber into
multiple fractionations.
[0160] Programming DEP Force Field for DEP-FFF Separation
[0161] In one embodiment, voltage signals at an amplitude V.sub.1
and a frequency f.sub.1 may be applied for a certain time period
(any time ranging from about 10 seconds to about 60 minutes).
During this signal mode, the difference in particle height and
velocity may be maximized by reducing the velocity of one particle
type as much as possible (ideally, down to zero) and increasing the
velocity of other particle types to large values (typically 10
cm/min). After some time, sufficient separation between the
particle in the chamber may be achieved or the fast-moving particle
type will have already exited the chamber. Then, a different
voltage signal at an amplitude V.sub.2 and a frequency f.sub.2 may
be applied. In this case, the velocities of both particle types may
be increased, so that both particle types are eluted quickly from
the chamber while a good separation between the two populations is
maintained.
[0162] In another embodiment, a signal having a constant amplitude
but a sweeping frequency (for instance, frequency linear modulation
by a triangular waveform) may be first applied to electrodes. Such
a signal, while it may not affect the elution characteristics of
certain particle types having relatively-large polarization factors
in the applied frequency range, may be important for particle types
having a polarization factor close to zero. Such sweep-frequency
signals may reduce the number of the particle that would be trapped
on the electrode plane and ensure that these particles move slowly.
Such a sweep-frequency voltage signal, after being applied for some
time, may lead to separation of the particle subpopulations in the
chamber. After such a frequency-sweep step, signals of fixed
frequency may be applied to elute all the particle types remaining
in the separation chamber.
[0163] In another embodiment, for separating particle mixtures
(e.g., cell mixtures) having multiple subpopulations, electrical
signals of constant amplitude may be applied with the signal
frequency being changed in a step-wise fashion. An example of this
embodiment may involve a sample containing three subpopulations
(S.sub.1, S.sub.2 and S.sub.3), having different dielectric
properties and different polarization factors. Signals of frequency
f.sub.1 would be applied to elute subpopulation S.sub.1, whilst
ensuring slow movement of subpopulations S.sub.2 and S.sub.3. After
subpopulation S.sub.1 is eluted from the chamber or is moved far
ahead of the other two subpopulations, subpopulation S.sub.2 may be
eluted by changing the signal frequency to f.sub.2 whilst still
ensuring slow movement of subpopulation S.sub.3. After another time
period, subpopulation S.sub.2 may be eluted or moved sufficiently
ahead of S.sub.3. Finally, a signal of frequency f.sub.3 may be
applied to rapidly elute subpopulation S.sub.3. With the benefit of
the present invention, those having skill in the art will recognize
that such an approach may be modified in numerous ways to separate
cells or particles having multiple subpopulations. Furthermore,
alternative or additional step-wise changes in signal amplitude,
amplitude and frequency, sweeping frequency, or any other property
of the signal may be used to improve separation as appropriate for
each application. For instance, signals having two or more
components (e.g., having frequency f.sub.1 and f.sub.2 and
intensities V.sub.1 and V.sub.2) may be applied simultaneously and,
programmably for certain periods of time.
[0164] In the embodiments disclosed herein, programming a DEP force
field as a function of time for DEP/FFF separation may not only
maximize particle separation but also may allow for fast
separation. The sweeping frequency has an important advantage in
that it may allow many particles of the same type to be levitated
slightly even though their dielectric properties are quite
different. Without using such a sweep-frequency signal, some
particles may be levitated, but others may experience positive DEP
forces that cause them to be trapped on one or more electrodes
(undesired effects).
[0165] As described above, the programming DEP force field as a
function of time may allow the flexible control for the DEP-FFF
separation process. For example, the frequency of the voltage
signals may be gradually and step-wisely reduced to allow the
elution of various cell sub-populations. The programming of a DEP
force field may be readily achieved by controlling one or more
signal generators with a computer or another suitable controller.
In this way, optimal separation conditions may be found by testing
various signal combinations.
[0166] Two Outlet Ports for DEP-FFF Chambers
[0167] A DEP-FFF chamber was constructed, in accordance with one
embodiment of the present invention, with two outlet ports, one
each on the top and the bottom walls. Particles or cells exiting
through the bottom outlet port were detected by flow cytometry. In
one embodiment, the total fluid flow rate in the DEP-FFF chamber
was between 0.5 and 2 ml/min, but it will be recognized that the
rate may vary widely. In this embodiment, to ensure that the fluid
flow rate through the flow cytometer was less than 0.1 ml/min., up
to about 95% of the fluid exiting the chamber was drawn from the
top outlet-port. So, for example, at a total fluid flow rate of 0.5
ml/minute, fluid was withdrawn from the top outlet port by a
syringe pump at a rate of 0.4 m/min, lowering a residual flow to
the flow cytometer at 0.1 ml/min. Again, the inlet and two outlet
flow rates may be programmed so that they vary with time.
[0168] In choosing operating conditions for a two outlet port
system with one detector at the bottom outlet port, it may be
important to make sure that all particles (or cells) exit from the
bottom port. Otherwise, some particles (or cells) may go undetected
or uncollected if they are eluted from the top outlet port. In this
two-port DEP-FFF embodiment, the 2nd outlet port is used to reduce
the fluid pressure at the bottom outlet port so that particles or
cells may be detected using apparatus such as flow cytometer.
Another benefit of such an embodiment is that the separated
particles or cells are "concentrated", in that diluting fluid is
withdrawn from the top outlet port.
[0169] Embodiments described herein allow for programmable DEP
force fields via programmable voltage signals, multiple outlet
ports, and continuous DEP-FFF particle separation. These
embodiments, and combinations thereof, provide for several
significant advantages. For instance, better particle (or cell)
separation may be achieved. In one embodiment, purity of separated
populations was increased from 70% using a single-frequency field
to greater than 99% with a sweep-frequency field. Separation may be
performed faster and more flexibly. In one embodiment, a separation
time was improved from 40 minute using a single-frequency field and
a single outlet port to 5 minutes with a programmed DEP force field
on a chamber having two outlet ports. A significant reduction of
fluid pressure and flow requirements of any down-stream particle
(or cell) detector or collector may be achieved with the two outlet
port arrangement. In one embodiment, greater than a 10 fold
reduction was achieved in fluid pressure. DEP-FFF separation may be
operated at higher flow-rates so that fast separation may be
performed. Two ports may allow for lower dilution factors for
separated cells. In one embodiment, concentration was increased by
a factor greater than ten. Continuous separation embodiments may
allow for the processing of large quantities of cell samples and
may be particularly applicable to the case where one cell type to
be separated is of low concentration (such as separating metastatic
cancer cells from mononuclear cells, or white blood cells from red
blood cells).
[0170] Such advantages are applicable to all DEP-FFF separation
problems, including synthetic particle separation (including
surface activated beads) and cell separation (including, but not
limited to, blood cell differential analysis, erythrocytes from
leukocytes, nucleated erythrocytes from erythrocytes/leukocytes,
fetal erythrocytes from maternal blood, activated cells from
non-activated cells, malarial-infected cells from normal blood
cells, metastatic cancer cells from blood, cancer cells from bone
marrow, cancer cells from normal counterpart cells, and leukemia
cells from blood).
[0171] The following additional examples are included to
demonstrate illustrative embodiments of the invention. It should be
appreciated by those skilled in the art that the apparatus and
techniques disclosed in the examples which follow represent devices
and techniques discovered by the inventor to function well in the
practice of the invention, and thus can be considered to constitute
preferred modes for its practice. However, those of skill in the
art should, in light of the present disclosure, appreciate that
many changes can be made in the specific embodiments which are
disclosed and still obtain a like or similar result without
departing from the spirit and scope of the invention. While the
following examples use the term particle, the skilled artisan will
realize that the present apparatus and methods are suitable to
solubilized matter as well.
EXAMPLE 1
[0172] FIG. 1A shows one exemplary embodiment of an apparatus
according to the present invention. In this figure, the electrode
array 5 is placed on the bottom wall 14 of a chamber 10; however it
is contemplated that the electrode array may be placed on the top
wall 12 and/or bottom wall 14 and/or side walls 16 of a chamber
constructed in accordance with the present invention. As shown in
FIG. 1B, the electrode array 5 may be placed along a chamber wall
in a position normal to a flow of fluid 35 through the chamber 10.
It is to be noted that the array may be adapted at any angle with
respect to the fluid flow, for example, parallel or at any other
angle. In this embodiment, the walls are aligned to create a thin
chamber. The walls are spaced apart by a spacer 20, which may be,
for example, constructed of the same material as the chamber walls,
or a TEFLON spacer, a sealing compound, or any other dielectric or
conductive material Electrical signals applied to the electrode
array create an inhomogeneous alternating electric field that
varies with the frequency and magnitude of the input signal. In a
particular embodiment, the electrode element 5 may be adapted to be
substantially normal to the fluid flow 35, as shown in FIG. 1B.
Further electric conductors, which may be electrode buses 40 and 45
may provide electrical signals to alternate elements of electrode
array 5. The strength of the electric field is dependent on the
applied voltage, the position within the chamber, and the size and
spacing of electrode elements. For manipulation of mammalian cells,
the field strength may be on the order of or less than
approximately 10.sup.6 V/m, although this may be much higher for
matter placed in an oil medium. The particulate matter desired to
be discriminated is introduced into the chamber in a carrier medium
that flows into at least one inlet port 15. There may be more than
one inlet port however, which permits input of the carrier medium.
The carrier medium may be input by a digital syringe pump, a manual
syringe, a peristaltic pump, a gravity feed catheter, or the like.
As discussed above, the particulate matter may include, for
example, biological molecules and non-biological molecules. Also,
the matter may include solubilized matter. The carrier medium may
be, for example, an eluate consisting of a cell-free suspension
buffer, including a mixture of sucrose and dextrose, tissue culture
medium, non-ionic or zwitter ionic solutes, or other suspension
mediums or non-biological oils, solvents such as phenol alcohol,
CCl.sub.4, ethylene glycol, or others known in the art.
Alternately, one or more ducts 25 may be provided to input a fluid
which may be flowed through chamber 10.
[0173] The carrier medium is caused to flow through the chamber and
thereby create a laminar flow profile in which the fluid flow
velocity increases with increasing distance from the chamber top
and bottom walls, and reaches its maximum at the center. However,
by adjusting the shape of the chamber, for example, a flow profile
may be created in which the maximum is at a location other than the
center of the chamber. In an exemplary embodiment, the total flow
rate through the chamber may be on the order of about 0.01 ml/min.
to about 5000 ml/min., and more preferably about 1 ml/min. to about
20 ml/min. The electric field applied to the electrode elements 5
creates dielectrophoretic forces on the particles in accordance
with their dielectric and conductive properties as well as those of
the carrier medium.
[0174] By controlling the frequency and/or intensity and/or
waveform of the applied electric signals, the component of the
dielectrophoretic force that is normal to the flow direction of the
carrier medium is controlled so as to cause the particles to
equilibrate at characteristic distances from the electrode element
5 creating the electric field. These characteristic distances are
referred as equilibrium heights at which the DEP forces in the
vertical direction are balanced by gravitational forces and
hydrodynamic lifting forces acting on the particles. The
dielectrophoretic force operates in conjunction with the action of
the combined hydrodynamic lifting forces and gravitational forces
as shown in FIG. 1C. FIG. 1C shows how these forces act on matter
within the chamber 10. Specifically, FIG. 1C shows the
gravitational force 50 acting in a downward direction and the
dielectrophoretic force 55 and hydrodynamic lifting force 52 acting
in an upward direction. The flow velocity profile 65 along the
vertical plane has a maximum at the center of the chamber, and a
minimum velocity at the top and bottom of the chamber. Since the
dielectrophoretic force acting on each individual particle depends
upon its dielectric permittivity and electrical conductivity at the
applied frequency, as well as upon its volume, particles having
different properties will be positioned at different distances from
the electrode element creating the electric field. Because the
fluid at different heights above the chamber bottom wall flows at
different velocities, particles having differing physical
properties will travel through the chamber 10 at different speeds
and emerge at an outlet port 30 at different times. It is to be
understood that there may be more than one outlet port from which
to collect the particulate matter which exits the chamber 10.
EXAMPLE 2
[0175] FIG. 2D is a three-dimensional view of a second apparatus
according to the present invention, as shown more fully in FIGS. 2A
through 2C. FIG. 2A shows a second embodiment of an apparatus
according to the present invention that includes a chamber 10
having two facing electrode arrays 5, as shown in FIGS. 2B and 2C,
on opposite surfaces of the chamber. The chamber is turned so that
the electrode planes 5 stand substantially vertical. In this
embodiment, the chamber is arranged so that the thin sides of the
chamber are vertically arranged. It is understood, however, that
the electrode planes need not be only vertical, and the present
invention contemplates adapting the apparatus at varying angles.
Different electrical signals (frequency, magnitude and waveforms)
are applied to the facing electrodes from the signal generator so
that particles experience different DEP forces from the field
produced by each array 5. Further, within each facing electrode
array 5, different electrical signals may be provided by the signal
generator to create an inhomogeneous alternating electric
field.
[0176] This alternate apparatus may have, for example, a top wall
12, a bottom wall 14, and two side walls 16. Further, the apparatus
may have one inlet port 15 adapted to receive the particulate
matter to be discriminated. The inlet port 15 may be located, for
example, close to the top of one end of the chamber 10. This
apparatus may also include one or more ducts 25 to introduce a
fluid that travels through the chamber 10. The ducts 25, which may
be arranged substantially along the entire width of the input end
of the chamber 10, serve to introduce a sheet of fluid that travels
throughout the chamber 10 in a substantially linear direction.
[0177] The introduced fluid carries the particulate matter through
the chamber 10. This fluid may be, for example an eluate, such as a
cell-free suspension having a mixture of dextrose and sucrose,
tissue culture medium, non-ionic or zwitterionic solutions, or
other suspension mediums or non-biological oils, solvents such as
phenol, alcohol, CCl.sub.4, ethylene glycol, or others known in the
art. Following transit through the chamber 10, fluid leaves at the
opposite end through an exit port. This exit end of the chamber 10
may include, for example, one or more exit ports 30, which may be
arranged in one or more arrays of ports as shown in FIG. 2A. In the
absence of an applied field, that is, when no electrical signal is
applied to the electrode elements 5, particles move through the
chamber 10 under the influence of fluid flow. Further, based on the
geometrical design of the chamber 10, the fluid may exhibit, for
example, a laminar flow-velocity profile, in which the speed of the
flow is fastest towards the center of the chamber 10 as shown in
FIG. 2E. That is, the hydrodynamic flow profile is along a
horizontal plane. Simultaneous to the influence of the fluid flow,
the particles undergo sedimentation due to gravitational forces on
the particles, so that they exit the chamber 10 at characteristic
heights determined by their sedimentation rates as shown in FIG.
2B.
[0178] When electrical signals are applied, however, the particles
experience DEP forces that cause them to move to characteristic
distances, known as an equilibrium position, from the side walls of
the chamber 10 where the electrode arrays 5 are arranged. At
equilibrium positions, DEP forces due to the electrical fields
generated by the two facing electrode arrays balance each other.
Interaction of the gravitational and dielectrophoretic forces on
matter are shown on FIG. 2E. FIG. 2E shows that the
dielectrophoretic forces 70 act in a horizontal direction and the
gravitational force 75 in a vertical direction. The flow velocity
profile 90 along the horizontal plane has a maximum at the center
of the chamber, with the velocity diminishing at the sides of the
chamber. In this embodiment, different electrical signals
(frequency or magnitude or both or waveform) are applied to
electrode elements 5 on each of the side walls. Particles having
different dielectric and conductive properties equilibrate at
different characteristic distances (or equilibrium positions) from
the side walls of the chamber, based on the synergism of the
differing electrical signals, which create an inhomogeneous
electric field, causing DEP forces on the particles. Such particle
equilibration positions with respect to the electrode elements on
the chamber side-walls depends on the dielectric and conductive
properties of the particles, the magnitude and frequency of the
electrical fields applied to the electrodes on the facing chamber
walls, and the fluid density, viscosity and flow rate as shown in
FIG. 2B. Particles introduced into chamber 10 moves to different
equilibrium positions from electrode array 5. The velocities of the
different particles within the fluid are controlled by the velocity
profile of the fluid and their positions within such a velocity
profile. Because fluid flowing through a thin chamber sets up a
velocity profile, particles that have equilibrated at different
distances from the chamber walls will be carried at different
velocities and therefore take varying amounts of time to traverse
the chamber. The fluid flows at a maximum velocity towards the
center of the chamber, with this velocity proportionately
diminishing as distance to the side walls decreases. Depending on
applications and chamber dimensions, the overall fluid flow rate
through the chamber may be between about 0.01 ml/min. and about
5000 ml/min., and more preferably between about 1 ml/min. and about
100 ml/min.
[0179] The skilled artisan will recognize, however that variations
in the dimensions of the apparatus will affect the fluid flow rate,
and that the indicated flow rates are illustrative for the
dimensions of the present apparatus.
[0180] Gravity forces act on the particles during their transit
through the chamber. The distance that particles sediment during
their passage across the chamber will depend upon their transit
time and the sedimentation rate (or velocity). Consequently
different particles will sediment to different depths based upon
the particle's transit time through the chamber 10 and the
particle's sedimentation rate. Particle sedimentation rate may
depend on particle characteristics, such as size, mass, and volume,
for example. As described above, the time required for particles to
travel across the entire length of the chamber is controlled by the
fluid flow profile and the positions of particles within the
flow-velocity profile. The placement of particles within the fluid
flow profile is in turn determined by the synergism of the
differing electrical signals. Thus, particles with different
characteristics (e.g.: dielectric property, size) may be placed at
different positions in the flow profile and therefore exhibit
different transit times. The combination of differences in transit
time and in sedimentation velocity between particles of different
properties (e.g. dielectric property, density, size) may lead to
different sedimentation depths for these particles. They may exit
the chamber through different exit ports which are placed at
different heights along the entire outlet end. Discrimination may
be accomplished either in "batch mode" or in "continuous mode." In
batch mode, an aliquot of particles is injected and collected with
respect to the time of transit for the particles and the height of
exit at the outlet ports 30. In continuous mode, a constant stream
of particles is injected into the inlet port, and particles
emerging at different heights are continuously collected.
[0181] In an apparatus according to the present invention, it is
possible to vary the carrier fluid characteristics at different
heights with respect not only to flow rate but also to fluid
density, dielectric permittivity, pH and conductivity. In this way
additional particle characteristics may be exploited for particular
separation applications.
[0182] In the general case, the device may be oriented at any angle
to take advantage of discriminating aspects of the horizontal and
vertical cases described above. In this generalized situation the
particle density, sedimentation rate and dielectric properties,
together with all components of the DEP force are utilized.
Separation in continuous or batch mode is possible. Different
embodiments of an apparatus according to the present invention may
have additional components connected to the outlet ports 30. For
example, particles emerging from the exit ports 30 of the apparatus
of the present invention may be collected by one or more fraction
collectors, or the like. Additionally, the matter may be measured
by one or more measuring or characterizing structures, such as a
cytometer, for example. Furthermore, when necessary, particles may
be transferred to collection wells containing appropriate solutions
or media, such as neutral salt buffers, tissue culture media,
sucrose solutions, lysing buffers, solvents, fixatives and the like
to trap cells exiting the chamber. The collected cells may be
cultured for further analysis.
Methods of Operation
[0183] The following descriptions detail construction of an
apparatus and methods of operation according to the present
invention.
[0184] In one embodiment, an apparatus according to the present
invention was constructed using two glass slides (1"-1.5", for
example) as chamber walls. These walls may be spaced Teflon
spacers; however other methods of separating chamber walls, such as
glue, polymer gaskets, or mechanical precision clamps may be used,
for example. The distance of separation between walls may be
between about 0.1 microns and about 10,000 microns, and more
preferably between about 10 microns and about 600 microns. In
studies using the present apparatus, the distance of separation was
127 microns. One wall of the chamber supported a microelectrode
array consisting of about 20 microns wide parallel electrode
elements spaced about 20 microns apart. The electrode elements may
run along the entire length of the chamber from the input port to
the output port. It is understood that the length, width, thickness
and spacing of electrode arrays may be altered to create electric
fields of differing intensities and different inhomogeneity. It is
also to be understood that an array of electrodes may be used with
the present invention, or a single electrode element may be
sufficient for certain applications, if combined with a ground
plane. Further, it is to be understood that the electrode array may
not be parallel, and other geometric configurations, such as
interdigitated castellated electrodes, serially arranged
electrodes, linear, polynomial, interleaved, three-dimensional and
the like may be utilized.
[0185] In an exemplary embodiment, alternate electrode elements are
connected to electrode buses along the two opposite long edges of
the chamber wall. These electrode buses are connected to an
electrical signal generator, which may be, for example, a function
generator. Other suitable signal generators may include, for
example, oscillators, pulse generators, digital output cards,
klystrons, RF sources, masers, or the like. The electrode array may
be fabricated using standard microlithography techniques, as are
known in the art. For example, the electrode array may be
fabricated by ion beam lithography, ion beam etching, laser
ablation, printing, or electrodeposition. The electrode array of
the exemplary embodiment described herein consisted of 100 nm gold
over a seed layer of 10 nm chromium. It is understood that the
present invention contemplates using electrical signals in the
range of about 0 to about 50 V and about 0.1 kHz to about 180 MHz,
and more preferably between about 10 kHz and about 10 MHz. In
studies which are described below, the signals were provided by a
HP 8116A function generator. The present invention may utilize a
fluid flow of about 0.01 ml/min. to about 500 ml/min., and more
preferably between about 1 ml/min and about 50 ml/min. In studies
described below, fluid flow in the range of about 1-100 ml/min, was
provided by a digital syringe pump.
[0186] Field Flow Fractionation
[0187] Cell mixtures in the studies discussed below consisted of
blood cells (collected by venipuncture from healthy volunteers and
diluted with 90 parts Ca.sup.2+/Mg.sup.2+-free PBS containing 5 mM
hemisodium EDTA) mixed in a ratio of 3:2 with HL-60 leukemia cells
that had been cultured under standard conditions and harvested by
centrifugation. Cell mixtures were washed twice in isotonic (8.5%)
sucrose containing 3 mg/ml dextrose and resuspended at a final
concentration of 2.times.10.sup.7 malignant cells and
3.times.10.sup.7 normal blood cells per ml in this same medium. The
suspension conductivity was adjusted to 10 mS/m by addition of
hemisodium EDTA to a final concentration of approximately 0.7 mM.
It is contemplated by the present invention that other methods of
obtaining and preparing samples are acceptable. Further, different
ratios of the mixture may be used. For example, cell mixtures may
be washed twice in an isotonic solution of 8.5% sucrose and 0.3%
dextrose, resuspended at a final concentration of 1.times.10.sup.7
malignant cells and 3.times.10.sup.7 normal blood cells per ml in
this same medium, and adjusted to a conductivity of 10 mS/m with a
final concentration of .about.0.7 mM hemisodium EDTA.
[0188] FIG. 3A shows the results of field flow fractionation on a
sample of HL-60 cells (ATCC) cultured in a medium of RPMI 1640 10%
FBS 22 mM HEPES in an apparatus as described above. The
fractionation occurred at a flow rate of 200 ul/min. As shown in
FIG. 3A, a sharp rise in HL-60 cells exiting the apparatus occurs
at approximately 10 minutes after the flow of cells began. After
this rise, the cell count rapidly tapers to a lower level which
continues for approximately 50 minutes. FIG. 3B similarly shows the
results of field flow fractionation of HL-60 cells at a flow rate
of 100 ul/min. As shown in FIG. 3B, a sharp rise in HL-60 cells
exiting the chamber occurs at approximately 30 minutes after the
flow of cells began. Again, after this rise, the cell count rapidly
tapers to a lower level which continues for approximately 30
minutes.
[0189] FIG. 3C shows the results of field flow fractionation on a
mixture of HL-60 and human whole blood in a medium of 8.5% sucrose
and 0.3% dextrose adjusted to a 10 mS/m conductivity, in an
apparatus as described above. The fractionation occurred at a flow
rate of 100 ml/min. As shown in FIG. 3C, a sharp rise in the HL-60
cells exiting the chamber occurred at approximately 20 minutes
after the flow began. Thereafter, a second rise in the number of
cells exiting occurred at approximately 60 minutes, which
correlated to the exit of the human blood cells. However, it is
noted that cells continue to exit before and after the peaks. Thus,
separation by field flow fractionation is not capable of a complete
separation. Therefore, FIGS. 3A, 3B, and 3C demonstrate that
although field flow fractionation may discriminate and separate
some particles of different characteristics, there is needed
greater discrimination capabilities.
[0190] Three types of studies utilizing the apparatus of the
present invention were performed that caused cDEP (conventional
dielectrophoretic) forces on the particulate matter:
[0191] (1) Levitation of Cells Caused by cDEP Force
[0192] The levitation of DS-19 murine erythroleukemia cells (kindly
supplied by M. Rifkind) supported in 8.5% sucrose +0.3% dextrose
solution having a conductivity of 56 mS/m was investigated as a
function of the frequency and voltage of signals applied to the
electrode array in the absence of fluid flow. It is to be
understood that various solutions having conductivities in the
range of about 10 mS/m to about 2 S/m, such as tissue culture
medium or the like, may be used. Further, it is possible to utilize
a collection of cells only. Other solutions may be used so long as
their electrical conductivity and osmolality are adjusted according
to the particular application.
[0193] The results of this study are shown in FIG. 4A and FIG. 4B.
In the frequency range 1 kHz-40 kHz, DS19 cells were levitated to
about 20 microns at an applied voltage of 4V peak to peak (p-p), as
shown in FIG. 4A. Above 40 kHz, the levitation height dropped
rapidly, and when the frequency reached 140 kHz and above, cells
were no longer levitated but were instead attracted to electrode
edges by positive cDEP.
[0194] At an applied frequency of 50 kHz, levitation of DS19 cells
occurred when the applied voltage was above about 0.5 V p-p, as
shown in FIG. 4B. Above this threshold, the cells levitated and the
height of levitation increased with increasing voltage. This
behavior was consistent with that predicted by cDEP theory, the
dielectric properties of the cells as measured using the technique
of electrorotation, and the density of the cells and their
supporting medium.
[0195] (2) cDEP/FFF studies
[0196] A second study using the apparatus discussed above involved
the velocity of HL-60 human promyelocytic leukemia cells supported
in 8.5% sucrose +0.3% dextrose solution having a conductivity of
about 10 mS/m with an established fluid flow in the chamber, as a
function of the frequency of the voltage signals applied to the
electrode array. When no voltage signal was applied, the cell
velocity was about 10 microns per second as they were transported
under the influence of an applied fluid flow rate of 10 ul/min
(FIG. 5A). The fluid flow may be either the solution including the
cells to be tested, or it may be another fluid, or the same fluid
without the cells. Additionally the solution may be ramped over
time to alter, for example, the pH, or conductivity of the
solution.
[0197] Addressing the electrodes with voltage signals affected the
height at which the cells traveled above the chamber bottom wall
and thereby altered their position and velocity in the laminar
flow. As shown in FIG. 5A, below 10 kHz, cell velocity increased to
about 50 microns per second with an applied voltage of 3 V p-p. As
the frequency was increased in the range of about 10 kHz to about
25 kHz, the cell velocity gradually fell as the levitation height
was reduced. Above 30 kHz, these cells were attracted to the
electrode and thus they ceased moving. This response with
increasing frequency agreed with the behavior expected from the
measured electrical properties of the cells.
[0198] As shown in FIGS. 5B and 5C, similar results were obtained
for studies using other cells having different cell properties.
Specifically, FIG. 5B shows the results for MDA 468 cells (kindly
supplied by Janet Price) in a solution of 8.5% sucrose 0.3%
dextrose conductivity at 10 ms/m at a flow rate of 40 ul/min at 3 V
p-p. FIG. 5C shows the results for MDA-435 cells (kindly supplied
by Janet Price) in the same solution at a flow rate of 40 ul/min at
3 V p-p. FIG. 5D shows the results for MDA-435 cells at a flow rate
of 40 ul/min at a frequency of 31.6 kHz. As noted in FIG. 5D, the
velocity of cells increases approximately linearly with
voltage.
[0199] (3) cDEP/FFF on Mixture of HL-60 and Human Blood Cells
[0200] The chambers of the apparatus were preloaded with a mixture
of HL-60 and human blood cells in the ratio 1:10 at a total
concentration of 5.times.10.sup.7 cells/ml. The cells were
supported in 8.5% sucrose +0.3% dextrose solution having a
conductivity of 10 mS/m. A voltage of 3 V p-p at 40 kHz was applied
to the electrodes and fluid flow at the rate of 10 ul/min was
started. All of the HL-60 cells were trapped at the edges of the
electrode elements, while the human blood cells (mainly
erythrocytes) were levitated and were transported by the fluid. By
adjusting the frequency in the range of 8-15 kHz, HL-60 cells were
also released and their rate of transport controlled relative to
the erythrocytes. When HL-60 cells were levitated to heights above
or below the erythrocytes, they moved correspondingly more quickly
or more slowly than these blood cells depending on their position
in the field flow.
[0201] The following is an additional study performed according to
the present invention. Fluids were injected and removed through
slots at each end of the chamber. The outlet port was finished with
a well to trap cells exiting the chamber. Prior to performing
studies, the chamber was soaked for 5 minutes with 20% (w/v) bovine
serum albumin solution to render the glass surfaces less adherent
to cells. Alternately the glass surfaces may be air blown, or
washed and treated with silane. Dielectrophoretic forces were
generated by connecting alternate electrodes to sinusoidal voltages
of fixed or swept frequencies, and were monitored using an
oscilloscope. Forces to remove cells from the separation chamber
were provided by laminar flow of an eluate buffer, controlled by
two digital syringe pumps connected in push-pull configuration
between the inlet and outlet ports of the chamber. A bubble-free
path of fluid was maintained between the pumps at all times.
[0202] Following injection of approximately 30 ul of the cell
mixture (about 1.2.times.10.sup.6 cells) to half fill the chamber,
a 200 kHz signal of 5 V peak-peak was applied to the electrode
array for 30 sec to collect all cells by positive DEP at the
high-field regions of the electrode tips. It is not required,
however, to only half-fill the chamber, and a larger chamber may
allow for better discrimination. Flow of eluate (consisting of
cell-free suspension buffer, which may also be a mixture of 8.5%
sucrose plus 3 mg/ml dextrose having a conductivity of 10 mS/m),
was then started at 5 ml/min. This flow may be accomplished under
the control of two digital syringe pumps operating in a push-pull
configuration between the inlet and outlet ports of the chamber.
Alternately, the flow may be controlled by a peristaltic pump,
gravity flow, blood pressure, or the like. The frequency of the
applied electric signal was lowered until the tumor cells were
selectively retained while the blood cells were eluted and trapped
in the collection well. After 20 minutes, cells were removed from
the well by cross-flow between two additional syringe ports without
disturbing the tumor cells still on the electrodes. The voltage was
then turned off to release the cells held by DEP and these were
eluted and collected separately.
EXAMPLE 3
[0203] Separation of Polystyrene Microbeads Using Dielectrophoretic
Field-Flow-Fractionation
[0204] The characterization of a dielectrophoretic/gravitational
field-flow-fractionation (DEP/G-FFF) system using model polystyrene
(PS) microbeads has been achieved.
[0205] Separations of PS beads of different surface
functionalization (COOH and none) and different sizes (6, 10 and 15
.mu.m in diameter) have been demonstrated. To investigate the
factors influencing separation performance, particle elution times
were determined as a function of particle suspension conductivity,
fluid flow rate, and applied field frequency and voltage.
Experimental data were analyzed using a theoretical model (Huang et
al., 1997) and good agreement between theory and experiment was
found. It was shown that separation of PS beads was based on the
differences in their effective dielectric properties. Particles
possessing different dielectric properties were positioned at
different heights in a fluid-flow profile in a thin chamber by the
balance of DEP and gravitational forces, were transported at
different velocities under the influence of the fluid flow, and
were separated. To explore hydrodynamic (HD) lift effects,
velocities of PS beads were determined as a function of fluid flow
rate in the separation chamber when no DEP field was applied. In
this case, particle equilibrium height positions were governed
solely by the balance of HD lift and gravitational forces. It was
concluded that under the experimental conditions reported in this
example that the DEP and gravitational forces were the dominant
factors in controlling particle equilibrium height and that HD lift
force played little role in DEP/G-FFF operation.
[0206] DEP-FFF technique reported in this Example exploits the
balance between dielectrophoretic and gravitational
(sedimentational) forces. In this method negative DEP forces are
produced by microelectrodes on a chamber and levitate particles to
equilibrium positions in a flow-velocity profile. Particles at
different heights in the flow stream move at different velocities
and can be fractionated based upon their different retention times
in the chamber. We term this technique
dielectrophoretic/gravitational-Field-flow-fractionation
(DEP/G-FFF), a subtechnique of DEP-FFF. Balancing DEP forces with
other types of physical forces (e.g. electrophoretic, crossflow or
DEP forces generated from different electrodes in the chamber)
results in other subtechniques such as DEP/electrophoretic-FFF.
[0207] The construction and characterization of a complete
DEP/G-FFF system consisting of a syringe pump, a
fluid-sample-injector, a DEP/G-FFF chamber and a particle detector
at the chamber outlet has been achieved. The separations of model
particles of polystyrene (PS) microbeads having different
surface-functionalizations (COOH and none) and different sizes (6,
10 and 15 .mu.m in diameter) have been demonstrated. Separation
performance has been shown to be a function of the suspension
conductivity, the fluid flow rate, and the voltage and frequency of
the signals applied to the electrode. A theoretical analysis of the
results has revealed that separation of PS microbeads is based upon
the differences in the effective dielectric properties of different
bead types.
[0208] Method and Materials DEP/G-FFF system. A schematic
representation of the DEP/G-FFF system used in this example is
shown in FIG. 13. FIG. 13 reveals a Teflon spacer with a flow
channel cut into it. The operation of the injection valve is as
follows: the sample is first loaded into the loop through the path
"syringe.fwdarw.5.fwdarw.4.fwdarw.loop.fwdarw.1.fwd-
arw.6.fwdarw.waste" with fluid flow in the second path; in the
injection mode, the fluid flow path is "syringe
pump.fwdarw.2.fwdarw.1.fwdarw.loop.- fwdarw.4.fwdarw.3.fwdarw.
chamber". Parallel microelectrode arrays having 50 .mu.m widths and
gaps were fabricated on 50.times.50 mm glass substrates using
standard photolithographic methods. Eight 50.times.50 mm electrode
plates were glued end-to-end onto a supporting glass plate to form
an electrode of area 50.times.400 mm. A Teflon spacer (H
0.4.times.W 50.times.L 400 mm) was cut to provide an open channel
with dimensions of 388 mm from tip to tip and 25 mm in width except
at the tapered ends. This was sandwiched between the bottom
electrode plate and a top glass plate to form the DEP/G-FFF
chamber. The chamber was firmly assembled with 36 Nylon
screw-clamps (Bel-Art Products, NJ). The top and bottom plates were
drilled with 0.0625 in.-diameter holes to fit inlet and outlet
tubing at positions coincident with the points of the tapered
opposite ends of the cutout channel. Microelectrode arrays, each
having two 4-mm wide electrical conductor buses running along the
edges, were connected in parallel to a lab-built PA05-based power
amplifier (Apex Microtechnology, AZ). The amplifier could deliver
up to 10 W of power into a 2-ohm load with a bandwidth of DC to 400
kHz.
[0209] A digital syringe pump (KD Scientific, MA) was used to
provide continuous flow of carrier medium through the DEP/G-FFF
chamber at a rate selectable between 1 .mu.l/min and 70 ml/min. A
sample injection valve (Rheodyne Model 7010, CA) allowed measured
sample introduction from a 10-.mu.l loop. A 5 cm length of PEEK
tubing (0.0625 in. O.D., 0.010 in I.D.) having a void volume of 2.5
.mu.l served as the inlet connection between the injection valve
and the chamber.
[0210] Two different methods were employed to characterize particle
responses in the DEP/G-FFF chamber. The first approach was to
manually gauge the dynamics of particle separation by counting
particles that passed by several specific inspection locations
along the length of the chamber as a function of time with the aid
of video microscopy. The second method was to monitor particles
exiting the chamber with an UV detector. To accomplish this, the
chamber outlet was connected to the 3 .mu.l flow cell of an UV
spectroscopic detection system (ISCO Model UA-6, NE) via a 5 cm
length of PEEK tubing (0.0625 in. O.D., 0.020 in I.D.). The
detector was operated at a wavelength of 254 nm and its output
voltage signal, proportional to light attenuation by particles in
the flow-cell, was fed to a chart recorder (Goetz, Austria).
[0211] Polystyrene (PS) bead preparation. Two types of experiments
were conducted: (1) separation of PS beads (Polysciences, PA) of
similar density (1050 kg/m.sup.3) and size (9.44.+-.0.95 vs
10.57.+-.1.03 .mu.m in diameter) but possessing different surface
functionalizations (COOH and none); and (2) separation of
non-functionalized PS (NF-PS) beads of similar density (1050
kg/m.sup.3) but of different sizes (6.14.+-.0.45, 10.57.+-.1.03 and
15.5.+-.1.84 .mu.m). Surface-carboxylated (COOH-PS) beads were
characterized by the manufacturer as having a surface charge of
0.12 meq (COO.sup.-)/g of polymer. While there are other methods
suitable for the separation of PS beads, these beads were chosen as
model particles in this Example to aid in the characterization and
development of the DEP/G-FFF system because they were relatively
homogeneous in terms of size, density and other structural and
compositional characteristics.
[0212] A DEP buffer, consisting of 8.5% (w/v) sucrose and 0.3%
(w/v) dextrose, was used as the FFF carrier fluid and
particle-suspending medium. Electrical conductivity of the buffer
was brought to 2.2 or 10 mS/m with aliquots of 300 mM EDTA
(adjusted to pH 7.0 with NaOH). The final pH of the buffer was
found to be .about.6.8. To ensure that no air bubbles were present
in the DEP/G-FFF chamber during separation, the sucrose/dextrose
buffer was degassed under vacuum for several minutes. Sample
mixtures were prepared by diluting aliquots of
Polysciences-supplied microbead suspensions with the
sucrose/dextrose buffer to achieve particle concentrations of
1.5.times.10.sup.7, 3.times.10.sup.6 and 4.times.10.sup.5 particles
per ml for PS beads of nominal diameter 6, 10 and 15 .mu.m,
respectively.
[0213] Bead separation protocol. The DEP/G-FFF chamber was first
loaded with carrier medium (sucrose/dextrose buffer) using the
syringe pump; precautions were taken to ensure that no air bubbles
were introduced into the chamber. Appropriate voltage signals
(between 0.5 and 1 V RMS at 50 kHz) were then applied to the
microelectrodes so that PS beads would be levitated to equilibrium
positions upon injection into the chamber, thereby minimizing
contact and possible adherence of the beads to the electrode
surface. Next, a mixture of PS bead types was introduced into the
chamber. To accomplish this, the injection valve was first set in
the "load" mode and the 10 .mu.l loop was filled with sample using
a manually operated syringe. The valve was then switched to the
"injection" mode and 35 .mu.l sucrose/dextrose buffer was pumped
through the loop by the syringe pump operating at 50 .mu.l/min to
flush the beads into the DEP/G-FFF chamber. The valve was then
switched back to the "load" mode, ready for the next sample
loading.
[0214] After PS microbeads had been loaded into the inlet port of
the chamber, they were allowed to relax for some minutes (up to 30
min.) in order to attain equilibrium height positions where the
sedimentation and DEP levitation forces were balanced. Following
relaxation, flow of the carrier medium was initiated in the chamber
from the syringe pump which was operated at a desired flow rate in
the range 20-2000 .mu.l/min. As PS beads were carried along the
chamber length, their kinetics were observed with video microscopy
(Nikon Microphot-SA microscope, Hamamatsu XC-77 CCD camera) and
results were recorded on a VCR (Panasonic: AG-7350). Finally, PS
beads exiting the chamber were monitored by the UV detection
system.
[0215] Results
[0216] Separation dynamics. To examine the dynamic process of
DEP/G-FFF separation, particle trajectories in the chamber were
followed by monitoring the number of particles that passed by
several inspection windows along the chamber as a function of time.
From these data, we constructed three-dimensional representations
were constructed of separation dynamics where the number (Z-axis)
of particles was plotted as a function of time (X-axis) at
different inspection positions (Y-axis) along the chamber. A
typical example is shown in FIG. 14 for a DEP/G-FFF separation of
NF-PS and COOH-PS beads. Conditions for FIG. 14 include:
Voltage--1.6 V RMS at 50 kHz; parallel electrode arrays--50 .mu.m
electrode widths and gaps. After injection, beads were allowed to
relax to their equilibrium height positions in the chamber for 10
minutes prior to the application of the fluid flow. To begin
separation, flow of sucrose buffer of electrical conductivity 10
mS/m was initiated at 200 .mu.l/min. Clearly, the two
subpopulations of beads traveled at different velocities and became
more and more separated as they moved further along the chamber. A
single peak was only visible at Position #1. Thereafter bifurcation
of the peak occurred until two distinct (non-overlapping) peaks
were observed at Position #5 (165 mm from the chamber inlet). This
indicates that complete separation of the two bead subpopulations
may be achieved with a chamber only 165 mm long. By the time the
beads reached Position #8 (close to the chamber outlet and 360 mm
from the inlet), the two subpopulation peaks were separated by a
time interval of >1 min.
[0217] DEP/G-FFF fractograms. To characterize the separation
performance, particles exiting the DEP/G-FFF chamber were monitored
using the UV detector. Typical fractograms displaying the time
dependence of the UV absorbance are shown in FIGS. 15A and 15B.
FIG. 15A depicts the separation of COOH-PS and NF-PS beads with the
two peaks occurring 11.7-12.4 and 12.8-13.4 minutes after the
initiation of fluid flow, respectively. Conditions for FIG. 15A
include: Voltage--1.24 V RMS at 50 kHz; parallel electrode
arrays--50 .mu.m electrode widths and gaps. Beads were allowed to
relax to their equilibrium height positions in the chamber for 10
minutes after injection and prior to the application of fluid flow.
Sucrose buffer of electrical conductivity 10 mS/m was pumped
through the chamber at 800 .mu.l/min. In order to associate elution
times with specific microbead types, DEP/G-FFF experiments were
performed on pure NF-PS or COOH-PS beads and then on several
mixtures of these bead types at different concentration ratios. By
comparing the elution peak times in these experiments, it has been
determined that the COOH-PS beads eluted ahead of the NF-PS beads.
FIG. 15B shows the separation of NF-PS beads of three different
sizes (nominal diameters 6, 10 and 15 .mu.m). Conditions for FIG.
15B include: Voltage--0.53 V RMS at 100 kHz; parallel electrode
arrays--50 .mu.m electrode widths and gaps. Beads were allowed to
relax for 25 minutes after injection and prior to the application
of fluid flow. Sucrose buffer of electrical conductivity 2.2 mS/m
was pumped through the chamber at 800 .mu.l/min. Direct observation
of particle motion under the microscope revealed that larger beads
moved faster than smaller ones. Thus, the three elution peaks in
time range of 9.9-10.8, 11.6-12.4 and 12.7-15 minutes corresponded
to populations of 15, 10 and 6 .mu.m diameters, respectively.
[0218] Separation of PS and COOH-PS beads. We (Huang et al., 1997)
and others; (Williams et al., 1992) have previously reported that
particles experience a hydrodynamic (HD) lifting force that pushes
them away from the chamber walls as they are carried along in a
fluid flow profile. This lifting force was shown to increase with
the fluid flow rate (Williams et al., 1992). To determine the
influence of the HD lifting force on overall particle kinetics,
separation experiments were conducted as a function of the fluid
flow rate for a specified DEP field condition. As shown in FIGS.
16A and 16B, the elution peak times for NF-PS and COOH-PS beads
were inversely proportional to the flow rate in the range of 100 to
1000 .mu.l/min. Separation effectiveness, as characterized by the
ratio of the elution peak times for two PS bead populations, was
not compromised even at the high flow rate of 1000 .mu.l/min. These
results indicate that the hydrodynamic lifting force played little
role in the separation process for the flow rate range investigated
here.
[0219] The importance of DEP forces in DEP/G-FFF separation is
illustrated in FIG. 17A where elution peak times for NF-PS and
COOH-PS beads are shown as a function of the applied DEP voltage
signals. Increasing the applied voltage from 0.07 V to 2.65 V RMS
resulted in faster elution of both NF-PS and COOH-PS beads. This is
expected when it is considered that larger applied voltages
levitate particles to higher equilibrium position the flow velocity
profile (Gascoyne et al., 1996; Huang et al., 1997). Separation
effectiveness was observed to be a function of the applied voltage
(FIG. 17B). The best separation of the two bead populations, as
characterized by a maximum value of .about.1.65 for the ratio of
the two elution peak times, was attained at an applied voltage of
0.21 V RMS. Increasing or decreasing the applied voltage resulted
in a gradual convergence of the two elution-peaks.
[0220] Separation of NF-PS beads of three different sizes. The
dependence of elution times on the applied DEP voltage is shown in
FIG. 18 for PS beads of nominal diameter 6, 10 and 15 .mu.m. As for
the separation of NF-PS and COOH-PS beads, an increase in the
applied voltage resulted in decreased bead elution times and
decreased separation between the three elution-peaks. Separation of
NF-PS beads of 6, 10 and 15 .mu.m was also studied as a function of
the applied field frequency for constant fluid flow and applied
voltage conditions (FIG. 19). The elution-peak time for 15 .mu.m
beads remained nearly constant in the frequency range 1 to 400 kHz.
For 10 .mu.m beads, the elution time was nearly constant in the
frequency range 5 to 200 kHz but became larger at lower (1-2 kHz)
or higher (400 kHz) frequencies. 6 .mu.m beads exhibited a strong
frequency dependency with a maximum elution time at 50 kHz. Optimum
separation of these beads was achieved for this example at
.about.50 kHz.
[0221] Bead relaxation. A common feature for most FFF operations
(Liu et al., 1991) is the relaxation process in which particles to
be separated are allowed to relax to their equilibrium positions
with respect to the two major surfaces of the separation chamber
before the fluid-flow is applied. The equilibrium positions are
determined by the balance of physical forces acting on the
particles. The relaxation process ensures that the differential
positions of the particles in the fluid-flow profile and the
corresponding particle velocities and transit times across the
chamber depend only on the physical properties of the particles,
not on their initial positions after introduction into the chamber.
Therefore, PS beads were allowed to sediment to their equilibrium
heights with appropriate DEP electrical fields applied after they
were introduced into the DEP/G-FFF chamber. The time (t.sub.r)
required for a particle of radius r and density .rho..sub.p to
sediment a distance H can be readily derived from the formula 14 t
r = 9 H 2 ( p - m ) r 2 g ( 16 )
[0222] where g is the acceleration due to gravity, .rho..sub.m and
.eta. are the density and viscosity of the suspending medium,
respectively. Equation 16 reveals that small particles take longer
to relax than larger particles. For example, the relaxation times
for PS beads of 6, 10 and 15 .mu.m diameter (density 1.05
g/cm.sup.3) are about 4, 9 and 25 minutes, respectively, for a
relaxation distance of 400 .mu.m in a medium of density 1.033
g/cm.sup.3 and viscosity 1.26.times.10.sup.-3
kg/(m.multidot.s).
[0223] DEP/G-FFF chamber surface treatment. As discussed above, PS
beads should theoretically settle to their equilibrium height
positions as determined by the balance of sedimentation and applied
DEP forces during relaxation. However, the DEP levitation force is
larger above the electrode edges of the electrode array and smaller
over the centers of the electrodes and gaps (FIG. 20). As a result
of this as well as certain imperfections such as occasional open
circuits in electrode elements, some PS beads may settle to the
chamber bottom surface. These may adhere to the chamber wall, where
they can disturb the laminar flow profile when fluid flow is
started, and impair the separation performance. Therefore, after
several experiments, we realized that, in this example, an
appropriate conditioning of the chamber bottom surface to inhibit
particle adherence was critical to achieving optimum separation
results.
[0224] The following procedures were developed for chamber surface
treatment. Electrodes were first washed in 1% (w/v) Alconox
detergent (Alconox Inc., NY), rinsed thoroughly with deionized
water and air-dried before chamber assembly. To remove any residual
water in the chamber, it was filled with ethanol and then dried
with filtered, low pressure N.sub.2. The chamber was then filled
with Sigmacote (Sigma, Mo.) for 15 minutes and dried again with
N.sub.2. Each Sigmacote treatment, which applied a hydrophobic
coating to the chamber walls, lasted for about twenty experiments.
After each day's usage, the chamber was flushed with 60 ml of 1%
(w/v) Alconox plus 0.05% (w/v) NaOCl (Clorox Inc., CA) solution at
a flow rate 2 ml/min for 30 min. For overnight storage, the chamber
was filled with this solution to ensure there was no growth of
micororganisms. It has been found that the chamber can be used for
many hundreds of experiments without noticeable changes in its
separation performance, provided the electrode surface treatment
described above is performed regularly.
[0225] Particle kinetics. As illustrated in FIG. 21, a particle
traveling in a DEP/G-FFF chamber experiences several forces. In the
vertical direction, DEP levitation, sedimentation (gravitation) and
HD lift forces act to determine the particle height in the
fluid-flow profile. As a particle travels across the electrode
array, the net force it experiences in the vertical direction
alternates around zero. As a result, the particle will move up and
down, and the magnitude of these height perturbations will depend
on the instantaneous DEP levitation forces, the fluid viscosity,
and the rate of travel across the electrode elements. We found that
the oscillation of particle heights was quite small (<2 .mu.m)
at moderate or high fluid flow rates (>200 .mu.l/min) because
particles had a short time (<20 ms) to respond to the variations
in the vertical DEP force component.
[0226] In the fluid flow direction, a particle experiences fluid
drag as well as a horizontal component of DEP forces from the
electrodes (Wang et al., 1998). Although the net fluid drag would
be zero if a particle moved at constant velocity at a fixed height
in the fluid flow profile in the absence of an applied electrical
field, the horizontal DEP force component causes the particle
velocity to suffer perturbations. Nevertheless, because of the
electrode periodicity, the average horizontal DEP force over a
complete electrode/gap period is zero FIG. (Wang et al., 1998).
Thus horizontal DEP forces have no effect on the average velocity
of the particle. As a corollary, the velocity of particles depends
only on their equilibrium height positions in the flow profile as
determined by the balance of the average DEP levitation force and
the sedimentation and HD lift forces; the horizontal DEP component
does not influence particle velocities. This is one of the major
differences between DEP/G-FFF and the early separation approach of
DEP-retention. In that case, the horizontal DEP force component was
used to compete with the fluid flow forces and thereby determine
the particle elution rate (Becker et al., 1995; Markx et al., 1994;
Talary et al., 1995).
[0227] Hydrodynamic (HD) lift forces. To investigate the HD lift
effect, we conducted gravitational field-flow-fractionation
experiments where no DEP forces were applied so that particle
equilibrium positions were determined solely by sedimentation and
HD lift forces. In these cases, based on experimental particle
elution data and a theoretical analysis (Wang et al. 1998), we
found that particle heights were essentially independent of the
fluid flow rate and HD lift forces levitated PS beads only slightly
and distances between particle peripheries and the chamber bottom
wall were between only 0.4 and 0.7 .mu.m. On the other hand,
experimental particle elution data demonstrated that DEP forces can
levitate particles much higher (up to 2 orders of magnitude)
equilibrium positions that HD effects. We therefore conclude that
hydrodynamic lift forces play little or no role in the DEP/G-FFF
separations described here.
[0228] Optimization of DEP/G-FFF separation. We have found that
that the particle elution time may be determined by a number of
operational parameters including the chamber height H and length L;
the average fluid flow rate (V.sub.m); the electrode periodic
distance d; the applied voltage U, the particle radius r, the
particle dielectric polarization parameter Re(f.sub.CM), and the
particle and suspension densities .rho..sub.p and .rho..sub.m.
Separation by DEP/G-FFF can therefore exploit differences in
particle size, density and dielectric properties.
[0229] As in most FFF applications, we have assumed that a
parabolic flow profile exists in the vertical direction of the
DEP/G-FFF chamber and that the shape of this profile is determined
by the chamber height H. For achieving better separation
performance, we have found that the chamber height H should be
chosen so as to maximize the fluid velocity gradient, given by, 15
V m h = 6 V m H 2 ( H - 2 h ) . ( 17 )
[0230] The gradient .sup.dV.sub.m/dh increases as the particle
height h is decreased in the flow profile so that better velocity
differentiation may be achieved for particles equilibrated closer
to the chamber bottom surface. For particles having a maximum
levitation height h.sub.max, the choice of the chamber height H may
depend on the specific optimization criteria. For example, to
maximize the average velocity gradient between 0 and h.sub.max, the
chamber height should be 2h.sub.max; to maximize the gradient at
h.sub.max the chamber height should be 4h.sub.max.
[0231] The particle elution time and the degree of separation
between different particle types are proportional to the chamber
length L, thus better separation may be achieved by increasing L.
The particle elution time is inversely proportional to the averaged
fluid velocity <V.sub.m> so that increasing the flow rate may
result in faster separation. Nevertheless, at sufficiently high
values of the fluid flow rate, the HD lift may impair particle
separation. Other undesirable effects may also come into play at
very high flow rates.
[0232] Electrode arrays with large d values may be preferred for
increasing the resolution of dielectric discrimination. Electrodes
having large d values may, however, require a much higher field
strength (E) to generate sufficiently strong DEP levitation forces
(proportional to E.sup.2/d). A higher field strength may cause, in
turn some undesirable effects such as Joule heating of the
suspending medium.
[0233] As shown in FIGS. 17A, 17B, and 18 the applied voltage U is
an important variable for DEP/G-FFF operation. Generally, large
voltages levitate particles to higher equilibrium positions where
the gradient of the fluid velocity is reduced. To exploit the
region of large velocity gradient in the fluid flow profile for
better particle separations, small voltages may be preferred. On
the other hand, HD lift effects at low positions in the profile may
complicate particle kinetic behaviors and small voltages also
result in longer separation times. Clearly the applied voltages
should be optimized for each application.
[0234] The density .rho..sub.m of suspending medium is another
important variable for DEP/G-FFF. For stable positioning of
particles in the flow profile, negative dielectrophoretic forces
and negatively buoyant particles should be used. .rho..sub.m should
therefore be smaller than the densities (.rho..sub.p) of the
particles (Gascoyne et al., 1996; Huang et al., 1997). The value
for .rho..sub.m may be chosen based on the following criteria. If
.rho..sub.m is much smaller than .rho..sub.p, then a large voltage
and field strength may have to be applied to levitate the
particles. On the other hand, if .rho..sub.m is just slightly below
.rho..sub.p, then a long time may be necessary for particles to
relax to their equilibrium positions after introduction into the
chamber.
[0235] The applied field frequency f and the dielectric properties
(electrical conductivity and permittivity) of suspending medium are
important factors in determining the dielectric polarization factor
Re(f.sub.CM) of the particles (Gascoyne et al., 1997; Wang et al.,
1997), and should be optimized by maximizing the differences in
Re(f.sub.CM) the real component of the Clausius-Mossotti factor
that reflects the magnitude and direction of field-induced
polarization in the particle at frequency f, between particles to
be separated. As long as the applied DEP levitation forces (a
function of the field frequency f and dielectric properties of
suspending medium) are effective in controlling particle
equilibrium heights in the flow profile, the DEP/G-FFF system may
be used for particle characterization and separation. In contrast
to particle separations using DEP-retention (Wang et al., 1993,
Becker et al., 1995) where different particles must have different
polarities for Re.sub.CM), DEP/G-FFF separations require that
different particles have different negative Re(f.sub.CM) values. As
shown previously in DEP/G-FFF (Huang et al., 1997), particle
velocity (and therefore the retention time) is very sensitive to
Re(f.sub.CM), suggesting considerably higher particle
discriminations for DEP/G-FFF than for the DEP-retention
method.
[0236] Conclusion
[0237] This example has shown that dielectrophoretic/gravitational
field-flow fractionation is an effective method for particle
separation. It may be readily applied for the separation of
particles of .about.1 .mu.m to several hundred micrometers. It
exploits not only differences in particle size and density, as in a
number of other FFF techniques, but also, and most significantly,
the particle dielectric properties. For biological cells, DEP/G-FFF
separation may be based on differences in cell size, membrane
capacitance and conductance (Gascoyne et al., 1997; Huang et al.,
1997) and cell interior dielectric properties. For colloidal
particles such as polystyrene beads, DEP/G-FFF separation may
utilize differences in particle size, particle surface properties
(such as surface charge) and bulk dielectric properties.
[0238] DEP levitation forces, generated by applying a relatively
small AC voltage (<10 V p-p) to microelectrodes on the bottom
surface of the separation chamber, may be used to balance the
gravitational (sedimentation) forces acting on the particles so as
to position them in a flow velocity profile. Particles possessing
different dielectric and density properties equilibrate at
different heights and are carried at different velocities in a flow
profile. As a result different particles elute from the separation
chamber at different times. The separation method is flexible
because it depends on a number of operational parameters including
the density and dielectric properties of the particle suspending
medium and the voltage and frequency of the applied DEP field.
These parameters may be varied to optimize separation performance
for specific applications. The operational field frequency is
typically above 1 kHz. This minimizes several undesired effects
including electrode polarization and water electrolysis at
electrode surfaces. The separation chamber may be readily
miniaturized for applications demanding the use of even minute
quantities of samples. Finally, DEP/G-FFF can be used to study the
physical properties of particles. For example, the particle
dielectric properties can be derived by determining the
dependencies of their elution times on the frequency and voltage of
the applied DEP field.
EXAMPLE 4
Cell Separation on Microfabricated Electrodes Using
Dielectrophoretic Field-Flow-Fractionation
[0239] Dielectrophoretic/gravitational field-flow-fractionation
(DEP/G-FFF) was used to separate cultured human breast cancer
MDA-435 cells from normal blood cells mixed together in a
sucrose/dextrose medium. An array of microfabricated,
interdigitated electrodes of 50 um widths and spacings, and lining
the bottom surface of a thin chamber (0.42 mm H.times.25 mm
W.times.300 mm L), was used to generate DEP forces that levitated
the cells. A 10-uL cell-mixture sample containing .about.50,000
cells was introduced into the chamber, and cancerous and normal
blood cells were levitated to different heights according to the
balance of DEP and gravitational forces. The cells at different
heights were transported at different velocities under the
influence of a parabolic flow profile that was established in the
chamber and were separated. Separation performance depended on the
frequency and voltage of the applied DEP field and the fluid-flow
rate. It took as little as 5 min to achieve cell separation. An
analysis of the dependency of cell elution-time and equilibrium
height on the DEP field frequency revealed that the separation
exploited the difference in dielectric and density properties
between cell populations. The significance of DEP/G-FFF technique
for cell processing has been recognized, particularly in relation
to the development of integrated microfluidic systems.
[0240] Experimental Details
[0241] The DEP/G-FFF System. The experimental setup for the
DEP/G-FFF system is similar to that shown in FIG. 13 and has been
described in detail in the article by Yang et al (1999a). Briefly,
.backslash.interdigitated microelectrodes, 50 .mu.m in width and
gaps, were fabricated on 50.times.50-mm glass substrates using
standard photolithography. Six electrodes were glued end-to-end
onto a supporting glass plate to form one electrode plate. The
DEP/G-FFF chamber was constructed by sandwiching a Teflon spacer
(0.42 mm H.times.50 mm W.times.300 mm L) between a top glass plate
and the bottom electrode plate with 36 Nylon screw-clamps (Bel-Art
Products, Paquannock, N.J.). The spacer was cut to provide an open
channel with dimensions of 288 mm from tip to tip and 25 mm in
width except at the tapered ends. The microelectrodes, each having
two 4-mm wide electrical conductor buses running along the edges,
were connected in parallel to a lab-built PA-05 based power
amplifier (Apex Microtechnology, Tucson, Ariz.). The signals to the
amplifier were produced from a function generator (model 33120A,
Hewlett-Packard, Santa Clara, Calif.) and monitored by an
oscilloscope (model RDS 320; Tektronix, Pittsfield, Mass.).
[0242] The top and bottom plates were drilled with 0.0625
in-diameter holes to fit inlet and outlet tubing at positions
coincident with the points of the tapered ends of the DEP/G-FFF
channel. To allow sample introduction, PEEK tubing, having a void
volume of 2.5 .mu.L, served as the connection between an injection
valve (model 7010 equipped with a 10-(L loop; Rheodyne, Rohnert
Park, Calif.) and the chamber inlet port. A digital syringe pump
(Daigger, Wheeling, Ill.) was used to provide a continuous flow of
carrier medium through the channel. Cells exiting the outlet tubing
of the chamber were collected with a fraction collector (model
Cygent 68-2170; Isco, Lincoln, Nebr.). The fraction collector can
operate by collecting sample fractions at fixed time intervals or
fixed drop number intervals. Cell kinetic behaviors in the chamber
were monitored under a microscope (model Microphot-SA; Nikon,
Melville, N.J.) equipped with a CCD camera (Hamamatsu, Bridgewater,
N.J.) and a video monitor. Cell equilibrium heights were determined
by differences in the microscope focal positions of the electrode
plane and the cells, multiplied by the refractive index of the
fluid in the chamber.
[0243] Cell Preparation. Cultured human breast cancer MDA-435 cells
mixed with normal human peripheral blood cells (mainly
erythrocytes) were used as a model system in this study. MDA-435
cells, derived from a pleural effusion of a patient with metastatic
breast cancer (Cailleau et al. 1978; Zhang & Fidler 1991), were
cultured in minimum essential/F12 medium supplemented with 10%
fetal bovine serum, 1 mM glutamine, and 20 mM HEPES (Life
Technologies, Gaithersburg, Md.), plus 0.5% penicillin and
streptomycin (Sigma Chemical Co., St. Louis, Mo.), and were
maintained in 75-cm.sup.2 plastic flasks under a 5% CO.sub.2/95%
air atmosphere at 37.degree. C. in a humidified incubator. The
cells were harvested at .about.80% confluence 48 h after seeding by
brief exposure to 0.25% trypsin-0.02% EDTA followed by
approximately 15 min recovery in complete medium. Cell mixtures
were prepared by adding an aliquot of fresh EDTA-anticoagulated
human blood to a suspension of harvested MDA-435 cells, and then
washed twice in an isotonic 8.5% (w/v) sucrose plus 0.3% (w/v)
dextrose buffer. The electrical conductivity of the sucrose buffer
was adjusted to 56 mS/m with minimum essential medium, as
determined with a conductivity meter (EC19101-00; Cole-Parmer
Instrument, Chicago, Ill.). The final concentration of cells in the
sucrose/dextrose buffer was 5.times.10.sup.6 cells/mL at a nominal
ratio of 2:3 for cancer to normal blood cells. It is noted that the
above cell preparation procedure removed the human blood plasma
from the cell mixture. However, removal of the plasma components is
not critical for the DEP/G-FFF separation described in this work.
Separation experiments were conducted in which the cell mixture was
prepared by adding diluted human blood to a suspension of breast
cancer cells. The separation performance was not affected, provided
the electrical conductivity of the final cell suspension was
maintained at an appropriate value.
[0244] Specific density values were determined for erythrocytes and
MDA-435 cells as 1.095 and 1.072 kg/dm.sup.3, respectively, with a
centrifugally generated continuous Percoll density gradient
(Pharmacia, Uppsala, Sweden) calibrated by density marker beads.
Cell Separation Protocol. The DEP/G-FFF chamber was first loaded
with the isotonic sucrose/dextrose buffer. The cell mixture sample
was then introduced into the inlet port of the chamber through the
injection valve. To accomplish this, the 10 .mu.L-loop on the
injection valve was first loaded with cell sample using a manually
operated syringe. The valve was then switched to the "injection"
mode and 35 .mu.L sucrose/dextrose buffer was flushed through the
loop by the syringe pump operating at 50 .mu.L/min to move all the
cells into the DEP/G-FFF chamber. Appropriate voltage signals were
applied to the microelectrodes during the sample introduction so
that the cells would be levitated in the chamber by DEP forces,
thereby preventing possible adherence of the cells to the bottom
surface of the chamber. Following injection, cells were allowed 5
min to attain equilibrium heights at which the opposing DEP and
gravitational forces acting on them were balanced. Flow of carrier
medium through the chamber was then started using the syringe pump,
and, because of geometrical characteristics, a parabolic
hydrodynamic flow profile was established inside the chamber. Under
the influence of fluid drag, cells were caused to move at different
velocities according to their relative positions in the flow
profile. Cells were collected as they exited the chamber using a
fraction collector and then characterized. To evaluate separation
performance, cell fractograms were obtained by counting cells under
video microscopy as they passed through a detection window at the
outlet end of the chamber. The different cell types were identified
by size (MDA-435 cells and erythrocytes are .about.15 .mu.m and 7
.mu.m in diameter, respectively).
[0245] Based on their fractograms, two parameters were defined for
each cell type to describe the elution characteristics of the
cells, namely, the elution-time and the elution-peak width. These
parameters were determined from the integral of cell number with
time, the accumulated count of cells as they passed through the
detection window. Elution-time was taken as the time at which the
integral reached 50% of its maximum. Elution-peak width was defined
as the time taken for the integral to increase from 12.5% to 87.5%
of its maximum. The elution-peak width definition corresponded to
the width at half-height for peaks having triangular or normal
distributions in time.
[0246] Theory
[0247] DEP/G-FFF Principle. FIG. 22 gives a schematic
representation of dielectrophoretic/gravitational
field-flow-fractionation principle. The bottom surface of the
chamber is lined with an interdigitated microelectrode array. The
DEP levitation force caused by the interdigitated electrodes acting
on an elliptical cell of volume V is given by (Huang et al, 1997,
Wang et al., 1998)
F.sub.DEPz=1.5V.di-elect cons..sub.m.alpha..sub.DEP
(f)U.sup.2p(f)Aexp(-2.pi.h/d) (18)
[0248] where U is the applied RMS voltage at a frequency f,
.di-elect cons..sub.m is the dielectric permittivity of the medium,
and .alpha..sub.DEP(f) (=Re(f.sub.CM)) is a factor characterizing
the field-induced polarization in the cell. The parameter p(f) is
included to correct for electrode polarization (Schwan, 1992). DEP
forces fall approximately exponentially with height h above the
electrode plane, with a decay constant that is characterized by the
periodic distance d of the electrode array and a unit-voltage force
coefficient A. The gravitational force is given by
-V(.rho..sub.c-.rho..sub.m)g . Here .rho..sub.c and .rho..sub.m are
the densities of the cell and its suspending medium, respectively,
satisfying the relationship .rho..sub.c>.rho..sub.m. The balance
of gravitational and DEP levitation forces positions the cell at a
stable equilibrium height, given by 16 h eq = d 4 ln ( 3 m Up ( f )
2 g A DEP ( f ) ( c - m ) ) . ( 19 )
[0249] Equilibrium heights are dependent on the dielectric property
(as characterized by the dielectric polarization factor
.alpha..sub.DEP) and density (.rho..sub.c) of the cell, of the
electrode dimensions (A and d), of the applied DEP field strength
(U) and frequency (f), and of the electrode polarization parameter
p(f).
[0250] The velocity with which a cell located at a height h.sub.eq
from the chamber bottom surface is carried along by the parabolic
flow profile in the chamber is given by (Williams et al., 1992) 17
V c = K r 6 V m h eq ( H - h eq ) H 2 ( 20 )
[0251] where H is the chamber thickness, and (V.sub.m) is the mean
fluid velocity. k.sub.r (<1) is a coefficient that characterizes
a retardation effect (Williams et al, 1992) that occurs when
particles are close to the chamber wall. Thus, by careful selection
of the DEP field conditions, cells having different dielectric and
density properties can be levitated to different heights above the
electrode surface and thereby be caused to move at different
velocities under the influence of the flow profile.
[0252] Cell Dielectric Modeling. For an elliptical particle, the
dielectric polarization factor .alpha..sub.DEP(f) along the j-axis
of the ellipsoid is given by (Kakutani et al, 1993) 18 DEP ( f ) =
Re ( ( c * - m * ) ( 3 ( c * - m * ) A j + 3 m * ) ) ( 21 )
[0253] where .di-elect cons..sub.c* and .di-elect cons..sub.m* are
the frequency-dependent complex dielectric permittivities of the
cell and its suspending medium, respectively, and A.sub.j is the
depolarization factor along the j-axis (j=x, y, z). In this work,
human breast cancer MDA-435 cells are modeled as spherical
particles (A.sub.j=0.333) consisting of an internal homogeneous
dielectric sphere (radius r) surrounded by a poorly conducting
plasma membrane of thickness d. The effective complex permittivity
is then given by (Fuhr & Hagedorn, 1996; Irimajiri et al, 1979;
Huang et al, 1992) 19 c * = mem * ( ( r + d ) 3 r 3 + 2 ( int * -
mem * ) int * + 2 mem * ( r + d ) 3 r 3 + int * - mem * int * + 2
mem * ) ( 22 )
[0254] where .di-elect cons..sub.mem* and .di-elect cons..sub.int*
refer to the complex permittivities of the cell membrane and
interior, respectively. Erythrocytes are modeled as single-shell
oblate ellipsoids having semi-axes of a, b, and c, satisfying the
relationship a=b>c. The shell and interior refer to the cell
membrane and cytoplasm, respectively. The cell effective
permittivity for such ellipsoids is given by (Kakutani et al, 1993)
20 c * = mem * ( mem * + ( int * - mem * ) ( A j + ( 1 - A j ) )
mem * + ( int * - mem * ) ( A j + A j ) ) . ( 23 )
[0255] where .upsilon. is the volume fraction of the cell interior
.upsilon.=(c-d)(a-d).sup.2/ca.sup.2. The depolarization factor is
given by
A.sub.j=0.5(e.sup.2a
tan(e.sup.2-1).sup.0.5-(e.sup.2-1).sup.0.5)/(e.sup.2-- 1)1.5
(24)
[0256] where e=a/c>1. Therefore, with the knowledge of the cell
dielectric parameters in the shell models, it is possible to
calculate their complex permittivities using Equations (22) and
(23).
[0257] Results A typical cell fractogram illustrating the time
dependency of the number of MDA-435 cells and erythrocytes passing
through the detection window is shown in FIG. 23 for an applied DEP
field frequency of 5 kHz. The two cell-populations were well
separated with an elution-time difference of 12 min. This result
was in agreement with the visual inspection of cell motion under
video-microscopy, revealing that MDA-435 cells moved almost twice
as fast as erythrocytes. The elution-peak widths for MDA-435 cells
and erythrocytes depended on several factors. The elution-time for
each cell depended on its velocity and travel distance through the
DEP/G-FFF chamber. Since it was not possible to ensure that all the
cells were positioned the same distance from the chamber outlet
port during sample loading, a small spread in elution-times was
expected even for the cells having the same velocity. Furthermore,
different cells of the same type exhibited different velocities
because of inherent population heterogeneity in the cell dielectric
properties caused by differences in cell composition, morphology,
and structural organization (**Huang et al., 1996; Fuhr &
Hagedorn, 1996; Pethig & Kell 1987; Becker et al, 1995;
Gascoyne et al, 1997.). The apparent double-peak for MDA-435 cells
appears to be associated with a real distribution of cell
dielectric parameters.
[0258] The dependency of cell elution-time on the frequency of the
applied DEP field is shown in FIG. 24A. At 2 kHz, MDA-435 cells and
erythrocytes were well separated with an elution-time difference of
20 min. In the frequency range of 2 to 10 kHz, the elution-time
difference between the two cell-populations was between 11 and 20
min. The elution-peak widths were quite narrow (1.7-3.5 min.),
leading to good cell separation even though the two elution peaks
were closer together at 10 kHz. Elution-time decreased with
increasing frequency, reflecting an increase in cell velocities. As
the frequency was increased to 50 kHz, elution-times and
elution-peak widths increased slightly for erythrocytes, and much
more for MDA-435 cells, indicating that the velocities of MDA-435
cells not only dropped but also exhibited a wide distribution. The
two populations were not well separated at 40 and 50 kHz because of
a broadening of the peaks.
[0259] Differences in elution-time between the two populations
resulted from the differential velocities of the cells, which in
turn reflected the differences in the cell equilibrium heights in
the fluid-flow profile. To verify the height-velocity relationship
in the DEP/G-FFF chamber, equilibrium heights for individual
MDA-435 cells and erythrocytes were measured under light microscopy
for a slow flow (<V.sub.m>)=16 .mu.m/sec) at a number of DEP
field frequencies (FIG. 24B). As anticipated from their larger
velocities, MDA-435 cells were found to be levitated to higher
positions than erythrocytes. Depending on the frequency, the
difference in the mean cell height between MDA-435 cells and
erythrocytes was as large as 15 micrometers. For each cell
population, the dependency of equilibrium height on DEP field
frequency was in qualitative agreement with the peak-time data
shown in FIG. 24A. For example, low equilibrium heights with a
large deviation for MDA-435 cells at 50 kHz accounted for the large
elution-time and elution-peak width observed under these
conditions.
[0260] Cell equilibrium heights were determined by the balance of
DEP levitation forces and gravitational forces. To understand the
basis for the difference in equilibrium heights between MDA-35
cells and erythrocytes, we calculated the frequency-dependency of
the dielectric factor .alpha..sub.DEP (FIG. 24C) for the two cell
populations from observed levitation heights using standard
equations with the knowledge of DEP field conditions and cell
density values. Whereas .alpha..sub.DEP for erythrocytes exhibited
little variation over the frequency range of 2 to 50 kHz, the
magnitude of .alpha..sub.DEP for MDA-435 cells displayed a
frequency dependency. The difference in .alpha..sub.DEP between the
two cell types at frequencies below 20 kHz indicated that the
greater levitation (and correspondingly larger velocity) exhibited
by MDA-435 cells were caused not only by their smaller density
(1.072 vs 1.095 kg/dm.sup.3) but also by their larger polarization
factors (-0.36 vs -0.21).
[0261] To illustrate the basis for the difference in dielectric
properties of MDA-435 cells and erythrocytes, the dielectric factor
.alpha..sub.DEP was calculated by cell dielectric modeling. Based
on their geometry, we modeled MDA-435 cells and erythrocytes as
single-shell spheroids and oblate ellipsoids, respectively, where
the outer shells corresponded with the plasma membranes. Compared
with erythrocytes, .alpha..sub.DEP for MDA-435 cells was larger in
the frequency range of 2 to 20 kHz and exhibited a frequency
dispersion above 40 kHz. These differences originated from the fact
that the size and membrane capacitance of MDA-435 cells were larger
than those of erythrocytes and that MDA-435 cells were spherical,
whereas erythrocytes were double-discoid. Therefore, we have
concluded that the DEP/G-FFF separation of MDA-435 cells and
erythrocytes reported here exploited differences in cell density,
size, shape, and membrane electrical properties as separation
criteria.
[0262] Cells exiting the DEP/G-FFF chamber were collected with a
fraction collector. At a DEP field frequency of 10 kHz and a
fluid-flow rate of 1 mL/min, the cells were collected into 15
fractions at a time interval of 2 min. Fractions 5 to 7 and 9 to 13
included >98% of MDA-435 cells and >99% of erythrocytes,
respectively. Furthermore, Trypan blue staining of the collected
cells revealed that >99% of the cells excluded the stain,
demonstrating that membrane integrity was maintained during
DEP/G-FFF separation.
[0263] Operational conditions such as DEP field voltage and
fluid-flow rate were changed to examine their influences on
separation performance. FIG. 25A shows the voltage dependency of
the elution-time for the MDA-435 cells and erythrocytes at 20 kHz.
Evidently, the larger the applied voltage, the shorter the
elution-time. This result was predicted because larger voltages
increased DEP levitation force and thereby caused cells to be
positioned higher in the fluid-flow profile where they were carried
faster. FIG. 25A further shows that the elution-peak width
increased significantly with decreasing applied voltage. An
important factor influencing the elution-peak width is the
heterogeneity in cell dielectric properties. Individual cells of
the same type having different dielectric properties were
positioned at different heights in the flow profile and therefore
traveled through the DEP/G-FFF chamber at different velocities.
With decreasing voltage, the cells were positioned lower in the
fluid-flow profile where larger velocity gradients existed. Thus,
the cells with slightly different dielectric properties were
carried by the fluid flow at velocities with larger differences,
leading to increased elution-peak widths.
[0264] FIG. 25B demonstrates the effect of the fluid-flow rate on
cell elution-time. The elution-time was found to be inversely
proportional to the flow rate, indicating that the cell equilibrium
heights were not affected by the change in the flow rate. Efficient
separations of the two populations could be achieved in less than 8
min at a flow rate of 2 mL/min. FIG. 25B further shows that the
elution-peak width increased with decreasing fluid-flow rate.
Nevertheless, the ratio of the elution-peak width to elution-time
remained almost unchanged for different flow rates, supporting the
conclusion that the flow rate did not influence cell equilibrium
heights.
[0265] We have found that the cell equilibrium heights in the
DEP/G-FFF chamber were almost independent of the flow rate, which
confirms that the applied DEP levitation forces were much stronger
than this hydrodynamic lift force. This finding is in agreement
with theoretical analyses of the hydrodynamic lift force (Yang et
al, 1999a).
[0266] To further demonstrate the general applicability of
DEP/G-FFF to cell-separation problems, experiments were conducted
in which cultured human leukemia HL-60 cells were separated from a
mixture with normal blood cells and MDA-435 cells were separated
from a mixture with purified human T-lymphocytes (unpublished
data). The results achieved were consistent with those in our
MDA-435/erythrocyte study, indicating that this method may have
applications to a number of cell separation needs.
[0267] When the cells were initially loaded into the chamber, they
exhibited a wide distribution of heights. In order for the cells to
reach equilibrium positions, they were allowed 5 min to sediment
before the fluid flow was applied. Allowing sufficient time for
this initial sedimentation (the so called relaxation-time) was
important for ensuring good separation performance.
[0268] We were able to sort .about.50,000 cells in each experiment
at a rate of several thousands per minute. Because the electrode
array has no moving parts and a single amplifier can be used to
power it, the DEP/G-FFF device may easily be scaled up as necessary
for routine cell separation in biological and clinical labs,
handling 10.sup.6 or more cells in each separation. Furthermore,
the DEP/G-FFF principle may be readily implemented in microfluidic
systems for processing cell samples in the volume range of
nanoliters to microliters. The only issue associated with
miniaturization of the DEP/G-FFF device is that the chamber has to
be long enough to provide sufficient resolution for the desired
cell separation. Our work (unpublished data in the lab) has shown
that the total length may be as short as 5 cm and that under our
flow conditions, this may be achieved in a space-efficient
serpentine configuration. The miniaturized DEP/G-FFF device may be
interfaced to other microflume components including micro PCR and
capillary electrophoresis devices, cell counters, and
electrochemical detectors. In addition, the DEP/G-FFF method
exploits cell dielectric and density properties, adding a new
dimension to cell separation. The capability of DEP/G-FFF
separation according to density properties suggests that it may be
used in integrated microflume systems as a substitute for
centrifugation, which is currently used as a basic step in cell
sample preparation. Finally, the technique is noninvasive and does
not rely on the interaction of antibodies with cell-surface
antigens, making it potentially attractive for applications such as
the separation of leukocyte subpopulations without the potential
problems of cell activation inherent in immuno-selective
methods.
EXAMPLE 5
[0269] The principle of cell characterization and separation by
dielectrophoretic field flow fractionation has been achieved. The
operational device in this example took the form of a thin chamber
in which the bottom wall supported an array of microelectrodes. By
applying appropriate AC voltage signals to these electrodes,
dielectrophoretic forces were generated to levitate cells suspended
in the chamber and to affect their equilibrium heights. A laminar
flow profile was established in the chamber so that fluid flowed
faster with increasing distances from the chamber walls. A cell
carried in the flow stream attained an equilibrium height, and a
corresponding velocity, based on the balance of dielectrophoretic,
gravitational and hydrodynamic lift forces it experienced. We have
described a theoretical model (Huang et al., 1997) for this system
and have shown that the cell velocity is a function of the mean
fluid velocity, the voltage and frequency of the signals applied to
the electrodes, and, most significantly, the cell dielectric
properties. The validity of the model has been demonstrated using
human leukemia (HL-60) cells subjected to a parallel electrode
array, and the application of the device in separating HL-60 cells
from peripheral blood mononuclear cells has been achieved.
[0270] The operational principle of DEP-FFF may be summarized as
follows: by applying appropriate voltage signals to electrodes,
particles having different dielectric and/or density properties may
be levitated to different heights and thereby caused to move at
different velocities under the influence of the flow profile.
Particles preloaded at the chamber inlet will then exit the chamber
at different times where they can be collected separately. In this
way, the times taken for particles to transit the chamber directly
reflect their dielectric and density properties, and this
dependency can be utilized for particle characterization and
separation. In DEP-FFF, particles are positioned in different
planes throughout the hydrodynamic flow profile above the electrode
surface as a result of the balance between sedimentation, vertical
DEP, and hydrodynamic-lift forces. Thus particles may be eluted in
a continuous fashion at different velocities under the influence of
horizontal fluid drag acting in their respective planes. In this
way, DEP-FFF utilizes the full range of fluid velocities within the
flow profile, and more importantly, exploits the three dimensional
capacity of the separation chamber.
[0271] Material and Methods
[0272] Cells. The human leukemia HL-60 cell line was used as a
model system in this study. Cells were cultured in RPMI 1640 medium
supplemented with 10% fetal bovine serum, 1 mM glutamine and 20 mM
HEPES (Life technologies, Gaithersburg, Md.), 0.5% penicillin and
streptomycin solution (Sigma, St. Louis, Md.), and were maintained
in 75 cm.sup.2 plastic flasks under a 5% CO.sub.2/95% air
atmosphere at 37.degree. C. in a humidified incubator. HL-60 cells
were harvested at a density of 2.times.10.sup.6/ml in the
exponential growth phase by gently rocking the flask 48 h after
seeding. Cell suspensions were found to have >98% viability by
Trypan blue dye exclusion. Cells were harvested from complete
medium by centrifugation at 100 g for 10 minutes and were then
resuspended at a density .about.10.sup.6/ml in isotonic 8.5% (w/v)
sucrose plus 0.3% (w/v) dextrose buffer. The conductivities of the
final suspensions were adjusted with culture medium to a nominal
value of 50 mS/m, and were then measured with a conductivity meter
(EC19101-00, Cole-Parmer Instrument, Chicago, Ill.). Peripheral
blood mononuclear (PBMN) cells were prepared from a buffy coat by
standard density gradient separation. In order to quantify cell
levitation effects, the specific densities of HL-60 cells were
assessed to be 1.071.+-.0.003 g/cm.sup.3 with
centrifugally-generated continuous Percoll density gradients
(Pharmacia, Uppsala, Sweden) calibrated by Percoll density marker
beads. The specific density of the cell suspending medium was
measured as be 1.033 g/cm.sup.3 with a hydrometer (VWR Scientific,
Greenbelt, Md.).
[0273] Electrode chambers. Parallel electrode arrays, shown in FIG.
26, were fabricated using standard photolithography. In brief,
gold-coated (thickness 250 A over a 100 A titanium seeding layer)
glass blanks (Thin Film Technology, Buellton, Calif.) were
spin-coated at 3000 rpm with S1830 photoresist (Shipley,
Marlborough, Mass.) to .about.1 .mu.m thickness. The photoresist
was polymerized by baking on a hot-plate at 110.degree. C. for 1
minute and then exposed to UV light through a positive mask image
(Process Technologies, Oak Creek, Wis.) of the electrode array
using a mask aligner (AB Manufacturing, San Jose, Calif.). The
exposed photoresist was developed with MF351 developer and the
exposed gold and titanium region was then etched. Finally, the
photoresist covering the unetched regions of the electrodes was
removed with acetone. Arrays of parallel electrode elements having
equal widths and gaps of 20 and 50 .mu.m were both used in this
study.
[0274] Chambers of dimensions 200 .mu.m (H).times.25 mm
(L).times.17 mm (W) were constructed of parallel top and bottom
glass plates separated by a Teflon spacer, as shown in FIG. 26. The
arrays of parallel electrodes were on the inner surface of the
bottom plates. Polyethylene tubes (I.D.: 0.87 mm; O.D.: 1.22 mm)
glued into holes drilled through the top glass plate with a diamond
drill allowed for the introduction and removal of cell suspensions
and eluate buffer.
[0275] Cell DEP-FFF kinetics. After introduction of a cell sample
into the chamber inlet, cells were allowed to settle onto the
electrode plane for about 20 s prior to the application of
electrical signals. Sinusoidal voltages between 10 kHz and 1 MHz
and up to 3 V (RMS) from a function generator (HP33120, Hewlett
Packard, Santa Clara, Calif.) were then applied to the electrode
elements through co-axial cables, and fluid flow was started by
pumping the sucrose/dextrose buffer through the inlet port using a
digital syringe pump (Daigger, Wheeling, Ill.) at flow rates of 20,
40 or 80 .mu.l/min. Cell kinetic behaviors were viewed by looking
upwards through the chamber bottom with a Nikon (Melville, N.J.)
TMD inverted microscope equipped with a Hamamatsu (Bridgewater,
N.J.) CCD video camera and recorded on video tape. Long working
length objective lenses of 4.times. to 40.times. provided final
magnifications of between 140 and 1400 on the TV monitor. Cell
velocities were analyzed by measuring the time taken for individual
cells to move at least 120 .mu.m (.gtoreq.3 electrode+gap periods).
In cell levitation experiments, the fluid flow was stopped and
heights of the cells above the electrode plane were measured to an
accuracy .+-.2.mu.m by subtracting the corresponding readings on
the focusing dial when focusing first on the electrode plane and
then on the cells and correcting for the refractive index (1.33) of
the suspending medium.
[0276] Experimental Results
[0277] DEP levitation. FIG. 27 shows the typical frequency
dependency of levitation height for HL-60 cells at a suspension
conductivity of 56 mS/m for an applied voltage of 1.06 V (RMS). As
the frequency of the voltage was increased from 2 to 30 kHz, the
cell levitation height steadily rose. However, increasing the
frequency above 30 kHz, led to a sharp drop off in levitation
height until above 300 kHz the cell became trapped at the electrode
edges. The levitation characteristics of other cell types including
murine erythroleukemia DS19 and human breast cancer MDA-MB-231
cells (data not shown) exhibited qualitatively similar frequency
dependencies except that the sharp transition from cell levitation
to trapping occurred in characteristically different frequency
ranges for each cell type. FIG. 28 shows the typical voltage
dependency of HL-60 levitation at a frequency of 17.8 kHz. Clearly,
the levitation height exhibited a sublinear, monotonic relationship
to the applied voltage.
[0278] DEP-FFF velocity. The effectiveness of applying DEP forces
to control particle velocities in a fluid flow profile was studied
by measuring the velocities of HL-60 cells in a DEP-FFF chamber as
a function of the frequency and magnitude of the signals applied to
the electrode array. It was not possible to measure these
dependencies for single cells due to the impracticality of tracking
them throughout an entire experiment. Instead, the velocities of
about 20 individual cells chosen at random were determined for each
experimental condition. FIG. 29 illustrates the frequency
dependencies of the mean velocity of HL-60 cells for three
different rates of fluid flow. Cells moved faster with increasing
the flow rate, thus at an applied frequency of 17.8 kHz, the mean
velocity increased from 77 (.+-.4.1) to 292 (.+-.32) .mu.m/s as the
flow rate was varied from 20 to 80 .mu.l/min. The overall frequency
dependency did not appear to change with the flow rate, and
application of voltage signals caused HL-60 cells to move at either
higher or smaller velocities. For a flow rate of 20 .mu.l/min, in
the applied frequency range of 10 to 50 kHz cell velocities were
about four times higher than with no field applied. In the narrow
frequency band from 100 to 300 kHz, cell velocities decreased
sharply to those observed with no field applied. At 100 kHz,
individual HL-60 cells exhibited vastly different velocities and
fast-moving cells traveled five times faster than the slowest ones.
Under these conditions, the variance of cell velocities was
.about.30% in contrast to .about.10% for frequencies below 100 kHz.
Increasing the field frequency above 300 kHz resulted in a further
decrement in mean velocity to values much smaller than those
observed in the absence of an electrical field. For example, at 1
MHz, the mean velocities were about 40% and 73% of the zero-field
values for flow rates of 20 and 80 .mu.l/min, respectively. For the
small flow rate of 20 .infin.l/min, some HL-60 cells were even
trapped on electrode edges.
[0279] The voltage dependencies of the mean velocity are shown in
FIG. 30 for HL-60 cells at a fixed frequency of 31.6 kHz for three
different flow rates, and as in FIG. 29, increasing the flow rate
led to aster cell elution. For a given flow rate, the mean velocity
increased steadily with applied voltage.
[0280] Separation of HL-60 cells from peripheral blood mononuclear
(PBMN) cells. Two approaches, namely DEP retention and DEP-FFF,
were used to separate HL-60 cells from a mixture with PBMN cells
using a parallel electrode chamber. The device was the same as in
FIG. 26, except that it had dimensions of 375 .mu.m (H).times.150
mm (L).times.24 mm (W) and electrode widths and spacings of 50
.mu.m. Cell mixtures in the ratio of 1:5 for HL-60:PBMN cells
(predominately lymphocytes) were prepared in isotonic
sucrose/dextrose buffer containing 10.sup.6 HL-60 cells/ml at a
conductivity 10 mS/m. About 250 .mu.l of cell mixture was loaded
into the chamber with a syringe for each separation experiment and
cells were allowed to settle on to the electrode plane for about 20
s. For DEP retention, signals of 0.88 V (RMS) at 50 kHz were then
applied to the electrode arrays while the sucrose/dextrose eluate
buffer was pumped through the chamber at a flow rate of 160
.mu.l/min. All HL-60 cells were trapped at the electrode edges
whilst PBMN cells were carried away with the fluid and were
collected at the chamber outlet. The voltage was then switched off
to release the HL-60 cells which were subsequently collected.
[0281] For DEP-FFF, voltage signals of 25 kHz of 0.88 V (RMS) were
applied to the electrode arrays and an eluate flow in the chamber
was established at a rate 160 .mu.l/min. Almost all the PBMN cells
and HL-60 cells were levitated and were caused to move under the
influence of the fluid. Using the significant difference in cell
size as the basis for identification, it was observed that PBMN
cells were levitated to higher positions and traveled at about
twice as fast as HL-60 cells. Therefore PBMN cells were eluted from
the chamber in about (15 min) half the time (26 min) taken by HL-60
cells and excellent separation was thereby obtained.
[0282] Conclusion
[0283] In this example, an inhomogeneous
dielectrophoretic-levitation force acted to balance the homogeneous
gravitational force, and provided an effective mechanism for
controlling positions of cells and their corresponding velocities
in a fluid flow profile. Cell velocities and their corresponding
transit time across a DEP-FFF chamber directly reflected their
individual dielectric characteristics and may be exploited for both
characterization and separation purposes.
[0284] Using HL-60 cells in thin chambers equipped with parallel
electrode arrays on their bottom walls, we demonstrated the
validity of a theoretical model for DEP-FFF system. The cell
levitation height and corresponding velocity in an eluate flow
profile were shown to be very sensitive to cell dielectric
properties. This ability is significant for biological and clinical
problems where cell subpopulations having subtle differences must
be separated. Finally, we demonstrated that HL-60 cells could be
separated from normal PBMN cells using both DEP-FFF and DEP
retention approaches.
[0285] We found that the electric field distribution critically
determines the sensitivity of the dependence of cell levitation
height, and thus traveling velocity, on cell dielectric
properties.
EXAMPLE 6
[0286] Purging Human Breast Cancer Cells from CD34+ Stem Cells by
Dielectrophoretic Field-Flow-Fractionation
[0287] In this Example, the purging of cultured human breast cancer
MDA-435 cells from CD34.sup.+ cells using a dielectrophoretic-field
flow fractionation (DEP-FFF) technique that separates cells
according to cell dielectric properties is demonstrated. An array
of interdigitated microelectrodes lining the bottom surface of a
thin chamber was used to generate dielectrophoretic forces that
levitated the cell mixture in a fluid flow profile. CD34.sup.+ stem
cells were levitated higher, were carried faster by the fluid flow,
and exited the separation chamber earlier than the cancer cells.
Using on-line flow cytometry, efficient separation of the cell
mixture was observed in less than 12 min, and CD34.sup.+ stem cell
fractions with a purity >99.2% were obtained. The method of
dielectrophoretic field-flow-fractionation is potentially
applicable to many biomedical cell separation problems including
microfluidic-scale diagnosis and preparative-scale purification of
cell subpopulations.
[0288] Several important features of this Example include the
following. First, there were two outlet ports on the chamber top
and bottom plates. The separated cells exited the chamber from the
bottom outlet port while the majority of the carrier medium exited
the chamber from the top outlet port. Secondly, the separated cells
were detected by on-line flow cytometry. Because of the two outlet
port arrangement, the fluid pressure and flow rate at the flow
cytometry were decreased but the concentration of the cells at the
flow cytometry was increased compared with typical one outlet port
arrangement such as those shown in FIGS. 13 and 26. Furthermore,
this allows the separation being operated at relatively higher flow
rates, that are not limited by the maximum flow rate at the flow
cytometer. Thirdly, two separation protocols , namely,
trap-and-release and sweep-frequency DEP-FFF have been explored,
and their separation performance in terms of purity, speed and
recovery rate have been examined. Each of the separation protocol
consisted of several segments during which electrical signals of
different amplitude, frequency or waveforms were applied to the
microelectrode array.
[0289] Material and Methods
[0290] Cell Preparation. MDA-435 cells, originally derived from a
pleural effusion of a patient with metastatic breast cancer
(Cailleau et al., 1978; Zhang and Fidler, 1991), were maintained
under standard tissue culture conditions in minimum essential/F12
medium containing 10% fetal bovine serum (FBS), 1 mM glutamine, and
20 mM HEPES, plus 0.5% penicillin and streptomycin. The cells were
harvested at 80% confluence 48 hr after seeding by a brief exposure
to 0.25% trypsin/0.02% EDTA, washed once and re-suspended in a
isotonic 8.5% (w/w) sucrose plus 0.3% (w/w) dextrose buffer at a
concentration of 6.times.10.sup.6 cells /mL. The electrical
conductivity of the sucrose buffer was adjusted to 10 mS/m with
RPMI 1640 medium.
[0291] Mobilized peripheral blood samples were collected from
leukapheresis products of patients after mobilization using rhG-CSF
(Neupogen, Amgen Corp., Thousand Oaks, Calif.) at a dose of 5
.mu.g/kg subcutaneously twice daily. Leukapheresis were performed
using the COBE Spectra Version 4.7 cell separator (COBE BCT, Inc.,
Lakewood, Colo.) for mononuclear collection. CD34.sup.+ cells were
then obtained using magnetic-activated cell-sorting system with
CD34 isolation kit according to the protocol provided by the
company (Miltenyi Biotech). The purified CD34.sup.+ (>99%) cells
were washed once and re-suspended in a PBS buffer containing 2%
FBS, 0.1% sodium azide at 2.times.107 cells/ml. For flow cytometry
detection, the CD34.sup.+ cells were stained with phycoerythrin
(PE)-conjugated CD34 antibodies (anti-HPCA-2, Becton Dickinson, San
Jose, Calif.) by adding 20 .mu.L antibody to every 50 .mu.L
cell-suspension and incubating the mixture for 30 min at 4.degree.
C. in dark. The labeled CD34.sup.+ cells were then washed once and
re-suspended in above sucrose buffer. The final cell mixtures were
prepared by adding MDA-435 to CD34.sup.+ cells in the sucrose
buffer for a total concentration of 6.times.10.sup.6 cells /ml at a
ratio of 1:1 for MDA-435 to CD34.sup.+ cells.
[0292] DEP-FFF System Setup. The experimental DEP-FFF used in this
Example is shown in FIG. 31. Interdigitated microelectrodes, 50
.mu.m in width and gaps, were fabricated on 50.times.50 mm glass
substrates using standard photolithography. Eight electrodes were
glued end-to-end onto a supporting glass plate to form one
electrode plate. The chamber was constructed by sandwiching a
Teflon spacer (H 0.42.times.W 50.times.L 400 mm) between a top
glass plate and the bottom electrode plate with 36 Nylon
screw-clamps. The spacer was cut to provide an open channel with
dimensions of 388 mm from tip to tip and 25 mm in width except at
the tapered ends. The microelectrodes, each having two 4-mm wide
electrical conductor buses along the edges, were connected in
parallel to a lab-build power amplifier. The voltage signals were
monitored on an oscilloscope. The top and bottom plates were
drilled with 0.0625 in-diameter holes to fit inlet and outlet
tubing at positions coincident with the points of the tapered ends
of the chamber. A 5 cm length of PEEK tubing, having a void volume
of 3 .mu.L, served as the inlet connection between the chamber and
an injection valve equipped with a 50 .mu.L loop. A digital syringe
pump was used to provide continuous flow of carrier medium through
the chamber. At the outlet end, the cells exited the chamber
through the tubing fitted to the bottom plate and were fed to a
flow cytometer (BRYTE HS, Bio-Rad, Hercules, Calif.) for detection.
To reduce the fluid pressure to the flow cytometer, a second
syringe pump was connected to the tubing from the top-plate hole
and was operated to pull 95% of the fluid out of the chamber.
[0293] DEP-FFF Operation Protocol. The DEP-FFF chamber was first
loaded with the sucrose buffer. The cell mixture sample was then
introduced into the inlet port of the chamber through the injection
valve, as described previously (Wang et al., 1998). Total injection
volume was 60 .mu.L. Voltage signals (4 V p-p) at an appropriate
frequency were applied to the electrodes during the sample
introduction so that cells would be levitated in the chamber by the
DEP forces, preventing possible adherence of the cells to the
bottom surface of the chamber. After injection, cells were allowed
5 min to attain equilibrium heights at which the DEP and
gravitational forces acting on them were balanced. Flow of the
carrier medium at the rate of 2 mL/min through the chamber was then
started using the syringe pump, and because of the geometrical
characteristics, a parabolic hydrodynamic flow profile was
established inside the chamber. Under the influence of the fluid
drag, cells were transported at different velocities according to
their relative positions in the flow profile. Cells exiting the
chamber were detected by the flow cytometry. Four-parameter
measurements were performed on individual cells, including time,
fluorescence (PE filter set), and forward and side size
scatter.
[0294] Two protocols of DEP field application were developed for
the separation of MDA-435 and CD34.sup.+ cell mixtures. In the
trap-and-release DEP-FFF protocol, the DEP field was first applied
for certain time at a frequency to trap MDA-435 cells at the
electrodes but simultaneously to levitate and elute CD34.sup.+
cells. The field was then changed to a frequency to release and
elute the previously trapped MDA-435 cells. In the second protocol,
the frequency of the DEP field was swept over a range repetitively
to maximize the differences in equilibrium levitation heights (and
thus the velocities) between MDA-435 and CD34.sup.+ cells. After
certain time, a single-frequency DEP field was applied to speed up
the elution of all the cells.
[0295] Result
[0296] Cell DEP-FFF Responses. To determine separation conditions,
DEP-FFF responses of MDA-435 and CD34.sup.+ cells were studied
separately as a function of the frequency of the applied field.
FIGS. 32-33 shows the DEP-FFF fractograms for the two cell types at
different frequencies. For MDA-435 cells at 10 kHz, cell elution
spanned between 3 and 8 min and peaked at 6 min. With increasing
frequency, the response was characterized by rapid broadening of
the fractogram with no obvious elution peak observed at frequencies
above 15 kHz. The total eluted cell number decreased at 20 kHz,
indicating that some cells were trapped in the chamber by DEP
forces. The cell trapping was confirmed by microscopic inspection
of the chamber and by detecting cells when the frequency was
switched back to 10 kHz. In contrast to MDA-435 cells, CD34.sup.+
cells exhibited DEP-FFF fractograms having single and narrow peaks.
Between 10 and 40 kHz, CD34.sup.+ cells eluted the chamber from 4
to 7 min with peaks at .about.4.5 min. With increasing frequency,
the elution fractogram gradually broadened with the peak position
shifted to -8 min at 60 kIIz.
[0297] To quantify these differences between MDA-435 and CD34.sup.+
cells, two parameters were defined to describe the elution
characteristics of the cells at different frequencies, namely, the
elution time and the elution peak width. These parameters were
determined from cell fractograms based on the integral of cell
number with time, as described previously in Yang et al., 1999a.
FIG. 34 shows the frequency dependency of these parameters for
MDA-435 and CD34.sup.+ cells, providing the information for
choosing the DEP-field frequency range to promote the separation of
these two types of cells. Both elution time and elution peak width
increased much more rapidly with frequency for MDA-435 cells than
CD34.sup.+ cells, reflecting significant differences in their
DEP-FFF responses.
[0298] Separation of MDA-435 cells from CD34.sup.+ cells. Two
separation protocols, namely, trap-and-release and sweep-frequency
DEP-FFF, were developed. Following the introduction to the chamber,
the cells were allowed 5 min to attain equilibrium height positions
under a 10 kHz DEP-field prior to the initiation of the fluid-flow.
In the trap-and-release protocol, as the fluid-flow started, the
DEP-field frequency was changed to 40 kHz at which CD34.sup.+ cells
were levitated by DEP forces and transported under the influence of
the fluid-flow, simultaneously, MDA-435 cells were trapped by DEP
forces. This field condition was maintained for 7 min during which
majority of CD34.sup.+ cells eluted the chamber. The DEP-field
frequency was then switched to 5 kHz to allow the levitation and
elution of the previously trapped MDA-435 cells. A typical
fractogram for this separation is shown in FIG. 35, where the first
and second peak corresponded to CD34.sup.+ and MDA-435 cells, as
identified by the fluorescence measurement and size-scatter in flow
cytometry. Flow cytometry detection further indicates that the
CD34.sup.+ peak between 4.5 and 5.5 min contained 99.5% CD34.sup.+
cells, and 0.5% MDA-435 cells. MDA-435 peak between 9 and 12 min
contained 96% MDA-435 cells and 4% CD34.sup.+ cell. In the
sweep-frequency protocol, the DEP-field frequency was swept between
15 and 40 kHz repetitively at a 5 sec period when the fluid-flow
was commenced. The sweep-frequency field was applied for 7 min.
during which CD34.sup.+ cells were levitated and majority of them
eluted the chamber whilst MDA-435 cells were levitated slightly and
moved slowly through the chamber. As in the trap-and-release
protocol, the frequency was then switched to 5 kHz to elute all the
remaining MDA-435 cells. A fractogram for the separation is
depicted in FIG. 36 showing that the separation was completed
within 12 min and the elution-peak difference between the two cell
types was >5 min. Flow cytometry detection further indicates
that the CD34.sup.+ peak between 3 and 4 min contained 99.2%
CD34.sup.+ cells, and 0.8% MDA-435 cells. MDA-435 peak between 7
and 10 contained 99% MDA-435 cells and 1% CD34.sup.+ cell.
[0299] A contour plot fluorescence vs. time for all the cells, as
measured by the flow cytometry, is shown in FIG. 37. Clearly,
CD34.sup.+ cells, having large fluorescence signals because of
staining with CD34-PE, exited ahead of the unstained MDA-435 cells.
The small cluster of unstained cells that exited before CD34.sup.+
cells were dead cell debris (based on smaller light scatter).
[0300] Discussion
[0301] Dependency of Cell DEP-FFF Responses on Their Membrane
Dielectric Properties. According to DEP-FFF theory, the balance
between DEP levitation force and gravitational force positioned
cells at equilibrium heights in the fluid-flow profile, and thus
determined their velocities and elution times (Huang et al., 1997;
Wang et al, 1998; Yang et al., 1999a). Cell elution time is a
function of the voltage of the applied DEP-field, of an electrode
geometry factor, and of cell dielectric polarization factor
.alpha..sub.DEP and cell density. Since MDA-435 and CD34.sup.+
cells have similar cell densities, the difference in frequency
dependencies of elution time (FIG. 34) between MDA-435 and
CD34.sup.+ cells reflected their different .alpha..sub.DEP values.
For CD34.sup.+ cells, the gradual-increase in elution time in the
frequency range of 10 to 60 kHz indicated a steady-drop in the
polarization factor .alpha..sub.DEP. On the other hand, elution
time for MDA-435 cells increased rapidly with frequency, suggesting
a sharp decrease in .alpha..sub.DEP.
[0302] To confirm these, cell dielectric properties were determined
from electrorotation (ROT) measurements. In ROT, cells were
subjected to a rotating electrical field and were induced to rotate
as a result of the interaction between field-induced polarization
and the rotating field. Cell rotational rate was measured as a
function of the field frequency (FIG. 38A) and the spectra were
fined with appropriate models to derive cell dielectric parameters.
The frequency dependency of .alpha..sub.DEP, calculated using the
mean dielectric parameters (Table 1), is given in FIG. 38B for
MDA-435 and CD34.sup.+ cells. Between 5 and 40 kHz, REP varied from
-0.5 to -0.3 for CD34.sup.+ cells. On the other hand,
.alpha..sub.DEP changed from -0.4 to 0 between 5 and 15 kHz for
MDA-435 cells. These results are in general agreement with the
above discussion regarding the frequency dependency of
.alpha..sub.DEP for the two cell types.
[0303] It is well established that the dielectric polarization
factor .alpha..sub.DEP below several hundred kHz is determined by
the cell membrane electrical properties (Wang et al., 1994; Pethig
and Kell, 1987; Arnold and Zimmerman, 1988). Indeed, the observed
difference in .alpha..sub.DEP between MDA-435 and CD34.sup.+ cells
is related to their different membrane capacitances (10.7 and 23.0
mF/m.sup.2 for CD34.sup.+ cells and MDA-435, respectively). Thus
the DEP-FFF separation of MDA-435 and CD34.sup.+ cells described
here exploited the difference between their membrane dielectric
properties.
[0304] Another important difference in DEP-FFF responses between
the two cell types is the large elution-peak-width for MDA-435
cells at 20 kHz (FIG. 34), as evidenced by the broad elution
fractogram (FIGS. 32-33). The elution peak widths depended on these
factors. The elution time for individual cell depended on its
velocity and travel distance through the DEP-FFF chamber. Since it
was not possible to ensure that all the cells were positioned the
same distance from the chamber outlet port during sample loading, a
small spread (<10%) in elution time was expected even for the
cells having the same velocity. Secondly, different cells of the
same type exhibited different velocities because of inherent
population heterogeneity in the cell dielectric parameters (see
Table 1 for-standard deviations of dielectric parameters). Indeed,
at 15 kHz, .alpha..sub.DEP for MDA-435 cells was around zero, and
small variations in .alpha..sub.DEP could lead to significant
difference in cell velocity (and thus elution time) between
individual cells because of the extreme sensitivity of cell
velocity for small .alpha..sub.DEP values. Thus, the widespread of
the elution fractogram for MDA-435 cells at 15 kHz was related to
the heterogeneity in their dielectric properties.
[0305] Flow Cytometry Detection of Cells. Individual cells exiting
the chamber were analyzed using a flow cytometer and cell elution
fractogram was constructed. To operate DEP-FFF separation at a high
flow rate (>0.1 mL/min, the maximum flow rate for the cytometer)
and achieve an increased cell concentration at the cytometer, a
second outlet port was introduced at the top plate of the chamber
to elute cell suspension buffer. In such a configuration, it is
important to ensure that no cells elute from the top outlet port by
varying the flow rates at the inlet and outlet ports. Under a
typical operating condition, a syringe pump drove the fluid flow in
the chamber from the inlet port at a flow rate 2 mL/min. A second
syringe pump was operated at 1.8 mL/min, pulling the fluid out of
the chamber from the top outlet port. Thus, only 10% of the fluid,
corresponding to a height of 80 .mu.m in the parabolic flow profile
from the chamber bottom, was eluted into the cytometer. The maximum
equilibrium heights for individual MDA-435 and CD34.sup.+ cells
measured under light microscope for a slow rate (10 .mu.L/min) were
less than 65 .mu.m. Thus, no cells exited from the top outlet port.
This was further confirmed experimentally, as the total cell number
detected by the cytometer for such a 10% elution was the same as
that for a 20% elution.
[0306] Separation Approaches. Separation between cell populations
using DEP-FFF approach exploits differential cell velocities.
According to FIG. 32, all the CD34.sup.+ cells eluted the DEP-FFF
chamber in <10 min at 40 kHz, whilst <10% of MDA-435 cells
eluted the chamber at the time 12 to 18 min. Thus, separation of
the two populations can be achieved at 40 kHz. Indeed, as shown in
FIG. 38B, the DEP mean crossover frequencies were found to be 10
kHz and .about.60 kHz for MDA-435 and CD34.sup.+ cells,
respectively. Thus, at 40 kHz, MDA-435 cells experienced positive
DEP forces, tending to trap them at the electrode edges, whilst
CD34.sup.+ cells were still levitated by negative DEP forces and
released. After all the CD34.sup.+ cells were eluted, the frequency
of the voltage signals were changed to 5 kHz to cause a fast
elution of the MDA-435 cells in the chamber. Thus, the inventors
effectively programmed the DEP field condition in order to improve
the separation performance. And as MDA-435 cells were "trapped" and
"released" in the two time segments of the DEP field application,
the inventors termed this approach trap-and-release protocol.
[0307] While the trap-and-release protocol achieved a desired
separation between the two populations, a disadvantage associated
with this is that during the "trap" voltage segment, many MDA-435
cells were trapped on the electrode edges. Such a direct
cell-electrode contact may not be ideal for certain applications.
For example, cells may nonspecifically bind to the electrodes, and
are not eluted even if a negative DEP force is applied. Also,
MDA-435 cells may experience large AC field as a result of a direct
cell-electrode contact. Furthermore, because of cell heterogeneity,
some MDA-435 cells adhered to electrodes whilst others were caused
to move under the fluid flow. Thus a large spread-out may occur
during the application of the "trap" voltage segment. Such a spread
out would result in a broadening of the final elution peak for
MDA-435 cells.
[0308] For these reasons, a sweep-frequency protocol was developed.
Thus, a signal having a fixed voltage and linearly sweeping
frequency (10 to 40 kHz) was applied. Under the application of such
a DEP field, CD34.sup.+ cells were eluted very similarly to the
condition of a fixed 10 kHz. On the other hand, such a signal,
effectively averaging DEP force across the sweep-frequency-band,
should greatly reduce the number of the MDA-435 cells being trapped
at the electrodes, and allow most MDA-435 cells slightly levitated
and moved slowly under the influence of the fluid flow.
Furthermore, because of the force averaging, large differences in
kinetic responses between individual MDA-435 cells observed during
single "trap" frequency application should be reduced, leading to a
small spread out. After all the CD34.sup.+ cells were eluted, the
voltage signals were switched to a lower frequency, at which
MDA-435 cells were levitated and eluted (with tight and clean
elution peaks).
1TABLE 1 Dielectric Parameters for MDA-435 and CD34+ Cells Cell
Type C.sub.specific,mF/m.sup.2 .sigma..sub.int, S/m
.epsilon..sub.int MDA-435 23.0 .+-. 7.1 0.55 .+-. 10.10 107.0 .+-.
29.5 CD34+ 10.2 .+-. 1.5 0.71 .+-. 0.11 141.2 .+-. 28.0
EXAMPLE 7
[0309] Dielectrophoretic field-flow-fractionation (DEP-FFF), which
exploits differences in cell dielectric properties, was applied to
several clinically-relevant cell separation problems. These
included the purging of human breast cancer cells from normal
T-lymphocytes, the separation of the major leukocyte
subpopulations, and the enrichment of leukocytes from blood. Cell
separations were achieved in a thin chamber equipped with a
microfabricated, interdigitated electrode array on its bottom wall
that was energized with AC electric signals. Cells were levitated
by the balance between DEP and sedimentation forces to equilibrium
heights that were dependent on cell density and dielectric
properties. When carrier medium was flowed through the chamber, the
resulting flow velocity profile transported cells that had been
levitated to different heights at different velocities and thereby
separated them. Cell separations achieved by DEP-FFF were evaluated
by on-line flow cytometry, revealing high separation performances.
For example, for a starting mixture of 2:3 for breast cancer cells
: T-lymphocytes, DEP-FFF separation produced fractions of the two
populations having purities of 99.2% and 92%, respectively. This
bulk-separation technique adds dielectric properties to the catalog
of physical characteristics that can be applied to cell
discrimination. It is applicable not only to existing clinical and
biomedical cell separation problems but also to the sample
preparation needs of microfluidic devices for diagnostic and
environmental detection purposes.
[0310] Materials and Methods
[0311] Cell preparation. Human breast cancer MDA-435 cells were
cultured in MEM supplemented with 10% fetal bovine serum under
standard tissue culture conditions (Becker 1995; Wang et al 1994).
Leukocyte subpopulations (T-, B-lymphocytes, monocytes and
granulocytes) were derived from buffy coat preparations (Gulf Coast
Regional Blood Bank, Houston, Tex.) using density-gradient
centrifugation, MACS sorting and erythrocyte lysis, as described
previously (Yang et al, 1999a). To allow for flow cytometry
detection of the cells eluted from DEP-FFF chamber, B-lymphocytes,
and monocytes were labeled with PE- or FITC-conjugated CD3, and
CD14 antibodies (Becton Dickinson, San Jose, Calif.), respectively,
by incubating the cell suspension with the antibody solution
(volume ratio 5:2) for 30 min at 4.degree. C. in the dark. Labeled
cells were then washed once and re-suspended in an isotonic buffer
(8.5% w/v sucrose plus 0.3% w/v dextrose) buffer having an
electrical conductivity of 10 mS/m, which was adjusted by adding
culture medium. Appropriate cell populations were then mixed in the
sucrose buffer. Cell mixtures included MDA-435 cells with
T-lymphocytes, B-lymphocytes with monocytes. The final cell
concentration was .about.1.2.times.10.sup.6/mL.
[0312] For DEP-FFF enrichment of leukocytes from blood, human blood
was taken from healthy volunteers and stained with PE-CD45 antibody
solutions to label leukocytes. The cell samples were then diluted
in the isotonic sucrose buffer to achieve the final cell
concentration .about.5.times.10.sup.6/mL.
[0313] DEP-FFF system setup. The DEP-FFF chamber and experimental
setup are shown in FIG. 39. Interdigitated microelectrodes having
50 .mu.m width and spacing were fabricated on 50.times.50 mm glass
substrates using standard photolithography. A Teflon spacer, which
was cut in the center to provide a separation channel (H
0.42.times.W 25.times.L 388 mm), was sandwiched between a top glass
plate and a long electrode plate (consisting of eight electrode
substrates in series). The microelectrodes were connected in
parallel to a lab-built PA05-based power amplifier (Apex
Microtechnology, Tucson, Ariz.). The top and bottom plates were
drilled with 1.6-mm-diameter holes to fit inlet and outlet PEEK
tubing (0.0625 in. OD, 0.010 in. ID, Upchurch Scientific, Oak
Harbor, Wash.). An infusion syringe pump (Daigger, Wheeling, Ill.)
was connected to the chamber through an injection valve (Rheodyne
Model 7010, Rheodyne, Cotati, Calif.) equipped with a 50-.mu.L loop
to provide continuous flow of the carrier medium in the chamber. A
second syringe pump was connected to the top outlet port of the
chamber and was operated to withdraw the cell-free portion of the
carrier fluid that constituted up to 95% of the total flow through
the chamber. Cells exited the chamber through the bottom outlet
port and were fed directly to the injection needle of a flow
cytometer (BRYTE HS, Bio-Rad, Hercules, Calif.) for detection,
bypassing the normal cytometer sample handling fluidics.
[0314] DEP-FFF Operation Protocol. The DEP-FFF chamber was first
loaded with sucrose buffer. An aliquot of the cell mixture (50
.mu.L) was then introduced into the inlet port of the chamber
through the injection valve, as described previously (Wang et al,
1998). A DEP signal (4 V p-p) at 10 kHz was applied to the
electrodes during sample injection so that cells were levitated in
the chamber by DEP forces and thereby prevented from adhering to
the bottom surface of the chamber. Different operation protocols
were then applied for separating different cell mixtures after
sample injection. The protocols for separating MDA-435 cells from
T-lymphocytes consisted of the following steps:
[0315] (1) Prior to the application of the fluid flow, a 10 kHz
field was applied for 10 min to allow the cells to reach their
equilibrium height positions.
[0316] (2) A flow velocity profile was established in the chamber
by starting the injection and withdrawal syringe pumps at rates of
2 and 1.6 mL/min, respectively. The DEP field was switched to 40
kHz (or swept between 15 and 35 kHz at a cycle period of 5 see).
This condition was maintained for 5 or 7 min so that all the
T-lymphocytes were eluted from the chamber and identified and
counted by the on-line flow cytometer.
[0317] (3) The DEP field was changed to 5 kHz so that the
previously retained MDA-435 cells were now levitated, eluted from
the chamber and detected by the flow cytometer.
[0318] For separating other cell mixtures, different DEP field
conditions were applied during the 2.sup.nd segment of the protocol
and are summarized in Table 1.
[0319] Results
[0320] Separation of breast cancer cells from normal T-lymphocytes.
The isolation and enumeration of cancer cells circulating in
peripheral blood is potentially an important screening tool for
early detection of cancer and allows for the genetic and
biochemical characterization of cancer cells for diagnosis and
prognosis. Current methods, which can detect one cancer cell per
.about.10.sup.6 mononuclear cells, involve separation of
mononuclear cells from the blood, enrichment of cancer cells, and
finally flow cytometric or PCR detection. To demonstrate the
usefulness of the dielectrophoretic approach to this problem, we
investigated the DEP-FFF separation of cultured, human breast
cancer MDA-435 cells from T-lymphocytes, which constitute
approximately 80% of peripheral blood mononuclear cells.
[0321] Cell dielectric properties and DEP behaviors depend
sensitively on the frequency of the applied electrical field
(Becker et al, 1995; Fuhr et al, 1996; Pethig, 1996; Gascoyne et
al, 1997). Therefore, to establish suitable cell separation
conditions, we measured the DEP-FFF responses of breast cancer
cells and T-lymphocytes separately as a function of frequency. FIG.
40 shows cell elution fractograms for the two populations. While
both cell types exhibited narrow, single elution peaks at 5 kHz,
the fractogram for the breast cancer cells quickly broadened as the
field frequency was increased above 10 kHz. At 20 kHz, only
.about.35% of the breast cancer cells were eluted; the rest were
retained in the chamber by positive DEP forces. In contrast, the
elution peak width for T-lymphocytes changed more gradually, and
almost all were eluted at frequencies up to 50 kHz. These
differences suggested that the two cell types should be separable
at frequencies between 20 and 50 kHz.
[0322] To examine this possibility under demanding conditions, we
prepared a cell mixture at a ratio of 2:3 (breast cancer: T
lymphocyte) at a concentration of 1.2.times.10.sup.6 cells/mL and
applied DEP field frequencies around 30 kHz. As the DEP-FFF
fractograms in FIG. 41 show, the mixture was separated in 11 min
and purities above 92% for the two populations, and a total cell
recovery of .about.70% were achieved (Table 2). The separation
occurred because at frequencies around 30 kHz, T-lymphocytes were
levitated well above the chamber bottom wall by DEP forces and
transported under the influence of the fluid flow. Meanwhile,
breast cancer cells were either barely levitated (and, therefore,
carried slowly by the slower-moving fluid near the chamber bottom
wall) or were trapped at the electrodes and immobilized by positive
DEP forces. After T-lymphocytes were eluted from the chamber, DEP
field was switched to 5 kHz and breast cancer cells were then
levitated and quickly eluted.
[0323] Separation of the major leukocyte subpopulations.
Purification of major leukocyte subpopulations (i.e. T- and
B-lymphocytes, monocytes and granulocytes) is important in many
clinical and biomedical applications. The differential diagnosis of
bacterial, viral and parasitic infections, and of mononucleosis and
leukemia, for example, require the enumeration of leukocyte
subtypes. In research, purified leukocyte sub-populations are
required for the study of molecular signaling between leukocyte
subpopulations by the interleukins and for the analysis of
immunological capacities of distinctive cell types.
[0324] The major leukocyte subpopulations have significantly
different dielectric properties (Yang et al, 1999b). To determine
the feasibility of purifying them by DEP-FFF, we mixed T- (or B-)
lymphocytes with monocytes, T- (or B-) lymphocytes with
granulocytes, and monocytes with granulocytes. To illustrate the
DEP-FFF separations, we will consider the mixture of B-lymphocytes
and monocytes, which was typical of all the blood cell mixtures. To
obtain these cells, B-lymphocytes and monocytes were purified from
a leukocyte-enriched buffy-coat preparation using the MACS method
(Yang et al, 1999b). The DEP-FFF characteristics of the individual
cell subpopulations suggested that a swept frequency field between
25 and 45 kHz would be a suitable separation condition and this was
applied for DEP-FFF to a cell mixture (1:2 for monocytes:
B-lymphocytes) at a total concentration of 1.2.times.10.sup.6 cells
/mL. A typical DEP-FFF fractogram is shown in FIG. 42 under these
conditions. DEP-FFF separation resulted in monocyte and
B-lymphocyte fractions having purities of 94% and 92%, respectively
(Table 2).
[0325] Enrichment of leukocytes from blood. Separation of
erythrocytes and leukocytes from blood, typically performed using
centrifugation or filtration, is a basic requirement for many
biomedical procedures such as erythrocyte transfusion. We therefore
attempted to use DEP-FFF to enrich leukocytes (and erythrocytes)
from blood diluted 1:1000 in a sucrose buffer. Based on the DEP-FFF
characteristics of erythrocyte and leukocyte subpopulations, a DEP
field at 10 kHz was applied for DEP-FFF. A typical fractogram is
shown in FIG. 43, leukocytes were enriched 35-fold and erythrocyte
purity was increased from 99.8% in the blood sample to >99.99%
in the final erythrocyte fraction. In this case, we believe that
the differential cell velocities exploited in the DEP-FFF
separation arose not only from levitation height differences
between leukocytes and erythrocytes but also from differences in
cell-hydrodynamic interactions caused by different cell shapes
since erythrocytes are double-discoid while leukocytes are
generally round.
2TABLE 2 DEP-FFF separation performance summary Experimental
systems Purify after Total-cell Separation (Protocol).sup.a Cell
types separation.sup.d recovery.sup.f time (min) MDA-435 vs
T-lymphocytes (2:3).sup.b MDA-435 99.2% 69% 11 (40k Hz, 5 min;
2/1.6 mL/min).sup.a T-lymphocytes 92% MDA-435 vs T-lymphocytes
(2:3).sup.b MDA-435 98% 75% 11 (15-35 kHz, 5 min; 2/1.6
mL/min).sup.a T-lymphocytes 92% Monocytes vs B-lymphocytes
(1:2).sup.b Monocytes 94% 73% 15 (20-40 kHz, 7 min; 2/1.9
mL/min).sup.a B-lymphocytes 92% Enrichment of leukocytes from blood
Leukocytes 5%.sup.e 55% 25 (10 kHz, 25 min; 0.5/0.4 mL/min).sup.c
Erythrocytes 99.99% .sup.aSingle frequency or swept frequency
(cycle period 5 s) of DEP-field used during the 2.sup.nd segment of
the protocol (see Materials and Methods). The two flow rates
correspond to the infusion and withdrawal syringe pumps at the
chamber inlet and outlet ends, respectively. .sup.bThe ratio
between the two cell populations in initial mixtures. .sup.cThe
enrichment of leukocytes from blood used a DEP field at 10 kHz for
25 min. .sup.dPurity after separation was determined by the flow
cytometry for the corresponding elution peaks. .sup.eLeukocyte to
erythrocyte ratio was increased from 1:700 to 1:19 after DEP-FFF
enrichment. .sup.fCell recovery was defined as the ratio of the
total cell number detected by flow cytometry at the DEP-FFF chamber
outlet end to the targeted total cell number that was calculated
based on the cell concentration and the injection loop volume.
[0326] Discussion
[0327] Cell dielectric separation criteria. The DEP-FFF method
exploits cell dielectric and density properties as the basis of
separation. In the frequency range used for the separations
described here, cell dielectric properties are determined by the
extent to which the applied field penetrates the cell interior via
the capacitance of the plasma membrane. At low frequencies, the
field penetration is small and the entire applied field appears
across the poorly-conducting membrane. Thus, cells are less
polarizable than the suspending medium and tend to be repelled from
strong electrical field regions by negative DEP forces. In our
electrode configuration, this causes levitation. At higher
frequencies, the field penetrates the plasma membrane into the cell
interior, which is more conductive than the suspending medium under
our conditions. Cells become more polarizable than the medium, the
DEP forces become positive, and cells are attracted to the strong
field regions and immobilized at the electrodes (Becker et al,
1995; Fuhr et al, 1996; Kaler & Jones, 1990; Pethig 1996). Take
breast cancer MDA-435 cells and T-lymphocytes as an example.
Because of differences in cell membrane morphology and composition,
T-lymphocytes have a mean membrane capacitance approximately half
that of breast cancer cells (Gascoyne et al, 1997; Huang et al,
1996; Huang et al, 1999), and the frequency range in which
sufficient field penetration occurred for the DEP force to become
positive was therefore higher for T-lymphocytes. Thus in the
frequency range from 15 to 35 kHz, T-lymphocytes were strongly
levitated by negative DEP forces and were transported quickly
through the chamber under the influence of the fluid flow. Breast
cancer cells, however, were only weakly levitated into the slow
moving fluid close to the chamber bottom wall or were trapped by
positive DEP forces at the electrodes. The differential velocities
for T-lymphocytes and the breast cancer cells resulted in the
DEP-FFF separation of the two populations. It follows that cell
membrane dielectric properties, determined by membrane composition
and morphological structures (Wang et al, 1994; Huang et al, 1999),
were DEP-FFF separation criteria exploited here.
[0328] Over the last 15 years, the dielectric properties of many
normal and cancerous cell types have been shown to be distinct and
to depend not only on cell type but also on the biological state.
For this reason, we have introduced the concept of cell dielectric
phenotype (Becker et al, 1995; Gascoyne et al, 1997). The
dielectric phenotypes of T-lymphocytes allow them to be
distinguished from breast cancer cells, monocytes and granulocytes,
and leukemia cells can be distinguished from normal leukocytes.
Other examples include significant membrane dielectric alterations
accompanying temperature-sensitive transformation of rat kidney
cells (Huang et al, 1996), drug-induced differentiation in leukemia
cells (Wang et al, 1994), mitogenic stimulation of human
lymphocytes (Huang et al, 1999), fertilization of rabbit oocytes
(Arnold et al, 1989), and changes in cell environment such as
exposure to heavy metals (Arnold et al, 1986), organic toxins
(Arnold, 1988), or hypo-osmotic media (Sukorukov, 1993). These
examples suggest that by exploiting such dielectric phenotypes, the
DEP-FFF technique may be applied for discrimination,
characterization and separation of cell subpopulations in many
biomedical problems.
[0329] It is contemplated that the apparatus and methods according
to the present invention may be used for cell or particle
characterization, as a diagnostic tool to identify, for example,
cancer cells or other cells that are desired or of interest to the
clinician, and as a therapeutic tool to purge a patient sample of
undesired cells or other particle.
[0330] For example, the methods according to the present invention
may be used to characterize the physical properties of an unknown
particulate matter. A sample including an unknown biological or
organic or mineral sample may be input into the chamber and
separated according to the procedures set forth above. Following
separation and removal of extraneous particles, the unknown
particle may be collected at an output port of the chamber. The
particle can then be analyzed using standard particle
characterization techniques known in the art, such as those used in
diagnostic microbiology and in histology, for example, electron
microscopy. After determining characteristics that are unique to a
particle, an investigator may then compare these characteristics to
the known characteristics of a particle. Therefore, the researcher
may determine whether the unknown particle is the same as a known
particle, or whether it has similar properties.
[0331] In addition, the invention contemplates the characterization
of known particles, which may then be used as a reference tool for
determining unknown particles based on similar trapping
frequencies, voltages, flow rates, and other parameters set forth
above. The sample may be introduced into the chamber of the present
invention and then be subjected to the separation methods detailed
above. By performing these separation techniques, the trapping
frequency and release frequency of the particle can be determined.
These values are then useful in comparing similar parameters of an
unknown sample to this known sample. Certain clinical applications
requiring separation of a known particle from an unknown particle
would require such values to complete the methods of
separation.
[0332] A clinical application of the present invention would be to
use the present apparatus and methods as a diagnostic tool to
screen unknown samples for the presence or absence of various cell
types. First, as set forth previously, a patient's sample may be
placed in the apparatus, and various cell types may be separated
based on previously determined parameters or characteristics. These
cells may include cancer cells, or cells infected with bacteria,
viruses, protozoans, or parasites, bacteria, viruses protozoans, or
they may include cells that are deficient in certain enzymes or
cell organelles, altered biopsies, plaques and scrape tests
including Pap smears and so forth. Thus, it is well within the
scope of the invention to separate all types of particles that have
differential sedimentation rates in a fluid stream, based on size,
density, dielectric strength, and conductivity, for example.
Therefore, the present invention may be used to diagnose the
presence of a condition, for example, a cancer, or other cellular
disorder.
[0333] Another clinical application would be to use the apparatus
and methods of the present invention to separate unwanted cells,
such as cancerous cells, from a cell population including wanted or
normal cells. For example, once a cancer has been detected, for
instance in bone marrow, a patient's bone marrow may be input into
an apparatus according to the present invention to separate the
cancer cells, or preneoplastic cells, from normal cells. These
normal cells may then be collected at the output of the chamber and
returned to the patient, while the unwanted cancer cells may be
later collected at the output of the chamber and characterized,
utilized in further studies, or discarded. In this manner, unwanted
cells are purged from a normal cell population, while at the same
time a particular cell type is enriched, such as tumor cells,
normal cells, progenitor cells, etc.
[0334] The apparatus and methods disclosed and claimed herein can
be made and executed without undue experimentation in light of the
present disclosure. While the apparatus and methods of this
invention have been described in terms of preferred embodiments, it
will be apparent to those of skill in the art that variations may
be applied to the apparatus, methods and in the steps or in the
sequence of steps of the method described herein without departing
from the concept, spirit and scope of the invention. All
substitutes and modifications apparent to those skilled in the art
are deemed to be within the spirit, scope and concept of the
invention as defined by the appended claims.
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