BACKGROUND OF THE INVENTION1. Field of the Invention
The present invention relates generally to fluidic processing and,
more particularly, to methods and apparatuses concerning an integrated fluidic device
capable of enriching and isolating a suspect cell subpopulation from a suspension
of cells and quantitatively analyzing that subpopulation for marker proteins and
mRNAs for the purpose of detection and diagnosis of conditions such as cancer.
2. Description of Related Art
The identification of increasing numbers of genes that influence disease
states and the approach of the post-genomic era make evident the need for faster
and automated technologies that will allow dissemination of the gains of molecular
diagnosis. If sufficiently small, automatic and inexpensive devices can be developed
for molecular screening, they would not only revolutionize the diagnosis and prognosis
of cancer and other diseases but also would enable molecular methods to be disseminated
completely - even to the point of care.
Although some devices such as gene chips and chip embodiments of the
polymerase chain reaction (PGR) are beginning to enter use, many of the methods
developed so far are labor intensive and are not readily suited to automated, continuous
monitoring, or high throughput applications. Clearly, a wide range of enabling technologies
is needed before integrated instruments capable of automated sample preparation
and molecular analysis of clinical samples become a reality.
SUMMARY OF THE INVENTION
Technology that is the subject of the present addresses issues related
to the creation of multiple-use diagnostic systems for combined sample preparation
and detection of molecular markers. Disclosed herein are systems, methods, and devices
capable of performing fully automated assays. These devices offer the advantages
of small size, low sample volume requirements, and the potential for mass production
at low cost. Such low-cost systems are applicable to reusable or disposable medical
In one embodiment, such a system may include the following subsystems:
(1) a prefilter stage to concentrate suspect cells; (2) a high discrimination separator
stage to fractionate cell subpopulations; (3) a stage to burst cells and mobilize
molecular components; and (4) a stage for automated analysis of protein and mRNA
molecular diagnostic markers.
Important technologies for the development of such a system, and others
made apparent by the present disclosure include the following: a prefiltering methodology
to trap suspected cancer cells from blood or dispersed lymph node cells; a force
balance method that exploits dielectric properties of the suspect cells, and, if
needed, their immunomagnetic labeling properties, to fractionate them into a microfluidic
isolation and analysis chamber; and a dielectric indexing and manipulation method
for carrier beads that, when combined with certain established molecular assay methods,
allows for the parallel quantification of multiple molecular markers.
U.S. Patent No. 5,858,192 entitled "Method and Apparatus for Manipulation
Using Spiral Electrodes" discloses the preamble of the independent claim 1.
As certain technology disclosed herein builds upon work involving
dielectrophoretic trapping, dielectrophoretic field-flow fractionation (DEP-FFF),
traveling wave methods, and other work performed by the inventors, United States
Patent No. 5,993,630 entitled "Method and Apparatus for Fractionation Using Conventional
Dielectrophoresis and Field Flow Fractionation"; U.S. Patent No. 5,888,370 entitled
"Method and Apparatus for Fractionation Using Generalized Dielectrophoresis and
Field Flow Fractionation"; U.S. Patent No. 5,993,632 entitled "Method and Apparatus
for Fractionation Using Generalized Dielectrophoresis and Field Flow Fractionation";
United States Application No. 09/249,955 filed February 12, 1999 and entitled "Method
and Apparatus for Programmable Fluidic Processing"; United States Application No.09/395,890
filed September 14, 1999 and entitled "Method and Apparatus for Fractionation Using
Generalized Dielectrophoresis and Field Flow Fractionation"; United States Provisional
Application No. 60/211,757 filed June 14, 2000 and entitled "Method and Apparatus
for Combined Magnetophoretic and Dielectrophoretic Manipulation of Analyte Mixtures";
United States Provisional Application No. 60/211,515 filed June 14, 2000 and entitled
"Dielectrically-Engineered Microparticles"; United States Provisional Application
No. 60/211,516 filed June 14, 2000 and entitled "Apparatus and Method for Fluid
Dielectric indexing represents a new approach to identifying individual
molecular tests in a parallel molecular analysis scheme that substitutes dielectric
indexing of carrier beads for the spatial indexing used on a gene chip. This new
approach allows different subpopulations of beads, each carrying a probe of a different
molecular marker, to be identified and manipulated within the carrier medium using
a dielectric fingerprint unique to each bead/probe type. The need to immobilize
different molecular probes in a tightly specified pattern on a fixed substrate as
demanded, for example, by gene chip technology, is thereby eliminated. Mixtures
of probes, each probe carried on a separately indexed bead type, may be injected
into and flushed from a reusable assay system in order to examine any desired panel
of molecular markers.
The use of bead dielectric properties as an indexing parameter not
only provides the capability of manipulating beads through dielectrophoresis or
another suitable manipulation force, but also offers a new alternative to optical
or fluorescent bead indexing methods that might interfere with low light emissions
in fluorescent probe assays.
Technology disclosed herein builds upon and synthesizes aspects of
many disciplines including field-flow fractionation (physical chemistry), dielectrophoresis
and magnetophoresis (physics), microfluidics (mechanical and fluid engineering),
microfabrication (photolithography, MEMS and magnetic materials science), control
electronics (electrical engineering), antibody and nucleic acid binding and linking
(immunology and molecular biology), cell biology (cell culture and cytology), flow
cytometry, and oncology.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and
are included by way of example and not limitation 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 which like references indicate similar elements,
in combination with the detailed description of specific embodiments presented herein.
FIG. 1 is graph showing different DEP crossover frequencies. It compares
the crossover frequencies for nine human tumor cell types and normal peripheral
blood mononuclear cells.
FIGS. 2A-2D are pictures showing the removal of cultured breast cancer
cells from blood by cDEP affinity trapping.
FIG. 3 is a schematic illustrating some operating principles of cDEP/FFF
FIG. 4 is a chart summarizing DEP-FFF separation data for various cell
FIG. 5 is a picture showing a spiral electrode array that may be used
to focus cells by twDEP.
FIGS. 6A-6B are charts showing field/frequency bursting characteristics
of (A) T-lymphocytes, and (B) MDA-MB-435 breast cancer cells.
FIG. 7 is a graph showing magnetic field strength emerging from two opposing
FIG. 8 is a flow chart illustrating functional stages of a device for
cell isolation and analysis.
FIG. 9 is a schematic of an integrated fluidic system, including a prefilter
stage, a separator stage, and an isolator and analysis stage.
FIG. 10 is a schematic showing a short section of a DEP-MAP-FFF chamber.
FIG. 11 is an end view of a magnetophoresis assembly. The magnets are
SmCo or NdFeB. The separation chamber sits in the magnetic flux gradient just above
the sintered iron spheres. Sintered iron spheres may be replaced by iron wedges
or filaments to produce different desired flux gradient characteristics.
FIG. 12 is a schematic of one embodiment of the integrated fluidic system,
including a prefilter stage, a separator stage, and an isolator and analysis stage
that includes a programmable fluidic processor.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
The presently disclosed systems, methods and apparatuses provide many
advantages (a few of which are the following). They permit for cell pre-filtering
that may be used to separate tumor cells from peripheral blood mononuclear cells
(PBMNCs). They allow for Dielectrophoretic-magnetophoretic field-flow fractionation
(DEP-MAP-FFF), allowing for combined dielectrophoretic and immunomagnetic cell separation.
They allow for the dielectric indexing of beads, the linkage of antibody and oligonucleotide
probes to bead surfaces, and for the simultaneous assays for two molecular markers
using a mixture of two different bead/probe types. They allow for the quantification
of the association of targets with the beads and identifications of the bead types
by dielectric measurements using impedance sensing methods. They allow for DEP-MAP-FFF
fractionation of cells according to their surface receptor concentrations. They
allow for DEP focusing of samples using swept frequency traveling fields applied
to spiral electrode arrays that can be used to concentrate isolated cell fractions
to ~ 109 cells/ml. They allow for the electro-mediated bursting of cells. They also
allow for the mixtures of different bead/probe combinations to be used to perform
parallel assays with dielectric indexing.
Areas that may benefit from this technology include, but are not limited
to: blood and urine assays, pathogen detection, pollution monitoring, water monitoring,
fertilizer analysis, the detection of chemical and biological warfare agents, food
pathogen detection, quality control and blending, massively parallel molecular biological
protocols, genetic engineering, oncogene detection, and pharmaceutical development
Because the present disclosure deals, in part, with the combination
of a number of technologies that may be discussed separately, it is useful to begin
the discussion with some theoretical underpinnings and considerations relating to
some of the individual techniques disclosed herein. In the Examples section, discussion
will focus more upon the combination of the techniques to form systems and apparatuses
(and associated methodology) according to embodiments of this disclosure.
Certain techniques of this disclosure use molecular recognition and
sensing elements that are attached to bead carriers so that a new aliquot of sensitized
beads can be used for each and every assay. By disposing of the beads afterwards,
by running a "blank" between each sample, and by allowing for cleaning cycles, calibration
issues can be addressed and the absence of carryover and cross-contamination can
Placing biologically active components on beads also means that a
single, fluidic device may be applied to a wide range of sample preparation and
molecular analysis problems by using different bead/probe combinations. Finally,
because no biological components need be attached to fixed surfaces within the device
of one embodiment, those surfaces may be PTFE coated, for example, to reduce biomolecular
adhesion and carryover issues. It follows that the use of beads enhances the potential
applicability of the technology by allowing a single device to have multiple applications.
Although molecular amplification techniques enjoy widespread use,
methods such as PCR have drawbacks including sensitivities to hard-to-control factors
that can render them only marginally quantitative. Furthermore, molecular amplification
bathes the reaction surfaces with high concentrations of the molecules to be detected.
The resultant carryover problem is so severe that all wetted surfaces in molecular
amplification experiments are typically made disposable. For these reasons, this
disclosure avoids direct molecular amplification steps in designing reusable devices
and focuses on detecting small numbers of molecules trapped directly on beads. Nevertheless,
having the benefit of this disclosure, those having skill in the art recognize that
the bead-based indexing technology described here is also compatible with molecular
amplification protocols should they be required.
Any in-situ hybridization assay may be adapted to operate on
the surface of a carrier bead including methods for detecting DNA, RNA and proteins.
In this disclosure, the established body of hybridization and immuno-fluorescent
molecular techniques may be used along with new methods for indexing bead carriers
so that individual bead types within a complex mixture of bead types are identifiable,
amenable to selective manipulation, and, if desired, to isolation. Assays using
dielectrically-engineered beads require minimal quantities of sample. For example,
a bead of about 5 µm diameter has the relatively large surface area of approximately
78 µm2 yet occupies a volume of only 65 fL, about 1/15 that of a typical
tumor cell. 100 tumor cells and 250 beads comprised of 10 different bead types may
be packed into spherical region of 50 µm diameter using DEP-mediated focusing. This
is the equivalent of almost 109 cells/ml held in contact with 2 x 109
beads/ml carrying the molecular probes. The time for hybridization of target mRNA's
to cDNA probes on magnetic bead surfaces has been shown to be just a few minutes
in concentrated cell lysates; therefore, the bead-based approach of this disclosure
may enable rapid assays for molecular markers in an integrated system.
The bead-based, dielectric indexing technology of this disclosure
is not meant to replace large gene-chip array methods designed for massively parallel
analysis of the expression of 10,000 or more genes. Those methods permit the identification,
in the first place, of key markers of specific cellular events. Instead, this disclosure
represents a technology in which a reduced panel of 10 or so key molecular markers
may be selected from a library of available markers for the purpose of screening
for specific subsets of suspected disease states. By combining sample preparation
and molecular analysis into a single, automated process, this system allows the
exploitation of gene-chip-derived molecular epidemiological data and renders it
accessible to a wide population.
This disclosure addresses the isolation of suspect cells from mixed
cell suspensions and the manipulation of mixtures of dielectrically indexed beads,
all in an integrated device. Achieving these steps ultimately depends upon ways
of moving matter with respect to the solution that suspends it, a problem to which
dielectrophoresis, or another suitable manipulation force, is ideally suited.
Dielectrophoresis (DEP) is the movement of a material or an object
caused by a spatially non-uniform electrical field. Completely distinct from the
well-known phenomenon of electrophoresis, DEP only arises when the object has a
different tendency to become electrically polarized relative to its surroundings.
If the object is more polarizable than its surroundings, it will be pulled-towards
higher field regions ("positive DEP"); conversely it will be repelled towards weak
field regions ("negative DEP") if it is less polarizable. Positive DEP is known
to most of us as the attraction of uncharged bits of paper to a charged plastic
comb. Magnetophoresis is the magnetic analog of dielectmphoresis, the collection
of magnetically polarizable particles in a spatially non-uniform magnetic field.
This force is responsible for the familiar collection of iron filings at the fringing
fields at the edges of a magnetic pole. Far from being restricted to electrostatic
fields, DEP also occurs in alternating (AC) fields even at optical frequencies.
An example is when a laser tweezers is used to trap a cell having a higher refractive
index (larger electronic polarizability) than its suspending medium at the high
field gradient focal region of the laser beam. (There is also a second, light pressure
term in this extreme case). At lower frequencies DEP can be used to impose forces
on cells that depend on their low-frequency spectral properties. Differences in
these spectral properties can be exploited to impose different or even opposing
forces on different cell types in a cell mixture. For techniques of this disclosure,
relatively low frequencies may be used, from about 10 kHz to about 10 MHz, at which
cell membrane and bead coating properties dominate the particle dielectric properties.
The essential characteristic ofDEP is the movement of objects with
respect to their suspending medium. For example, objects can be concentrated to
a focal point by negative DEP and/or trapped by positive DEP. In addition, different
particle types can be moved apart from one another in three dimensions under appropriate
field conditions. These basic manipulations can be used to sort, isolate, and trap
cells and beads, and to change the reagents in which they are suspended.
Of particular relevance to this disclosure is the extensive DEP work
on normal and cancer cells in which the inventors and others have shown that different
cell types have distinct dielectrophoretic fingerprints and may be used (in accordance
with embodiments disclosed herein) to characterize, manipulate, fractionate, isolate,
trap, and selectively burst them.
Summarizing, DEP is a force that:
1. arises when a particle having dielectric properties distinct from its carrier
medium is subjected to a spatially non-uniform electrical field anywhere from DC
to optical frequencies;
2. in complete contrast to electrophoresis, completely ignores any net charge
on the particle (this is a critical consideration when performing assays with highly
charged biomolecules such as nucleic acids);
3. can be used to trap, focus, fractionate and isolate cells;
4. depends specifically on the dielectric fingerprint of each cell type. In
principle, DEP can be used to exploit any spectral differences between cells but
this disclosure focuses on low frequency differences dominated by plasma membrane
5. can be produced by an AC electrical field that typically has a frequency
between 10 kHz and 1 MHz for cell isolation experiments. No electrolysis occurs
at these frequencies and cells are not damaged unless the field is deliberately
increased above a high threshold magnitude to achieve controlled cell bursting;
6. can be produced by an array of micro electrodes that are inexpensive to fabricate
according to methods known in the art;
7. can be produced at AC frequencies even if the electrodes carry a thin coating
ofPTFE or other insulator;
8. is controlled via the frequency and/or voltage of the signal applied to the
electrodes. The electronics are straightforward, can be incorporated in a box the
size of a pocket calculator, are inexpensive, and can be kept separate as is all
known in the art so that DEP chambers may be disposable while the electronics are
9. is ideal for meso- and microfluidic-scale applications because electrodes
can line the floor and/or walls of fluidic channels and chambers;
10. allows cells, beads, or other targets to be selectively manipulated within
their carrier medium or held in place while a new carrier medium is washed over
In one embodiment, high discrimination sample preparation of suspect
tumor subpopulations is accomplished through a separation technique called hyperlayer
field-flow fractionation. The underlying principle is straightforward: the velocity
of fluid flowing through a flat channel increases from zero at the floor and ceiling
to a maximum at the center. If different cell types are positioned at different
characteristic heights above the channel floor then they will be carried at different
velocities by the fluid and separated as the cell mixture travels through the channel.
The different types can then be isolated and trapped as they emerge from the far
end of the channel. Separation does not depend on the interaction of cells with
any material other than the carrier fluid, reducing non-specific binding, carryover,
and contamination effects that are inherent in chromatographic methods, for example.
To position different cell types characteristically in the separation
channel, one may balance dielectrophoretic and gravitational forces on cells. Additionally,
magnetophoretic forces may be used as well for positioning cells if desired. In
this way, immunomagnetic labeling can be used as an additional feature to discriminate
between different cell types. The DEP-MAP-FFF method is equally applicable to cells,
which have their own intrinsic dielectric properties, and to beads that can act
as molecular marker carriers. When a cell subpopulation has intrinsic dielectric
differences that distinguish if from other cell types in a mixture, it is not necessary
to use magnetic labeling and the method may revert to a DEP-FFF scheme.
The continuous MAP-sorting of immunomagnetically labeled cells in
a laminar flow profile subjected to a quadrupole magnetic field configuration has
been demonstrated. While the sorting of cells according to surface receptor density
was achieved, the method has the disadvantage that the MAP force is unbalanced.
Consequently, separation is flow-rate dependent. Furthermore, heavily labeled cells
may collide with the sides of the flow chamber only to become trapped or to suffer
remixing with other cell types. The DEP-MAP-FFF design of the present disclosure,
however, balances opposing DEP and MAP forces to place cells in equilibrium positions
in the flow profile. In this way, the pitfalls of unbalanced forces, which are likely
to be of even greater concern when sorting inherently inhomogeneous tumor cell subpopulations,
may be avoided.
In addition to cell sorting, DEP may be used to prefilter cells when
large numbers of cells need to be processed, to trap cells after they emerge from
the DEP-MAP-FFF separator, to concentrate the cell isolates and beads, to lyse the
cells, and to hold beads in place while reagents are changed in molecular analysis
protocols. In this way, dielectmphoresis provides for the ability to realize an
automated device that will integrated a sample prefilter, a DEP-MAP-FFF separator,
a cell fraction isolation and lysis stage, and a molecular analysis stage.
In one embodiment, a DEP-MAP-FFF system may take a sample of about
20 µL of cell suspension containing a maximum of 2 x 105 cells when performing
high resolution separations. A lower detection limit of 20 cancer cells in the molecular
analysis stage requires an incidence of 1 or more cancer cells per 1000 normal cells.
While this level of discrimination is adequate for biopsy samples of putatively
tumorous tissue, in other applications, such as the detection of residual disease,
of metastatic cells in bone marrow harvests, or of micrometastases in sentinel lymph
nodes, the goal is to detect 1 tumor cell per 106 or more normal nucleated
cells. To provide 20 tumor cells for analysis in such applications, there is the
need to sort > 2 x 107 normal cells, a number that far exceeds the
capacity of DEP-MAP-FFF separator stage because to achieve high discrimination this
stage needs to operate at cell concentrations where cell-cell interactions are negligible.
To sort high numbers of cells, a stage that will execute a DEP prefiltering
step for suspect cancer cells may therefore be needed. While prefiltering does not
provide a pure population of suspect cells, it does provide a sample that is suitable
for the DEP-MAP-FFF stage of the device (which is explained and illustrated, in
one embodiment, in the Examples section of this disclosure). In one embodiment,
the prefilter may process ~20 x 106 cells and extract ~2 x 105
cells enriched in the suspect cell subpopulation. Those 2 x 105 cells
may then be routed to a high discrimination DEP-MAP-FFF separator stage. If the
lower limit of molecular analysis in the last stage of the integrated device is
20 cancer cells, then the integrated device may achieve a detection limit of 1 cancer
cell per 106 starting nucleated cells.
It has been shown that the DEP force acting on a particle due to an
imposed electrical field, E(ω), can be written as
fCM(ε*p,ε*m,ω)=ε*p(ω)-ε*m(ω) / (ε*p(ω)+2ε*m(ω)).
is the Clausius-Mossotti factor that embodies the frequency-dependent dielectric
of the particle and its suspending medium, respectively. ω is the angular
frequency and E(rms) is the rms value of the applied electric field.Ei0
and ϕi (i=x; y; z) are the magnitudes and phases, respectively,
of the field components in the principal axis directions. Equation (1), which is
sufficient for the present discussion, shows there are two independent force contributions
to DEP motion:
(i) A field inhomogeneity component: the left hand term depends on thereal
(in-phase, or capacitative) component Re(fCM) of the induced
dipole moment in the particle and the spatial nonuniformity, ▿E(rms)2,
of the field magnitude. This force pushes particles towards strong or weak field
regions, depending upon whether Re(fCM) is positive or
negative. This is the DEP force that allows cells to be attracted or repelled from
electrode edges. It is the only DEP force component that can act when an electrode
array is energized by single or dual phase signals.
(ii) A traveling field component: the right hand term depends on theimaginary
(out-of-phase, or lossy) component Im(fCM) of the induced
dipole moment and the spatial nonuniformity (▿ϕx, ▿ϕy
and ▿ϕz) of the field phase. This force pushes
the particle in the same or the opposite direction to which the field is traveling
depending on the sign of Im(fCM). It allows cells to be
swept along by an electric field that travels over an electrode array. At least
three excitation phases must be provided for this force to arise.
These force components act independently but, by appropriate electrode
array design, can be applied simultaneously to levitate cells above an electrode
array while moving them over it, for example.
Cell Dielectric Properties
At low frequencies cells exhibit negative DEP (repulsion from electrode
tips) but at higher frequencies, above their so-called DEP crossover frequencies,
they exhibit positive DEP (attraction towards electrode tips). Different cell types
have different crossover frequencies. At frequencies between about 104
and 3 x 104 Hz breast cancer cells will experience positive DEP trapping
while blood cells will experience negative DEP repulsion. These dielectric differences
between the cancer and blood cell types can be used as a basis for cell identification,
discrimination and separation. Cell sizes, cell compositions, and especially cell
membrane morphologies all contribute to the dielectric differences between the cells;
i.e. different cells have different dielectric phenotypes.
The inventors have found that the dielectric phenotype of every transformed
cell type they have examined is significantly different from that of a more normal
cell of origin, or from the same cell type following induced differentiation. This
results from greater cell surface morphological complexity and a correspondingly
higher membrane capacitance in the transformed cell types. Furthermore, tumor cells
are normally much larger that blood cells. The effect of these combined differences
is that the dielectric properties of transformed cells differ very significantly
from normal blood cells. Of particular relevance to this disclosure, the inventors
have measured the DEP crossover frequencies of 9 human cancers comprising 5 human
breast cancer cell lines, an ascites sample taken from a patient with breast cancer,
and two colon cancer cell lines. The DEP crossover frequencies of these cancer cell
types suspended in solutions of 100 mS/m conductivity are shown in FIG. 1 in comparison
with data for normal peripheral blood mononuclear cell types. The tumor cells all
exhibit much lower crossover frequencies. These differences may be exploited for
isolating populations of suspect cells from PBMNCs and lymph cell dispersions.
Prefiltering by DEP Trapping of Cells
Exploitation of dielectric differences for cell separation may be
accomplished in several ways. The simplest though least discriminating method is
to apply a frequency that repels one cell type from one or more electrodes by negative
DEP while attracting and trapping a different cell type by positive DEP. FIG. 2A
shows a mixture of MDA-MB-231 human breast cancer cells and human peripheral blood.
The larger breast cancer cells, about 12 µm in diameter, are readily identifiable.
In FIG. 2B, a 2.5 x 104 Hz AC signal has been applied between neighboring
gold electrodes (dark patterns) and fluid flow has been started from left to right.
The human breast cancer cells are attracted to the electrode tips and trapped (FIG
2B&C). Blood cells, on the other hand, are repelled from the electrodes and
carried off by the fluid. They emerge downstream, where no cell mixture was loaded,
free of cancer cells, (FIG. 2D). This DEP trapping approach works well when there
are large differences in the dielectric properties of target cells and other cell
types in the starting mixture. For example, the inventors have demonstrated that
it is possible to recover 100% of human breast tumor cells from PBMNCs even at the
most dilute concentration tested in preliminary experiments, one tumor cell per
3 x 105 PBMNCs.
After flushing out the blood cells, tumor cells may be recovered by
lowering the frequency below 10 kHz causing them to be repelled from the electrodes
by negative DEP and released from the chamber. The inventors have found that some
normal cells may be associated with the tumor cells during the trapping phase such
that while recovery efficiency may be extremely good, purity may not be so good.
It should be noted that at higher applied frequencies (200kHz or more) all viable
cells have been found to become trapped by positive DEP regardless of type. Therefore,
DEP may be used quite generally to immobilize cells within a stream of reagents
without regard to cell type if required.
In applications involving rare cancer cells, a prefilter system may
be used having a surface area of about 60cm2 over which suspensions of
nucleated cells can be passed at the rate of about 3.6x106 cells/min.
This may be operated for about 6 minutes with suspensions of cells from lymph nodes
and whole blood to screen 20 x 106 nucleated cells for the presence of
tumor cells. Suspect cells, at a purity of >0.1%, may then be passed for high
discrimination separation by the DEP-MAP-FFF in and, after subsequent isolation,
for downstream molecular analysis in the integrated device (discussed in more detail
in the Examples section of this disclosure).
To allow high discrimination separation of tumor cells from biopsy
samples or from lymph node or blood cell samples prefiltered by DEP trapping, a
fractionation method termed DEP-MAP-FFF may be used. Such a method may also use
immunomagnetic capabilities when needed. Instead of trapping target cells, DEP-FFF
uses parallel electrodes without castellated edges to levitate cells above the electrode
plain using fringing fields. The strength and inhomogeneity of the electrical field
decreases with increasing height above the electrode plane and the DEP force on
cells falls exponentially with height. If a frequency for which cells experience
negative DEP is applied to the electrode array, cells will be levitated to a height
at which the repulsive DEP force balances the sedimentation force. Cells having
differences in density and/or dielectric properties will therefore be levitated
to characteristic heights as illustrated in FIG. 3. This equilibrium height is given
for a parallel electrode geometry, where U is the electrical potential applied
to the electrode array, A is a geometrical term, p is the proportion
of the applied field unscreened by electrode polarization (p~1 at frequencies >
50 kHz), and (ρc-ρm)g
is the sedimentation force.
To exploit this equilibrium levitation effect for cell fractionation,
fluid flow is initiated in the channel. Fluid flows through the channel in a parabolic
profile - slowest at the chamber top and bottom walls, and fastest in the middle
(at about half height according to one embodiment). The velocity at height
heq, is given by
where H is the chamber height and <ν> is the mean fluid velocity.
The fluid will then carry cells at a velocity corresponding to their levitation
height. Mixed cell types starting at one end of a long chamber will therefore be
separated according to their dielectric and density properties.
The family of techniques that exploits hydrodynamic flow profiles
for separation is termed field-flow fractionation (FFF); hence the inventors term
this method DEP-FFF. The discriminating power of DEP-FFF is extremely high in the
frequency range where the cell dielectrophoretic force approaches zero (i.e. near
the crossover frequencies shown in FIG. 1). Less discriminating power can be selected
electrically by employing a lower frequency or by using modulated frequencies.
The inventors have made several DEP-FFF separators ranging in size
from about 45 cm x 2 cm to the size of a microscope slide (see section below concerning
microfabrication). With the benefit of the present disclosure, those having skill
in the art recognize that other sizes may be used as well. DEP-FFF separation normally
take from 4 to 15 minutes to complete, but this time may vary significantly depending
on the size of the device and other parameters such as sample size. For different
separation times for different cell types, under different experimental parameters,
see FIG. 4.
In one embodiment, a modified form of DEP-FFF may be employed in which
an additional vertical force component is added that depends on immunomagnetic labeling
of the cells. This may address potential concerns that some tumor cell types might
not have intrinsic dielectric properties like those shown in FIG. 1 that permit
their separation from normal cells by DEP-FFF alone. The inventors feel that exploitation
of cell intrinsic properties, when possible, may be more desirable than requiring
a labeling step; therefore, they have designed DEP-MAP-FFF separators so that exploitation
of immunomagnetic labeling is an available, though non-essential, option: in the
absence of immunomagnetic labeling, the device may function as a DEP-FFF separator
that can discriminate cells by dielectric properties alone.
A particle of volume ν and magnetic permeability µp
subjected to an inhomogeneous magnetic field will experience a MAP force that is
the magnetic analog of the DEP force given in equation (1)
Here, µs and µp are the magnetic permeability
of the suspending medium and particle, respectively, R is the particle radius and,
is the Magnetic Clausius-Mossotti factor describing the magnetic polarizability
of the particle with respect to its suspending medium. In the static fields typically
used for MAP cell sorting, ωH, the frequency of the applied
magnetic field, has the value 0 and µs and µp
become static magnetic permeability parameters. Furthermore, the magnetic permeability
of the aqueous suspension in an immunomagnetic labeling experiment can be approximated
as that of free space and the net polarizability of a labeled cell can be assumed
to result from the combined effect of n identical labels that are bound to it. Finally,
for a fixed geometry, the magnetic field gradient may be written as a geometry term
GMAP times the applied magnetic field strength,
B0. Hence, in a biological labeling experiment we may simplify
the MAP force equation to
FMAP = nϕ GMAPB20
where ϕ is a constant for a given magnetic label type. This is the fundamental
equation that determines magnetic capture of cells in MACS; however, the goal of
the present disclosure is not to magnetically trap cells. By appropriate design
of the magnetic elements that create the magnetic field and its inhomogeneity characteristics
embodied in GMAP, a MAP force may be provided that is essentially
constant throughout a separation chamber and directed towards the chamber floor.
We indicated earlier that the DEP force above a parallel electrode
array falls off exponentially with height h as FDEP
= FDEP 0e-h/hDEP.
When the electrical field conditions are chosen to provide repulsive DEP, as in
DEP-FFF, the MAP force will pull an immunomagnetically labeled cell toward the electrode
plane until the sum of the downward MAP and sedimentation forces are balanced by
the levitating DEP force. Writing the electrical field gradient in terms of an electrode
geometry termGDEP and the applied RMS voltage
V0 applied to the electrode array, the balance of forces
that determines the particle equilibrium height will be given by eq. 7 below:
where mlabel is the mass of each immunomagnetic label.
If the magnetic labeling is negligible (n→0), this equation reduces
to that given earlier for plain DEP-FFF. On the other hand, if magnetic labeling
dominates the downward force then the decrease in h becomes approximately
proportional to the logarithm of the number n of magnetic labels attached to the
cell. Since in this context "dominate" means to provide a MAP force significantly
in excess of the small cell sedimentation force, it will be appreciated that much
smaller magnetic forces are needed in DEP-MAP-FFF than for magnetic trapping against
a flow stream as used in MACS.
Note also that V0 can always be chosen to
ensure that no cells are pulled all the way to the chamber floor. Because, according
to one embodiment, cells are separated in a FFF scheme according to their characteristic
heights h in the fluid flow profile, one may separate them according to the
extent of immunomagnetic labeling and, as is familiar in fluorescently-activated
cell sorting (FACS), the logarithmic relationship may be very convenient for ensuring
a good dynamic range when sorting different classes of cells. Therefore, when needed,
MAP provides an ideal additional level of discrimination for sorting suspect tumor
cell subpopulations by, for example, epithelial surface markers or receptors such
as for EGF.
DEP-Mediated Cell Focusing
Cells can be manipulated simultaneously by DEP, which attracts or
repels them from electrode edges, and twDEP, which transports them parallel to the
plane of the electrodes. A spiral electrode configuration may be used to exploit
these effects simultaneously for concentrating cells and achieving electrically
stimulated cell lysis. The spiral array in one embodiment includes four parallel
electrode elements that are energized by signals of the same frequency but phases
of 0°, 90°, 180°, and 270° to create a concentric traveling field that sweeps towards
the center of the spiral. Excitation by phases 0°, 270°, 180°, and 90° results in
a field that sweeps outward towards the periphery of the spiral. Signals of 0°,
180°, 0°, and 180° phases produce a stationary field pattern that can be used for
DEP trapping, levitation, or, at very high field strengths, cell bursting.
An example of cell trapping and focusing is shown in FIG. 5 where
HL-60 human promyelytic leukemia cells have been focused from a scattered state
to the center of a spiral in about 15 seconds. In one embodiment, the spiral arms
of the electrode array may be extended until they almost touch at the center of
the spiral allowing greatly increased cell concentrations to be achieved. The inventors
have applied this technique to trap and focus murine erythroleukemia and human breast
cancer cell lines from a flow stream, and separate breast and leukemia cells from
blood cells. Also the inventors have successfully separated erythrocytes parasitized
by the malarial agent Plasmodium falciparum from their uninfected counterparts
with this technique.
In one embodiment, five spiral array segments may be used to trap
cell subpopulations as they emerge at different times from a DEP-MAP-FFF separator
stage of an integrated device. By injecting assay beads into the stream of cells
as they emerge from the separator and before they are trapped, and by then applying
a swept field to the spiral electrodes, cells and beads may be focused to the center
concurrently to form a highly concentrated mixture.
Electro-Mediated Lysis of Cells
Once a target cell population has been successfully isolated, subsequent
molecular analyses normally require that the cells be disrupted to release intracellular
proteins, RNA, and DNA. Approaches to this include exposure to detergents or other
lysing reagents. Although these methods can be used in systems and devices disclosed
herein, cells may be lysed electrically using large AC electrical fields. DEP manipulations
typically involve local electrical fields less than 104 V/m and the inventors
have shown that cells can sustain prolonged (40 minutes and longer) exposure to
such fields without loss of viability or activity. Depending on the electrode geometry,
voltages of the order of 1 V RMS are used to achieve this.
However, higher AC voltages may be applied to create fields that can
burst cells. Depending on the cell type, at about 5 x 104 V/m, temporary
membrane electropermeabilization occurs, and this can be used to load reagents into
cells. Above about 2 x 105 V/m, instantaneous destruction of the cell
membranes occurs. The inventors have found that different cell types have characteristically
different susceptibilities to destruction. FIG. 6A illustrates the field intensity
vs. frequency dependency for the disruption of human T-lymphocytes and FIG. 6B shows
results for human MDA-MB-435 breast cancer cells. Clearly the cells burst in characteristic,
and distinct, frequency and field ranges. A useful feature is the ability to select
electrically whether to reversibly permeabilize or totally disrupt all, or select
subpopulations, of cells that have been trapped on an isolation electrode.
In one embodiment, electro-mediated cell lysis may be utilized at
the center of the spiral isolation segments to release molecular species from target
cells into the immediate vicinity of the assay beads mixed and concentrated with
Electrode arrays for use in, for instance, a separation according
to embodiments of the present disclosure may be made by microlithography as is known
in the art. The inventors have built DEP chambers and separators over a wide range
of sizes from about 200 µm - 45 cm with capacities of 10 µL to 4 mL. The use of
silicon and glass and micromachining methods may be used for cases where integrated
electronics and sensor capabilities are required that other fabrication methods
cannot provide. In other cases, a combination of flat glass and injection-molded
polymers may be used to fabricate the devices disclosed herein by methods known
in the art. Small devices may be made by silicon and glass micromachining, and can
be reproduced by single layer lithography on a flat glass substrate (for the electrodes)
with all fluidic channels molded into a clear polydimethylsiloxane (PDMS) top. Molding
PDMS has been suggested as a much more cost effective approach than micromachining
glass and silicon; it comes as a clear liquid that can be cast or injected into
a mold. Devices of the present disclosure may be designed to handle not only small
(about 20 µL) samples but also larger volumes (~ 10 mL or more). To accomplish this,
a microfluidic front-end is clearly unsuitable because it would be unable to process
large samples at reasonable rates. In one embodiment, the sample may be enriched
as it passes through the device and to simultaneously reduce its volume. In this
way a microfluidic stage, with its advantages of small sample requirements and rapid
processing capabilities, may be seamlessly interfaced to the macroscopic world to
complete the molecular analysis.
Magnetic Field Generation
The MAP force to be used in conjunction with DEP-FFF requires a magnet
having rather unusual properties, namely the product of the magnetic field strength
and its inhomogeneity need to be effectively constant over the entire length of
the separator. To achieve this, one may use several flat magnets of SnCo or NdFeB
materials placed a parallel configuration in an opposing pole orientation. FIG.
7 shows two magnets in this configuration. The field lines experience compression
in the space between the opposing poles and emerge in a relatively homogeneous distribution.
Controlled inhomogeneity in the field may be created by using a composite material
made of sintered iron spheres in the field path.
The field strength and homogeneity (in the absence of the sintered
iron elements) has been tested for two 6 mm thick SnCo magnets having 25 mm x 25
mm pole faces and a "free field" of 0.22 T in air. The field of the opposing pole
configuration was measured with a directional Hall probe. Field strengths in excess
of 0.4 T were measured (FIG. 7) for pole spacings of 4 mm or less and the horizontal
field component was below 5%. Based upon the inventors' measurements of the magnetic
fields used in small MACS separators, these intensities are more than sufficient
to achieve magnetic positioning of immunomagnetically-labeled cells in DEP-MAP-FFF.
The following examples are included not for limitation but, rather,
to demonstrate specific embodiments of the invention. It should be appreciated by
those of skill in the art that the techniques disclosed in the examples which follow
represent techniques discovered by the inventors to function well in the practice
of the invention, and thus can be considered to constitute specific 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.
Example 1 - Design Issues
In one embodiment, the present disclosure is directed to an integrated
fluidic device able to sort, isolate and burst target cells from clinically relevant
samples and to execute molecular marker assays on them rapidly and automatically.
FIG. 8 shows a functional block diagram of a complete integrated device and FIG.
9 shows a design for the system.
FIG. 9 shows the design of the prefilter and DEP-MAP-FFF cell fractionation
stages of the device. The prefilter is essentially a scaled-up version of a DEP
cell trapping device. Its purpose is to cope with the huge numbers of cells that
need to be sorted in rare cell detection applications. It is aimed at capturing
all cancer suspect cells even at the expense of retaining some normal cells. The
scaled-up prefilter is designed, in one embodiment, to process a sample of ≤10
ml volume containing ≤ 2 x 107 cells in ≤ 10 minutes at a maximum
rate of 3.6 x 106cells per minute. It is designed to extract from that
sample suspect cells that will be passed to the second, high discrimination cell
fractionation stage (discussed below).
Samples may be exemplified by peripheral blood from which erythrocytes
have been lysed, dispersions of lymph node tissue, or dispersed biopsy cells. To
achieve sorting in ≤10 minutes, the prefilter may sort ≤1000 µL of cell suspension
per minute. This may be accomplished by a DEP trapping array lining the floor of
a chamber 20 mm wide, 400 µm high and 30cm long. These dimensions ensure that (1)
suspect cancer cells in the mixture are guaranteed sufficient time when traversing
the chamber to sediment close enough to the DEP electrode array to be trapped by
an applied 50 kHz field while normal blood cells are repelled; (2) hydrodynamic
forces experienced by trapped cells remain sufficiently weak not to dislodge them
from the electrode array; and, (3) cell density remains sufficiently low that suspect
cells are not knocked away from electrodes by collisions with an over-abundance
of other cells.
After processing the starting suspension, clean eluate may be passed
through the prefilter at about 400 µL/min to wash away remaining untrapped cells.
During this rinsing phase, the DEP prefilter trapping electrode may be de-energized
while the secondary trapping stage remains energized. Suspect cells in the prefilter
stage may be released and carried to the secondary trap. This consolidation of trapped
cells is made possible by the removal of the large concentration of normal cells
from the system. Throughout these early phases, emerging eluate may be sent to waste.
After the consolidation step, the secondary trapping stage may contain
the suspect cancer cells together with some entrapped blood cells. Based on the
inventors' experience, this stage is expected to collect a total of no more than
2 x 105 "suspect" cells at this stage. The suspect cells may include
a limited number of monocytes, some macrophages, and any other large circulating
cells including all of the true cancer cells. This number of cells is ideal for
high discrimination sorting by DEP-MAP-FFF because the cell density has been sufficiently
reduced for cell-cell interactions to be ignored. A major advantage of the prefilter
design is its relative tolerance of such cell-cell interactions.
Magnetic Antibody Labeling
The cells in the secondary trapping stage may be incubated with magnetically
labeled antibodies if MAP separation is to be utilized in the next step of cell
isolation. Also, fluorescent antibodies, appropriate for surface marker detection
much further downstream in the device, may be added at this point. To accomplish
labeling, antibodies may be injected into the port provided for this purpose while
the cells are held in place on the electrode by DEP forces from a field of, in embodiment,
about 250 kHz. Once fluid flow has stopped, a DEP field of about 3 V peak-peak may
be alternated between about 10 kHz and about 250 kHz at about 10 second intervals
to alternately levitate and trap the cells, gently stirring them with the antibodies.
Following incubation, the DEP field may be switched to about 250 kHz to trap the
cells while the antibodies are washed away and the cells are rinsed with fresh buffer.
Following the optional antibody-labeling steps, a 0.5 V, 10 kHz signal
may be applied to release the suspect cells from the secondary trapping electrode
without levitating them. Fluid flow may be initiated in the prefilter stage and
the cells may be flushed into the DEP-MAP-FFF stage via the fluid splitter. Because
of the dimensions of the chambers and the splitter position, the suspect cells may
be carried into the DEP-MAP-FFF stage in 20 µL of eluate. A syringe pump at the
end of the DEP-MAP-FFF stage may be used to control the sample flow.
In applications such as analysis of fine needle aspiration biopsy
samples, the starting cell count may be about 2 x 105 cells or less,
and the prefiltering step becomes superfluous because the DEP-MAP-FFF fractionator
can handle such small samples without undesirable cell-cell interactions. Such samples
may be injected into the preconcentrated sample loading port at the concentrator
injection stage for the optional antibody labeling steps and thence directly into
the DEP-MAP-FFF sorter.
During and after injection of the cell sample from the prefilter stage,
the DEP electrode array in the DEP-MAP-FFF separator stage may be energized with
a frequency appropriate for separation, typically in the 20-80 kHz range. With flow
stopped, cells may be allowed sufficient time to reach equilibrium heights at which
the magnetic, DEP and gravitational force fields acting on them are balanced. Based
on DEP-FFF experiments this so-called relaxation time need not exceed five minutes.
Following relaxation, fluid flow through the DEP-MAP-FFF stage may be initiated
and cells may be carried through the chamber at characteristic velocities in accordance
with their positions in the parabolic flow profile controlled by the balance of
DEP, MAP and gravitational forces. Based on DEP-FFF experiments, this separation
step should take, in one embodiment, 12 minutes or less.
Trapping of Cell Fractions
As at the interface of the prefilter and DEP-MAP-FFF stages, a split
flow may be used between the DEP-MAP-FFF stage and the isolator and analysis stage
so that only flow close to the bottom of the separator, in which cells may emerge,
is passed through. The remaining eluate may be extracted from above and sent to
waste. A controlled flow of analysis beads may be injected into the flow stream
as it emerges from the DEP-MAP-FFF separator and enters the isolation and analysis
stage. This may mix analysis beads with the emerging cell fractions.
The cell isolation stage may be divided into 5 separate electrode
array segments, each capable of trapping and concentrating a separate fraction of
cells that emerges from the separator. Before any cells have emerged, a non-traveling
10kHz field may energize the first 4 segments of the isolation stage. This may levitate
both cells and beads by negative DEP and prevent them from settling on those segments.
However, the fifth segment may energized at 500 kHz, a frequency at which all cell
types and the beads may become trapped. Therefore, the first cells to emerge, and
the beads mixed with them, may be carried across the first 4 segments and be trapped
on the fifth by positive DEP. After an appropriate time span to isolate the first
fraction of cells on the fifth segment, the 4th segment of the trap may be energized
at 500 kHz so that cells emerging subsequently may be trapped there together with
the beads that were mixed with them. At appropriate time intervals, the 3rd, then
the 2nd, and finally the 1st trap may be similarly energized at 500 kHz.
After completion of this process 5 different cell fractions may have been isolated.and
trapped, each containing cells that emerged from MAG-DEP-FFF separation between
different time limits together with beads that were mixed with them. Although here
described with respect to five segments, those having skill in the art recognize
that any number of segments may be used.
Based on the inventors' knowledge of DEP-FFF and predictions about
MAG-DEP-FFF, cells combining the smallest sizes, most uncomplicated surface morphologies
and lowest concentrations of magnetically-labeled surface markers may emerge early
and be trapped in segment 5. Conversely, cells combining large size, complex surface
morphology and high concentrations of surface markers may emerge last and be trapped
in segment 1.
Histological Analysis of Cell Isolates
Optionally, the cells trapped in the different segments of the isolation
and analysis stage may be treated with antibodies or stains by injecting these through
the reagent port provided for this purpose. So long as the histological reagents
do not affect cell viability, the cells may be held in place by positive DEP during
perfusion and treatment. Several staining steps can be used and excess reagents
or antibodies washed away, as needed. Glass and/or clear PDMS may be used for constructing
the separation chambers. Therefore, after staining, cells isolated in the five segments
may be compared and contrasted in situ by optical and/or fluorescence microscopy
by a pathologist. If desired, additional reagents for the next step of cell analysis
may be added at this point.
Having trapped cells and beads on the five segments of the isolator
stage, optionally examined them with histological stains, and perfused them with
the reagents needed for the next step in analysis, the cells may be focused to form
a dense mixture with the beads. To accomplish this, the spiral electrodes in all
five segments of the isolation stage may be energized with a four-phase field swept
in frequency from 10 kHz to 200 kHz to provide a twDEP force directed towards the
center of each of the five spirals. Because of the established dielectric properties
of mammalian cells and the customized dielectric properties of the beads, this may
sweep cells and beads of all types towards the center of the spiral on which they
were originally trapped. It is believed that this process should take no more than
1 minute and should result in a dense conglomeration of cells and beads at the center
of each spiral. In this way, each isolated fraction may be concentrated to a density
of ~109 cells/ml together with ~1010 beads/ml suspended in
the reagent mixture that was perfused prior to focussing.
Once the cells and beads are concentrated, electro-mediated lysis
of the cells can occur. This may be achieved by applying a strong AC voltage to
the spiral electrode (e.g. 15 V peak to peak). Those having skill in the art recognize,
however, that any other voltage suitable to cause bursting may be used.
The liberation of intracellular components following cell lysis may
allow their reaction with the perfused reagents and their interaction with the surfaces
of beads (if present). Based on experiments reported in the literature for the hybridization
of rare mRNA's in concentrated cell lysates with probes carried on beads, these
reactions occur very rapidly, typically within a few minutes.
After an incubation time of 15 minutes, the target mRNA's should have
hybridized with complimentary probes on beads. The spiral electrode segments may
be energized with a 500 kHz non-travelling field to trap the beads at this point.
Cell debris is not attracted by positive DEP and may be washed away from the beads.
Indeed, relatively harsh reagents can be added to clean up the beads at this point
providing those do not degrade the mRNA's bound to different bead types or damage
the beads. After washing the beads free of debris and unhybridized molecules, the
beads may be perfused with secondary fluorescent probes for target mRNA sequences.
In this way, target sequences on the bead surfaces may be fluorescently labeled.
Following additional washing steps to remove unbound secondary labels, the spiral
electrodes may be energized with a 10 kHz signal to release the beads. At this point,
eluate flow may be commenced through one spiral segment after another and the beads
may be examined as they pass through the proximal impedance sensors.
Simultaneous fluorescence analysis may be used to quantify the amount
of mRNA secondary label bound to each bead, and the AC impedance characteristics
may be used to identify each bead/probe combination (and hence index the mixed assays).
This process should take about 15 minutes.
Total Analysis Time
If all steps shown above were to be undertaken, the entire analysis
from start to finish may take about 2 hours. This would include prefiltering cells
from a starting mixture with a detection limit that should approach 1 cancer cell
per 106 normal cells; isolating tumor cells based on their dielectric
properties and, optionally, surface immunomagnetic markers; histological analysis
of the cells in comparison with other isolates; and molecular analysis for up to
10 different mRNA's.
Alternatively, if immunomagnetic markers and histology steps were
omitted, the cell sorting, isolation, and molecular analysis would take about 45
minutes from start to finish.
Example 2 - Fabrication IssuesFabrication of Electrodes
Electrode arrays may be fabricated using standard microphotolithographic
techniques. Briefly, one may start with a clean glass substrate coated with 70Å
titanium and 1000Å gold. Coating to NNN-S-450 specification may be done either commercially
by Thin Film Technology, Inc., and guaranteed to be of uniform deposition, pinhole-free
quality and able to withstand 10,000 psi lifting force, or using sputtering. The
resulting gold blanks (up to 125 mm x 125 mm in size) may be spin coated with Shipley
photoresist which is exposed to UV light through a mask using a mask aligner (AB
Manufacturing, San Jose). The resulting pattern is developed and inspected and the
gold and titanium layers are then etched in two steps with KI/I2 and
hot HCl, respectively. Masks are designed by an IC CAD layout package (Design Workshop).
Masks are either made commercially by the e-beam method (masks up to 6" x 6" and
features down to <1µm) or else produced by photographically reducing a 10 x version
of the mask printed on, for instance, a Hewlett-Packard DesignJet 2500CP printer
at 600 dpi (final mask size up to 4.8" x 4.8" and features down to 4µ m).
To prevent cell sticking, electrodes may be silanized to produce a
hydrophobic coating or else coated with TEFLON. Silanization is routinely accomplished
with SigmaCote. TEFLON coating is accomplished by solvent deposition from a fluomcarbon
carrier and subsequent baking onto silanized electrodes or by sputtering (in collaboration
with the Stanford Microfabrication Laboratory).
Device structural fabrication
The glass substrate of the electrode array constitutes the lower wall
of the device. Two approaches may be taken to construct device tops. In the first,
the top wall consists of 4 mm glass into which holes are drilled for inlet and outlet
port connections using a triple-tipped diamond drill. PEEK or TEFLON tubes are glued
into the holes and cut off flush on what may become the inside surface of the device
to form fluid interconnects. The two facing walls of the device are either sealed
along their long edges with UV-curing epoxy glue, held in place by multiple small
plastic clamps, or clamped by a single metal frame machined for the purpose. Fluid
flow paths inside the device are defined in this construction method by a gasket
of between 50 and 400 µm thickness, as required, having a slot cut wherever fluid
flow is desired. The inventors have successfully used gaskets of PTFE, Gore-Tex,
RTV and PDMS polymers. This method is adequate for simple flow paths but for the
more complex flow paths in the integrated microffuidic component required for the
multiple-segment spiral isolation and impedance sensing stage, a method using injection
molded seals may be used. Seals may be made for this purpose in a separate mold
and then sandwiched between a plain top and bottom as described above or the top
of the device may be machined from Lucite and have seals injection molded directly
into it. In this case the seals are made to extend above the surface of the top
plate by a distance equal to the desired channel thickness. Simply pressing the
device top plate against the device bottom then forms the required flow path and
this allows for easy disassembly and cleaning without damaging a gasket. The molding
material used to form the seals is PDMS, a resilient polymer that is durable, biologically
inert, sufficiently compressible to form a good seal against fluids even with limited
compression force, and transparent. In order to realize complex seal patterns, the
inventors use a small Sherline CNC milling machine that operates directly from a
CAD layout. In this way, flow paths that are mathematically defined can be cut directly
into device top blanks under computer control. This allows well-defined, smooth
fluidic pathways to be fabricated quickly and reproduced easily.
Fluid flow control
Fluid flow may be controlled by digital syringe pumps (KD scientific,
Boston, Mass) each capable of holding two syringes of different barrel sizes. The
inventors have found that the useful flow rate from these pumps (i.e. for which
there is an effective absence of pulsations due to stepper motor action) extends
over 7 decades from 0.01 µl/min to 70.57 ml/min. For the fully integrated system
as many as four pumps may be needed to allow automated sample control in the DEP
prefilter, DEP-MAP-FFF stage and isolator. The pumps can be daisy-chained for convenient
serial control by computer or manually controlled. Flow valves may be needed to
control some waste and outlet lines. These can all be mounted off the fluidic device.
Low dead volume valves from Lee may be used for these fluid control needs.
Conductivity measurements of suspending medium solutions may be made
with a Cole-Parmer 19101-00 electronic conductivity meter using either a flow-through
or dip electrode cell with platinum black coated platinum electrodes.
Devices under test may be mounted on the stage of a Zeiss Axiovert
S-100 inverted microscope (magnification X5 - X600) equipped with video recording
and image analysis capabilities. This allows direct observation of any section of
the transparent-walled devices and permits manual or automated visualization of
cells. The microscope is equipped with epifluorescence and a sensitive three color
CCD camera that is used for fluorescence microscopy. By quantifying the signal with
software, fluorescence of molecular probes may be accomplished. For detection of
molecular probe fluorescence signals, the inventors have an Oriel MS257 high sensitivity
fiber optic tuneable dye laser spectrometer system and a Zeis Axiovert 405M inverted
microscope equipped with a Photometrics CH210 liquid nitrogen cooled photon-counting
Electrical fields for DEP/FFF and DEP trapping may be provided from
2 Hewlett-Packard 33120 signal generators (up to 15 V peak-peak, frequencies up
to 50 MHz) with FM and AM sweeping capabilities. For twDEP focusing on the spiral
electrode, four sine signals in quadrature are required and a digitally synthesized
source based on a quadrature-phase numerically controlled oscillator chip may be
used. This may be interfaced to a computer to provide quadrature signals up to 12
MHz and up to 12 V peak-peak with modulation characteristics that can be software
controlled. Signals may be monitored with a Tektronix 200 MHz.digital oscilloscope.
An important task in developing the DEP-MAP-FFF method is designing
magnetic components to provide field distributions that achieve an appropriate distribution
of B▿.B throughout the separation chamber. The
design for the magnet system is shown in FIGS. 10 and 11. This arrangement of magnetic
pole pieces may allow the field to be produced over the large area needed for a
full sized DEP-MAP-FFF separator. Parallel SmCo or NdFB permanent magnets (e.g.,
0.5 Tesla) may be used to provide fields closer to 1 Tesla. The field enhancement
may be accomplished by exploiting boundary conditions on B and
H at the iron surface. The enhancement is controlled by the shape
of the Fe component and, in particular, by the size of the effective pole face.
Field inhomogeneity may be controlled by the sintered iron particles underneath
the DEP-MAP-FFF separation channel. In fact, principles used for creating MAP forces
in the DEP-MAP-FFF separator are the same as used in existing MACS separators. However,
the iron field enhancer and shapers may rely upon a well-defined microgeometry rather
than the random geometries used in present day MACS separators. It should be borne
in mind that the MAP forces needed to control the height of cells in a flow stream
are about an order of magnitude less than those needed to trap cells in a column
against hydrodynamic forces. For this reason the inventors believe that SmCo or
NdFB magnets may be adequate.
Magnetic simulations may be undertaken while magnets are being built
and tested using directional Hall probes to ascertain the field strengths and spatial
inhomogeneity properties. In this way, design, simulation, construction, testing
and refinement steps may go hand-in-hand to produce magnets suitable for the MAP
requirements of this project.
The distribution of the electrical and magnetic fields within the
fluid between the chamber walls determines the DEP and MAP forces experienced by
cells. Although the inventors' early electric field calculations were performed
by the charge density method, implemented by FORTRAN, more recently the inventors
have used the ANSYS multiphysics finite element analysis package to compute field
distributions and have used the post-processing capabilities ofMATLAB to derive
the corresponding DEP force distributions.
DEP electrode geometries known in the art may be used. To achieve
optimalB▿.B distributions for DEP-MAP force balance,
however, one may need to use the ANSYS package to do simulations as a function of
the size, shape and placement of the magnets, the iron field concentrator, and the
sintered iron components. The ANSYS package allows simultaneous electrical and magnetic
computations so that it is ideal for modeling the behavior of the DEP-MAP force
balance properties of various geometries.
Finally, the ANSYS package also allows modeling of hydrodynamic characteristics
of flow channels and the inventors plan to model the behavior of the fluid and cells
as they pass though the integrated device, particularly in the fluid inlet and egress
regions. This may be important in the interface regions between stages of the system
to ensure the design allows efficient sample transport without "dead" spaces in
which cells may settle.
Where needed, a 500 kHz field at 5 V p-p may be used to trap cells
by DEP. This frequency is sufficiently high to penetrate the cell membranes efficiently
without causing damage and induces a strong DEP body force on the cells, trapping
them efficiently against fluid flow. DEP trapping may be used in four ways within
the integrated system: (1) for cells being concentrated in the second segment of
the prefilter following elution of normal cells and for small samples injected directly
before the DEP-MAP-FFF stage; (2) for cell subpopulations that are isolated in the
spiral electrode segments after elution from the DEP-MAP-FFF stage; and, (3) for
holding cells in place during reagent perfusion at several steps in processing;
(4) for holding beads in place for reagent perfusion following cell lysis and hybridization
Based on the inventors' experience with DEP-FFF, up to 2 x 105
cells can be analyzed without cell concentration becoming so large as to cause perturbing
cell-cell interactions in the size of DEP-MAP-FFF fractionator chosen here. For
samples expected to have a high concentration of suspect cells, such as dispersed
cells from biopsies of suspected tumors or fine needle aspiration biopsies, 2 x
105 cells are sufficient to ensure that tumor cells, if present, may
be sufficient for molecular analysis. In such cases, up to 20 µL of cell suspension
may be injected via the preconcentrated sample loading port. For samples in which
the concentration of suspect cells is expected to be so low that there is unlikely
to be sufficient suspect cells in a 2 x 105 cell sample, prefiltering
may be necessary. Samples such as peripheral blood mononuclear cells or dispersed
lymph node cell populations fall into this category.
Following injection of a 20 µL sample or prefiltering, as appropriate,
the secondary trapping electrode may be energized at 250 kHz frequency and 5 V p-p.
All cell types may be trapped from the flow stream by DEP on the electrode in the
entrance region of the DEP-MAP-FFF separator stage. Sample injection into the DEP-MAP-FFF
stage may now occur with an appropriate DEP levitation signal applied. After cells
have been given time to reach equilibrium heights (2-5 minutes) under the influence
of DEP, MAP and gravitational forces, carrier medium flow may be started from a
digital syringe pump (KD scientific, Boston, Mass). The first cell subpopulations
should begin emerging from the DEP-FFF fractionator approximately 2-5 minutes after
the initiation of fluid flow. Frequencies from 10 kHz to 500 kHz, voltages from
0.5 V p-p to 3 V p-p, and carrier fluid conductivities from 5-1000 mS/m may be used.
Cell fractionation, isolation, concentration and bursting may be investigated
in the integrated devices. Cultured breast tumor cells may be mixed with PBMCs to
provide a well-characterized and reproducible model system for investigating the
performance and optimal operating conditions for the component parts of the integrated
system. To assist in tracking the cell subpopulations, one may initially prelabel
the breast cancer cells to facilitate tracking. This may be done in two ways. Initially,
cells may be incubation for 10 mins in 25 µg/ml BCECF-AM (Molecular Probes), a fluorescein
probe that is irreversibly accumulated by cells through the action of nonspecific
esterases. BCECF is only accumulated by viable cells and simultaneously acts as
a viability indicator. In experiments, such labeling allowed convenient tracking
of tumor cells which appeared as brilliant spheres against a dark field of unlabelled
cells, allowing even a single tumor cell within a very large unlabelled population
(> 105 cells) to be instantly identified. This tracking technique
may be used to study the cells by fluorescent microscopy while they are undergoing
separation and manipulations in the device.
Secondly, FTTC-conjugated human epithelial antigen (HEA) antibody
may be used to prelabel breast cancer cells prior to adding them to PBMNC mixtures.
The fluorescence of this labeling procedure is much weaker than BCECF, however cells
emerging from the separator stages can be passed directly into a flow cytometer
and definitively identified as being of epithelial origin by this method.
Cell and cell culture: For model studies, one may use MDA-MB-435,
MDA-MB-453, MDA-MB-236, and MDA-MB-468 human breast cancer lines originally established
by Cailleau et al. as well as MCF-7 originally from the Michigan Cancer Foundation.
These have formed the basis for investigations into many aspects of tumomgenesis
and metastasis, are well characterized, and are available from ATTC to other researchers
for follow-up studies. MDA-MB-453 shows a 64-fold enhancement in mRNA level of HER2/neu
compared with MDA-MB-231 and a comparable increase in cell surface concentration
of the corresponding protein and is therefore suitable for both immunological and
mRNA assays. Tumor cells are cultured in RPMI 1640 medium supplemented with 10%
fetal bovine serum, 1 mM glutamine and 20 mM HEPES buffer in 25-cm2 vented
culture flasks (Costar) at 37°C under a 5% CO2/95% air atmosphere. Cultures
are free of, and are periodically checked by radionucleic acid hybridization assay
(Gen-Probe, Inc.) for, mycoplasma. Cells are harvested from 50-70% confluent cultures
by brief exposure to 0.25% trypsin-0.02% EDTA solution. Viability is determined
by trypan blue dye exclusion.
Samples for DEP fractionation and manipulation may be prepared by
suspending cells in sucrose/dextrose solution to yield suspensions having a specified
conductivity of between 10 and 1000 mS/m and physiological osmolarity (300 mOs/kg).
If necessary, conductivity is adjusted with additional culture medium.
Cell samples can be incubated with antibodies for markers prior to
loading into the separation stages, while at the interface between the prefilter
and DEP-MAP-FFF fractionator stages, and after trapping in the spiral electrode
isolator stage prior to concentration. A series of DEP levitation/trapping cycles
can be applied to "stir" the antibody/cell mixture at each of these steps. Following
labeling, cells may be trapped by positive DEP and washed free of antibodies by
perfusing them with rinsing reagents as many times as needed. Fluorescently, magnetically
or enzymically labeled antibodies can be used. Fluorescence microscopy can be used
to detect fluorescence of the antibodies or of their catalytic by-products. Immunomagnetic
labels may modify the DEP-MAP-FFF properties of cell types in accordance with their
surface marker concentrations. One may use antibodies for human epithelial antigen
(HEA) because this is a useful marker for identifying epithelial cells in blood
and lymph node cell dispersions, and EGF receptor antibody since this is a relevant
prognostic marker for breast cancer. Clearly, these examples are merely exemplary
of the more general applicability of the technology and surface markers relevant
to any different application could be used instead.
twDEP focusing/concentration of cells
The twDEP properties of blood and cultured breast cancer cell lines
are known in the art. A traveling wave field applied to the spiral electrode array
at a frequency that both levitates and translates a cell subpopulation may allow
it to be focused at the center of the spiral. A swept frequency may be applied to
ensure that all cell and bead types on each spiral isolation segment may be swept
to the center to form a highly concentrated mixture. Traveling waves in the frequency
range 10 kHz to 500 kHz, voltages from 0.5 V p-p to 5 V p-p, and carrier fluid conductivities
from 5-1000 mS/m may be used.
In one embodiment, the pumps and signal generators used to operate
the system are all computer controllable. Image processing may use a dual-Pentium
II PCI/EISA mother board. The image grabber may include a real-time image processor
(Image Series 640+Neighborhood Processor with on-board 4 MB memory, Matrox Electronic
Systems Ltd., Dorval, Canada) that is used to acquire images and to accelerate image
operations. Appropriate software known in the art performs real-time process control
of the serial and HPIB devices (pumps, valves, signal sources, digital camera) used
to operate the system and a real-time imaging library (MIL-32 3.10, Matrox Electronic
Systems Ltd., Dorval, Canada) used in conjunction with Labview software may be exploited
for system control and fluorescence detection.
Bursting of cells
Following the trapping of cell fractions on the spiral electrode segments
and their concentration by twDEP, the voltage and frequency applied to the spiral
electrode may be changed to burst the target cells. A further level of cell discrimination
is possible at this stage because targeted bursting can be done on cell mixtures
if desired. Breast cancer cells are typically in the 10-12 µm diameter range and
have specific membrane capacitances of ~20 mF/m2. These parameters in
conjunction with the suspending medium conductivity define the optimum bursting
conditions. These may be examined for target cultured breast cancer and human specimen
cells for carrier fluid conductivities from 5-1000 mS/m. Optimum field conditions
for rapidly bursting all cells on the spiral electrode may also be determined. Voltages
from 10 V peak-peak to 20V peak-peak and frequencies from 10 kHz to 100 kHz may
be used, including swept frequencies.
While the present disclosure may be adaptable to various modifications
and alternative forms, specific embodiments have been shown by way of example and
described herein. However, it should be understood that the present disclosure is
not intended to be limited to the particular forms disclosed. Rather, it is to cover
all modifications, equivalents, and alternatives falling within the spirit and scope
of the disclosure as defined by the appended claims. Moreover, the different aspects
of the disclosed apparatus and methods may be utilized in various combinations and/or
independently. Thus the invention is not limited to only those combinations shown
herein, but rather may include other combinations.
Example 3 - Programmable Fluidic Processor
In one embodiment of the present invention, a programmable fluidic
processor (PFP) may be coupled to the array isolator that may coupled to the electrode
array isolator that is used to trap cells after they exit from the field-flow fractionation
separator. Various embodiments of the PFP are discussed in pending U.S. Application
No. 09/249,955, which has been previously incorporated herein by reference.
As previously indicated, the array isolator may consist of a plurality
of spiral traps. The PFP may be coupled to the spiral traps by a variety of means
known in the art. For example, the PFP may be coupled to the spiral traps by means
of a channel, or the PFP may be integral with the spiral traps. There may be one
or more PFPs. Each spiral trap may have its own PFP, or multiple spiral traps may
be connected to a single PFP.
Once the cells have been trapped on the spiral traps, they may
be moved to the PFP for further analysis. Once the cells have been transferred,
the PFP may be used to programmably manipulate the cells in a variety of ways. FIG.
12 shows one embodiment of the present invention that includes a PFP. As shown in
FIG. 12, a single PFP may be connected to each of the spiral traps.
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Fluidvorrichtung für die Analyse von Zellen, wobei die Vorrichtung aufweist:
(a) einen dielektrophoretischen Feldströmungs-Fraktionierungsseparator, der
einen Fluidkanal und eine Anordnung von Mikroelektroden aufweist, die dafür angeordnet
sind, Zellen zu unterscheiden, indem eine dielektrophoretische Kraft durch eine
Schwerkraft kompensiert wird, um unterschiedliche Zelltypen auf unterschiedlichen
Höhen über dem Boden des Fluidkanals innerhalb eines Geschwindigkeitsprofil in dem
Separator zu positionieren, so dass unterschiedliche Zelltypen durch den Fluidkanal
bei unterschiedlichen Geschwindigkeiten zwecks Trennung transportiert werden, wobei
die getrennten unterschiedlichen Zelltypen aus einem entfernten Ende des Kanals
(b) einen Mehrfachelement-Elektrodenanordnungsisolator, der mehrere Elektrodenelemente
aufweist, die mit dem Separator verbunden sind und so konfiguriert sind, dass jedes
Elektrodenanordnungselement eine getrennte Fraktion von Zellen als eine Funktion
der Austrittszeit der Zellen aus dem Separator einfängt.
Vorrichtung nach Anspruch 1, wobei die Elektrodenelemente Spiralen-Elektrodenelemente
Vorrichtung nach Anspruch 1, wobei die Vorrichtung ferner aufweist:
einen programmierbaren Fluidprozessor, der mit dem Elektrodenanordnungsisolator
Vorrichtung nach Anspruch 2 oder 3, wobei die Vorrichtung ferner aufweist:
ein mit dem Separator verbundenes dielektrophoretisches Vorfilter, wobei das
Vorfilter eine oder mehrere Einfangelektroden aufweist, die dafür konfiguriert sind,
wenigstens einen Teil von den Zellen mit einer dielektrophoretischen Kraft einzufangen.
Vorrichtung nach Anspruch 2, wobei der Separator ferner einen Magneten aufweist,
der dafür konfiguriert ist, mit einer magnetophoretischen Kraft die Zellen auf Positionen
innerhalb des Geschwindigkeitsprofils in dem Separator zu verschieben.
Vorrichtung nach Anspruch 5, wobei der Magnet aus mehreren flachen Magneten
aus SmCo oder NdFeB Materialien besteht, die in einer parallelen Konfiguration in
einer gegenüberliegenden Polorientierung angeordnet sind.
Vorrichtung nach Anspruch 1, wobei jedes von den mehreren Spiral-Elektrodenelementen
dafür konfiguriert ist, dass es durch ein Signal mit nur einer Frequenz erregt wird,
wobei aber die Phase des Signals für jedes von den mehreren Elektrodenelementen
Vorrichtung nach Anspruch 7, wobei die Vorrichtung ferner vier Elektrodenelemente
aufweist, und wobei die Phasen des Signals 0°, 90°, 180°, 270° sind.
Vorrichtung nach Anspruch 4, wobei die Vorrichtung ferner aufweist:
einen Reagens-Anschluss, der dafür konfiguriert ist, die Injektion von Reagenzien
auf die auf den Spiral-Elektrodenelementen eingefangenen Zellen zu ermöglichen.
Verfahren zur Zellisolation und Analyse, wobei das Verfahren die Schritte aufweist:
Einführen von Zellen in den dielektrophoretischen Feldströmungs-Fraktionierungsseparator
gemäß Definition in Anspruch 1;
Unterscheiden der Zellen in dem Separator, wobei der Unterscheidungsschritt
den Schritt der Kompensation einer dielektrophoretischen Kraft mit einer Schwerkraft
umfasst, um die Zellen auf Positionen innerhalb eines Geschwindigkeitsprofils in
dem Separator zu verschieben; und
Einfangen wenigstens eines Teils von aus dem Separator austretenden den Zellen
mit dem Mehrfachelement-Elektrodenanordnungsisolator, der mehrere Spiral-Elektrodenelemente
aufweist, gemäß Definition in Anspruch 1, als eine Funktion der Austrittszeit
der Zellen aus dem Separator.
Verfahren nach Anspruch 10, wobei das Verfahren ferner den Schritt aufweist:
Manipulation der Zellen mit einem programmierbaren Fluidprozessor, der mit dem
Elektrodenanordnungsisolator verbunden ist.
Verfahren nach Anspruch 10, wobei das Verfahren ferner den Schritt der Lösung
der durch den Mehrfachelement-Elektrodenanordnungsisolator eingefangenen Zellen
aufweist, wobei insbesondere der Schritt der Lösung die Verwendung von elektrischen
Verfahren nach Anspruch 10, wobei wenigstens ein Teil der Zellen zu Beginn mit
der Oberfläche eines Trägerteilchens verbunden ist.
Verfahren nach Anspruch 10 oder 11, wobei das Verfahren ferner die Schritte
Einführen der Zellen in das dielektrophoretisches Vorfilter gemäß Definition
in Anspruch 2, um dadurch wenigstens einen Teil von den Zellen mit einer
dielektrophoretischen Kraft einzufangen, und Weiterleiten der von dem Vorfilter
eingefangenen Zellen in einen mit dem Vorfilter verbundenen Separator.
Verfahren nach Anspruch 14, wobei mehrere Analyseteilchen mit den Zellen gemischt
werden, nachdem die Zellen aus dem Separator austreten.
Verfahren nach Anspruch 15, wobei das Verfahren ferner den Schritt der Konzentration
der Zellen auf den mehreren Spiral-Elektrodenelementen aufweist, der Schritt der
Konzentration der Zellen den Schritt der Erregung der mehreren Spiral-Elektrodenelementen
mit einem mehrphasigen Feld aufweist, wobei insbesondere das mehrphasige Feld vier
Phasen aufweist und eine Frequenz zwischen 10 kHz bis 200 kHz aufweist.
Verfahren nach Anspruch 10, wobei die Zellen mit magnetisch markierten Antikörpern
A fluidic device for the analysis of cells, the device comprising:
(a)a dielectrophoretic field-flow fractionation separator comprising a fluidic
channel and an array of micro-electrodes configured to discriminate cells by balancing
a dielectrophoretic force with a gravitational force to position different cell
types to different heights above the floor of the fluidic channel within a velocity
profile in the separator so that different cell types are carried through the fluidic
channel at different velocities to become separated, the separated different cell
types emerging from a far end of the channel; characterized by
(b)a multi-element electrode array isolator comprising a plurality of electrode
elements coupled to the separator and configured so that each electrode array element
traps a separate fraction of cells as a function of the cells' time of emergence
from the separator.
The device of claim 1, wherein the electrode elements are spiral electrode elements.
The device of claim 1, the device further comprising
a programmable fluidic processor coupled to the electrode array isolator.
The device of claim 2 or 3, the device further comprising
a dielectrophoretic prefilter coupled to the separator, the prefilter comprising
one or more trapping electrodes configured to trap at least a portion of the cells
with a dielectrophoretic force.
The device of claim 1, wherein the separator further comprises a magnet configured
to displace with a magnetophoretic force the cells to positions within the velocity
profile in the separator.
The device of claim 5, wherein the magnet are several flat magnets of SmCo or
NdFeB materials placed at a parallel configuration in an opposing pole orientation.
The device of claim 1, wherein each of the plurality of spiral electrode elements
is configured to be energized by a signal of a single frequency, but wherein the
phase of the signal is different for each of the plurality of electrode elements.
The device of claim 7, the device further comprising four electrode elements,
and wherein the phases of the signal are 0°, 90°, 180°, 270°.
The device of claim 4, the device further comprising
a reagent port configured to allow for the injection of reagents onto the cells
trapped on the spiral electrode elements.
A method for cell isolation and analysis, the method comprising the steps of:
introducing cells into the dielectrophoretic field-flow fractionation separator
as defined in claim 1;
discriminating the cells in the separator, the step of discriminating comprising
the step of balancing a dielectrophoretic force with a gravitational force to displace
the cells to positions within a velocity profile in the separator; and
trapping at least a portion of the cells emerging from the separator with the
multi-element electrode array isolator comprising a plurality of spiral electrode
elements, as defined in claim 1, as a function of the cells' time of emergence from
The method of claim 10, the method further comprising the step of
manipulating the cells with a programmable fluidic processor coupled to the electrode
The method of claim 10, the method further comprising the step of lysing the
cells trapped by the multi-element electrode array isolator, in particular wherein
the step of lysing comprises using AC electrical fields.
The method of claim 10, wherein at least a portion of the cells is initially
coupled to the surface of a carrier bead.
The method of claim 10 or 11, the method further comprising the steps of
introducing the cells into the dielectrophoretic prefilter as defined in claim
3, thereby trapping at least a portion of the cells with a dielectrophoretic force,
directing the cells trapped from the prefilter into the separator coupled to
The method of claim 14, wherein a plurality of analysis beads is mixed with
the cells after the cells emerge from the separator.
The method of claim 15, the method further comprising the step of concentrating
the cells on the plurality of spiral electrode elements, the step of concentrating
the cells comprising the step of energizing the plurality of spiral electrode elements
with a multi-phase field, in particular wherein the multi-phase field comprises
four phases, and comprises a frequency between 10 kHz to 200 kHz.
The method of claim 10, wherein the cells are incubated with magnetically labeled
Appareil basé sur le flux pour l'analyse de cellules, l'appareil comprenant
(a) un séparateur par fractionnement par couplage flux-force diélectrophorétique
comprenant un canal de flux et une rangée de micro-électrodes configurée pour différencier
les cellules en équilibrant une force diélectrophorétique avec une force gravitationnelle
pour positionner différents types cellulaires à différentes hauteurs au-dessus de
la base du canal de flux à l'intérieur d'un profil de vitesse dans le séparateur
de manière à ce que différents types cellulaires soient portés le long du canal
de flux à différentes vitesses pour qu'ils se séparent, les différents types cellulaires
séparés sortant d'une extrémité éloignée du canal ;caractérisé par
(b) un isolateur à rangée d'électrodes à plusieurs éléments, comprenant une
pluralité d'éléments d'électrodes couplés avec le séparateur et configurés de manière
à ce que chaque élément de la rangée d'électrodes piège une fraction séparée de
cellules en fonction du temps de sortie du séparateur des cellules.
Appareil selon la revendication 1, dans lequel les éléments d'électrodes sont
des éléments d'électrodes en spirale.
Appareil selon la revendication 1, l'appareil comprenant de plus
un processeur fluidique programmable couplé à l'isolateur à rangée d'électrodes.
Appareil selon la revendication 2 ou 3, l'appareil comprenant de plus
un pré-filtre diélectrophorétique couplé au séparateur, le pré-filtre comprenant
une ou plusieurs électrodes de piégeage configurées pour piéger au moins une portion
des cellules avec une force diélectrophorétique.
Appareil selon la revendication 1, dans lequel le séparateur comprend de plus
un aimant configuré pour déplacer les cellules avec une force magnétophorétique
à des positions à l'intérieur du profil de vitesse dans le séparateur.
Appareil selon la revendication 5, dans lequel l'aimant est constitué de plusieurs
aimants plats de matériau SmCo ou NdFeB placés dans une configuration en parallèle
dans une orientation à pôles opposés.
Appareil selon la revendication 1, dans lequel chacun de la pluralité d'éléments
d'électrodes en spirale est configuré pour être stimulé par un signal à une seule
fréquence, mais, dans lequel la phase du signal est différente pour chacun des différents
Appareil selon la revendication 7, l'appareil comprenant de plus quatre éléments
d'électrodes, et dans lequel les phases du signal sont de 0°, 90°, 180°,270°.
Appareil selon la revendication 4, l'appareil comprenant de plus
un port de réactifs configuré pour permettre l'injection de réactifs sur les
cellules piégées sur les éléments d'électrodes en spirale.
Procédé pour isoler et analyser des cellules, le procédé comprenant les étapes
consistant à :
introduire des cellules dans le séparateur par fractionnement par couplage flux-force
diélectrophorétique tel que défini dans la revendication 1 ;
distinguer des cellules dans le séparateur, l'étape de distinction comprenant
l'étape d'équilibrage d'une force diélectrophorétique avec une force gravitationnelle
pour déplacer les cellules jusqu'à des positions à l'intérieur d'un profil de vitesse
dans le séparateur ; et
piéger au moins une portion des cellules sortant du séparateur avec l'isolateur
à rangée d'électrodes à plusieurs éléments comprenant une pluralité d'éléments d'électrodes
en spirale, tel que défini dans la revendication 1, en fonction du temps de sortie
du séparateur des cellules.
Procédé selon la revendication 10, le procédé comprenant de plus les étapes
manipuler les cellules avec un processeur fluidique programmable couplé à l'isolateur
à rangée d'électrodes.
Procédé selon la revendication 10, le procédé comprenant de plus l'étape de
lyse des cellules piégées par l'isolateur à rangée d'électrodes à plusieurs éléments,
en particulier dans lequel l'étape de lyse comprend l'utilisation de champs électriques
de courant alternatif.
Procédé selon la revendication 10, dans lequel au moins une portion des cellules
est initialement couplée à la surface d'une bille porteuse.
Procédé selon la revendication 10 ou 11, le procédé comprenant de plus les étapes
introduire les cellules dans le pré-filtre diélectrophorétique tel que défini
dans la revendication 3, piégeant ainsi au moins une portion des cellules avec une
force diélectrophorétique, et
orienter les cellules piégées depuis le pré-filtre jusque dans le séparateur
couplé au pré-filtre.
Procédé selon la revendication 14, dans lequel une pluralité de billes d'analyse
sont mélangées avec les cellules après que les cellules sont sorties du séparateur.
Procédé selon la revendication 15, le procédé comprenant de plus l'étape de
concentration des cellules dans une pluralité d'éléments d'électrodes en spirale,
l'étape de concentration des cellules comprenant l'étape de stimulation de la pluralité
d'éléments d'électrodes en spirale avec un champ à phases multiples, en particulier
dans lequel le champ à phases multiples comprend quatre phases, et comprend une
fréquence entre 10kHz et 200kHZ.
Procédé selon la revendication 10, dans lequel les cellules sont incubées avec
des anticorps marqués de manière magnétique.