WO2013096304A1 - Appareils et procédés de séparation diélectrophorétique en flux continu - Google Patents

Appareils et procédés de séparation diélectrophorétique en flux continu Download PDF

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WO2013096304A1
WO2013096304A1 PCT/US2012/070333 US2012070333W WO2013096304A1 WO 2013096304 A1 WO2013096304 A1 WO 2013096304A1 US 2012070333 W US2012070333 W US 2012070333W WO 2013096304 A1 WO2013096304 A1 WO 2013096304A1
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fluid
chamber
cells
conductivity
flow
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PCT/US2012/070333
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Peter R. GASCOYNE
Thomas E. ANDERSON, Jr.
Sangjo SHIM
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Board Of Regents, The University Of Texas System
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/06—Investigating concentration of particle suspensions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C2201/00—Details of magnetic or electrostatic separation
    • B03C2201/26—Details of magnetic or electrostatic separation for use in medical applications
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C5/00—Separating dispersed particles from liquids by electrostatic effect
    • B03C5/005—Dielectrophoresis, i.e. dielectric particles migrating towards the region of highest field strength
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C5/00—Separating dispersed particles from liquids by electrostatic effect
    • B03C5/02—Separators
    • B03C5/022—Non-uniform field separators
    • B03C5/026—Non-uniform field separators using open-gradient differential dielectric separation, i.e. using electrodes of special shapes for non-uniform field creation, e.g. Fluid Integrated Circuit [FIC]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N2001/4038—Concentrating samples electric methods, e.g. electromigration, electrophoresis, ionisation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/49—Blood
    • G01N33/491—Blood by separating the blood components

Definitions

  • the present disclosure relates generally to discrimination and/or separation of target matter (e.g., from a biological sample). More particularly, but not by way of limitation, the present disclosure relates to continuous-flow dielectrophoretic separation of target matter.
  • cell separation may have numerous applications in areas such as medicine, biotechnology, biomedical research, environmental monitoring and bio/chemical warfare defense.
  • cell separation can make possible certain life-saving procedures such as autologous bone marrow transplantation for the remediation of advanced cancers where the removal of cancer-causing metastatic cells from a patient's marrow is necessitated (Fischer, 1993).
  • highly purified cell subpopulations permit studies that would otherwise be impossible.
  • a rotational electrical field is applied and the interaction between the cells' polarization and the applied field can result in cell rotation. If that field is inhomogeneous, then the cells can experience a lateral dielectrophoretic (DEP) force, the frequency response of which is a function of their intrinsic electrical properties (Gascoyne el al., 1992). In turn, these properties may depend strongly on cell composition and organization, features that generally reflect cell morphology and phenotype. Cells differing in their electrical polarizabilities can thus experience differential forces in the inhomogeneous electric field (Becker et al., 1994; Becker el al., 1995).
  • DEP dielectrophoretic
  • DEP force may induce separation between particles of different characteristics.
  • DEP has been used on a microscopic scale to separate bacteria from erythrocytes (Markx et al., 1994), viable from nonviable yeast cells (Wang et al., 1993), and erythroleukemia cells from erythrocytes (Huang et al., 1992).
  • the differences in the electrical polarizabilities of the cell types in those various mixtures were greater than those to be expected in many typical cell sorting applications.
  • FFF Field flow fractionation
  • the technique can be used to separate different types of matter, for example, having a size from about 1 nm to more than about 100 micrometers, which may include, for example, biological and non-biological matter. Separation according to field flow fractionation generally occurs by differential retention in a stream of liquid flowing through a channel. FFF techniques may combine elements of chromatography, electrophoresis, and ultracentrifugation, and may utilize a flow velocity profile established when the fluid is caused to flow through the channel. Such a velocity profile may be, for example, linear or parabolic.
  • a field may be applied at right angles to the flow in the channel, and the field may serve to drive the matter into different positions within the flow velocity profile.
  • the matter being displaced at different positions within the velocity profile can then be carried with the fluid flow through the chamber at differing velocities.
  • Fields may be based on sedimentation, crossflow, temperature gradient, centrifugal forces, and the like.
  • DEP-FFF dielectrophoretic field flow fractionation
  • CTCs circulating tumor cells
  • PBMNs peripheral blood mononuclear cells
  • target cells do not need to be trapped by the electric field.
  • the maximum practical specimen size for high isolation efficiency isolation of target cells is generally less than 5 million cells in each run or batch.
  • the present disclosure includes apparatuses and methods for continuous flow dielectrophoretic separations. Embodiments can, for example, be optimized based on DEP and FFF.
  • Some embodiments of the present apparatuses comprise: a body defining a chamber having one or more inlets and one or more outlets, where the one or more inlets are configured to receive a first fluid including target matter and a second fluid.
  • the chamber can include: a first portion having a first end, and a second end between the one or more inlets and the one or more outlets; and a second portion between the second end of the first portion and at least one of the one or more outlets.
  • the apparatus can also include one or more electrodes configured to generate a non-uniform electric field in the second portion of the chamber.
  • the apparatus is configured such that if a first fluid including target matter and a second fluid are introduced into the chamber through the one or more inlets: the first fluid and the second fluid can flow substantially laminarly through the first portion to permit diffusion of solutes between the first fluid and the second fluid; and the one or more electrodes can generate a dielectrophoretic force on the target matter in the second portion to extract the target matter from the first fluid.
  • the apparatus is further configured such that during the substantially laminar flow through the first portion, the cross-sectional area of the first fluid is smaller than the cross-sectional area of the second fluid.
  • the length of the first portion is large enough to permit the first fluid and the second fluid to substantially reach diffusion equilibrium in the first portion, where the diffusion in the first portion reduces the conductivity of the first fluid.
  • the chamber includes a bottom surface and side surfaces, and the one or more electrodes are disposed on or beneath the bottom surface.
  • the width of the chamber is larger than the height of the chamber.
  • the chamber is configured to direct the first and second fluids from the one or more inlets to the one or more outlets according to a predetermined velocity profile.
  • the second fluid may include one or more osmolytes configured to facilitate diffusion between the first fluid and the second fluid to reduce the conductivity of the first fluid.
  • the first portion of the chamber does not include electrodes that are configured to generate a non-uniform electric field.
  • the one or more inlets comprise a first inlet configured to receive the first fluid and a second inlet configured to receive the second fluid; and, optionally, where the first inlet is angled relative to (e.g., perpendicular to) the direction of flow in the first portion of the chamber.
  • the one or more outlets comprise a first outlet configured to permit the target matter to exit the chamber and a second outlet configured to permit the remainder of the first and second fluids to exit the chamber; and, optionally, where the first outlet is angled relative to (e.g., perpendicular to) the direction of flow in the second portion of the chamber.
  • the apparatus further comprises a signal generator coupled to the one or more electrodes, the signal generator configured to generate an electric signal with a frequency and voltage for the non-uniform electric field.
  • the apparatus includes a conductivity sensor configured to measure a conductivity of a fluid in the chamber and a controller configured to calculate one or more target properties of the non-uniform electric field based on at least one of measured conductivity of the first fluid and a property of the target matter.
  • the controller is further configured to adjust the electric signal generated by the signal generator to cause the non-uniform electric field to substantially include the one or more target properties.
  • the apparatus includes a current sensor configured to measure a current of the electric signal and a controller coupled to the current sensor and configured to compare the measured current to a target current, and the controller is further configured to adjust the electric signal based on the comparison.
  • adjusting the electric signal includes changing at least one of the frequency and the voltage of the electric signal.
  • Some embodiments of the present methods includes steps necessary to carry out the functions described above with respect to the operation of the described apparatus.
  • Some embodiments of the present methods comprise: introducing a first fluid including target matter and a second fluid into a chamber of an apparatus, the chamber having one or more inlets and one or more outlets.
  • the chamber includes: a first portion having a first end, and a second end between the one or more inlets and the one or more outlets; and a second portion between the second end of the first portion and at least one of the one or more outlets.
  • the apparatus also includes one or more electrodes configured to generate a non-uniform electric field in the second portion of the chamber.
  • the method includes causing the first and second fluids to flow substantially laminarly through the first portion of the chamber such that diffusion between the first fluid and the second fluid lowers the conductivity of the first fluid, and applying an electric signal to the one or more electrodes to generate a dielectrophoretic force on the target matter to extract the target matter from the first fluid.
  • the method also includes outputting the target matter through a first outlet of the chamber and outputting the remainder of the first and second fluids through a second outlet of the chamber; and, optionally, where: the first fluid is introduced through a first inlet that is angled relative to the first portion of the chamber; and/or the first outlet is angled relative to the direction of flow in the second portion of the chamber.
  • the method includes controlling the flow rate of the first fluid and the second fluid such that the first fluid and the second fluid flow through the first and second portions of the chamber according to a target velocity profile, where the target velocity profile is such that the first fluid and the second fluid substantially reach diffusion equilibrium in the first portion of the chamber.
  • the method includes measuring conductivity of the first fluid; calculating one or more target properties of the non-uniform electric field based on at least one of: measured conductivity of the first fluid and a property of the target matter; and adjusting the electric signal to include the one or more target properties.
  • the method includes measuring a current of the electric signal; comparing the measured current to a desired current; and adjusting the electric signal based on the comparison.
  • adjusting the electric signal includes changing at least one of the frequency and the voltage of the electric signal.
  • the method includes adding one or more osmolarity-altering components, density-altering components, and/or viscosity-altering components to the first and/or second fluids to achieve desired osmolarity, density and/or viscosity relationships between the first and second fluids.
  • compositions related to antibody- independent isolation of circulating tumor cells by continuous-flow dielectrophoresis are provided.
  • dielectrophoresis is employed to discriminate between blood and tumor cells, for example, on the basis of cell size and/or membrane morphology that contribute to differences in cell membrane area and consequently to cell dielectric properties.
  • the separation criterion is independent of cell surface protein markers.
  • DEP allows isolation of CTCs that are applicable to a broad range of cancers.
  • dielectric properties of a cancer cell type are significantly different from those of PBMNs and allow their isolation from blood.
  • Exemplary cancer types include lung, breast, prostate, pancreas, brain, skin, kidney, colon, liver, endometrium, thyroid, heart, nerve, stomach, spleen, testicular, ovarian, gall bladder, head and neck, blood, bone, liver, and so forth.
  • Some embodiments of the present methods are configured for and/or are capable of efficiently isolating cultured cells from PBMNs of healthy donors or efficiently isolated CTCs from clinical specimens derived from cancer patients.
  • Some embodiments of the present methods allow continuous flow microfluidic processing at rates of at least 10 6 nucleated cells/minute; in some cases, the rate is up to 10 7 cell/min.
  • a continuous flow microfluidic processing chamber is utilized into which a peripheral blood mononuclear cell fraction of a clinical specimen is slowly injected, deionized by diffusion, and then subjected to a balance of DEP, sedimentation and hydrodynamic lift forces, for example.
  • these forces cause tumor cells to be transported close to the floor of a chamber of an apparatus while blood cells are carried about three cell diameters above them.
  • the tumor cells may be isolated by skimming them from the bottom of the chamber while the blood cells flow to waste, in certain embodiments.
  • circulating tumor cells there is isolation of circulating tumor cells from a clinical specimen and verification of the tumor origin of these cells by molecular analysis. Upon verification of the tumor origin of the cells, a medical provider may then draw prognostic inferences about the disease state of the individual from whom the specimen was derived and/or provide suitable therapy to the individual from whom the specimen was derived, including chemotherapy, surgery, immunotherapy, radiation, and so forth, that is tailored for the specific cancer.
  • a medical provider may then infer that the individual from whom the specimen was derived has cancer.
  • circulating tumor cells there is isolation of circulating tumor cells from a clinical specimen withdrawn for screening purposes from a patient who is in remission from cancer. Upon verification of the tumor origin of the cells, a medical provider may then infer that the individual from whom the specimen was derived has relapsed and once again has cancer.
  • specimens utilized in embodiments of the present methods may originate from a variety of sources; such specimens may come from an individual, a hospital or medical facility, or a repository, for example.
  • the specimens may or may not have been stored prior to processing of the specimens with one or more methods of the invention.
  • the processing methods may be performed by the individual that collected the specimen(s) or they may be performed by a separate entity.
  • the specimens may originate from a healthy individual or an individual that has cancer, is at risk for having cancer, or is in remission from cancer, or is suspected of having cancer, for example because of analysis from the same or other specimen(s) using an alternative method (biopsy, histology, immunoanalysis, and so forth).
  • any embodiment of any of the present methods or apparatuses can consist of or consist essentially of - rather than comprise/include/contain/have - any of the described steps, elements, and/or features.
  • the term “consisting of or “consisting essentially of” can be substituted for any of the open-ended linking verbs recited above, in order to change the scope of a given claim from what it would otherwise be using the open-ended linking verb.
  • FIG. 1A is a schematic diagram illustrating one embodiment of an apparatus for optimized continuous flow dielectrophoretic separations.
  • FIG. IB is a schematic diagram illustrating one embodiment of a body of an apparatus for optimized continuous flow dielectrophoretic separations.
  • FIG. 2 is a schematic flow chart illustrating one embodiment of a method optimized continuous flow dielectrophoretic separations.
  • FIGS. 3A and 3B are schematic flow charts illustrating additional steps for one embodiment of a method optimized continuous flow dielectrophoretic separations.
  • FIG. 4 depicts a side view of DEP-FFF isolation of tumor cells from PBMNs in an embodiment of the present continuous-flow chambers.
  • the relative vertical scale of the extremely thin chamber is exaggerated by ⁇ 120-fold for clarity.
  • FIG. 5 depicts COMSOL Multiphysics simulations of the fluid flow behavior at the DEP-FFF chamber withdrawal slot for different geometries and flow rates: (a) when the withdrawal slot width d ou , is small compared with the chamber height H, optimal skimming behavior is observed with negligible vortices or regions of low flow rate; (b) as the relative withdrawal slot width is increased, a vortex forms within the slot, streamlines from the main channel are depressed into the slot region, and zones of low flow rate appear; (c) the skimming height h s accurately follows that predicted by simple Poiseuille flow theory. Vorticity (d) and depression of the streamlines from the main channel (e) increase with increasing slot width.
  • FIG. 6 depicts COMSOL-multiphysics simulations of the conductivity distribution in the flow stream (a) at the specimen injection zone (with a specimen-containing first fluid entering a first, smaller inlet and having a conductivity of 1400 mS.m “1 , and a second fluid entering a larger, second inlet having a conductivity of 30 mS.m '1 ), (b) at the midpoint of the cell settling and ion diffusion zone (in which diffusion has occurred and conductivity varies from about 64 mS.m "1 at the bottom of the chamber to about 30 mS.m '1 at the top of the chamber), and (c) at the cell skimming zone (in which conductivity is a substantially uniform 61.5 mS.m '1 throughout the height of the chamber).
  • FIG. 7 depicts height distributions of MDA- B-231 human breast cancer cells and PBMNs as they flowed through the chamber in force-balance equilibrium during batch mode DEP-FFF separation.
  • the height distribution was mapped from the cell elution times assuming that the transit velocities of the cells reflected their heights in the Poiseuille hydrodynamic flow profile inside the DEP-FFF chamber.
  • FIG. 8 depicts flow cytometric (FACS) scattergrams showing the recovery of tumor cells from PBMNs spiked with (a) 6000, (b) 2000 and (c) 500 MDA-MB-435 cultured cells prelabeled with CellTracker Green fluorescent dye.
  • FIG. 9 depicts an image of circulating tumor cells collected by continuous flow DEP- FFF from the peripheral blood of a patient with colon cancer. The green (lighter) fluorescence reveals staining of cytokeratin in the tumor cells by F1TC -conjugated CK3-6H5 antibodies. PBMNs show only blue (darker) fluorescence due to DAPI staining of their nuclei. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
  • Coupled is defined as connected, although not necessarily directly, and not necessarily mechanically; two items that are “coupled” may be unitary with each other.
  • the terms “a” and “an” are defined as one or more unless this disclosure explicitly requires otherwise.
  • the term “substantially” is defined as largely but not necessarily wholly what is specified (and includes what is specified; e.g., substantially 90 degrees includes 90 degrees and substantially parallel includes parallel), as understood by a person of ordinary skill in the art. In any disclosed embodiment, the terms “substantially,” “approximately,” and “about” may be substituted with "within [a percentage] of what is specified, where the percentage includes .1 , 1 , 5, and 10 percent.
  • an apparatus, or a component of an apparatus, that is configured in a certain way is configured in at least that way, but it can also be configured in ways than those specifically described.
  • the present methods and apparatuses may be suitable for many applications in which it may be desirable to separate and/or isolate certain particles or packets or matter having certain properties (which may be referred to in this disclosure as “target matter” or “target particles”), from a mixture containing additional particles having one or more different properties (which may be referred in this disclosure as “background particles”).
  • target matter or target particles
  • background particles additional particles having one or more different properties
  • the present apparatuses and methods can be applied to the isolation of circulating tumor cells from the peripheral blood of cancer patients, and/or isolation of infected cells and/or infectious agents from the blood, urine, saliva or lavage specimens of individuals carrying disease or exposed to disease or to disease agents.
  • the present apparatuses and methods can also be applied to the isolation of stem and/or progenitor cells or of other types of target cells from mixtures containing other cell and/or particle types.
  • the present apparatuses and methods can be applied to purifying particles having desired conductive and/or dielectric properties from particles lacking desired conductive and/or dielectric properties.
  • the present apparatuses and methods can be applied to isolating pathogens, biowarfare or bioterrorism agents from liquid suspensions containing mixtures of particles that may contain dirt, pollen, and/or other non-target particles.
  • the present apparatuses and methods can be applied to isolate one type of living organism from non living particles and/or from other types of living organisms (e.g., isolating target bacteria from dirt and/or from non-target bacteria).
  • the present apparatuses and methods can be applied to isolating metals and/or minerals having desired conductive and/or dielectric properties from particles lacking desired conductive and/or dielectric properties.
  • the present apparatuses and methods can be applied to the isolation of desired foodstuffs from non-desired contaminants, and/or to the isolation of pathogens and/or potential pathogens from foodstuffs in order to facilitate the detection and/or identification of said pathogens.
  • Embodiments of the present apparatuses and methods allow for adjustment of properties of a suspending medium (e.g., a first fluid) including target matter (e.g., cells) via diffusion between the first fluid and a second fluid in a chamber immediately prior to the first fluid entering a DEP portion or section of the chamber.
  • a suspending medium e.g., a first fluid
  • target matter e.g., cells
  • this temporal proximity of the adjustment of properties of the first fluid to the DEP sorting can result in reduced damage (e.g., cell rupture, reduced activity, and/or the like) to target matter (e.g., cells) than might otherwise be caused by traditional cell preparation.
  • particles are subjected to DEP, sedimentation and hydrodynamic lift forces as they move through a chamber towards one or more exits or outlets. At least one of these forces generally varies with distance from the chamber floor, and at least one of these forces is configured to oppose the other forces.
  • the flow conditions are generally selected to enable particles to spend enough time traveling under the influence of the applied forces to attain equilibrium heights over the DEP electrode array. Attainment of equilibrium heights can assure that particles exit the chamber at a height that reflects their physical properties (e.g., independent of the position at which they entered the chamber).
  • Fractionation in such continuous-flow DEP-FFF apparatuses and methods can be achieved by withdrawing or permitting fluid to exit through two or more ports or outlets at the exit end or portion of the chamber.
  • the ports can be arranged to skim off fluid that contains particles within defined height ranges above the chamber floor.
  • the flow rate through a first exit port may be configured to cause particles up to a defined height above the chamber floor to be captured through a first exit port
  • the flow rate through a second exit port may be configured to cause particles travelling above said the height of the first exit port to exit through a second exit port.
  • DEP-FFF can be configured to allow the particles to reach an equilibrium height above the chamber floor at which the DEP, sedimentation (gravitational), and hydrodynamic lift forces substantially balance. If the particles travel through the chamber without contacting the chamber floor (so-called hyperlayer mode DEP-FFF), the height h above the accumulation wall at which a particle moves is determined by the balance of vertical DEP, sedimentation and hydrodynamic lift (HDL) forces:
  • F ml is the sedimentation force j ⁇ ⁇ R 3 (p p - p )g , where p p and p x are the densities of the particle and the DEP-FFF elutant medium, respectively, and g is the acceleration due to gravity.
  • the cell density ⁇ for a given particle type is usually considered to be a fixed parameter, but alterations in suspension conditions such as osmolarity may impact it dynamically for cells, for example. It is evident from the expression for the sedimentation force that the density of the suspension medium p s is a parameter that will generally impact the force balance in equation [1] and thereby influence the behavior of microparlicles and/or cells in DEP-FFF.
  • Equation [2] is an approximation, and it will be understood by those skilled in the art that more complex expressions may govern the hydrodynamic lift properties of microparticles and/or cells in specific cases. Nevertheless, the more complex expressions are also expected to depend upon the viscosity 3 ⁇ 4 and shear rate v 0 of the suspending medium. It follows that the viscosity 3 ⁇ 4 is a parameter that will impact the force balance in equation
  • F n I> arises from the dielectric response of a particle to an imposed inhomogeneous electric field.
  • the field induces electric polarization whereby equal and opposite charges build up on opposite sides of the particle to form an electric dipole. Because the electric field varies spatially, the field intensities on the opposing charges on either side of the particle are different, imposing coulombic forces that do not balance.
  • the residual net force called the dielectrophoretic (DEP) force, acts on the particle even though it retains zero net charge. Because reversal of the field also causes reversal of the particle polarization, the DEP force direction is independent of the field sense.
  • the buildup of charges in DEP is not spontaneous, but depends on both particle dielectric and geometric characteristic and the medium. Particle DEP responses to fields of different frequencies may be used to infer the particle properties and to impose separation forces on different particle types.
  • Suitable inhomogeneous electric fields for DEP can be created by an array of phased electrodes and, depending on the configuration and excitation phases, the electric field pattern may move through space (a so-called traveling wave) or form a fixed field pattern.
  • DEP forces for many field types have been explored, in FFF F D ! , is usually produced by a fixed electric field distribution created by energizing, in anti-phase, two interdigitated arrays of microelectrodes patterned all over the accumulation wall of the FFF chamber.
  • the electrode array consists of parallel plain microclectrode strips of equal width and spacing s , the vertical component of the DEP force due to fringing fields above the electrode plane may be written as
  • V is the AC voltage of frequency / that energizes the microelectrode array to provide the electric field. V and may be adjusted to program the DEP response.
  • P cff ( ) which ideally approaches unity, defines the proportion of the applied excitation voltage that is effective in imposing a DEP force on particles within the eluate.
  • P eJJ ( ) accounts for frequency-dependent voltage drops caused not only by electrode polarization at the electrode-eluate interface but also by electrode imperfections and stray impedance in the leads, buses and electrodes downstream of the voltage measurement point.
  • Equation [3] shows that the voltage V is a parameter that will impact the force balance in equation [1J and thereby influence the behavior of microparticles and/or cells in DEP- FFF. Furthermore, P eg ⁇ f) is a parameter that will impact the force balance in equation [1].
  • Equation [3] shows further that it is possible to compensate for changes in P eff (/ ) by adjusting
  • Equation [5] applies specifically to viable cells.
  • f 0 is a characteristic crossover frequency at which a given cell type exhibits a null DEP response. f 0 depends both on the cell properties and on the conductivity of the medium in which the cell is suspended. More precisely, for a cell having a low plasma membrane conductivity, where R is the cell radius, C mem is the cell plasma membrane capacitance per unit area of membrane, and the cell is suspended in an eluate of conductivity cr ( .
  • ⁇ 0 — , the cell crossover frequency per unit conductivity of the suspending medium.
  • ⁇ 0 may be written in terms of C nKm and of the cell total plasma membrane capacitance C lol , respectively, as
  • the parameter ⁇ 0 is different for different cell types and the value of ⁇ 0 for a given cell type is a characteristic of that cell type that may be used to identify and predict its behavior in DEP analysis.
  • the frequency /of the applied electric field that creates the DEP forces must be controlled so that it bears a precise relationship to the characteristic ⁇ 0 values of the cell types in the mixture that is being processed.
  • the crossover frequency of a given cell type having a crossover frequency per unit suspending medium conductivity of ⁇ 0 is
  • the applied DEP frequency in the DEP process is to bear a well-defined relationship to the crossover frequency of the cells to achieve optimal separation of different cell types then the applied frequency must be adjusted to take account of the suspending medium conductivity ⁇ ⁇ ⁇ It follows that the frequency / is a parameter that will impact the force balance in equation
  • the conductivity of the cytoplasm of a mammalian cell approaches 1 S.m "1 and has an osmolarity of -320 mOs.
  • cells are usually suspended in a medium of conductivity ⁇ ⁇ . that is much lower than 1 S.m "1 .
  • the eluate is typically chosen to have a conductivity in the range lO to lOO mS. m "1 .
  • the osmolarity of an aqueous solution containing only ions and having a conductivity of between 10 mS/m and 100 mS/m is very small compared with the physiological osmolarity of 290 mOs.kg "1 needed to support mammalian cells without inflicting damage on them.
  • low conductivity eluate may be supplemented with a non-ionic osmolyte to adjust its osmolarity to a target osmolarity O x that lies in the range of osmolarities within which cells have desired properties.
  • the osmolyte supplement used to adjust the osmolarity to desired conditions in low conductivity DEP eluate suspension buffers is typically a cell membrane-impermeant sugar such as 9.5% sucrose, 5.1% mannitol, another sugar, or a zwitterionic osmolyte such as glycine.
  • the conductivity of the final suspending medium may be adjusted to a target operating conductivity with salts such as sodium chloride or potassium chloride.
  • Salts such as sodium chloride or potassium chloride.
  • Alternative osmolyte and ion combinations may be used to achieve the same purpose using principles and recipes well known in the art.
  • steps must be taken to reduce the ionic conductivity and to compensate the osmolarity of the suspending medium before DEP-FFF fractionation can occur. These steps may involve centrifuging the cell specimen to remove cells from their physiological medium and resuspending the cells in a low conductivity medium containing an appropriate osmolyte. The resultant cell suspension maybe injected into a chamber for DEP analysis. [0059]
  • this approach presents difficulties when a large quantity of cells is to be processed. In particular, cells cannot be kept in a low-conductivity medium for an extended period of time without their biological and dielectric properties changing.
  • ion leakage occurs from the cell cytoplasms into the suspending medium and non-ideal osmotic responses of the cells towards the osmolyte used to compensate the suspension to a target osmolarity O s may alter cell size, shape and physiological function.
  • the cell dielectric properties may alter and this, in turn, may modify the cell responses to DEP and confound the discrimination and separation of cells by DEP methods.
  • DEP-FFF runs should be completed on cells that have been suspended in low conductivity medium for «1000 seconds and ideally every cell analyzed should be suspended in low conductivity medium for the same period of time at the time it undergoes DEP processing to insure that the dielectric properties of all cells processed from a specimen are consistent.
  • the large number of background cells leads to specimen processing times considerably in excess of 1000 seconds. Therefore, it is disadvantageous to replace the suspending medium for a whole specimen with low conductivity medium prior to starting the DEP-FFF process because the properties of cells undergoing DEP processing late in the DEP-FFF run may have dielectric properties that differ from cells processed early in the run as a result of the different cell exposure times to the low conductivity medium.
  • solute exchange is a common requirement in biological processing.
  • the most common approaches to solute exchange are the replacement of the suspending medium following centrifugation or filtration (for particles of sufficiently high density of large enough to be retained by filtration), dialysis across a semipermeable membrane (in the case of removing small solutes from blood and proteins, for example), or diafiltration in which a new solute is infused with the particle mixture while the particle suspension travels along a tube perforated by small pores that allow the suspending mixture to leak out yet retain particles and/or cells.
  • These approaches are difficult to realize in microfluidic applications.
  • An alternative approach is the use of solute diffusion across fluid junctions.
  • a thin layer of fluid will be referred to as a fluid lamina.
  • fluid flow in the DEP separation chamber is laminar and fluid streams from two or more inlet ports may be merged into a composite flow in which the two individual fluid flow stream move as separate fluid laminas travelling in parallel and in contact with one another throughout their passage through the chamber (see Figure 1).
  • a transfer of solutes across the interface between the two parallel fluid laminas may occur by diffusion.
  • Fick's first law of diffusion relates the diffusive flux of a solute to its concentration and, in one dimension, the flux may be written as [9 ⁇ dx
  • J is the diffusion flux [(moles of solute) m 2 .s " ']
  • D is the diffusion coefficient [m 2 .s ']
  • ⁇ is the solute concentration in dimensions of [moles.m -3 ]
  • x is the position [m]
  • D is proportional to the squared velocity of the diffusing molecules of the solute, which in turn depends on the temperature, viscosity of the fluid and the size of the particles.
  • the Einstein- Smolokowskii diffusion equation describes the relationship between diffusion distance, time and effective size of diffusing entities as kTAt
  • r is approximately equal to the mean radius for microparticles and cells. For macromolecules and solutes, r may be approximated in terms of the molecular weight M of the diffusing entity in
  • the design parameters for this type of separation including the relative thickness and flow rates of the first and second flow laminas and the length of the interface region between them, may be chosen based on the diffusion coefficient of the undesired solute to insure that the undesired solute may be sufficiently depleted to meet desired conditions for DEP. Note that the maximum depletion of a diffusible, undesired solute in this scheme will occur when the concentration of the undesired solute is the same as it would have been had the first flow lamina been mixed completely with the second flow lamina.
  • a great advantage of the microfluidic approach shown here is that no dialysis membrane is required. This not only greatly simplifies the arrangement needed to achieve solute depletion but also greatly accelerates the rate of solute depletion relative to dialysis because there is no need for the solute to cross a semipermeable membrane. Diffusion across a membrane is typically orders of magnitude slower than unimpeded diffusion across a fluid interface.
  • the height of the first fluid lamina containing the microparticles and/or cells should be small.
  • the rate of diffusion through a region of space depends inversely on the square of the distance across it.
  • Sodium ions in water at room temperature for example, will diffuse about 50 micrometers in one second yet they will take about 100 seconds to cross 500 micrometers and 10,000 seconds to diffuse 5mm. It follows that laminar flow-based solute depletion will benefit greatly from reduction of the thickness of the fluid lamina that contains the microparticles and/or cells and undesired solute.
  • the heights of the laminas may be found from the equations of lamina flow. Assume that the first fluid lamina arises from fluid injected at a flow rate /J, into the bottom inlet slot and joins the faster eluate flow rate B 2 from the top inlet that forms the second fluid lamina.
  • An analogous expression describes the height h x from which fluid will be skimmed through an outlet port that withdraws fluid at a rate B sklm .
  • the height at which cells and/or micoparticles traverse the microelectrode array in DEP-FFF depends on the DEP frequency and voltage and the suspension medium conductivity and density. Providing the cell and/or microparticle suspension injection rate is sufficiently small for the cells and/or microparticles to undergo solute depletion at an eluate flow rate B 2 such that the injected cells and/or microparticles reach desired equilibrium heights over the microelectrode array before reaching the outlet port, target microparticle and/or cell populations may be collected by adjusting B skm appropriately.
  • apparatus 10 comprises a body 14 that defines a chamber 18 having a first portion 22 and a second portion 26.
  • chamber 18 has a bottom 30, and two sides 34 (only one shown) that define a substantially U-shaped chamber or channel.
  • a width (between and perpendicular to sides 34) of the chamber may be larger than a height 38 of the chamber.
  • the chamber 18 may also include one or more inlets and one or more outlets.
  • body 14 includes a first inlet 42, a second inlet 46, a first outlet 50 and a second outlet 54.
  • first portion 22 includes a first end 58 and a second end 62 between the one or more inlets (e.g., inlets 42 and 46, in the embodiment shown) and the one or more outlets (e.g., outlets 50 and 54, in the embodiment shown).
  • second portion 26 is disposed between second end 62 of first portion 22 and at least one of the outlets (e.g., both of outlets 50 and 54, in the embodiment shown).
  • apparatus 10 further includes one or more electrodes 66 configured to generate a non-uniform electric field in second portion 26 of chamber 18.
  • apparatus 10 comprises a plurality of electrodes 66. Electrodes 66 may, for example, be disposed on or beneath bottom 30 of chamber 18.
  • first portion 22 does not include electrodes 66 that are configured to generate a non-uniform electric field.
  • first portion 22 may include detection electrodes that are configured to detect or measure one or more properties of fluids (e.g., liquids) in chamber 18 (e.g., in conjunction with a controller or sensor coupled to such detection electrodes).
  • first inlet 42 is configured to receive a first fluid 70 including a target matter (e.g., cells, malignant cells, and/or other particles or packets of matter), and second inlet 46 is configured to receive a second fluid 74.
  • Second fluid 74 may include one or more components configured to diffuse, or facilitate diffusion, between first fluid 70 and second fluid 74 (e.g., to reduce the conductivity of the first fluid 70).
  • apparatus 10 may be configured such that if a first fluid 70 (including target matter) and a second fluid 74, are introduced into chamber 18 through inlets 42 and 46, first fluid 70 and second fluid 74 can flow substantially laminarly through first portion 22 to permit diffusion of solutes between first fluid 70 and second fluid 74.
  • the chamber 18 may be configured to direct first and second fluids 120 and 122 from inlets 42 and 46, respectively, to outlets 50 and 54 according to a predetermined velocity profile.
  • the predetermined velocity profile may be such that first fluid 70 and second fluid 74 substantially reach diffusion equilibrium in first portion 22 of chamber 18.
  • a length 78 of first portion 22 is large enough to permit first fluid 70 and second fluid 74 to substantially reach diffusion equilibrium in first portion 22.
  • length 78 of first portion 22 is greater than length 82 of second portion 26.
  • lengths 78 and 82 may be substantially equal, or length 82 may be greater than length 78.
  • Diffusion between first and second fluids 70 and 74 in first portion 22 may, for example, reduce the conductivity of the first fluid 70 (e.g., to improve the sensitivity of separation of the target matter in second portion 26).
  • apparatus 10 is configured such that first fluid 70 flows below second fluid 74 through chamber 18.
  • apparatus 10 can be configured such that during the substantially laminar flow through first portion 22, the cross-sectional area of first fluid 70 is smaller than the cross-sectional area of second fluid 74.
  • the lamina of first fluid 70 has a height 86 that is shorter than (e.g., less than or between any of: 5%, 10%, 15%, 20% of) height 90 of the lamina of second fluid 74.
  • apparatus 10 is configured such that electrodes 66 can generate a non-uniform or spatially inhomogeneous electric field in second portion 26 to generate a dielectrophorelic force on target matter (in first fluid 70) in second portion 26, such that the target matter is extracted from first fluid 70.
  • apparatus 10 includes a signal generator 94 coupled to electrodes 66, and the signal generator is configured to generate an electric signal with a designated frequency and voltage for delivery to electrodes 66 to cause the electrodes to generate the non-uniform electric field in second portion 26 for dielectrophoretic separation of target matter from first fluid 70.
  • first outlet 50 is configured to permit the target matter to exit the chamber
  • second outlet 54 is configured to permit the remainder of first and second fluids 70 and 74 to exit the chamber.
  • apparatus 10 can include a conductivity sensor 98 that is configured to measure the conductivity of the fluid in the chamber 18(e.g., first fluid 70 after at least some— up to and including all— of the diffusion in first portion 22 takes place).
  • conductivity sensor 98 is configured to measure the conductivity of first fluid 70 in second portion 26; but in other embodiments, may be configured to measure the conductivity of the fluid in first portion 22 and/or in second portion 26.
  • the fluid of which the conductivity is measured may be in contact with electrodes 66, and/or one or more other detection electrodes may be included that are configured to contact the fluid.
  • apparatus 10 can also include a controller 102 (e.g., a microcontroller, processor, and/or the like) configured to perform one or more functions that support operation (e.g., semi- and/or fully-automated operation) of the apparatus.
  • controller 102 is configured to calculate one or more target properties of the non-uniform electric field (e.g., one or more properties of the electric field that are likely to result in separation of the target matter from the first fluid in second portion 26 of the chamber) based on at least one of the measured conductivity of first fluid 70 and a property of the target matter (e.g., conductivity, density, and/or the like).
  • controller 102 can also be configured to adjust the electric signal generated by signal generator 94 to cause the non-uniform electric field to substantially include the one or more target properties.
  • the controller may calculate one or more properties of the electric field that are likely to cause separation of the target matter in a fluid having the measured conductivity, and send a control signal to signal generator 94 so that signal generator 94 adjusts the generated electric signal accordingly.
  • Such an adjustment of the electric signal may include, for example, the frequency and/or the voltage of the electric signal.
  • apparatus 10 can include a current sensor 106 that is configured to measure the current of the electric signal generated by signal generator 94.
  • controller 102 e.g., coupled to current sensor 106
  • controller 102 can be configured to compare the measured current to a target current, and to to adjust the electric signal based on the comparison (e.g., can increase frequency and/or voltage of the electric signal if the measured current is below a target current).
  • Some embodiments of the present apparatuses do not include either of conductivity sensor 98 or current sensor 106, or include only one of conductivity sensor 98 or current sensor 106.
  • FIG. 1 B illustrates an alternative embodiment of a body 12' for some embodiments of the present apparatuses.
  • body 12' differs from body 12 in that body 12' includes first and second outlets 50' and 54' that differ in configuration relative to first and second outlets 50 and 54 of body 12.
  • first outlet 50' diverts target matter in a direction that is angularly disposed relative to direction 1 10 of flow through the chamber.
  • the direction of first outlet 50' is substantially perpendicular to direction 1 10.
  • the direction of first outlet 50' may be disposed at any suitable angle (e.g., greater than or between any two of: 15, 30, 45, 60, 75, and/or 90 degrees) relative to direction 1 10.
  • length 78' of first portion 22' is larger than length 78
  • length 82' of second portion 26' is shorter than length 82.
  • the ratio of length 78' to length 82' is larger than the ratio of length 78 to length 82.
  • FIG. 1 B also illustrates the separation of target matter from the remainder of first fluid 70 and second fluid 74 that is representative of target matter that may also be isolated or separated in the embodiment of FIG. 1 A.
  • first fluid 70 includes a blood sample that includes blood cells 1 14 and malignant cells 1 18, with malignant cells 1 18 being the target matter that is removed from the blood sample through first outlet 50'.
  • FIG. 2 illustrates one embodiment of a method 200 for optimized continuous flow dielectrophoretic separations.
  • the method 200 may include a step 202 of introducing a first fluid 70 including target matter (e.g., a blood sample including malignant cells) and a second fluid 74 into a chamber (e.g., 18) of an apparatus.
  • the second fluid may include one or more osmolytes configured to facilitate the diffusion between the first fluid and the second fluid to reduce the conductivity of the first fluid.
  • the apparatus may, for example, be one that is depicted in and described with reference to FIG. 1 A or 1 B.
  • the chamber 18 of apparatus 10 may include one or more inlets (e.g., first and second inlets 42 and 46) and one or more outlets (e.g., first and second outlets 50 and 54).
  • the chamber 18 may also include a first portion 22 having a first end 58 , and a second end 62 between the first and second inlets (42 and 46) and the f irst and second outlets (50 and 54), and a second portion 26 between second end 62 of first portion 22 and at least one the first and second outlets (e.g., first outlet 50).
  • Apparatus 10 may also include one or more electrodes (e.g., electrodes 66) configured to generate a nonuniform electric field in the second portion (26) of the chamber.
  • the heights and/or sizes of the inlets (42 and 46) may be configured such that the input streams of first and second fluids 70 and 74 are thin enough to achieve rapid diffusion between the first and second fluids 70 and 74 (e.g., such that first and second fluids 70 and 74 substantially reach diffusion equilibrium in first portion 22).
  • method 200 may also include a step 204 of causing first and second fluids 70 and 74 to flow substantially laminarly through first portion 22 of the chamber such that diffusion between first fluid 70 and second fluid 74 lowers the conductivity of first fluid 70.
  • first and second fluids 70 and 74 flows continuously through first and second portions 22 and 26 of chamber 18.
  • length 78 of first portion 22 of the chamber may be large enough to permit first fluid 70 and second fluid 74 to substantially reach diffusion equilibrium in first portion 22, and during the laminar flow through first portion 22, the cross-sectional area of first fluid 70 may be smaller than the cross-sectional area of second fluid 74.
  • method 200 may further include a step 206 of applying an electric signal to the one or more electrodes 66 of the apparatus to generate a dielectrophoretic (DEP) force on the target matter to extract the target matter from the first fluid.
  • Electrodes 66 of the apparatus e.g., when an appropriately configured electric signal is applied
  • the DEP force applied on the target matter in an appropriate direction may balance out the sedimentation force, hydrodynamic lift force, and/or on the target matter, and the residual DEP force may act on the target matter to direct them to exit from a desired outlet (e.g., first outlet 50) of the chamber.
  • Method 200 may also include a step 210 of outputting the target matter through a first outlet of the chamber and the remainder of the first and second fluids through a second outlet of the chamber.
  • the sedimentation force acting on microparticles and/or cells may be at orders of magnitude greater than the sedimentation force acting on electrolytes and osmolytes. Therefore, if the first fluid 70 containing the target matter (e.g. cells and/or microparticles) is configured so as to flow underneath the second fluid 74 containing the osmolyte, then the target matter may tend to sediment away from the interface between the fluids and cause the target matter to be confined within the lower fluid. This may facilitate bringing target matter into juxtaposition with the on or more electrodes 66 that imposes DEP forces, thereby facilitating a stronger influence of DEP on cell behavior.
  • the target matter e.g. cells and/or microparticles
  • method 200 may further include an optional step 208 that can include one or more of various components illustrated in FIGS. 3A-3B. As illustrated in FIGS.
  • method 200 can include a sub-step 302 of controlling the flow rate of the first fluid and the second fluid (e.g., at a target flow rate).
  • the target flow rate may be such that first fluid 70 and second fluid 74 flow through first and second portions 22 and 26 of the chamber according to a target velocity profile.
  • the target velocity profile may, for example, be such that first fluid 70 and second fluid 74 substantially reach diffusion equilibrium in first portion 22 of the chamber.
  • the diffusion between the first and second fluids may, for example, adjust the conductivity of the first fluid.
  • method 200 can include a substep 304 of measuring the conductivity of the first fluid (e.g., with conductivity sensor 98); a substep 306 of calculating (e.g., with controller 102) one or more target properties of the non-uniform electric field based on at least one of measured conductivity of the first fluid and a property of the target matter; and/or a substep 308 of adjusting (e.g., with controller 102) the electric signal to include the one or more target properties.
  • the adjustment of the electric signal may include changing at least one of the frequency and the voltage of the electric signal.
  • an optimal frequency f 0 can be computed from a product of a target frequency-per-unit conductivity parameter ⁇ 0 ⁇ and the measured fluid conductivity ⁇ ⁇ .
  • An electric signal may then be generated at the optimal frequency f 0 and applied to the one or more electrodes 66 to provide the DEP forces to the target matter in the chamber 18.
  • method 200 can include a substep 310 of measuring a current of the electric signal (e.g., with current sensor 106); a substep 312 of comparing the measured current to a desired current (e.g., with controller 102); and/or a substep 314 of adjusting the electric signal based on the comparison (e.g., with controller 102).
  • the adjustment of the electric signal may include changing at least one of the frequency and the voltage of the electric signal.
  • the electric current l me that is flowing through the DEP electrodes 66 as a result of an applied signal of frequency f 0 and voltage V from the signal generator 94 may be detected and compared to a demand current that is known to likely to provide optimal DEP performance at the fluid conductivity cr and frequency f Q . If the measured current l mp differs from the demand current, then a voltage correction may be computed and the signal generator may be adjusted through a feedback network so as to provide an electric signal at a compensated voltage V that brings l DEV to the demand current value.
  • the processes of conductivity sensing and current sensing may be carried out at the same time to achieve a desired (e.g., improved and/or optimal) frequency and voltage for the electric signal generated by the signal generator 94.
  • the conductivity (J v of the fluid that flows over electrodes 66 may be determined by conductivity sensor 98.
  • the optimal frequency f 0 may be computed from a product of a target frequency per unit conductivity parameter ⁇ 0 ⁇ and the fluid conductivity a s .
  • the signal generator frequency may be adjusted to f 0 through a first feedback scheme thereby insuring that the frequency of the electric signal applied to the electrodes 66 is appropriate for the fluid conductivity ⁇ ⁇ .
  • the electric current l mv that is flowing through DEP electrodes 66 as a result of the applied signal of frequency f 0 and voltage V from the signal generator maybe sensed by current sensor 106 and compared by controller 102 to a demand current that is likely to provide desired (e.g., optimal) DEP voltage at the fluid conductivity cr v and frequency f 0 . If the measured current l DEl , differs from the demand current, then signal generator 94 may be adjusted through a second feedback scheme that compensates the DEP signal voltage V in order to bring I DEr to the demand current value.
  • the two feedback schemes insure that both the applied frequency and voltage from signal generator 94 are desired (e.g., optimal) and automatically compensated for changes in the conductivity of the fluid and the condition of electrodes 66.
  • method 200 can include a substep 316 of adding one or more osmolarity-altering components to first fluid 70 (e.g., such that the osmolarity of first fluid 70 bears a desired relationship to the osmolarity of second fluid 74); and/or a substep 318 of adding one or more osmolarity-altering components to second fluid 74 (e.g., such that the osmolarity of second fluid 74 bears a desired relationship to the osmolarity of first fluid 70).
  • the osmolarity- altering components may, for example, include salts such as sodium chloride or potassium chloride.
  • Osmolarity adjustment may, for example, achieved by flowing first fluid 70 adjacent to second fluid 74 to allow an osmolyte contained in one of the first and second fluids to diffuse to the other of the first and second fluids across the interface between the first and second fluids, thereby altering the osmolarity in first fluid 70.
  • method 200 can include a substep 320 of adding one or more density-altering components to first fluid 70 such that the density of first fluid 70 bears a desired relationship to the density of second fluid 74; and/or a substep 322 of adding one or more density-altering components to second fluid 74 such that the density of second fluid 74 bears a desired relationship to the density of first fluid 70.
  • the desired density relationship may be such that convection is reduced and/or substantially prevented between first and second fluids 70 and 74.
  • undesired flow patterns of and between the first and second fluids may be reduced and/or obviated by adjusting the density of the first and/or second fluids such that a desired density relationship exists between the first and second fluids (e.g., that inhibits convection).
  • undesired sedimentation behavior of microparticles and/or cells in the flow channel may be reduced and/or obviated by adjusting the density of the first and/or second fluids such that a desired density relationship exists between the first and second fluids (e.g., in accordance with and/or compatible with the density characteristics of such microparticles and/or cells).
  • method 200 can include a substep 324 of adding one or more viscosity-altering components to first fluid 70 such that the viscosity of first fluid 70 bears a desired relationship to the viscosity of second fluid 74; and/or a substep 326 of adding on or more viscosity -altering components to second fluid 74 such that the viscosity of second fluid 74 bears a desired relationship to the viscosity of first fluid 70.
  • the desired density relationship may be such that convection is reduced and/or substantially between the first and second fluids.
  • the blood of patients having the disease of cancer may contain cancer cells, and it may be desirable to isolate said cancer cells from the blood for purposes of prognosis, diagnosis, treatment and/or management of the cancer.
  • cancer cells may be circulating in the blood at a very small concentration compared with the many normal blood cells within the peripheral blood stream of the patient.
  • cancer cells mixed with the blood are generally referred to as "circulating tumor cells" or CTCs.
  • CTCs circulating tumor cells
  • the isolation of CTCs from the peripheral blood of cancer patients is generally considered to be of importance for the prognosis and treatment of breast, prostate, ovarian, colon, and other cancers.
  • the CTC concentrations in the peripheral blood of patients vary in relation to the stage of the disease but are often extremely low compared with the background count of PBMNs.
  • Eluate consisting of 9.5% sucrose at 30 mS/m was flowed from the inlet to the outlet end of the chamber at a rate of 1 mL/min and met the influx of cell suspension to form an upper fluid lamina (that is, a thin layer of fluid) .
  • the blood cell suspension had the same density as the eluate and filled the bottom 40 um of the chamber while the eluate flowed above it.
  • the cell suspension flowed over a DEP electrode from the moment it entered the chamber, however the first 40 mm of the electrode was not energized.
  • the target CTCs experienced a positive DEP force because the applied signal was above their DEP crossover frequency. This DEP force pulled the CTCs towards the electrode but was not sufficient to overcome HDL forces. Therefore the CTCs were not trapped on the DEP electrode by DEP forces but instead moved slowly at a height of about 5 micrometers above the chamber floor towards the first outlet or exit port. Simultaneously, the applied electric signal frequency was well below the crossover frequency for the PBMNs and these background cells were levitated about 22 micrometers above the chamber floor and moved rapidly towards the second outlet or exit port. Fluid was withdrawn through the first outlet or exit port at 15 uL/min by a syringe pump and was collected on a filter. The target CTCs were thereby captured. The vast majority of PBM background cells passed over the first outlet or exit port and was carried out of the second outlet or exit port to waste.
  • the same strategy used to isolate CTCs from PBMNs may also be used to eliminate cancer cells from desired blood cells.
  • the target cells are the blood cells and the undesired cells are the cancer cells.
  • the blood cells passing over the first outlet or exit port are collected at the second, downstream or exit port.
  • the cancer cells withdrawn through the first outlet or exit port may be then used for diagnostic, prognostic and/or or risk assessment purposes or discarded.
  • the strategies described above for isolating desired cancer cells from blood cells or for isolating desired blood cells from cancer cells may also be used to eliminate other types of undesired cells from desired target cells providing the desired and undesired cells have different DEP crossover frequencies. In general for both strategies, the cell type with the lower crossover frequency will always be collected at the first outlet or withdrawal port.
  • Example 2 Overview of Separation Strategy
  • the specimen forms a thin lamina of height h in flowing adjacent to the floor of the chamber beneath the main eluate flow stream.
  • the thin specimen lamina travels along the chamber floor in the Ion-diffusion Region, cells find themselves subjected to sedimentation and weak hydrodynamic lift forces and settle to equilibrium heights very close to the chamber floor (see FIG. 4(c)).
  • ions diffuse from the thin specimen lamina into the main eluate flow stream while non-ionic osmolytes counter-diffuse from the eluate into the specimen lamina.
  • the high conductivity of the specimen lamina is reduced while the osmolarity near the cells is maintained at a physiological level.
  • the ion concentration, and resultant electrical conductivity of the medium becomes independent of height in the chamber and small enough for DEP separation to be undertaken.
  • the flow then enters the DEP Separation Region, where microelectrodes on the chamber floor are energized by an appropriate AC voltage to impose DEP forces on the cells.
  • tumor cells are pulled towards the chamber floor by positive DEP forces while PBMNs are repelled and levitated by negative DEP forces. Cells eventually move to heights at which the DEP, sedimentation and hydrodynamic lift forces balance (see FIG. 4(d)).
  • one embodiment for continuous flow DEP-FFF is to transport the specimen along the floor of the separation chamber in a thin lamina that flows beneath the main eluate stream.
  • the thickness of this specimen lamina, /?, administrat, above the chamber floor is related to the injection flow rate, B / and the eluate inlet rate / ? according to the expression Bi l(Bi + , where H is the height of the chamber.
  • H is the height of the chamber.
  • a thin lamina of fluid may be skimmed from the chamber floor up to a height h s by withdrawing fluid at a rate 3 ⁇ 4 / Struktur, where h s « //(&* «» / 3/? composite 1H ) 2 . Downstream from the withdrawal port, the residual fluid flows at a rate B ou
  • the inventors simulated flow profiles for various chamber heights, slot widths and flow rates using COMSOL Multiphysics software (Stockholm, Sweden) assuming that the fluid was incompressible and had a density of 1036 kg.m "3 and a dynamic viscosity of 1.31 x 10 "3 Pa.s, reflecting the properties of the eluate medium used in the inventors' cell-isolation experiments.
  • the results for the withdrawal slot simulations arc shown in FIG. 5.
  • the behavior at the inlet slot region is a mirror image of that at the withdrawal slot.
  • Desirable skimming behavior was observed when the slot width d ou , was small compared with the chamber height H (FIG. 5 (a)).
  • a well-defined separation region at the slot opening cleanly split the thin lamina of height h s at the chamber bottom from the main chamber flow. Streamlines above the skim height h s travelled essentially horizontally across the slot and no regions of low flow rale or significant vortices were generated in the separation region.
  • tumor cells close to the chamber floor would be skimmed cleanly into the withdrawal slot, while PBMNs in the fluid above the skim height would be rapidly carried over the slot without being thrown into the slot by inertial forces or having the opportunity to sediment into the slot from regions of low flow rate.
  • the simulations establish criteria for designing efficient withdrawal slots and show, in particular, that a withdrawal slot of width d out ⁇ H/3, regardless of the flow rate, exhibits negligible depression of the main chamber streamlines into the mouth of the withdrawal port (FIG. 5(e)) as well as low vorticity (FIG. 5 (d)). Similar principles apply to the injection slot design to prevent the potential for cell accumulation in the mouth of the injection slot. For the inventors' experiments, a chamber height of 314 ⁇ and injection and withdrawal slot widths of 127 ⁇ were chosen, corresponding to the flow conditions shown in FIG. 5(a).
  • the injection and withdrawal flows were provided by 1 mL syringes driven by digital syringe pumps ( DS210, D Scientific, Holliston, Massachusetts) and the eluate flow by a gear pump (Ismatec, Glattbrugg, Switzerland).
  • Tumor cells flow directly downward through the withdrawal slot in the chamber floor while PBMNs travel horizontally. Furthermore, microelectrodes are positioned on the chamber floor on both the upstream the downstream sides of the withdrawal slot in the design. These continue to levitate PBMNs by DEP forces in the slot region and on the far side of the slot. This helps reduce still further the likelihood PBMNs will fall into the withdrawal slot and contaminate the tumor cell isolate. In these ways, the ⁇ configuration improves significantly upon the H-filter design concept for applications involving sedimentary particles such as cells.
  • the cell suspending medium conductivity must be much lower than the cell cytoplasmic conductivity (Gascoyne and Vykoukal, 2002).
  • the starting conductivity of the specimens was 1.4 S.m " ', approximately the same as that of the cell cytoplasms, and this needed to be lowered to a target value of about 60 mS.m "1 before the cells could be subjected to DEP separation.
  • the reduction in conductivity was accomplished by using diffusion to deplete the ions in the specimen as it moved through the Cell Settling and Ion Diffusion Zone shown in FIG. 4 (c).
  • the length of the Ion Diffusion Zone was chosen in accordance with the chamber height H and flow rate Ih to insure that ions had sufficient time to diffuse throughout the chamber height before the sample entered the DEP zone.
  • the required mixing length L mix for a diffusible species has been analyzed for the H-filter microfluidic configuration (Bruus, 2008) and, by analogy for the ⁇ configuration device, may be written as L NM 3 ⁇ 4 H( I + where Wis the width of the chamber in the direction perpendicular to the plane of FIG. 4 and D is the diffusion coefficient of the diffusing species.
  • the cell specimens contained between 20 x 10 6 and 40 x 10 6 PBMNs (containing trace levels of tumor cells) collected from whole blood by centrifugation over Histopaque 1077 (Cat 10771 -l OOmL, Sigma-Aldrich, St Louis, USA) and were suspended in 1 mL RPMI medium that had been adjusted to a density of 1036 kg.m '3 by adding iodixanol (OptiprepTM Density Medium D 1556, Sigma-Aldrich, St Louis) to a concentration of 1 1 % (Gascoyne et al., 2009).
  • iodixanol OptiprepTM Density Medium D 1556, Sigma-Aldrich, St Louis
  • the inventors used the eluate recipe developed in earlier DEP-FFF studies (Gascoyne et al., 2009; Shim et al., 201 1 ) composed of an aqueous solution of 9.5% sucrose (S7903, Sigma-Aldich, St Louis, MO), 0.1 mg mL " 1 dextrose (S73418-1 , Fisher, Fair Lawn, NJ), 0.1 % pluronic F68 (P I 300, Sigma-Aldich, St Louis, MO), 0.1 % bovine serum albumin (A7906, Sigma-Aldich, St Louis, MO), 1 mM phosphate buffer pH 7.0, 0.1 mM CaAcetate, 0.5 mM MgAcetate and 100 units mL " 1 catalase (C30, SigmaAldich, St Louis, MO).
  • the Na + , K + and CI " ions that dominated the high conductivity of the specimen diffused throughout the eluate.
  • Sucrose counter diffused from the eluate to maintain the osmolarity of the cells at a physiological level so they were not osmolically stressed, which would have impacted cell size and membrane dielectric characteristics (Shim et al., 201 1 ).
  • FIG. 6 The inventors simulated the diffusion of ions and sucrose in the depicted embodiment of continuous-flow devices using the COMSOL Multiphysics software and the results for the conductivity distributions in key zones of the chamber are shown in FIG. 6.
  • the specimen injection zone FIG. 6 (a)
  • a very large conductivity gradient exists where the specimen stream ( 1400 mS.m "1 ) first joins the main eluate flow (30 mS.m *1 ).
  • the cell settling and ion diffusion zone FIG.
  • the ion conductivity is clearly spreading upwards through the chamber and by the time the flow reaches the DEP equilibration zone 40mm downstream from the specimen inlet and the tumor cell skimming zone 80mm downstream from the specimen inlet (FIG. 6 (c)), ion diffusion is essentially complete and the conductivity is homogeneous throughout the chamber height at about 61 .5 mS. m "1 . Sucrose diffusion is also completed before the specimen reaches the DEP equilibration zone.
  • the inventors measured the AC current drawn by the microelectrode array. This current depends much more sensitively on the conductivity immediately adjacent to the microelectrode that on the conductivity high in the chamber.
  • the microelectrode current was the same under continuous specimen injection conditions as when the chamber was filled with a homogeneous medium having a conductivity of 61.5 mS.m "1 . This showed that the ion concentration at the microelectrode plane was the same in both cases, proving that diffusion was complete.
  • the inventors conducted an experiment in which fluid leaving the ion diffusion region was skimmed from the chamber floor up to different heights by altering the withdrawal flow rate. The conductivity of the withdrawn fluid was 61.5 mS.m "1 regardless of the skim height, also showing that diffusion was complete.
  • the frequency and magnitude of the electrical signal applied to the microelectrodes in the DEP equilibration zone are chosen so that the tumor cells are pulled towards the chamber floor while the PBMNs are repelled high above it. This is possible because the direction of the DEP force is determined by whether the applied electric field frequency is greater or less than the characteristic "crossover frequency" - ⁇ " of a given cell type (Pethig, 2010; Jones and Kallio, 1979; Chan et l., 1997).
  • the DEP force imposed on cells depends on the square of the applied voltage V, suggesting it might be advantageous to use a high DEP voltage to increase the height differential between tumor and PBMNs leaving the DEP equilibration zone (Shim et al., 2013).
  • the electric field that gives rise to the DEP force also induces a transmembrane potential difference that can stress cells and cause them to become leaky towards ions and/or electroporated, which could alter their DEP properties and confound their separation characteristics.
  • the magnitude of the induced transmembrane potential difference depends upon the electric field strength, the cell diameter and the applied electric field frequency (Wang et al., 1999).
  • the cell elution times could be mapped directly to the cell equilibrium height distribution in the chamber.
  • the results are shown in FIG. 7 and it may be seen that the MDA-MB-231 cells traveled through the DEP-FFF chamber between 5 and 13 ⁇ above the chamber floor while the PBMNs were transported at between 14 and >40 ⁇ height.
  • Tumor cells may be isolated from PBMNs in continuous flow DEP-FFF by using a skim height that is between the tumor cell and PBMNs transport heights under the prevailing DEP field conditions.
  • the batch mode DEP-FFF chamber had a microelectrode array area of 7500 mm and the power output capacity of the signal generator limited the maximum DEP voltage to 2.8 V p-p.
  • the chamber had a microelectrode array area only one third that size, which permitted the inventors to use the higher operating voltage of 4 V p-p.
  • the DEP-FFF force balance equation (Shim et al., 2013; Shim et al., 201 1 ; Gascoyne, 2012) showed that the levitation height for PBMNs under those conditions was > 27 ⁇ .
  • fluid convection cells could form adjacent to energized DEP electrodes as the result of temperature gradients associated with Joule heating of the fluid.
  • Convection cells on the periodic DEP microelectrode array would be expected to have the same spatial periodicity as the DEP force field and it might in certain aspects it is possible for convection and DEP fields to act in combination to alter the height at which cells are transported over the microelectrode array and thereby impact the cell isolation characteristics. This possibility was neglected in the analytical equations we used to describe DEP-FFF and the effect, if present, seems to be relatively small because one result of it would be to shift the apparent crossover frequency at which the direction of the DEP force on cells appears to reverse in DEP-FFF.
  • Example 6 Exemplary Tests with Clinical Specimens
  • Tests were run of continuous flow DEP-FFF isolation of MDA-MB-435, MDA-MB- 231 and other cultured tumor cells spiked into PBMNs from healthy donors to compare with earlier batch-mode DEP-FFF experiments (Gascoyne et al., 2009). Measurements were conducted using a DEP-FFF chamber 160 mm long having a width of 25 mm, a height of 314 ⁇ , and an inlet to outlet slot spacing of 90 mm. The chamber floor was lined by a microelectrode array based on the design detailed earlier (Vykoukal et al, 2008) in which parallel gold-on-copper microelectrodes of 50 ⁇ width and spacing were patterned on a kaptan substrate.
  • Peripheral blood specimens were obtained as part of the Initiative for Molecular Profiling in Advanced Cancer Therapy (IMPACT) Trial at The University of Texas M.D. Anderson Cancer Center with informed patient consent and the approval of Institutional Biosafety Committee. Specimens of at least 7 mL volume were collected from patients in 10 mL BD purple cap (EDTA) vacutainers and processed within 3 hours. The PBM s, putatively containing CTCs, were separated from the patient specimens over Histopaque 1077 and subjected to continuous DEP-FFF also using the experimental conditions described in the sections above. The isolate from the withdrawal slot was collected in a 1 mL syringe for each specimen over the approximately 40 minute processing time.
  • IMPACT Initiative for Molecular Profiling in Advanced Cancer Therapy
  • EDTA BD purple cap
  • the FACS approach is generally not feasible for analyzing CTCs isolated from clinical specimens because a 10 mL blood specimen may contain as few as ten CTCs and very rarely more than hundreds and downstream immunostaining in cell suspension (as opposed to fluorescent dye preloading used to prepare cells before spiking experiments) followed by subsequent FACS analysis are too lossy to use to count such small cell populations accurately. Therefore, a Cytopro rM instrument (Wescor Model 7620, Logan, Utah) was used to mount the cells from this isolate onto two microscope slides.
  • DNA was extracted from the slide using PicoPure (cat 1 1815-00, Applied Biosystems) then further cleaned with a QIAamp DNA Micro Kit (cat 56304, Qiagen).
  • the DNA was preamplified using the following primers: Forward: ATGACTGAATA ' I AAACTTGTGGTAGTTGGA (SEQ ID NO: l ), Reverse: GAATTAGCTGTATCGTCAAGGCACT (SEQ ID NO:2), Vic Reporter: CTTGCCTACGCCACCAG (SEQ ID NO:3), FAM Reporter: CTTGCCTACGTCACCAG (SEQ ID NO:4).
  • the Taqman Pre- Amp Master Mix (Cat 4391 128) was employed according to the protocol specified by Fluidigm.
  • the sample from the slide, together with a positive and a negative control were tested using a Fluidigm 48.48 Genotyping Array (cat BMK-M-48.48GT, Fluidigm) according to Fluidigm's protocol.
  • a Fluidigm 48.48 Genotyping Array catalog BMK-M-48.48GT, Fluidigm
  • MDA-MB-231 was used as a positive control because it possesses this mutation.
  • the specimen exhibited a positive result with a signal intensity that indicated approximately 10% of the cells on the slide had the KRAS G13D mutation, mirroring the proportion of cells that stained positively for cytokeratin in FIG. 9.
  • Vykoukal DM Gascoyne PR
  • Vykoukal J Dielectric Characterization of Complete Mononuclear and Polymorphonuclear Blood Cell Subpopulations for Label-Free Discrimination. Integr Biol 1 :477-484.

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Abstract

La présente invention comprend des appareils et des procédés de séparation diélectrophorétique en flux continu (par exemple, amélioré et / ou optimisé) de la matière cible (par exemple, des cellules) de liquide (par exemple, un échantillon de sang). Dans certains modes de réalisation, l'appareil peut comprendre un corps définissant une chambre (par exemple, une chambre ayant: une ou plusieurs entrées et une ou plusieurs sorties, une première partie et une seconde partie entre une extrémité de la première partie et les sorties) et une ou plusieurs électrodes configurées pour générer un champ électrique non uniforme dans la seconde partie de la chambre. L'appareil peut être configuré de telle sorte que si un premier fluide, comprenant la matière de la cible et un second fluide sont introduits dans la chambre par les orifices d'entrée, le premier fluide et le deuxième fluide peuvent s'écouler de manière sensiblement laminaire à travers la première partie pour permettre la diffusion de solutés entre le premier fluide et le second fluide, et les une ou plusieurs électrodes peuvent générer une force diélectrophorétique sur la matière cible dans la seconde partie pour extraire la matière cible à partir du premier fluide.
PCT/US2012/070333 2011-12-18 2012-12-18 Appareils et procédés de séparation diélectrophorétique en flux continu WO2013096304A1 (fr)

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CN105723207A (zh) * 2013-11-24 2016-06-29 凯米罗总公司 分析含有固体物质的颗粒的液体样品的方法和系统以及此种方法和系统的用途
WO2021050086A1 (fr) * 2019-09-14 2021-03-18 Hewlett-Packard Development Company, L.P. Séparation de particules par tourbillon
US11946902B2 (en) 2018-10-11 2024-04-02 Hewlett-Packard Development Company, L.P. Dielectrophoresis separator cross-over frequency measurement systems

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105723207A (zh) * 2013-11-24 2016-06-29 凯米罗总公司 分析含有固体物质的颗粒的液体样品的方法和系统以及此种方法和系统的用途
CN105723207B (zh) * 2013-11-24 2019-11-15 凯米罗总公司 分析含有固体物质的颗粒的液体样品的方法和系统以及此种方法和系统的用途
US11946902B2 (en) 2018-10-11 2024-04-02 Hewlett-Packard Development Company, L.P. Dielectrophoresis separator cross-over frequency measurement systems
WO2021050086A1 (fr) * 2019-09-14 2021-03-18 Hewlett-Packard Development Company, L.P. Séparation de particules par tourbillon

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