WO2018009892A1 - Diélectrophorèse isomotrice pour analyse diélectrique de sous-populations de particules - Google Patents

Diélectrophorèse isomotrice pour analyse diélectrique de sous-populations de particules Download PDF

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WO2018009892A1
WO2018009892A1 PCT/US2017/041238 US2017041238W WO2018009892A1 WO 2018009892 A1 WO2018009892 A1 WO 2018009892A1 US 2017041238 W US2017041238 W US 2017041238W WO 2018009892 A1 WO2018009892 A1 WO 2018009892A1
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electrode
particle
isodep
dielectrophoresis
particles
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Stuart J. Williams
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University Of Louisville Research Foundation, Inc
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    • 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]
    • 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
    • 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

Definitions

  • the presently-disclosed subject matter generally relates to methods and devices for particle analysis.
  • certain embodiments of the presently-disclosed subject matter relate to methods and devices for isomotive dielectrophoresis (isoDEP) particle analysis.
  • Dielectrophoresis is the translation of a polarizable particle by applied non-uniform electric fields. Unlike electrophoresis, particles do not need to carry a charge in DEP.
  • the polarization of a particle with DEP is based, in part, on the interfacial polarization at the interface of the liquid/particle boundary under an applied electric field.
  • Selectivity of DEP sorting occurs due to differences in the particles' dielectric properties (conductivity, permittivity) and resultant polarization when subjected to an electric field. This selectivity may be used to either attract or repel the particles from regions of greater field strength.
  • an AC field is typically used for dielectrophoresis to either (i) mitigate electrophoretic movement and/or (ii) translate negligibly charged neutral particles.
  • DEP has been able to capture, sort, concentrate, and characterize a variety of particles and biological entities as small as a few nanometers.
  • the polarization behavior of a particle observed over a spectrum of AC frequencies also provides insight into its state and composition, a characteristic utilized in electrorotation.
  • an important characteristic of DEP is that by tracking the magnitude and direction of manipulated particles' velocities over a range of applied AC frequencies, the dielectric properties of the particles can be extracted and subsequently used for particle identification or analysis.
  • DEP has been used to differentiate subpopulations of cells including live and dead bacteria, healthy and damaged phytoplankton, breast cancer sublines, circulating tumor cells from non-transformed cells, leukocyte subpopulations (T lymphocytes, B lymphocytes, granulocytes, monocytes), and blood types.
  • the primary disadvantage of current dielectrophoretic systems is that the applied DEP force is spatially non-uniform, especially in three dimensions, which complicates or prevents comprehensive cellular sub-population analysis.
  • DEP requires a nonuniform electric field to provide a non-zero net force on the particle.
  • the magnitude of the DEP force is a proportional to the gradient of the field-squared (Vi ⁇ ms) and, therefore, is highly dependent on the electrode geometry and layout.
  • the most prevalent DEP systems use co-planar metal electrodes.
  • the DEP force is inherently greatest close to the surface of the electrodes, but exponentially decreases with height above the electrode plane. Therefore, samples are exposed to highly non-uniform DEP forces. For example, using co-planar electrodes with a 25 ⁇ gap, the magnitude of the DEP force is approximately 100 times greater for a particle 1 ⁇ above the electrode edge compared to when it is 10 ⁇ away - a distance comparable to the diameter of some biological cells.
  • ACEO AC electro-osmosis
  • ET electrothermal
  • ET flow is proportional to E 2 (external heating) or E 4 (Joule heating) and becomes negligible at high AC frequencies; ET flow can be reduced through proper heat transfer design to eliminate regions of large temperature gradients.
  • Joule heating in electrokinetic chips is proportional to E 2 and the conductivity of the media ( ⁇ ). Joule heating can been avoided by using low conductivity media ( ⁇ 1.0 S/m).
  • An alternate technique includes electrorotation (ROT).
  • Cellular ROT involves the use of rotating fields generated, typically, by a four-electrode system that are designed to isolate single cells (FIG. 1, left).
  • the direction and rate of particle spin is a function of the field spatial configuration and its AC frequency as well as the dielectric properties of the media and cell.
  • ROT can discriminate dielectric differences between single cells and has been used to differentiate cells with different morphologies and other biological characteristics (FIG. 1, right).
  • ROT systems provide detailed electrokinetic measurements of cell subpopulations, though its most significant disadvantage is its limited throughput which results from its inherent design of trapping and analyzing individual cells (FIG. 1, left). For example, Han, et al. used ROT to differentiate between six cell types but acquired ROT data using an average of nine cells for each cell type (54 cells total). Further, the physics of laminar microfluidic flows makes it nontrivial to position individual cells within an electrode region whose geometry is on the same scale as the diameter of the cell (FIG. 1, left). Analyzing a larger cell subpopulation will provide additional insight into how that specific cell type is affected by external stimuli (e.g., environmental factors).
  • external stimuli e.g., environmental factors
  • Flow cytometry is a laser-based technique used for cell counting, cell sorting, and biomarker detection that can process thousands of cells per second. Scattered light is detected from a fluorescently labeled cell as it passes through a laser beam (or multiple beams).
  • EIS allows the quantitative measurement of the inherent electrical and dielectric properties of cells and can be integrated with other lab-on-a-chip technologies.
  • EIS systems measure the electrical impedance of cells at a particular AC frequency.
  • EIS systems There are two types of EIS systems, dynamic and static.
  • Dynamic EIS systems provide higher throughput, although they also have limited sensitivity (i.e., measurements are typically acquired at one specific AC frequency).
  • Static EIS systems trap individual cells and obtain detailed impedance spectra (i.e. over a range of AC frequencies) of the cell enabling highly sensitive measurements.
  • a method of analyzing particles comprising providing an isomotive dielectrophoresis device; positioning a sample within the device, the sample including at least one particle; applying an electric field to the device, the electric field inducing a constant dielectrophoresis force on the at least one particle of the sample; and monitoring a translation of the at least one particle.
  • the at least one particle includes a cell.
  • positioning the sample within the device comprises injecting the sample into the device until bulk fluid motion is halted.
  • the constant dielectrophoresis force comprises a constant force within an analytical space of the device.
  • applying the electric field to the device comprises applying an AC signal across a channel in the device.
  • the AC signal is between 100 Hz to 100 MHz, 1 kHz and 100 MHz, 1 kHz and 50 MHz, 1 kHz and 10 MHz, or any suitable combination, subcombination, range, or sub-range thereof.
  • applying the electric field to the device comprises applying an AC signal through a channel in the device.
  • the method further comprises extracting dielectric properties of the at least one particle.
  • the method further comprises determining a cell physiology from the dielectric properties.
  • an isomotive dielectrophoresis device comprising a first electrode having a first surface geometry; a second electrode having a second surface geometry; and an electrically insulating material at least partially surrounding the first electrode and the second electrode.
  • the first electrode and the second electrode are arranged and disposed to provide a constant dielectrophoresis force within an analytical space of the device.
  • the first electrode and the second electrode are arranged and disposed to provide a constant gradient field-squared within the analytical space of the device.
  • the first electrode and the second electrode define a microchannel therebetween.
  • the constant dielectrophoresis force is applied across the microchannel.
  • the analytical space of the device comprises a portion of the microchannel.
  • a method of forming the isomotive dielectrophoresis device comprises selecting a curvature of the first electrode and a curvature of the second electrode based upon the equation V -
  • an isomotive dielectrophoresis device comprising a first insulative feature having a first surface geometry, a second insulative feature having a second surface geometry, and at least one electrode positioned upstream and at least one electrode positioned downstream of an analytical space.
  • the first insulative feature and the second insulative feature are arranged and disposed to form a microchannel extending from the inlet to the outlet of the device, and the first surface geometry of the first insulative feature and second surface geometry of the second insulative feature are arranged and disposed to provide a constant dielectrophoresis force within an analytical space of the device.
  • the first electrode and the second electrode are arranged and disposed to provide a constant electrical field gradient within the analytical space of the device.
  • the constant dielectrophoresis force is applied through the
  • the analytical space of the device comprises a portion of the microchannel.
  • FIG. 1 shows an image of a typical four-electrode electrorotation (ROT) system (left) and a graph illustrating a sample spectra demonstrating subpopulation differentiation of six cell types (right).
  • ROT four-electrode electrorotation
  • FIG. 2 shows a multilayered spherical model with labeled dielectric properties of the liquid solution (s), membrane (m), and cytoplasm (c) (left) and associated Re[K CM ] and Im[K CM ] spectra.
  • FIGS. 3A-E show graphs and images of an electrode-based isomotive dielectrophoresis system according to an embodiment of the disclosure.
  • B shows an electrode-based isoDEP system using extruded two-dimensional electrodes, with the respective equipotential lines (solid lines) and electric field lines (dashed lines) illustrated therein.
  • C shows an electric field being applied across the microchannel of an electrode- based isoDEP system.
  • D shows a cross-section view of the isoDEP system of FIGS. 3A-C.
  • FIGS. 4A-E show graphs and images of an insulator-based isomotive dielectrophoresis system according to an embodiment of the disclosure.
  • A shows an isoDEP system using insulative microchannel curvature, with the respective equipotential lines (solid lines) and electric field lines (dashed lines) illustrated therein.
  • C shows an electric field being applied through the channel.
  • D shows a cross-section view of the isoDEP system of FIGS.
  • (E) shows a cross-section view of the isoDEP system including electrodes embedded upstream and downstream of the analytical space, according to an embodiment of the disclosure.
  • FIG. 5 shows that the magnitude of velocity that a particle undergoes during
  • dielectrophoresis is a function of its dielectric properties. It is either repelled from the Origin' of the device (positive DEP) or attracted towards the device (negative DEP). For either case, particle translation moves radially to/from the origin.
  • FIG. 6 is a schematic view of an isoDEP process including injecting a sample, applying a field, and acquiring an image.
  • FIGS. 7A-C show images illustrating fabrication of an isoDEP system.
  • A shows a top view of features milled into a copper sheet.
  • B shows the sheet subsequently plated with nickel/gold and diced.
  • C shows the metal sheet sealed between two acrylic substrates, one containing fluid access ports. General-use epoxy and conductive epoxy will be used to seal the microchannels and create electrical connections, respectively.
  • FIG. 8 shows 8 ⁇ polystyrene particles trapped in a 3D quadropole electrode machined with a 100 ⁇ bit.
  • FIG. 9 is Photolithographic mask for the fabrication of proof-of-concept isomotive DEP devices.
  • the red lines denote final device dimensions of approximately 11.0 mm x 11.0 mm.
  • the yellow areas denote the regions that will be etched via deep reactive ion etching (DRIE).
  • DRIE deep reactive ion etching
  • the grid in the figure provides a 5.0 mm scale.
  • FIGS. 10A-B shows images of isoDEP devices according to an embodiment of the disclosure.
  • A shows diced 4" wafer illustrating the final isomotive DEP devices.
  • B shows an individual device with two identical channels next to a penny for scale.
  • FIGS. 11A-B shows images demonstrating consistent radial translation of particles in an isoDEP device.
  • A shows radial translation using polystyrene particles exhibiting negative DEP (overlaid particle images are 1.25 seconds apart).
  • B shows radial translation using silver-coated particles exhibiting positive DEP (overlaid particle images are 0.67 seconds apart).
  • FIGS. 12A-B show images illustrating the path of individual particles undergoing nDEP towards the origin.
  • FIGS. 13A-B show images of particle scaled velocity and trajectory.
  • B shows the angle at which the particle's trajectory deviates from the theoretical direction (i.e., pure radial trajectory).
  • FIGS. 14A-B show images illustrating negative and positive DEP with an insulative isoDEP device according to an embodiment of the disclosure.
  • A shows negative DEP with the insulative isoDEP device at 300 V and 2 kHz.
  • B shows positive DEP with the insulative isoDEP device at 300 V and 2 kHz.
  • Polystyrene particles were used in (A) with overlaid particle images 2.0 seconds apart.
  • Silver-coated hollow glass particles were used in (B) with overlaid particle images 0.33 seconds apart.
  • the image insert in (B) shows pearl chaining in the direction of the applied electric field.
  • the dashed line denotes a 120° angle with the system origin labeled with a black circle.
  • the term "about,” when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ⁇ 20%, in some embodiments ⁇ 10%, in some embodiments ⁇ 5%, in some embodiments ⁇ 1%, in some embodiments ⁇ 0.5%, and in some embodiments ⁇ 0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.
  • ranges can be expressed as from “about” one particular value, and/or to "about” another particular value. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.
  • an optionally variant portion means that the portion is variant or non-variant.
  • polarizable particle means a particle that forms concentrations of charge when subjected to a field.
  • concentrations of charge include the formation of a dipole (two poles of charge) in the direction of the field. These poles form at the interface between the particle/fluid.
  • the devices and methods include electrokinetic analysis that provides detailed dielectric measurements of individual cells.
  • the detailed dielectric measurements of individual cells facilitate and/or provide identification of cell sub-populations.
  • the detailed dielectric measurements of individual cells facilitate and/or provide measurement of variants within a cell population.
  • the presently-disclosed subject matter includes a method of generating a constant dielectrophoretic (DEP) force.
  • the time-averaged dielectrophoretic force of a homogeneous spherical particle is:
  • F DEP 2ne m a 3 Re[K CM ]VE ⁇ ms (1)
  • s permittivity and the subscript m refers to fluid medium properties
  • a is the particle radius
  • E rms is the root mean square electric field
  • KCM refers to the Clausius-Mossotti factor, which is defined as:
  • a particle's resultant DEP motion is a function of particle radius (known or measured optically using established methods), Clausius-Mossotti factor (a function of the applied AC frequency, fluid conductivity, fluid permittivity, particle conductivity, and particle permittivity), and gradient of electric field squared (Vi ⁇ ms ) ⁇
  • the Clausius-Mossotti factor is assumed unknown, can be measured through quantifying particle translation, is different for various cell and particle types (conductive vs insulative, live vs dead, cancerous vs noncancerous, etc.), and a detailed frequency sweep can provide detailed insight into cell/particle composition and/or state (e.g., compromised cell membrane).
  • the gradient of electric field squared is assumed known from electrode geometry.
  • Equation (1) also assumes that the particle is spherical and homogeneous.
  • Equation (1) may be modified for multi-shelled and non-spherical particles which can be used to describe biological particles.
  • DEP and ROT experimentation can yield Re [K CM ] and Im[K CM ] spectra (arbitrary representative curves shown in FIG. 1).
  • the spectra is shifted with changes in cell morphology (ex: membrane thickness) and dielectric properties ( ⁇ , ⁇ ).
  • a dead cell may have a leaky membrane such that the cytoplasm is released - such changes can be identified with these spectra.
  • Such cell dielectric characteristics can be extracted from fitting acquired measurements to an assumed geometrical cell model.
  • the dielectrophoretic force is dependent on the real part of the Clausius- Mossotti factor and governs the attraction or repulsion of particles from regions of high field strength. If Re [K CM ] is negative, the particle experiences negative DEP (nDEP) and is repelled from larger field regions; vice versa for a positive value of Re [K CM ], when a particle experiences positive DEP (pDEP) it is attracted to high field regions.
  • the range of values for Re [K CM ] is between +1 and -0.5.
  • the experimentally altering the medium changes the direction of DEP.
  • dielectrophoretic (DEP) force includes providing a potential according to one of the following forms (in cylindrical coordinates):
  • isomotive dielectrophoresis systems arranged and disposed to provide isomotive dielectrophoresis (isoDEP).
  • isoDEP refers to a dielectrophoretic device where the dielectric properties of particles (e.g., cells) are extracted from particle tracking or particle velocimetry measurements.
  • particles e.g., cells
  • an electric field is applied and polarizable particles with negligible net charge are translated due to dielectrophoresis (DEP), or the translation of particles in a non-uniform field.
  • the isoDEP devices and/or methods described herein apply a uniform DEP force to all particles within the optical viewing area, providing uniform (isomotive) microparticle translation.
  • the isoDEP systems disclosed herein generate an electric field such that the induced DEP force is constant for all particles within the optical viewing area (i.e., analytical space).
  • the applied DEP force can be measured and the dielectric properties of individual particles can be obtained.
  • several AC frequencies can be applied to acquire a comprehensive dielectric response.
  • isoDEP is accomplished through a unique geometry where the gradient of the field-squared (Vi ⁇ ms) is constant.
  • applying a uniform DEP force includes arranging and disposing one or more electrodes to apply the uniform DEP force.
  • the electrodes are configured to provide a constant value of Vi ⁇ ms m the viewing area when an electric field is applied thereto.
  • the isoDEP devices described herein include a three-dimensional electrode geometry. Additionally, while Pohl describes a two-dimensional solution for particle sorting, which requires continuous fluid flow, in some embodiments, the presently-disclosed subject matter provides individual particle analysis where a flow of the sample is stopped during an analysis period.
  • the three-dimensional electrode geometry includes any suitable geometry and/or profile capable of providing a constant DEP force within a desired analytical space.
  • FIGS. 3A-B illustrate two curves representing equipotential boundaries to an isomotive field solution, where Equation (4a) represents the equation of each line of voltage, V.
  • Equation (4a) represents the equation of each line of voltage, V.
  • the resultant electric field (dashed lines) produces a constant gradient of the field squared, according to Equation (5).
  • this uniform electric field is applied between the electrodes and across the microchannel formed therebetween (FIG. 3C). Therefore the DEP force is constant in the region between two such boundaries.
  • Suitable geometries and/or profiles include, but are not limited to, straight-walled, curved, rounded, angled, oblique, or a combination thereof.
  • opposing surfaces of the one or more electrodes may include the same, substantially the same, or different geometrical shapes and/or sizes.
  • the surface of one electrode includes two flat or substantially flat portions intersecting at an obtuse angle while an opposing surface of another electrode includes a curved geometry substantially corresponding to the angle of the surface of the first electrode.
  • the curvature of the two electrodes follows the profile defined in Equation 5.
  • Electrodes 3B following a 120° angle with the system's origin at its bend is provided.
  • These three-dimensional electrodes are contained by electrically insulating material, such as glass, and provide a two-dimensional field throughout the depth of a chamber formed therein.
  • the electrodes are positioned between electrically insulative layers, and are positioned to directly contact a sample flowing through the microchannel formed therebetween (FIG. 3D).
  • the geometry, positioning, and/or arrangement of the electrodes is determined and/or selected to provide a constant value of VE ⁇ ms sufficient for tracking particles within the isoDEP device.
  • selection and/or determination of the geometry, positioning, and/or arrangement of the electrodes is based, at least in part, upon the size and/or shape of the particles being tracked. For example, in FIG. 3A, Electrode 1 has a minimum radius of curvature of 50 ⁇ while Electrode 2 is positioned such that a 10 V potential applied between the electrodes induces a value of VE ⁇ ms sufficient to provide adequate passage of suspended particles and/or simultaneous observation of multiple cells having a diameter of between about 5 and 50 ⁇ .
  • applying a higher voltage facilitates the application of isoDEP to smaller cells.
  • a voltage amplifier may be used to apply voltages greater than 10 V, which facilitates tracking of smaller cells, widens the microchannel, and/or provides simultaneous viewing of more cells.
  • decreasing the spacing between Electrode 1 and Electrode 2 increases the value of VE ⁇ ms and facilitates application of isoDEP to smaller cells, such as, but not limited to, bacteria and/or mammalian cells having a diameter of between about 1 and 10 ⁇ .
  • the extruded electrodes follow the curvature of the equipotential lines and/or form the wall of the microchannel itself.
  • Three-dimensional electrodes may be fabricated in a variety of ways, including sub-millimeter
  • CNC Computer Numeric Control
  • DRIE deep reactive ion etching
  • the microchannel defining electrodes of the system shown in FIGS. 3A-E are replaced with insulative features.
  • the insulative features include similar or identical geometry, positioning, and/or arrangement as the microchannel defining electrodes described above. That is, geometrically, the same or similar boundary curvature is applied for both types of devices; with electrodes serving as sidewalls in FIGS. 3A-E and insulative material serving as sidewalls in FIGS. 4A-E.
  • the electrodes are positioned upstream and downstream of the analytical space.
  • these electrodes may include any suitable shape and/or size, including, but not limited to, shapes and sizes other than those described for the sidewall electrodes of the system shown in FIGS. 3A-E.
  • the term “upstream” refers to any location in a sample pathway before the analytical space, as determined with respect to a direction of flow through the device or system.
  • the term “downstream,” as used herein, refers to any location in a sample pathway after the analytical space, as determined with respect to a direction of flow through the device or system.
  • the electrodes are positioned at a sample inlet and sample outlet of the system (FIG. 4C).
  • the upstream electrode is positioned anywhere between the sample inlet and the analytical space
  • the downstream electrode is positioned anywhere between the analytical space and the sample outlet (FIG. 4E).
  • Equation 4a The equation of each line of voltage in the isoDEP field of the system including inlet and outlet electrodes is provided by replacing the sinusoidal term in Equation (4a) with a cosine term, giving Equation (4b).
  • Equation (4b) The resultant equipotential lines (solid lines) and electric field (dashed lines) are illustrated in FIG. 4A.
  • the resultant field will also produce a constant gradient of field squared, as in Equation (5).
  • the inlet and outlet electrodes apply an electric field through the channel (FIG. 4A).
  • a greater voltage is provided to apply the electric field through the channel as compared to the voltage provided to apply the electric flied across the channel.
  • the resultant dielectrophoretic force is in the radial direction for both devices; particles will translate inward towards the origin for positive DEP and in the opposite direction for negative DEP.
  • the microchannel follows a similar curvature to that of the microelectrode design.
  • the insulative isoDEP system includes two curves that are more straightforward to fabricate.
  • the variable L refers to the distance between the origin and the electrode for insulative isoDEP devices (FIG. 5).
  • FIG. 5 also illustrates the resulting translation of a particle within an isoDEP environment. A particle experiencing nDEP will translate in the radial direction towards the origin; pDEP particles will translate radially in the opposite direction.
  • a particle size (i.e., radius, a) is extracted through image analysis, leaving the Clausius-Mossotti factor (KCM) as the only unknown parameter in Equation 1 above.
  • the particle reaches its terminal velocity within milliseconds of the application of field and the Clausius-Mossotti factor is extracted through DEP-induced particle velocimetry measurements (FIGS. 6A-C).
  • FIGS. 6A-C DEP-induced particle velocimetry measurements
  • a similar process including injecting a sample, applying a field, and acquiring an image may be used with a system including an insulative defined channel.
  • the particle's velocity changes as the AC frequency is swept over a specified range.
  • the dielectric properties of the particle(s) are obtained by tracking each particle individually for a variety of applied AC field frequencies.
  • the devices and/or methods described herein include determining and/or obtaining a detailed "particle velocity'V'frequency" spectrum for each tracked particle, which is similar to a "rotation speed'V'frequency" ROT spectrum (FIG. 1, right). That is, in some embodiments, the isoDEP device and/or method is and/or provides a translational equivalent to the rotational analysis of ROT. Thus, in another embodiment, the isoDEP device and/or method facilitates and/or provides simultaneous monitoring of the translation of many particles. In a further embodiment, this simultaneous monitoring improves DEP analytical throughput of individual particles and/or cells by at least one to two orders of magnitude, as compared to ROT.
  • the dielectric properties of the particle may be obtained by any suitable particle tracking method.
  • tracking each particle individually includes single particle tracking velocimetry/spectroscopy.
  • Particle tracking velocimetry is a technique to track a particle (typically in a liquid media) from acquired digital images.
  • a microparticle typically reaches terminal velocity within a microfluidic environment within milliseconds. If an external (unknown) force is applied to a particle, hydrodynamic drag forces will counter it. Therefore, velocimetry measurement may be used to quantify an unknown external force.
  • the radius of a particle needs to be determined, which can be estimated through various means including microscopic imaging.
  • Additional optical characterization techniques e.g., cell staining and fluorescent tagging
  • additional optical stimuli e.g., optical tweezers, infrared photostimulation, etc.
  • optical tweezers e.g., infrared photostimulation, etc.
  • isoDEP provides an electrokinetic analytical tool that is not meant to trap, divert, or concentrate particle samples.
  • electrokinetic techniques provide increased portability and/or decreased cost as compared to fluorescent techniques (e.g., flow cytometry).
  • the presently-disclosed subject matter may be applied to portable, remote diagnostic techniques, such as, for example, lab-on-chip technologies (e.g., first responders, remote environment monitoring, impoverished healthcare facilities).
  • advantages of the presently-disclosed subject matter include, but are not limited to, (i) providing a uniform DEP force that permits many particles to be observed simultaneously; (ii) providing application of straightforward microchannel injection without the need for precise particle placement relative to an electrode feature; (iii) permitting application of existing particle tracking velocimetry methods to extract K C M information for each tracked cell simultaneously, and (iv) providing increased throughput.
  • isoDEP provides high-throughput particle and/or cell analysis, including extraction of electrical and dielectric properties of each particle and/or cell (membrane permittivity/conductivity, cytoplasm permittivity/conductivity, etc.), which directly correlate to the cell physiology. Additionally or alternatively, the devices and/or methods described herein detect statistically significant variances in a particle sample (dielectric and/or geometry). In one embodiment, the devices and/or methods described herein provide analysis of cell subpopulations (i.e., individual analysis of cells). Analysis of cell subpopulations includes, but is not limited to, differentiation between different cell species, differentiation between physiological conditions (e.g., healthy and unhealthy), or a combination thereof.
  • Suitable cell subpopulations include, but are not limited to, live and dead bacteria, healthy and damaged phytoplankton, breast cancer sublines, circulating tumor cells from non- transformed cells, leukocyte subpopulations (e.g., T lymphocytes, B lymphocytes, granulocytes, monocytes), and blood types.
  • leukocyte subpopulations e.g., T lymphocytes, B lymphocytes, granulocytes, monocytes
  • isoDEP provides cell manipulation without or substantially without damaging cells from exposure to the applied field.
  • the devices and/or methods may include the use of low-conductivity (e.g., ⁇ 1 S/m) media and/or effective heat transfer design to decrease or eliminate sample heating.
  • the devices and/or methods described herein include autonomous sample injection, image acquisition, and/or analyses.
  • the devices and/or methods include isoDEP device integration, fluid handling (injection and valve control), visualization (camera optics, illumination, image acquisition), and/or data processing.
  • the devices and/or methods provide measurement of one hundred individual particles in less than ten minutes, which is an acquisition rate of approximately two orders of magnitude greater than ROT methods.
  • the method for individual particle analysis includes providing an isoDEP device, positioning a sample within the device, stopping or substantially stopping a flow of the sample within the device, applying an electric field to the device, and monitoring a translation of one or more particles in the samples.
  • applying the electric field to the device provides a constant value of i ⁇ ms m a viewing area thereof.
  • the method includes determining one or more properties of the one or more particles based upon the translation of the particle(s). In some embodiments, the method facilitates and/or provides simultaneous monitoring of the translation of many particles.
  • a method of forming the isoDEP device illustrated in FIGS. 3A-E includes fabricating the electrodes through computer numeric controlled (CNC) machining (e.g., milling).
  • CNC machining includes any suitable sized and/or shaped bit to form the desired shape of the one or more electrodes.
  • Suitable bits include, but are not limited to, bits having a diameter of 0.1 mm or smaller, such as, but not limited to, bits of between about 25 ⁇ and 100 ⁇ .
  • the curvature of the inner electrode boundary (Electrode 1, FIG. 3A) is limited by a diameter of the micrometer end-mill used during fabrication.
  • the resultant electrode perimeter is shown as "Electrode 1 boundary" in FIG. 3A.
  • the size of the bits is selected such that the depth of the milled feature does not exceed twice the diameter of the bit.
  • the machining provides straight electrode sidewalls ( ⁇ 5° taper) with a milled profile that matches the desired electrode boundary (FIG. 3A) to within 2 ⁇ .
  • fabricating the electrodes includes forming one or more microfluidic channels therein.
  • the electrodes of the isoDEP device simultaneously serve as the microchannel walls. Accordingly, the electrodes may include any material that is suitable for conducting electricity and/or compatible with the liquid sample. Suitable materials include, but are not limited to, copper, silver, gold, aluminum, doped silicon, or a combination thereof.
  • the presently-disclosed subject matter is not limited to the examples described above and, in some embodiments, may include any other conductive material that is compatible with a liquid sample being used.
  • the method may also include scaling an analytical solution to facilitate designing of the isoDEP device for a particular cell diameter.
  • forming the isoDEP device includes electroplating the electrode material with one or more electroplating materials.
  • Suitable electroplating materials include, but are not limited to, nickel, copper, chromium, zinc, tin, silver, gold, or a combination thereof.
  • the electroplating increases a biocompatibility of the electrodes and/or the isoDEP device.
  • the method includes sandwiching and/or at least partially surrounding the electrode with a substrate material.
  • the method includes spinning a thin film of UV epoxy onto two acrylic substrates, one of which includes fluid access ports, and sandwiching the machined material between the substrates.
  • the UV epoxy is cured and the substrate is diced into individual devices, isolating the two electrodes (FIG. 7).
  • Conductive and non-conductive epoxy is then applied around a perimeter of the device to make electrical connections and seal the channels, respectively.
  • any other suitable fabrication method may be used to form the isoDEP device described herein.
  • an additional high-precision micromilling machine with nanometer resolution and an assortment of 25 ⁇ to 100 ⁇ carbide square end mills is used.
  • metallic laser cutting may be used.
  • highly-doped silicon wafers patterned via deep reactive ion etching (DRIE) is used; though etching is typically conducted in a clean room environment and is more costly.
  • DRIE deep reactive ion etching
  • the isoDEP system including the insulative feature defined channel is formed from a mold.
  • the mold is formed from Su-8, which is spun and pattered onto a base, such as a glass wafer.
  • the Su-8 is pattered with any suitable geometry for the isoDEP platform, and includes a microchannel gap of any suitable size.
  • inlet and outlet well features are connected with the microchannel to form the mold.
  • forming the isoDEP system includes pouring an insulative material over the mold and then curing the insulative material, such as, for example, by baking the material over the mold.
  • the insulative material is peeled from the mold and bonded to a planar insulative piece.
  • the fluid inlets and outlets are then formed by any suitable method and the electrodes are provided therein.
  • hollow needles are inserted into the cured material to form the fluid inlets and outlets and provide the electrodes in a single step.
  • Fabrication is not restricted to soft-lithography. There exists different insulative dielectrophoresis platforms that have been fabricated, typically, out of polymer (like PDMS) or glass. Insulative microfluidic devices can be fabricated via soft lithography, chemical etching, hot embossing, injection molding, CNC milling, or other established methods.
  • insulative dielectrophoretic methods may incorporate isoDEP features in their system.
  • One example is the incorporation of DC insulator DEP to introduce electrokinetic flow (combined electrophoresis and electro-osmosis).
  • contactless DEP cDEP
  • cDEP contactless DEP
  • insulative dielectrophoretic systems decrease cost and/or reduce complexity of device fabrication as compared to those containing microelectrodes.
  • the insulative isoDEP features are relatively large for microfluidic devices, including channel widths that are generally greater than 0.1 mm while still conforming to the curvature of the analytical solution (FIG. 4A).
  • the preceding theoretical treatment of the insulative isoDEP system suggests that k, may be, interestingly, independent of the microchannel gap (r 60 ) thus enabling flexibility in its design.
  • This Example describes formation of an analytical DEP system capable of measuring the dielectric properties of individual particles.
  • the goveming isoDEP equations were derived as disclosed above and applied to two different isoDEP prototypes: (i) one fabricated from deep reactive ion etching (DRIE) of a conductive silicon wafer (1-10 ⁇ -cm) whose patterned features served as electrodes and microchannel sidewalls simultaneously; (ii) a second where the electric field is applied lengthwise through a PDMS microchannel whose geometry follows a specific curvature.
  • DRIE deep reactive ion etching
  • the isoDEP platform is capable of analyzing multiple particles simultaneously, providing greater throughput than traditional electrorotation platforms.
  • This example illustrates the required length scale of a microelectrode-based isoDEP device.
  • Equation (1) assumes a stationary field. However, if there is a spatial non- uniformity in the electric field's phase ( ⁇ ), the extended DEP force is given by
  • ROT electrorotation
  • Examples 4-10 are directed to the isoDEP system including the electrode defined channel
  • isoDEP provides a detailed, sensitive measurement for each cell which is as sensitive as ROT and static EIS. Additionally, although the processing rate of isoDEP is may be less than that of flow cytometry and dynamic EIS, it is at least one to two orders of magnitude greater than ROT and static EIS. Furthermore, in certain embodiments, the cost of isoDEP device fabrication and operation is significantly lower than the other techniques.
  • isoDEP devices can be fabricated using a benchtop micromill as opposed to cleanroom fabrication methods (unlike ROT, EIS microfluidic chips) which comparatively require significant infrastructure and facilities.
  • isoDEP operation requires a benchtop waveform generator and a digital camera which is a significant reduction in operation cost compared to ROT (four-channel waveform generators), static EIS (impedance analyzers), and, especially, flow cytometry. Reduced costs also enable more frequent tests which is important for applications that require frequent monitoring. Another advantage is the portability of isoDEP. Flow
  • Table 1 General overview comparing various cell subpopulation analytical techniques. $: Requires specialized fabrication facilities (i.e. cleanroom) or specialized equipment for operation. $$:
  • Phytoplankton growth is a function of light ()d a p er s econnd temperature, buoyancy, inorganic nutrient availability, interactions with organic compounds, organic micronutrients, and competition and
  • Neochloris oleoabundans has been sorted and concentrated for biofuel production using DEP. Electrorotation has been used to distinguish between viable-healthy and non-viable Tetraselmis cells in marine environments. Insulator-based DEP has been used to examine the dielectric properties of the cell membranes of viable and non-viable Selenastrum capricornutum, a common freshwater species.
  • isoDEP methods described herein would (1) decrease the cost of electrokinetic characterization of cells as compared to other methods, and (2) provide portability in the field (onto water bodies) for real-time analysis of the physiological status of phytoplankton assemblages in situ. This information would give important insight into the overall health of an aquatic ecosystem.
  • the tested species include two species of green algae, Chlorella vulgaris and
  • Microcystis toxin causes severe and acute toxicological responses in other aquatic organisms when subjected to bloom conditions. All identified species are ubiquitous in freshwater phytoplanktonic assemblages and are relatively easy to culture and maintain.
  • Phytoplankton cultures used in this project are maintained under sterile conditions in Percival incubators at Hancock Biological Station (HBS), Murray State University (Murray, KY). Maintenance of actively metabolizing and growing phytoplankton cultures takes place in accordance with methods of Lorenz et al. The physiological status of cultures is monitored and cells will be prepared for experimentation using methods of Maclntyre and Cullen. Appropriate QA/QC procedures are adhered to for cultures over the course of this study. Comparisons of the physiological response of healthy cells by isoDEP are made with cells that are senescing (out of log-phase growth) or killed (by boiling or poisoning).
  • isoDEP By measuring the electrokinetic response of several freshwater phytoplankton species (Table 2), isoDEP provides identification and differentiation thereof. Rapid, accurate detection of potentially harmful algal blooms and degraded water quality using isoDEP methods indicates a positive outcome from these experiments that will contribute to other applications in unhealthy or otherwise compromised aquatic systems (e.g., polluted, contaminated, eutrophic).
  • An advantage of using isoDEP technology in water quality studies is that it is rapid, accurate, and can detect subtle changes in algal cell health (and, thus, ecosystem health), and can be taken into the field (on shipboard) for instant results without having to collect, maintain, and transport live samples back to a land-based laboratory for testing.
  • Example 6 Fabrication and system operation.
  • a commercially available miniature end mill (Mini-Mill 1, Minitech Machinery, Norcross, GA), which has a spindle accuracy of 0.8 ⁇ and spindle speed up to 60,000 rpm, is used for this example.
  • a 100 ⁇ end mill is used to create the electrodes from copper sheets as outlined in FIG. 7.
  • the milled features include the electrode geometry as well as microfluidic channels (FIG. 7A).
  • the metal is electroplated with nickel then gold to provide a robust finish to their product. Further, the plated layer of gold aids in electrode
  • a thin film of UV epoxy is spun onto two acrylic substrates (one of which contains fluid access ports) that are used to sandwich the machined metal sheet. After curing, the substrate is diced into individual devices (FIG. 7B) which subsequently isolates the two electrodes (FIG. 7C). Finally, conductive and non-conductive epoxy is applied around the perimeter of the device to make electrical connections and seal the channels, respectively (FIG. 7C).
  • a milled DEP quadropole device was formed using a copperclad PCB substrate.
  • the quadropole electrode from this work is shown in FIG. 8, and was created using a 100 ⁇ bit with the copperclad substrate and subsequent DEP trapping of 8 ⁇ particles.
  • a particle velocimetry map of the microchannel will be obtained to determine if identified velocimetry inconsistencies are specific to a particular microchannel region or feature. Alternatively, velocimetry inconsistencies may be attributed to the statistically inhomogeneous distribution of particle diameters (results will be compared accordingly).
  • Fluorescent imaging will be used for more precise particle tracking (i.e. tracking the individual particle's fluorescent peak).
  • fluorescent analysis is nontrivial to implement for portable micro-devices and alternative methods may need to be sought for future development.
  • particle shadow velocimetry uses a pulsed LED and has demonstrated velocimetry resolution comparable to fluorescent/excited PIV methods.
  • PIV particle shadow velocimetry
  • Proper cell imaging cannot be overlooked since morphological information for each particle can be extracted accordingly and, further, the cell's size gives insight into its DEP force (which is directly proportional to the volume of the particle).
  • Example 8 Demonstrate bead subpopulation identification.
  • the Clausius-Mossotti factor may be extracted from isoDEP measurements.
  • frequency-dependent Re [f CM ] measurements of polystyrene and silica particles are compared to those in literature (these particle types are found extensively in DEP experimental research) and evaluated for similarity.
  • Example 9 Assess unwanted electrokinetic effects in isoDEP devices.
  • Example 10 Design and fabrication.
  • a proof-of-concept design was fabricated at the University of Louisville Micro/Nano Technology Center.
  • the electrodes for the final device were constructed from a highly doped silicon wafer (0.35 mm thick, B-doped to a resistivity of 1 -10 ⁇ -cm).
  • any two arbitrary equipotential lines can be chosen as long as they are of the form (using polar coordinates)
  • V - kr 3 ' 2 sin(3 e / 2 ) (4a)
  • the curvature of Electrode 2 is determined through an assumed applied voltage. For an assumed voltage difference of 100 V, the curvature of Electrode 2 is
  • the curvatures chosen for one of the devices is Vi and V 2 and those of the second device is Vj and Vs. Any electrode profiles can be chosen following this method.
  • the 4" silicon doped wafer was anodically bonded to a 4" diameter borosilicate glass wafer (0.7 mm thick) followed by DRIE of the pattem.
  • a second glass wafer was anodically bonded on the other side of the silicon wafer.
  • the wafer was diced to produce the final devices. The diced wafer is shown in FIG. 10A and an individual device is shown in FIG. 10B with two identical channels.
  • the devices were tested using polystyrene particles (24.9 ⁇ diameter, carboxylate modified, Spherotech) and silver coated hollow glass spheres (5-30 ⁇ diameter, Cospheric). The particles were suspended in a deionized water solution with an electrical conductivity less than 1 mS/m. Electrical wires were affixed to the electrodes at the perimeter of the device using conductive epoxy. The sample was inj ected manually with a syringe and was adjusted until bulk fluid motion halted. An AC signal was then applied across the channel (1 kHz to 10 MHz, up to 100 V rms ). The device was viewed with an inverted microscope (Nikon Ti) using a 2X objective lens.
  • FIG. 11A shows a composite image of polystyrene particles traveling towards the origin exhibiting negative DEP (nDEP) for an AC signal of 100 kHz and 34.4 V.
  • nDEP negative DEP
  • the shown overlaid particle images represent their respective position after several 1.25 second intervals.
  • Preliminary qualitative observations suggest that the velocity of each particle is nearly constant (i.e. their spacing between each time step is consistent) and, further, particles translate in a radial direction; a more detailed particle tracking analysis is ongoing.
  • FIG. 11B shows a composite image of silver coated particles translating away from the origin exhibiting positive DEP (pDEP) for an AC signal of 500 kHz at 33 V.
  • the shown overlaid particle images represent their respective position after 0.67 second intervals. Consistent with the negative DEP results, translation is away from the origin of the device at a consistent rate. Consistent with DEP theory, larger particles translate at a greater velocity.
  • FIGS. 12A-B show the path and linear fit for 110 individual particles undergoing nDEP as in FIG. 11A.
  • FIGS. 13A-B show scaled particle velocity and deviation of particle trajectory from the theoretical direction, respectively.
  • Examples 11-12 are directed to isoDEP systems including the insulative feature defined channel
  • Example 1 1 - Design and fabrication.
  • a PDMS microfluidic device was fabricated from a master mold of Su-8, a common procedure for microfluidic devices.
  • a single layer of Su-8 50 approximately 100 ⁇ thick, was spun and pattered onto a soda-lime glass wafer.
  • Inlet and outlet well features of 2.0 mm diameter were connected with the microchannel; their center was approximately 3.0 mm from the device origin.
  • An illustration of the Su-8 mold geometry is shown in FIG. 4B.
  • PDMS was poured over the master mold and baked in an oven at 65 °C for at least two hours. After curing, the PDMS was peeled from the mold and plasma bonded to a planar PDMS piece. Fluid inlets and outlets were created by inserting 22 gauge stainless steel needles; the needles themselves also simultaneously served as device electrodes (FIG. 4B).
  • the AC signal originated at an arbitrary waveform generator (Keithley 3390) and was amplified using an in-house custom-built amplifier; the output signal was monitored using a benchtop oscilloscope.
  • FIGS. 14A-B demonstrates the trajectory of translating particles undergoing negative DEP (FIG. 14A) and positive DEP (FIG. 14B) at 300 V and 2 kHz using an insulating isoDEP device with a r 60 gap of 500 ⁇ .
  • the particles translated in the radial direction in accordance with expected theory. Particle spacing between overlaid images (2.0 seconds apart for FIG. 14A, 0.33 seconds for FIG. 14B) is consistent, suggesting relatively constant dielectrophoresis-induced velocity.
  • the insert in FIG. 14B shows the chaining of two particles aligned in the direction of the applied field; interestingly, inherent to the nature of this insulative isoDEP system, the direction of the
  • FIG. 15 The trajectories of 18 polystyrene particles subjected to negative dielectrophoresis are shown in FIG. 15. Compared to previous results using the microelectrode isoDEP device (FIG. 3B), the trajectories of the insulative isoDEP device were not significantly influenced when the particles were in close proximity to the wall, this was likely due to the smooth contour of the microchannel. In some cases bulk fluid motion was observed and, to some extent, compromised the trajectory of the particles; future versions of the isoDEP system will incorporate to eliminate bulk fluid motion during
  • insulative isoDEP devices are primarily constructed from the same material (PDMS for this work). Since the thickness of the device is much smaller than its footprint (millimeters compared to centimeters), conductive heat transfer through its thin boundaries will be the primary mode of heat transfer. Therefore, significant temperature gradients will be generated orthogonal to the direction of the electric field, a desirable system characteristic in order to reduce the effects of electrothermal flow. As such, similar to the previous isoDEP device, electrothermal hydrodynamic motion was observed under certain experimental conditions.
  • Examples 13-18 are directed to isoDEP systems including both the electrode based and insulative feature defined channel
  • Example 13 Design and fabrication of the electrode based isoDEP device.
  • the device was mounted to a microscope slide with quick-drying epoxy; the epoxy was also used to seal the "Electrode Gaps" (FIG. 3E) that prevented electrical shorting between the adjacent electrodes.
  • a diamond bur (0.9 mm diameter, 801L/009C, Crosstech) was subsequently used to drill holes into the upper glass substrate and enable fluid access.
  • pieces of single-sided rubber adhesive FDA-compliant silicone rubber, #8991K52, McMaster-Carr
  • Electrical connections to the device were accomplished by applying conductive epoxy (CW2400, Circuit Works) to the perimeter of the device at the exposed portions of the doped silicon electrodes; thin wires were embedded in the conductive epoxy and cured in a 60 °C oven for 20 minutes.
  • conductive epoxy CW2400, Circuit Works
  • Example 14 Design and fabrication of the insulator-based isoDEP device
  • a PDMS microfluidic device was fabricated via soft lithography from a master mold of Su-8, a common procedure for microfluidic devices.
  • a single layer of Su-8 50 approximately 120 ⁇ thick, was spun and pattered onto a soda-lime glass wafer.
  • the centers of the inlet and outlet wells (2 mm in diameter) were 3.0 mm from the device origin.
  • PDMS was poured over the master mold and baked in an oven at 65 °C for at least two hours.
  • the PDMS was peeled from the mold and plasma bonded to a planar PDMS piece. Fluid inlets and outlets were created by inserting 22 gauge stainless steel needles; the needles themselves also simultaneously served as device electrodes (FIG. 4B).
  • the AC signal originated at an arbitrary waveform generator (Keithley 3390) and subsequently amplified using either a commercial benchtop amplifier (2100HF, Trek; up to 150 V, 1 MHz) or an in-house custom-built amplifier (up to -800 V, 10 kHz).
  • the signal output was monitored with a benchtop digital multi-meter (Agilent 34405 A).
  • Flow was manually adjusted until one or more particles were observed in the viewing area, at that time a quarter-turn flow valve was closed to halt flow. Particles were allowed to sediment ( ⁇ 1 min.) before the AC field was applied.
  • TrackMate a particle tracking plug- in for ImageJ (imagej.net/TrackMate), was used to measure particle diameter and position for each video frame. Particles that were adhered to a surface or collided with an electrode wall were omitted from particle tracking analysis.
  • a custom MATLAB program determined particle velocity and trajectory from particle position data.
  • all particle tracking data was cropped to a common length (165 frames or 11 seconds for microelectrode isoDEP, 60 frames or 4.0 seconds for insulative isoDEP).
  • a common length 165 frames or 11 seconds for microelectrode isoDEP, 60 frames or 4.0 seconds for insulative isoDEP.
  • its initial position within that frame was extracted (x,, y,) as well as its coordinates for the next four subsequent frames (x ;+ i, 3 ⁇ 4 +1 ; x,+2, y ⁇ +2, xm, i+3; x ; +4, i+4).
  • the applied frequencies for these cases were arbitrarily chosen as the observed velocities of the particles did not change over a range of tested frequencies (1 kHz - 1 MHz, data not shown); this was expected as the Clausius-Mossotti factor would be relatively independent of AC frequency for large (> 2 ⁇ ) homogeneous particles.
  • FIGS. 11A-B and 14A-B illustrate nDEP of polystyrene particles and pDEP of silver- coated hollow glass particles using both isoDEP devices.
  • the particles translated in the radial direction, either towards (nDEP) or away (pDEP) from the origin. Further, qualitatively, particle translation appears to be constant during its trajectory as demonstrated with consistent spacing between particle images. Refer to Supporting Information for experimental videos demonstrating isoDEP particle translation using negative and positive DEP.
  • polystyrene particles were tracked for quantitative evaluation of the devices' performance.
  • Particle trajectories (FIGS. 12A and 15) were consistently migrating towards the device origin.
  • DRIE inherently does not produce smooth features; as such, rough electrode features will distort the electric field in close proximity to the wall and, therefore, compromise particle trajectory, detracting it from the theorized motion.
  • the trajectories of the insulative isoDEP device were not significantly influenced when the particles were in close proximity to the wall; this was likely due to the smoother contour of the PDMS microchannel.
  • the nature of Su-8 photolithography produces sidewalls that are slightly tapered, though that did not appear to significantly influence results; however, DRIE could be used to create a master mold with vertical walls, as demonstrated previously with other electrokinetic microfluidic chips, and may be considered for future insulative isoDEP devices.
  • Example 18 - Observed electrohydrodynamics Electrohydrodynamic motion was observed at larger applied potentials (> 50V for microelectrode isoDEP, > 400 V for insulator isoDEP); a more comprehensive investigation will be conducted in future work to determine the impact of such fluid motion on particle trajectory.
  • Electrohydrodynamic motion in general, is either AC electro-osmosis (ACEO) and/or electrothermal (ET) flow.
  • ACEO occurs when the electric field acts upon the charges accumulated on the surface of a polarized electrode, inducing hydrodynamic slip. It is expected that this isoDEP device will produce negligible ACEO flow as the tangential component of the electric field at the electrode surface is significantly weaker compared to other studied ACEO microelectrode geometries like interdigitated electrodes. Therefore, it is hypothesized that ET flow is the cause of the observed electrohydrodynamic motion. In ET flow the electric field acts upon dielectric gradients (permittivity, conductivity) in the fluid caused by Joule heating.
  • the silicon in the microelectrode isoDEP device may serve as a heat sink as well as provide more efficient heat transfer (compared to the glass boundaries) and, thus, propagate temperature gradients in the same direction as the applied field.
  • Insulative isoDEP systems are likely more favorable from a heat transfer perspective as temperature gradients are more orthogonal to the field direction relative to the silicon-based devices, thereby reducing the effects of electrothermal flow. Future work will study the heat transfer and resulting electrohydrodynamics of isoDEP devices.
  • isoDEP features are relatively large when compared to traditional microfluidic devices as microchannel widths are generally greater than 0.1 mm; however, conforming to the curvature of the analytical solution (FIGS. 3B and 4A) is important for proper device performance.
  • Particular to insulative isoDEP devices k may be, interestingly, independent of the microchannel gap (for L » r 60 ), thus enabling flexibility in its design.
  • isoDEP isoDEP into their system.
  • DC insulator-based DEP to introduce electrokinetic flow (combined electrophoresis and electro-osmosis).
  • contactless DEP may be used to prevent direct contact between the electrode and fluid sample. The suppression of unwanted flow (bulk flow, electrohydrodynamics) coupled with ideal electrode fabrication (smooth parallel sidewalls) will result in unhindered particle trajectory.
  • isoDEP isoDEP
  • this platform provides optical access to the sample enabling the integration of optically -based manipulation (ex: optical tweezers) and analysis (ex: immunostaining) enabling comprehensive single-cell analysis.
  • V Ar n sin(n0) (D2) where A and n are constants.
  • E —VV the equation is:
  • V(£ 2 ) 2 (n - l n 2 A 2 r 2n ⁇ 3 r (D6)
  • V 2 / 3 k r 3 / 2 sin(30/2) (4a)
  • Pethig, R., Dielectrophoresis Status of the theory, technology, and applications. Biomicrofluidics, 2010. 4: p. 022811. Sun, T., Morgan, H., and Green, N.G., Analytical solutions of ac electrokinetics in interdigitated electrode arrays: electric field, dielectrophoretic and traveling-wave dielectrophoretic forces. Phys Rev E Stat Nonlin Soft Matter Phys, 2007. 76(4 Pt 2): p. 046610. Muller, T., Gradl, G., Howitz, S., Shirley, S., Schnelle, T., and Fuhr, G., A 3-D microelectrode system for handling and caging single cells and particles.

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Abstract

La présente invention concerne des procédés d'analyse de particules et des dispositifs de diélectrophorèse isomotrice, et des procédés de formation de dispositifs de diélectrophorèse. Le procédé d'analyse de particules comprend la fourniture d'un dispositif de diélectrophorèse isomotrice ; le positionnement d'un échantillon dans le dispositif, l'échantillon comprenant au moins une particule ; l'application d'un champ électrique au dispositif, le champ électrique induisant une force de diélectrophorèse constante sur l'au moins une particule de l'échantillon ; et la surveillance d'une translation de l'au moins une particule. Un dispositif de diélectrophorèse isomotrice comprend une première électrode ayant une première géométrie de surface ; une deuxième électrode ayant une deuxième géométrie de surface ; et un matériau électriquement isolant entourant au moins partiellement la première électrode et la deuxième électrode.
PCT/US2017/041238 2016-07-08 2017-07-07 Diélectrophorèse isomotrice pour analyse diélectrique de sous-populations de particules WO2018009892A1 (fr)

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WO2022238235A1 (fr) * 2021-05-11 2022-11-17 Ceidos Sa Système de surveillance de culture cellulaire et cartouche de diélectrophorèse

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