EP2039433A1 - Dielektrophoretische Vorrichtung zur Verarbeitung von Teilchen - Google Patents

Dielektrophoretische Vorrichtung zur Verarbeitung von Teilchen Download PDF

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Publication number
EP2039433A1
EP2039433A1 EP07115462A EP07115462A EP2039433A1 EP 2039433 A1 EP2039433 A1 EP 2039433A1 EP 07115462 A EP07115462 A EP 07115462A EP 07115462 A EP07115462 A EP 07115462A EP 2039433 A1 EP2039433 A1 EP 2039433A1
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Prior art keywords
electrodes
array
particles
dielectrophoretic
neighboring
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EP07115462A
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English (en)
French (fr)
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Koninklijke Philips NV
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Koninklijke Philips Electronics NV
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Priority to EP07115462A priority Critical patent/EP2039433A1/de
Priority to PCT/IB2008/053417 priority patent/WO2009027927A2/en
Publication of EP2039433A1 publication Critical patent/EP2039433A1/de
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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
    • B03C5/005—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]
    • 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
    • B03C5/028—Non-uniform field separators using travelling electric fields, i.e. travelling wave dielectrophoresis [TWD]

Definitions

  • the present invention relates to dielectrophoretic devices. More particularly the present invention relates to a dielectrophoretic device for manipulating particles, to a method for forming such a device and to a method for manipulating particles using such a device.
  • the methods and devices according to embodiments of the invention may, for example, be used for identifying and/or separating and/or sorting particles, e.g. cells, or for lysis or electroporation of cells.
  • the ability to identify and separate cell sub-populations from a heterogeneous cell mixture is essential in many biomedical applications.
  • the simplest methods known for such purposes are based on filtration and centrifugation and rely on differences in cell size or density. More advanced methods exploit specific binding of antibodies to antigens on a cell surface to target a particular cell population. Examples of such methods are magnetically activated cell sorting (MACS), where antibody-functionalized magnetic beads are attached to the cells and sorted in a magnetic field, or fluorescence-activated cell sorting (FACS), where cells are labeled with fluorescent antibodies and separated by electrostatically deflecting charged liquid droplets containing the cells.
  • MCS magnetically activated cell sorting
  • FACS fluorescence-activated cell sorting
  • Current FACS analyzers are very versatile instruments and allow cell separation on the basis of multiple simultaneous markers, cell size, and scattering properties. However, they are large and expensive instruments and can only be operated by trained personnel.
  • DEP dielectrophoresis
  • DEP has been applied to manipulate and separate a variety of cells including bacteria, yeast, and mammalian cells in microsystems.
  • DEP has been used to separate cancer cells from blood, isolate CD34+ stem cells from blood, bacteria from blood and to separate various cell sub-populations of blood.
  • Hyperlayer dielectrophoretic field-flow fractionation is a significant step towards a more refined separation of cell populations.
  • a linear array of microelectrodes is used to levitate cells by negative DEP.
  • a non-constant flow profile causes cells levitated at different heights to emerge from the separation channel at different times.
  • electrosmear has been developed to allow collection of cells onto characteristic zones on a substrate.
  • DEP-FFF this method is also based on the combination of liquid flow and cell levitation by negative DEP.
  • the cells are introduced at one end of an electrode array, which provides a levitation force that opposes cell sedimentation and prevents cells from adhering to the substrate.
  • the voltage applied to the electrodes varies along the array. The cells flow along the channel until the DEP forces are no longer sufficient to levitate them, at which point they touch down and adhere to the substrate, which is coated with a binding agent.
  • a device comprising a channel defined by two rows of electrodes separated by the channel and wherein the width of the channel progressively decreases in the direction of particle migration.
  • the spacing between successive electrodes in each of the rows may be the same ( Fig. 1(a) ) or the spacing between successive electrodes in each of the rows may progressively decrease in the direction of particle migration ( Fig. 1(b) ).
  • Traveling wave dielectrophoresis (twDEP) is used to transport the particle through the channel.
  • a particle moving under twDEP, in EP 0 815 942 referred to as traveling wave field migration will be accelerated as a result of the field increasing as the channel width decreases.
  • the present invention provides a dielectrophoretic device for manipulation of particles.
  • the device comprises:
  • An advantage of embodiments of the invention is that different types of particles even with small differences in properties may be manipulated, e.g. separated, with the dielectrophoretic device according to embodiments of the invention.
  • the dielectrophoretic device according to embodiments of the invention is efficient and sensitive.
  • the dielectrophoretic device according to embodiments of the invention can be used for performing particle manipulation from small, non-flowing volumes of particle suspensions.
  • the array of electrodes may be a one-dimensional array.
  • the direction of decrease of the width of the space in between two neighboring electrodes may be oriented in the direction of the average longitudinal direction of the neighboring electrodes.
  • the direction may be a radial direction, for linear arrays, the direction may be the longitudinal direction of the electrodes.
  • the electrodes may be funnel-shaped.
  • the array of electrodes may be a radial array in which the electrodes are arranged as segments of a circle extending from a center of the circle to its circumference.
  • the array of electrodes may be a linear array and the electrodes are funnel-shaped.
  • the driving means may be adapted for driving the electrodes with a mutual phase difference of 360°/n between two neighboring electrodes, preferably between every two neighboring electrodes, with n being higher than 2.
  • n may be 4 and the driving means may be adapted for driving the electrodes with a mutual phase difference of 90° between two neighboring electrodes.
  • the dielectrophoretic device may furthermore comprise transport means for transporting particles toward the array of electrodes.
  • the transport means may be formed by a linear array of parallel electrodes.
  • the dielectrophoretic device may furthermore comprise collection means for collecting particles.
  • the collection means may be formed by an array of collection channels.
  • the collection channels may be microfluidic channels.
  • the collection means may furthermore comprise impedance measurement means for determining an amount of particles in the collection means.
  • the particles may be biological particles (e.g. cells), solid dielectric particles, or engineered particles.
  • the present invention also provides the use of the dielectrophoretic device according to embodiments of the invention for particle separation or sorting.
  • the present invention also provides the use of the dielectrophoretic device according to embodiments of the invention for cell lysis or cell electroporation.
  • a method for forming a dielectrophoretic device for manipulation of particles comprises:
  • An advantage of the method according to embodiments of the invention is that it allows forming a dielectrophoretic device in which different types of particles even with small differences in properties may be manipulated, e.g. separated, with the dielectrophoretic device according to embodiments of the invention.
  • Providing an array of electrodes may be performed such that the array is a one-dimensional array.
  • Providing an array of electrodes may furthermore be performed such that the direction of decrease of the width of the space in between two neighboring electrodes, preferably in between every two neighboring electrodes is oriented in an average longitudinal direction of the two neighboring electrodes.
  • the same direction may be a radial direction
  • the same direction may be the longitudinal direction of the electrodes.
  • providing an array of electrodes may be performed such that a radial array is formed in which the electrodes are arranged as segments of a circle extending from a center of the circle to its circumference.
  • providing an array of electrodes may be performed such that a linear array is formed with electrodes having a funnel-shape.
  • the method may furthermore comprise providing transport means for transporting particles toward the array of electrodes.
  • the method may furthermore comprise providing collection means for collecting particles.
  • the method may furthermore comprise providing impedance measurement means to the collection means for determining an amount of particles in the collection means.
  • a method for manipulating particles using a dielectrophoretic device comprising an array of electrodes wherein the space in between two neighboring electrodes, preferably in between every two neighboring electrodes, has a decreasing width in a longitudinal direction of the electrodes.
  • the method comprises applying a voltage signal to the electrodes with a mutual phase difference of 360°/n between two neighboring electrodes, preferably between every two neighboring electrodes, with n being higher than 2.
  • n may be 4 and a voltage signal may be applied to the electrodes with a phase difference of 90° between two neighboring electrodes, preferably between every two neighboring electrodes.
  • the method may furthermore comprise transporting the particles towards the array of electrodes for allowing them to be manipulated.
  • the method may furthermore comprise collecting particles in a collection means after manipulation of the particles.
  • the method may furthermore comprise counting the amount of particles in the collection means.
  • Manipulating particles may comprise separating particles based on a difference in dielectric properties.
  • Manipulating particles may comprises lysis or electroporation of cells.
  • the present invention also provides the use of the method according to embodiments of the invention in molecular diagnostics, biological sample analysis or chemical sample analysis.
  • the present invention also provides the use of the method according to embodiments of the invention for particle separation.
  • the present invention also provides the use of the method according to embodiments of the invention for cell lysis or cell electroporation.
  • a controller for controlled driving of electrodes of an array wherein the space in between two neighboring electrodes, preferably in between every tow neighboring electrodes, has a decreasing width in a longitudinal direction of the electrodes.
  • the controller comprises a control unit for controlling a driving means for applying a voltage to the electrodes with a mutual phase difference of 360°/n between two neighboring electrodes, preferably in between every tow neighboring electrodes, with n being higher than 2.
  • n may be 4 and the controller may be adapted for controlling the driving means such that a voltage signal is applied to the electrodes with a phase difference of 90° between two neighboring electrodes, preferably between every two neighboring electrodes.
  • the present invention furthermore provides a computer program product for performing, when executed on a computing means, a method according to embodiments of the invention.
  • the present invention also provides a machine readable data storage device for storing the computer program product according to embodiments of the invention.
  • the present invention also provides a transmission of the computer program product according to embodiments of the invention over a local or wide area telecommunications network.
  • the dielectrophoretic device according to embodiments of the invention is efficient and sensitive.
  • the dielectrophoretic device according to embodiments of the invention can be used for performing manipulation of particles in small, non-flowing volumes of particle suspensions.
  • the dielectrophoretic device may be used for manipulating different types of particles. According to embodiments of the invention, different types of particles can be separated, even with small differences in properties, e.g. shape, size, or composition.
  • the dielectrophoretic device according to embodiments of the invention can also be used to separate particles with different degrees of positive or negative dielectrophoresis. Hence the present invention can be used for cell sorting.
  • top, bottom and the like in the description and the claims are used for descriptive purposes and not necessarily for describing relative positions. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other orientations than described or illustrated herein.
  • an element described herein of an apparatus embodiment is an example of a means for carrying out the function performed by the element for the purpose of carrying out the invention.
  • the present invention provides a dielectrophoretic device for manipulation of particles, a method for manufacturing such a dielectrophoretic device and a method for manipulating, e.g. identifying, sorting, separating, lysis or electroporation, of particles, e.g. cells using such a dielectrophoretic device.
  • the device and methods according to embodiments of the invention may be used for manipulation of dielectric particles such as microparticles, nanoparticles, cells, or to any other suitable particles having dielectrophoretic properties.
  • suitable particles which may be used with embodiments of the present invention may be solid dielectric particles such as e.g. polystyrene or latex beads or carrier beads (beads to which molecules or cells can be bound), engineered particles such as e.g. particles with a conductive core and an insulating shell, or vice versa, biological particles such as cells, bacteria, viruses, large molecules e.g. large proteins, complexes of molecules.
  • the device and method for manipulation of particles according to embodiments of the present invention need only rely on electric field induced effects to achieve manipulation of particles, e.g. separation of particles, on a macroscopic level.
  • particles e.g. cells are transported electrically through a stationary fluid. Therefore, in accordance with embodiments of the present invention, the need for liquid flow generation and thus for pumping mechanisms is eliminated. This allows, in principle, using very small volumes of suspensions comprising the particles.
  • the dielectrophoretic device and methods according to embodiments of the invention can be used for performing particle manipulation from small, non-flowing volumes of particle suspensions. With small volumes is meant volumes of between 0.5 and 50 ⁇ l, for example 10 ⁇ l.
  • the device and methods according to embodiments of the invention may be used in a variety of applications including molecular diagnostics, biological sample analysis or chemical sample analysis.
  • the device and methods according to embodiments of the invention may be used for separating or sorting particles with different dielectric properties.
  • Particle types may differ in size, shape and/or composition, which will lead to different dielectric properties and thus to different dielectrophoretic responses.
  • dielectric particles e.g. cells
  • dielectric particles may be separated according to their dielectric properties into distinct zones on the device. This facilitates visual or automatic inspection of a sample under investigation.
  • particles, e.g. cells can be collected into separate microfluidic channels for subsequent analysis or processing. This includes, for example, counting cells in various collection channels, statistically analyzing particle distributions, or further fractionating a target sub-population.
  • the dielectrophoretic device allows a more refined particle, e.g. cell, separation.
  • particles, e.g. cells, with different degrees of positive (or negative) DEP can be separated in a single step.
  • particles having a different degree of positive (negative) DEP is meant that these particles undergo positive (or negative) DEP, but the force has a different magnitude. Due to long interaction distances achieved with the dielectrophoretic device according to embodiments of the invention, even small variations in the dielectric properties of particles, e.g. cells, can be resolved.
  • the dielectrophoretic device and method for manipulation of particles, e.g. cells, according to embodiments of the invention may be used for lysis or electroporation of cells.
  • a dielectrophoretic device for manipulation of particles.
  • the dielectrophoretic device comprises an array of electrodes, there being a space in between every pair of neighboring electrodes.
  • the array of electrodes can be a one-dimensional array.
  • the array may be a linear array or a radial array.
  • the dielectrophoretic device may comprise at least two one-dimensional arrays placed next to each other.
  • Each electrode has a longitudinal direction.
  • Two neighboring electrodes e.g. every pair of neighboring electrodes, have an average longitudinal direction, being a direction defined by the average of the longitudinal directions of the two neighboring electrodes.
  • the array of electrodes is formed such that the space between two neighboring electrodes, for example between every two neighboring electrodes, has a width which is decreasing in the longitudinal direction of the electrodes. The width may be decreasing continuously. Preferably the width of the space between two neighboring electrodes may be measured in a direction perpendicular to the average longitudinal direction of the neighboring electrodes.
  • the space in between two neighboring electrodes e.g. every two neighboring electrodes, may be funnel-shaped.
  • the direction of the decrease of the width of the spaces in between the two neighboring electrodes may be oriented in a same direction, i.e. the average longitudinal direction of the two neighboring electrodes may be the same (linear array).
  • the direction of the decrease of the width of the spaces in between every two neighboring electrodes may be radially oriented to a center point of the array (radial array).
  • the dielectrophoretic device is based on traveling wave dielectrophoresis (twDEP) in combination with either negative (repulsive) or positive (attractive) DEP. Therefore, the dielectrophoretic device furthermore comprises driving means adapted for driving the electrodes of the array to generate a twDEP force to be exerted on the particles.
  • twDEP traveling wave dielectrophoresis
  • An array of electrodes e.g. microelectrodes
  • twDEP a relatively long distance by twDEP.
  • the present invention proposes the use of an electrode array with a space in between every two neighboring electrodes having a width which is decreasing in a longitudinal direction of the electrodes.
  • the space in between every neighboring electrode may be referred to as funnel-shaped, hence the space may also be referred to as a funnel.
  • Configurations of the array according to embodiments of the invention allow, in addition to generating a propelling twDEP force, generating a weaker DEP component perpendicular to the traveling direction.
  • the magnitude and direction of such component varies for different types of particles with differences in dielectric properties, e.g. sub-populations of cells, and allows them to be manipulated, e.g. transported or separated or sorted.
  • configurations of electrode arrays according to embodiments of the invention may generate a strongly inhomogeneous electric field. Particles experiencing a positive DEP force may be pulled towards locations in the space in between neighboring electrodes where the width of the space is smaller, and thus where the electric field is stronger.
  • the degree or extent to which they move to the smallest part of the space depends on the strength of the positive DEP force, which in turns depends strongly on the characteristics and properties of the particles, e.g. cells, for example on their size, shape and/or composition. Particles, e.g. cells, undergoing negative DEP on the contrary, are directed towards the entrance of the funnel, or where the space in between neighboring electrodes is broadest, and thus where the electric field is weaker.
  • the dielectrophoretic device according to the present invention will be described by means of different embodiments.
  • the dielectrophoretic device will be described by means of the particles being cells. It has to be understood that this is not intended to limit the invention in any way and that the invention also applies for particles other than cells, such as e.g. microparticles, nanoparticles, solid dielectric particles, engineered particles or biological particles, as described above.
  • the electrodes 1 may have a length which is at least 2 times larger than their average width.
  • the longitudinal direction of the electrodes 1 is in their length direction.
  • the dielectrophoretic device may comprise a radial array 10 of electrodes 1.
  • radial array 10 is meant that the electrodes 1 are arranged as segments of a circle extending from a center 4 of the circle to its circumference, thereby forming spaces 2 in between every two neighboring electrodes 1, the spaces 2 having a width which is decreasing along the longitudinal direction of the electrodes 1.
  • the width of the space 2 between every two neighboring electrodes may be measured in a direction perpendicular to the average longitudinal direction of the neighboring electrodes, being a direction defined by the average of the longitudinal direction of the two neighboring electrodes.
  • the space 2 in between neighboring electrodes 1 may also be referred to as funnel-shaped space or as funnel.
  • the decreasing width of the space 2 in between every two neighboring electrodes 1 may be radially oriented to a center point of the radial array 10, as indicated by arrow 3.
  • the electrodes 1 may have a length of between 50 ⁇ m and 1 mm.
  • the average distance between neighboring electrodes 1 may be such that high electrical fields of between 10 3 V/m and 10 6 V/m may be obtained by applying acceptable voltages of between 1V and 10V.
  • the average distance between neighboring electrodes 1 may be between 10 ⁇ m and 500 ⁇ m.
  • the funnel-shaped space 2 in between neighboring electrodes 1 results from the radial arrangement of the electrodes 1.
  • the electric field generated when driving the electrodes 1 will vary along the longitudinal direction of the electrodes 1, i.e. in the direction indicated with reference number 3. More particularly, the electrical field will become stronger toward the center 4 of the electrode array 10.
  • Neighboring electrodes 1 in the array 10 may be energized or driven with power signals, e.g. voltage signals, adapted in order to obtain twDEP.
  • power signals e.g. voltage signals
  • generating twDEP requires a phase shift of 360°/n between neighboring electrodes, with n being higher than 2.
  • n may be chosen to be 3 or 4.
  • n may kept low because in these cases less different signals are required and thus simpler electronics is needed to drive the dielectrophoretic device.
  • n may be 4 and neighboring electrodes 1 in the array 10 may be energized or driven with power signals, e.g. voltage signals, with a mutual phase difference of 90° in order to obtain twDEP.
  • the phase differences of voltage signals applied to four of the electrodes 1 are indicated in Fig. 2 . Because of these applied voltage signals, cells 5 present at the electrode array 10 will experience a tangential twDEP force. Simultaneously to the tangential twDEP force, the cells 5 will also experience a radial force due to the inhomogeneity of the electric field. For cells 5 undergoing positive DEP, the radial force may be directed towards the center 4 of the array 10, where the electric field is stronger, as the electrodes 1 are closer to each other. As a result, the cells 5 will be propelled along a spiral trajectory, indicated by arrows 6 and 7. In other words, cells 5 undergoing positive DEP will follow a contracting spiral towards the center 4 of the array 10.
  • the trajectory, and in particular, the degree of penetration into the 'spiral', will, for a fixed driving voltage amplitude, depend on the cell characteristics, such as shape, size and/or composition.
  • Cells 5 undergoing negative DEP will be attracted towards the outside of the radial array 10, where the electric field is weaker, and will therefore follow an expanding spiral, i.e. the cells 5 will be propelled along a spiral trajectory in the opposite direction as indicated by arrows 6 and 7. For clarity reasons, this case is not illustrated in Fig. 2 .
  • cells 5 of different types may be collected on distinct zones of the substrate on which the electrode array 10 is formed.
  • the dielectrophoretic device may comprise driving means for driving or energizing the electrodes 1 of the array 10.
  • the driving means may be adapted for driving the electrodes 1 with a mutual phase difference of 360°/n between every two neighboring electrodes 1, with n being higher than 2.
  • n may be 4 and the driving means may be adapted for driving the electrodes with a mutual phase difference of 90° between every two neighboring electrodes 1.
  • the electrodes 1 of the array 10 may, according to embodiments of the invention, be driven by active matrix electronics, e.g. LTPS (low-temperature polycrystalline silicon) technology.
  • LTPS low-temperature polycrystalline silicon
  • the dielectrophoretic device may furthermore comprise transport means 8 for transporting cells 5 toward the array 10 of electrodes 1.
  • the transport means 8 may comprise a linear array of parallel electrodes to transport the cells 5 by twDEP, as indicated in Fig. 2 .
  • This linear array 8 may also be referred to as conveyor array.
  • the conveyor array 8 may comprise any suitable number of electrodes as is required to transport the cells 5 toward the electrode array 10.
  • the conveyor array 8 may comprise between at least 3 electrodes, and may, for example, comprise 20 electrodes.
  • the dielectrophoretic device may comprise collection means 16 for collecting cells 5 (not shown in Fig. 2 ; however, illustrated in the embodiment of Fig. 3 ).
  • the collection means 16 may comprise collection channels, e.g. microfluidic collection channels. Such channels 16 can, for example, be placed adjacent the electrode array 10.
  • the collection channels 16 may comprise additional electrodes for performing impedance measurements. Such additional electrodes may, for example, form particle or cell counters sometimes called Coulter counters to count the cells 5 collected in each of the collection channels 16.
  • active matrix electronics based on large area electronics, e.g., LTPS technology
  • the same active matrix electronics may also be used to drive the electrodes 1 of the electrode array 10 of the dielectrophoretic device.
  • the radial arrangement of the electrodes 1 in the array 10 in the dielectrophoretic device according to the first embodiment of the invention provides an interaction distance of the cells 5 with respect to the electrodes 1 in the array 10 in the order of millimeters or even centimeters while maintaining a compact size of the dielectrophoretic device in the order of, for example, 100 mm 2 . Because of the high interaction distance achievable with the radial design, the dielectrophoretic device according to the first embodiment may be able to resolve even small variations in the dielectric properties of the cells 5.
  • the device according to the first embodiment of the invention may be used for the following experiment.
  • the experiment may be started with a homogeneous distribution of particles 5, e.g. cells.
  • the device is turned on by applying a voltage signal to the electrodes 1 as described above.
  • the distribution of particles in the device can be monitored by taking images of the device at certain time intervals (e.g. 1 s) or by recording a video.
  • the particles 5, e.g. cells will start to rotate but particles 5, e.g. cells of different types will follow different trajectories.
  • Video processing can be used in real time to detect when a fractionation, i.e. separation in sub-groups between different types of particles 5, e.g. cells is visible.
  • the assay can be stopped and the device can be turned off.
  • particles 5, e.g. cells present in each of the sub-groups can be counted and statistics can be performed.
  • various populations or sub-groups can be examined e.g. by means of a microscope by a pathologist. If necessary, the various populations or sub-groups can be fixed to a slide and stained with standard histochemical techniques to facilitate identification.
  • the dielectrophoretic device may comprise a linear array 10 of funnel-shaped electrodes 1, also referred to as triangular-shaped electrodes. This is illustrated in Fig. 3 .
  • the electrodes 1 may have a length of between 50 ⁇ m and 1 mm.
  • the average distance between neighboring electrodes 1 may be such that high electrical fields of between 10 3 V/m and 10 6 V/m may be obtained by applying acceptable voltages of between 1V and 10V.
  • the average distance between neighboring electrodes 1 may be between 10 ⁇ m and 500 ⁇ m.
  • the funnel-shaped space 2 in between neighboring electrodes 1 results, differently from the first embodiment, from the shape of the electrodes 1.
  • the electric field generated when driving the electrodes 1 will vary along the longitudinal direction of the electrodes 1. More particularly, the electrical field will become stronger toward the location in the array 10 where the width of the electrodes 1 is the highest and thus where the electrodes 1 are closest together, in the example given at the top 9 of the electrode array 10.
  • the dielectrophoretic device may comprise driving means which may be adapted for driving the electrodes 1 with a mutual phase difference of 90° between every two neighboring electrodes 1.
  • the driving means may be adapted for driving the electrodes 1 with a mutual phase difference of 360°/n between every two neighboring electrodes 1, with n being higher than 2.
  • the electrodes 1 of the array 10 may be driven by active matrix electronics such as e.g. LTPS (low-temperature polycrystalline silicon) technology.
  • twDEP Under the influence of twDEP (indicated by arrow 12 in Fig. 3 ), cells 5 are transported along the electrode array 10. Simultaneously to the lateral twDEP force, the cells 5 will experience a second force substantially perpendicular to the twDEP force and due to the inhomogeneity of the electrical field in between every two neighboring electrodes 1. For cells 5 undergoing positive DEP, the second force is directed towards the location where the electrical field is the strongest, in the example given towards the top 9 of the electrode array 10. For cells 5 undergoing negative DEP, the second force is directed towards the location of the electrode array 10 where the electrical field is weaker, in the example given, towards the bottom 11 of the electrode array 10.
  • the trajectory of the cell 5 may, for example, be indicated by arrows 13 (cells 5 undergoing strong positive DEP), 14 (cells 5 undergoing strong negative DEP) and 15 (cells 5 undergoing weak positive DEP).
  • the dielectrophoretic device according to the second embodiment may comprise transport means 8 for transporting cells 5 toward the array 10 of electrodes 1 (not shown in Fig. 3 ).
  • the transport means 8 may comprise a linear array of substantially parallel electrodes to transport the cells 5 by twDEP.
  • This linear array 8 may also be referred to as conveyor array.
  • the conveyor array 8 may comprise any suitable number of electrodes as is required to transport the cells 5 toward the electrode array 10.
  • the conveyor array 8 may comprise at least 3 electrodes, and may, for example, comprise 20 electrodes.
  • the electrode array 10 can extend over a relatively long distance, i.e. in the order of millimeters or even centimeters. Due to such long interaction distance, even small variations in the dielectric properties of the cells 5 can be resolved. Cells 5 of different types, i.e. with different dielectric properties, can then be trapped onto characteristic zones on the dielectrophoretic device.
  • the dielectrophoretic device may comprise collection means 16 for collecting cells 5.
  • different cell sub-populations, or cells 5 with different dielectric properties may be sorted into subsequent microfluidic collection channels 16, as illustrated in Fig. 3 .
  • Such channels 16 can, for example, be placed adjacent the electrode array 10.
  • the collection channels 16 may comprise additional electrodes (not shown in the figure) for performing impedance measurements.
  • additional electrodes may, for example, form Coulter counters to count the cells 5 collected in each of the collection channels 16.
  • active matrix electronics based on large area electronics, e.g., LTPS technology
  • the same active matrix electronics may also be used to drive the electrodes 1 of the electrode array 10 of the dielectrophoretic device.
  • a method for manipulating particles 5 using a dielectrophoretic device according to embodiments of the present invention.
  • the method comprises applying voltage signals to the electrodes 1 with a mutual phase difference of 360°/n between every two neighboring electrodes 1, with n being higher than 2.
  • applying voltage signals with a mutual phase difference of 360°/n between every two neighboring electrodes 1 is performed to obtain twDEP.
  • n may be 4 and neighboring electrodes 1 may be driven with a mutual phase difference of 90°.
  • the cells 5 are moved over the electrode array 10 and, based on their properties, e.g. size and/or dielectric properties are differently manipulated.
  • the particles 5 may be separated and collected, e.g. sorted, in collection means 16 where they can be counted or treated.
  • the particles 5 may be moved towards a location, such as a electric field processing region (see further), in the array 10 where the electric field is the highest and where electric lysis or electroporation of cells 5 can be performed.
  • the method for manipulating particles 5 may furthermore comprise transporting the particles 5 towards the array 10 of electrodes 1. This may be done by transport means 8.
  • transport means 8 may comprise a linear array of parallel electrodes to transport the particles 5 by twDEP.
  • the linear array of parallel electrodes may comprise any suitable number of electrodes as is required to transport the particles 5 toward the electrode array 10.
  • a first experiment an electrode array 10 similar to the one described in the second embodiment was used. This configuration allowed verification that cells 5 undergoing positive DEP are pulled towards the location on the array where the electric field is the highest, i.e. to the top 9 of the electrode array 10 as illustrated in Fig. 3 , while cells 5 undergoing negative DEP are pulled towards locations on the electrode array 10 where the electric field is the smallest, i.e. to the bottom 11 of the electrode array 10 as illustrated in Fig. 3 .
  • Fig. 4 illustrates the principle of the dielectrophoretic device according to embodiments of the invention for mouse cancer cells 5 undergoing positive DEP.
  • CM clausius-mossotti
  • a suitable frequency range may be between 10 kHz and 5 MHZ. It has to be noted that it is difficult to obtain twDEP at high conductivities. Therefore, preferably particles 5, e.g. cells are suspended in a low conductivity medium.
  • the cells 5 that were looked at are surrounded by a white circle.
  • the electrodes 1 were driven with a mutual phase difference of 90° between neighboring electrodes 1. It can be seen from the subsequent figures (a), (b) and (c) of Fig. 4 that the cells 5 are moved along the longitudinal direction of the electrodes 1. The cells 5 are pulled towards locations in the space 2 between two neighboring electrodes 1 where the width is smallest, i.e. where the electric field is the highest.
  • a radial array 10 of electrodes 1 according to the first embodiment was used with mouse cancer cells 5.
  • the experiment is illustrated in Fig. 5 which shows successive steps 1 to 4 in the movement of mouse cancer cells 2 in a radial electrode array 10.
  • Cells 5 move in a circle (indicated with arrow 17) when the electrodes 1 in the array 10 are energized or driven with twDEP voltage signals as described above. The movement was observed to be fast, i.e.
  • cells 5 close to the center 4, for example less than 10 ⁇ m from the center 4, of the radial array 10 can do one revolution in 10 à 20 seconds when the electrodes 1 are driven at a voltage of 2V which may lead to an electric field of about 400 kV/m for a distance between the electrodes 1 of about 5 ⁇ m, whereas cells 2 located further away from the center 5 of the radial array move slower.
  • about 1 minute is required to move them toward the center 4.
  • the movement of the cells 5 was observed to depend strongly on the properties of the cells 5, such as their size, shape and/or composition. Changes in the dielectric properties of the cells 5 also had an effect on the cell trajectory. For example, in several occasions cells 5 were observed to invert the sense of rotation immediately prior to lysis. This is due to the fact that a strong electric field disrupts the cell membrane. This dramatically changes the dielectric properties of the cell (also expressed by means of the Clausius-Mossotti (CM) factor (see above)). If the imaginary part of the CM factor changes sign, the rotation sense is inverted.
  • CM Clausius-Mossotti
  • the direction of the rotation of the cells 5 can be inverted by varying the relative phase of the applied voltage signals or, alternatively, by varying the frequency of the signals across the DEP cross-over frequency, i.e. the frequency where a cell 5 makes the transition from negative to positive DEP.
  • perpendicular DEP component a force substantially perpendicular to the twDEP force, also referred to as perpendicular DEP component, which is due to configuration of the electrode array 10 according to embodiments of the invention, was also verified during this experiment. Although spiral trajectories (both expanding and contracting) could be observed for short periods of time, the perpendicular component was in general too weak to lead to a consistent spiral trajectory over several revolutions when using cells. To overcome this problem, the dimensions of the device may be optimized.
  • the dielectrophoretic device can be used to separate cells 5, or in general to separate particles 5, on the basis of their dielectric properties, e.g. to collect or sort such cells or particles.
  • particles e.g. cells
  • cell separation or sorting on the basis of cell type has important applications, such as the isolation of stem cells from blood or bone marrow for regenerative therapy, or the isolation of circulating tumor cells from blood, so that they can be further examined in detail by e.g. a pathologist.
  • the differences in dielectric properties between the particles 5, e.g. cells that are to be separated are small.
  • An advantage of the dielectrophoretic device according to embodiments of the present invention compared to other separation or sorting methods based on DEP is that the interaction time of the particles 5, e.g. cells, with the electrical field is much longer in case of the device according to embodiments of the invention, i.e. may be from 10s up to several minutes. Hence, even particles 5, e.g. cells, with very small differences in dielectric properties can be separated.
  • the application area of the dielectrophoretic device according to embodiments of the invention can be expanded greatly in cases where the differences in the dielectrophoretic properties of the particles 5, e.g. cells can be artificially enhanced, e.g. by labeling specific cells with polymeric beads.
  • This allows particles 5, e.g. cells, to be sorted on the basis of the presence of certain proteins on the membrane.
  • This approach could be useful in clinical applications, such as stem cell enrichment and tumor cell isolation, but also in biotech applications such as protein engineering. For instance, libraries of (genetically altered) cells can be screened to develop peptide and antibody affinity reagents.
  • the dielectrophoretic device according to embodiments of the invention can also be used in electrical field lysis or electroporation of cells 5.
  • Cell lysis i.e. breaking open a cell membrane to release sub-cellular material
  • a total homogeneous lysate i.e. contents released from a lysed cell
  • the requirements for a device suitable for cell lysis include speed, high efficiency, selectivity (e.g. breaking down cell membranes while protecting organelles) and integration with other micro fluidic devices.
  • cells 5 are lysed by either chemical or mechanical means.
  • the cell membrane consists of a lipid double-layer, which can be dissolved by buffers containing detergents. In general, such buffers also dissolve the organelle membranes and are therefore not suitable for assays where organelle integrity is critical. Moreover, additional steps are needed to introduce and/or remove the lysis buffer.
  • Mechanical breakdown of the cell membrane can be induced by shear stress, wear or by applying ultrasound (sonication). Such methods also do not generally provide the discrimination needed for organelle analysis and are difficult to integrate into microfluidic systems.
  • Thermal lysis can also be used but is only compatible with nucleic acids analysis because proteins are denatured by heat.
  • electric field lysis has attracted considerable attention as it enables sub-cellular fractionation without the complications of chemical and mechanical lysis and can be easily integrated into microfluidic systems.
  • an external electric field disrupts the cell membrane by inducing changes in the trans-membrane potential.
  • the membrane can be disrupted permanently (lysis) or reversibly (electroporation).
  • transient pores are created in the membrane, either to release intracellular material or to introduce macromolecules such as DNA or drugs.
  • the applied voltage can be a pulsed DC voltage or an AC voltage to avoid electrolysis and gas formation.
  • the dielectrophoretic device according to embodiments of the invention has several advantages for electric field lysis.
  • the principle of cell lysis will be described by means of a dielectrophoretic device wherein the electrodes 1 of the array 10 are arranged radially as described with respect to the first embodiment (see Fig. 7 ).
  • the dielectrophoretic device according to the second embodiment of the invention can be used for electric cell lysis.
  • the magnitude of the electrical field created by the electrodes 1 increases gradually towards the center 4 of the electrode array 10 as illustrated in Fig. 2 and Fig. 7 .
  • the cells 5 When operating under positive (attractive) DEP conditions, the cells 5 will experience a radial force directed towards the center 4 of the array 10. As they move towards the center 4 of the array 10, the cells 5 are exposed to a stronger and stronger electric field until they arrive in the high field region (indicated with reference number 19), also referred to as lysis region, and are eventually lysed. Lysed cells are indicated in Fig. 7 with reference number 20. Electric lysis of cells 5 using the dielectrophoretic device according to embodiments of the invention may have the following advantages:
  • Cells 5 can be collected over a large area and transported to the processing region such as a lysis region 19, resulting in high efficiency.
  • Electrodes 1 To create high electrical fields on the order of 10 6 V/m with acceptable voltage signals, i.e. with voltage signals of between 1V and 10 V, it is necessary to have electrodes 1 close together. For example, for a voltage signal of 1V the distance between neighboring electrodes may be 1 ⁇ m and for a voltage signal of 10V the distance between neighboring electrodes may be 10 ⁇ m. This can have two negative effects for the device. A first one is that the chance of electrolysis may increase because the resistance between the electrodes 1 is low and a second one is that the capacitance may increase which results in higher power consumption. Unlike conventional structures for electric lysing, such as e.g.
  • the configuration according to the first embodiment of the invention only has electrodes 1 close together near the center 4 of the radial array 10.
  • the electrodes 1 are only close together at one end, in the example of Fig. 3 at the top 9 of the array 10.
  • the average electrode distance of the configurations according to embodiments of the invention may be larger than in prior art devices, the overall capacitance may be lower and therefore lower power may be required. The larger average distance also increases the resistance and therefore reduces the likelihood of electrolysis.
  • the proper voltage for cell lysis is obtained automatically, and does not have to be adjusted for different types of cells 5.
  • the intracellular material is released.
  • the electric field amplitude at the location of the lysis region 19 will not be sufficient to disrupt organelles membranes, which will therefore remain intact.
  • a determination is made of the position where lysing of individual cells 5 occurs.
  • the radial coordinate where lysing occurs defines the electric field that is required.
  • the present invention also provides a system controller 30 for use in dielectrophoretic device for controlling driving of the electrodes 1 of an array 10 in a dielectrophoretic device according to embodiments of the present invention.
  • the system controller 30, which is schematically illustrated in Fig. 8 , may comprise a control unit 31 for controlling driving means 32 for applying a voltage signal to the electrodes 1 with a mutual phase difference of 360°/n between every two neighboring electrodes 1, with n being higher than 2.
  • n may be 4 and the controller may be adapted for controlling driving means 32 for applying a voltage signal to the electrodes 1 with a mutual phase difference of 90° between every two neighboring electrodes 1
  • the system controller 30 may include a computing device, e.g. microprocessor, for instance it may be a micro-controller.
  • a programmable controller for instance a programmable digital logic device such as a Programmable Array Logic (PAL), a Programmable Logic Array, a Programmable Gate Array, especially a Field Programmable Gate Array (FPGA).
  • PAL Programmable Array Logic
  • FPGA Field Programmable Gate Array
  • the use of an FPGA allows subsequent programming of the microfluidic system, e.g. by downloading the required settings of the FPGA.
  • the system controller 30 may be operated in accordance with settable parameters, such as driving parameters, for example temperature and timing parameters.
  • Fig. 9 shows one configuration of processing system 40 that includes at least one programmable processor 41 coupled to a memory subsystem 42 that includes at least one form of memory, e.g., RAM, ROM, and so forth.
  • the processor 41 or processors may be a general purpose, or a special purpose processor, and may be for inclusion in a device, e.g., a chip that has other components that perform other functions.
  • one or more aspects of the method according to embodiments of the present invention can be implemented in digital electronic circuitry, or in computer hardware, firmware, software, or in combinations of them.
  • the processing system may include a storage subsystem 43 that has at least one disk drive and/or CD-ROM drive and/or DVD drive.
  • a display system, a keyboard, and a pointing device may be included as part of a user interface subsystem 44 to provide for a user to manually input information, such as parameter values.
  • Ports for inputting and outputting data, e.g. desired or obtained flow rate, also may be included. More elements such as network connections, interfaces to various devices, and so forth, may be included, but are not illustrated in Fig. 9 .
  • the various elements of the processing system 40 may be coupled in various ways, including via a bus subsystem 45 shown in Fig.
  • the memory of the memory subsystem 42 may at some time hold part or all (in either case shown as 46) of a set of instructions that when executed on the processing system 40 implement the steps of the method embodiments described herein.
  • the present invention also includes a computer program product which provides the functionality of any of the methods according to the present invention when executed on a computing device.
  • Such computer program product can be tangibly embodied in a carrier medium carrying machine-readable code for execution by a programmable processor.
  • the present invention thus relates to a carrier medium carrying a computer program product that, when executed on computing means, provides instructions for executing any of the methods as described above.
  • carrier medium refers to any medium that participates in providing instructions to a processor for execution. Such a medium may take many forms, including but not limited to, non-volatile media, and transmission media.
  • Non-volatile media includes, for example, optical or magnetic disks, such as a storage device which is part of mass storage.
  • Computer readable media include, a CD-ROM, a DVD, a flexible disk or floppy disk, a tape, a memory chip or cartridge or any other medium from which a computer can read.
  • Various forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.
  • the computer program product can also be transmitted via a carrier wave in a network, such as a LAN, a WAN or the Internet.
  • Transmission media can take the form of acoustic or light waves, such as those generated during radio wave and infrared data communications. Transmission media include coaxial cables, copper wire and fiber optics, including the wires that comprise a bus within a computer.
EP07115462A 2007-08-31 2007-08-31 Dielektrophoretische Vorrichtung zur Verarbeitung von Teilchen Ceased EP2039433A1 (de)

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PCT/IB2008/053417 WO2009027927A2 (en) 2007-08-31 2008-08-26 Dielectrophoretic device for manipulation of particles

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WO2022112474A1 (de) * 2020-11-27 2022-06-02 Karlsruher Institut für Technologie Anordnung, system und verfahren zur erzeugung von flüssigkeitsströmen

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DE102018211001A1 (de) 2018-07-04 2020-01-09 Robert Bosch Gmbh Vorrichtung zum dielektrophoretischen Einfang von Teilchen
CN112871229B (zh) * 2021-01-21 2022-06-28 中国科学技术大学 一种用于水体介电泳细菌分选的芯片
WO2022176048A1 (ja) * 2021-02-17 2022-08-25 日本電信電話株式会社 粒子配列運搬デバイスおよび方法

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WO2022112474A1 (de) * 2020-11-27 2022-06-02 Karlsruher Institut für Technologie Anordnung, system und verfahren zur erzeugung von flüssigkeitsströmen

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