EP1744831B1 - Method and device for collecting suspended particles - Google Patents
Method and device for collecting suspended particles Download PDFInfo
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- EP1744831B1 EP1744831B1 EP05747443A EP05747443A EP1744831B1 EP 1744831 B1 EP1744831 B1 EP 1744831B1 EP 05747443 A EP05747443 A EP 05747443A EP 05747443 A EP05747443 A EP 05747443A EP 1744831 B1 EP1744831 B1 EP 1744831B1
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- particles
- compartment
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- electrode
- collecting area
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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]
Definitions
- the invention relates to a method for collecting particles suspended in a liquid, in particular for collecting suspended biological objects, such as biological cells, in a fluidic microsystem, having the features of the preamble of claim 1.
- the invention also relates to a device for implementation such a method and its applications.
- Electro-hydrodynamic flows can be generated in a liquid-filled compartment by electro-osmosis using planar electrodes, which are exposed to high-frequency alternating voltages.
- KF Hoettges et. al. describe in the publication "Optimizing Particle Collection for enhanced surface-based biosensors" (see IEEE ENGINEE-RING IN MEDICINE AND BIOLOGY MAGAZINE “, November / December 2003, p. 68 ) the use of circulating electrohydrodynamic flows to collect in the liquid suspended particles.
- a vortex flow 30 ' is formed, which rotates about an axis 31' parallel to the orientation of the side surface 11 '.
- a flow-calmed region is formed, which represents a collection region 40' for the particles brought in between the electrodes 21 'by the turbulence 30'.
- the by K. F. Hoettges et. al. described technique has in particular for the application in biology, biochemistry and medicine several disadvantages.
- the circulating vortex flow has a relatively small catchment area for the particles to be collected.
- the particles can only be collected directly adjacent to the electrodes.
- contact with the electrodes may be detrimental to the particles, especially if the particles comprise biological materials.
- relatively large electrodes are required to form correspondingly large collection areas. At large-area electrodes, however, occurs an undesirable heating.
- Hoettges et al. This technique is based on electroosmosis and positive electrophoresis and is thus limited to low frequencies and low conductivities of the solutions used. Therefore, it is not possible with this method to investigate cells in physiological solutions.
- the object of the invention is to provide improved methods for collecting suspended in a liquid particles, in particular for the collection of suspended biological objects, which overcomes the disadvantages of conventional methods and in particular a collection of an enlarged catchment area and without damage to allow the collected particles.
- Another object of the invention is to provide improved devices for Collection of particles suspended in a liquid, in particular for implementing the method according to the invention.
- the invention is based in particular on the technical teaching of collecting suspended particles in at least one collection area in a compartment with circulating flows which extend at least partially along a longitudinal extent of at least one electrode on a side surface of the compartment.
- the collection area is the volume into which the flow carries the particles and in which the particles can collect in particular by a local flow reduction.
- the circulating flows generated according to the invention by an interaction of the liquid with high-frequency electric fields at the electrodes run in a plane parallel to the respective side face.
- the inventors have found that the limitation on the collection efficiency of the conventional techniques can be overcome and the catchment area of the flows circulating at the electrodes can be increased if the flows do not revolve about an axis parallel to the orientation of the side face as before, but a local axis of rotation perpendicular to this Have side surface.
- Another important advantage of the invention is that with the flows even the smallest particles, such as viruses can be effectively collected.
- the particles according to a preferred embodiment of the invention are collected in the collection area without mechanical contact with a wall or other part of the compartment, there may be advantages for the manipulation of biological particles, such as biological cells, which respond to mechanical contact with undesirable state changes would.
- the particles may be placed in the collection area with a touch of a side surface of the compartment. This can advantageously be simplified by a Kompartimentwand a measurement. Even if the collection takes place with a contact of the side surface, in contrast to the conventional electro-osmotic techniques, an electrode contact and thus an undesired electrode reaction can be avoided.
- the collection area may be formed by a part of the side surface in which the wall material of the compartment is exposed and no electrodes are present.
- a plurality of locally circulating flows are generated at least one electrode, of which at least one branch of the local circulation is directed to the at least one collection area. Run along the electrode for example, two currents.
- this increases the effectiveness of the collection.
- a further enlargement of the catchment area of the collection can advantageously be achieved if, according to the method according to the invention, a plurality of locally circulating flows are generated at a plurality of electrodes. This allows in particular that the particles are guided from several directions to the at least one collection area. If the flows relative to each other are formed so symmetrically, in particular point-symmetrical to the collection area that this flow-calmed or substantially flow-free, can be advantageously achieved that the particles conveyed from one side to the collection area the collection area in another direction, for. B. on the opposite side not leave again.
- the catchment area can advantageously be widened with elongate, band or strip-shaped electrodes which preferably extend radially from the collection area in different directions.
- the particles are collected from a catchment area of the compartment whose volume is 10 2 to 10 9 times greater than the volume of the collection area.
- This ratio shows that with the method according to the invention particles can not only be collected, but concentrated or enriched with a high factor.
- the collection area of a single vertebra may have a volume of up to 10 ⁇ l and the collection area a volume of 1 femtoliter up to 50 picoliters, so that the invention can be advantageously implemented with fluidic microsystems.
- high-frequency electric fields are also utilized for directly exerting a predetermined dielectrophoretic driving force on the particles.
- the particles are moved by negative dielectrophoresis to the collection area.
- this further enhances the indirect hydrodynamic force effect. It is particularly preferred if, according to the invention, high-frequency electric fields are generated which are used for the electrodynamic flow generation and simultaneously for the dielectrophoretic manipulation of the particles.
- the collection efficiency can be further increased if at least one dielectrophoretic field cage with a potential minimum, which is located in the collection area, is generated with the high-frequency electric fields.
- the dielectrophoretic forces in the field cage depend on the particle size.
- particles that are so small that the capture forces of the field cage would be too weak for effective collection can be connected to the electrohydrodynamic flows to larger aggregates so that field forces are sufficient, which are sufficient for a safe catch in the field cage.
- the field cage is closed in two (funnel-shaped field cage) or three (all-sided field cage) spatial directions.
- the field cage can be formed with 6, 8 or more electrodes.
- electrodes are arranged and with high-frequency electrical Are applied voltages that several field cages are formed, advantageously the catchment area of the particle collection according to the invention can be further increased.
- an inner and an outer field cage are provided whose potential minima have the same position in the collection area.
- the field cages are arranged concentrically to each other, wherein the respective outer field cage moves particles by negative dielectrophoresis towards the inner field cage.
- At least one further force acts on the particles in the collection area.
- This can advantageously be achieved additional support and / or manipulation of the particles in the collection area.
- the generation of an optically effective force may have advantages in the combination of the inventive technique with an optical measurement in the collection area and for a selective particle manipulation.
- the generation of a dielectrophoretic force may have advantages for effective interaction with a dielectrophoretic barrier of the field cage.
- An additional magnetic force offers advantages in the manipulation of magnetic particles.
- the at least one additional force may be a force mediated by ultrasound, for example, nodes of an ultrasound field may be formed in the collection area.
- a start object is located in the collection area, eg a bead, which can also be functionalized.
- the particles are not only influenced by dielectric interactions, but also possibly by a specific binding to the bead or caused by the starting object hydrodynamic foreclosure.
- at least one measurement of the collected particles takes place in the collection area. This may in particular result in advantages in the manipulation or evaluation of collected biological particles.
- the measurement preferably comprises an electrical, electrochemical and / or optical measurement known per se from the art of fluidic microsystems.
- the measurement is directed to the detection of a receptor-ligand binding event.
- the side area of the compartment in the region of the at least one collection area can be functionalized with detection spots in the form of receptor molecules (eg proteins, antibodies, DNA, viruses (for transfection experiments), etc.), as is conventional microarrays or biochips is known, so that a specific receptor - ligand interaction takes place with particles or molecules accumulated in the collection area.
- the interaction can then be determined in a known manner e.g. can be detected by electrical, electrochemical or optical readout.
- the concentration of analyte particles or molecules in the vicinity of the detection spots can be increased (increase in sensitivity) and the detection process can be accelerated compared to purely diffusive transport of analyte particles or molecules to the detection spots.
- the functionalized receptor array can e.g. B. be applied to a flat electrode and together with a Forming electrodes containing second substrate form a micro-chamber. After enrichment of the analyte particles or molecules by the method according to the invention and the binding of the same to the immobilized receptors on the array, the collection structure can then be removed again. It can also be used several times accordingly.
- the particles are collected in several collection areas in the compartment, there may be advantages for parallel enrichment of the particles from multiple catchment areas in the compartment and parallel manipulation or evaluation of the collected particles.
- the collection can take place not only from a catchment area with a stationary suspension liquid, but even dynamically from a moving suspension liquid out.
- the compartment can be penetrated, for example, by a laminar flow, which is superimposed on the electrodes according to the invention with the locally circulating flow.
- a mutual superimposition of several locally circulating flows can be provided in the compartment.
- a first circulating flow may just direct the particles into a collection area that is part of another downstream circulating flow. This makes it possible to arrange a multiplicity of cascade-like circulations in which particles are led out of an extended catchment area into a single collection area.
- the inventive method is particularly well suited for the collection of particles with a diameter below 1 micron.
- it is thus advantageously possible in particular to use cells, viruses, bacteria, proteins, cell components and / or biological macromolecules, eg. B. DNS are collected.
- the currents circulating locally at the electrodes are intensified by a local temperature gradient in the liquid.
- the temperature gradient can be formed by local heating of the liquid, which is preferably carried out by irradiation of the liquid and / or side surfaces of the compartment with light and its corresponding absorption and / or embedded ("buried") thermocouples in the walls.
- the temperature gradient can alternatively or additionally be formed by a local, targeted cooling of the liquid.
- the local heating of the liquid may advantageously be used in addition to excitation of chemical reactions. Due to the high local temperatures in the collection area can be targeted z. Thermally activated reactions, e.g. an aggregation or precipitation.
- a collection device for collecting suspended particles which, in particular in a compartment for receiving a liquid on a side surface, has electrodes for generating a plurality of locally circulating flows in the liquid with which suspended particles are at least one predetermined collection area can be performed in the compartment, wherein the collection device is adapted to at least creating a flow such that a portion of the flow extends along the longitudinal extent of the electrode and the flow circulates about an axis perpendicular to the respective adjacent side surface aligned with the electrode.
- the collection area may be arranged at a distance from the side surfaces of the compartment or so that the collection area is in contact with one of the side surfaces.
- the electrode on each of which a circulating flow can be generated, connected to a voltage source for providing predetermined high-frequency electrical voltages.
- the at least one electrode used to generate the circulating flow is also referred to as a collecting electrode.
- the collection device when generating a plurality of circulating flows directed to one or more collection areas, respectively includes a plurality of collection electrodes forming a collection electrode array.
- the collection device is adapted to exert on the particles to be collected not only electrohydrodynamic but also dielectrophoretic forces, the collection efficiency can be improved by the additional force effect.
- the dielectrophoretic force action is exerted by the interaction of the particles with high-frequency electric fields generated in the compartment with at least one electrode, which is referred to below as the cage electrode.
- the cage electrode If the aforementioned one-sided or all-around field cages are to be generated, the compartment is equipped with a cage electrode array.
- the collecting and cage electrodes are identical.
- the collecting electrode and cage electrode arrays are formed by a common electrode arrangement. In this case, the structure of the collecting device and the driving of the electrodes are simplified.
- a particular advantage of the collection device according to the invention is its miniaturization.
- the compartment of the collection device is preferably part of a fluidic microsystem.
- the collection function according to the invention can be combined with collection, sorting, evaluation or measurement functions of the microsystem.
- the collection device is, for example, arranged in the channel of a fluidic microsystem which forms the said compartment with the flow generator.
- the collecting device according to the invention can also be used to collect particles in the throughflow channel.
- a plurality of collection areas along a longitudinal direction of the channel are arranged in rows.
- the collection device is equipped with a magnetic field device for exerting a magnetic holding force in said collection region and / or a measuring device for detecting electrical, electrochemical or optical properties of particles in the collection region.
- the flow generator may additionally comprise a heating device and / or a light source.
- the application of the invention is not limited to the fluidic microsystems for dielectrophoretic particle manipulation, but also in other cases, in which in particular for biochemical tasks suspended particles in liquid-filled compartments, for. As laboratory vessels to be collected, are applied.
- FIG. 1 11 illustrates, in an enlarged schematic sectional view, a part of a channel or another section of a fluidic microsystem through which the compartment 10 of the collection device according to the invention is formed.
- an electrode arrangement 20 with eight electrodes 21 is arranged on the channel walls, which represent side surfaces 11 of the compartment 10. It is on the lower side surface (Bottom surface) and on the upper side surface (top surface) each have four electrodes 21 arranged (see also FIGS. 2, 3 ).
- the electrode assembly 20 is formed as it is known per se from electrode arrangements for generating dielectrophoretic field cages.
- Each electrohydrodynamic flow generating electrode is in the form of a strip or ribbon of a length (see also US Pat FIGS. 2, 3 ), which is much larger than the electrode width.
- the aspect ratio electrode width: electrode length is selected in the range of 1:10 to 1: 100.
- the dimensions of the electrode 21 are, for example, 10 .mu.m.times.500 .mu.m.
- the elongate electrode shape defines a longitudinal orientation of the electrode 21.
- Each electrode 21 is arranged so that the longitudinal orientation to a collection area 40 in the middle between the side surfaces 11 or the vertical projection from the collection area to the respective side surface 11 has.
- the electrodes 21 are electrically connected in a known manner with a voltage source for generating high-frequency electrical voltages, preferably with predeterminable amplitudes, frequencies and phase ratios.
- a voltage source for generating high-frequency electrical voltages, preferably with predeterminable amplitudes, frequencies and phase ratios.
- Reference numeral 50 refers to a measuring device, for example a microscope with a CCD camera, with which, for example, fluorescence-marked particles in the collection region can be optically measured and evaluated.
- a measuring device for example a microscope with a CCD camera, with which, for example, fluorescence-marked particles in the collection region can be optically measured and evaluated.
- at least one optically transparent window is provided in the side surface 21 of the channel (see FIG. 5 ).
- a measuring device may alternatively or additionally at least one more Electrode be provided for impedance measurements in the collection area 40.
- FIG. 2 the state of the collection device is illustrated just before the start of an electrohydrodynamic collection.
- particles 1 are randomly distributed as long as the electrodes 21 are de-energized or subjected to a relatively low voltage ( ⁇ 1 V).
- the flows 30 (for illustrative purposes also in FIG FIG. 2 shown).
- one or two locally circulating flows 32, 33 are generated.
- a first flow branch of each flow runs along the longitudinal direction of the electrode 21 and parallel to the side surface 11 through the compartment 10 substantially in the direction of the collection area 40, as shown in FIGS FIGS. 2 and 3 is illustrated.
- Another branch of the circulating flow 30 leads back over the electrode 21 in the opposite direction.
- the circulation takes place about an axis 31, which is perpendicular to the plane in which the electrodes are arranged. With the flows 30, the particles 1 are led out of the outer space outside the electrode arrangement 20 into the inner collection area 40, where they form an aggregate ( FIG. 3 ).
- the cause of the electrohydrodynamic flow 30 is in FIG. 4A illustrated.
- FIG. 4A In the left part of FIG. 4A are the temperatures in the xz plane (according to FIG. 1 ) and in the xy-plane (according to FIG. 2 ).
- a temperature profile results such that the collection area 40 between the electrodes 21 is warmer than the ambient solution.
- the electrical conductivity and the dielectric constant are temperature-dependent, the medium becomes dielectrically inhomogeneous in the collection region. Thereby The electric field exerts on the liquid polarization forces, which lead to the formation of the desired flow vortex. Since the flow vortices are formed on all electrodes, a symmetrical inflow to the center of the cage takes place into the collection area 40.
- FIG. 4A (left part), the temperature conditions are shown in a liquid initially residing in the compartment. Surprisingly, the formation of the circulating flows indicative of the collection area also occurs if the liquid flows in the compartment. The liquid forms a carrier stream at a velocity which is less than the liquid velocity in the circulating streams.
- the selection of the voltage amplitude required for generating the electrohydrodynamic flow takes place as a function of the dielectric properties of the suspension fluid and the geometric properties of the electrode arrangement. An empirical selection by experiment may also be provided.
- the high-frequency electric fields are selected so that only negative dielectrophoresis acts on the particles.
- the in the FIGS. 2 and 3 shown collection can be realized for the collection of 1 micron particles, for example, with the following operating parameters.
- the particles are suspended in KCl (concentration: 12.5 mM).
- the electrodes 21 are subjected to a high-frequency electrical voltage (frequency: 8 MHz, amplitude: 3.5 V).
- the distance between the electrodes (tip-to-tip), which are opposite one another in a plane, is 40 ⁇ m.
- hepatitis A viruses Under the following operating conditions, an accumulation of hepatitis A viruses (diameter about 30 nm) could be achieved within 10 minutes. High-frequency alternating voltages with frequency: 7.4 MHz, amplitude: 4 V rms ), electrode spacing: 5 ⁇ m. The starting concentration of the viruses in the compartment was approx. 10 9 to 10 10 / ml. Enrichment of the fluorescently labeled hepatitis A virus is observed at various observation times FIG. 4B shown. After 2 minutes, a first small aggregate was formed from the viruses, which had a diameter of approx. 4 ⁇ m (9 min.) Growth. In a catchment area of approx. 100 ⁇ m * 100 ⁇ m * 10 ⁇ m (channel height) this corresponds to a concentration of approx. 10 3 .
- FIG. 5 schematically illustrates the formation of a series of collection areas 41, 42, 43,... in the channel of a fluidic microsystem, with only the electrodes 21 of the electrode arrangements on one of the side surfaces of the channel and the associated connection lines being shown for clarity, via which the electrodes 21 are connected to a voltage source.
- the left part of the opposite phase control of each adjacent electrodes in a single field cage 20 is symbolically illustrated, with the the desired flow vortices can be generated at each collection area 41, 42, 43,.
- a measuring device outside the fluidic microsystem is a measuring device (not shown), with which the particles in the collection areas 41, 42, 43, ... are measured through a window 51 along a scanning line 52.
- a fluorescence correlation measurement FCS is performed to detect receptor-ligand binding events in the collected particles.
- FIG. 6 A cascade-shaped combination of a plurality of circulating flows is schematically shown in FIG FIG. 6 illustrated.
- a flux directed towards the collection area 40 is generated with the electrode assembly 20 over a relatively large area.
- a plurality of collecting electrodes 21, 22 pointing radially to the collecting area 40 are provided.
- the innermost electrodes 23 at the same time form collecting and cage electrodes, correspondingly FIG. 2 form a field cage.
- particles are transported, for example. With the vortex 34 at the first collecting electrode 21 in the vortex 35 of the second collecting electrode 22, from which the further transport to the vortex 36 of the collecting and cage electrode 23 takes place. With this, the particles are conveyed into the central collection area 40.
- FIG. 6 illustrates that each two vortices are formed on a strip-shaped electrode, wherein the axis 31 (offset drawn) of the flow circulation is aligned perpendicular to the adjacent side surface with the electrodes.
- the electrodes can be used in the FIG. 6 shown embodiment of the invention or in the embodiments described above have a conical shape, in which the width of the electrode strip widened with increasing radial distance from the collection area to the outside. By this design, the catchment area of the collecting currents can be extended. It is alternatively possible for the electrodes to have a straight strip shape and for the electrodes to become larger at a radial distance from the collection area to the outside. For example, inside narrow, small electrodes and outside wide, large electrodes are provided, wherein outwardly z. B. the aspect ratio of the electrodes increases.
- FIG. 7 illustrates an embodiment of the collection device according to the invention with an electrode assembly 20 having an outer cage 20.1, in the capture area an inner cage 20.2 is formed.
- Each of the inner and outer field cages 20.1 and 20.2 is a closed 8-electrode field cage.
- the associated electrode arrangements are offset relative to each other by 45 °, whereby the interaction of the two field cages is improved.
- FIG. 8 illustrated in the embodiment according to FIG. 7 resulting flow profiles (numerical simulation).
- the flow profiles are shaped so that the catchment area of the electrode arrangement 20 is increased and also the central rest or particle collection zone is widened.
- the outer field cage 20.1 alone would provide less flow and hence less effective particle transport, whereas the inner field cage 20.2 alone would have a smaller catchment area and a smaller quiet zone.
- the individual electrodes and their connection lines to the voltage sources are electrically isolated from each other.
- the insulation is achieved by a multi-level structure of electrode and insulation layers.
- the collection device may be provided with a cooling device, e.g. B. a Peltier element to avoid unwanted total heating of the collection device.
- a cooling device e.g. B. a Peltier element to avoid unwanted total heating of the collection device.
Abstract
Description
Die Erfindung betrifft ein Verfahren zur Sammlung von in einer Flüssigkeit suspendierten Partikeln, insbesondere zur Sammlung von suspendierten biologischen Objekten, wie zum Beispiel biologischen Zellen, in einem fluidischen Mikrosystem, mit den Merkmalen des Oberbegriffs von Anspruch 1. Die Erfindung betrifft auch eine Vorrichtung zur Umsetzung eines derartigen Verfahrens und deren Anwendungen.The invention relates to a method for collecting particles suspended in a liquid, in particular for collecting suspended biological objects, such as biological cells, in a fluidic microsystem, having the features of the preamble of
Es ist bekannt, in einer Flüssigkeit suspendierte Partikel in fluidischen Mikrosystemen in einem dielektrophoretischen Feldkäfig zu fangen oder zu sammeln (siehe zum Beispiel Publikation "
Mit planaren Elektroden, die mit hochfrequenten Wechselspannungen beaufschlagt werden, können in einem flüssigkeitsgefüllten Kompartiment durch Elektroosmose elektrohydrodynamische Strömungen erzeugt werden. K. F. Hoettges et. al. beschreiben in der Publikation "Optimizing Particle Collection for enhanced surface-based biosensors" (siehe "
Die von K. F. Hoettges et. al. beschriebene Technik besitzt insbesondere für die Anwendung in der Biologie, Biochemie und Medizin mehrere Nachteile. Die zirkulierende Wirbelströmung besitzt einen relativ geringen Einzugsbereich für die zu sammelnden Partikel. Des Weiteren können die Partikel ausschließlich unmittelbar an die Elektroden angrenzend gesammelt werden. Der Kontakt mit den Elektroden kann für die Partikel jedoch schädlich sein, insbesondere wenn die Partikel biologische Materialien umfassen. Außerdem sind relativ großflächige Elektroden erforderlich, um entsprechend große Sammlungsbereiche zu bilden. An großflächigen Elektroden tritt jedoch eine unerwünschte Erwärmung auf. Schließlich besteht ein wesentlicher Nachteil der von Hoettges et al. beschriebenen Technik darin, dass diese auf Elektroosmose und positiver Elektrophorese beruht und somit auf niedrige Frequenzen und niedrige Leitfähigkeiten der verwendeten Lösungen beschränkt ist. Daher ist es nicht möglich mit diesem Verfahren Zellen in physiologischen Lösungen zu untersuchen.The by K. F. Hoettges et. al. described technique has in particular for the application in biology, biochemistry and medicine several disadvantages. The circulating vortex flow has a relatively small catchment area for the particles to be collected. Furthermore, the particles can only be collected directly adjacent to the electrodes. However, contact with the electrodes may be detrimental to the particles, especially if the particles comprise biological materials. In addition, relatively large electrodes are required to form correspondingly large collection areas. At large-area electrodes, however, occurs an undesirable heating. Finally, there is a significant disadvantage of Hoettges et al. This technique is based on electroosmosis and positive electrophoresis and is thus limited to low frequencies and low conductivities of the solutions used. Therefore, it is not possible with this method to investigate cells in physiological solutions.
Es ist des Weiteren bekannt, Viren 1' unter Verwendung von elektrohydrodynamischen Strömungen 30' in den Fangbereich eines trichterförmigen, dielektrischen Feldkäfigs 50' zu führen, wie es in
Weitere fluidische Mikrosysteme zur Manipulation und Vermessung suspendierter Partikel unter der Wirkung hochfrequenter elektrischer Felder sind in
Strömungen in fluidischen Mikrosystemen können auch durch hohe elektrische Feldstärken induziert werden (elektrisches Heizen). Dieses Prinzip, das z. B. bei Wanderwellenpumpen in Mikrochips genutzt wird (siehe Publikation "
Die Aufgabe der Erfindung ist es, verbesserte Verfahren zur Sammlung von in einer Flüssigkeit suspendierten Partikeln, insbesondere zur Sammlung von suspendierten biologischen Objekten, bereitzustellen, mit denen die Nachteile der herkömmlichen Verfahren überwunden werden und die insbesondere eine Sammlung aus einem vergrößerten Einzugsbereich und ohne Schäden für die gesammelten Partikel ermöglichen. Eine weitere Aufgabe der Erfindung ist es, verbesserte Vorrichtungen zur Sammlung von in einer Flüssigkeit suspendierten Partikeln, insbesondere zur Umsetzung der erfindungsgemäßen Verfahren bereitzustellen.The object of the invention is to provide improved methods for collecting suspended in a liquid particles, in particular for the collection of suspended biological objects, which overcomes the disadvantages of conventional methods and in particular a collection of an enlarged catchment area and without damage to allow the collected particles. Another object of the invention is to provide improved devices for Collection of particles suspended in a liquid, in particular for implementing the method according to the invention.
Diese Aufgabe wird mit Verfahren und Vorrichtungen mit den Merkmalen der Patentansprüche 1 und 23 gelöst. Vorteilhafte Ausführungsformen und Anwendungen der Erfindung ergeben sich aus den abhängigen Ansprüchen.This object is achieved by methods and apparatus having the features of
Verfahrensbezogen beruht die Erfindung insbesondere auf der technischen Lehre, suspendierte Partikel in mindestens einem Sammlungsbereich in einem Kompartiment mit zirkulierenden Strömungen zu sammeln, die wenigstens teilweise entlang einer Längsausdehnung mindestens einer Elektrode auf einer Seitenfläche des Kompartiments verlaufen. Der Sammlungsbereich ist das Volumen, in das die Strömung die Partikel führt und in dem sich die Partikel insbesondere durch eine lokale Strömungsverminderung sammeln können. Vorteilhafterweise verlaufen die erfindungsgemäß durch eine Wechselwirkung der Flüssigkeit mit hochfrequenten elektrischen Feldern an den Elektroden erzeugten, zirkulierenden Strömungen in einer Ebene parallel zur jeweiligen Seitenfläche. Die Erfinder haben festgestellt, dass die Beschränkung der Sammlungseffektivität der herkömmlichen Techniken überwunden und der Einzugsbereich der an den Elektroden zirkulierenden Strömungen vergrößert werden können, wenn die Strömungen nicht wie bisher um eine Achse parallel zur Ausrichtung der Seitenfläche umlaufen, sondern eine lokale Drehachse senkrecht zu dieser Seitenfläche aufweisen. Ein weiterer wichtiger Vorteil der Erfindung besteht darin, dass mit den Strömungen auch kleinste Partikel, wie zum Beispiel Viren effektiv gesammelt werden können.In terms of method, the invention is based in particular on the technical teaching of collecting suspended particles in at least one collection area in a compartment with circulating flows which extend at least partially along a longitudinal extent of at least one electrode on a side surface of the compartment. The collection area is the volume into which the flow carries the particles and in which the particles can collect in particular by a local flow reduction. Advantageously, the circulating flows generated according to the invention by an interaction of the liquid with high-frequency electric fields at the electrodes run in a plane parallel to the respective side face. The inventors have found that the limitation on the collection efficiency of the conventional techniques can be overcome and the catchment area of the flows circulating at the electrodes can be increased if the flows do not revolve about an axis parallel to the orientation of the side face as before, but a local axis of rotation perpendicular to this Have side surface. Another important advantage of the invention is that with the flows even the smallest particles, such as viruses can be effectively collected.
Der Netto-Flüssigkeitsstrom in den zirkulierenden Strömungen ist null, da im Sammlungsbereich keine Quelle oder Senke existiert und die Flüssigkeit inkompressibel ist. Dennoch wird ein Netto-Teilchenstrom von außen nach innen beobachtet. Dies kann damit erklärt werden, dass durch negative Dielektrophorese die Teilchenkonzentration zwischen den Elektroden (Flüssigkeitsstrom nach außen gerichtet) kleiner ist, als in der Umgebung der Elektroden (Flüssigkeitsstrom nach innen gerichtet).The net fluid flow in the circulating flows is zero because there is no source or sink in the collection area exists and the liquid is incompressible. Nevertheless, a net particle flow is observed from outside to inside. This can be explained by the fact that by negative dielectrophoresis, the particle concentration between the electrodes (liquid flow directed outwards) is smaller than in the vicinity of the electrodes (fluid flow directed inwards).
Wenn die Partikel gemäß einer bevorzugten Ausführungsform der Erfindung im Sammlungsbereich ohne eine mechanische Berührung einer Wand oder eines anderen Teils des Kompartiments gesammelt werden, können sich Vorteile für die Manipulation biologischer Partikel, wie zum Beispiel biologischer Zellen ergeben, die auf mechanische Berührungen mit unerwünschten Zustandsänderungen reagieren würden. Falls ein mechanischer Kontakt jedoch gerade gewünscht ist, können die Partikel gemäß einer alternativen Ausführungsform der Erfindung im Sammlungsbereich mit einer Berührung einer Seitenfläche des Kompartiments angeordnet werden. Damit kann vorteilhafterweise eine Messung durch eine Kompartimentwand vereinfacht werden. Auch wenn die Sammlung mit einer Berührung der Seitenfläche erfolgt, kann im Unterschied zu den herkömmlichen elektroosmotischen Techniken eine Elektrodenberührung und damit eine unerwünschte Elektrodenreaktion vermieden werden. In diesem Fall kann der Sammlungsbereich durch einen Teil der Seitenfläche gebildet werden, in dem das Wandmaterial des Kompartiments freiliegt und keine Elektroden vorhanden sind.When the particles according to a preferred embodiment of the invention are collected in the collection area without mechanical contact with a wall or other part of the compartment, there may be advantages for the manipulation of biological particles, such as biological cells, which respond to mechanical contact with undesirable state changes would. However, if mechanical contact is desired, according to an alternative embodiment of the invention, the particles may be placed in the collection area with a touch of a side surface of the compartment. This can advantageously be simplified by a Kompartimentwand a measurement. Even if the collection takes place with a contact of the side surface, in contrast to the conventional electro-osmotic techniques, an electrode contact and thus an undesired electrode reaction can be avoided. In this case, the collection area may be formed by a part of the side surface in which the wall material of the compartment is exposed and no electrodes are present.
Gemäß einer besonders bevorzugten Ausführungsform der Erfindung werden an mindestens einer Elektrode mehrere lokal zirkulierende Strömungen erzeugt, von denen jeweils mindestens ein Zweig der lokalen Zirkulation auf den mindestens einen Sammlungsbereich gerichtet ist. Entlang der Elektrode verlaufen zum Beispiel zwei Strömungen. Vorteilhafterweise wird dadurch die Effektivität der Sammlung erhöht.According to a particularly preferred embodiment of the invention, a plurality of locally circulating flows are generated at least one electrode, of which at least one branch of the local circulation is directed to the at least one collection area. Run along the electrode for example, two currents. Advantageously, this increases the effectiveness of the collection.
Eine weitere Vergrößerung des Einzugsbereiches der Sammlung kann vorteilhafterweise erzielt werden, wenn gemäß dem erfindungsgemäßen Verfahrens an mehreren Elektroden eine Vielzahl von lokal zirkulierenden Strömungen erzeugt werden. Dies ermöglicht insbesondere, dass die Partikel von mehreren Richtungen zu dem mindestens einen Sammlungsbereich geführt werden. Wenn die Strömungen relativ zueinander derart symmetrisch, insbesondere punktsymmetrisch zum Sammlungsbereich ausgebildet werden, dass dieser strömungsberuhigt oder im Wesentlichen strömungsfrei ist, kann vorteilhafterweise erreicht werden, dass die von einer Seite zum Sammlungsbereich geförderten Partikel den Sammlungsbereich in einer anderen Richtung, z. B. auf der gegenüberliegenden Seite nicht wieder verlassen.A further enlargement of the catchment area of the collection can advantageously be achieved if, according to the method according to the invention, a plurality of locally circulating flows are generated at a plurality of electrodes. This allows in particular that the particles are guided from several directions to the at least one collection area. If the flows relative to each other are formed so symmetrically, in particular point-symmetrical to the collection area that this flow-calmed or substantially flow-free, can be advantageously achieved that the particles conveyed from one side to the collection area the collection area in another direction, for. B. on the opposite side not leave again.
Da gemäß der Erfindung die Strömung entlang der Längsausdehnung der jeweiligen Elektrode erzeugt wird, kann der Einzugsbereich vorteilhafterweise mit langgestreckten, band- oder streifenförmigen Elektroden erweitert werden, die sich vorzugsweise vom Sammlungsbereich radial in verschiedene Richtungen erstrecken.Since, according to the invention, the flow is generated along the longitudinal extent of the respective electrode, the catchment area can advantageously be widened with elongate, band or strip-shaped electrodes which preferably extend radially from the collection area in different directions.
Gemäß einer weiteren vorteilhaften Ausführungsform der Erfindung werden die Partikel aus einem Einzugsbereich des Kompartiment gesammelt, dessen Volumen 102 bis 109 -fach größer als das Volumen des Sammlungsbereiches ist. Dieses Verhältnis zeigt, dass mit dem erfindungsgemäßen Verfahren Partikel nicht nur gesammelt, sondern mit einem hohen Faktor konzentriert oder angereichert werden können. Beispielsweise können der Einzugsbereich eines einzelnen Wirbels ein Volumen von bis zu 10 µl und der Sammlungsbereich ein Volumen von 1 Femtoliter bis zu 50 Picoliter besitzen, so dass die Erfindung vorteilhafterweise mit fluidischen Mikrosystemen implementierbar ist.According to a further advantageous embodiment of the invention, the particles are collected from a catchment area of the compartment whose volume is 10 2 to 10 9 times greater than the volume of the collection area. This ratio shows that with the method according to the invention particles can not only be collected, but concentrated or enriched with a high factor. For example, the collection area of a single vertebra may have a volume of up to 10 μl and the collection area a volume of 1 femtoliter up to 50 picoliters, so that the invention can be advantageously implemented with fluidic microsystems.
Gemäß einer besonders bevorzugten Ausführungsform der Erfindung werden hochfrequente elektrischen Felder auch zur direkten Ausübung einer vorbestimmten dielektrophoretischen Vortriebskraft auf die Partikel ausgenutzt. Unter der Wirkung der hochfrequenten elektrischen Felder werden die Partikel durch negative Dielektrophorese zum Sammlungsbereich bewegt. Vorteilhafterweise wird dadurch die indirekte hydrodynamische Kraftwirkung noch verstärkt. Besonders bevorzugt ist, wenn erfindungsgemäß hochfrequente elektrische Felder erzeugt werden, die zur elektrodynamischen Strömungserzeugung und simultan zur dielektrophoretischen Manipulation der Partikel verwendet werden.According to a particularly preferred embodiment of the invention, high-frequency electric fields are also utilized for directly exerting a predetermined dielectrophoretic driving force on the particles. Under the effect of high-frequency electric fields, the particles are moved by negative dielectrophoresis to the collection area. Advantageously, this further enhances the indirect hydrodynamic force effect. It is particularly preferred if, according to the invention, high-frequency electric fields are generated which are used for the electrodynamic flow generation and simultaneously for the dielectrophoretic manipulation of the particles.
Die Sammlungseffektivität kann weiter gesteigert werden, wenn mit den hochfrequenten elektrischen Feldern mindestens ein dielektrophoretischer Feldkäfig mit einem Potentialminimum erzeugt wird, das sich im Sammlungsbereich befindet. Die dielektrophoretischen Fangkräfte im Feldkäfig sind abhängig von der Partikelgröße. Vorteilhafterweise können Partikel, die so klein sind, dass die Fangkräfte des Feldkäfigs für ein effektives Sammeln zu schwach wären, mit den elektrohydrodynamischen Strömungen so zu größeren Aggregaten verbunden werden, dass Feldkräfte erreicht werden, die für ein sicheres Fangen im Feldkäfig ausreichend sind. Erfindungsgemäß ist der Feldkäfig in zwei (trichterförmiger Feldkäfig) oder drei (allseitiger Feldkäfig) Raumrichtungen geschlossen. Der Feldkäfig kann mit 6, 8 oder mehr Elektroden gebildet werden.The collection efficiency can be further increased if at least one dielectrophoretic field cage with a potential minimum, which is located in the collection area, is generated with the high-frequency electric fields. The dielectrophoretic forces in the field cage depend on the particle size. Advantageously, particles that are so small that the capture forces of the field cage would be too weak for effective collection, can be connected to the electrohydrodynamic flows to larger aggregates so that field forces are sufficient, which are sufficient for a safe catch in the field cage. According to the invention, the field cage is closed in two (funnel-shaped field cage) or three (all-sided field cage) spatial directions. The field cage can be formed with 6, 8 or more electrodes.
Wenn gemäß einer vorteilhaften Variante der Erfindung Elektroden so angeordnet und mit hochfrequenten elektrischen Spannungen beaufschlagt werden, dass mehrere Feldkäfige gebildet werden, kann vorteilhafterweise der Einzugsbereich der erfindungsgemäßen Partikelsammlung noch vergrößert werden. Es sind vorzugsweise ein innerer und ein äußerer Feldkäfig vorgesehen, deren Potentialminima die gleiche Position im Sammlungsbereich aufweisen. Die Feldkäfige sind konzentrisch zueinander angeordnet, wobei der jeweils äußere Feldkäfig Partikel durch negative Dielektrophorese hin zum inneren Feldkäfig bewegt.If, according to an advantageous variant of the invention, electrodes are arranged and with high-frequency electrical Are applied voltages that several field cages are formed, advantageously the catchment area of the particle collection according to the invention can be further increased. Preferably, an inner and an outer field cage are provided whose potential minima have the same position in the collection area. The field cages are arranged concentrically to each other, wherein the respective outer field cage moves particles by negative dielectrophoresis towards the inner field cage.
Erfindungsgemäß kann vorgesehen sein, dass im Sammlungsbereich mindestens eine weitere Kraft auf die Partikel wirkt. Damit kann vorteilhafterweise eine zusätzliche Halterung und/oder Manipulation der Partikel im Sammlungsbereich erzielt werden. Die Erzeugung einer optisch wirksamen Kraft kann Vorteile bei der Kombination der erfindungsgemäßen Technik mit einer optischen Messung im Sammlungsbereich und für eine selektive Partikelmanipulation besitzen. Die Erzeugung einer dielektrophoretischen Kraft kann Vorteile für ein effektives Zusammenwirken mit einer dielektrophoretischen Barriere des Feldkäfigs besitzen. Eine zusätzliche magnetische Kraft bietet Vorteile bei der Manipulation magnetischer Partikel. Schließlich kann die mindestens eine weitere Kraft eine durch Ultraschall vermittelte Kraft sein, beispielsweise können Knoten eines Ultraschallfeldes im Sammlungsbereich gebildet werden.According to the invention, it can be provided that at least one further force acts on the particles in the collection area. This can advantageously be achieved additional support and / or manipulation of the particles in the collection area. The generation of an optically effective force may have advantages in the combination of the inventive technique with an optical measurement in the collection area and for a selective particle manipulation. The generation of a dielectrophoretic force may have advantages for effective interaction with a dielectrophoretic barrier of the field cage. An additional magnetic force offers advantages in the manipulation of magnetic particles. Finally, the at least one additional force may be a force mediated by ultrasound, for example, nodes of an ultrasound field may be formed in the collection area.
Zur Ausübung einer weiteren Kraft besteht ferner die Möglichkeit, dass sich im Sammlungsbereich ein Startobjekt befindet, z.B. ein Bead, welches auch funktionalisiert werden kann. Durch dieses Startobjekt werden die Partikel nicht nur durch dielektrische Wechselwirkungen beeinflusst, sondern auch ggf. durch eine spezifische Bindung an das Bead oder eine durch das Startobjekt hervorgerufene hydrodynamische Abschottung. Gemäß einer weiteren bevorzugten Ausführungsform der Erfindung erfolgt im Sammlungsbereich mindestens eine Messung an den gesammelten Partikeln. Damit können sich insbesondere Vorteile bei der Manipulation oder Evaluierung gesammelter biologischer Partikel ergeben. Die Messung umfasst vorzugsweise eine zum Beispiel aus der Technik fluidischer Mikrosysteme an sich bekannte elektrische, elektrochemische und/oder optische Messung.For exercising another force, there is also the possibility that a start object is located in the collection area, eg a bead, which can also be functionalized. Through this starting object, the particles are not only influenced by dielectric interactions, but also possibly by a specific binding to the bead or caused by the starting object hydrodynamic foreclosure. According to a further preferred embodiment of the invention, at least one measurement of the collected particles takes place in the collection area. This may in particular result in advantages in the manipulation or evaluation of collected biological particles. The measurement preferably comprises an electrical, electrochemical and / or optical measurement known per se from the art of fluidic microsystems.
Gemäß einer bevorzugten Anwendung der Erfindung ist die Messung auf die Detektion eines Rezeptor-Ligand-Bindungs-Ereignisses gerichtet. Für diese Messung kann erfindungsgemäß die Seitenfläche des Kompartiments im Bereich des mindestens einen Sammlungsbereiches mit Detektionsspots in Form von Rezeptormolekülen (z.B. Proteinen, Antikörpern, DNA, Viren (für Transfektionsexperimente), usw.) funktionalisiert sein, wie es an sich von konventionellen Mikroarrays oder Biochips bekannt ist, so dass eine spezifische Rezeptor - Ligand - Interaktion mit im Sammlungsbereich akkumulierten Partikeln oder Molekülen stattfindet. Die Interaktion kann dann in bekannter Weise z.B. über elektrische, elektrochemische oder optische Ausleseverfahren nachgewiesen werden kann.In accordance with a preferred application of the invention, the measurement is directed to the detection of a receptor-ligand binding event. For this measurement, according to the invention, the side area of the compartment in the region of the at least one collection area can be functionalized with detection spots in the form of receptor molecules (eg proteins, antibodies, DNA, viruses (for transfection experiments), etc.), as is conventional microarrays or biochips is known, so that a specific receptor - ligand interaction takes place with particles or molecules accumulated in the collection area. The interaction can then be determined in a known manner e.g. can be detected by electrical, electrochemical or optical readout.
Vorteilhafterweise können mit dem erfindungsgemäßen Verfahren die Konzentration von Analytpartikeln oder -molekülen in der Nähe der Detektionsspots erhöht (Sensitivitätssteigerung) und der Detektionsprozeß im Vergleich zu rein diffusivem Antransport von Analytpartikeln oder -molekülen an die Detektionsspots beschleunigen werden.Advantageously, with the method according to the invention, the concentration of analyte particles or molecules in the vicinity of the detection spots can be increased (increase in sensitivity) and the detection process can be accelerated compared to purely diffusive transport of analyte particles or molecules to the detection spots.
Das funktionalisierte Rezeptorarray kann z. B. auf einer flächigen Elektrode aufgebracht sein und zusammen mit einem die Sammelelektroden enthaltenden zweiten Substrat eine Mikrokammer bilden. Nach Anreicherung der Analytpartikel oder -moleküle durch das erfindungsgemäße Verfahren und die Bindung derselben an die immobilisierten Rezeptoren auf dem Array kann die Sammelstruktur dann wieder entfernt werden. Sie kann entsprechend auch mehrfach verwendet werden.The functionalized receptor array can e.g. B. be applied to a flat electrode and together with a Forming electrodes containing second substrate form a micro-chamber. After enrichment of the analyte particles or molecules by the method according to the invention and the binding of the same to the immobilized receptors on the array, the collection structure can then be removed again. It can also be used several times accordingly.
Wenn gemäß einer weiteren Modifizierung der Erfindung die Partikel in mehreren Sammlungsbereichen im Kompartiment gesammelt werden, können sich Vorteile für eine parallele Anreicherung.der Partikel aus mehreren Einzugsgebieten im Kompartiment und eine parallele Manipulation oder Evaluierung der gesammelten Partikel ergeben.According to a further modification of the invention, if the particles are collected in several collection areas in the compartment, there may be advantages for parallel enrichment of the particles from multiple catchment areas in the compartment and parallel manipulation or evaluation of the collected particles.
Für die Anwendung in fluidischen Mikrosystemen ist es von besonderem Vorteil der Erfindung, dass die Sammlung nicht nur aus einem Einzugsbereich mit einer ruhenden Suspensionsflüssigkeit, sondern sogar dynamisch aus einer bewegten Suspensionsflüssigkeit heraus erfolgen kann. Das Kompartiment kann zum Beispiel von einer laminaren Strömung durchsetzt werden, die an den Elektroden erfindungsgemäß mit der lokal zirkulierenden Strömung überlagert wird.For use in fluidic microsystems, it is of particular advantage of the invention that the collection can take place not only from a catchment area with a stationary suspension liquid, but even dynamically from a moving suspension liquid out. The compartment can be penetrated, for example, by a laminar flow, which is superimposed on the electrodes according to the invention with the locally circulating flow.
Des Weiteren kann im Kompartiment eine gegenseitige Überlagerung von mehreren lokal zirkulierenden Strömungen vorgesehen sein. Eine erste zirkulierende Strömung kann die Partikel gerade in einen Sammlungsbereich führen, der Teil einer weiteren, nachgeordneten zirkulierenden Strömung ist. Dies ermöglicht eine Anordnung einer Vielzahl von Zirkulationen nach Art einer Kaskade, bei der aus einem ausgedehnten Einzugsbereich Partikel in einen einzigen Sammlungsbereich geführt werden.Furthermore, a mutual superimposition of several locally circulating flows can be provided in the compartment. A first circulating flow may just direct the particles into a collection area that is part of another downstream circulating flow. This makes it possible to arrange a multiplicity of cascade-like circulations in which particles are led out of an extended catchment area into a single collection area.
Das erfindungsgemäße Verfahren eignet sich besonders gut zur Sammlung von Partikeln mit einem Durchmesser unterhalb von 1 µm. Für biologische Anwendungen können somit vorteilhafterweise insbesondere Zellen, Viren, Bakterien, Proteine, Zellbestandteile und/oder biologische Makromoleküle, z. B. DNS gesammelt werden.The inventive method is particularly well suited for the collection of particles with a diameter below 1 micron. For biological applications, it is thus advantageously possible in particular to use cells, viruses, bacteria, proteins, cell components and / or biological macromolecules, eg. B. DNS are collected.
Gemäß weiteren Varianten der Erfindung kann vorgesehen sein, dass die an den Elektroden lokal zirkulierenden Strömungen durch einen lokalen Temperaturgradienten in der Flüssigkeit verstärkt werden. Der Temperaturgradient kann durch eine lokale Aufheizung der Flüssigkeit gebildet werden, die vorzugsweise durch eine Bestrahlung der Flüssigkeit und/oder von Seitenflächen des Kompartiments mit Licht und dessen entsprechende Absorption und/oder durch in die Wände eingebettete ("vergrabene") Thermoelemente erfolgt. Der Temperaturgradient kann alternativ oder zusätzlich durch eine lokale, gezielte Abkühlung der Flüssigkeit gebildet werden.According to further variants of the invention it can be provided that the currents circulating locally at the electrodes are intensified by a local temperature gradient in the liquid. The temperature gradient can be formed by local heating of the liquid, which is preferably carried out by irradiation of the liquid and / or side surfaces of the compartment with light and its corresponding absorption and / or embedded ("buried") thermocouples in the walls. The temperature gradient can alternatively or additionally be formed by a local, targeted cooling of the liquid.
Die lokale Aufheizung der Flüssigkeit kann vorteilhafterweise zusätzlich zu Anregung von chemischen Reaktionen verwendet werden. Durch die lokal hohen Temperaturen im Sammlungsbereich können hier gezielt z. B. thermisch aktivierte Reaktionen ablaufen, z.B. ein Aggregation oder eine Fällung.The local heating of the liquid may advantageously be used in addition to excitation of chemical reactions. Due to the high local temperatures in the collection area can be targeted z. Thermally activated reactions, e.g. an aggregation or precipitation.
Vorrichtungsbezogen wird die oben genannte Aufgabe der Erfindung durch eine Sammlungsvorrichtung zur Sammlung von suspendierten Partikeln gelöst, die insbesondere in einem Kompartiment zur Aufnahme einer Flüssigkeit an einer Seitenfläche Elektroden zur Erzeugung von mehreren lokal zirkulierenden Strömungen in der Flüssigkeit aufweist, mit der suspendierte Partikel zu mindestens einem vorbestimmten Sammlungsbereich im Kompartiment geführt werden können, wobei die Sammlungsvorrichtung dazu eingerichtet ist, die mindestens eine Strömung so zu erzeugen, dass sich ein Teil der Strömung entlang der Längsausdehnung der Elektrode erstreckt und die Strömung um eine Achse umläuft, die senkrecht zu der jeweils angrenzenden Seitenfläche mit der Elektrode ausgerichtet ist.With respect to the apparatus, the above object of the invention is achieved by a collection device for collecting suspended particles which, in particular in a compartment for receiving a liquid on a side surface, has electrodes for generating a plurality of locally circulating flows in the liquid with which suspended particles are at least one predetermined collection area can be performed in the compartment, wherein the collection device is adapted to at least creating a flow such that a portion of the flow extends along the longitudinal extent of the electrode and the flow circulates about an axis perpendicular to the respective adjacent side surface aligned with the electrode.
Gemäß vorteilhaften Varianten der Erfindung kann der Sammlungsbereich mit einem Abstand von den Seitenflächen des Kompartiments oder so angeordnet sein, dass der Sammlungsbereich in Kontakt mit einer der Seitenflächen steht.According to advantageous variants of the invention, the collection area may be arranged at a distance from the side surfaces of the compartment or so that the collection area is in contact with one of the side surfaces.
Vorzugsweise ist die Elektrode, an der jeweils eine zirkulierende Strömung erzeugt werden kann, mit einer Spannungsquelle zur Bereitstellung von vorbestimmten hochfrequenten elektrischen Spannungen verbunden. Die mindestens eine Elektrode, die zur Erzeugung der zirkulierenden Strömung verwendet wird, wird auch als Sammelelektrode bezeichnet. Die Sammlungsvorrichtung umfasst bei der Erzeugung einer Vielzahl zirkulierender Strömungen, die auf einen oder mehrere Sammlungsbereiche gerichtet sind, entsprechend eine Vielzahl von Sammelelektroden, die ein Sammelelektroden-Array bilden.Preferably, the electrode, on each of which a circulating flow can be generated, connected to a voltage source for providing predetermined high-frequency electrical voltages. The at least one electrode used to generate the circulating flow is also referred to as a collecting electrode. The collection device, when generating a plurality of circulating flows directed to one or more collection areas, respectively includes a plurality of collection electrodes forming a collection electrode array.
Wenn die Sammlungsvorrichtung gemäß einer bevorzugten Ausführungsform der Erfindung dazu eingerichtet ist, auf die zu sammelnden Partikel nicht nur elektrohydrodynamische, sondern auch dielektrophoretische Kräfte auszuüben, kann durch die zusätzliche Kraftwirkung die Sammlungseffektivität verbessert werden. Die dielektrophoretische Kraftwirkung wird durch die Wechselwirkung der Partikel mit hochfrequenten elektrischen Feldern ausgeübt, die im Kompartiment mit mindestens einer Elektrode erzeugt werden, die im Folgenden als Käfigelektrode bezeichnet wird. Wenn die oben genannten ein- oder allseitig geschlossenen Feldkäfige erzeugt werden sollen, ist das Kompartiment mit einem Käfigelektroden-Array ausgestattet. Gemäß einer besonders bevorzugten Variante der Erfindung sind die Sammel- und Käfigelektroden identisch. Die Sammelelektroden- und Käfigelektroden-Arrays werden durch eine gemeinsame Elektrodenanordnung gebildet. In diesem Fall wird der Aufbau der Sammlungsvorrichtung und die Ansteuerung der Elektroden vereinfacht.If the collection device according to a preferred embodiment of the invention is adapted to exert on the particles to be collected not only electrohydrodynamic but also dielectrophoretic forces, the collection efficiency can be improved by the additional force effect. The dielectrophoretic force action is exerted by the interaction of the particles with high-frequency electric fields generated in the compartment with at least one electrode, which is referred to below as the cage electrode. If the aforementioned one-sided or all-around field cages are to be generated, the compartment is equipped with a cage electrode array. According to a particularly preferred variant of the invention, the collecting and cage electrodes are identical. The collecting electrode and cage electrode arrays are formed by a common electrode arrangement. In this case, the structure of the collecting device and the driving of the electrodes are simplified.
Ein besonderer Vorteil der erfindungsgemäßen Sammlungsvorrichtung besteht in deren Miniaturisierbarkeit. Das Kompartiment der Sammlungsvorrichtung ist vorzugsweise Teil eines fluidischen Mikrosystems. Vorteilhafterweise kann die erfindungsgemäße Sammlungsfunktion mit Sammlungs-, Sortier-, Evaluierungs- oder Messfunktionen des Mikrosystems kombiniert werden. Die Sammlungsvorrichtung ist bspw. im Kanal eines fluidischen Mikrosystems angeordnet, der das genannte Kompartiment mit dem Strömungsgenerator bildet. Überraschenderweise kann mit der erfindungsgemäßen Sammlungsvorrichtung auch im durchströmten Kanal eine Sammlung von Partikeln erfolgen.A particular advantage of the collection device according to the invention is its miniaturization. The compartment of the collection device is preferably part of a fluidic microsystem. Advantageously, the collection function according to the invention can be combined with collection, sorting, evaluation or measurement functions of the microsystem. The collection device is, for example, arranged in the channel of a fluidic microsystem which forms the said compartment with the flow generator. Surprisingly, the collecting device according to the invention can also be used to collect particles in the throughflow channel.
Zur Erhöhung der Sammlungsaktivität kann gemäß einer Abwandlung der Erfindung vorgesehen sein, dass mehrere Sammlungsbereiche entlang einer Längsrichtung des Kanals reihenförmig angeordnet sind.To increase the collection activity can be provided according to a modification of the invention that a plurality of collection areas along a longitudinal direction of the channel are arranged in rows.
Besondere Vorteile für einen erweiterten Anwendungsbereich der Sammlungsvorrichtung ergeben sich, wenn diese mit einer Magnetfeldeinrichtung zur Ausübung einer magnetischen Haltekraft im genannten Sammlungsbereich und/oder einer Messeinrichtung zur Erfassung von elektrischen, elektrochemischen oder optischen Eigenschaften von Partikeln im Sammlungsbereich ausgestattet ist.Particular advantages for an extended field of application of the collection device arise when it is equipped with a magnetic field device for exerting a magnetic holding force in said collection region and / or a measuring device for detecting electrical, electrochemical or optical properties of particles in the collection region.
Gemäß weiteren Varianten der Erfindung kann der Strömungsgenerator zusätzlich eine Heizeinrichtung und/oder eine Lichtquelle umfassen.According to further variants of the invention, the flow generator may additionally comprise a heating device and / or a light source.
Weitere Einzelheiten und Vorteile der Erfindung werden aus der folgenden Beschreibung von Ausführungsbeispielen und den beigefügten Zeichnungen ersichtlich. Es zeigen:
-
Figur 1 : eine schematische Schnittansicht einer Ausführungsform einer erfindungsgemäßen Sammlungsvorrichtung, -
: verschiedene Phasen der Sammlung von Partikeln mit dem erfindungsgemäßen Verfahren,Figuren 2, 3 -
Figuren 4A, 4B : Illustrationen von Feld- und Temperaturbedingungen in einer erfindungsgemäßen Sammlungsvorrichtung und von experimentellen Ergebnissen, die mit einer erfindungsgemäßen Sammlungsvorrichtung erzielt wurden, -
Figur 5 : eine Ausführungsform einer erfindungsgemäßen Sammlungsvorrichtung mit einer Reihe von Sammlungsbereichen, -
Figur 6 : eine weitere Ausführungsform einer erfindungsgemäßen Sammlungsvorrichtung mit einer Kaskade von Sammlungsbereichen, -
Figur 7 : eine weitere Ausführungsform einer erfindungsgemäßen Sammlungsvorrichtung mit einer Kaskade von Sammlungsbereichen, -
Figur 8 : eine Illustration der Strömungsbedingungen in einer Sammlungsvorrichtung gemäßFigur 7 , und -
Figuren 9, 10 : Illustrationen herkömmlicher Sammlungstechniken (Stand der Technik).
-
FIG. 1 FIG. 2 is a schematic sectional view of an embodiment of a collection device according to the invention, FIG. -
FIGS. 2, 3 : different phases of the collection of particles with the method according to the invention, -
FIGS. 4A, 4B : Illustrations of field and temperature conditions in a collection device according to the invention and of experimental results obtained with a collection device according to the invention, -
FIG. 5 an embodiment of a collection device according to the invention with a number of collection areas, -
FIG. 6 FIG. 2 shows a further embodiment of a collection device according to the invention with a cascade of collection areas, FIG. -
FIG. 7 FIG. 2 shows a further embodiment of a collection device according to the invention with a cascade of collection areas, FIG. -
FIG. 8 FIG. 2: an illustration of the flow conditions in a collection device according to FIGFIG. 7 , and -
FIGS. 9, 10 : Illustrations of conventional collection techniques (prior art).
Die Ausführungsbeispiele der Erfindung werden im Folgenden unter Bezug auf die Anwendung der Erfindung in fluidischen Mikrosystemen zur dielektrophoretischen Partikelmanipulation beschrieben. Derartige fluidische Mikrosysteme, ihre Komponenten und ihre Betriebsverfahren sind an sich bekannt und werden daher im Folgenden nicht beschrieben. Die Erfindung wird im Folgenden beispielhaft unter Bezug auf eine Gestaltung erörtert, bei der Elektroden sowohl zum Sammeln als auch zur Ausübung einer dielektrophoretischen Vortriebskraft verwendet werden, bei der also die Sammel- und Käfigelektroden identisch sind. Es wird betont, dass die Umsetzung der Erfindung nicht auf diese Ausführungsform beschränkt ist. Vielmehr können erfindungsgemäß Sammelelektroden ausschließlich zur Erzeugung einer elektrohydrodynamischen Strömung vorgesehen sein und nicht Teil eines dielektrischen Feldkäfigs bilden, wie dies bspw. in den
Jede Elektrode zur elektrohydrodynamischen Strömungserzeugung besitzt die Form eines Streifens oder Bandes mit einer Länge (siehe auch
Das Bezugszeichen 50 bezieht sich auf eine Messeinrichtung, zum Beispiel ein Mikroskop mit einer CCD-Kamera, mit der zum Beispiel fluoreszenzmarkierte Partikel im Sammlungsbereich optisch gemessen und ausgewertet werden können. Hierzu ist in der Seitenfläche 21 des Kanals wenigstens ein optisch transparentes Fenster vorgesehen (siehe
In
Die Ursache der elektrohydrodynamischen Strömung 30 ist in
In
Unter der Wirkung der hochfrequenten Felder im Kompartiment 10 werden auf die Partikel auch dielektrophoretischen Kräfte ausgeübt. Im rechten Teil von
Die Auswahl der zur Erzeugung der elektrohydrodynamischen Strömung erforderlichen Spannungsamplitude erfolgt in Abhängigkeit von den dielektrischen Eigenschaften der Suspensionsflüssigkeit und den geometrischen Eigenschaften der Elektrodenanordnung. Es kann auch eine empirische Auswahl durch Experimente vorgesehen sein. Vorzugsweise werden die hochfrequenten elektrischen Felder so gewählt, dass auf die Partikel ausschließlich negative Dielektrophorese wirkt. Die in den
Unter den folgenden Betriebsbedingungen konnte eine Anreicherung von Hepatitis-A-Viren (Durchmesser rd. 30 nm) innerhalb von 10 Minuten erreicht werden. Hochfrequente Wechselspannungen mit Frequenz: 7.4 MHz, Amplitude: 4 Vrms), Elektrodenabstand: 5 µm. Die Startkonzentration der Viren im Kompartiment betrug rd. 109 bis 1010/ml. Die Anreicherung der fluoreszenzmarkierten Hepatitis-A-Viren ist für verschiedene Beobachtungszeiten in
Außerhalb des fluidischen Mikrosystems befindet sich eine Messeinrichtung (nicht dargestellt), mit der die Partikel in den Sammlungsbereichen 41, 42, 43, ... durch ein Fenster 51 entlang einer Abtastzeile 52 gemessen werden. Zur Detektion von Rezeptor-Ligand-Bindungsereignissen in den gesammelten Partikeln erfolgt beispielsweise eine Fluoreszenz-Korrelations-Messung (FCS).Outside the fluidic microsystem is a measuring device (not shown), with which the particles in the
Eine kaskadenförmige Kombination einer Vielzahl von zirkulierenden Strömungen ist schematisch in
Bei den in den
Gemäß einer weiteren Modifizierung der Erfindung kann die Sammlungsvorrichtung mit einer Kühleinrichtung, z. B. einem Peltier-Element ausgestattet sein, um eine unerwünschte Gesamterwärmung der Sammlungsvorrichtung zu vermeiden.According to a further modification of the invention, the collection device may be provided with a cooling device, e.g. B. a Peltier element to avoid unwanted total heating of the collection device.
Die in der vorstehenden Beschreibung, den Zeichnungen und den Ansprüchen offenbarten Merkmale der Erfindung können sowohl einzeln als auch in Kombination für die Verwirklichung der Erfindung in ihren verschiedenen Ausgestaltungen von Bedeutung sein.The features of the invention disclosed in the foregoing description, drawings and claims may be significant to the realization of the invention in its various forms both individually and in combination.
Claims (36)
- A method for collecting particles (1, 2) which are suspended in a liquid, comprising the steps:- providing the liquid containing the suspended particles (1, 2) in a compartment (10) having lateral surfaces (11), wherein a plurality of electrodes (21, 22, 23) is arranged on at least one of the lateral surfaces (11), and- generating high-frequency electric fields by means of said electrodes (21, 22, 23)
characterized in that- by the action of said high-frequency electric fields a plurality of circulating flows (30, 34, 35, 36) are formed, by means of which the particles (1, 2) are guided to at least one predetermined collecting area (40) within the compartment (10),whereby- said flows (30, 34, 35, 36) are formed in such a way that at least one branch of the flow runs along a longitudinal extension of the electrode (21, 22, 23), and the flow (30, 34, 35, 36) circulates about an axis (31) which is oriented perpendicular to the respectively adjacent lateral surface (11) provided with said electrode (21, 22, 23), and- said electrodes (21, 22, 23) are arranged on different sides of the collecting area (40), from which sides the particles (1, 2) are respectively guided to the collecting area (40) by means of said plurality of flows (30, 34, 35, 36). - The method according to Claim 1, in which the particles are arranged in the collecting area (40) without a contact with the lateral surface (11) of the compartment (10).
- The method according to Claim 1, in which the particles are arranged in the collecting area (40) in such a way that they make contact with one of the lateral surfaces (11) of the compartment (10).
- The method according to at least one of the preceding claims, in which a plurality of circulating flows (32, 33) are generated at one electrode (21) in each case, by means of which flows the particles are guided to the at least one collecting area (40).
- The method according to Claim 5, in which the flows are generated in an essentially symmetrical manner relative to the collecting area (40).
- The method according to at least one of the preceding claims, in which, due to the high-frequency electric fields, forces are exerted on the particles by means of negative dielectrophoresis, said forces being directed towards the collecting area (40).
- The method according to at least one of the preceding claims, in which the high-frequency electric fields are generated by strip-shaped electrodes of an electrode arrangement (20) for generating at least one dielectrophoretic field cage, said electrodes being arranged on the lateral surfaces (11) of the compartment.
- The method according to Claim 7, in which the dielectrophoretic field cage is generated with a potential minimum located in the collecting area (40).
- The method according to Claim 8, in which a dielectrophoretic field cage which is closed in at least two spatial directions is generated.
- The method according to at least one of Claims 6 to 9, in which the high-frequency electric fields are generated by electrodes of an electrode arrangement (20) with an outer dielectrophoretic field cage (20.1) and an inner dielectrophoretic field cage (20.2), wherein the electrodes are arranged on the lateral surfaces (11) of the compartment.
- The method according to at least one of the preceding claims, in which the particles are guided into the collecting area (40) from a catchment area of the compartment (10), the volume of which catchment area is 102 to 109 times greater than the volume of the collecting area (40).
- The method according to Claim 11, in which the catchment area has a volume of up to 50 µl and the collecting area (40) has a volume of from 40 µl up to 1 fl.
- The method according to at least one of the preceding claims, in which, in the collecting area (40), at least one further force acts on the particles.
- The method according to Claim 13, in which the force is an optically active force, a dielectrophoretic force or a magnetic force.
- The method according to at least one of the preceding claims, in which, in the collecting area (40), a measurement is carried out on the collected particles.
- The method according to Claim 15, in which the measurement comprises an electrical, electrochemical or optical measurement.
- The method according to Claim 16, in which the measurement comprises a detection of a receptor/ligand binding event.
- The method according to at least one of the preceding claims, in which, in the compartment, there are arranged a plurality of collecting areas (41, 42, 43, ...) in which particles are collected.
- The method according to at least one of the preceding claims, in which, in the compartment, a laminar flow or an ultrasonic field is generated, which overlays the circulating flow (30).
- The method according to at least one of the preceding claims, in which, in the compartment (10), a plurality of circulating flows (34, 35, 36) are generated, which are overlaying one another.
- The method according to at least one of the preceding claims, in which particles with a diameter of less than 1 µm are collected.
- The method according to Claim 21, in which particles which comprise cells, viruses, bacteria, proteins, cell constituents or biological macromolecules are collected.
- A collecting device for collecting particles which are suspended in a liquid, comprising:- a compartment (10) confined by lateral surfaces (11) for containing the liquid containing the suspended particles, and- a plurality of electrodes (21, 22, 23) which are arranged on at least one of the lateral surfaces (11) and by means of which high-frequency electric fields can be generated in the compartment (10),
characterized in that- said electrodes (21, 22, 23) have an elongated shape with an aspect ration between electrode width and electrode length which is choosen to be in the range from 1:10 to 1 : 100, said electrodes being designed to generate high-frequency electric fields for forming a plurality of circulating flows (30) by means of which the particles (1, 2) can be guided to at least one predetermined collecting area (40) within the compartment (10), and to form the flows (30) in such a way that a branch of each flow (30) runs along the elongate shape, and- at least one axis (31), about which the flow (30) circulates, is oriented perpendicular to the respectively adjacent lateral surface (11) provided with said electrode. - The device according to Claim 23, in which the collecting area (40) is arranged at a distance from the lateral surfaces (11) of the compartment (10).
- The device according to Claim 23, in which the collecting area (40) makes contact with one of the lateral surfaces (11) of the compartment (10).
- The device according to at least one of Claims 23 to 25, in which an electrode arrangement (20) comprising a plurality of electrodes (21, 22, 23) is designed to generate a plurality of circulating flows (32, 33).
- The device according to at least one of Claims 23 to 26, in which cage electrodes for generating at least one dielectrophoretic field cage are arranged on the lateral surfaces (11) of the compartment.
- The device according to Claim 27, in which the cage electrodes form part of the electrode arrangement (20).
- The device according to Claim 27, in which the cage electrodes form an outer dielectrophoretic field cage (20.1) and an inner dielectrophoretic field cage (20.2) which is arranged inside the outer dielectrophoretic field cage (20.1).
- The device according to at least one of Claims 23 to 27, in which a heating device, a cooling device and/or a light source are provided.
- The device according to at least one of Claims 23 to 30, in which the collecting area (40) is equipped with a magnetic field device.
- The device according to at least one of Claims 23 to 31, which is equipped with a measuring device (50) for detecting electrical or optical properties of particles in the collecting area (40).
- The device according to at least one of Claims 23 to 32, in which at least one of the lateral surfaces of the compartment, in the region of the at least one collecting area, is functionalized with detection spots in the form of receptor molecules.
- The device according to at least one of Claims 23 to 33, in which a plurality of collecting areas (41, 42, 43) are provided.
- The device according to at least one of Claims 23 to 34, in which the compartment forms part of a channel of a fluidic microsystem.
- The device according to Claim 35, in which the collecting areas (41, 42, 43) are arranged in a row along a longitudinal direction of the channel.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE102004023466A DE102004023466B4 (en) | 2004-05-12 | 2004-05-12 | Method and device for collecting suspended particles |
PCT/EP2005/004925 WO2005110605A1 (en) | 2004-05-12 | 2005-05-06 | Method and device for collecting suspended particles |
Publications (3)
Publication Number | Publication Date |
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EP1744831A1 EP1744831A1 (en) | 2007-01-24 |
EP1744831B1 true EP1744831B1 (en) | 2010-11-17 |
EP1744831B8 EP1744831B8 (en) | 2011-09-07 |
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Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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EP05747443A Not-in-force EP1744831B8 (en) | 2004-05-12 | 2005-05-06 | Method and device for collecting suspended particles |
Country Status (5)
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US (1) | US7879214B2 (en) |
EP (1) | EP1744831B8 (en) |
AT (1) | ATE488301T1 (en) |
DE (2) | DE102004023466B4 (en) |
WO (1) | WO2005110605A1 (en) |
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JP2008003074A (en) * | 2006-05-26 | 2008-01-10 | Furuido:Kk | Micro fluid device, measuring device, and micro fluid stirring method |
DE102006052925A1 (en) * | 2006-11-09 | 2008-05-15 | Evotec Technologies Gmbh | Field cage and associated operating method |
EP1935498A1 (en) | 2006-12-22 | 2008-06-25 | Universität Leipzig | Device and method for contactless manipulation and alignment of sample particles in a measurement volume with the aid of an inhomogeneous electrical alternating field |
KR101947233B1 (en) * | 2016-09-26 | 2019-02-12 | 울산과학기술원 | Electrode for separating particles based on dielectrophoresis and electroosmosis, and an apparatus for separating particles including the same |
JP6742618B2 (en) * | 2018-06-11 | 2020-08-19 | シャープ株式会社 | Bioparticle observation device and bioparticle observation method |
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US4440638A (en) * | 1982-02-16 | 1984-04-03 | U.T. Board Of Regents | Surface field-effect device for manipulation of charged species |
DE19653659C1 (en) | 1996-12-20 | 1998-05-20 | Guenter Prof Dr Fuhr | Electrode arrangement for field cages |
DE19983263T1 (en) * | 1998-05-29 | 2001-05-31 | Ind Res Ltd | Method and device for concentrating and / or positioning particles or cells |
DE19859459A1 (en) * | 1998-12-22 | 2000-06-29 | Evotec Biosystems Ag | Microsystems for cell permeation and fusion |
DE19903001A1 (en) * | 1999-01-26 | 2000-08-24 | Evotec Biosystems Ag | Method and device for the detection of microscopic objects |
DE10055921A1 (en) * | 2000-11-10 | 2002-05-29 | Evotec Ag | Method and device for generating micro convections |
DE10059152C2 (en) * | 2000-11-29 | 2003-03-27 | Evotec Ag | Microsystem for the dielectric and optical manipulation of particles |
DE10224150B4 (en) * | 2002-05-27 | 2004-04-01 | Siemens Ag | Reactor for the treatment of a sample medium |
ATE285590T1 (en) * | 2002-10-25 | 2005-01-15 | Evotec Technologies Gmbh | METHOD AND APPARATUS FOR CAPTURING THREE-DIMENSIONAL IMAGERY OF FLOATED MICROOBJECTS USING HIGH-RESOLUTION MICROSCOPY |
DE10255858A1 (en) | 2002-11-29 | 2004-06-17 | Evotec Oai Ag | Fluidic microsystem with field-forming passivation layers on microelectrodes |
DE10320869A1 (en) * | 2003-05-09 | 2004-12-16 | Evotec Technologies Gmbh | Methods and devices for liquid treatment of suspended particles |
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- 2004-05-12 DE DE102004023466A patent/DE102004023466B4/en active Active
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2005
- 2005-05-06 DE DE502005010554T patent/DE502005010554D1/en active Active
- 2005-05-06 EP EP05747443A patent/EP1744831B8/en not_active Not-in-force
- 2005-05-06 AT AT05747443T patent/ATE488301T1/en active
- 2005-05-06 US US11/568,895 patent/US7879214B2/en not_active Expired - Fee Related
- 2005-05-06 WO PCT/EP2005/004925 patent/WO2005110605A1/en active Application Filing
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US20070221501A1 (en) | 2007-09-27 |
DE502005010554D1 (en) | 2010-12-30 |
ATE488301T1 (en) | 2010-12-15 |
EP1744831A1 (en) | 2007-01-24 |
DE102004023466B4 (en) | 2008-11-13 |
WO2005110605A1 (en) | 2005-11-24 |
US7879214B2 (en) | 2011-02-01 |
EP1744831B8 (en) | 2011-09-07 |
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