EP1603678B1 - Procedes et dispositifs pour separer des particules dans un ecoulement de liquide - Google Patents

Procedes et dispositifs pour separer des particules dans un ecoulement de liquide Download PDF

Info

Publication number
EP1603678B1
EP1603678B1 EP04721159A EP04721159A EP1603678B1 EP 1603678 B1 EP1603678 B1 EP 1603678B1 EP 04721159 A EP04721159 A EP 04721159A EP 04721159 A EP04721159 A EP 04721159A EP 1603678 B1 EP1603678 B1 EP 1603678B1
Authority
EP
European Patent Office
Prior art keywords
particles
microsystem
compartment
electrodes
potential
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
EP04721159A
Other languages
German (de)
English (en)
Other versions
EP1603678A1 (fr
Inventor
Torsten Müller
Thomas Schnelle
Rolf Hagedorn
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Revvity Cellular Technologies GmbH
Original Assignee
Evotec OAI AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Evotec OAI AG filed Critical Evotec OAI AG
Publication of EP1603678A1 publication Critical patent/EP1603678A1/fr
Application granted granted Critical
Publication of EP1603678B1 publication Critical patent/EP1603678B1/fr
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Images

Classifications

    • 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

Definitions

  • the present invention relates to methods for separating particles in a fluidic microsystem, in particular under the action of electrophoresis, and fluidic microsystems adapted to carry out such methods.
  • FIGS. 10A, B Two conventional separation principles, which basically differ according to the type of electrical separation forces, are illustrated schematically in FIGS. 10A, B.
  • Figure 10A shows schematically the separation by means of negative dielectrophoresis (see, for example, DE 198 59 459).
  • a fluidic microsystem 100 ' particles having different dielectric properties flow through a first channel 30'.
  • an electrode arrangement 40 ' By means of an electrode arrangement 40 ', a field barrier extending transversely across the channel 30' is generated by application of high-frequency electric fields which, depending on the dielectric properties of the particles, has a permeable or laterally deflecting effect in cooperation with the flow forces.
  • Particles 22 'having a low dielectric constant (or conductivity) compared to the medium are deflected into an adjacent channel 30A', while particles 21 'having a higher dielectric constant (or conductivity) continue in channel 30'.
  • dielectrophoresis depends on the particle size (see T. Schnelle et al., In “Naturburgen” Bd., 83, 1996, pp. 172-176), even with the same dielectric Characteristics of a separation of the particles made according to the size.
  • Conventional dielectrophoretic particle separation may have disadvantages in terms of separation reliability, especially for particles with similar dielectric constants, and the complexity of the channel design.
  • the reliability of the separation may be limited especially in the separation of biological cells of the same type into different subtypes (eg macrophages, T lymphocytes, B lymphocytes).
  • Figure 10B illustrates an electrophoretic separation of particles, e.g. Molecules in a microstructured channel (see T. Pfohl et al., In “Physik Journal", Vol. 2, 2003, pages 35-40).
  • electrodes 41', 42 ' are arranged, which form an electrophoresis field when subjected to a DC voltage in the channel 30'.
  • the drift velocity of the molecules in the electrophoresis field depends on their molecular weight and charge. In the wider sections of the channel 30 ', the drift velocity of the larger molecules is lower, so that in the course of the separation first the small molecules and later the large molecules arrive at the end of the separation distance.
  • the electrophoretic separation in fluidic microsystems has the advantage that it can be dispensed with the use of a separation gel as in macroscopic electrophoresis.
  • the principle shown in FIG. 10B has the disadvantage that a separate microsystem with adapted geometrical parameters must be provided for each separation task and in particular each particle type.
  • Another disadvantage is that the separation takes place in the dormant liquid, because this is associated with a high expenditure of time and additional measures for adaptation to flow systems.
  • the combination of dielectrophoresis and electrophoresis on the closed field cage is limited to relatively large, single particles. Disadvantages may arise in the measurement of, for example, macromolecules, since in these the effect of the negative dielectrophoresis is significantly lower than that of the electrophoresis, so that it can lead to an undesired attachment of the macromolecules to the electrodes. Particle groups can not be measured with this technique, since all particles require their own correction movement. Separation of particles would also be hampered by a dipole-dipole effect (see T. Schnelle et al., "NaturBiben", Vol. 83, 1996, pp. 172-176), which promotes particle aggregation.
  • DE 198 59 459 also discloses the combination of alternating and direct voltages in fluidic microsystems for targeted cell fusion or fusion. In this technique, the effect of the DC voltage on the fusion or poration is limited, a particle separation is not provided.
  • the object of the invention is to provide improved methods for the separation of particles in liquid flows in fluidic microsystems, with which the disadvantages of conventional techniques are avoided.
  • Processes according to the invention are to be distinguished, in particular, by a broader field of application with a multiplicity of different particles and increased reliability in particle separation.
  • the object of the invention is also to provide improved microsystems for implementing such methods, in particular improved microfluidic separation devices, which are characterized by a simplified construction, a high reliability, a simplified control and a wide range of applications for various particles.
  • the present invention is based on the general technical teaching of the method and apparatus, at least one particle suspended in a liquid by means of a combined exercise of separation forces, on the one hand focusing dielectrophoretic separation forces and on the other hand distracting separation forces, such as electrophoretic separation forces to move in a state of continuous flow within the liquid, so relative to the flowing liquid.
  • the at least one particle can be directed into at least one separating device in the fluidic microsystem, depending on its geometric, electrical, magnetic or derived properties in a certain flow range during the pre-accession.
  • the flow region may comprise a specific flow path within the flow cross-section of the liquid or a downstream or downstream portion of the flow.
  • the movement of the particle into a particular flow area allows separation of particle mixtures during the continuous flow of the particle suspension, for example, through a group of multiple electrodes.
  • the release effect is based on the specific reaction of different particles to the different deflecting and focusing field effects.
  • a separation distance can be traversed, whereby the reliability of the targeted movement of individual particles can be increased, for example, to specific, preferably two, flow paths.
  • the effect of the electric fields can be tuned by adjusting the field characteristics (in particular frequency, voltage amplitudes, clock, etc.) to the parameters of the particles to be separated.
  • the invention enables a simplified construction of the electrophoretic separator, since no gels for embedding electrophoresis electrodes or special channel shapes are needed. Furthermore, a gas formation by suitable control of the electrodes in combination with the permanent Flow can be avoided.
  • the invention also has advantages particularly in terms of reliability and selectivity in particle separation into different flow paths, and high efficiency and high throughput of separation.
  • a separation of particles in a compartment in particular a channel of a fluidic microsystem, through which particles flow in the suspended state, wherein at least a portion of the particles or particles of at least one type under the action of a deflecting potential from the sample to be separated in a predetermined deflecting direction (first reference direction, for example to the edge of the compartment) are moved to the effect that simultaneously or temporally and / or spatially alternating under the action of an opposite potential by dielectrophoresis, in particular negative or positive dielectrophoresis an opposite movement of the particles (second reference direction , for example, away from the walls or as a rally in the canal center).
  • first reference direction for example to the edge of the compartment
  • particles with different electrical, magnetic or geometric properties experience the potential effects as separation forces in various ways, so that different effective forces (potential minima) form due to the combined application of the potentials to which the particles migrate.
  • the potential minima are z. B. in the flow cross-section of the liquid, so that a separation in the flow to different flow paths is possible.
  • the focusing, dielectrophoretic potential is preferably formed towards the center of the channel acting. If, in the channel cross-section, the electrodes are arranged essentially on a circular line, the focusing potential with respect to the flow direction in the channel can advantageously be formed radially symmetrically.
  • the particles which are preferably separated or separated from one another by the technique according to the invention generally comprise colloidal or individual particles with a diameter of z. B. 1 nm to 100 microns.
  • synthetic particles eg, latex beads, superparamagnetic particles, vesicles
  • biological particles eg, cell groups, cell constituents, cell debris, organelles, viruses
  • hybrid particles made of synthetic and biological, different synthetic or different are constructed biological particles, subjected to the separation process of the invention.
  • the distinction of the subtypes represents a particular advantage of the invention, since these are poorly distinguishable with conventional dielectrophoretic separation methods.
  • the selectivity is increased especially for cells of the same type.
  • the separation method can be advantageously used for cleaning a suspension sample with suspended biological material.
  • the material which, for example, is inhomogeneously composed after cultivation and, for example, complete cells, dead cells, living cells or fragments of cells, such as, for example, cells.
  • organelles cell residues or protein clumps can be purified by the method according to the invention.
  • the Unwanted fragments of cells can be removed from the microsystem via certain flow paths. An adverse effect on the following structural elements in the microsystem, such. As a clogging of channels by cell components can be avoided.
  • the deflecting potential can be generated by electrical, magnetic, optical, thermal and / or mechanical forces and thus adapted to a wide variety of applications and particle types.
  • Mechanical forces include, for example, forces transmitted by sound, additional currents or inertia.
  • the deflecting potential can in particular be given by a gravitational field, wherein according to the invention the movement of the particles in the focusing potential (by high-frequency electric fields) is superimposed with a sedimentation movement of the particles.
  • the deflecting separation forces comprise electrical forces under the effect of which the particles are drawn by electrophoresis from the liquid towards the edge thereof, there may be advantages in terms of the separation result.
  • the combination of electrophoresis and dielectrophoresis for particle separation can in particular have advantages in the separation of biological materials which, for example, react very differently to electrophoresis and dielectrophoresis depending on the material or particle size and can therefore be separated with high selectivity.
  • the DC fields for the electrophoretic particle movement can additionally be used for an electrical treatment of the particles.
  • biological cells can be lysed in static electric fields. Lysis involves an electrically induced change, for example destruction of the cells.
  • the lysis serves For example, the preparation of cell material for PCR procedures. Since the effect of the lysis is field-strength-dependent, it is provided according to a particularly preferred embodiment of the invention that certain cells are deflected from a cell mixture by electrophoresis in a flow region near the electrodes, where due to the smaller distance from the electrodes, the field strength is higher and thus the lysis is carried out simultaneously to the process of particle separation.
  • the selectivity can also be flexibly adjusted by a suitable AC voltage control.
  • the dielectric potential can be shaped differently in the case of negative dielectrophoresis.
  • the DC voltage control pH profiles that affect the electrophoretically or dielectrically effective potential.
  • the separating devices for generating the opposing potentials can advantageously be formed by a common unit.
  • the separator comprises electrodes which are disposed on walls of the channel and which are supplied with electric fields for the production of dielectrophoresis and electrophoresis. Advantages for the control of the separation may in particular arise if the electric fields comprise high-frequency AC components and DC components which are generated simultaneously or alternately.
  • the deflecting separation forces may comprise electrical forces which, like the focusing potential, are generated by high-frequency electric fields.
  • the deflection can thus also be generated by suitable dielectrophoretic forces formed by high-frequency electrical signals, z. B. sine or square wave signals are superimposed with suitable frequency components.
  • the deflecting and focusing potentials may be formed alternately in time in at least a portion of the channel.
  • the particles effectively have a potential that corresponds to the superposition of both potentials.
  • control of the at least one separating device can be simplified.
  • the two potentials can be generated alternately in different, successive sections of the channel.
  • the structure of the microsystem can be simplified.
  • a further separation according to the principle of the invention for example, a combined exercise of electrophoretic and dielectrophoretic field effect takes place.
  • advantageously hierarchical separation principles can be realized with a separation in coarse and subsequently in fine fractions.
  • sequence of several separation processes in the manner of a cascade into different fractions is not necessarily to the provision bound to separate compartments. Rather, the realization of the separation cascade with flow paths in a common, sufficiently wide channel of the microsystem is possible.
  • the flow in the microsystem can be directed so that particles pass through a separation stage several times, so that advantageously the separation result can be improved even more.
  • the detection comprises, for example, a known optical measurement (fluorescence measurement or transmitted light measurement) or a known impedance measurement.
  • control parameters of the deflecting and focusing potentials are adjusted so that improves the separation effect.
  • the effectiveness of the separation according to the invention can advantageously be increased if the particles first pass past a dielectrophoretic or hydrodynamic line-up element.
  • individual particles or a group of particles are strung on a particular flow path on which they pass at the separators, for example, the electrodes for performing dielectrophoresis and electrophoresis.
  • a pH gradient is generated in the channel of the microsystem in which the particle separation takes place, advantages for the separation effect can result. Due to the pH gradient, the effect of the distracting Potentials such. B. the electrophoretic cell particle movement location-dependent. This allows a particle deflection into different flow paths as a function of the particle position along the flow direction through the channel.
  • a particularly simple design of the microsystem results when the pH gradient is generated electrochemically using the electrodes, which are also used to form the DC field for electrophoresis.
  • Another advantage of the invention is that the particle separation can take place simultaneously in several spatial directions.
  • a plurality of deflecting potentials with different effective directions can be generated with the focusing potential, which is then preferably acting towards the center of the channel, in order to simultaneously separate the particles to be separated with respect to two different features, such as e.g. B. to separate dielectric and magnetic properties.
  • Another object of the invention is a fluidic microsystem, which is adapted to implement the method according to the invention and in particular comprises at least one separating device for exercising focusing dielectrophoretic separation forces and deflecting separation forces.
  • a fluidic microsystem with at least one compartment for example a channel for receiving a flowing liquid with suspended particles and a first separator for generating a deflecting, the particles in the first reference direction, for example, from the center of the flow pulling potential is in particular with a second separator equipped for generating at least one focusing, opposite potential. Under the action of high-frequency electric fields, the particles with the second separator become by dielectrophoresis from the lateral Walls of the channel and / or disposed thereon electrodes or other parts of separating devices repelled.
  • the first separating device is designed to generate electrical, magnetic, optical and / or mechanical forces. It comprises, for example, an electrode device with electrodes or electrode sections and in this case forms a common deflection unit with the second separation device.
  • the first separator comprises a magnetic field device, a laser or an ultrasound source.
  • the deflection unit preferably comprises electrodes, which are constructed like micro-electrodes known per se in fluidic microsystems.
  • the electrodes can be controlled alternately in time.
  • the electrodes for combined dielectrophoresis and electrophoresis are preferably arranged on insides of the walls of the compartment. In this design, there may be advantages in terms of the effectiveness of the field effect.
  • the separation devices can act alternately or temporally and / or spatially alternately, so that particles are directed to different flow paths depending on the effective time potentials, it is advantageously possible for the first and second separation devices to be separate in different, consecutive Sections of the compartment are arranged.
  • the separation devices comprise, for example, electrode sections which can each be activated for dielectrophoresis or electrophoresis.
  • FIG. 1 and 2 show a detail of an inventive fluidic microsystem 100 in an enlarged schematic plan view and cross-sectional view.
  • the microsystem 100 includes a channel 30 bounded by the lateral channel walls 31, 32, the channel bottom 33 (top view in FIG. 1) and the top surface 34.
  • electrodes 40 are formed as a separator.
  • funnel electrodes 51, 52 of a dielectric alignment element 50 are provided.
  • the structure of the microsystem 100 and the formation of the electrodes and their electrical connection are known per se from microsystem technology.
  • the channel has, for example, a width of approx. 400 ⁇ m and a height of approx. 40 microns (these ratios are not shown to scale in the figures).
  • the lateral electrode spacing in the planes of the channel bottom 33 and the top surface 34 is, for example, 70 ⁇ m, while the vertical distance between the opposing electrodes corresponds to the channel height rd. 40 microns.
  • the electrodes 40 comprise straight electrode strips extending longitudinally of the channel 30, i. extend in the flow direction through the channel.
  • the electrodes 40 are divided into individual electrode segments 41, 42,... In each case a group of electrode segments forms an electrode section, which can be controlled separately.
  • Each segment has a width of about 50 microns and in the flow direction a length of z. B. 1000 microns.
  • Each electrode section is connected to a controller 70 (shown here only for the electrodes 41, 42).
  • the control device 70 is designed to act on the electrodes 40 with voltages such that the passing particles in an electrode section (for example 45-48, see FIG. 2) are exposed to repulsion from the electrodes by means of negative dielectrophoresis and / or electrophoretic drift motion perpendicular to the flow direction become.
  • the controller includes an AC generator 71 and / or a DC generator 72 connected to the electrodes.
  • the AC generator 71 may be equipped with an actuator, with which the amplitudes of high-frequency AC voltages can be adjusted at the electrodes.
  • the suspension liquid 10 (carrier liquid) flows with particles 20 through the channel 30.
  • the flow rate of the suspension liquid 10, which can be adjusted with a syringe pump is z. B. 300 microns / s.
  • the particles 20 are preferably lined up with the dielectric line-up element 50.
  • a hydrodynamic Auf Herbertlement be provided, in which the particles 20 are focused with additional enveloping streams.
  • the potentials acting on the particles are schematically illustrated in FIG.
  • a DC voltage field is generated which generates a potential P1 which drops transversely to the flow cross-section. Particles experience in the potential P1 an outward force (deflecting potential, deflection direction transverse to the flow direction).
  • the high-frequency activation of the electrodes generates an opposite, inwardly directed, focusing potential profile P2a or P2b.
  • the negative dielectrophoresis is based on a particle polarization, which has a stronger effect on the large particles than on the small particles. In the high-frequency field, therefore, the large particles 21 experience the potential P2a and the small particles 22 the shallower potential P2b.
  • Electrodes in FIG. 2 Phase of the high frequency alternating voltage Potential DC voltage 47 0 ° Dimensions 48 180 ° pulse 45 0 ° pulse 46 180 ° Dimensions
  • the electrode drive can take place, for example, according to the following scheme (rotating electric field): Electrodes in FIG. 2 Phase of the high frequency alternating voltage Potential DC voltage 47 0 ° Dimensions 48 90 ° pulse 45 270 ° pulse 46 180 ° Dimensions
  • Figure 1 shows schematically a separator 40A (shown in phantom)
  • the separator 40A provided in or outside the duct wall is, for example, a magnetic device for applying magnetic forces, a laser device for applying optical forces analogous to the principle the laser tweezer or a sound source for the exercise of mechanical forces z. B. by ultrasound.
  • Figure 4 shows features of modified embodiments of the invention. Notwithstanding Figure 1 may be provided that the flow path 11 is displaced from the center of the channel 30 to the outside, in which the potential minimum of the dielectrophoresis is shifted by appropriate asymmetric activation of the electrodes 40. Furthermore, it can be provided that the flow paths 11, 12 open into separate compartments 35, 36 of the channel 30, which are separated from one another by channel walls or (as illustrated) by an electric field barrier. The electric field barrier is created by at least one barrier on the electrode 60 which extends in the channel direction.
  • electrodes 41, 42 for electrophoresis are located in a channel 30 laterally on the channel walls 31, 32 and / or on the bottom surface 33, and at least one electrode 43 for dielectrophoresis is centrally located.
  • the electrode 43 is provided in a manner known per se with an electrically insulating passivation layer 43a.
  • the passivation layer 43a has two functions. First, it prevents field loss of the DC field for electrophoresis, second, it prevents permanent attachment and thus possibly associated denaturation of particles or electrochemical reactions at the electrodes.
  • the electrodes 41, 42 and 43 are each connected to a DC voltage source and an AC voltage source.
  • the channel edge can be realized by porous materials (eg hollow fibers). This makes it possible to impose additional external chemical gradients (eg a pH profile).
  • the at least one electrode 43 and the electrodes 41, 42 may be arranged offset in the flow direction for electrophoresis.
  • micro-objects for example macromolecules
  • the central electrode 43 For particle separation, flushed-in micro-objects (for example macromolecules) are pulled to the central electrode 43 by positive dielectrophoresis. Simultaneously or with alternating activation of the electrodes, the microobjects are drawn by electrophoresis to the edge of the channel 30.
  • the separation is based on the principles described above of a differential effect of the combination of dielectrophoresis and electrophoresis on the different particles.
  • the following procedure can be realized.
  • the particles are first collected at the central electrode 43.
  • the lateral flow 10 is stopped by the channel 30 and carried out a separation of the micro-objects via electrophoresis.
  • the flow 10 is continued.
  • the essential advantage of the interruption of the flow transport through the channel which is optionally provided according to the invention during the electrophoresis, is that an increased selectivity of the electrophoresis can be achieved by the previously defined start conditions.
  • electrodes 43.1 to 43.5 are provided for dielectrophoresis, the structure shown in FIG. 6 results.
  • the electrodes 41, 42 for electrophoresis and on the bottom surface of the electrodes 43.1 to 43.5 for dielectrophoresis (electrical leads not shown).
  • On the top surface are dielectrophoresis electrodes in the same number and arrangement as the electrodes 43.1 to 43.5.
  • the electrodes 43.1 to 43.5 are subjected to signals which are phase-shifted between adjacent electrodes (for example 43.1, 43.2) by 180 ° and for in-phase electrodes (for example 43.1 and opposite electrode on the top surface) in phase are.
  • the particles 20 sparged with the flow 10 include two types, one type of which is not addressed by electrophoresis.
  • the particles 20 first arrange themselves dielectrophoretically (negative dielectrophoresis) in the intermediate space of the electrodes standing one above the other (hidden in the top view). It is only when passing the electrophoretic field that the particles of one type are deflected, while the other type remains unaffected.
  • many optionally passivated electrodes 43.1 to 43.11 for dielectrophoresis are arranged between the electrodes 41, 42 for electrophoresis.
  • On the top surface are dielectrophoresis electrodes in the same number and arrangement as the electrodes 43.1 to 43.11.
  • the first dielectrophoresis electrode pair 43.1, 43.2 is provided with a dielectric line-up element 50 for increasing the selectivity.
  • the DC electrophoresis field is aligned parallel to the flow direction of the liquid 10 (see arrow) through the compartment 30.
  • the particles 20 arrange between the electrodes (negative dielectrophoresis).
  • the dielectrophoresis electrodes form a periodic i.
  • the asymmetric modulation of the dielectrophoresis fields means that alternately higher or lower field strengths are set between adjacent electrode strips of the array 43.1 to 43.11.
  • the electrophoresis potential between the electrodes 41, 42 is not kept constant in time, but switched periodically or randomly.
  • Brown's ratchet a highly sensitive separation according to the principle of the so-called Brown's ratchet ("Brownian ratchet” or Rüttelratsche, see H. Linke et al., Physikalische full Bd. 56, No. 5, 2000, pp 45-47) realize.
  • Brown's ratchet the migration speed of particles through Brownian motion is highly dependent on particle size.
  • the separation takes place in different flow sections in the flow direction depending on the different migration velocities of the particles.
  • a particular advantage of this procedure is that the separation over several adjustable parameters can be sensitively controlled by the superposition of Brownian motion, electrophoresis and dielectrophoresis.
  • This embodiment of the invention is particularly suitable for molecular separation (eg, separation of DNA molecules or DNA fragments that are all negatively charged in a physiological environment).
  • the input channel with the array element 50 should be centered on the array of dielectrophoresis electrodes so that different charge objects are electrophoretically moved in different directions.
  • planar structures can also realize asymmetric potential for positive dielectrophoresis, z. B. by applying asymmetric, so relative to the channel longitudinal direction, for example, different thick passivation layers.
  • FIG. 8 illustrates, like FIG. 2, a cross-sectional view of a fluidic microsystem 100 with four electrodes 45-48. With these electrodes, a focusing potential is generated whose potential minimum lies in the middle of the channel.
  • a first electrical potential acting in the x direction is generated for an electrophoretic field effect, and additionally in the y direction a magnetic field gradient for forming a second deflecting potential.
  • the magnetic field gradient is with a magnetic field generating element 49th formed, for example, a permanent magnet or a liquid-insulated, current-carrying conductor comprises.
  • the magnetic field generating element may be arranged at a distance from the channel.
  • This embodiment of the invention is used, for example, for the separation of latex-coated, superparamagnetic particles with the aim of obtaining fractions with high monodispersity.
  • the graph in FIG. 9 illustrates the dielectrophoretic force f diel normalized to the respective volume, which acts on a particle in the alternating field as a function of the frequency of the alternating field.
  • the symbolically illustrated electrodes are arranged analogously to FIG. 1 and are applied alternately or superimposed with a signal which contains frequency components below 100 kHz and above 1 MHz.
  • the low-frequency and higher-frequency signal components are generated, for example, with equal amplitudes in time-quadratic mean, but different phase relationships illustrated in the image feeds.
  • the higher-frequency signal focuses the particles by negative dielectrophoresis towards the center of the channel.
  • the low-frequency signal acts as a function of the particle size by positive or negative dielectrophoresis, which is superimposed on the focusing effect of the higher-frequency signal.
  • the smaller particles are deflected to the top left, while the larger particles (eg 5 ⁇ m) collect on a diagonal line at the bottom right.
  • Corresponding Particles of different sizes enter different flow paths within the flow through the channel.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Electrochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Electrostatic Separation (AREA)
  • Cyclones (AREA)
  • Physical Or Chemical Processes And Apparatus (AREA)

Claims (33)

  1. Procédé pour la séparation de particules (20, 21, 22) dans un compartiment (30) d'un microsystème fluide (100), comprenant les étapes :
    - de déplacement d'un liquide (10) où les particules (20, 21, 22) sont suspendues avec une direction d'écoulement définie au travers du compartiment (30), et
    - de génération d'un potentiel de dérivation où au moins une partie des particules (20, 21, 22) est déplacée dans une direction de dérivation, par rapport au liquide,
    caractérisé par les autres étapes :
    - de génération d'au moins un potentiel de focalisation, de manière à déplacer par diélectrophorèse sous l'effet des champs électriques à haute fréquence au moins une partie des particules à l'opposé de la direction de dérivation, par rapport au liquide, et
    - de conduction de particules présentant différentes propriétés électriques, magnétiques ou géométriques vers différentes zones d'écoulement (11, 12) du liquide.
  2. Procédé selon la revendication 1, où la direction de dérivation diffère de la direction d'écoulement et présente une composante transversale à la direction d'écoulement.
  3. Procédé selon la revendication 2, où la direction de dérivation s'étend perpendiculairement à la direction d'écoulement jusqu'à au moins une des parois latérales du compartiments, où le potentiel de dérivation est généré par des forces électriques, magnétiques, optiques, thermiques et/ou mécaniques, et où les zones d'écoulement comprennent des voies d'écoulement (11, 12) correspondant à différents minima de potentiel formés pour les particules correspondantes par superposition des potentiels de dérivation et de focalisation pendant la traversée du compartiment en moyenne temporelle.
  4. Procédé selon la revendication 3, où le potentiel de dérivation est formé par un champ de tension continue sous l'effet duquel les particules sont attirées par électrophorèse vers au moins une des parois latérales du compartiment (30).
  5. Procédé selon la revendication 4, où les particules comprennent des cellules biologiques dont au moins une partie est lysée sous l'effet du champ de tension continue.
  6. Procédé selon la revendication 3, où le liquide (10) comprend une suspension de matière biologique contenant des cellules biologiques et des composants de cellules, les cellules étant séparées des composants de cellules sous l'effet du champ de tension continue.
  7. Procédé selon la revendication 4, où des électrodes (40) sont disposées sur les parois (31-34) du compartiment (30), lesquelles sont exposées à des champs électriques pour la génération de la diélectrophorèse et de l'électrophorèse.
  8. Procédé selon l'une des revendications précédentes au moins, où les potentiels de dérivation et de focalisation sont générés en alternance temporelle dans au moins une partie du compartiment (30) ou en alternance géométrique dans différentes parties successives du compartiment (30).
  9. Procédé selon les revendications précédentes 5 et 6, où les champs électriques comprennent des parts de tension alternative et des parts de tension continue, générées simultanément ou en alternance.
  10. Procédé selon la revendication 7, où une pluralité de potentiels de focalisation est générée au moyen d'un réseau d'électrodes (43.1 à 43.11) entre les deux électrodes (41, 42), les particules étant conduites sur les différentes voies d'écoulement (11, 12) en fonction de leurs propriétés électriques ou géométriques.
  11. Procédé selon l'une des revendications précédentes 2 à 9 au moins, où les particules (20, 21, 22) sont conduites sur au moins deux voies d'écoulement (11, 12) séparées.
  12. Procédé selon la revendication 11, où les deux voies d'écoulement (11, 12) au moins débouchent dans d'autres compartiments (35, 36) séparés du microsystème (100).
  13. Procédé selon la revendication 12, où au moins deux voies d'écoulement (11, 12) débouchent dans des compartiments (35, 36) séparés du microsystème (100), lesquels sont séparés par des parois de compartiment ou des barrières électriques (60).
  14. Procédé selon la revendication 1, où la direction de dérivation s'étend parallèlement à la direction d'écoulement, où plusieurs potentiels de focalisation sont générés, lesquels sont asymétriquement modulés parallèlement à la direction de dérivation, et où les particules traversent le potentiel de dérivation à des vitesses différentes.
  15. Procédé selon l'une des revendications précédentes au moins, où les particules (20, 21, 22) passent devant les électrodes en s'écoulant contre un élément chauffant (50) diélectrophorétique ou hydrodynamique.
  16. Procédé selon l'une des revendications précédentes au moins, où un gradient de pH est généré dans le canal (30).
  17. Procédé selon la revendication 16, où le gradient de pH est généré par des champs de tension électrique continue prévus pour la séparation électrophorétique des particules.
  18. Procédé selon l'une des revendications précédentes au moins, où une détection des particules a lieu après conduction des particules sur les différentes voies d'écoulement (11, 12).
  19. Procédé selon l'une des revendications précédentes au moins, où le potentiel de dérivation et le potentiel de focalisation sont formés par plusieurs tensions alternatives superposées avec différentes fréquences.
  20. Procédé selon l'une des revendications précédentes au moins, où au moins deux potentiels de dérivation sont générés avec différentes directions de dérivation.
  21. Microsystème fluidique, comprenant :
    - au moins un compartiment (30) où s'écoule un liquide avec des particules (20, 21, 22) dans une direction d'écoulement définie, et
    - un premier dispositif de séparation pour la génération d'un potentiel de dérivation où les particules (20, 21, 22) sont déplacées dans une direction de dérivation,
    caractérisé par
    - un deuxième dispositif de séparation avec des électrodes (40) pour la génération d'au moins un potentiel de focalisation, de manière à déplacer par diélectrophorèse les particules à l'opposé de la direction de dérivation.
  22. Microsystème selon la revendication 21, où la direction de dérivation diffère de la direction d'écoulement.
  23. Microsystème selon la revendication 21 ou 22, où le premier dispositif de séparation est équipé pour la génération de forces électriques, magnétiques, optiques et/ou mécaniques.
  24. Microsystème selon la revendication 23, où le premier dispositif de séparation comprend des électrodes pour électrophorèse, un dispositif de champ magnétique, un laser ou une source d'ultrasons.
  25. Microsystème selon l'une des revendications précédentes 21 à 24 au moins, où le premier et le deuxième dispositif de séparation sont séparément disposés dans différentes parties successives du compartiment (30).
  26. Microsystème selon la revendication 21, 23 ou 25, où le premier et le deuxième dispositif de séparation forment une unité de dérivation commune comprenant les électrodes (40).
  27. Microsystème selon la revendication 26, où l'unité de dérivation est commandable avec des tensions alternatives et continues en alternance temporelle.
  28. Microsystème selon la revendication 24, où un réseau d'électrodes (43.1 à 43.11) en barres d'électrodes, individuellement commandables avec des tensions alternatives à haute fréquence, est disposé entre les électrodes pour électrophorèse (41, 42).
  29. Microsystème selon la revendication 21, où la direction de dérivation s'étend parallèlement à la direction d'écoulement.
  30. Microsystème selon l'une des revendications précédentes 21 à 29 au moins, où les électrodes (40) sont disposées sur des faces intérieures des parois du compartiment (30).
  31. Microsystème selon l'une des revendications précédentes 21 à 30 au moins, où le compartiment (30) débouche dans des compartiments séparés (35, 36) du microsystème (100).
  32. Microsystème selon la revendication 31, où les compartiments (35, 36) du microsystème (100) sont séparés par des parois de compartiment ou des barrières électriques (60).
  33. Microsystème selon l'une des revendications précédentes 21 à 32 au moins, où un élément chauffant (50) diélectrophorétique ou hydrodynamique est disposé dans le compartiment (30) en amont des dispositifs de séparation.
EP04721159A 2003-03-17 2004-03-17 Procedes et dispositifs pour separer des particules dans un ecoulement de liquide Expired - Lifetime EP1603678B1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10311716A DE10311716A1 (de) 2003-03-17 2003-03-17 Verfahren und Vorrichtung zur Trennung von Partikeln in einer Flüssigkeitsströmung
DE10311716 2003-03-17
PCT/EP2004/002774 WO2004082840A1 (fr) 2003-03-17 2004-03-17 Procedes et dispositifs pour separer des particules dans un ecoulement de liquide

Publications (2)

Publication Number Publication Date
EP1603678A1 EP1603678A1 (fr) 2005-12-14
EP1603678B1 true EP1603678B1 (fr) 2006-07-19

Family

ID=32980607

Family Applications (1)

Application Number Title Priority Date Filing Date
EP04721159A Expired - Lifetime EP1603678B1 (fr) 2003-03-17 2004-03-17 Procedes et dispositifs pour separer des particules dans un ecoulement de liquide

Country Status (5)

Country Link
US (2) US8262883B2 (fr)
EP (1) EP1603678B1 (fr)
AT (1) ATE333323T1 (fr)
DE (2) DE10311716A1 (fr)
WO (1) WO2004082840A1 (fr)

Families Citing this family (47)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2524646C (fr) 2003-05-08 2012-02-21 The University Court Of The University Of St Andrews Fractionnement de particules
US7704362B2 (en) * 2005-03-04 2010-04-27 Wisconsin Alumni Research Foundation Apparatus for transport and analysis of particles using dielectrophoresis
DE102005012128A1 (de) * 2005-03-16 2006-09-21 Evotec Technologies Gmbh Mikrofluidisches System und zugehöriges Ansteuerverfahren
DE102005046657B4 (de) * 2005-09-29 2008-10-09 Bruker Daltonik Gmbh Stationäres Trennsystem für Gemischkomponenten
US20070105206A1 (en) * 2005-10-19 2007-05-10 Chang Lu Fluidic device
CN100457239C (zh) * 2005-12-30 2009-02-04 财团法人工业技术研究院 多样品微流体介电泳分离装置
GB0618605D0 (en) 2006-09-21 2006-11-01 Univ St Andrews Optical sorting
GB0618606D0 (en) 2006-09-21 2006-11-01 Univ St Andrews Optical sorting
DE102006052925A1 (de) * 2006-11-09 2008-05-15 Evotec Technologies Gmbh Feldkäfig und zugehöriges Betriebsverfahren
US8698031B2 (en) * 2007-04-17 2014-04-15 Dynamic Connections, Llc Separation and manipulation of a chiral object
US7935906B2 (en) * 2007-04-17 2011-05-03 Dynamic Connections, Llc Separation and manipulation of a chiral object
EP2027929A3 (fr) * 2007-08-20 2013-03-27 Olympus Corporation Dispositif de séparation cellulaire et procédé de séparation cellulaire
US8740600B1 (en) * 2007-10-09 2014-06-03 Isopur Technologies, Inc. Apparatus for agglomerating particles in a non-conductive liquid
KR101338349B1 (ko) * 2007-11-30 2013-12-06 연세대학교 산학협력단 미세입자 분리 장치 및 이의 제작 방법
JP2011516867A (ja) * 2008-04-03 2011-05-26 ザ レジェンツ オブ ザ ユニヴァースティ オブ カリフォルニア 細胞、小胞、ナノ粒子およびバイオマーカーを分離および単離するためのエキソビボの多次元システム
KR101023040B1 (ko) * 2008-11-13 2011-03-24 한국항공대학교산학협력단 고속 입자분리 장치 및 그 방법
DE102011050254A1 (de) * 2011-05-10 2012-11-15 Technische Universität Dortmund Verfahren zur Separation polarisierbarer Biopartikel
CA2845713A1 (fr) * 2011-08-19 2013-02-28 David Martin Morrow Separation par dielectrophorese de gradients (grads) avec therapie lumineuse concomitante
EP2754709A4 (fr) * 2011-09-07 2015-01-21 Panasonic Healthcare Co Ltd Cellule de mesure de quantité de microorganismes et dispositif de mesure de quantité de microorganismes contenant celle-ci
JP5924276B2 (ja) * 2012-04-03 2016-05-25 ソニー株式会社 流路デバイス、粒子分取装置及び粒子分取方法
CA2870160C (fr) 2012-04-16 2021-08-24 Biological Dynamics, Inc. Preparation d'un echantillon d'acide nucleique
US8932815B2 (en) 2012-04-16 2015-01-13 Biological Dynamics, Inc. Nucleic acid sample preparation
US9192944B2 (en) * 2012-09-30 2015-11-24 Arizona Board Of Regents, A Body Corporate Of The State Of Arizona Acting For And On Behalf Of Arizona State University Methods, systems and apparatus for size-based particle separation
US9731293B2 (en) * 2012-10-03 2017-08-15 The United States Of America, As Represented By The Secretary Of The Navy Paired laser and electrokinetic separation, manipulation, and analysis device
US20160025610A1 (en) * 2013-03-26 2016-01-28 Sony Corporation Measurement apparatus and measurement method
KR101501983B1 (ko) * 2013-06-05 2015-03-13 한국항공대학교산학협력단 음의 유전 영동력 기반의 고효율 다단 세포 분리장치
US10954546B2 (en) * 2013-09-24 2021-03-23 Ait Austrian Institute Of Technology Gmbh Method for pathogen isolation
US10928404B2 (en) 2014-02-26 2021-02-23 The Brigham And Women's Hospital, Inc. System and method for cell levitation and monitoring
US20150273231A1 (en) * 2014-03-31 2015-10-01 Electronics And Telecommunications Research Institute Plasma system
WO2015157217A1 (fr) 2014-04-08 2015-10-15 Biological Dynamics, Inc. Dispositifs améliorés pour la séparation de matériaux biologiques
US10657869B2 (en) 2014-09-10 2020-05-19 E Ink Corporation Methods for driving color electrophoretic displays
EP3191892B1 (fr) 2014-09-10 2020-01-01 E Ink Corporation Dispositifs d'affichage électrophorétiques colorés
US20160327549A1 (en) * 2015-05-04 2016-11-10 Biological Dynamics, Inc. Particle based immunoassay with alternating current electrokinetics
AU2017237187B2 (en) 2016-03-24 2022-12-08 Biological Dynamics, Inc. Disposable fluidic cartridge and components
US10629833B2 (en) * 2016-11-21 2020-04-21 Northwestern University Flashing ratchets
CN106323727B (zh) * 2016-11-21 2023-07-14 大连海事大学 一种基于微通道内液体拉力效应的颗粒分离装置及方法
US10596567B2 (en) * 2017-03-27 2020-03-24 International Business Machines Corporation Microfluidic ratchets for displacing particles
IL270445B1 (en) 2017-05-08 2024-02-01 Biological dynamics inc Methods and systems for processing information on tested material
CN107262288B (zh) * 2017-07-27 2023-03-21 重庆科技学院 带有金属颗粒的静电式净化装置
CN107442285B (zh) * 2017-07-27 2023-03-24 重庆科技学院 静电式滤清装置
EP3727693A4 (fr) 2017-12-19 2021-08-25 Biological Dynamics, Inc. Procédés et dispositifs de détection d'analytes multiples à partir d'un échantillon biologique
WO2019195196A1 (fr) 2018-04-02 2019-10-10 Biological Dynamics, Inc. Matériaux diélectriques
US11454583B2 (en) 2019-12-27 2022-09-27 Imec Vzw Field-array free flow fractionation
EP3842146A1 (fr) * 2019-12-27 2021-06-30 Imec VZW Procédé de séparation continue des composants d'un échantillon
US11846863B2 (en) 2020-09-15 2023-12-19 E Ink Corporation Coordinated top electrode—drive electrode voltages for switching optical state of electrophoretic displays using positive and negative voltages of different magnitudes
AU2021345023B2 (en) 2020-09-15 2023-12-21 E Ink Corporation Four particle electrophoretic medium providing fast, high-contrast optical state switching
WO2022060700A1 (fr) 2020-09-15 2022-03-24 E Ink Corporation Tensions de commande améliorées pour affichages électrophorétiques en couleurs évolués et affichages à tensions de commande améliorées

Family Cites Families (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SE339122B (fr) * 1968-05-10 1971-09-27 Lkb Produkter Ab
DE4127405C2 (de) 1991-08-19 1996-02-29 Fraunhofer Ges Forschung Verfahren zur Trennung von Gemischen mikroskopisch kleiner, in einer Flüssigkeit oder einem Gel suspendierter dielektrischer Teilchen und Vorrichtung zur Durchführung des Verfahrens
DE19500683B4 (de) * 1994-12-10 2007-03-22 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Trapping von Molekülen und Mikropartikeln in Feldkäfigen
DE19540683A1 (de) 1995-11-01 1997-05-07 Behr Gmbh & Co Wärmeüberträger zum Kühlen von Abgas
US5993630A (en) * 1996-01-31 1999-11-30 Board Of Regents The University Of Texas System Method and apparatus for fractionation using conventional dielectrophoresis and field flow fractionation
DK0925494T3 (da) 1996-09-04 2002-07-01 Scandinavian Micro Biodevices Mikrostrømningssystem til partikelseparation og analyse
EP1089824B1 (fr) * 1998-06-26 2003-11-05 Evotec OAI AG Dispositif a electrodes destine a la deviation electrophoretique de particules
JP2000061472A (ja) * 1998-08-18 2000-02-29 Kurita Water Ind Ltd 水中微粒子の除去方法および装置
DE19859459A1 (de) 1998-12-22 2000-06-29 Evotec Biosystems Ag Mikrosysteme zur Zellpermeation und Zellfusion
AU4773400A (en) * 1999-05-28 2000-12-18 Leisamon (Israel), Ltd. Methods and apparatus for nonlinear mobility electrophoresis separation
DE19952322C2 (de) 1999-10-29 2002-06-13 Evotec Ag Verfahren und Vorrichtung zur Partikeltrennung
DE60130052T2 (de) * 2000-04-13 2008-05-15 Wako Pure Chemical Industries, Ltd. Elektroden-Bau für dielektrophoretische Anordnung und dielektrophoretische Trennung
CA2413634A1 (fr) * 2000-06-14 2001-12-20 Peter R. C. Gascoyne Procede et dispositif permettant de combiner les manipulations par magnetophorese et dielectrophorese dans le cas de melanges d'analysats
US20020121443A1 (en) * 2000-11-13 2002-09-05 Genoptix Methods for the combined electrical and optical identification, characterization and/or sorting of particles
DE10136275C1 (de) 2001-07-25 2002-12-12 Fraunhofer Ges Forschung Verfahren und Vorrichtung zur Probentrennung
US6783647B2 (en) * 2001-10-19 2004-08-31 Ut-Battelle, Llc Microfluidic systems and methods of transport and lysis of cells and analysis of cell lysate
US7156970B2 (en) * 2003-06-12 2007-01-02 Palo Alto Research Center Incorporated Distributed multi-segmented reconfigurable traveling wave grids for separation of proteins in gel electrophoresis
WO2007102839A2 (fr) * 2005-10-27 2007-09-13 Applera Corporation Separation optoelectronique de biomolecules

Also Published As

Publication number Publication date
DE502004000991D1 (de) 2006-08-31
DE10311716A1 (de) 2004-10-14
EP1603678A1 (fr) 2005-12-14
US8262883B2 (en) 2012-09-11
ATE333323T1 (de) 2006-08-15
US9149813B2 (en) 2015-10-06
US20060289341A1 (en) 2006-12-28
WO2004082840A1 (fr) 2004-09-30
US20120305398A1 (en) 2012-12-06

Similar Documents

Publication Publication Date Title
EP1603678B1 (fr) Procedes et dispositifs pour separer des particules dans un ecoulement de liquide
EP1089824B1 (fr) Dispositif a electrodes destine a la deviation electrophoretique de particules
EP1141264B1 (fr) Microsystemes pour la permeation cellulaire et la fusion cellulaire
EP1069955B1 (fr) Procede et dispositif permettant de manipuler des microparticules contenues dans des ecoulements fluidiques
EP0718038B1 (fr) Appareil pour la séparation de mélanges de particules diélectriques de taille microscopique suspendues dans un fluide ou un gel
DE19544127C1 (de) Verfahren und Vorrichtung zur Erzeugung von Resonanzerscheinungen in Partikelsuspensionen und ihre Verwendung
DE60014391T2 (de) Beeinflussung von partikeln in flüssigen medien
EP1286774B1 (fr) Dispositif et procede permettant de manipuler de petites quantites de matiere
DE60019761T2 (de) Apparat und verfahren für dielektrophoresis
WO2002082053A2 (fr) Procede et dispositif pour manipuler de faibles quantites de liquide et/ou des particules contenues dans ce liquide
EP1140343B1 (fr) Procede et dispositif pour deplacer des liquides par convection dans des microsystemes
EP1335198A1 (fr) Composant microfluidique et procédure pour le triage de particules dans un fluide
EP1089823A1 (fr) Dispositif a electrodes destine a la production de barrieres de champ fonctionnelles dans des microsystemes
WO2007039209A1 (fr) Procede et dispositif de manipulation de particules en sedimentation
DE102009028493A1 (de) Mikrofluidische Zelle
EP1525449A1 (fr) Mesure d'impedance dans un microsysteme fluidique
DE19860118C1 (de) Elektrodenanordnungen zur Erzeugung funktioneller Feldbarrieren in Mikrosystemen
DE102009028496A1 (de) Mikrofluidische Zelle
EP1744831B8 (fr) Procedes et dispositif pour recueillir des particules en suspension
EP1858645A1 (fr) Systeme microfluidique et procede de commande correspondant
DE19500683B4 (de) Trapping von Molekülen und Mikropartikeln in Feldkäfigen
DE19860117A1 (de) Elektrodenanordnung zur dielektrophoretischen Partikelablenkung
EP1279953A2 (fr) Dispositif et méthode de séparation au sein d'échantillons
DE10117771A1 (de) Verfahren und Vorrichtung zur Manipulation kleiner Flüssigkeitsmengen und/oder darin enthaltener Teilchen
DE102018210993A1 (de) Vorrichtung zum simultanen dielektrophoretischen Einfang von mehreren Teilchen verschiedener Art

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20050912

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL LT LV MK

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

DAX Request for extension of the european patent (deleted)
AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PL PT RO SE SI SK TR

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT;WARNING: LAPSES OF ITALIAN PATENTS WITH EFFECTIVE DATE BEFORE 2007 MAY HAVE OCCURRED AT ANY TIME BEFORE 2007. THE CORRECT EFFECTIVE DATE MAY BE DIFFERENT FROM THE ONE RECORDED.

Effective date: 20060719

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20060719

Ref country code: NL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20060719

Ref country code: PL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20060719

Ref country code: IE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20060719

Ref country code: SI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20060719

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20060719

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20060719

Ref country code: CZ

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20060719

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

Free format text: NOT ENGLISH

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

Free format text: LANGUAGE OF EP DOCUMENT: GERMAN

REF Corresponds to:

Ref document number: 502004000991

Country of ref document: DE

Date of ref document: 20060831

Kind code of ref document: P

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BG

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20061019

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20061019

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20061019

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20061030

RAP2 Party data changed (patent owner data changed or rights of a patent transferred)

Owner name: EVOTEC TECHNOLOGIES GMBH

REG Reference to a national code

Ref country code: CH

Ref legal event code: PUE

Owner name: EVOTEC TECHNOLOGIES GMBH

Free format text: EVOTEC OAI AG#SCHNACKENBURGALLEE 114#22525 HAMBURG (DE) -TRANSFER TO- EVOTEC TECHNOLOGIES GMBH#MEROWINGERPLATZ 1A#40225 DUESSELDORF (DE)

Ref country code: CH

Ref legal event code: NV

Representative=s name: RITSCHER & PARTNER AG

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20061219

NLT2 Nl: modifications (of names), taken from the european patent patent bulletin

Owner name: EVOTEC TECHNOLOGIES GMBH

Effective date: 20061108

NLV1 Nl: lapsed or annulled due to failure to fulfill the requirements of art. 29p and 29m of the patents act
GBT Gb: translation of ep patent filed (gb section 77(6)(a)/1977)

Effective date: 20070103

REG Reference to a national code

Ref country code: GB

Ref legal event code: 732E

REG Reference to a national code

Ref country code: IE

Ref legal event code: FD4D

EN Fr: translation not filed
PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

26N No opposition filed

Effective date: 20070420

BERE Be: lapsed

Owner name: EVOTEC TECHNOLOGIES GMBH

Effective date: 20070331

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20070331

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20070331

REG Reference to a national code

Ref country code: CH

Ref legal event code: PCAR

Free format text: RITSCHER & PARTNER AG;RESIRAIN 1;8125 ZOLLIKERBERG (CH)

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20070511

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20061020

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: AT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20070317

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20060719

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20060719

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20060719

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20070317

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: TR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20060719

Ref country code: HU

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20070120

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DE

Payment date: 20100527

Year of fee payment: 7

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: CH

Payment date: 20100525

Year of fee payment: 7

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GB

Payment date: 20100525

Year of fee payment: 7

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

GBPC Gb: european patent ceased through non-payment of renewal fee

Effective date: 20110317

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20111001

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20110331

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20110331

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GB

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20110317

REG Reference to a national code

Ref country code: DE

Ref legal event code: R119

Ref document number: 502004000991

Country of ref document: DE

Effective date: 20111001