EP1973660A1 - Système microfluidique et procédé d'exploitation - Google Patents

Système microfluidique et procédé d'exploitation

Info

Publication number
EP1973660A1
EP1973660A1 EP07702840A EP07702840A EP1973660A1 EP 1973660 A1 EP1973660 A1 EP 1973660A1 EP 07702840 A EP07702840 A EP 07702840A EP 07702840 A EP07702840 A EP 07702840A EP 1973660 A1 EP1973660 A1 EP 1973660A1
Authority
EP
European Patent Office
Prior art keywords
particles
microfluidic system
operating method
field
feldkafige
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.)
Withdrawn
Application number
EP07702840A
Other languages
German (de)
English (en)
Inventor
Torsten Müller
Thomas Schnelle
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.)
PerkinElmer Cellular Technologies Germany GmbH
Original Assignee
Evotec Technologies GmbH
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 Technologies GmbH filed Critical Evotec Technologies GmbH
Publication of EP1973660A1 publication Critical patent/EP1973660A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0668—Trapping microscopic beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0645—Electrodes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
    • B01L2400/0421—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic electrophoretic flow

Definitions

  • the invention relates to an operating method for a microfluidic system and a correspondingly configured microfluidic system according to the dependent claims.
  • Microfluidic systems with dielectrophoretic electrode arrangements for the manipulation of suspended particles are known, for example, from Muller, T. et al. : "A 3D micro-electrode for handling and caging smgle cells and particels", Biosensors and Bioelectronics 14, 247-256 (1999).
  • the dielectrophoretic electrode arrangements may, for example, be field cages ("cages") for fixing the suspended particles.
  • the conventional micro fluidic systems enable a targeted investigation of suspended particles by being rolled into the microfluidic system and fixed in a field cage. In the fixed state, the suspended particles can then be examined for example by impedance spectroscopy or optically.
  • a disadvantage of the known micro-fluidic systems described above is the fact that the particles of interest in each case can only be examined individually. In contrast, it is often desirable to investigate the chemical or biochemical interaction between different particles, which is possible only with great effort with the conventional microfluidic systems. For example, there is an interest in studying the differentiation of stem cells or immune cells depending on one certain cell stimulation by chemical or trigger substances. Another example is the monitoring of cell-cell interactions.
  • the invention is therefore based on the object to improve the known microfluidic systems to the effect that the interaction of different particles can be examined. Furthermore, the object of the invention is to specify a corresponding operating method for a micro-fluidic system.
  • the invention comprises the general technical teaching of loading the field cage in a microfluidic system successively with particles of different particle types, so that subsequently at least one of the field-cage particles of different particle types is located, which enables a particle-specific interaction of the different particle types.
  • the invention thus enables a targeted investigation of the interaction between different particles with which the field cages are loaded, which is of interest, for example, in pharmaceutical research.
  • a variety of particle-particle interactions can be studied to determine, for example, pharmacologically active substances.
  • the invention makes it possible to selectively stimulate particles with specific trigger substances in order to trigger a reaction, which is known, for example, in stem cell research.
  • the field cages can excite certain trigger substances to reach and observe a particular cell differentiation.
  • the individual Feldkafige be selectively loaded with the particles of a first type of particle by the individual Feldkafige selectively be electrically energized during loading.
  • the selective electrical control of the individual Feldkafige here can act repulsive, so that the affected Feldkafige repel the insufflated particles and thereby prevent loading of the affected Feldkafige with the respective particle.
  • the alternative there is also the alternative
  • the suspended particles After loading the field cages with the suspended particles, the suspended particles preferably adhere to the respective field cages, i. the particles stick in the
  • adhesion is used here in a general sense and encompasses both the actual active process of living cells and passive attachment, which can occur both in living cells and in other particles.
  • Adhering of the particles in the field cage is, however, not absolutely necessary in the context of the invention. Rather, it is u.U. also when the suspended particles are held in the respective Feldkafig by negative dielectrophoresis just above the channel wall of the carrier flow channel.
  • the adhe- vated particles in the field cages can then be removed again by introducing a surface-liberating substance into the microfluidic system.
  • the superficial substance for releasing the adhdated particles can, for example, act enzymatically. Examples of such surface-sparing substances are trypsin, verses, Accumax, Accutase or chelate images, in particular EDTA (ethylenediaminetetraacetic acid).
  • EDTA ethylenediaminetetraacetic acid
  • the surface-flattening substance preferably changes the surface tension of the carrier fluid and thereby causes the detachment of the adhdated particles.
  • the invention is not limited to the above examples with regard to the surface-liberating substance, but can in principle also be realized with other substances.
  • the surface-liberating substance is then preferably ejected from the microfluidic system after an exposure time, wherein the detached particles are fixed in the field cages by an electrical control of the field cages and therefore are not initially unrolled.
  • the detachment of the adhdated particles can be achieved by a temperature change of the adhesive surface (eg of the carrier flow channel), whereby the adhesive surface changes its surface property due to the temperature change, for example from hydrophilic to hydrophobic or vice versa Resistance heating) or cooling (eg by means of Peltier elements) are effected, which are integrated into the microfluidic system.
  • the already mentioned above loading the Feldkafige with the particles of the second particle type is preferably carried out by the individual Feldkafige selectively be electrically energized during loading to either repel or attract the particles.
  • the Feldkafige be controlled for loading with the particles of the second particle type so that flow vortices arise, which claim the particles of the second particle type in the Feldkafige.
  • certain particles can then be selected in the associated field cages.
  • the selected particles are preferably the particles with which the Feldkafige first were loaded.
  • the selected particles it is alternatively also possible for the selected particles to be the Pa. delt, with which the Feldkafige were last loaded.
  • the particles of the different types of particles can undergo a reaction, which can lead to a permanent connection of the particles, for example.
  • the one particle is a stem cell
  • this stem cell may undergo certain cell differentiation due to the interaction with the other particle.
  • the invention enables a real-time observation or a real-time investigation of the process of cell differentiation. The selection can then be made depending on whether and, if appropriate, how the cell differentiation takes place.
  • the particles selected in this way can then be removed from the microfluidic system, for example by unwinding the particles.
  • negative dielectrophoresis is used for selective loading of the field cage.
  • negative dielectrophoresis generally repels the particles.
  • particles that are close enough, preferably only one particle reach the potential minimum of the field cage, with the other particles experiencing only the repulsive forces. If the particles, cells brought close enough to the channel / Gefubbwand, they adharieren. A once adhdated cell is stable bound independently of the field and can only be discharged by "Losesch".
  • a second particle preferably reaches the first particle by switching off the field. If the field remains switched on, the repulsive forces predominate and the second particle does not get into the field cage.
  • the Feldkafige can thus be controlled so that the particles are taken up by means of negative di- electrophoresis of the Feldkafigen.
  • the Feldkafige to be loaded are then turned on while loading, while the other Feldkafige can be switched off.
  • the particles are attracted to the Feldkafigen by means of positive dielectrophoresis.
  • the particles are deposited on the edge of the ring electrodes.
  • a punctiform electrode is arranged within the ring electrodes, e.g. in the middle of the ring structure.
  • the Feldkafige for selective loading are controlled so that the suspended particles are repelled by dielectrophoresis selectively from the Feldkafigen.
  • the field cages to be loaded can then be switched off during loading, while the other field cages are switched on and then act repulsively.
  • both the Feldkafige to be loaded are electrically controlled and the Feldkafige that should not be loaded.
  • the Feldkafig to be loaded are then controlled so that the particles are attracted.
  • the other field cages are controlled repulsively, so that the particles are repelled.
  • the speed of the individual field cells in the Belac particles of the second particle type is controlled to be different and / or different, so that a gradient of the particles of the second particle type is formed between the field cages.
  • any spatial concentration distributions of the particles of the second particle type can be achieved within the microfluidic system.
  • the invention is not limited to a loading of the individual Feldkafige with particles of two different particle types. Rather, it is within the scope of the invention also the possibility that the individual Feldkafige be loaded with more than two particles or with particles of more than two types of particles. Thus, the loading of the field cages with three different particles allows an investigation of the interaction between these three particles.
  • the possibility of marking the suspended particles for example by a fluorescent label or by a radioactive label. This advantageously facilitates the tracking of the particle reactions or of the particles themselves.
  • both the particles of the first particle type and the particles of the second particle type can be very different particles.
  • examples include biological cells, stem cells and immune cells.
  • the particles are magnetic or magnetizable particles, in particular particles with a magnetic or magnetizable core.
  • Particles also antigens, antibodies, hormones, Vir * technically modified viruses in question.
  • the particles to be examined within the scope of the invention may also be biological cells, macromolecules, in particular immunoglobulin, particles with a coated target substance in the particle interior, eg RNA, siRNA, DNA, or particles which have a target structure on their particle surface , in particular in the form of molecules, such as biological cells, stem cells or nanostructures.
  • biological cells macromolecules, in particular immunoglobulin, particles with a coated target substance in the particle interior, eg RNA, siRNA, DNA, or particles which have a target structure on their particle surface , in particular in the form of molecules, such as biological cells, stem cells or nanostructures.
  • the particles are 2-phase systems, such as, for example, droplets.
  • 2-phase systems may comprise an aqueous phase and an oil phase, or an oil phase and a water phase or phase, and a solvent phase, wherein the solvent may be, for example, perfluorotripentylamine (FC70).
  • FC70 perfluorotripentylamine
  • a two-phase system may have a gas phase and a water phase.
  • a particle consists of several areas / phases (inhomogeneous).
  • a cell could be in a drop of aqueous medium, which in turn is suspended in, for example, FC70.
  • Application can find this system in combinatorial chemistry and for setting (in the array of different) defined concentrations.
  • a drop of an aqueous medium is caught in an FC70 solution in Feldkafig and then loaded with other known droplets in which a chemical substance (compound) is dissolved in a known concentration. After the merger, the chemical substance is well-known in the now trapped drop dilute concentration.
  • These drops can be fused elsewhere in the chip with another drop containing one cell. This can be used for kinetics studies, for example for IC 50 determination.
  • the invention is not limited to the above particle types with respect to the particles to be used, but in principle also with other types of particles feasible.
  • the invention makes it possible to study an interaction of any desired combinations of the above-mentioned particle types.
  • the first particles of the first type of particle which have been wound in first are larger than the particles of the second particle type which are subsequently injected.
  • the first particles of the first particle type, which are first injected it is also possible within the scope of the invention for the first particles of the first particle type, which are first injected, to be smaller than the subsequently wetted particles of the second particle type.
  • the particles of the different particle types are the same size.
  • the possibility that the particles of the different types of particles are moved by magnetic forces relative to each other.
  • the particles of the different particle types can be moved toward each other by the magnetic forces during loading of the field cages so that the field cages are loaded with particles of different particle types.
  • the particles of the different particle types are affected by the magnetic forces during the unwinding of the particles. from the microfluidic system.
  • the magnetic forces during loading of the field cage are set lower than the dielectrophoretic forces of the field cage, so that the dielectrophoretic forces generated by the field cage dominate via the external magnetic forces.
  • the magnetic forces during the unwinding of the particles from the microfluidic system to be set lower than the binding forces between the particles of the types of particles that have been shown.
  • this examination can be carried out by means of measuring electrodes which, for example, carry out an impedance spectroscopic examination.
  • the suspended particles can be deposited directly on the measuring electrodes, which is particularly advantageous in the case of the so-called patch-clamp technique.
  • the measuring electrodes are preferably set at the frequency of the capture field to a potential level that corresponds to the potential level that would prevail even without the measuring electrodes in their place.
  • the settling of the particles to be examined directly on the measuring electrodes offers the advantage that electrical measurements can be carried out much more sensitively.
  • the measuring electrodes can be used within the scope of the invention manipulation electrodes, beisp particle fusion, cell-cell fusion or for particle cultivation.
  • the field cages are not electrically activated during the examination, in order to avoid a falsification of the electrical examination by the control of the field cages.
  • the capture field is switched off while the measurement field is switched on.
  • the invention relates not only to the operating method described above for a micro-fluidic system, but also to a correspondingly designed microfluidic system itself.
  • microfluidic system is to be understood generally and not limited to microfluidic systems, which are integrated ⁇ chip, having a closed support: and having leads and output leads. Rather, the term of a microfluidic system also includes a flat or curved plate on which at least one Feldkafig is arranged. Another example of a microfluidic system in the sense according to the invention is a (micron) titer plate.
  • the loading of the individual Feldkafige can also be done by pipetting, so that the Trager- power supply is formed by a pipetting device.
  • the microfluidic system according to the invention preferably provides a carrier flow channel as a carrier current feed, via which a carrier flow with particles suspended therein can be supplied.
  • a carrier flow channel as a carrier current feed, via which a carrier flow with particles suspended therein can be supplied.
  • the term of a Tragerstromkanals used in the invention is to be understood in general and not limited to narrow channels with a polygonal cross-section. Rather, the carrier flow channel can also have a flat extension, so that a plurality of FeId- kafigen can be arranged in the Tragerstromkanal in a common plane.
  • the microfluidic system has a plurality of dielectric field cages, which are arranged spatially separated from one another in the carrier flow channel and either fix or repel the suspended particles as a function of their electrical activation. By means of a corresponding electrical activation of the field cage, these can then be loaded selectively with the particles of the different particle types.
  • the individual field cages can be, for example, three-dimensional, as described in the publication cited above. Muller T. et al .: "A 3D microelectrode and caging single cells and particles" described that the content of this publication is fully within the scope of the present description with respect to the structural design and operation of the field cages.
  • the individual Feldkafige thus each have eight Kafigelektroden, which are arranged cubic.
  • the microfluidic system according to the invention can have at least one auxiliary electrode arrangement, which is arranged in the carrier flow channel upstream or downstream behind at least one of the field cages for the fixation or repulsion of the particles.
  • auxiliary electrodes may, for example, be funnel-shaped
  • the individual Feldkafige are arranged on a carrier, which is for example plate-shaped.
  • the carrier consists essentially of glass, ceramic, a semiconductor, in particular silicon, or of plastic. 1, however, is not limited to the materials mentioned above with regard to the material for the wearer, but can also be realized with other materials.
  • the carrier for the Feldkafige this forms boundary surfaces for the Tragerstromkanal.
  • the electrodes of the individual Feldkafige may be attached to the upper and lower channel wall of the Tragerstromkanals.
  • the electrodes of the Feldkafige are attached to the opposite side channel walls of the Tragerstromkanals, wherein the spatial orientation data refer to the gravitational field of the earth.
  • the individual Feldkafige each annular and / or closed, wherein the Feldkafige downstream may have a weakening, which may for example consist of a passivation layer, which is known per se.
  • the passivation layer then allows the coupling-out of the capture field for the fixation of the suspended particles, whereas the passivation layer has a shielding effect for measurement signals (for example DC measurement signals or low-frequency measurement signals as in patch clamp measurements or membrane impedance measurements).
  • the field cells may have electrodes that are curved in the opposite direction to the direction of flow.
  • the electrodes of the Feldkafige each semicircular or arcuate, the electrode ends facing the direction of flow.
  • the individual Feldkafige matri rows and columns are arranged, which can be selectively controlled individually preferably by row control lines and column control lines.
  • Such a matrix-like arrangement of a multiplicity of field cages is described, for example, in German Patent Application 10 2006 002 462, so that the content of this patent application is to be fully added to the present description with regard to the matrix-like arrangement of the field cages.
  • the microfluidic system has connection contacts which enable a detachable electrical connection of the field cage with a separate driver circuit (generator).
  • a releasable electrical contact can be realized by spring contacts.
  • the Feldkafige an outer ring electrode and an inner ring electrode, wherein the outer ring electrode, the inner ring electrode preferably concentrically surrounds.
  • the two ring electrodes can each be planar. In this case, there is the possibility that the planar ring electrodes are arranged in a common electrode plane. However, it is alternatively possible for the planar ring electrodes to be arranged in parallel but mutually offset electrode planes.
  • At least one measuring electrode is arranged within the inner ring electrode in order to measure the particles fixed in the field cage.
  • the individual measuring electrodes can optionally be round, in particular o-val, or angular, in particular rectangular.
  • the arrangement of the measuring electrodes within the ring electrodes offers the advantage that the dielectrophoretic barrier is reduced upwards and thus facilitates the capture of particles from the carrier flow, in particular when the particles are heavier than the carrier fluid, which is typical in biological cells.
  • the use of at least one internal electrode enables the process of catching and loading in a non-adsorbed state, which is advantageous for suspension cells such as blood cells. The whole works with only one inner electrode
  • Rmg-Rmg-Dot structure If settling onto the substrate is necessary or desirable, it is preferably set to the potential at which the electrode area would, on average, even without an internal electrode, be in switched ring electrodes. Measurements or manipulations can then take place between the central electrode and the inner ring electrode. Another advantage of the internal electrode is that both positive and negative dielectrophoresis can be used. For pDEP Positiomerung about a field of appropriate frequency between the central electrode and the inner and / or outer ring is switched.
  • the ring electrodes may have a passivation layer, wherein the passivation layer is preferably formed so strongly that the passivation layer allows a coupling out of an electric field for particle fixation, whereas the passivation layer for shielding direct current signals and low-frequency signals.
  • the fixation of the individual particles in the field can be additionally supported by negative pressure.
  • the microfluidic system has a vacuum connection which emits in a flexible cage in order to additionally fix the particles located there.
  • the vacuum connection can also be located between the two measuring electrodes in order to fix the particles there for a measurement.
  • the measuring electrodes in the field cage can be arranged either symmetrically or asymmetrically.
  • FIG. 1A is a plan view of a field cage of a microfluidic system according to the invention.
  • FIG. 1B shows a cross-sectional view through the field cage according to FIG. 1A along the line A-A
  • FIG. 2A shows a microfluidic system according to the invention with a plurality of field cells during a selective loading with particles, wherein one field cage is loaded with particles, while the other field cage repulses the inserted particles
  • FIG. 2B shows the microfluidic system according to the loading of the field cages
  • FIG. 3A shows an alternative exemplary embodiment of a microfluidic system according to the invention, in which the loading of the field cage is effected from above,
  • FIG. 3B shows the microfluidic system according to FIG. 3B after the loading
  • FIG. 4A shows an alternative exemplary embodiment of a field cage for use in a microfluidic system according to the invention
  • FIG. 4B shows a cross-sectional view of the field cage according to FIG. AA along the line A-A
  • FIG. 5 shows a matrix-like arrangement of a plurality of field cages in a microfluidic system according to the invention
  • FIG. 6A shows a plan view of an alternative embodiment of a field cage for use in a microfluidic system according to the invention
  • FIG. 6B shows a cross-sectional view of the field cage according to FIG. 6A along the lines A-A,
  • FIG. 7A, 7B show the method according to the invention for a microfluidic system in the form of a flow chart
  • 8 shows an alternative embodiment of a field cage according to the invention with coplanar ring electrodes and two measuring electrodes inside the inner ring electrode
  • FIG. 8 shows an alternative embodiment of a field cage according to the invention with coplanar ring electrodes and two measuring electrodes inside the inner ring electrode
  • FIG. 9A shows the field distribution E 2 in the field cage according to FIG. 9A
  • FIG. 8 in the sectional plane A-A, wherein the inner ring electrode and the outer ring electrode are driven in opposite directions with the same voltage while the measuring electrodes are grounded, FIG.
  • FIGS. 10A-10C show a perspective view of a field cage with eight cage electrodes, wherein the capture field is modified for loading with the particles of the second particle type, as well as FIGS. 10A-10C
  • Figures 11A-11C is a perspective view of a Feldkafigs with eight Kafigelektroden, wherein for loading with the particles of the second Parti- keltyps Stromungswirbel be generated.
  • FIGS. 1A and 1B show an exemplary embodiment of a dielectrophoretic field cage 1 which can be used in a microfluidic system according to the invention, such as it is shown for example in Figures 2A and 2B and 3B.
  • the Feldkafig 1 has in this exemplary embodiment, two planar ring electrodes 2, 3, which are arranged coplanar in a common electrode plane, as can be seen from the cross-sectional view in Figure IB.
  • the two ring electrodes 2, 3 are in this case arranged concentrically on a plate-shaped carrier 4 made of glass and can be actuated electrically independently of each other.
  • a particle 5 shown here only schematically can enter the field box 1 by means of negative dielectrophoresis and be fixed in the field cage 1, the particle 5 adhering to the carrier 4 in the fixed state. ren and then after a shutdown of Feldkafigs 1 sticks to the carrier 4.
  • the Feldkafig 1 can, however, also be controlled electrically such that the particle 5 is repelled by the Feldkafig 1 by means of dielectrophoresis, whereby a loading of the Feldkafigs 1 with the particle 5 and thereby also adharie ren of the particle 5 inside the inner ring electrode 3 prevented becomes.
  • Selective activation of the field cage 1 thus makes it possible, within the inventive microfluidic system described below, optionally to load the field cage 1 with the particle 5 or to repel the particle 5 in order to prevent the field cage 1 from being loaded with the particle 5.
  • FIGS 2A and 2B show detail of a Inventions geaireses microfluidic system with a plurality of cages FeId- 1 ', l' 1 corresponding to the figures IA and IB, in which For the sake of simplicity, only two of the drawings I 1 , I 1 'are shown in the drawings.
  • the carrier 4 in this case runs a Tragerstromkanal through which flows in the direction of arrow Tragerstrom with particles 6 suspended therein.
  • FIG. 2A shows a state of the microfluidic system in which the particles 5 are already fixed in the two field cage 1 'and I 1 ' and adhere to the carrier 4 there.
  • the right field cage 1 "is electrically driven in this state in such a way that it repels the particles 6 by means of dielectrophoresis in order to prevent the particles 6 from attaching to the particles 5.
  • the two particles 5, 6 in this case belong to different particle types.
  • the particle 5 is a biological cell, while the particles 6 are viruses that act on the particles 5.
  • FIG. 2B shows a state in which the two field boxes I 1 , I ' 1 are switched off.
  • the particles 5 then remain adhered to the carrier 4 and can later be dissolved with an upper flattening substance such as trypsin and let out of the microfluidic system.
  • the particles 5 are then examined with the particles 6 deposited there in order to investigate the interaction between the particles 5, 6.
  • particles 5 could be selectively extracted and reused.
  • FIGS. 3A and 3B show an alternative exemplary embodiment of a microfluidic system according to the invention, which largely corresponds to the exemplary embodiment described above and illustrated in FIGS. 2A and 2B, so that reference is made to the above description to avoid repetition, with corresponding references being made Parts the same reference numerals are used.
  • a special feature of this exemplary embodiment is that the loading of the Feldkafige I 1 and l ' 1 is not parallel to the electrode plane, ie parallel to the carrier 4, but at right angles thereto parallel to gravity, as illustrated by the drawn direction of gravity g.
  • FIG. 3B shows a state of the microfluidic system according to the invention after loading when the two field batteries 1 'and I 1 ' are switched off.
  • a multiplicity of the particles 6 has then deposited, which applies in particular to the interior of the field cage 1', where the particles 5, 6 directly adjoin one another. which adjoin so that their interaction can take place.
  • the individual particles 5 can thus be brought together specifically with certain particles 6 in order to investigate their interaction.
  • FIGS. 4A and 4B show an alternative exemplary embodiment of a field cage for use in the microfluidic system according to the invention. This embodiment is partly in accordance with the exemplary embodiments of a field cage described above and shown in FIGS. 1A and 1B, so that reference is made to the above description to avoid repetition, the same reference numerals being used for corresponding components.
  • a special feature of this exemplary embodiment is that the ring electrodes 2, 3 are not closed annularly, but only semicircular, wherein the two semicircular ring electrodes 2, 3 are curved against the flow direction.
  • Figure 5 shows a matrixformige arrangement of a plurality of Feldkafige 1 according to Figures 4A and 4B, wherein the individual Feldkafige 1 can be selectively controlled by a plurality of column control lines 7 and a plurality of row control lines 8.
  • the column control lines 7 are in each case with the inner ring electrode 3 of the Feldkafige 1 of the column concerned, line control lines 8 are each connected to the outer de 2 of the field cages 1 of the relevant row.
  • the procedure is, for example, as follows.
  • Cells are wound from the left by means of a flow.
  • the column control line 7 and the row control lines 8 are grounded except for the column control lines 6 having the indices imax and imax-1.
  • the next two columns (imax-2) and (imax-3) are subsequently activated by additional activation of the column control lines 7 filled with the indices (imax-2) and (imax-3).
  • This process can be continued column-by-column until the array is completely filled and has the advantage that the upstream structures do not hinder the loading of the downstream ones.
  • Loading with the second particles takes place by means of flow from the right, wherein all column and row control lines 7, 8 (for example, inverse phase) are activated except for the cells to be loaded (1, M), the column and row control lines 6, 7 (1, M) are grounded.
  • the optional detachment of target cells is carried out by an analog control by means of a flow over a transverse channel. It is particularly advantageous if the base electrode structure has been produced as shown in FIG. 1, but has been provided on one side with a passivation layer which, at a sufficiently low frequency, corresponds dielectrically to the structure according to FIG. Then one became the initial loading with cells, for example with the lower frequency, the targeted triggering and the discharge of the target cells with d ⁇ the cells can then be discharged in the channel direction (to or from the left).
  • FIGS. 6A and 6B show an alternative exemplary embodiment of a field cage 1 for use in a microfluidic system according to the invention. This embodiment is partly in accordance with the embodiment described above and shown in Figures IA and IB, so that reference is made to avoid repetition of the above description, wherein the same reference numerals are used for corresponding parts.
  • a special feature of this exemplary embodiment is that the field cage 1 does not have the outer ring electrode 2, as is the case in the exemplary embodiment according to FIGS. 1A and 1B.
  • the field cage 1 in this exemplary embodiment has a funnel-shaped electrode arrangement 9 ("funnel") upstream of the field cage, through which the particles 5 are directed into the field cage 1 in a targeted manner.
  • the inner ring electrode 3 is not closed, but is open on its upstream side and merges into the funnel-shaped electrode assembly 9.
  • a Tra biological cells suspended therein are fed into the visible system.
  • the individual FeId- kafige are then loaded in the microfluidic system with the cells insisted by the individual Feldkafige be selectively controlled.
  • the field cages which are to be loaded with the insulatated cells, are for this purpose controlled so that the cells are fixed by means of negative dielectrophoresis.
  • the other field cages, which are not to be loaded with the biological cells, are electrically controlled so that the biological cells are rejected by negative dielectrophoresis.
  • step S3 the biological cells then adhere to the previously selectively loaded field cages, so that the adha ⁇ erten cells not loose after a subsequent shutdown of the individual Feldkafige, but remain in the loaded Feldkafigen.
  • triggering particles for example antigens are then fed into the carrier flow channel of the microfluidic system.
  • the Feldkafige are then loaded m in a further step S5 selectively with the trigger particles, which by a speaking selective electrical control of the means of positive or negative Dielektrophores is.
  • the individual Feldkafige be loaded with both biological cells and with trigger particles, the interaction between the first zugechtten cells and the subsequent zugeschreibten trigger particles in a further step S6 is examined, which can be done for example by optical or impedance spectroscopy. However, the examination can also be carried out by a patch clamp measurement or by a direct current measurement.
  • step S7 specific cells are then selected as a function of the investigation of the interaction between the biological cells and the added trigger particles.
  • the cells loaded in the field cage can be dissolved by application of a surface-insoluble substance (for example trypsin), in which case a sufficient exposure time of the surface-opacifying substance is awaited in a step S9.
  • a surface-insoluble substance for example trypsin
  • the cells loaded in the field cage cells are then fixed to the field cage again in a step S1O, in order to prevent the cells from being unwound by the carrier current.
  • the Feldkafige be electrically controlled in a suitable manner.
  • a further step Sil the previously added surface-liberating substance is then ejected from the microfluidic system with simultaneous fixation of the cells in the field cages.
  • specific field cages which contain the previously selected cells are then switched off in a selective manner. This selective deactivation of the field cage causes the cells contained therein to be reeled out of the microfluidic system by the carrier current in a further step S13, while the other cells remain fixed in the still connected other field cage.
  • the cells are selectively ejected from the microfluidic system, which have previously shown a certain interaction with the carrier substance.
  • a last step S14 the cells selected and extracted in this way are then collected outside the microfluidic system for further use.
  • the microfluidic system has further channels and the particles are first subjected to manipulation (for example sorting) before the particles are then collected.
  • FIG. 8 shows a further exemplary embodiment of a field box for use in the microfluidic system according to the invention.
  • This exemplary embodiment is largely identical to that described above and shown in Figures IA and IB Darge ⁇ presented exemplary embodiment uberein, so reference is made to avoid repetition of the foregoing description, reference numerals are used for corresponding parts the same reference numerals.
  • a special feature of this exemplary embodiment is that within the inner ring electrode 3, two measuring electrodes The or manipulation electrodes 10, 11 angeordni an impedance spectroscopic examination of i inner ring electrode 3 adharwholesome particles 5 and 6 allow.
  • manipulation electrodes 10, 11 allow cell fusion.
  • one of the two manipulation electrodes 10, 11 is held to ground, while the other manipulation electrode 10 or 11 is acted upon by short DC or AC pulses.
  • the two measuring electrodes 10, 11 are arranged symmetrically on the opposite side of the middle point of the field box 1.
  • the Feldkafig 1 in this exemplary embodiment an intake opening 12, via which in the Feldkafig 1, a negative pressure can be generated, which sucks the particles 5 and 6 and thereby supports the loading of Feldkafigs 1 with the particles 5 and ⁇ .
  • the Ansaugo réelle 12 is in this case arranged between the two measuring electrodes 10, 11, so that the particles 5, 6 are fixed in the loading of Feldkafigs 1 between the two measuring electrodes 11, 12, which is advantageous for a subsequent measurement.
  • FIG. 9A shows the field distribution E 2 within the field cage 1 according to FIG. 8, wherein the inner ring electrode 3 and the outer ring electrode 2 are driven with opposite-phase electrical signals of the same voltage U, while the two measuring electrodes 10, 11 are grounded.
  • FIG. 9B shows the field distribution E 2 in the field cage 1 according to FIG. 8, wherein the inner ring electrode 3 and the outer ring electrode 2 are connected in phase opposition with the same voltage U. be controlled while the two Meßelektroi are driven in phase with the inner ring electrode 3 with Vietnamese of 0.26 "U.
  • FIGS. 10A to 10C show an alternative exemplary embodiment of a field cage 13, which is arranged in a microfluidic system according to the invention and is impinged by a carrier flow in the direction of the arrow.
  • the field cage 13 has eight cage electrodes 14, with the cage electrodes 14 being arranged cubically, as described, for example, in Muller, T. et al .: "A 3D microelectrode for handling and caging single cells and particles", biosensors and Bioelectronics 14, 247-256 (1999).
  • the Kafigelektroden 14, whose electrical control is modified, for example, by switching off, attenuation or control with a changed phase position are reproduced without hatching, while the Kafigelektroden 14 whose drive remains unchanged, here are shown hatched.
  • FIG. 10A shows the state after the loading of the field cage 13 with a particle 15 of a first particle type.
  • the Feldkafig 13 generates a trapping field, which fixes the particle 15 in the Feldkafig 13 and hold other particles 16 of a second particle type outside the Feldkafigs 13.
  • FIG. 10B shows the loading of the field cage 13 with the particles 16 by modifying the activation of the upstream cage electrodes so that the particles 16 of FIG can be the carrier flow in the direction of arrow in the Feldkafii.
  • FIG. 10C shows a state in which the various particles 15, 16 are fixed together in the field cage 13 by a capture field in order, for example, to investigate the interaction between the particles 15, 16.
  • FIGS. 11A to 11C show an alternative control of the field cage 13 for loading with the particles 16.
  • the cage electrodes 14 are activated in accordance with the loading of the particle 15 shown in FIG Figure HB is shown and described for example in WO 2005/110605 Al.
  • These turbulences then carry the particles 16 into the field cage 13, where they are finally fixed together with the particle 15 by a conventional trapping field, as shown in Figure HC.

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Abstract

L'invention concerne un procédé d'exploitation d'un système microfluidique consistant à: introduire un flux porteur contenant des particules en suspension (5) d'un premier type dans le système microfluidique ; à charger plusieurs cages de champ électrique (1', 1'') dans le système microfluidique contenant les particules (5) du premier type ; à céder un flux porteur contenant les particules en suspension (6) d'un deuxième type de particules dans le système microfluidique ; et à charger les cages de champ (1', 1'') dans le système microfluidique contenant les particules (6) du deuxième type de telle façon que, dans au moins une cage de champ (1', 1''), se trouvent une particule (5) du premier type de particules et une particule (6) du deuxième type. L'invention concerne enfin un système microfluidique correspondant.
EP07702840A 2006-01-18 2007-01-17 Système microfluidique et procédé d'exploitation Withdrawn EP1973660A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE202006010646 2006-01-18
DE102006033889A DE102006033889A1 (de) 2006-01-18 2006-07-21 Mikrofluidisches System und zugehöriges Betriebsverfahren
PCT/EP2007/000389 WO2007082737A1 (fr) 2006-01-18 2007-01-17 Système microfluidique et procédé d'exploitation

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US20110192726A1 (en) * 2008-10-31 2011-08-11 Agency For Science ,Technology And Research Device and method for detection of analyte from a sample
DE102009043527B4 (de) * 2009-09-30 2021-06-10 Boehringer Ingelheim Vetmedica Gmbh Anordnung und Verfahren unter Verwendung von Mikrosensoren zum Messen von Zell-Vitalitäten
DE102011050254A1 (de) * 2011-05-10 2012-11-15 Technische Universität Dortmund Verfahren zur Separation polarisierbarer Biopartikel
DE102011054659A1 (de) * 2011-10-20 2013-04-25 AeroMegt GmbH Verfahren und Vorrichtung zum Messen von Aerosolen in einem großen Volumenstrom
EP2802416B1 (fr) 2012-01-09 2016-04-06 Sophion Bioscience A/S Amélioration de l'adhérence cellulaire dans la région du patch
DE102016223029A1 (de) * 2016-11-22 2018-05-24 Leibniz-Institut Für Festkörper-Und Werkstoffforschung Dresden E.V. Dreidimensionaler tomograf
WO2018213562A1 (fr) * 2017-05-17 2018-11-22 University Of Cincinnati Utilisation de forces électrocinétiques pour manipuler des particules en suspension
DE102017121326B4 (de) * 2017-09-14 2021-01-14 Leica Microsystems Cms Gmbh Sammeleinrichtung und Verfahren zum Sammeln dissektierter oder ablatierter Proben und Mikroskop mit einer solchen Einrichtung

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EP0746408B1 (fr) 1994-02-24 1998-09-23 Fraunhofer-Gesellschaft Zur Förderung Der Angewandten Forschung E.V. Formage de microparticules dans des cages a champs electriques
DE4434883A1 (de) 1994-02-24 1995-08-31 Stefan Fiedler Formen von Mikropartikeln in elektrischen Feldkäfigen
DE19916921A1 (de) 1999-04-14 2000-10-19 Fraunhofer Ges Forschung Elektrisches Sensorarray
AU2002307218A1 (en) 2001-03-24 2002-10-08 Aviva Biosciences Corporation Biochips including ion transport detecting structures and methods of use
US7018819B2 (en) 2001-11-30 2006-03-28 Cellectricon Ab Method and apparatus for manipulation of cells and cell-like structures focused electric fields in microfludic systems and use thereof
EP1595140A2 (fr) 2003-02-18 2005-11-16 Board Of Regents The University Of Texas System Focalisation de particules dielectriques
WO2005075958A1 (fr) * 2004-02-04 2005-08-18 Evotec Technologies Gmbh Systeme microfluidique et procede correspondant pour le faire fonctionner

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US8128797B2 (en) 2012-03-06
WO2007082737A1 (fr) 2007-07-26

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