EP1226419A1 - Procede et dispositif de separation de particules - Google Patents

Procede et dispositif de separation de particules

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Publication number
EP1226419A1
EP1226419A1 EP00975936A EP00975936A EP1226419A1 EP 1226419 A1 EP1226419 A1 EP 1226419A1 EP 00975936 A EP00975936 A EP 00975936A EP 00975936 A EP00975936 A EP 00975936A EP 1226419 A1 EP1226419 A1 EP 1226419A1
Authority
EP
European Patent Office
Prior art keywords
particles
optical
cages
target
functional elements
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Withdrawn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
EP00975936A
Other languages
German (de)
English (en)
Inventor
Rolf Günther
Gabriele Gradl
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.)
Evotec OAI AG
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 EP1226419A1 publication Critical patent/EP1226419A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • H—ELECTRICITY
    • H05—ELECTRIC TECHNIQUES NOT OTHERWISE PROVIDED FOR
    • H05H—PLASMA TECHNIQUE; PRODUCTION OF ACCELERATED ELECTRICALLY-CHARGED PARTICLES OR OF NEUTRONS; PRODUCTION OR ACCELERATION OF NEUTRAL MOLECULAR OR ATOMIC BEAMS
    • H05H3/00—Production or acceleration of neutral particle beams, e.g. molecular or atomic beams
    • H05H3/04—Acceleration by electromagnetic wave pressure
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C5/00—Separating dispersed particles from liquids by electrostatic effect
    • B03C5/02—Separators
    • B03C5/022—Non-uniform field separators
    • B03C5/026—Non-uniform field separators using open-gradient differential dielectric separation, i.e. using electrodes of special shapes for non-uniform field creation, e.g. Fluid Integrated Circuit [FIC]
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C5/00—Separating dispersed particles from liquids by electrostatic effect
    • B03C5/02—Separators
    • B03C5/022—Non-uniform field separators
    • B03C5/028—Non-uniform field separators using travelling electric fields, i.e. travelling wave dielectrophoresis [TWD]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1429—Signal processing
    • G01N15/1433—Signal processing using image recognition
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1484—Optical investigation techniques, e.g. flow cytometry microstructural devices
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography

Definitions

  • the invention relates to a method for separating particle mixtures according to certain particle properties, in particular a separation method for mixtures of microscopic particles in fluidic microsystems, and a separation device for carrying out the method.
  • a task frequently used in biological or medical examinations, in diagnostics and in biotechnological processes is the separation of suspension mixtures by distributing or sorting cells or micro-particles from a large starting quantity into specific groups, each with the same properties.
  • the transfection of a cell line can often only be carried out incompletely, so that the successfully transfected cells must be separated from the unsuccessfully processed cells before further processing or use of this cell line.
  • the sorting out of rarely occurring individuals places very high demands on the separation process with regard to the purity and homogeneity of the target fraction.
  • Another example in the medical field is bone marrow transplantation, in which stem cells have to be separated from a large number of other cells.
  • fetal cells when analyzing fetal cells in maternal blood, a few fetal cells must be separated from the cell mixture that is in the maternal blood.
  • An important task in tumor diagnosis is the detection, sorting out and analysis of metastatic cancer cells in the patient's blood.
  • Various methods for separating cell mixtures are known, but they only solve the separation task incompletely. These include in particular affinity chromatography, separation processes on the basis of fluorescent labels such as, for example, fluorescence-activated cell sorting (FACS), on the basis of labeling with ferro-magnetic particles, magnetic-activated cell sorting (MACS) and microscopic methods.
  • FACS fluorescence-activated cell sorting
  • MCS magnetic-activated cell sorting
  • Affinity chromatography as well as MACS are restricted to separation processes in which the cells on the cell surface express a molecule which has an affinity for a further, known or unknown substance.
  • affinity chromatography this substance is applied to a chromatography matrix, for example based on beads. A suspension of the cells to be separated is placed over this matrix so that the expressing cells adhere to it while the other cells are being rinsed away.
  • the disadvantage of this method in addition to the restriction to cellular systems with a surface expression of molecules, is the high mechanical load on the cells and the low selectivity of the chromatography. The selectivity is poor because many cells with low affinity remain unspecifically attached to the matrix, which actually do not belong to the cells to be separated.
  • affinity chromatography is limited to the separation of bacteria that are not mechanically sensitive.
  • the cell suspension is mixed with micrometer-sized magnetic particles to which the affine substance is applied, and the magnetic particles are separated off together with the target cells using a magnet located outside the reaction vessel.
  • the carryover of unspecifically adhering cells to the magnetic particles is a considerable limitation of the method with regard to the purity of the target fraction.
  • FACS fluorescence-activated cell separation
  • the disadvantage of fluorescence-activated cell separation is that it is a serial process.
  • the optical effort for the measuring section is so great that a parallelization is too expensive for practical applications.
  • the measurement time In order to be able to analyze 10,000 cells per second, for example, the measurement time must be limited to 100 ms or less in a serial procedure. Measurements are possible in such short time ranges, but they also have relatively large errors, so that the selectivity of the method is adversely affected.
  • Microscopic cell separation is based on forming a two-dimensional layer of cells (so-called cell lawn) and scanning it with a microscope. An image of each cell is recorded, which is subjected to an image evaluation to determine predetermined cell properties. Depending on the evaluation result, a picking device is used to remove individual cells from the cell lawn after the measurement. and deliver it to a destination. A particularly high resolution in image acquisition is achieved by using confocal microscopy. This method also has restrictions, particularly with regard to the throughput of the picking device. Without damaging the cells, a picking process can be carried out at a maximum of about 10 seconds, which in addition represents a mechanical load on the cells to be separated.
  • a fluidic microsystem 100 ′ contains a first channel structure 10 ′ and a second channel structure 20 ′, which include channels or chambers that border one another, for example, arranged next to one another or one above the other.
  • a particle mixture 1 'suspended in a liquid is moved in the first chamber structure 10' to a field cage 30 '.
  • a deflection electrode 40 ' is actuated depending on the measurement result in such a way that the particle m is the second channel structure 20 'transferred or remains for further movement m of the first channel structure 10' (see arrows).
  • This method is disadvantageous because of the serial separation method. If the separation method according to FIG. 5 is to be carried out with several field cages, then several deflection electrodes had to be detuned accordingly. However, due to the electrical interaction of neighboring structures, this cannot be reliably carried out in time ranges of practical interest, and undesirable fault separations have occurred. Another disadvantage is the limitation to the measurement electrical properties of the particles in the microsystem.
  • the object of the invention is to provide an improved separation process which is distinguished by a high throughput, a high selectivity and a low mechanical load on the particles to be separated.
  • the object of the invention is also to provide a separating device for implementing such a method.
  • the basic idea of the invention is to carry out a particle separation in a fluidic microsystem, in which the particles to be separated are first of all, in particular functional elements, individually or in groups in at least one receiving structure on a multiplicity of functional elements which are set up to at least temporarily hold the particles or particle groups the basis of field cages or other suitable field barriers through positive or negative Dielectrophoresis are positioned and measured at this observation position (or: m this observation field).
  • this observation position or: m this observation field.
  • predetermined properties of the particles to be separated and, accordingly, the positions of the particles which are to be separated from the mixture or the positions of the particles which belong to the residual mixture are determined.
  • the particles to be separated are also called target particles and the particles belonging to the rest of the mixture are called residual particles.
  • Temporary holding is understood here to mean positioning the particles at a predetermined location in the microsystem for the duration of the respective measurement or manipulation.
  • the target or residual particles are subjected to laser radiation successively or simultaneously to form optical cages after the measurement.
  • the target or residual particles are preferably simultaneously subjected to laser radiation to form optical cages.
  • the laser irradiation can take place, for example, within a field cage, on a field camera or in the flow direction immediately behind a field cage or a field barrier. Accordingly, the target or residual particles are exposed to optical forces in addition to the electrical field forces.
  • the target or residual particles are transmitted by shifting the optical cages m the adjacent channel structure or by opening the electrical field cages to the adjacent channel structure while simultaneously holding the optical cages. This transfer is preferably carried out simultaneously for several target or residual particles.
  • the target particles are collected accordingly in the adjacent or in the original channel structure of the microsystem and processed further. Groups of particles from the observation fields can also be will wear.
  • the target and residual particles are divided into two groups.
  • the target and residual particles can include one or more types of particles.
  • the target or residual particles 2, 3 are preferably transferred to m different collecting containers after the separation.
  • a device for particle separation in fluidic microsystems in particular comprises one or more first receiving structure (s) with devices for at least temporarily holding the particles, in particular with electrode devices for forming a large number of functional elements (for example dielectric field cages), one or more, in particular adjacent, second ones Pick-up structure (s), which are or are connected to the first or the first pick-up structure (s) via openings, preferably at the positions of the functional elements, one or more laser devices (s) used to form one or more optical cages on each function - Element, in particular in each dielectric field cage, is or are formed, and one or more light modulator (s) with which one or more optical cages can be switched on or off in any predetermined position.
  • the optical cages can preferably be switched on or off individually. Furthermore, it is preferred to form an optical cage on each functional element.
  • the device preferably has one or more loading device (s) for positioning particles on the functional elements.
  • the receiving structures are in particular channel or chamber structures of the fluidic microsystem.
  • the openings between see the receiving structures can be temporarily closed with flaps.
  • the transmission of the target particles takes place by switching on the optical cages in or behind the field cages or field barriers with the target particles, capturing the target particles in the optical cages and moving the optical cages into the second channel structure, while the remaining particles are in the first Channel structure remain.
  • the second channel structure is either offset in the y direction parallel to the side of the first (relative to the flow direction of the particle stream).
  • the particles held in the optical cages are moved by moving the device in the xy direction relative to the optical axis of the laser beam.
  • the second channel structure lies above the first channel structure in the z direction along the optical axis. In this case, the particles are moved to the second level by adjusting the focus of the optical cages.
  • the target particles are also held in optical cages in the respective dielectric field cages, while the remaining particles in the remaining dielectric field cages remain unilluminated and thus free of optical forces.
  • all field cages are detuned in such a way that dielectric repulsive forces are formed which are directed onto the particles in the field cages towards the second channel structure.
  • the residual particles are then transferred into the second channel structure, while the target particles remain in the first channel structure under the action of the optical forces despite the field cage detuning.
  • An essential feature of the invention is a combination of parallelized holding forces, in particular electrical ⁇ field forces, for position control with parallelized optical forces for sorting.
  • Arbitrary positions m of the device according to the invention can be addressed simultaneously by the special optical structure with the laser radiation, without moving the device.
  • the combination with reproduced field cages provides for the simultaneous observation and evaluation of a large number of particles, which are held at predetermined positions, for example by dielectric forces.
  • the sorting process is then carried out by the parallelized and addressable laser irradiation for this large number of particles, preferably in one step, ie at the same time. This enables a high throughput during sorting.
  • Optical cages are formed on the observation fields by means of parallel optics for simultaneous addressing of all desired observation fields.
  • the invention has the following advantages. With the principle according to the invention of the simultaneous or successive measurement of a large number of particles with a subsequent simultaneous or successive transmission of the target particles, the throughput of the particle separation is increased considerably in comparison to the conventional techniques.
  • the target particles are preferably transmitted simultaneously.
  • the measurement of the particles z. B. m the dielectric field cages can be carried out gently without adverse effects on the particles. Sufficient time is available for the measurement to reliably determine the properties of the particles in question. In particular, the sorting out of rarely occurring particles in a large initial population can be carried out with great reliability, since all particles can be addressed individually.
  • the dielectric field cages or field barriers spatially separate the individual particles or groups of particles from one another during the procedure.
  • the optical cages enable individual particles to be captured without crosstalk to other particles.
  • the sharpness of particle separation is considerably improved.
  • the measurement can relate to the electrical and / or optical properties of the particles.
  • the invention can be implemented with any particles of natural or synthetic origin that can be manipulated in fluidic microsystems. Another advantage is the compatibility of the separation process with the available microsystem technology.
  • FIG. 1 is a schematic plan view of a microsystem with a separating device.
  • FIG. 2 shows a schematic side view of a microsystem according to FIG. 1,
  • FIG. 3 shows a schematic top view of a microsystem with a further separating device according to the invention
  • FIG. 4A is a schematic side view of a microsystem according to FIG. 3 with superimposed channels
  • 4B is a schematic top view of a microsystem with channels stored next to one another
  • 5 shows a schematic top view of a microsystem with a two-dimensional, flat matrix arrangement of field cages and a flow profile typical for the embodiment according to the invention
  • 6 shows a schematic top view of a microsystem with a two-dimensional, flat matrix arrangement of field barriers
  • Fig. 7 is a schematic plan view of a conventional Emzelpartteilverteiler (prior art).
  • a first embodiment of the invention which is illustrated in FIGS. 1 and 2, is based on the combination of dielectric field cages with adjustable optical cages.
  • a separation device 200 according to the invention which is integrated in a fluidic microsystem 100, comprises a plurality of dielectric field cages 30, in each of which an optical cage with an adjustable focus 41 is formed with the laser device 40 and the optics 42.
  • Reference numeral 43 indicates a light modulator with which the optical cages in the field cages 30 can be selectively switched on or off.
  • optical cages and their use for manipulating microscopic particles is known per se (see, for example, A. Ashkm et al. In “Nature”, Vol. 330, 1987, pp. 796 ff., And G. Weber et al. In “ International Review of Cytology ", vol. 133, 1992, pp. 1 ff.).
  • Details of the laser operation (e.g. power, wavelength, etc.) and the beam focusing can be adapted to the specific separation task when realizing the system according to the invention on the basis of the known techniques.
  • the microsystem 100 comprises at least two channel structures 10, 20.
  • the channel structures 10, 20 each comprise microchannels or microchambers, e.g. B. open reservoirs or wells such as m nano or microtiter plates, with an application-specific shape. Combinations of microchannels and microchambers in a microsystem are also possible.
  • Microchannels are preferably provided. In the illustrated For example, two channels arranged one above the other in the operating function are provided. However, they can also be arranged next to one another (see FIG. 4B).
  • the channels are made, among other things, of plastic, glass or semiconductor materials using methods known per se from microsystem technology and are designed to be flowed through by liquids with suspended particles.
  • the particles suspended in a liquid are preferably conveyed through the microsystem using a liquid transport system.
  • the channels have characteristic dimensions which, depending on the application, are selected so that an unimpeded suspension throughput is possible. If the microsystem is used, for example, to manipulate biological cells (characteristic size: 10 ⁇ m), each channel has a typical cross section that is larger than 10 ⁇ m and, for example, up to 50 ⁇ m. For separating synthetic particles (beads), for example in applications in combinatorial chemistry, the channels can also have characteristic cross-sectional dimensions in the range from 100 to 200 ⁇ m. In order to ensure optical particle measurement in the field cages, the walls of the microsystem 100 are correspondingly designed to be at least partially transparent.
  • a plurality of field cages 30 are formed in the first channel structure 10 of the microsystem 100.
  • Each field cage 30 comprises a group of schematically represented microelectrodes 31 which are attached to the top and bottom surfaces of the respective channel structure.
  • the microelectrodes 31 are designed to be subjected to high-frequency fields in such a way that a field distribution results between the microelectrodes 31, which polarization forces are generated on dielectric particles and hold the particles in the interior of the field cage. Details of the construction and control of dielectric field cages and field barriers are known per se. known (see, for example, G. Fuhr et al. in "Electromanipulation of Cells", CRT Press Inc., 1996, pp. 259 ff.).
  • a charging device 50 is also preferably provided in the microsystem 100, which is designed to distribute particles to the field cages 30 of the separating device 200.
  • a charging device 50 the function of which is explained in detail below, is used with particular advantage.
  • this form of the loading device is not a mandatory feature of the invention and can alternatively be replaced by other loading or straddling elements.
  • Several charging devices can also be provided.
  • the observation positions or fields can also be loaded passively, for example, by transporting the particle suspension over the laminar liquid flow.
  • the flow profile in the microchannels of the device is not parabolic, but runs straight across most of the channel - with the exception of the outermost edge regions (see FIG. 5), since the channel is considerably wider than it is high. In this way, the particles are transported uniformly quickly across the major part of the channel and the field cages or field barriers are statistically evenly loaded.
  • Deflection electrodes 63 on the edge of the channel serve to convey particles from the flow-calming edge zones of the channel into zones with a uniform flow.
  • the deflection electrodes can also be part of field cage or field barrier electrodes. If an observation position remains vacant, this is irrelevant for the further procedure.
  • the channel structures 10, 20 are separated from one another by a continuous wall.
  • the task of the separation method now consists in the first channel structure 10 corresponding to the Particle mixture 1 which has flowed in in the direction of the arrow A into target particles 2, which are to be transferred to the second channel structure 20 in this embodiment of the invention, and to separate residual particles 3 which are to remain in the first channel structure 10.
  • the particles are first loaded individually or in groups, for example using the loading device 50, into the observation positions (field cages or field barriers) 30 arranged in a row (see FIG. 1).
  • the charging device 50 comprises, for example, two straight, strip-shaped microelectrodes which are arranged on the upper and lower channel rarities and which, when exposed to high-frequency electric fields, exert repulsive forces on the inflowing particles under the action of negative dielectrophoresis and thus a field barrier which runs obliquely to the direction of flow (arrow A) form.
  • Individual particles are considered here as examples.
  • the implementation of the invention with groups of particles is carried out analogously.
  • the field barrier is switched on, the first inflowing particle la runs first along the arrow direction B to the end of the charging device 50 and then into the field cage 30a (see FIG. 2).
  • the field cage 30a is open on the upstream side, so that the particle freely reaches the center 31a of the field cage 30a.
  • the field cage 30a is also closed on the upstream side.
  • the remaining field cages 30b, 30c, ... are loaded analogously by inflowing particles.
  • the loading device 50 is actuated in synchronism with the loading condition of the field cages. On the loading device 50, further alignment elements, field cages or barriers are optionally provided for arranging the inflowing particles.
  • the measurement is carried out to determine physical and / or biochemical parameters of the particles.
  • This measurement is carried out, for example, optically (for example fluorescence or scattered light measurement) and / or electrically (for example electrorotation measurement in the field cages).
  • a laser device which is also designed to form the optical cages, is preferably used for the optical measurements. Provision can also be made to use the suspension liquid in the microsystem to coil additives to the field cages, the interaction of which with the held particles is detected optically and / or electrically.
  • the field cages in which the target particles with predetermined parameters are located are recorded in preparation for the following separation step.
  • the light modulator 43 is actuated in such a way that laser radiation is released into the detected field cages with the target particles.
  • An optical cage is formed by this irradiation, the focus 41 of which coincides with the center (eg 31a) of the dielectric field cage 30.
  • the target particles are caught in the optical cages like with laser tweezers.
  • the focus 41 is simultaneously shifted through the opening 11 between the channel structures (arrow C). This shift takes place, for example, by adjusting the optics 42.
  • the laser radiation is switched off with the light modulator 43.
  • the target particles are thereby released in order to be subjected to a further movement and / or processing of the second channel structure 20.
  • the field cages 30 m of the first channel structure 10 are opened on the downstream side, so that the remaining particles 3 are also released.
  • the target or residual particles 2, 3 can each be subjected to one or more further separations according to the principle explained in order to sort them into a plurality of To make particle groups.
  • the separating device 200 comprises in particular the laser device 40, the light of which is bundled with the optics 42 to form the focus 41, and the light modulator 43.
  • the laser device 40 comprises a laser diode arrangement or a single laser with a beam expansion optics. A plurality of laser beams can also be provided to form an optical cage in each case. In the laser diode arrangement, a large number of laser diodes are aligned in accordance with the position of the field cages or field cameras in the microsystem. When using a single laser, however, the entire arrangement of the field cages is illuminated with the single expanded beam.
  • the laser device 40 is preferably operated at a wavelength which is particularly well suited for the formation of effective optical cages with the lowest possible light output. For most applications, the wavelength is selected in the infrared or red spectral range.
  • the light modulator 43 is a transmission or reflection modulator.
  • Known microshutter arrangements can be used as the transmission modulator, in which a large number of microshutters are aligned according to the position of the field cages. Mechanical microshutters or switchable Flussigk ⁇ stall arrangements are used.
  • a switchable matrix of mirror elements can be used as the reflection modulator.
  • the laser device 40 is designed with a laser diode arrangement, it is also possible to integrate the light modulator m with the laser device 40. In this case, the light modulator is a control circuit with which the individual laser diodes can be switched on or off individually.
  • the optics 42 comprise a microlens arrangement comprising a multiplicity of microlenses, which preferably correspond to the positions cages are aligned. With the optics according to the invention, it is also possible to detect several partial areas of a field cage in parallel and to direct laser tweezers onto these areas. This is particularly advantageous if groups of particles are contained in the field cages and individual particles or subgroups from the groups of target or residual particles 2, 3 are to be taken over from the observation fields. There is particular interest in this embodiment for applications for enriching particle types.
  • the microlens arrangement is mounted to move the focus 41 in a vertically movable manner, as is known per se from laser tweezers.
  • the particles in the field cages are measured using an optical detector device, for example an array detector based on CCD, CID (charge injection device), CMOS, APD (avalanche photodiode) ) or PD - (photodiode) arrangements is formed.
  • the detector device is preferably attached on the side of the microsystem 100 that is opposite to the laser device 40.
  • the detector elements of the detector device are preferably designed for wavelength-selective light detection.
  • filter devices which may be integrated in the detector device, are provided. The wavelength-selective measurement is used in particular to measure the fluorescence on the particles.
  • Another embodiment of the invention which can also be implemented with the arrangement illustrated in FIGS. 1 and 2, is based on the combination of optical cages with adjustable dielectric field cages.
  • the particles of the starting mixture 1 are first positioned and measured in the field cages 30.
  • the laser radiation with the light modulator 43 released the laser radiation with the light modulator 43. This traps each target particle in an optical cage.
  • the entire field cages 30 are now detuned in such a way that a resulting dielectric force is exerted on the particles through the opening 11 into the second channel structure 20.
  • This force effect is followed by all particles which are not additionally held by the optical cage, so that a corresponding collection m of the second channel structure 20 results.
  • the illuminated particles eg target particles
  • the target and residual particles in the first or second channel structure 10, 20 are transported further.
  • FIGS. 3, 4A and 4B A further modified embodiment of the invention, which can be operated in accordance with one of the two combination principles mentioned above, is illustrated in FIGS. 3, 4A and 4B.
  • the microsystem 100 in turn contains two channel structures 10, 20 arranged one above the other or next to one another with at least partially transparent channel walls.
  • application-dependent microelectrodes are formed to produce field cages and / or barriers.
  • the separating device 200 in this embodiment comprises a flat matrix arrangement of field cages 30 in straight rows and columns. Thus, several rows of observation positions are arranged one behind the other in the direction of flow.
  • this has the advantage that individual particles can be evaluated several times in succession, which considerably improves the reliability of the analysis and thus also the separation.
  • such a two-dimensional array of observation fields significantly increases the throughput of particles that be evaluated at the same time.
  • the channels can be arranged one above the other or next to one another, as illustrated in FIGS. 4A and 4B.
  • FIG. 5 A further modified embodiment of the invention with regard to the shape of the electrodes for forming field cages 60 is shown in FIG. 5.
  • the individual field cages are formed by tine or comb-like electrodes (61a, 61b, 61c ).
  • the particles 62a, 62b, 62c ... are arranged in such an arrangement in the spaces between the tines.
  • This type of electrode has advantages above all in terms of simple electrode processing with connected leads.
  • FIG. 6 A further modification of the invention is demonstrated in FIG. 6.
  • field bamers 70 with the aid of electrodes as shown at 70a, 70b, 70c ...
  • the particles 71a, 71b, 71c ... are printed against the dielectric field barriers by a steady flow of liquid 72 and are thus held at the respective observation positions.
  • the irradiation takes place from the underside of the microsystem, i.e. from the side of the first channel structure 10.
  • at least the channel floor 12 consists of a transparent material.
  • the particles trapped in the field cages 30 can also be optically measured from the underside of the microsystem 100 or from the opposite side.
  • the light from the laser device 40 is in turn focused via a light modulator 43 and an optics 42 m the field cages 30.
  • a row of field cages is provided as the loading device 50.
  • the particles 1 flowing in the first channel structure 10 are first positioned in the loading device 50.
  • the entire row of field cages 51 and the adjacent row of field cages 30a are controlled in such a way that the particles are taken over into field cages 30a.
  • the field cages 51 of the loading device 50 are then loaded again.
  • the particles are again passed on to the field cages 30a and from there to the row of the field cages 30b until all rows of the field cages 30 are filled step by step.
  • the particles are then measured and separated according to the principles explained above.
  • FIGS. 1 to 6 An important advantage of the embodiments of the invention shown in FIGS. 1 to 6 is that all dielectric field cages are controlled simultaneously. Interference fields or crosstalk between the field cages are thereby avoided, as a result of which the accuracy of the separation according to the invention, particularly in comparison with the conventional technology explained with reference to FIG. 7, is considerably improved.
  • the particle separation takes place exclusively under the action of optical forces.
  • the particles to be separated are not positioned in dielectric field cages, but rather on mechanically designed holders in the microsystem. Such brackets are formed, for example, by holes in the channel wall.
  • the group of target particles m is picked up in corresponding optical cages and lifted away from the holders into the adjacent channel structure.
  • three channel structures are arranged adjacent to one another, the positioning and measurement of the particles in the central channel structure and the transfer of target particles to the adjacent channel structures taking place by firstly a first group of target particles, for example m the upper channel structure and then another group of target particles the lower channel structure is transmitted.
  • the particles are held or positioned by mechanical or acoustic forces.
  • brackets can, for. B. can be formed with micromanipulators or ultrasonic devices.

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Abstract

L'invention concerne des procédés et dispositifs de séparation de particules. Dans un microsystème fluidique composé d'un mélange particulaire (1) de nombreuses particules, des particules cibles (2) présentant des propriétés particulaires prédéterminées sont séparées de particules restantes (3). Ledit procédé consiste à positionner les particules du mélange particulaire (1) dans des champs d'observations sur des éléments fonctionnels (30), dans au moins une première structure réceptrice (10) du microsystème (100), les éléments fonctionnels étant conçus pour maintenir les particules au moins temporairement, à mesurer les particules dans les champs d'observation pour la détermination des propriétés particulaires, à relever les champs d'observation contenant les particules cibles (2) ou les particules restantes (3), à créer des cages optiques sur les champs d'observation contenant les particules cibles ou les particules restantes avec utilisation d'au moins un modulateur de lumière permettant de commuter au moins une cage optique, et à transmettre simultanément ou successivement les particules cibles ou restantes de leurs champs d'observation respectifs vers au moins une deuxième structure réceptrice (20) du microsystème, des forces optiques des cages optiques et des forces des éléments fonctionnels (30) étant en interaction.
EP00975936A 1999-10-29 2000-10-27 Procede et dispositif de separation de particules Withdrawn EP1226419A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19952322A DE19952322C2 (de) 1999-10-29 1999-10-29 Verfahren und Vorrichtung zur Partikeltrennung
DE19952322 1999-10-29
PCT/EP2000/010586 WO2001031315A1 (fr) 1999-10-29 2000-10-27 Procede et dispositif de separation de particules

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EP1226419A1 true EP1226419A1 (fr) 2002-07-31

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EP (1) EP1226419A1 (fr)
DE (1) DE19952322C2 (fr)
WO (1) WO2001031315A1 (fr)

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US6797942B2 (en) * 2001-09-13 2004-09-28 University Of Chicago Apparatus and process for the lateral deflection and separation of flowing particles by a static array of optical tweezers
US7699767B2 (en) 2002-07-31 2010-04-20 Arryx, Inc. Multiple laminar flow-based particle and cellular separation with laser steering
WO2004012133A2 (fr) * 2002-07-31 2004-02-05 Arryx, Inc. Systeme et procede de tri de materiau utilisant une orientation par laser holographique
US11243494B2 (en) 2002-07-31 2022-02-08 Abs Global, Inc. Multiple laminar flow-based particle and cellular separation with laser steering
DE10304653B4 (de) * 2003-02-05 2005-01-27 Evotec Technologies Gmbh Mehrparametrige Detektion in einem fluidischen Mikrosystem
DE10320956B4 (de) * 2003-02-05 2005-02-17 Evotec Technologies Gmbh Untersuchungsverfahren für biologische Zellen und zugehörige Untersuchungseinrichtung
DE10311716A1 (de) 2003-03-17 2004-10-14 Evotec Oai Ag Verfahren und Vorrichtung zur Trennung von Partikeln in einer Flüssigkeitsströmung
ES2544944T3 (es) 2003-05-08 2015-09-07 The University Court Of The University Of St. Andrews Fraccionamiento de partículas
DE502005002217D1 (de) * 2004-02-04 2008-01-24 Evotec Technologies Gmbh Mikrofluidisches system mit einer elektrodenanordnung und zugehoriges ansteuerungsverfahren
WO2006063335A2 (fr) * 2004-12-10 2006-06-15 Arryx, Inc. Extraction et purification automatique d'echantillons a l'aide de pinces optiques
DE102005012128A1 (de) * 2005-03-16 2006-09-21 Evotec Technologies Gmbh Mikrofluidisches System und zugehöriges Ansteuerverfahren
US7574076B2 (en) * 2005-04-08 2009-08-11 Arryx, Inc. Apparatus for optically-based sorting within liquid core waveguides
ITBO20050643A1 (it) * 2005-10-24 2007-04-25 Si Bio S R L Metodo ed apparato per la manipolazione di particelle in soluzioni conduttive
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
DE102008018170B4 (de) 2008-04-03 2010-05-12 NMI Naturwissenschaftliches und Medizinisches Institut an der Universität Tübingen Mikrofluidisches System und Verfahren zum Aufbau und zur anschließenden Kultivierung sowie nachfolgender Untersuchung von komplexen Zellanordnungen
US10908066B2 (en) 2010-11-16 2021-02-02 1087 Systems, Inc. Use of vibrational spectroscopy for microfluidic liquid measurement
US8961904B2 (en) 2013-07-16 2015-02-24 Premium Genetics (Uk) Ltd. Microfluidic chip
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US11331670B2 (en) 2018-05-23 2022-05-17 Abs Global, Inc. Systems and methods for particle focusing in microchannels
EP4245140A3 (fr) 2019-04-18 2024-01-17 ABS Global, Inc. Système et procédé d'ajout en continu de cryoprotecteur
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