EP1092144A1 - Method and device for manipulating particles in microsystems - Google Patents

Method and device for manipulating particles in microsystems

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Publication number
EP1092144A1
EP1092144A1 EP19990931204 EP99931204A EP1092144A1 EP 1092144 A1 EP1092144 A1 EP 1092144A1 EP 19990931204 EP19990931204 EP 19990931204 EP 99931204 A EP99931204 A EP 99931204A EP 1092144 A1 EP1092144 A1 EP 1092144A1
Authority
EP
European Patent Office
Prior art keywords
particles
microsystem
ƒ
ñ
gem
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Withdrawn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
EP19990931204
Other languages
German (de)
French (fr)
Inventor
Günter FUHR
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.)
Evotec OAI AG
Original Assignee
Evotec Biosystems 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
Priority to DE19828919 priority Critical
Priority to DE19828919 priority
Priority to DE1998153658 priority patent/DE19853658A1/en
Priority to DE19853658 priority
Application filed by Evotec Biosystems GmbH filed Critical Evotec Biosystems GmbH
Priority to PCT/EP1999/004468 priority patent/WO2000000816A1/en
Publication of EP1092144A1 publication Critical patent/EP1092144A1/en
Application status is Withdrawn legal-status Critical

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Classifications

    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027Containers 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
    • B01L3/50273Containers 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 characterised by the means or forces applied to move the fluids
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027Containers 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
    • B01L3/502761Containers 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
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B03SEPARATION 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
    • B03CMAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C5/00Separating dispersed particles from liquids by electrostatic effect
    • B03C5/02Separators
    • B03C5/022Non-uniform field separators
    • B03C5/026Non-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]
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06Fluid handling related problems
    • B01L2200/0647Handling flowable solids, e.g. microscopic beads, cells, particles
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06Fluid handling related problems
    • B01L2200/0647Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0652Sorting or classification of particles or molecules
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/08Geometry, shape and general structure
    • B01L2300/0809Geometry, shape and general structure rectangular shaped
    • B01L2300/0825Test strips
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00Moving or stopping fluids
    • B01L2400/04Moving fluids with specific forces or mechanical means
    • B01L2400/0403Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0409Moving fluids with specific forces or mechanical means specific forces centrifugal forces
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00Moving or stopping fluids
    • B01L2400/04Moving fluids with specific forces or mechanical means
    • B01L2400/0403Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0415Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00Moving or stopping fluids
    • B01L2400/04Moving fluids with specific forces or mechanical means
    • B01L2400/0403Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0415Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
    • B01L2400/0424Dielectrophoretic forces
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00Moving or stopping fluids
    • B01L2400/04Moving fluids with specific forces or mechanical means
    • B01L2400/0403Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0457Moving fluids with specific forces or mechanical means specific forces passive flow or gravitation
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S977/00Nanotechnology
    • Y10S977/902Specified use of nanostructure
    • Y10S977/904Specified use of nanostructure for medical, immunological, body treatment, or diagnosis

Abstract

The invention relates to a method and a device for manipulating particles in a fluidic microsystem (15). The particles are moved in a predetermined reference direction in a suspension liquid. According to the invention, the microsystem (15) is closed at least at the end (17a, 17b) which lies in the reference direction. The particles move under the influence of centrifugal and/or gravitational forces in the suspension liquid which is at rest in relation to the microsystem (15). The centrifugal and/or gravitational forces are essentially parallel to the reference direction. The particles are also exposed to deflection forces in the microsystem (15), the direction of said deflection forces deviating from the reference direction.

Description

Method and apparatus for manipulation of particles in microsystems

The invention relates to a method for the manipulation of particles in fluidic microsystems, in particular for moving particles in microsystems along predetermined, at least sectionally straight paths, and apparatus for implementing such a method, in particular a fluidic microsystem, in which synthetic or biological particles in a suspension liquid be manipulated, and applications of such a microsystem.

Fluidic microsystems with liquid flows structures (eg, channels) in which micro-electrodes for influencing particles (for example, biological cells) are mounted in the flow-through channels by high-frequency fields on the basis of negative or positive Dielekrophorese, for example, in the publication of G. Fuhr et al , in 'Science "(vol. 81, 1994, p 528 ff.) described.

Usually fluidic microsystems are traversed by a fluid for propelling the particles. The on both channel longitudinal sides (top, bottom) applied microelectrode lead to compartmentalization of the channel by means of high-frequency electric fields, with those of the suspended particles in the desired manner, for example via branches in adjacent channels or other structural elements may be deflected. Difficulties are especially Einspülungen the particles each at one end of the channel and the setting of generally low flow rates (some ul / h), which always bring with increasing miniaturization serious limitations. A general disadvantage of conventional fluidic microsystems is that a Losungsstromung is required for directional and controllable particle motion, the controller (for example, the flow velocity) causes problems.

The publication of MJ Madou et al. in "SPIE" Vol 3259, 1998, p 80 et seq., discloses a Zentπfugal flow system, are not set in the liquid flows m a microsystem with conventional pumps and valves, but under the action of centrifugal forces. For this purpose, the micro-system is in a disk-shaped transducer in the form of a CD-ROM disc. Analogously to the operation of CD storage media is provided to the transducer to be rotated at high speed (in the range of 100 to 10,000 revolutions per minute). The liquids in the microsystem move under the action of centrifugal forces radially outward. Simultaneous to this Flussigkeitsbewegung done certain biochemical reactions in microsystems. It is also envisaged that the particle transport Flussigkeitsbewegung how to use m a conventionally pumped Flussigkeitsstromung.

The centrifugal technology by MJ Madou et al. has the following disadvantages. Both the obtaining with enough Flussigkeitsbewegung and a disability-free as possible entrainment of particles with the liquid in the disk-shaped, planar rotor inevitably require the aforementioned high rotational speeds of the carrier. This results in a limitation of the conventional Zentrifugaldurchflußsystems to certain basic functions of the conventional centrifuging or achieving biochemical reactions. The above-mentioned micro-electrode technology for generating high-frequency electric fields m the microstructures is not applicable. Another drawback is related to the realized with the conventional centrifugal particle sorting and censuses. These are only possible by micro channels are made with a size corresponding to the size of the particles to be processed. For a given microsystem is always limited to a specific particle size. Also, with the handling of biological particles (cells, cell components) fast to interactions between the particles and the channel wall, which lead to channel clogging.

There are further centrifuge systems generally known in which not only the centrifugal forces but also, in addition, for example, magnetic or electrical forces are exposed to the sample material in the centrifuge in order to achieve specific effects, depending on the separation ratio of the centrifugal force and the additional forces. However, this centrifuge systems can not be used to manipulate biological objects. Biological objects (eg, cells) are in fact handled in relatively highly conductive solutions or suspensions (0.5 to 3 Siemens / m conductivities in the range approx.). In such conductivities would occur in the conventional centrifuge systems with relatively large electrode surfaces to undesired heating phenomena. The conventional centrifuge systems are therefore on conductivities of approx. 0.1 Siemens / m limited.

The object of the invention is to provide an improved method for manipulating particles in fluidic microsystems, with which the disadvantages of conventional micro systems are overcome and which has an extended range of applications. The object of the invention is furthermore to provide an improved micro-fluidic system with a directed particle motion, which is simplified and can be set with high accuracy. The object of the invention is also to provide such an improved application of a microsystem. These objects are achieved by methods and devices having the features according to claims 1 and 10 respectively. Advantageous embodiments and applications of the invention result from the dependent claims.

A first important aspect of the invention is to move unlike the conventional Zentrifugaldurchflußsystem with moving fluids to a method, wherein the move particles to be manipulated in a fluidic microsystem, under the effect of centrifugal forces exclusively, with essentially no liquid flows or movements in the microsystem occur. For this purpose, a number of measures are implemented which comprise in particular the use of at least one side closed fluidic microsystem, the attachment of such a microsystem to a swing rotor centrifuge means and the operation of this centrifuge instrument at a predetermined speed at which the particles in the microsystem in the desired manner move.

The inventive method enables Zentrifugierungsvor- gears at low speeds. Because of the use of an oscillating rotor system, in which a rotor as a carrier for the micro-system from a vertical orientation (at standstill or low speeds) to a horizontal orientation erects (at high speed), whether the gravitational force influence at decreasing speeds increasing the movement of the particles in microsystems. According to another aspect of the invention, a particle motion is described in at least one side closed microsystems that are at a standstill with a vertical orientation of the microsystem. The particle movement is as sedimentation under the action of gravity. According to the invention, in particular such micro systems that are equipped with micro-electrode devices for dielectrophoretic manipulation of the particle motion, combined with the principle of centrifugation. The suspended particles move due to the centrifugal force through the microchannels or other micro-structures in a micro system, in which they are separated under the effect of electrical polarization forces, for example, brought into a predetermined position, merged, sorted or permeates (without being able to escape).

An important advantage of the invention is that is possible to dispense with complex structured microsystems with dielektropho- retischer Teilchenbeeinflussung the use of difficult to control and prone to failure pumps or valves for the first time, without limiting the functionality of the microsystem occurs. There are no restrictions in terms of the channel cross dimensions. It is possible to put the micro system simultaneously with the associated control electronics in rotation. Interactions of particles (in particular biological particles) with wall areas of the microsystem can be achieved in a predetermined manner readily avoided or else with a corresponding structuring for the study of binding events.

An important advantage of the invention is that all particles are equally exposed to the centrifugal force and move according to a reference direction along vorbestim- mer channels and the separation z. As is obtained in different sub-channels or reservoirs only via deflection forces which act independently of the centrifugal force particulate specific. The deflection forces have differing from the reference direction, the angle difference is preferably less than 90 °. the particle velocity is only set via the centrifugal force. After separation, the additional forces can be turned off without having the particles mix again. It is an unexpected and important feature that the contact of particles can be avoided by the use Probenkammerwandungen a swing rotor centrifuge, which is particularly in biological specimens is important.

Details and further advantages of the invention will be described below with reference to the accompanying drawings. Show it:

Fig. 1 is a schematic perspective view of a construction according to the invention of a centrifuge with a microsystem

Fig. 2 is a schematic plan view of an inventive micro system that is set up for particle separation, and

Fig. 3 is a schematic plan view of a programmable loading microsystem according to another embodiment of the invention.

The herein described embodiments of the invention relate to the combination of a microsystem, which is equipped with a micro-electrode means for applying negative or positive dielectrophoresis (dielectrophoretic micro System), with an oscillating rotor centrifuge device. Both the dielectrophoretic microsystem (apart from at least one-sealability of channel structures) and the swing rotor centrifuge device are respectively known per se, so that will not be discussed here on the technical details. It is emphasized that the concept of the vibrating rotor centrifuge device is to be understood here to the effect in the broadest sense, that each Zentrifugenein- direction is incorporated with at least one drehzahlabhangig erectile rotor, which itself forms the microsystem and the corresponding control, in which the micro-system and associated control integrated or to which the micro-system and associated control system are mounted.

The erfmdungsgemaß manipulated particles include synthetic particles or biological objects. The synthetic particles are, for example membranumhullte structures such as liposomes or vesicles, or so-called beads or macromolecules. The biological properties include, for example, biological cells or components thereof (eg cell organelles), bacteria or viruses. The particles can be aggregates or agglomerates of such particles and / or objects. The invention is preferably implemented with cell-physiologically or medically relevant fluids with conductivities below 5 Siemens / m.

Fig. 1 is a schematic overview representation of a ER- findungsgemaßen device to illustrate the application of a dielectrophoretic system to a centrifuge device.

there are four receptacles 12, in which are respectively inserted snugly and the applied rotational speeds corresponding to a micro system 15 and an electronic control system 13 for driving the micro-system with high-frequency AC signals of different phase and amplitude on a conventional or anwendungsabhangig modified rotor of a centrifuge, with the rotation axis. 11 The control electronics is connected otherwise cable 14, plug or the microsystem 15th The power supply of the controller is preferably via an electrical connection (circumferential contact) with the solid laboratory system. The microsystem has an input repository 16, which may be different anwendungsabhangig large in size, is filled with a particle or cell suspension prior to centrifugation. Depot from the input 16 of a channel structure, details of which will be explained later runs, up to collection zones 17a, 17b, which form a coherent, at least during centrifugation end of the microsystem 15 °. This means that the end of the microsystem can be either permanently closed or opened by means of appropriate connecting elements at standstill of the apparatus and connected to predetermined additional systems for sample transfer. The microsystem 15 is arranged on the receptacle 12 so that in operation of the centrifuge device (rotation of the rotor about the rotational axis 11 with the rotational frequency ω) acting on the micro-system 15 and, in this located particles centrifugal forces in the reference direction from the entrance depot 18 toward the directed collection zones 17a, 17b. The receptacles 12 are pivoted on the rotor (not shown). When stoppage of the centrifuge, the receptacle 12 are aligned substantially vertically or with a small angle relative to the axis of rotation. When the centrifuge operation, the receptacles 12 direct function of the speed in a larger angle ranging in the horizontal direction perpendicular to the axis of rotation 11. Under the effect of the gravitational force bwz (at standstill of the centrifuge). the centrifugal forces through the particles, the electronically-controlled micro-channel system and collect in the collection zones (for example, at the closed end of the side facing away from the rotor axis part of the microsystem).

In this run, the particles according to predetermined programs (s. Below) treated. Since the particles perform a function of their density, various movements and occupy end positions, in the present invention, the advantage of the centrifugal separation and movement with the capabilities of the programmable dielectrophoresis combined. In general, negative dielectrophoresis, also used positive dielectrophoresis of the particles in exceptional cases. Another advantage of the invention is the control of the particle accelerator movement about the rotation speed (ω) of the rotor 11. Since in this case programmable variations may also be passed through, a second complex of predefined parameters is given in the particle manipulation.

The centrifuge device is provided with a (not shown) speed control, in particular, m is a reproducible and accurate Drehzahlemstellung low-speed areas is established. The speed is anwendungsabhangig selected depending on the desired speed of the particles to be manipulated and depending on the specific structure of the centrifuge. The particles of interest rates are for biological particles (such as cells) is below approx. 500 .mu.m / s (preferably in the range of 50 to 100 microns / s) and for synthetic particles (eg latex beads) at higher speeds (for example, a few mm / s). The rotational speed of the centrifuge device is selected according to the Zusammenhangen speed and centrifugal force depending on the size or bulk density of the particles. The following data refer to a distance of the microsystem from the rotor axis in the range of 1 to 10 cm. For particle diameter in the range of 50 to 600 nm (such as viruses) can be for example in the range from 1 to 1000 U / mm, the rotational speeds. For particles with a diameter of approx. 5 microns are preferred up to 100 U / mm speeds, but even higher speeds are adjustable. For particularly small particles, such as macromolecules, even higher speeds are possible. arising at a distance of about the microsystem for biological cells. 5 to 10 cm from the rotation axis 11 speeds in the range of a few revolutions per minute up to a few 100 (e.g., B. 600) revolutions per minute, preferably below 100 V / mm. The recoverable centrifugal range from pN to nN. The centrifuge device is however also designed for greater speeds, which can be set especially for small particles or for cleaning or Spulzwecke. These increased speeds can range up to the range of the rotational speeds of conventional laboratory centrifuge.

The speed of the centrifuge is also selected in dependence on the dielectrophoretic forces acting on the particles in the microsystem. Dielectrophoretic forces are dependent as a polarizing forces of the particle type and size of. The rotational speed is preferably selected so that the centrifugal forces are less than or equal to the dielectrophoretic forces on the particle. If they are not known, the speed can also be selected m reference to the following criterion. The particles have to move slowly through the channel structure that, when the pre Mikroelektrodenemrichtungen to allow sufficient time for dielectrophoretic deflection. The effectiveness or ineffectiveness of the dielectrophoretic deflection m dependence upon the speed can be detected optically or electrically by suitable sensors.

Fig. 2 shows schematically a micro-system for separating a particle mixture, consisting of large particles 21 (for example cells) and small particles 22 which are present in a suspension. The centrifugal forces acting direction of the arrow 23 (reference direction). The typical dimensions of the channel structure 24 are the following:

Width: to a few microns to several 10 mm

(Typically: 200 - 400 microns) long: several to several mm to cm

(Typically: 20 - 50 mm) High: several micrometers up to a few 100 microns

(Typically 50 microns) on the top side 25 and underside 26 of the channel 24 are micro-electrodes 27a, 27b arranged opposite, upon driving with an AC voltage (usually a frequency in the MHz range and an amplitude of a few volts) across the channel generate field barriers, which negative (due also positive) dielectrophoresis, the particles deflect (in the case shown here, the large particles).

The channel structure 24 extends from the entrance 28 to the depot closed channel ends 29a, 29b, in which just the branches in a central portion of channel. A first pair of micro-electrodes 27a, 27b is arranged immediately at the channel-side end of the input depots 28 to form a field barrier which obliquely protrudes into the channel and has the task to force the large particles 21 into the right in a plan view part of the channel 24th A second pair of micro-electrodes 27a, 27b is a field barrier which obliquely extends across the channel width to the 29b to the end of the channel leading junction and is provided immediately before the branching to the channel ends 29a, 29b and forming, the large particles 21 at this leading channel end.

An inventive manipulation method which is directed to a separation of the particles in this example, comprises the following steps.

Prior to centrifugation, the microsystem is filled with a suitable liquid. Here, the microsystem (1 s. Fig.) Is already in a receptacle 12 of the centrifuge incorporated. but the installation can also take place after the filling of the microsystem. the electrodes 27a, 27b are actuated shortly before the start of the centrifugation and in the depot input 28 for example is added at a pipetting the suspension of particles to be separated. The centrifuge device is initially in the rest state, that is, the microsystem is vertical or slightly inclined oriented to the vertical. The gravitational force acting on the particles, leads to a rapid drop different masseabhan- gig m the channel structure (sedimentation). The further movement of the particles towards the channel ends is done after the desired particle velocity e solely under the action of gravity or under the combined effect of the force of gravity and centrifugal forces. The centrifugation can thus be interpreted as sedimentation under the effect of artificially increased acceleration of gravity. The moving particles are großenabhangig separated by the electric field of the first pair of microelectrodes.

The illustration in Fig. 2 shows the conditions during the sedimentation or centrifugation. By precisely adjustable centrifugal forces on the rotational speed of the particles move into the lower part of the microsystem. According to the usual Zentrifugationsprinzipien sedimented animals the particles with the greatest density first. Because the particles are displaced 21 by the electric field barrier in the channel to the right, while the particles remain unaffected 22 thereof, it is apparent in the channel ends 29a, 29b of a separation of both particle types. The particles of each of the channel ends arrange themselves in addition as in the conventional centrifugal fugation according to their density. The microsystem shown can be considered as a basic form of an inventive shaped device, said basic form anwendungsabhangig enlarged, expanded, or may be combined with other microstructures. The advantage is that no Losungsstromung created yet addressed the particle movement and is adjustable. Such systems may also produce opposite movements, when the particles have a buoyancy. Starting from the basic form shown a erfmdungs- gemaßes microsystems can be extended, as it is known per se from the dielectrophoretic microsystems. Accordingly, the channel structure may in particular have a plurality of branches interconnected via Emzelkanale. The channels can be straight or curved. Curved channel shapes (eg, sheet, meanders, bends, angles, etc.) can be used to study differences binding of particles to the channel walls in particular.

According to a further modification of the microsystem can be connected to the receptacle 12 (s. Fig. 1) may be rotatably mounted. During a first Zentπfugationsvorganges takes place in a first orientation microsystems for example, a Te lchentrennung according to Fig. 2. Then the orientation of the micro-system is changed by 180 °, so that the gravitational and / or centrifugal forces opposite to the Pfeilπchtung 23 act. The channel ends 29a, 29b then take over the function of input depots, of which in the presence of suitable channel structures (additional lateral branches), a further distribution of the separated particles subgroups or a particular treatment (loaded with substances, electroporation u. The like.) Can be effected. There are also possible depending on the channel structure different orientation sands conclusions than the said 180 ° -Umkehr. There is also the possibility that the micro-system is rotated during centrifugation, to make the receptacle 12 so.

A further embodiment of the invention, namely a programmable micro loading system for cells or particles is shown in Fig. 3. Here is Zentrifugationskanal three parts 31a, 31b, 31c divided. In the intermediate walls are openings 32 extend through the back electrodes 33 on the top and bottom of the channel. The openings are adapted to the particle size (typically 5 to 20 times larger than the diameter). At the beginning of the channel members 31a to 31c are filled in each different solutions which serve to chemical change or loading of the particles. Thereafter (eg 31c here), the particles are inserted into a channel part. By centrifugation, the particles pass (for example, the black first, then the light) to the electrodes 33 and can thus be automatically transferred via the electric field barriers through the openings 32 in neighboring solutions.

Here, too, there is a sorting into the three channel ends 31d, 31e, 31f and at the same time an arrangement of the particles according to the mass differences.

Further characteristics of the micro-systems are that they can have openings (inflows, flow rates, drains) which can be closed, so that the particles can be easily removed or inserted after the centrifugation or before. Further, all of the microelectrodes elements (sustain electrodes for particles, micro field cages, etc.) can be incorporated, which are known for dielectrophoretic manipulation of particles per se and are used in conventional micro-systems which operate with flowing liquids. Because of the combination of gravitational and centrifugal forces with the dielekrophoretischen method of the invention is an electrically controlled or active centrifugation. In addition, combinations may be provided with the emission of optical power (laser tweezer), magnetic forces (acting on magnetic particles), or mechanical forces in the form of ultrasonic forces.

Applications of the invention are in particular:

Cell separation / fractionation, cell sorting, cell loading (molecular, nanoparticles, beads), cell discharge (molecular), cell permeation (so-called. Electroporation), cell fusion (so-called. Electrofusion), Zellparchenbildung, and cell aggregate formation.

The invention is not limited to certain sionsflussigkeiten Losungs- or suspensions. It is advantageous if the viscosity of the liquid contained in the micro-system is known. At known viscosity, the rotational speed let πthmus determine to a certain particle speed setting on the basis of table values, or by a Programmalgo-. Alternatively, however, it is also possible to detect the actual speed of the particles in the microsystem during centrifugation (for example, with an optical sensor) and the revolution speed for setting a specific Par tikelgeschw to regulate speed. It can be provided that, liquids are included with different viscosities in different portions of the channel structures, for example in parallel verlaufenen channels which are connected to each other only via an opening. In this case, however, viscosities are preferred where it is ensured that the diffusion of the fluids through the opening via the centrifugal fugationszeitraum is relatively small or negligibly small.

If the mass density of the particles is smaller than the fluid in the micro-system, the invention can be implemented modified accordingly by particles are optionally placed on the opposite side of the rotation axis side of the microsystem and under the effect of buoyancy or by combined action of the buoyancy and centrifugal forces on the other, wander the end of the microsystem. The microsystem is adjusted depending on the application with regard to the channel structure and the orientation of the electrode means. The channel transverse dimensions are substantially greater than the diameter of the individual particles in the rule. This clogging of the channels is avoided advantageously. are merely to manipulate particles with particularly small dimensions (eg bacteria or viruses, or cell organelles), the channel dimensions can be reduced correspondingly, such as to amounts below 10 microns.

The invention is implemented with a micro-system which is at least closed on one side. The closed end may be a closed channel end, a closed collection zone or even a closed cavity in the microsystem. In the inventive particle manipulation essentially no liquid movement is toward the closed end. This means, in particular in the realization of collecting zones or cavities at the closed end, that this as the entire microsystem is filled at the beginning of particle manipulation with the solution or suspension for the particles.

If it comes in manipulating the particles into clusters or temporary clogging of the channel structures, so the invention provides to increase the speed of the centrifuge for a short time so as to replace the cohesive particles and to continue to move.

Claims

PATENTANSPR├ £ CHE
1. A method for the manipulation of particles m a fluidic microsystem (15, 24, 31), wherein the particles (21, 22) are moved in a Suspensionsflussigkeit in a predetermined reference direction, characterized in that the microsystem daß (15, 24 , 31) at least at its end lying in the reference direction end (17a, 17b, 29a, 29b, 31d, 31e, 31f) is closed, the particles having an adjusted by predetermined centrifugal and / or in the relative speed Gravitationskräfte to the microsystem (15, 24, 31) move dormant Suspensionsflussigkeit, wherein the centrifugal and / or Gravitationskräfte extend substantially parallel to the reference direction, and the particles in the microsystem (15, 24, 31) are exposed Ablenkkraften whose direction differs from the reference direction.
2. The method gemäß claim 1, wherein the microsystem (15, 24, 31) mounted on a swing rotor centrifuge device, wherein the particle motion genemrichtung as sedimentation under the effect of the gravitational force and in operation of the Schwingrotorzentrifu- at standstill of the swing rotor centrifuge means under the action the Zentrifugalkräfte done.
3. The method gemäß claim 2, wherein the Ablenkkrafte include electrical polarization forces, optical Kräfte, magnetic Kräfte or ultrasonic forces.
4. The method gemäß claim 3, wherein the rotational speed of the Schwingrotorzentrifugenemrichtung is set so daß the forces acting on the particles Zentrifugalkräfte less than or equal than the Ablenkkräfte.
5. The method gemäß claim 3, wherein the speed of the swing rotor centrifuge device is set so the particles daß move so slowly daß under the action of Ablenkkräfte deflection of the particles from the reference direction.
6. The method gemäß claim 3, wherein the speed of the swing rotor centrifuge device is controlled in Abhängigkeit of the erfaßten with an optical or electrical sensor velocity of the particles.
7. The method of any preceding gemäß Ansprüche, wherein a plurality of particles movements take place under the action of Zentrifugalkräfte in separate centrifugation, wherein between the centrifugation an adjustment of the microsystem to veränderten orientation with respect to the Zentrifugalkrà NFTE is done.
8. The method of any preceding gemäß Ansprüche, wherein the speed of the swing rotor centrifuge device is gewählt in Abhängigkeit of the Größe or density of the particles.
9. The method of any preceding gemäß Ansprüche, in which the particles under the effect of Auftriebskräften opposite to the direction of the centrifugal and / or Gravitationskräfte move.
10. microsystem (15, 24, 31) with at least one channel of an input Depot (16, 28) to channel ends (17a, 17b, 29a, 29b, 31d, 31e, 31f) verl├ñuft, characterized da├ ƒ the microsystem (15, 24, 31) is adapted for mounting on the rotor of a centrifuge such da├ƒ during centrifuge operation, the Zentrifugalkr├ñfte acting on the particles in the channel, extend substantially parallel to the channel orientation and the channel ends (17a, 17b, 29a, 29b, 31d, 31e, 31f) or closed w├ñhrend centrifuge operation are verschlie├ƒbar.
11. Microsystem comprising gemäß claim 10, the device is a microelectrode, umfaßt the micro-electrodes for generating field barriers in the microsystem.
12. Microsystem gemäß claim 11, wherein the micro electrodes arranged on gegenüberliegenden Längsseiten of the channel and are adapted for application of a high-frequency alternating voltage.
13. Microsystem gemäß claim 12, wherein said microelectrodes are bandförmige electrodes schräg extend to the channel orientation and adapted to generate field barriers in the channel are.
14. Microsystem gemäß one of Ansprüche 10 to 13, which is pivotally mounted on the rotor of the centrifuge.
15. Microsystem gemäß one of the preceding Ansprüche 10 to 14, in which an electronic control of the micro-system is attached to the rotor of the centrifuge.
16. Use of a method or a device gemäß one of the preceding Ansprüche for separating, fractionating, sorting, loading, unloading, permeation, Fusion, Pärchenbildung and / or aggregate formation of synthetic particles and / or biological particles.
EP19990931204 1998-06-29 1999-06-28 Method and device for manipulating particles in microsystems Withdrawn EP1092144A1 (en)

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DE19828919 1998-06-29
DE19828919 1998-06-29
DE1998153658 DE19853658A1 (en) 1998-11-20 1998-11-20 Manipulation of biotic or abiotic particles suspended in fluid microsystem, useful for e.g. separation and aggregate formation of biological particles
DE19853658 1998-11-20
PCT/EP1999/004468 WO2000000816A1 (en) 1998-06-29 1999-06-28 Method and device for manipulating particles in microsystems

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