EP1603678A1 - Methods and devices for separating particles in a liquid flow - Google Patents

Abstract

Methods and devices for the separation of particles (20, 21, 22) in a compartment (30) of a fluidic microsystem (100) are described, in which the movement of a liquid (10) in which particles (20, 21, 22) are suspended with a predetermined direction of flow through the compartment (30), and the generation of a deflecting potential in which at least a part of the particles (20, 21, 22) is moved relative to the liquid in a direction of deflection are envisaged, whereby further at least one focusing potential is generated, so that at least a part of the particles is moved opposite to the direction of deflection relative to the liquid by dielectrophoresis under the effect of high-frequency electrical fields, and guiding of particles with different electrical, magnetic or geometric properties into different flow areas (11, 12) in the liquid takes place.

Description

 Method and device for separating particles in a liquid flow

The present invention relates to methods for separating particles in a fluidic microsystem, in particular under the action of electrophoresis, and fluidic microsystems which are set up to carry out such methods.

In biomedical and chemical-analytical technology, the separation of micro objects, such as B. particles of natural or synthetic origin or molecules in fluidic microsystems under the action of electrically or magnetically induced forces are becoming increasingly important. Two conventional separation principles, which differ fundamentally according to the type of electrical separation forces, are illustrated schematically in FIGS. 10A, B.

FIG. 10A schematically shows the separation by means of negative dielectrophoresis (see, for example, DE 198 59 459). In a fluidic microsystem 100 ', particles with different dielectric properties flow through a first channel 30'. With an electrode arrangement 40 ', exposure to high-frequency electrical fields creates a field barrier which extends across the channel 30' and which, depending on the dielectric properties of the particles, has a permeable effect or has a lateral deflecting effect in conjunction with the flow forces. Particles 22 'with a low dielectric constant (or conductivity) compared to the medium are deflected into an adjacent channel 30A', while particles 21 'with a higher dielectric constant (or conductivity) continue to flow in the channel 30'. Since dielectrophoresis depends on the particle size (see T. Schnell et al. In "Naturwissenschaften" Vol. 83, 1996, pp. 172-176), even with the same dielectric see properties a separation of the particles by size. Conventional dielectrophoretic particle separation can have disadvantages with regard to the reliability of the separation, especially with particles with similar dielectric constants and the complexity of the channel structure. The reliability of the separation can be limited, in particular when biological cells of the same type are separated into different sub-types (eg macrophages, T-lymphocytes, B-lymphocytes).

Another problem that is only solved to a limited extent in conventional dielectrophoretic particle separation can be the occurrence of undesired cell components in biological suspension samples. Cell components often cannot be distinguished from complete cells simply by their dielectrophoretic properties. Furthermore, in microsystems, they can lead to undesired accumulation of channel constrictions and blockages and even system failure. Finally, unwanted cell components can also interfere with measurements on cells, such as a patch clamp measurement. There is therefore an interest in an improved method for cleaning suspension samples which is more reliable than dielectrophoretic particle separation.

Figure 10B illustrates electrophoretic separation of particles, e.g. B. Molecules in a microstructured channel (see T. Pfohl et al. In "Physik Journal", Vol. 2, 2003, pages 35-40). At the ends of the channel 30 ', alternately formed with wide and narrow sections, electrodes 41', 42 'are arranged which, when subjected to a direct voltage, form an electrophoresis field in the channel 30'. The drift speed of the molecules in the electrophoresis field depends on their molecular weight and charge. In the wider sections of the channel 30 ', the drift speed of the larger molecules is wrestler, so that in the course of the separation, first the small molecules and later the large molecules arrive at the end of the separation path. Electrophoretic separation in fluidic microsystems has the advantage that it is not necessary to use a separating gel as in macroscopic electrophoresis. However, the principle shown in FIG. 10B has the disadvantage that a separate microsystem with adapted geometric parameters must be provided for each separation task and in particular each type of particle. It is also disadvantageous that the separation takes place in the still liquid because this is associated with a high expenditure of time and additional measures for adaptation to flow systems.

The separation principles mentioned above are also mentioned in WO 98/10267. In the channel of a fluidic microsystem, charged particles are e.g. B. electrophoretically drawn from a sample in a parallel flowing buffer solution. This technique is limited to samples with certain properties of the sample components. It is also disadvantageous because the particles can be drawn electrophoretically to the channel walls, which is particularly the case with biological materials, e.g. B. cells is undesirable.

The electrophoretic deflection of particles is also described in DE 41 27 405. Particles are moved in a still liquid under the effect of electric traveling waves. If they pass electrophoresis electrodes while moving, separation takes place according to the electrical properties of the particles. There are the same disadvantages as with the above. WO 98/10267.

It is also known to combine dielectrophoretic and electrophoretic field effects when manipulating particles in fluidic microsystems. According to DE 195 00 683, liquid-suspended particles are placed in an electrode arrangement. Supported, which forms a closed field cage (potential well) when exposed to high-frequency AC voltages by negative dielectrophoresis. In order to correct position variations due to thermal shocks during precision measurements, particles in the field cage are also shifted electrophoretically. The electrophoretic shift takes place within the framework of a control loop depending on the position variations of the particle, for example optically determined. The technique described in DE 195 00 683 is not suitable for particle separation since it represents a closed, stationary measuring system. Furthermore, the combination of dielectrophoresis and electrophoresis in a closed field cage is limited to relatively large, individual particles. Disadvantages can arise when measuring macromolecules, for example, since the effect of negative dielectrophoresis is significantly less than that of electrophoresis, so that the macromolecules can accumulate undesirably on the electrodes. Particle groups cannot be measured with this technique, since all particles require their own correction movement. A separation of particles would also be made more difficult by a dipole-dipole effect (see T. Schnell et al. In "Naturwissenschaften" Vol. 83, 1996, pp. 172-176), by means of which particle aggregation is promoted.

From DE 198 59 459 the combination of AC and DC voltages in fluidic microsystems for targeted cell fusion or -poration is also known. With this technique, the effect of the direct voltage on the fusion or poration is limited, and particle separation is not provided.

From the publication by S. Fiedler et al. in "Anal Chem." Vol. 67, 1995, pp. 820-828, it is known to generate temporal or local pH gradients detectable with fluorescent dyes by possibly pulsed direct voltage control of microelectrodes in aqueous electrolyte solutions. For pharmacological, analytical and biotechnological research, there is an interest not only in the separation of particle mixtures according to geometric (size, shape) or electrical properties (dielectric constant, conductivity), but also according to other parameters, such as. B. surface charges or charge-volume ratios. The occurrence of surface charges is described, for example, by N. Arnold et al. in "J. Phys. Chem." Vol. 91, 1987, pp. 5093-5098, L. Gorre-Talini et al. in "Phys. Rev. E" Vol. 56, 1997, pp. 2025-2034 and Maier et al. in "Biophysical J." 73, 1997, pp. 1617-1626.

The object of the invention is to provide improved methods for separating particles in liquid flows in fluidic microsystems, with which the disadvantages of conventional techniques are avoided. Methods according to the invention are to be distinguished in particular by an expanded field of application for a large number of different particles and increased reliability in particle separation. The object of the invention is also to provide improved microsystems for implementing such methods, in particular improved microfluidic separation devices, which are distinguished by a simplified structure, a high degree of reliability, a simplified control and a wide range of uses for different types of particles.

These objects are achieved by methods and devices with the features of claims 1 and 21. Advantageous embodiments and applications of the invention result from the dependent claims.

The present invention is based on the method and device based on the general technical teaching, at least one particle suspended in a liquid by a Combined exertion of separating forces, which comprise focusing dielectrophoretic separating forces on the one hand and deflecting separating forces on the other hand, such as electrophoretic separating forces, for example, in the state of a continuous flow within the liquid, ie to be shifted relative to the flowing liquid. The at least one particle can be directed into a specific flow region during the passage past at least one separation device in the fluidic microsystem depending on its geometric, electrical, magnetic or properties derived therefrom. Depending on the orientation of the deflecting separating forces (deflection direction) relative to the direction of movement of the liquid (flow direction), the flow region can comprise a specific flow path within the flow cross section of the liquid or a section of the flow that is front or rear in the flow direction.

The movement of the particle into a specific flow area enables a separation of particle mixtures during the continuous flow of the particle suspension, for example by a group of several electrodes. The separation effect is based on the specific reaction of different particles to the different deflecting and focusing field effects. In contrast to separation at field barriers, a separation section can be traversed, as a result of which the reliability of the targeted movement of individual particles, for example on certain, preferably two, flow paths can be increased. The effect of the electrical fields can be adjusted to the parameters of the particles to be separated by adjusting the field properties (in particular frequency, voltage amplitudes, clock, etc.). The invention enables a simplified construction of the electrophoretic separation device, since no gels for embedding electrophoresis electrodes or special channel shapes are required. Furthermore, gas formation can be controlled by suitable control of the electrodes in combination with the nent flow can be avoided. The invention also has advantages in particular with regard to the reliability and selectivity when separating particles into different flow paths and a high effectiveness and a high throughput of the separation.

According to the invention, a separation of particles in a compartment, in particular a channel of a fluidic microsystem, will flow through the particles in the suspended state, at least some of the particles or particles of at least one type being removed from the one to be separated under the action of a deflecting potential Sample in a predetermined direction of deflection (first reference direction, for example, to the edge of the compartment), further developed in such a way that at the same time or temporally and / or spatially alternating under the effect of an opposite potential by dielectrophoresis, in particular negative or positive dielectrophoresis, an opposite movement of the particles (second reference direction, for example away from the walls or as a collection in the middle of the channel). Particles with different electrical, magnetic or geometric properties advantageously experience the potential effects as separating forces in different ways, so that the combined exercise of the potentials results in different effective forces (potential minima) to which the particles migrate. The potential minima are e.g. B. spaced in the flow cross-section of the liquid, so that a separation in the flow on different flow paths is possible. The focusing, dielectrophoretically acting potential is preferably formed acting towards the center of the channel. If the electrodes are arranged essentially on a circular line in the channel cross section, the focusing potential can advantageously be formed radially symmetrically with respect to the flow direction in the channel. The particles which are preferably separated or separated by the technology according to the invention generally comprise colloidal or individual particles with a diameter of, for. B. 1 nm to 100 microns. There can be synthetic particles (eg latex beads, superparamagnetic particles, vesicles), biological particles (eg cell groups, cell components, cell debris, organelles, viruses) and / or hybrid particles made from synthetic and biological, different synthetic or different biological particles are built up, are subjected to the separation process according to the invention.

The electrophoretic mobility μ (v = μ ′ E) for cells advantageously depends not only on the composition of the external medium, that is to say the suspension liquid (in particular conductivity, ion composition, for example Ca ²⁺ content and pH value), but also from the cell type, so that different cell types within a cell group or different subtypes (e.g. macrophages, T-lymphocytes, B-lymphocytes) can be distinguished within a cell group with the technique according to the invention. The differentiation of the subtypes represents a particular advantage of the invention, since these are difficult to distinguish using conventional dielectrophoretic separation processes. The combination of dielectrophoretic focusing according to the invention increases the selectivity, in particular for cells of the same type.

If the particles to be separated are a mixture of biological cells and cell components, such as e.g. B. cell debris, the separation process can advantageously be used for cleaning a suspension sample with suspended biological material. The material which, for example, is inhomogeneously composed after cultivation and, for example, complete cells, dead cells, living cells or fragments of cells, such as, for. B. organelles, cell residues or protein clumps can be cleaned with the inventive method. The unwanted fragments of cells can be removed from the microsystem via certain flow paths. A disadvantageous influence on the following structural elements in the microsystem, such as B. clogging of channels by cell components can be avoided.

The deflecting potential can advantageously be generated by electrical, magnetic, optical, thermal and / or mechanical forces and thus be adapted to the most varied of applications and types of particles. Mechanical forces include, for example, forces that are transmitted through sound, additional currents or inertia. The deflecting potential can in particular be given by a gravitational field, the movement of the particles in the focussing potential (by high-frequency electrical fields) being superimposed with a sedimentation movement of the particles.

If, according to a preferred embodiment of the invention, the deflecting separating forces comprise electrical forces, under the effect of which the particles are drawn from the liquid towards the edge thereof by electrophoresis, there may be advantages with regard to the separating result. The combination of electrophoresis and dielectrophoresis for particle separation can have particular advantages in the separation of biological materials which, for example, react very differently to electrophoresis and dielectrophoresis depending on the material or particle size and can therefore be separated with a high degree of selectivity.

According to a further embodiment of the invention, the DC voltage fields for the electrophoretic particle movement can advantageously also be used for an electrical treatment of the particles. It is known that biological cells can be lysed in static electrical fields. Lysis involves an electrically induced change, for example cell destruction. The lysis serves For example, the preparation of cell material for PCR processes. Since the effect of the lysis depends on the field strength, according to a particularly preferred embodiment of the invention it is provided that certain cells from a cell mixture are deflected by the electrophoresis into a flow area near the electrodes, where the field strength is higher due to the smaller distance from the electrodes and so that the lysis occurs simultaneously with the process of particle separation.

The selectivity can still be adjusted flexibly using a suitable AC voltage control. By changing the phase position of fields, the dielectric potential can be shaped differently in the case of negative dielectrophoresis. In addition, the DC voltage control can be used to impress pH profiles which influence the electrophoretically or dielectrically effective potential.

In the combination of electrophoresis and dielectrophoresis according to the invention, the separation devices for generating the opposing potentials can advantageously be formed by a common unit. The separating device comprises electrodes which are arranged on the walls of the channel and which are acted upon by electrical fields for producing the dielectrophoresis and the electrophoresis. Advantages for the control of the separation can arise, in particular, if the electrical fields comprise high-frequency AC components and DC components that are generated simultaneously or alternately.

According to a modified variant of the invention, the deflecting separating forces can comprise electrical forces which, like the focusing potential, are generated by high-frequency electrical fields. The deflection can thus also be generated by suitably formed dielectrophoretic forces, in the high-frequency electrical signals, e.g. B. sine or square wave signals with suitable frequency components.

According to a preferred embodiment of the invention, the deflecting and focusing potentials can be alternately formed in at least one section of the channel. On average over time, the particles effectively have a potential that corresponds to the superposition of both potentials. Advantageously, the control of the at least one separation device can thus be simplified.

According to a further preferred embodiment of the invention, the two potentials can be generated alternately in different, successive sections of the channel. The structure of the microsystem can thus advantageously be simplified.

It can be particularly advantageous for the preservation of the separation result if the flow paths open into further, separate compartments of the microsystem. If the separated fractions have flowed into the subsequent compartments, subsequent mixing is impossible. This separation of the fractions can be particularly effective if the compartments are separated from one another by channel walls or electrical field barriers.

According to a further embodiment of the invention it can be provided that in the compartments there is a further separation according to the principle of the invention, for example a combined exercise of electrophoretic and dielectrophoretic field effects. In this way, hierarchical separation principles can advantageously be implemented with a separation into coarse and subsequently into fine fractions. However, the sequence of several separation processes in the manner of a cascade into different fractions is not absolutely necessary for the provision of the separate compartments. Rather, it is possible to implement the separation cascade with flow paths in a common, sufficiently wide channel of the microsystem.

According to a modification of the invention, the flow in the microsystem can be directed such that particles pass through a separation stage several times, so that the separation result can advantageously be improved.

Further advantages of the invention can result if, after the separation (deflection into different flow areas), a detection takes place in the flow areas to check the separation result. The detection includes, for example, an optical measurement known per se (fluorescence measurement or transmitted light measurement) or an impedance measurement known per se.

Advantageously, depending on the measurement result, e.g. For example, the control parameters of the deflecting and focussing potentials can be adjusted in such a way that the separation effect improves as a function of the separation quality or occurring separations.

The effectiveness of the separation according to the invention can advantageously be increased if the particles first pass a dielectrophoretic or hydrodynamic array element. Individual particles or a group of particles are lined up on this on a specific flow path, on which they pass the separation devices, for example the electrodes for performing dielectrophoresis and electrophoresis.

If, according to a further variant of the invention, a pH gradient is generated in the channel of the microsystem in which the particle separation takes place, advantages for the separation effect can result. The effect of the ab- steering potential, such as B. the electrophoretic cell particle movement depending on the location. This enables particle deflection into different flow paths depending on the particle position along the direction of flow through the channel. The microsystem is advantageously of a particularly simple construction if the pH gradient is generated electrochemically using the electrodes which are also used to form the DC voltage field for electrophoresis.

Another advantage of the invention is that the particle separation can take place simultaneously in several spatial directions. According to the invention, a plurality of deflecting potentials with different directions of action can be generated with the focusing potential, which is then preferably formed towards the center of the channel, in order to simultaneously separate the particles to be separated in relation to two different features, such as, for. B. to separate dielectric and magnetic properties.

Another object of the invention is a fluidic microsystem which is set up to implement the method according to the invention and in particular comprises at least one separating device for exercising focusing dielectrophoretic separating forces and deflecting separating forces. A fluidic microsystem with at least one compartment, for example a channel for receiving a flowing liquid with suspended particles and a first separating device for generating a deflecting potential which pulls the particles in the first reference direction, for example from the middle of the flow, is in particular with a second separating device equipped, which is set up to generate at least one focusing, opposite potential. Under the action of high-frequency electric fields, the particles are separated from the side by means of dielectrophoresis with the second separating device Walls of the channel and / or electrodes or other parts disposed thereon are repelled by separating devices.

According to a preferred embodiment of the invention, the first separating device is set up to generate electrical, magnetic, optical and / or mechanical forces. For example, it comprises an electrode device with electrodes or electrode sections and in this case forms a common deflection unit with the second separating device. Alternatively, the first separation device comprises a magnetic field device, a laser or an ultrasound source. According to the invention, these components are combined for the first time to separate flowing particles with dielectrophoretic manipulation.

If the separation devices form a common deflection unit, this advantageously results in a simplified structure of the microsystem. The deflection unit preferably comprises electrodes which, like microelectrodes known per se, are constructed in fluidic microsystems. The electrodes can be controlled alternately in time.

The electrodes for combined dielectrophoresis and electrophoresis are preferably arranged on the inside of the walls of the compartment. With this design, there can be advantages in terms of the effectiveness of the field effect.

Since the separating devices can act at the same time or alternating in time and / or space, so that particles are directed to different flow paths depending on the effective potentials acting on average over time, it is advantageously possible for the first and second separating devices to be separated in different, successive ones Sections of the compartment are arranged. The separators include, for example, electrode sections which can be controlled in each case for dielectrophoresis or electrophoresis.

Further details and advantages of the invention are described below with reference to the accompanying drawings. Show it:

FIG. 1: a schematic top view of a first embodiment of a microsystem according to the invention (detail),

FIG. 2 shows a cross-sectional view of the microsystem according to FIG. 1 along the line II-II,

FIG. 3: a cross-sectional view of the microsystem with schematically illustrated potential relationships,

FIGS. 4 to 7: schematic top views of further embodiments of microsystems according to the invention (detail), and

FIG. 8 shows a schematic cross-sectional view of an electrode arrangement to illustrate an embodiment of the invention, in which a plurality of deflecting potentials are generated,

FIG. 9 shows a graph to explain the generation of a distracting potential by superimposing dielectrophoretic forces,

FIGS. 10A, B: schematic illustrations of conventional microsystems with a dielectrophoretic (A) and an electrophoretic (B) separation. The invention is described below with reference to the separation of particles in the channel of a fluidic microsystem. Fluidic microsystems are known per se and are therefore not described in further detail. The implementation of the invention is not limited to the illustrated channel structures, for example in chip structures or in hollow fibers, but can generally also be implemented in differently shaped compartments.

The combination according to the invention of focusing and deflecting forces, the superimposition of which for the particles to be separated, depending on the particle properties, lead to different equilibrium positions (flow paths or sections) in the liquid flow, with two separating devices or with a separating device acting in a combined manner, with reference to the preferred exemplary embodiment Combination of dielectrophoresis and electrophoresis described. If the deflecting force has at least one vector component in a reference direction (deflection direction) perpendicular to the direction of liquid movement in the channel, the dielectrophoresis has a focusing effect from the walls of the channel into the interior of the flow cross section of the flowing liquid, while the electrophoresis reversed to the outer edge of the Flow profile, in particular directed towards electrodes on the walls. Analogous to the principles explained below, other deflecting forces can be used. If, on the other hand, the deflecting force runs parallel to the direction of the liquid flow, the dielectrophoresis has a focusing effect along the liquid flow, the particles in the electrophoresis field being moved at different speeds by modulating the dielectrophoretic effect.

FIGS. 1 and 2 show sections of a fluidic microsystem 100 according to the invention in an enlarged schematic plan view and cross-sectional view. The microsystem 100 contains a channel 30 which is delimited by the side channel walls 31, 32, the channel bottom 33 (top view in FIG. 1) and the top surface 34. Electrodes 40 are formed on the channel bottom 33 and the top surface 34 as a separating device. Furthermore, funnel electrodes 51, 52 of a dielectric alignment element 50 are provided. The structure of the microsystem 100 and the design of the electrodes and their electrical connection are known per se from microsystem technology. The channel has a width of approx. 400 μ and a height of approx. 40 μm (these relationships are not shown to scale in the figures). The lateral electrode distance in the planes of the channel bottom 33 and the top surface 34 is, for example, 70 μm, while the vertical distance of the electrodes lying opposite one another corresponds to the channel height approx. Is 40 μm.

The electrodes 40 comprise straight electrode strips which extend in the longitudinal direction of the channel 30, i.e. extend in the flow direction through the channel. The electrodes 40 are divided into individual electrode segments 41, 42, ... In each case a group of electrode segments forms an electrode section which can be controlled separately. Each segment has a width of around 50 microns and a length of z. B. 1000 microns. Each electrode section is connected to a control device 70 (shown here only for the electrodes 41, 42).

The control device 70 is set up to apply voltages to the electrodes 40 such that the particles flowing past in an electrode section (for example 45-48, see FIG. 2) are repelled by the electrodes by means of negative dielectrophoresis and / or an electrophoretic drift movement perpendicular to the Direction of flow are exposed. The control device contains an AC voltage generator 71 and / or a DC voltage generator 72, which are connected to the electrodes. The AC voltage generator 71 can be equipped with an actuating device, with which the amplitudes of high-frequency AC voltages on the electrodes can be set.

To carry out the method according to the invention, the suspension liquid 10 (carrier liquid) with particles 20 flows through the channel 30. The flow rate of the suspension liquid 10, which can be adjusted with a syringe pump, is z. B. 300 μm / s. First, the particles 20 are preferably lined up with the dielectric line-up element 50. The funnel electrodes 51, 52 are operated, for example, with a high-frequency alternating voltage (f = 2 MHz, U = 20 V _pp ) in order to place the particles 20 on a flow path 11 in the middle of the channel 30 to focus. Alternatively, a hydrodynamic alignment element can be provided, in which the particles 20 are focused with additional enveloping flows.

After the particles have been lined up, they reach the area of the electrodes 40. These are driven, for example, alternately with an AC voltage and a DC voltage with a clock frequency in the range from 1 to 10 Hz (AC voltage: f = 2.5 MHz, U = 20 V _pp , DC voltage U = 50 V, duration t = 80 μs). By matching the voltage and frequency parameters of the high-frequency AC voltage to the flow velocity and setting the DC voltage parameters (pulse time, voltage and clock frequency), the smaller particles can be removed from the original flow path 11 into an adjacent flow path 12 by a few 10 μm within a few seconds (see Figure 2) pull out while the larger particles remain in the original flow path 11.

The potentials acting on the particles are illustrated schematically in FIG. 3. For electrophoresis, a DC voltage field is generated, which generates a potential Pl falling transversely to the flow cross section. Particles experience an outward force in the potential Pl (distracting potential, steering direction transverse to the flow direction). The high-frequency control of the electrodes generates an opposite, inward, focusing potential curve P2a or P2b. Negative dielectrophoresis is based on particle polarization, which has a greater effect on the large particles than on the small particles. In the high-frequency field, therefore, the large particles 21 experience the potential P2a and the small particles 22 the flat potential P2b. The superimposition of the two cases with the focusing potential Pl gives the effective potentials Pa, Pb corresponding to the solid lines. While the low potential P2a is hardly changed by the electrophoresis, there is a shift of the potential minimum from the center of the channel to the outside for the flat potential P2b. For the large particles, the dielectrophoretic, focusing forces are so large that they each compensate for the electrophoretic deflection, while this is not the case for the small particles 21. The separate flow paths 11, 12 are formed accordingly. Different flow velocities can be given in the flow paths 11, 12. For example, with a laminar flow in the channel, the flow velocity near the channel wall is lower than in the middle of the channel. According to the invention, particles of different properties can thus be focused in areas with different flow velocities, which can improve the selectivity.

Analogous effects arise with particles with different relative dielectric constants or with different net charges, for example surface charges.

The separation with a mixture of particles 20 was shown experimentally, the smaller particles 21 with a diameter of 1 μm (“Fluospheres” sulfate microspheres, Molecular Probes) and larger particles 22 with a diameter of 4.5 μm (Polybeadpolystyrene, 17135, Polysciences ) include. As a suspension liquid, Cytocon Solution I (Evotech Technologies GmbH, Hamburg, Germany) was used. Since the negative dielectrophoresis has a considerably weaker effect on the small particles than on the large particles, the small particles can be pulled out of the middle flow path 11 by the electrophoretic force.

The electrode is controlled, for example, according to the following scheme:

Alternatively, the electrodes can be controlled according to the following scheme (rotating electric field):

To illustrate the combination of dielectrophoresis according to the invention with other deflecting forces, FIG. 1 schematically shows a separating device 40A (shown in dashed lines) the laser tweezer or a sound source to exert mechanical forces e.g. B. by ultrasound. FIG. 4 shows features of modified embodiments of the invention. Deviating from FIG. 1, it can be provided that the flow path 11 is also shifted outwards from the center of the channel 30 by shifting the potential minimum of the dielectrophoresis by correspondingly asymmetrical activation of the electrodes 40. Furthermore, it can be provided that the flow paths 11, 12 open into separate compartments 35, 36 of the channel 30, which are separated from one another by channel walls or (as illustrated) by an electrical field barrier. The electrical field barrier is created by at least one barrier on the electrode 60 that extends in the channel direction.

In the embodiment illustrated in FIG. 5, electrodes 41, 42 for electrophoresis and centrally at least one electrode 43 for dielectrophoresis are located in a channel 30 on the side of the channel walls 31, 32 and / or on the bottom surface 33. The electrode 43 is provided with an electrically insulating passivation layer 43a in a manner known per se. The passivation layer 43a has two functions. Firstly, it prevents a loss of the direct current field for electrophoresis, secondly, it prevents permanent accumulation and possibly associated denaturation of particles or electrochemical reactions on the electrodes. The electrodes 41, 42 and 43 are each connected to a DC voltage source and an AC voltage source.

Optionally, the channel edge can be realized using porous materials (e.g. hollow fibers). This makes it possible to apply additional external chemical gradients (eg a pH profile). Furthermore, the at least one electrode 43 and the electrodes 41, 42 can be arranged offset in the flow direction for electrophoresis. For particle separation, flushed-in micro objects (for example macromolecules) are drawn to the central electrode 43 by positive dielectrophoresis. Simultaneously or with alternate activation of the electrodes, the micro objects are drawn to the edge of the channel 30 by electrophoresis. The separation is based on the principles described above of a different impact of the combination of dielectrophoresis and electrophoresis on the different particles.

Alternatively, the following procedure can be implemented with the arrangement according to FIG. 5. The particles are first collected at the central electrode 43 by dielectrophoresis. The lateral flow 10 through the channel 30 is then stopped and the micro-objects are separated by electrophoresis. After the electrophoretic separation into different flow paths, the flow 10 is continued. The main advantage of the interruption of the flow transport through the channel, which is optionally provided during electrophoresis according to the invention, is that the electrophoresis can be made more selective by the previously defined starting conditions.

If several, possibly passivated, electrodes 43.1 to 43.5 are provided for dielectrophoresis, the structure shown in FIG. 6 results. Electrodes 41, 42 for electrophoresis and electrodes 43.1 to 43.5 for dielectrophoresis (electrical leads not shown) are located in channel 30 in three dimensions on the side walls. Dielectrophoresis electrodes are in the same number and arrangement as the electrodes 43.1 to 43.5 on the cover surface (not shown). The electrodes 43.1 to 43.5 are acted upon by signals which are 180 ° out of phase between adjacent electrodes (for example 43.1, 43.2) and in phase for electrodes one above the other (for example 43.1 and the opposite electrode on the cover surface) are. The particles 20 washed in with the flow 10 comprise, for example, two types, one type of which is not addressed by electrophoresis. The particles 20 are arranged electrophoretically (negative dielectrophoresis) initially in the space between the electrodes standing one above the other (covered in the view). The particles of one type are deflected only when they pass the electrophoretic field, while the other type remains unaffected.

In the embodiment according to FIG. 7, many, possibly passivated, electrodes 43.1 to 43.11 for dielectrophoresis are also arranged between the electrodes 41, 42 for electrophoresis. On the top surface (not shown) there are dielectrophoresis electrodes in the same number and arrangement as electrodes 43.1 to 43.11. The first pair of dielectrophoresis electrodes 43.1, 43.2 is provided with a dielectric alignment element 50 in order to increase the selectivity. In contrast to the embodiments described above, in FIG. 7 the direct voltage electrophoresis field (deflection direction) is aligned parallel to the flow direction of the liquid 10 (see arrow) through the compartment 30.

When the dielectrophoresis electrode array is activated with a 180 ° phase shift between adjacent and opposite electrodes or with a 90 ° phase shift, the particles 20 arrange themselves between the electrodes (negative dielectrophoresis). The dielectrophoresis electrodes form a periodic i. A. asymmetric modulated potential, on which the electrophoresis potential between the electrodes 41, 42 is superimposed. The asymmetrical modulation of the dielectrophoresis fields means that alternately higher or lower field strengths are set between adjacent electrode strips of the arrays 43.1 to 43.11. The electrophoresis potential between the electrodes 41, 42 is not kept constant over time, but switched periodically or randomly. With that leaves a highly sensitive separation based on the principle of the so-called Brown 'ratchet ("Brownian ratchet" or vibrating ratchet, see H. Linke et al. Physikalische Blätter Vol. 56, No. 5, 2000, pp. 45-47). In the Brown 'see ratchet, the traveling speed of particles through Brown' see movement strongly depends on the particle size. The separation takes place in different flow sections in the flow direction depending on the different migration speeds of the particles. A particular advantage of this procedure is that the separation can be sensitively controlled by superimposing the Brown movement, the electrophoresis and the dielectrophoresis using several adjustable parameters. This embodiment of the invention is particularly suitable for molecular separation (eg separation of DNA molecules or DNA fragments, which are all negatively charged in a physiological environment).

In the case of a mixed population of differing charges (+/-), the input channel with the alignment element 50 should lie in the center of the array of the dielectrophoresis electrodes, so that objects of different charges are moved in different directions electrophoretically. Asymmetric potential for positive dielectrophoresis can also be realized in planar structures, e.g. B. by applying asymmetrical, ie relative to the channel longitudinal direction, for example, different thickness passivation layers.

FIG. 8 illustrates, like FIG. 2, a cross-sectional view of a fluidic microsystem 100 with four electrodes 45-48. A focusing potential is generated with these electrodes, the potential minimum of which lies in the center of the channel. At the same time, analogously to FIG. 3, a first electrical potential acting in the x-direction for an electrophoretic field effect and additionally in the y-direction a magnetic field gradient is generated to form a second deflecting potential. The magnetic field gradient is generated with a magnetic field generating element 49 formed, which comprises, for example, a permanent magnet or a current-carrying conductor isolated from the liquid. In a departure from the illustrated embodiment, the magnetic field generating element can be arranged at a distance from the channel.

As the particles move through the channel in the z direction, they experience a deflection in both x and y spatial directions, the strength of which depends on the dielectric and magnetic properties of the particles to be separated. This embodiment of the invention is used, for example, to separate latex-coated, superparamagnetic particles with the aim of obtaining fractions with a high monodispersity.

The curve in FIG. 9 illustrates the dielectrophoretic force fdiei _* - normalized to the respective volume - which acts on a particle in the alternating field, as a function of the frequency of the alternating field. The simulation results relate to latex beads with diameters of 0.5 μm, 1 μm, 2 μm and 5 μm (curves from above) with a conductivity of 0.7 mS / m and DK = 3.5 in water. The symbolically illustrated electrodes are arranged analogously to FIG. 1 and are alternately or superimposed with a signal which contains frequency components below 100 kHz and above 1 MHz. The low- and higher-frequency signal components are generated, for example, with the same amplitude over time in the square mean, but different phase relationships illustrated in the picture inserts. The higher-frequency signal focuses the particles towards the center of the channel through negative dielectrophoresis. The low-frequency signal, on the other hand, acts depending on the particle size through positive or negative dielectrophoresis, which overlaps with the focusing effect of the higher-frequency signal. As a result, the smaller particles are deflected to the top left, while the larger particles (e.g. 5 μm) collect on a diagonal line at the bottom right. decision speaking particles of different sizes get into different flow paths within the flow through the channel.

The features of the invention disclosed in the above description, the drawings and claims can be of importance both individually and in combination for the implementation of the invention in its various configurations.

Claims

claims

1. A method for separating particles (20, 21, 22) in a compartment (30) of a fluidic microsystem (100), with the steps:

- Movement of a liquid (10) in which particles (20, 21, 22) are suspended with a predetermined flow direction through the compartment (30), and

- Generation of a deflecting potential in which at least some of the particles (20, 21, 22) are moved in a deflection direction relative to the liquid, characterized by the further steps:

Generation of at least one focusing potential, so that under the action of high-frequency electric fields at least some of the particles are moved relative to the liquid by dielectrophoresis in the opposite direction to the deflection direction, and

- Direction of particles with different electrical, magnetic or geometric properties in different flow areas (11, 12) in the liquid.

2. The method of claim 1, wherein the deflection direction deviates from the flow direction and has a component transverse to the flow direction.

3. The method according to claim 2, wherein the deflection direction is perpendicular to the flow direction towards at least one of the side walls of the compartment, the deflecting potential is generated by electrical, magnetic, optical, thermal and / or mechanical forces, and the flow areas are flow paths (11 , 12), which correspond to different potential minima, for the respective particles due to the superposition of the deflecting and focusing Potentials are formed during the passage through the compartment on average over time.

4. The method of claim 3, wherein the deflecting potential is formed by a DC field, under the action of which the particles are drawn to at least one of the side walls of the compartment (30) by electrophoresis.

5. The method of claim 4, wherein the particles comprise biological cells, at least some of which are lysed under the action of the DC field.

6. The method according to claim 3, wherein the liquid (10) comprises a suspension of biological material containing biological cells and cell components, the cells being separated from the cell components under the action of the DC voltage field.

7. The method according to claim 4, in which on the walls (31-34) of the compartment (30) electrodes (40) are arranged which are acted upon by electric fields for producing the dielectrophoresis and the electrophoresis.

8. The method according to at least one of the preceding claims, in which the deflecting and focussing potentials are generated alternately in time in at least one section of the compartment (30) or geometrically alternately in different, successive sections of the compartment (30).

9. The method according to the preceding claims 5 and 6, wherein the electrical fields comprise high-frequency AC components and DC components that are generated simultaneously or alternately.

10. The method according to claim 7, wherein a plurality of focusing potentials are generated with an electrode array (43.1 to 43.11) between the two electrodes (41, 42), the particles depending on their electrical or geometric properties on the different flow paths ( 11, 12) are steered.

11. The method according to at least one of the preceding claims 2 to 9, in which the particles (20, 21, 22) are directed onto at least two separate flow paths (11, 12).

12. The method according to claim 11, wherein the at least two flow paths (11, 12) open into further, separate compartments (35, 36) of the microsystem (100).

13. The method according to claim 12, wherein the at least two flow paths (11, 12) open into separate compartments (35, 36) of the microsystem (100), which are separated by compartment walls or electrical barriers (60).

14. The method according to claim 1, wherein the deflection direction runs parallel to the flow direction and a plurality of focusing potentials are generated, which are asymmetrically modulated parallel to the deflection direction and in which the particles pass through the deflecting potential at different speeds.

15. The method according to at least one of the preceding claims, wherein the particles (20, 21, 22) in front of the electrodes flow past a dielectrophoretic or hydrodynamic array element (50).

16. The method according to at least one of the preceding claims, in which a pH gradient is generated in the channel (30).

17. The method according to claim 16, wherein the pH gradient is generated by electrical DC fields which are provided for the electrophoretic separation of the particles.

18. The method according to at least one of the preceding claims, in which after the particles are directed onto the different flow paths (11, 12) the particles are detected.

19. The method according to at least one of the preceding claims, in which the deflecting and the focusing potential are formed by a plurality of superimposed alternating voltages with different frequencies.

20. The method according to at least one of the preceding claims, in which at least two deflecting potentials are generated with different deflection directions.

21. Fluidic microsystem, with:

- At least one compartment (30) through which a liquid with particles (20, 21, 22) flows in a predetermined flow direction, and

- A first separating device for generating a deflecting potential, in which the particles (20, 21, 22) are moved in a deflecting direction, characterized by

- A second separating device with electrodes (40) for generating at least one focusing potential, so that the particles are moved opposite to the deflection direction by dielectrophoresis.

22. The microsystem according to claim 21, wherein the deflection direction deviates from the flow direction.

23. Microsystem according to claim 21 or 22, in which the first separating device is set up to generate electrical, magnetic, optical and / or mechanical forces.

24. The microsystem according to claim 23, wherein the first separation device comprises electrophoresis electrodes, a magnetic field device, a laser or an ultrasound source.

25. Microsystem according to at least one of the preceding claims 21 to 24, in which the first and second separating devices are arranged separately in different, successive sections of the compartment (30).

26. Microsystem according to claim 21, 23 or 25, wherein the first and second separating means form a common deflection unit which comprises the electrodes (40).

27. Microsystem according to claim 26, in which the deflection unit can be controlled alternately with alternating and direct voltages.

28. Microsystem according to claim 24, in which an electrode array (43.1 to 43.11) of electrode strips is arranged between the electrophoresis electrodes (41, 42), which can be controlled individually with high-frequency alternating voltages.

29. Microsystem according to claim 21, in which the deflection direction runs parallel to the flow direction.

30. Microsystem according to at least one of the preceding claims 21 to 29, in which the electrodes (40) are arranged on the inside of the walls of the compartment (30).

31. Microsystem according to at least one of the preceding claims 21 to 30, in which the compartment (30) opens into separate compartments (35, 36) of the microsystem (100).

32. Microsystem according to claim 31, in which the compartments (35, 36) of the microsystem (100) are separated by compartment walls or electrical barriers (60).

33. Microsystem according to at least one of the preceding claims 21 to 32, in which a dielectrophoretic or hydrodynamic alignment element (50) is arranged in the compartment (30) in front of the separating devices.