Method and fluidic microsystem for dielectrophoretic manipulation of suspended particles

Abstract

The invention relates to a method for operating a fluidic microsystem (100) for the dielectrophoretic manipulation of suspended particles (1) having a particle diameter in a suspension liquid (2), wherein the microsystem (100) comprises: - a channel (10) having a longitudinal direction; - an electrode device (20) having an electrode (21), the longitudinal extent of which deviates from the longitudinal direction of the channel (10) and which has individually controllable electrode segments (22) for producing dielectrophoretic forces which act on the particles (1), each electrode segment (22) having a deflection angle α, relative to the longitudinal direction of the channel (10), and a segment length (si), which determine a segment offset (Di) perpendicular to the longitudinal direction of the channel (10); and - a control device (30). The method comprises: - producing a flow of the suspension liquid (2) with a flow velocity so that the particles (1) successively pass through an interaction region of the electrode (21), which interaction region is spanned by the electrode segments (22); and - activating the electrode segments (22) in order to deflect the particles (1) onto predetermined motion paths (4, 5), which are determined by a superposition of flow forces in the flow of the suspension liquid (2) and of the dielectrophoretic forces at the electrode segments (22). During the passage of each particle, each of the electrode segments (22) which are passed by the particle (1) is activated in a clocked manner for a predetermined activation duration, according to the desired motion path (4, 5), the activation duration of each electrode segment (22) being determined by the quotient of the segment length (si) of the electrode segment (22) and the flow velocity. The electrode segments (22) are dimensioned such that the segment offset (Di) of each electrode segment (22) is less than the particle diameter. For the deflection of each particle (1), at least two successive electrode segments (22) cooperate.

Description

[0001] The invention relates to a method for the dielectrophoretic manipulation of suspended particles, in particular for the sorting of suspended particles, such as biological cells or microcompartments, in a fluidic microsystem. Furthermore, the invention relates to a fluidic microsystem configured for the dielectrophoretic manipulation, in particular for the sorting, of suspended particles. Applications of the invention include, for example, the processing of particles, in particular biological cells, microcompartments, or other micro-objects, in chemistry, medicine, biology, or biochemistry.

[0002] The following prior art, which represents the technical background of the invention, is referred to in the present description: [1] M. Boutros et al. (2015): Microscopy-based high-content screening. Cell 163, 1314-1325; [2] N. Godino et al. (2019): Combining dielectrophoresis and computer vision for precise and fully automated single-cell handling and analysis. Lab Chip 19, 4016-4020; [3] C. - T. Ho et al. (2005): Micromachined electrochemical T-switches for cell sorting applications. Lab Chip 5, 1248-1258; [4] M. Kirschbaum et al. (2008): T cell activation on a single-cell level in dielectrophoresisbased microfluidic devices. J Chromatogr A. 1202 (1), 83-89; [5] B. Landenberger et al. (2012): Microfluidic sorting of arbitrary cells with dynamic optical tweezers. Lab Chip 12, 3177-3183; [6] M. Li et al. (2018): Cellular dielectrophoresis coupled with single-cell analysis. Analytical and Bioanalytical Chemistry 410, 2499-2 515; [7] G. Meineke et al. (2016): A microfluidic opto-caloric switch for sorting of particles by using 3D hydrodynamic focusing based on SLE fabrication capabilities.Lab Chip 16, 820-828; [8] N. Nitta et al. (2018): Intelligent Image-Activated Cell Sorting. Cell 175(1 ):266-276; [9] S. Sakuma et al. (2017): On-chip cell sorting by high-speed local-flow control using dual membrane pumps. Lab Chip 17, 2760-2767;

[10] Y. Shen et al. (2019): Recent advances in microfluidic cell sorting systems. Sensors & Actuators: B. Chemical 282, 268-281;

[11] DE 198 15 882 A1;

[12] DE 198 60 117 A1;

[13] DE 198 60 118 C1;

[14] US 2013 / 256197 A1;

[15] EP 3 410 107 A1;

[16] Kazemi Bahar et al. (2018) "Numerical simulation of dielectrophoretic particle separation using slanted electrodes", PHYSICS OF FLUIDS, Bd. 30, Nr. 10, S. 102003;

[17] US 2006 / 177815 A1; und

[18] US 2012 / 031759 A1. .

[0003] In biology and medicine, there is strong interest in the characterization and processing of heterogeneous particle samples, such as heterogeneous cell samples. It is well known that conventional flow cytometry allows for the characterization of large cell samples within a short time using very simple markers ("low-content" markers), such as size, granularity, or integral fluorescence intensity of the biological cells. To also capture the spatially resolved structural properties of individual cells or artificial microcompartments ("high-content" markers), microscopy techniques are typically used. The "high-content" markers are highly relevant for modern biomedicine, as, for example, biological processes are often determined by the spatial arrangement of cellular components [1]. Thus, important cellular properties, such as…the antigen specificity of immune cells, coagulation disorders of blood platelets or the metastasis potential of cancer stem cells, the strength and type of interaction between cells, the local protein distribution within the cell and / or the number and arrangement of cellular components.

[0004] For functional analyses, it is crucial not only to identify cells based on their phenotype but also to be able to sort them. This is possible using fluorescence-activated cell sorting (FACS) based on flow cytometry. However, currently available FACS instruments cannot be combined with microscopy techniques, which means that only low-content markers, and not high-content markers, can be detected. Due to the complexity of biological processes, FACS is therefore insufficient for many questions in biomedicine to adequately differentiate individual cell types or subpopulations.

[0005] A combination with microscopy techniques is possible for many fluidic microsystems with planar channel structures. Using these systems, microscope image data from microscopy techniques or measurement data from other complex measurement techniques can thus be directly used for the identification and sorting of cells based on high-content markers. The sorting of suspended cells moving sequentially in a channel of a fluidic microsystem can be achieved using micromechanical ([3]), optical ([5]), hydrodynamic ([7], [8] or [9]), electrokinetic [2] or other

[10] forces.

[0006] While micromechanical approaches are complex and expensive to manufacture, hydrodynamic forces can often only be applied with low precision and require large or complex micromechanical actuators, which hinders the simple parallelization of such methods. Optical forces are very weak and therefore only applicable at low flow velocities and within the field of view of a microscope, which severely limits their application. Electrokinetic forces have the advantage that they can be generated with electrodes integrated in the channel, in a highly parallel manner, and with high local precision and integration density, independent of the measurement of the cells, particularly the optical field of view of a microscope. The application of electrokinetic forces is based, for example, on dielectrophoresis, in which a force effect is generated by polarizing the cells in inhomogeneous high-frequency electric fields ([6]).

[0007] Typically, in a channel of a fluidic microsystem, electrodes arranged on the top and bottom of the channel are subjected to high-frequency electric fields to generate dielectrophoretic forces, so that repulsive forces are exerted on the cells and a field barrier is formed by the electrodes (negative dielectrophoresis, see e.g. [4] and

[11] to

[18] ).

[0008] An example of a channel 10' of a fluidic microsystem 100' with two pairs of electrodes 21A', 21B' is shown in Figure 2(Prior art, see e.g.

[11] ,

[12] and

[13] ) shown, with only the electrodes on the underside of the channel depicted. Depending on whether an electric field is applied to the electrodes 21A', 21B' or not, it is either impossible or possible for suspended cells 1' to pass through the respective field barriers. For example, the cells 1' can pass through the non-activated electrodes 21A' but cannot pass through the activated electrodes 21B'. Since the electrodes 21A', 21B' are arranged at an angle α' to the flow direction in the channel, the cells are guided to a different flow path in the channel by the superposition of the flow forces in the suspension fluid flow and the dielectrophoretic forces along the electrodes or field barriers, respectively, or they continue to follow their original flow path.

[0009] In a sorting application, exemplified in Figure 3(as shown in the state of the art, see e.g.

[11] ,

[12] and

[13] ), cells 1A', 1B' in the fluidic microsystem 100' are to be flushed successively through the planar channel 10' equipped with a microscope 40' and directed at a switch with an electrode 21' depending on properties of the cells 1A', 1B' detected with the microscope 40' to different sub-channels 11', 12' of the microsystem 100'.

[0010] The sequence of cells 1A', 1B' in channel 10' should be maintained or at least monitored by measurement at least during the time interval between image acquisition with the microscope 40' and the sorting process (especially during the period required for image processing), as otherwise a correct assignment between the captured, analyzed and sorted cell is hardly possible.

[0011] The reliability of the electrode function, with which the particles can be deflected from their hydrodynamic flow lines (paths of motion) at a given flow velocity, depends on the deflection angle of the deflecting electrode relative to the channel and flow direction (angle between the channel direction and the longitudinal direction of the electrode extension). The smaller the deflection angle, the more reliably the electrode operates. This means that, given a specific target offset (displacement of the particles perpendicular to the flow direction), the required interaction length (or: detection length) of the electrode increases with decreasing deflection angle. The reliability, in particular, of the sorting function of electrode 21' is thus related specifically to the interaction length L' of electrode 21' in the longitudinal direction (flow direction) of channel 10'. The electrode 21' according to Figure 3has a larger interaction length L' and therefore a greater reliability of the sorting function than electrode 21B' according to Figure 2 .

[0012] The interaction length L' also determines the minimum distance between the cells 1' flowing one after the other in channel 10', at which the individual cells 1' can still be handled independently of each other and their error-free sorting is possible without also detecting or influencing subsequent cells 1A'. For example, in the Figure 3 In the situation shown, the cell density is so high that the different cells 1A', 1B' cannot be separated as desired and directed to the different sub-channels 11', 12'.

[0013] There is an interest in implementing flow-through sorting processes with the highest possible throughput (number of cells processed per unit of time). The throughput is equal to the product of the flow velocity and the cell density of the sample. A conflict exists between these two parameters: a high flow velocity requires a large electrode interaction length to maintain the reliability of the sorting function, while a short electrode interaction length is desirable for a high cell density.

[0014] To achieve high throughput, i.e., for sorting with high cell density and high flow velocity, there is an interest in using electrodes with a short interaction length while simultaneously avoiding any impairment of electrode reliability at high flow velocities. However, conventional techniques, e.g., according to

[11] ,

[12] and

[13] , are characterized by a relatively long electrode interaction length, so that the aforementioned requirements cannot be met, or can only be met to a limited extent, when manipulating cells using dielectrophoretic forces.

[0015] From

[12] and

[13] , it is known to subdivide electrodes whose total length exceeds the particle diameter by a factor of 20 to 50 into individual electrode segments. The function of the conventional subdivision is to create additional degrees of freedom in shaping the field generated by an electrode or the shape of the field barrier. For example, when controlling electrode segments according to

[12] , individual electrode segments can be deactivated (switched off) to allow particles to pass through the microsystem channel on a specific trajectory, while all other electrode segments are activated together. In

[13] , a planar array of point-like electrode segments is described that allows flexible shaping of the field barrier by controlling selected electrode segments within the array.However, conventional electrode segmentation does not allow for a simultaneous increase in throughput and reliability of particle manipulation.

[0016] The aforementioned limitations of conventional techniques arise not only when manipulating biological cells, but also with non-biological particles, such as artificial microcompartments or carrier particles of chemical substances.

[0017] The object of the invention is to provide an improved method for operating a fluidic microsystem for the dielectrophoretic manipulation of suspended particles in a suspension liquid and / or an improved fluidic microsystem for the dielectrophoretic manipulation of suspended particles, thereby avoiding the disadvantages of conventional techniques. In particular, the dielectrophoretic manipulation of the suspended particles should be possible with an increased particle density without impairing the reliability of the electrode function.

[0018] This problem is solved by a method for operating a fluidic microsystem or by a fluidic microsystem having the features of the independent claims. Preferred embodiments and applications of the invention are set forth in the dependent claims.

[0019] According to a first general aspect of the invention, the above problem is solved by a method for operating a fluidic microsystem for the dielectrophoretic manipulation of suspended particles with a predetermined particle diameter in a suspension liquid. The fluidic microsystem comprises a channel with a longitudinal direction, an electrode assembly with an elongated electrode (deflection electrode) whose longitudinal extent deviates from the longitudinal direction of the channel and which has a plurality of individually controllable electrode segments (partial electrodes) for generating dielectrophoretic forces acting on the particles, each electrode segment having a deflection angle (electrode angle) ( )relative to the longitudinal direction of the channel and has a segment length (si ) which determines a segment offset (Di ) transverse to the longitudinal direction of the channel, and a control device with which the electrode segments can be controlled.

[0020] The method for operating the fluidic microsystem comprises the steps of generating a flow of the suspension fluid with a flow velocity in the channel such that the suspended particles successively pass through an interaction area of ​​the electrode, which is defined by the electrode segments, and controlling the electrode segments to deflect the particles in the channel onto predetermined paths of motion, which are determined by a superposition of flow forces in the flow of the suspension fluid and of the dielectrophoretic forces generated at the electrode segments.

[0021] As each particle passes through, each electrode segment, which the particle passes successively, is activated by the control unit for a predetermined activation duration, depending on the desired trajectory. The activation duration of each electrode segment is determined by the quotient of the segment length (si) of the electrode segment and the flow velocity.

[0022] According to the invention, the electrode segments are dimensioned such that the segment offset (Di) of each electrode segment is smaller than the particle diameter, and at least two successive electrode segments work together to deflect the particles.

[0023] According to a second general aspect of the invention, the above problem is solved by a fluidic microsystem designed for the dielectrophoretic manipulation of particles with a predetermined particle diameter in a suspension liquid. The microsystem comprises a longitudinally oriented channel, an electrode assembly with an elongated electrode whose longitudinal extent deviates from the longitudinal direction of the channel, and which has a plurality of individually controllable electrode segments for generating dielectrophoretic forces acting on the particles, each electrode segment having a deflection angle ( The channel has a segment length (si) relative to its longitudinal direction and a segment offset (Di) perpendicular to the channel's longitudinal direction, and a control device for controlling the electrode segments. The channel is configured to receive a flow of suspension fluid at a flow velocity such that the suspended particles successively traverse an interaction zone of the electrode defined by the electrode segments. The control device directs the electrode segments to deflect the particles along predetermined paths within the channel. These paths are determined by the superposition of flow forces in the suspension fluid and the dielectrophoretic forces generated at the electrode segments.

[0024] The control device is designed to activate each of the electrode segments, which the particle passes successively, in a timed manner depending on the desired trajectory, for a predetermined activation period, whereby the activation period of each electrode segment is determined by the quotient of the segment length (si ) of the electrode segment and the flow velocity.

[0025] According to the invention, the electrode segments are dimensioned such that the segment offset (Di) of each electrode segment is smaller than the particle diameter. Furthermore, according to the invention, the control device is configured to control the electrode segments such that at least two consecutive electrode segments interact to deflect the particles.

[0026] Preferably, the method according to the first general aspect of the invention or one of its preferred embodiments is carried out with the fluidic microsystem according to the second general aspect of the invention or one of its preferred embodiments.

[0027] Advantageously, the object of the invention is achieved in particular by activating the electrode segments of an electrode in a particle-specific manner, i.e., for a predetermined activation duration. The activation duration is at least equal to the time interval required for a particle to pass through an electrode segment. Each particle passes each of the electrode segments for a specific time interval, which is determined by the segment length and the flow velocity. Since the time intervals of each particle's passage through each of the electrode segments, and thus also the relative positions of the time intervals, are predetermined, the electrode segments can be individually and specifically controlled for the activation durations.

[0028] In contrast to

[12] , the invention provides for the continuous particle-specific control of all electrode segments. The control of the electrode segments moves with the particles that reach the electrode. The control of an electrode segment at a specific time affects only the particle that passes through the electrode segment in question. At the same time, the control of the electrode segment has no effect on particles that are located at other electrode segments of the electrode at that time. Accordingly, particles with a higher cell density (number of cells per unit length in the channel) can pass the electrode, thus increasing the throughput. The electrode has a specific, and possibly different, effect for each particle, even if several particles are located within the interaction length of the entire electrode.

[0029] Furthermore, since, in contrast to

[12] , the segment offset (D i ) of each electrode segment is smaller than the particle diameter and at least two successive electrode segments interact for the deflection of each particle, the flow velocity in the microsystem can be chosen to be relatively high according to the invention, without having to accept disadvantages due to the resulting high total interaction length of the electrode with respect to the particle density.

[0030] Advantageously, the aforementioned conflict between flow velocity and sample cell density is resolved. Samples with increased density can be reliably manipulated, and in particular sorted, even at high flow velocities.

[0031] The dielectrophoretic manipulation of the particles generally comprises the displacement of particles along predetermined paths within the channel of the microsystem by the interaction of dielectrophoretic forces and flow forces. This can be used, for example, to distribute the particles along these paths, to change the order of the particles, or preferably to sort the particles into different channels downstream of the electrode array. To move a particle from an initial path to another path within the channel of the microsystem, the control device sequentially activates the electrode segments that the particle successively passes over until the particle reaches the desired path through the segment offset at each activated electrode segment, and the electrode segment located within that path is deactivated.

[0032] Advantageously, the invention is applicable to various types of particles, including, for example, biological particles such as biological cells or their components, or non-biological particles such as carrier particles containing chemical substances or macromolecules. All particles can have the same diameter (homogeneous particle sample). Alternatively, the particles can each have different diameters (heterogeneous particle sample), in which case the segment offset is smaller than the smallest particle diameter. The particles generally comprise micro-objects with a characteristic size, e.g., a diameter that is preferably equal to or greater than 1 µm and / or equal to or less than 1 mm. This size range offers particular advantages with regard to the observability of the particles with sufficient optical resolution, the dielectrophoretic forces, and / or the maintenance of laminar flow conditions.The particles can include biological particles, such as animal or plant cells, bacteria, or cell clusters. The diameters of biological cells, for example, range from 5 µm to 25 µm, and the diameters of cell clusters, for example, range from 25 µm to 250 µm. Alternatively or additionally, the particles can include non-biological micro-objects, such as plastic particles and / or semiconductor particles. The suspension fluid is a liquid medium, such as an aqueous solution; in the case of manipulating biological cells, specifically a physiologically compatible liquid or a culture medium.

[0033] An "electrode" is typically formed from two congruent groups of electrode segments on opposing canal walls, e.g., the canal's underside and canal's upper surface. Alternatively, an electrode can comprise a single group of electrode segments on a single canal wall. The elongated electrode, whose longitudinal extent deviates from the longitudinal direction of the canal, typically has the form of a straight, segmentally straight, or curved strip composed of successively arranged electrode segments, enclosing the deflection angle with the longitudinal direction of the canal. The opposing ends of the electrode segments are separated and electrically insulated. The electrode segments can all have the same deflection angle or they can each have different deflection angles.In the case of curved electrode segments, the deflection angle can be determined by a tangent to the electrode segment, for example at one of its ends or its middle.

[0034] The electrode deflects the particles in the channel along predetermined paths. These paths are flow routes or trajectories of the particles, which are arranged side by side in the channel in the direction of flow. In the area of ​​the electrode assembly, the channel preferably has a straight course, and the paths run parallel to the flow profile of a laminar flow formed in the channel.

[0035] According to preferred embodiments of the invention, the following features can be provided individually or in combination. The segment lengths (si) of the electrode segments can be less than or equal to 10 times the particle diameter, in particular less than or equal to twice or even less than or equal to the particle diameter. Advantageously, this results in shorter local interaction lengths of the individual electrode segments than in the prior art. The detection length is reduced and the space requirement in the microsystem is decreased. The segment lengths (si) of the electrode segments can, for example, be less than or equal to 50 µm, in particular less than or equal to 10 µm. Furthermore, the segment lengths are preferably at least equal to or greater than one-tenth of the particle diameter. Alternatively or additionally, the deflection angles ( ) of the electrode segments less than 10°, in particular less than 5° to almost 0°. The deflection angles ( The deflection angles of the electrode segments are significantly smaller than those disclosed, for example, in

[12] . Advantageously, the small deflection angles improve the reliability of the field barrier formed by the electrode and the reliability of particle manipulation.

[0036] The activation durations of the electrode segments can be selected depending on the particle size and the flow velocity. For example, particularly with a particle diameter of 100 µm and a flow velocity of 1 mm / s, the activation durations of the electrode segments can be equal to or less than 100 ms, particularly equal to or less than 50 ms, and most preferably 20 ms or 30 ms or even less. Advantageously, such short activation durations of the electrode segments allow for an increase in the throughput of particle manipulation due to the higher selectable feed rate / flow velocity.

[0037] According to a further advantageous embodiment of the invention, a position detection system is provided for determining at least one particle position of each particle, and the electrode segments are controlled based on this position. Advantageously, the position detection system can determine a sequence of time intervals during which the respective particle passes the individual electrode segments. The activation durations of the electrode segments correspond to the detected time intervals. The electrode segments can be activated or deactivated for each particle for the duration of the respective time intervals.

[0038] Preferably, the fluidic microsystem is equipped with a position detection device with which the at least one particle position of each particle can be detected, wherein the control device for controlling the electrode segments is set up depending on the at least one particle position of each particle.

[0039] According to an advantageous embodiment, the position detection comprises observing the electrode's interaction area with a microscope, wherein the electrode segments over which the particle successively passes are directly detected by the microscope. Accordingly, the position detection device of the microsystem comprises the microscope, which is arranged for observing the electrode's interaction area and for directly detecting the electrode segments over which the particle successively passes. This embodiment is particularly advantageous if the position detection simultaneously allows for a sorting decision in real time, i.e., with no or negligible delay.

[0040] According to an alternative, particularly preferred embodiment, the position detection comprises observing an observation area upstream of the electrode's interaction area with a microscope. The observation area is spaced from each electrode segment by a predetermined channel length, and the electrode segments past which the particle successively passes are determined from the particle's observation time within the observation area, the channel lengths, and the flow velocity. Accordingly, the microscope constitutes a position detection device for the microsystem, arranged upstream of the electrode. Advantageously, the position detection can be combined with image processing for particle feature recognition, with the transit time of each particle along the channel length providing sufficient time for image processing and, for example, a sorting decision.

[0041] According to a further, particularly preferred embodiment of the invention, at least one particle property of each particle is detected, wherein the control of the electrode segments is carried out depending on the at least one particle property. Preferably, the control device for controlling the electrode is configured depending on at least one particle property. The particle property preferably comprises at least one particle structure and at least one particle substance.

[0042] The particle properties are determined, for example, using optical and / or photonic methods, such as scattering measurements, and / or lensless X-ray diffraction. Particularly preferably, the particle properties are determined using a microscope in conjunction with an image analysis device, with which at least one particle property can be determined from image data of each particle.

[0043] Image-based particle sorting, particularly cell sorting, with high throughput is of great benefit to all areas of the life sciences and cell-based medicine. Precise and safe cell isolation, especially in clinical settings, opens up new cell therapy pathways and standards with high societal and economic potential (e.g., in CAR-T cell therapy). Image-based cell sorting also has applications in basic biomedical research, particularly in drug development, such as drug screening, immuno-oncology, and stem cell generation.

[0044] Preferably, the particle distribution within the microsystem is chosen such that several particles are present in the electrode's interaction zone, with at most one particle being located on each electrode segment on average over time. Advantageously, the particle distribution can be determined simultaneously with the measurement of at least one particle property. Particles with such a small distance between them that at least two particles pass through an electrode segment simultaneously can be detected and discarded.

[0045] According to a preferred application of the invention in particle sorting, the channel of the microsystem is divided into several subchannels downstream of the electrode's interaction area, and each particle is moved into one of the subchannels by controlling the electrode segments depending on at least one particle property. In this case, the control device is configured to move each particle into one of the subchannels by controlling the electrode depending on at least one particle property. Advantageously, the sorting function of the electrode can be performed particle-specifically, even if several particles are located within the electrode's interaction length. Each electrode segment where a particle is currently located is controlled (activated or deactivated) according to the detected particle property and sorting decision.

[0046] According to a further advantageous embodiment of the invention, the flow velocity of the suspension is set to a predetermined constant value by a control loop. Preferably, the microsystem, in particular the control device, is coupled to the control loop. Advantageously, a constant flow velocity allows for increased accuracy in setting the activation duration of the electrode segments.

[0047] Further details and advantages of the invention are described below with reference to the accompanying drawings. The drawings show: Figure 1: a schematic illustration of features of embodiments of the method and fluidic microsystem according to the invention; and Figures 2 and 3: schematic illustrations of conventional fluidic microsystems.

[0048] Features of embodiments of the invention are described below by way of example with reference to the sorting of biological cells at a Y-junction of a channel of a fluidic microsystem into two sub-channels. It is emphasized that the application of the invention is not limited to this example, but is possible analogously for other manipulations of particles, e.g., for their displacement onto one of more than two paths of movement in the channel, e.g., for a redistribution or change in particle spacing. Instead of the manipulation of biological cells, the invention can also provide for the manipulation of other, in particular non-biological, particles. In practical implementation of the invention, the sizes and shapes of the parts of the microsystem can be selected depending on the requirements of the specific application.Details of the construction and operation of the fluidic microsystem, in particular the generation of high-frequency electric fields for electrode control, and the acquisition of particle properties, e.g. by processing measurement data from a microscope, are not described, as these are known from the prior art.

[0049] Figure 1Figure 1 shows a schematic top view of channel 10 of the fluidic microsystem 100 with an electrode assembly 20, a control unit 30, and a microscope assembly 40. Channel 10 extends straight along a longitudinal direction z to a branching point into sub-channels 11 and 12. Channel 10 has, for example, a rectangular cross-section with a width ranging from 20 µm to 1000 mm and a height ranging from 5 µm to 1 mm. The flat underside is also referred to as the channel bottom 13, and the flat top side as the cover surface (not shown). Cells 1 and 1A are suspended in a suspension fluid 2 within the channel. When a pump 14 is actuated, a flow of the suspension fluid is generated in channel 10 with a flow direction that coincides with the longitudinal direction z.

[0050] The electrode assembly 20 comprises the electrode 21, which is divided into a plurality of electrode segments 22. In the example shown, the electrode 21 has the shape of a straight strip formed by the arranged straight electrode segments 22. Figure 1 Only the electrode 21 on the channel floor 13 is shown. Preferably, another electrode (not shown) with the same size, shape, and orientation relative to the channel 10, or alternatively a flat counter electrode, is arranged on the top surface. The longitudinal extent of the electrode 21 forms a deflection angle with the longitudinal direction z of the channel 10. , which, due to the straight shape of the electrodes, determines the deflection angle Each electrode segment 22 forms a segment length si along the longitudinal direction z of the channel 10 and a segment offset D i perpendicular to the longitudinal direction z of the channel 10. Accordingly, each electrode segment 22 is assigned an interaction length I i. The interaction length (detection length) L of the entire electrode 21 results from the sum of the individual interaction lengths I i of the electrode segments 22 and their mutual distances. In the example shown, all electrode segments 22 have the same interaction lengths I i.

[0051] The electrode 21 is subdivided into electrode segments 22 to enable operation with the highest possible cell density at the smallest possible deflection angle. This allows for stepwise deflection of the cells with a minimal segment offset Di (sorting window). Ideally, the subdivision is such that the segment offset Di is on the order of the cell diameter. The individual electrode segments 22 can be switched on and off sequentially, so that only the electrode segment 22 corresponding to the cell being sorted is active at any given time. Even closely spaced cells can thus be handled independently of the preceding cell. In a specific embodiment, for example, there are 20 electrode segments 22, each with a segment length si of 20 µm and a deflection angle of . A 3° angle is provided, resulting in an interaction length L of the entire electrode 21 of 0.2 mm. The width of the electrode 21 is, for example, 10 µm.

[0052] The control unit 30 comprises an electrode voltage source 31 and a computer unit 32. The computer unit 32 controls the electrode voltage source 31, the microscope unit 40, and the pump unit 14. Furthermore, the computer unit 32 is designed to analyze image data from the microscope unit 40, to acquire particle properties of cells 1 and 1A, and to generate a sorting decision based on the acquired particle properties. The electrode voltage source 31 is designed to generate high-frequency electrical voltages for controlling the electrode 21. According to the invention, each electrode segment 22 is controlled individually. For this purpose, the electrode voltage source 31 has a group of output channels, the number of which is equal to the number of electrode segments 22.Each output channel is connected to one of the electrode segments 22 of the electrode 21 and to one of the electrode segments of the electrode not shown on the top surface of the channel 10.

[0053] The microscope apparatus 40 comprises, for example, a transmitted light or fluorescence microscope, which is arranged for image acquisition in an observation area upstream of the interaction area of ​​the electrode 21. In the observation area, the cover surface of the channel 10 is transparent. Simultaneously, the microscope apparatus 40 forms a position detection device with which the particle position of cells 1, 1A can be detected. For this purpose, the passage of cells 1, 1A through the observation area and the corresponding observation time are recorded. In conjunction with the flow velocity in the channel 10 and the segment lengths Ii, the time intervals at which the particles 1, 1A pass the electrode segments 22 are obtained.

[0054] In the time interval between the observation time and the reaching of the first electrode segment 22, the computer unit 32 performs the analysis of the image data of the microscope device 40, the acquisition of particle properties of the cells 1, 1A, such as size, shape, co-localization of fluorescently stained membrane proteins and the sorting decision.

[0055] For cell sorting in channel 10, cells 1, 1A suspended in the suspension fluid 2, such as cell culture medium, buffer solution, etc., flow through the observation area of ​​the microscope unit 40 to the electrode unit 20. Each cell is assigned a particle property and the time intervals of passage at the electrode segments 22. The electrode segments 22 are activated by applying high-frequency electrical voltages for activation durations equal to the respective time intervals of passage. If no field is generated at an electrode segment 22 (the electrode 21 is locally inactive), the cells can pass freely through the electrode segment 22. If a field is generated at an electrode segment 22 (the electrode 21 is locally active), the cells are prevented from passing through by negative dielectrophoresis and are guided onto a different path according to the electrode geometry and hydrodynamic propulsion.By dividing the electrode 21 into the electrode segments 22 with a relatively small deflection angle α i, the effective detection length of the electrode 21 is minimized, so that even closely spaced cells 1, 1A can be individually sorted and correctly separated to reach the subchannels 11, 12.

[0056] In contrast to previously described methods (e.g., [8]), the use of the described sorting function enables the processing of very dense cell samples. In combination with (easily implemented) parallelization of the system, the same throughput can therefore be achieved with significantly lower flow velocities, which facilitates optical image acquisition and the handling of dead times potentially caused by image processing. Furthermore, the complex microfluidic control elements required at high flow velocities are eliminated, further reducing the complexity of the method and thus considerably improving the compactness, cost, and operability of the system.

[0057] The features of the invention disclosed in the foregoing description, the drawings and the claims may be important for the realization of the invention in its various embodiments, both individually and in combination or sub-combination.

Claims

1. Method for operating a fluidic microsystem (100) for dielectrophoretic manipulation of suspended particles (1) having a predetermined particle diameter in a suspension liquid (2), wherein the fluidic microsystem (100) comprises: - a channel (10) having a longitudinal direction, - an electrode device (20) having an elongate electrode (21), the longitudinal extension of which deviating from the longitudinal direction of the channel (10) and which has a plurality of individually activatable electrode segments (22) for generating dielectrophoretic forces acting on the particles (1), wherein each electrode segment (22) has a deflection angle () relative to the longitudinal direction of the channel (10) and a segment length (si), which determine a segment offset (Di) transverse to the longitudinal direction of the channel (10), and - a control device (30) by means of which the electrode segments (22) can be activated, comprising the steps: - generating a flow of the suspension liquid (2) with a flow velocity in the channel (10), so that the suspended particles (1) in succession pass an interaction region of the electrode (21) which is spanned by the electrode segments (22), and - activating the electrode segments (22) in order to deflect the particles (1) in the channel (10) onto predetermined movement paths which are determined by a superposition of flow forces in the flow of the suspension liquid (2) and the dielectrophoretic forces generated at the electrode segments (22), wherein - with each particle passage, each of the electrode segments (22) which the particle (1) passes in succession is activated in a clocked manner by the control device (30) in dependence on the desired movement path in each case for a predetermined activation time, wherein the activation time of each electrode segment (22) is determined by the quotient of the segment length (si) of the electrode segment (22) and the flow velocity, characterized in that - the electrode segments (22) are so dimensioned that the segment offset (Di) of each electrode segment (22) is smaller than the particle diameter, and in each case at least two successive electrode segments (22) cooperate for the deflection of each particle (1).

2. Method according to claim 1, wherein - the segment lengths (si) of the electrode segments (22) are less than or equal to 10 times the particle diameter, in particular less than or equal to twice the particle diameter.

3. Method according to any one of the preceding claims, wherein - the deflection angles () of the electrode segments (22) are less than 10°, in particular less than 5°.

4. Method according to any one of the preceding claims, having the steps - position detection for determining at least one particle position of each particle, and - activation of the electrode segments (22) in dependence on the at least one particle position of each particle (1).

5. Method according to claim 4, wherein - the position detection comprises monitoring of the interaction region of the electrode (21) with a microscope device (40) with which the electrode segments (22) which the particle (1) passes in succession are detected directly.

6. Method according to claim 4, wherein - the position detection comprises observing of a monitoring region upstream of the interaction region of the electrode (21) with a microscope device (40), wherein the monitoring region is spaced apart from each of the electrode segments (22) by a predetermined channel length and the electrode segments (22) which the particle (1) passes in succession are determined from an observation time of the particles (1) in the monitoring region, the channel lengths and the flow velocity.

7. Method according to any one of the preceding claims, having the step - detection of at least one particle property of each particle, wherein - the activation of the electrode segments (22) takes place in dependence on the at least one particle property.

8. Method according to claim 7, wherein - the channel (10) is divided downstream of the interaction region of the electrode (21) into multiple subchannels (11,11',12,12'), wherein - each of the particles (1) is moved into one of the subchannels (11,11',12, 12') by the activation of the electrode segments (22) in dependence on the at least one particle property.

9. Method according to any one of the preceding claims, wherein - the flow velocity of the suspension liquid (2) is set at a predefined constant value by a control loop.

10. Method according to any one of the preceding claims, wherein - a distribution of the particles (1) is chosen such that multiple particles (1) are located in the interaction region of the electrode (21), wherein, when averaged over time, not more than one of the particles (1) is located at each electrode segment (22).

11. Fluidic microsystem (100) adapted for the dielectrophoretic manipulation of particles (1) having a predetermined particle diameter in a suspension liquid (2), comprising, - a channel (10) having a longitudinal direction, - an electrode device (20) having an elongate electrode (21), the longitudinal extension of which deviating from the longitudinal direction of the channel (10) and which has a plurality of individually activatable electrode segments (22) for generating dielectrophoretic forces acting on the particles (1), wherein each electrode segment (22) has a deflection angle () relative to the longitudinal direction of the channel (10) and a segment length (si), which determine a segment offset (Di) transverse to the longitudinal direction of the channel (10), and - a control device (30) by means of which the electrode segments (22) can be activated, wherein - the channel (10) is adapted to receive a flow of the suspension liquid (2) with a flow velocity such that the suspended particles (1) pass in succession through an interaction region of the electrode (21) which is spanned by the electrode segments (22), - the control device (30) is adapted to activate the electrode segments (22) in order to deflect the particles (1) in the channel (10) onto predetermined movement paths which are determined by a superposition of flow forces in the flow of the suspension liquid (2) and the dielectrophoretic forces generated at the electrode segments (22), and - the control device (30) is adapted, as the particles (1) pass, to activate in a clocked manner each of the electrode segments (22) which one of the particles (1) passes in succession in dependence on the desired movement path in each case for a predetermined activation time, wherein the activation time of each electrode segment (22) is determined by the quotient of the segment length (si) of the electrode segment (22) and the flow velocity, characterised in that - the electrode segments (22) are so dimensioned that the segment offset (Di) of each electrode segment (22) is smaller than the particle diameter, and - the control device (30) is adapted to activate the electrode segments (22) so that in each case at least two successive electrode segments (22) cooperate for the deflection of each particle.

12. Fluidic microsystem according to claim 11, having the features - the segment lengths (si) of the electrode segments (22) are less than or equal to 10 times the particle diameter, in particular less than or equal to twice the particle diameter, and / or - the segment lengths (si) of the electrode segments (22) are less than or equal to 100 µm, in particular less than or equal to 10 µm.

13. Fluidic microsystem according to any one of claims 11 to 12, which comprises - a position detection device with which at least one particle position of each particle can be detected, wherein - the control device (30) is adapted to activate the electrode segments (22) in dependence on the at least one particle position of each particle.

14. Fluidic microsystem according to claim 13, wherein - the position detection device comprises a microscope device (40) which is arranged to observe the interaction region of the electrode (21) and to directly detect the electrode segments (22) which the particle (1) passes in succession.

15. Fluidic microsystem according to claim 13, wherein - the position detection device comprises a microscope device (40) which is arranged to observe an monitoring region upstream of the interaction region of the electrode (21) with a microscope device (40), wherein the monitoring region is spaced apart from each of the electrode segments (22) by a predetermined channel length, wherein - the control device (30) is adapted to determine the electrode segments (22) which the particle (1) passes in succession from an observation time of the particles (1) in the monitoring region, the channel lengths and the flow velocity.

16. Fluidic microsystem according to any one of claims 11 to 15, wherein - the control device (30) is adapted to activate the electrode (21) in dependence on at least one particle property.

17. Fluidic microsystem according to claim 16, wherein - the channel (10) divides into multiple subchannels (11,11',12,12') downstream of the interaction region of the electrode (21), wherein - the control device (30) is adapted to move each of the particles (1) into one of the subchannels (11,11',12,12') by activation of the electrode (21) in dependence on the at least one particle property of the particle.

18. Fluidic microsystem according to any one of claims 11 to 17, comprising - a control loop with which the flow velocity of the suspension liquid (2) can be set at a predefined constant value.

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