Fluidic microsystem for manipulating particles in a fluid, and particle manipulation method

Abstract

A fluidic microsystem (100) for manipulating particles (1), (2), (3), for example biological cells, comprises a fluidic channel arrangement (10), which is designed to receive a flow of a fluid (4) with a flow direction S, in which fluid the particles (1), (2), (3) are arranged, and which contains, successively in the flow direction S, a first analysis section (11A), a first actuator section (12A) and a second analysis section (11B); a detector device (20) with a field of view (21A) which covers the first analysis section (11A) and the second analysis section (11B), wherein the detector device (20) is configured for radiation-based detection of at least one particle property of each of the particles (1), (2), (3) in the first analysis section (11A) and in the second analysis section (11B); and an actuator device (30) which is configured to subject each of the particles (1), (2), (3) in the first actuator section (12A) to a first actuator step, in particular comprising at least one of: executing a trigger action, taking a measurement and applying an actuating force. The fluidic channel arrangement (10) has a reversal section (13) having a first leg (13A), a reversal curve and a second leg (13B), wherein the first analysis section (11A) is arranged in the first leg (13A) and the second analysis section (11B) is arranged in the second leg (13B) adjacent to the first analysis section (11A) with respect to a direction transverse to the flow direction S. The invention also relates to a particle manipulation method for manipulating particles (1), (2), (3) in a fluidic microsystem.

Description

[0001] Fluidic microsystem for manipulating particles in a fluid and particle manipulation methods

[0002] The invention relates to a fluidic microsystem configured for manipulating, e.g., sorting and / or processing, particles, such as biological cells, in a fluid. Furthermore, the invention relates to a particle manipulation method for manipulating particles in a fluid within a fluidic microsystem. Applications of the invention include, for example, the processing of biological cells, cell groups or cell components, microcompartments, or other micro-objects in chemistry, medicine, biology, or biochemistry.

[0003] The following prior art, which represents the technical background of the invention, is referred to in the present description:

[0004] [1] Boutros et al. (2015): Microscopy-based high-content screening. Cell 163, 1314-1325;

[0005] [2] Gerling et al. (2023): High-precision, low-complexity, high-resolution microscopy-based cell sorting. Lab Chip, 23, 3172-3185;

[0006] [3] Godino et al. (2019): Combining dielectrophoresis and computer vision for precise and fully automated single-cell handling and analysis. Lab Chip 19, 4016-4020;

[0007] [4] Ho et al. (2005): Micromachined electrochemical T-switches for cell sorting applications. Lab Chip 5, 1248-1258;

[0008] [5] Kirschbaum et al. (2008): T cell activation on a single-cell level in dielectrophoresis-based microfluidic devices. J Chromatogr A. 1202 (1), 83-89;

[0009] [6] Landenberger et al. (2012): Microfluidic sorting of arbitrary cells with dynamic optical tweezers. Lab Chip 12, 3177-3183;

[0010] [7] Li and Anand (2018): Cellular dielectrophoresis coupled with single-cell analysis. Analytical and Bioanalytical Chemistry 410, 2499-2515;

[0011] [8] Meineke et al. (2016): A microfluidic opto-caloric switch for sorting af particles by using 3D- hydrodynamic focusing based on SLE fabrication capabilities. Lab Chip 16, 820-828;

[0012] [9] Nitta et al. (2018): Intelligent Image-Activated Cell Sorting. Cell 175(l):266-276.

[0013]

[0010] Nawaz et al., (2020) Nat Methods, 17, 595-599;

[0014]

[0011] Sakuma et al. (2017): On-chip cell sorting by high-speed local-flow control using dual membrane pumps. Lab Chip 17, 2760-2767;

[0015]

[0012] Schraivogel, et al., (2022) Science, ,375, 315-320;

[0013] Shen et al. (2019): Recent advances in microfluidic cell sorting systems. Sensors & Actuators: B. Chemical 282, 268-281;

[0016]

[0014] WO 1999 / 052 640 Al; und

[0017]

[0015] DE 10 2007 018 752 Al.

[0018] It is well known that the identification and isolation of individual cells or cell populations, for example with specific physiological properties, from heterogeneous cell samples in fluidic microsystems is an important procedure in biotechnology and biomedical research. For this purpose, the cells are first analyzed and classified using various methods before the cells corresponding to a target specification are actively extracted from the population. The analysis and classification of the cells is generally based on magnetic, acoustic, electrical, and / or radiation-based measurements. For radiation-based measurements, for example, microscopic imaging is used (see [1], [2], [9],

[0010] or

[0012] ).

[0019] The aforementioned methods are typically performed with cells suspended in liquids, using flow-through techniques. The liquid, containing the cells, flows through a flow cell or microchannel to be conveyed, ideally in a linear arrangement, to an analysis section containing an analytical unit, such as a radiation-based microscope. Here, the relevant information about the cells is acquired, and after processing, a sorting decision is transmitted to a sorting device in a downstream actuator section. There, if a cell is selected, forces are exerted on it by suitable actuator structures, deflecting it from its original path and thus separating it from the other cells.

[0020] The separation of individual cells from a stream of cells flowing consecutively in a microchannel can be achieved, for example, with micromechanical ([4]), radiation-based ([6]), hydrodynamic ([9]; [8];

[0011] ), electrokinetic ([3], [2]) or other (

[0013] ) forces. It is known from

[0014] and

[0015] to manipulate cells in fluidic microsystems. According to

[0014] , cells can be transferred between different channels under the influence of dielectrophoretic forces, although for fluid dynamic reasons, repeated transfer between channels with a reversing circular motion is excluded. In

[0015] , cells in a fluidic microsystem are subjected in particular to dielectrophoretic forces, whereby a measurement of the cells' velocity, but not of the cells' properties themselves, is provided. In the microsystem of

[0015] , a channel can be formed in a meandering shape to achieve space savings.

[0021] The precision of both the cell analysis by the analytical function in the analysis section and the separation of individual cells from the particle stream is subject to technical limitations that can affect the sorting result. For example, depending on the selected particle density, two or more cells may pass through the analysis section or the sorting unit simultaneously and therefore cannot be processed independently (see Supplementary Information of [2]). As a result, the sorting decision is applied to both cells in the same way, which, in the case of one target and one non-target cell, leads to a reduced purity of the sorted cell population. In this situation, it would be possible to discard both cells, thus leaving the purity of the sorting result unaffected. However, this would adversely reduce the yield.Furthermore, false-positive analysis results can lead to an erroneous activation of the sorting function, causing non-target cells to enter the target outlet.

[0022] Several approaches exist to address these problems. First, the cross-section of the analysis function can be improved by increasing the detector sensitivity and / or reducing the flow rate. Second, the precision of the sorting process can be improved by increasing the average cell spacing and / or reducing the size of the sorting window. However, these concepts are often not feasible without accepting further limitations in performance, complexity, cost, or robustness of the method.

[0023] Another way to improve the purity of a sorted sample is to apply an additional sorting step, involving further analysis and the activation of another actuator structure, to the sorting result obtained in the first sorting process. This allows the removal of non-target cells that were inadvertently or unavoidably captured along with the target cells during the first sorting step. However, repeating the entire sorting process, including cell manipulations at sample entry, during sample organization, and during arrangement for analysis, adversely increases the overall procedure time and can negatively affect the condition, such as cell viability, due to increased force. For a second sorting step, the second sorting process could be performed directly after the first, e.g.,in a channel downstream of the first actuator structure. This would require a second analysis unit for measurement in a second analysis section and the second actuator structure. However, the second analysis unit can be disadvantageous or undesirable due to space constraints. For example, in the case of microscope radiation-based cell analysis, a second microscope would incur high costs, assuming the installation of a second radiation-based setup was even possible due to space limitations. The latter problem can be avoided with an approach described, for example, in [3], in which a single microscope is used for multiple analyses in separate measurement sections (ROI, Region of Interest) within the microscope's field of view.

[0024] However, the placement of multiple regions of interest (ROIs), especially with typical separation distances of 1 mm or more in fluidic microsystems, requires a relatively large field of view from the microscope. This precludes the use of high-resolution optics with a field of view of, for example, less than 500 pm, which negatively impacts the sensitivity, accuracy, and / or reproducibility of cell analysis.

[0025] The problems mentioned above occur not only during the sorting of biological cells, but also generally during the processing of biological cells or the manipulation of other particles, such as synthetic nanoparticles, in fluidic microsystems. Furthermore, these problems arise not only when manipulating particles suspended in liquids, but also when manipulating particles dispersed in gases.

[0026] The object of the invention is to provide an improved fluidic microsystem for particle manipulation and / or an improved particle manipulation method for manipulating particles in a fluidic microsystem, thereby avoiding the disadvantages of conventional techniques. In particular, the particle manipulation should be possible using high-resolution imaging and / or enable improved sensitivity, accuracy, and / or reproducibility of the analysis and / or allow for improved sorting results with increased sample purity.

[0027] This problem is solved by a fluidic microsystem and / or by a particle manipulation method, which have the features of the independent claims. Preferred embodiments and applications of the invention are set forth in the dependent claims. According to a first general aspect of the invention, the above problem is solved by a fluidic microsystem configured for particle manipulation. The fluidic microsystem comprises a fluidic channel arrangement configured to receive a flow of fluid with a flow direction in which the particles are arranged, and which, in the flow direction, successively includes a first analysis section, a first actuator section, and a second analysis section. The fluidic microsystem is further equipped with a detector device having a field of view (radiation-based detection range, viewing range, e.g.,The fluidic microsystem is equipped with a camera field of view covering the first and second analysis sections, wherein the detector device is configured for radiation-based detection of at least one particle property of each of the particles in the first and second analysis sections. The fluidic microsystem further comprises an actuator device configured to subject each of the particles in the first actuator section to a first actuator step, for example, comprising at least one of performing a trigger action, performing a measurement, and applying an actuating force.

[0028] According to the invention, the fluidic channel arrangement has a reversing section with a first leg, a reversing curve and a second leg, wherein the first analysis section is arranged in the first leg and the second analysis section is arranged in the second leg adjacent to the first analysis section in the first leg with respect to a direction transverse to the flow direction (lateral direction, in particular transverse to the longitudinal direction of the fluidic channel in the area of ​​the analysis sections).

[0029] According to a second general aspect of the invention, the above problem is solved by a particle manipulation method for manipulating particles in a fluidic microsystem.The particle manipulation method comprises the steps of forming a flow of a fluid in which the particles are arranged, with a flow direction in a fluidic channel arrangement which, in the flow direction, successively contains a first analysis section, a first actuator section and a second analysis section, radiation-based detection of at least one particle property of each of the particles in the first analysis section and in the second analysis section with a detector device which has a field of view that covers the first analysis section and the second analysis section, and application of a first actuator step, for example comprising at least one of an exercise of a trigger action, a performance of a measurement and an exercise of an actuating force, to at least one of the particles in the first actuator section.According to the invention, the fluid channel arrangement used for the particle manipulation method has a reversing section with a first leg, a reversing curve and a second leg, wherein the first analysis section is arranged in the first leg and the second analysis section is arranged in the second leg adjacent to the first analysis section in the first leg with respect to a direction transverse to the flow direction, in particular transverse to the longitudinal direction of the fluid channel in the area of ​​the analysis sections.

[0030] Preferably, the method according to the second general aspect of the invention or one of its embodiments can be carried out with the fluidic microsystem according to the first general aspect of the invention or one of its embodiments.

[0031] The fluidic microsystem is generally a single- or multi-part body (microsystem chip) equipped with the fluidic channel array. The fluidic channel array is embedded (integrated) into the microsystem chip and / or exposed on its surface. At least in the case of the embedded fluidic channel array, the microsystem chip comprises a transparent material, in particular an optically transparent material such as plastic or glass, which allows radiation-based image acquisition with the detector device. The transparent material can also be provided in the case of the exposed fluidic channel array.

[0032] The fluidic channel arrangement can comprise one or more fluidic channels (or microchannels) dimensioned and arranged to accommodate the flow of the fluid containing the particles. The fluidic channel arrangement can include branches and / or intersections of fluidic channels and / or be equipped with at least one additional fluidic component, such as those known per se from fluidic microsystems technology, e.g., at least one fluid reservoir, at least one valve, and / or at least one additional functional element. The fluidic channel arrangement can preferably be equipped with at least one inlet and / or at least one outlet. At least one pump device can be provided, configured to generate a pressure gradient within the fluidic channel arrangement, thereby creating a flow of the fluid through the arrangement.

[0033] The at least one fluidic channel can have a rectangular cross-section, at least in the area of ​​the analysis and actuator sections, e.g., with a width in the range of 20 µm to 5 mm and a height in the range of 5 µm to 200 µm or more. The longitudinal direction of the at least one fluidic channel, in conjunction with the pumping device, determines the (local) flow direction of the fluid. Preferably, the fluidic channel arrangement and the pumping device can be dimensioned and operated such that a laminar flow is formed in the fluidic channel arrangement.

[0034] The analysis sections are generally regions of the at least one fluid channel located within the field of view of the detector device. The actuator section, or optionally several actuator sections, are generally regions of the at least one fluid channel equipped with the actuator device or in which the actuator device acts on particles suspended in the fluid. The first analysis section and the first actuator section are arranged sequentially in the direction of flow, and both sections may be overlapping, adjacent, or spaced apart with respect to the flow direction. Likewise, the second and optionally each subsequent analysis section, and each optionally second and subsequent actuator section, are arranged sequentially in the direction of flow.

[0035] The length between the first actuator section and the second analysis section along the fluidic channel—that is, the length of the fluidic channel between the first actuator section and the second analysis section—can be selected depending on the effective length of the first actuator step, such as the trigger action, the measurement, and the application of the actuating force. This length can be, for example, in the range of 200 pm to 20 mm. If the first actuator step is, for instance, a sorting step involving the application of a dielectrophoretic actuating force, the length can be, for example, 1 mm or more, and thus larger than the diameter of the detector's field of view.

[0036] Advantageously, the object of the invention is solved in particular by the fact that, by designing the at least one fluidic channel with at least one reversing section, which can be described as a meandering loop or U-shaped channel section, and the associated reversal of the fluid flow with respect to the fluidic microsystem, it is made possible that the first analysis section and the second analysis section are arranged adjacent to each other (return of the fluid to the field of view of the detector device), so that a first analysis can be carried out with the detector device in the first analysis section as well as a second analysis in the second analysis section after passing through the first actuator section, in particular after execution of the first actuator step.Since the first analysis section is located in the first leg (channel section of the fluidic channel of the reversing section upstream of the reversing section) and the second analysis section is located in the second leg (channel section of the fluidic channel of the reversing section downstream of the reversing section), and since the first and second legs are adjacent to each other, the first and second analysis sections can be positioned adjacent to each other in a direction transverse to the local flow direction in the fluidic channel without limiting the length between the first actuator section and the second analysis section along the fluidic channel. The first and second analysis sections can be separated only by a wall of the fluidic microsystem, the thickness of which is selected to be in the range of, for example, 50 pm to 1 mm.

[0037] The reversal section preferably forms a reversal of the channel direction by substantially 180°. At the reversal curve, the channel can preferably have a rounded curve or, alternatively, an angular shape. Preferably, the lateral distance between the legs immediately before the reversal curve of the reversal section can be greater than the lateral distance between the legs in the region of the adjacent analysis sections. Advantageously, this avoids an acute angle at the reversal of the channel direction and / or allows for a radius of curvature at the reversal of the channel direction that permits the maintenance of laminar flow along the reversal section. Furthermore, the length of a section of the partition between the two legs of the reversal section, where the analysis sections are adjacent and the partition is as thin as possible, can be reduced to a minimum.

[0038] The adjacent arrangement of the first and second analysis sections can mean that they are aligned side-by-side in the lateral direction (perpendicular to the local flow direction), which advantageously minimizes the field of view. Alternatively, the adjacent arrangement can also include configurations in which the first and second analysis sections overlap or are offset from each other in the flow direction. In either case, compared to conventional techniques, this allows the use of a limited field of view while simultaneously achieving high-resolution imaging.

[0039] The detector system can generally comprise a device for radiation-based particle detection, such as a camera or an imaging spectrometer. The detector system can be configured for radiation-based particle detection in the optical spectral range (UV, visible light, and / or IR) or in other spectral ranges, such as the X-ray range. The field of view is determined, in particular, by the optics of the detector system.

[0040] The detector device for radiation-based particle detection in the optical spectral range can also be referred to as a camera device. The camera device preferably comprises an optical element of a radiation-based microscope and is particularly preferably part of an optical microscope.

[0041] The field of view of the detector device covers both the first and second analysis sections, i.e., the field of view encompasses both sections, and it contains two separate measurement sections in which particles in the first and second analysis sections can be detected simultaneously. The detector device can preferably detect the first and second analysis sections directly and simultaneously; that is, the radiation path from the analysis sections to the detector device can preferably be free of any further radiation-directing elements, particularly optical ones, apart from the optics. Alternatively, additional radiation-directing elements, such as mirrors and / or lenses, can be provided between the analysis sections and the detector device so that the analysis sections are covered by the field of view of the detector device.

[0042] Preferably, in the case of microscopic imaging with high-resolution microscope optics, the detector unit can be equipped with an immersion objective with at least 40x magnification and a numerical aperture >1. Advantageously, this allows a field of view with a diameter of 550 pm or less to be achieved while still covering multiple analysis sections. For example, a 200 pm wide fluidic channel can be positioned so that both legs pass through the field of view of the detector unit's objective at a distance of 150 pm. If the particles, e.g., biological cells, are guided in each analysis section only in a portion of the fluidic channel (approximately half, a third, a quarter, or a smaller portion) on the side facing the other fluidic channel, wider channels or...Larger distances between the legs can be achieved, while simultaneously ensuring that the particles in both legs pass through the field of view of the lens.

[0043] The detector device can preferably be connected to an image processing device configured to provide an analysis result based on a captured particle image. The image processing device can provide at least one particle property.

[0044] An important advantage of the invention is that better performance parameters of a particle (e.g., cell) sorting process, such as improved throughput, purity, yield, biocompatibility and / or process duration, and / or a particle (e.g., cell) monitoring process can be achieved without having to implement the detector device as an analysis unit (and possibly also an actuator structure) multiple times, thus reducing costs and effort and / or avoiding other technical limitations.

[0045] The particles manipulated using the technique according to the invention can, for example, include biological particles such as biological cells, microorganisms, viruses, cell groups and / or cell components, and / or non-biological particles such as nanoparticles and / or carrier beads, and / or composite bio-synthetic particles such as carrier beads carrying biological cells.

[0046] The particles are arranged in the fluid and are fluidic with the fluid in the fluidic microsystem. The particles in the fluid form a dispersion, the fluid being either a gas or a liquid. The particle-gas dispersion can be referred to as an aerosol. The particle-liquid dispersion forms a suspension in which the particles are suspended in a suspension liquid. Accordingly, for preferred applications of the invention, the fluid can be referred to as a suspension liquid.

[0047] The manipulation of the particles, in particular the first and optionally each subsequent actuator step, can include, for example, the application of a trigger action, such as electrical, thermal and / or optical stimulation, a measurement, such as further electrical and / or optical detection, and / or the application of an actuating force, such as an electrical, magnetic or optical actuating force.

[0048] Advantageously, the fluidic channel arrangement can include branched fluidic channels, with the first analysis section and the second analysis section each being arranged in different sub-channels. Advantageously, the fluidic channel of the arrangement can thus have a branch into a sub-channel downstream of the first actuator section, with the second analysis section being arranged downstream of the branch in the sub-channel. Advantageously, the analysis and, optionally, the subsequent sorting of particles can take place in the sub-channel separately from the particles in the original fluidic channel. This embodiment can be particularly advantageous if the cells sorted out in a first sorting step are to be immediately removed from the system and the isolation of another particle type is provided in a second sorting step (3-way sorting).

[0049] According to a preferred embodiment of the invention, the fluidic channel arrangement can have a second actuator section following the second analysis section in the flow direction, wherein the actuator device is configured to subject each of the particles in the second actuator section to a second actuator step, in particular comprising at least one of a trigger action, a measurement, and the application of an actuating force. With regard to the particle manipulation method, the fluidic channel arrangement can have the second actuator section following the second analysis section in the flow direction, and in the second actuator section, a second actuator step, in particular comprising at least one of a trigger action, a measurement, and the application of an actuating force, can be applied to at least one of the particles.The functionality of the fluidic microsystem can be advantageously expanded by providing the second actuator section and integrating the actuator mechanism into both sections. Of particular benefit is the option to implement a multi-stage sorting process that yields a target cell population with increased purity.

[0050] The actuator assembly can accordingly comprise two separate actuator elements, each acting in one of the actuator sections. The actuator elements can be configured to apply the same type of actuator step or different types of actuator steps in each actuator section. For example, an actuating force can be applied in each actuator section, or a trigger action can be performed in one actuator section and an actuating force in the other.

[0051] Advantageously, the second (and optionally each subsequent) actuator element can have an area of ​​operation that extends only over a portion of the width of the fluidic channel, while the remaining width of the fluidic channel is unaffected by the actuator element. The second (and optionally each subsequent) actuator element can act exclusively on a portion of the flow paths in the fluidic channel. This embodiment of the invention enables two- or multi-stage actuator steps, e.g., two- or multi-stage sorting processes, in which the respective actuator step (e.g., sorting step) acts only on a portion of the upstream manipulated particles. Alternatively, the actuator assembly can comprise a single actuator element that acts simultaneously, covering both actuator sections. Depending on the sorting principle and the detection area of ​​the actuator assembly, the single actuator element can even be used for two sorting processes.

[0052] Preferably, the fluidic microsystem can be equipped with a control unit coupled to the detector and actuator units. The control unit can be configured to control at least the actuator step in the first actuator section, depending on the particle properties of the respective particle detected in the first analysis section. The control unit can, for example, comprise one or more computer units. The control unit can programmatically decide whether and / or which actuator step is executed by the actuator unit and / or whether the actuator unit is actuated. The image processing unit of the detector unit can be integrated into the control unit.

[0053] According to particularly preferred embodiments of the invention, the fluidic microsystem and / or the particle manipulation method can be configured such that a control device is provided which is coupled to the detector device and the actuator device, wherein the control device is configured to control the first actuator step depending on the particle property of the respective particle detected in the first analysis section, and the control device is further configured to control the second actuator step depending on the particle property of the respective particle detected in the second analysis section. Advantageously, the control device can be used to control at least two stages of the actuator device.

[0054] According to a preferred application of the invention, the fluidic microsystem and / or the particle manipulation method can be configured to sort the particles in the fluid into at least two particle fractions that differ by at least one particle property, wherein the actuator is configured to perform the first and second actuator steps by applying an actuating force to each of the particles. In this embodiment, the control device is further configured to apply and / or adjust the actuating force depending on the respective particle property detected, wherein the control device and the actuator are configured to displace particles in the first actuator section transversely to the flow under the influence of the actuating force.such that the at least two particle fractions are each formed by particles with the predetermined particle property (target particles) and particles without the predetermined particle property (other target particles or non-target particles) in different flow paths in the fluidic channel arrangement, and wherein the control device and the actuator device are configured to displace particles in the second actuator section transversely to the flow under the influence of the actuating force, so that individual particles from at least one of the two particle fractions are transferred to the other of the two particle fractions or to a further (third) particle fraction. Advantageously, in the second actuator section, a further sorting of at least one of the particle fractions or a separation of the further particle fraction from at least one of the particle fractions can take place.

[0055] If the actuator assembly according to a preferred embodiment of the invention comprises at least one electrode (sorting electrode) configured to apply the actuating force to the particles by means of negative dilectrophoresis, particular advantages arise for the application of the invention in the manipulation of particles, especially biological cells, and / or for a compact configuration of the fluidic microsystem. Advantageously, the actuating force can be exerted directly on the particles by means of negative dilectrophoresis. Electrodes for manipulating particles based on dilectrophoresis are known, for example, from [2]. These can be arranged in a fluidic channel in a small space and with high integration density.

[0056] Advantageously, the functionality of the fluidic microsystem can be further expanded by providing additional analyses and optionally additional actuator steps.According to advantageous variants of the invention, the fluidic microsystem and / or the particle manipulation method can be configured such that the fluidic channel arrangement, following the second actuator section in the flow direction, contains at least one further analysis section, the fluidic channel arrangement has at least one further reversal section with adjacent legs, wherein the at least one further analysis section in one of the legs is arranged adjacent to the second analysis section in the other leg with respect to a direction transverse to the flow direction, and the detector device is additionally configured for the radiation-based detection of the at least one particle property of particles in the at least one further analysis section of the fluidic channel arrangement, wherein the at least one further analysis section is covered by the field of view of the detector device together with the other analysis sections.Advantageously, this enables multi-stage analyses and manipulations of the particles. Preferably, the fluidic channel arrangement can have a further reversing section with a reversing curve of the fluidic channel, wherein the legs of the further reversing section are formed by the second leg of the first reversing section and a third leg, the further analysis section in the third leg being arranged adjacent to the second analysis section in the second leg with respect to a direction transverse to the flow direction.

[0057] In general, for each optionally additional analysis section, the fluidic channel arrangement can have a further reversing section, so that preferably all analysis sections are arranged on adjacent legs of the reversing sections, for example in a row, and are jointly detected by the one (single) field of view of the detector device.

[0058] According to further preferred embodiments of the invention, the fluidic microsystem and / or the particle manipulation method can be designed such that the fluidic channel arrangement, following the further analysis section in the direction of flow, contains at least one further actuator section, and a further actuator step, in particular comprising at least one of a trigger action, a measurement and an actuating force, is applied to at least one of the particles in the at least one further actuator section.

[0059] Preferred applications include image-based cell sorting, preferably with high performance parameters, in all areas of the life sciences and cell-based medicine. The invention offers, for example, precise and reliable isolation of biological cells, particularly in basic research, clinical applications, and / or drug development, such as in drug testing, immuno-oncology, or stem cell production. Alternative applications of the invention include the monitoring of particles in fluids, for example, multi-stage monitoring with additional controls in subsequent steps of an initial monitoring process.

[0060] Features disclosed in connection with the fluidic microsystem or its embodiments also constitute preferred features of the particle manipulation method according to the invention or of its embodiments. The aforementioned aspects and preferred features of the invention, particularly with regard to the structure of the fluidic microsystem and the dimensions and compositions of individual components described in relation to the fluidic microsystem, also apply to the particle manipulation method for its application. The preferred embodiments, variants, and features of the invention described herein are combinable with one another.

[0061] Further details and advantages of the invention are described below with reference to the accompanying drawings. The drawings schematically show:

[0062] Figure 1: Features of embodiments of the fluidic microsystem and particle manipulation method according to the invention;

[0063] Figure 2: another illustration of a section of a fluidic channel arrangement with two

[0064] Analysis sections and two actuator sections for a multi-stage sorting task; and

[0065] Figures 3 and 4: Features of modified embodiments of the fluidic microsystem according to the invention.

[0066] Features of embodiments of the invention are described below by way of example with reference to a fluidic microsystem designed for particle sorting, in particular the sorting of suspended biological cells in a suspension fluid. It is emphasized that the application of the invention is not limited to this example, but is possible analogously for other manipulation tasks, such as the electrical or light-induced stimulation of biological cells depending on their properties, and / or with other particle types and / or with other fluids, e.g., particles dispersed in gases (for example, aerosols), and / or with other detector devices. In the practical implementation of the invention, the sizes and / or 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 to generate dielectrophoretic actuating forces, 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.

[0067] Figure 1 schematically shows a top view of a fluidic channel arrangement 10 of a fluidic microsystem 100 with a fluidic channel 10A branching into two sub-channels 10B and 10C. Figure 2 shows further details of the fluidic microsystem 100 with a channel configuration implemented in practice. The fluidic microsystem 100 is designed for sorting a mixture of suspended cells 1 and 2 into two particle fractions with target cells (1) and non-target cells (2). The cells 1 and 2 are suspended in a fluid 4 (suspension fluid) which flows with the flow direction S under the action of a pump (not shown) from an inlet to outlets of the fluidic channel arrangement 10. As a result of the sorting process, the target cells 1 and the non-target cells 2 are directed into sub-channel 10B and sub-channel 10C, respectively.

[0068] Upstream of the branch into sub-channels 10B and 10C, the fluidic channel 10A is formed with a reversing section 13 comprising a first leg 13A, a reversing curve, and a second leg 13B. Regions of the fluidic channel 10A in the first leg 13A form a first analysis section 11A and a first actuator section 12A, and regions of the fluidic channel 10A in the second leg 13B form a second analysis section 11B and a second actuator section 12B.

[0069] The fluidic microsystem 100 is equipped with a schematically shown detector device 20, in particular a camera device, which includes, for example, an Olympus 1X83 type microscope with an immersion objective with, for example, 40x magnification and a numerical aperture of, for example, 1.4, and an integrated camera detector. The detector device 20 has a field of view 21 with a diameter of, for example, 500 pm. The field of view 21, shown as a circular example in Figure 1, can have a different shape, such as elliptical or rectangular (see Figure 2). If fluorescence is to be detected as a particle property, a fluorescence microscope, optionally with a separate excitation light source, can be used.

[0070] Furthermore, the fluidic microsystem 100 is equipped with a control unit 40, which includes, for example, a computer unit (see Figure 1). The control unit 40 is connected to the detector unit 20 and the actuator unit 30, in particular their electrodes 31A, 31B (see below), and is configured to receive and process image signals from the detector unit 20 and to control the electrodes of the actuator unit 30.

[0071] The first analysis section 11A and the second analysis section 11B are sections of the fluidic channel 10A that are detected by the field of view 21 of the detector unit 20. The detector unit 20 (in conjunction with the control unit 40 and / or a separate image processing unit) can detect at least one particle property of each of the cells 1, 2 in the first analysis section 11A and in the second analysis section 11B. Particle properties such as the size, shape, surface texture, and / or fluorescence of the cells 1, 2 can be detected.

[0072] In the first actuator section 12A and the second actuator section 12B, the fluidic channel 10A contains, as an actuator device, a strip-shaped electrode 31A, 31B (sorting electrodes, see Figure 2) which is connected to the control unit. The configuration is such that when the electrode 31A, 31B is energized with a high-frequency electrical voltage, a repulsive dielectrophoretic force is generated on cells in the first actuator section 12A or the second actuator section 12B by means of negative dielectrophoresis. Together with the mechanical flow force exerted by the fluid 4, this results in a displacement of the respective cell transversely to the flow direction S into an adjacent flow path within the fluidic channel 10A.

[0073] Additionally, the fluidic channel 10A contains strip-shaped electrodes 32 (guide electrodes, see Figure 2) which are arranged outside the analysis sections and the actuator sections for guiding and / or aligning the cells 1, 2 by means of negative dielectrophoresis.

[0074] To sort cells 1 and 2 into the desired particle fractions, cells 1 and 2 are lined up upstream of the first analysis section 11A with one of the electrodes 32 (see Figure 2) and introduced as a series of individual cells into the first analysis section 11A (first sorting station), where they are detected and classified into target cells and non-target cells. Depending on the result of the classification, the electrode in the first actuator section 12A is activated if a non-target cell 2 is detected, and not activated if a target cell 1 is detected. The discarded non-target cells 2 are redirected to a modified flow path, e.g., B. on the outside of the fluidic channel 10A, deflected, where after passing the reversing section 13 they are directed further to the sub-channel IOC ("discard"), without entering the effective area of ​​the downstream dielectrophoretic electrodes 31B in the second actuator section 12B.

[0075] The cells 1 identified as target cells 1 at the first analysis section 11A and separated from the remaining cells at the first actuator section 12A continue along the original flow path, e.g., on the inside of the fluidic channel 10A, also pass through the reversal section 13, and then reach the second analysis section 11B in the second leg 13B (second sorting point). There, they are analyzed and classified again, and if classified as target cells 1, they are directed to subchannel 10B ("Target"). If, due to misclassification, non-target cells 2 were able to pass through the first sorting point without being moved onto the altered flow path (see, e.g., 2A in Figure 1), such cells can be detected at the second sorting point and directed to subchannel IOC ("Discard") by electrode 31B, thus minimizing or eliminating misclassifications.

[0076] Advantageously, the analysis of cells 1 and 2 at the first and second sorting points, i.e., in the first analysis section 11A and the second analysis section 11B, is performed using the same analysis function, which is fulfilled by the detector device 20 in conjunction with the image processing. Due to the channel shape with the reversing section 13, the first analysis section 11A and the second analysis section 11B can be arranged with a minimal distance, defined solely by the thickness of the wall of the fluidic microsystem 100 between the legs 13A and 13B, so that both analysis sections 11A and 11B can be covered by the detector device 20 with a narrow field of view 21 of a high-resolution optic.

[0077] The shape of the fluidic channel arrangement 10 of the fluidic microsystem 100 is not limited to the geometry shown in Figures 1 and 2. Figure 3 shows a modified embodiment of the invention which differs from the embodiments of Figures 1 and 2 with respect to the shape of the fluidic channel arrangement 10 of the fluidic microsystem 100 and is otherwise identical to the embodiments of Figures 1 and 2.

[0078] As shown in Figure 3, at the branch point of fluidic channel 10A into sub-channels 10B and 10C, sub-channel 10C is reversed. The non-target cells 2 obtained after passing through the second actuator section 12B are guided into sub-channel 10C by a guide electrode (not shown), which is continuously controlled by the control unit (not shown), and from there to an output of the fluidic channel arrangement 10.

[0079] Advantageously, in addition to the first reversing section 13, the curved sub-channel 10C forms a further reversing section 14, so that a section of sub-channel 10C downstream of the branch can provide a further analysis section 11C, which can optionally be followed by a further actuator section 12C. This embodiment is shown schematically in Figure 4 with reference to an extended fluidic channel arrangement 10. According to Figure 4, the fluidic channel arrangement 10 comprises a fluidic channel 10A, which branches into sub-channels 10B and IOC, of ​​which sub-channel IOC branches into further sub-channels 10D and 10E. The fluidic channel arrangement 10 is shaped such that further reversing sections 14 and 15 are formed by sub-channels IOC and 10E after the first reversing section 13 of the fluidic channel 10A. The analysis sections 11A, 11B and 11C are formed in different parts of the fluidic channel arrangement 10, each representing a leg of the different reversal sections 13, 14 and 15.Each of the analysis sections in one of the legs of one of the reversal sections is arranged in a direction perpendicular to the flow direction adjacent to the following analysis section in the other of the legs of the associated reversal section.

[0080] This arrangement allows separate sorting points to be formed at the first analysis and actuator sections 11A, 12A, the second analysis and actuator sections 11B, 12B, and the third analysis and actuator sections 11C, 12C, as shown in Figure 4. Alternatively, another sorting point with analysis and actuator sections (not shown) can be provided in subchannel 10E.

[0081] The analysis and actuator sections 11A, 12A, 11B, 12B, 11C, and 12C are covered by the common field of view 21 of the detector assembly 20. Three regions of interest (ROIs) are formed within the field of view 21, in which simultaneously passing cells 1, 2, and 3 can be detected and classified. At each sorting station, with a pair of analysis and actuator sections, sorting into different cell fractions along different flow paths occurs depending on the classification result, as described above with reference to Figures 1 and 2. The electrodes 31A, 31B, and 31C provided for this purpose are each connected to the control unit (not shown). Furthermore, the fluidic channel assembly 10 includes guide electrodes (not shown, see Figure 2B) which are intended to guide the cells in the channels of the fluidic channel assembly 10 under the influence of negative dielectrophoresis.

[0082] For example, as shown in Figure 4, a three-stage sorting process can be provided into a first cell fraction of target cells 1 (electrode 31A), a second cell fraction of target cells 2 (electrode 31B), and a third cell fraction of non-target cells or further target cells 3 (electrode 31C). Sorting errors at one sorting stage are corrected at the downstream sorting stage. For example, sorting errors that result in target cells 2 being found in the first cell fraction of target cells 1 are corrected at the second sorting stage by the second analysis section 11B and the second actuator section 12B, where target cells 2 are deflected into the flow path towards subchannel 10C. The features of the invention disclosed in the foregoing description, the drawings, and the claims can be important for the realization of the invention in its various embodiments, both individually and in combination or subcombination.

Claims

Claims 1. Fluidic microsystem (100) configured for manipulating particles (1, 2, 3), for example biological cells (1, 2, 3), comprising: - a fluidic channel arrangement (10) with at least one fluidic channel (10A, 10B, IOC), wherein the fluidic channel arrangement (10) is configured to receive a flow of a fluid (4) with a flow direction (S) in which the particles (1, 2, 3) are arranged, and which, in the flow direction (S), successively includes a first analysis section (11A), a first actuator section (12A) and a second analysis section (11B), - a detector device (20) with a field of view (21) covering the first analysis section (11A) and the second analysis section (11B), wherein the detector device (20) is for radiation-based detection of at least one particle property of each of the particles (1, 2, 3) is set up in the first analysis section (11A) and in the second analysis section (11B), and - an actuator device (30) which is configured to subject each of the particles (1, 2, 3) in the first actuator section (12A) to a first actuator step, in particular comprising at least one of an exercise of a trigger action, a measurement and an application of an actuating force, characterized in that - the at least one fluidic channel (10A, 10B, 10C) of the fluidic channel arrangement (10) has a reversal section (13) with a first leg (13A), a reversal curve and a second leg (13B), wherein the first analysis section (11A) is arranged in the first leg (13A) and the second analysis section (11B) is arranged in the second leg (13B) in a direction transverse to the flow direction (S) adjacent to the first analysis section (11A).

2. Fluidic microsystem according to claim 1, wherein - the fluidic channel arrangement (10) contains branched sub-channels and the first analysis section (11A) and the second analysis section (11B) are each arranged in different sub-channels.

3. Fluidic microsystem according to one of the preceding claims, wherein - the fluidic channel arrangement (10) in the flow direction (S) following the second analysis section (11B) has a second actuator section (12B), and - the actuator device (30) is configured to subject each of the particles (1, 2, 3) in the second actuator section (12B) to a second actuator step, in particular comprising at least one of an exercise of a trigger action, a measurement and an application of an actuating force.

4. Fluidic microsystem according to claim 3, further comprising - a control device (40) coupled to the detector device (20) and the actuator device (30), wherein - the control device (40) is set up to control the first actuator step depending on the particle property of the respective particle detected in the first analysis section (11A), and - the control device (40) is set up to control the second actuator step depending on the particle property of the respective particle detected in the second analysis section (11B).

5. Fluidic microsystem according to claim 4, which is configured to sort the particles (1, 2, 3) into at least two particle fractions that differ by at least one particle property, wherein - the actuator device (30) is set up to perform the first and second actuator steps by applying an actuating force to each of the particles (1, 2, 3), and - the control device (40) is set up to apply and / or adjust the actuating force depending on the particle property detected in each case, wherein - the control device (40) and the actuator device (30) are configured to displace particles (1, 2, 3) in the first actuator section (12A) under the influence of the actuating force transversely to the flow, so that the at least two particle fractions are formed by particles (1, 2, 3) with the predetermined particle property and particles (1, 2, 3) without the predetermined particle property in different flow paths in the fluidic channel arrangement (10), and - the control device (40) and the actuator device (30) are configured to displace particles (1, 2, 3) in the second actuator section (12B) under the influence of the actuating force transversely to the flow, so that individual particles (1, 2, 3) from at least one of the two particle fractions are transferred to the other of the two particle fractions or to a further particle fraction.

6. Fluidic microsystem according to claim 5, wherein - the actuator device (30) has at least one electrode (31A, 31B) which is configured to apply the actuating force to the particles (1, 2, 3) by means of negative dielectrophoresis.

7. Fluidic microsystem according to any one of claims 3 to 6, wherein - the fluidic channel arrangement (10) following the flow direction (S) to the second actuator section (12B) contains at least one further analysis section (11C), - the fluidic channel arrangement (10) has at least one further reversing section (14, 15, 16) with adjacent legs, wherein the at least one further analysis section (11C) in one of the legs is arranged adjacent to the second analysis section (11A) in the other leg in a direction transverse to the flow direction (S), and - the detector device (20) is additionally equipped for radiation-based detection of at least one particle property of particles (1, 2, 3) in the at least one further analysis section (11C) of the fluidic channel arrangement (10), wherein the at least one further analysis section (11C) is covered by the field of view (21) of the detector device (20).

8. Fluidic microsystem according to claim 7, wherein - the fluidic channel arrangement (10) following the flow direction (S) to the further analysis section (11C) contains at least one further actuator section (12C), and - the actuator device (30) is configured to subject each of the particles (1, 2, 3) in the at least one further actuator section (12C) to a further actuator step, in particular comprising at least one of an exercise of a trigger action, a measurement and an application of an actuating force.

9. Particle manipulation method for manipulating particles (1, 2, 3), for example biological cells (1, 2, 3), in a fluidic microsystem, comprising the steps: - Formation of a flow of a fluid in which the particles (1, 2, 3) are arranged, with a flow direction (S) in a fluidic channel arrangement (10) with at least one fluidic channel (10A, 10B, 10C), wherein the fluidic channel arrangement (10) contains, in the flow direction (S), a first analysis section (11A), a first actuator section (12A) and a second analysis section (11B) successively, - radiation-based detection of at least one particle property of each of the particles (1, 2, 3) in the first analysis section (11A) and in the second analysis section (11B) using a detector device (20) having a field of view (21) covering the first analysis section (11A) and the second analysis section (11B), and - Application of a first actuator step, in particular comprising at least one of an exercise of a trigger action, a performance of a measurement and an exercise of an actuating force, to at least one of the particles (1, 2, 3) in the first actuator section (12A), characterized by the fact that - the at least one fluidic channel (10A, 10B, IOC) of the fluidic channel arrangement (10) has a reversal section (13) with a first leg (13A), a reversal curve and a second leg (13B), wherein the first analysis section (11A) is arranged in the first leg (13A) and the second analysis section (11B) is arranged in the second leg (13B) in a direction transverse to the flow direction (S) adjacent to the first analysis section (11A).

10. Particle manipulation method according to claim 9, wherein - the fluidic channel arrangement (10) in the flow direction (S) following the second analysis section (11B) has a second actuator section (12B), and - in the second actuator section (12B) a second actuator step, in particular comprising at least one of a trigger action, a measurement and an actuating force, is applied to at least one of the particles (1, 2, 3).

11. Particle manipulation method according to claim 10, further comprising - a control device (40) coupled to the detector device (20) and the actuator device (30), wherein - the first actuator step is controlled by the control unit (40) depending on the particle property of the respective particle detected in the first analysis section (11A), and - the second actuator step is controlled by the control unit (40) depending on the particle property of the respective particle detected in the second analysis section (11B).

12. Particle manipulation method according to claim 11, comprising sorting the particles (1, 2, 3) into at least two particle fractions that differ by at least one particle property, wherein - the actuator device (30) is set up to perform the first and second actuator steps by applying an actuating force to the particles (1, 2, 3), and - the application and / or adjustment of the actuating force by the control device (40) is carried out depending on the particle property detected in each case, wherein - the control device (40) and the actuator device (30) displace particles (1, 2, 3) in the first actuator section (12A) under the influence of the actuating force transversely to the flow, so that the at least two particle fractions are formed by particles (1, 2, 3) with the predetermined particle property and particles (1, 2, 3) without the predetermined particle property in different flow paths in the fluidic channel arrangement (10), and - the control device (40) and the actuator device (30) to displace particles (1, 2, 3) in the second actuator section (12B) under the influence of the actuating force transversely to the flow, so that individual particles (1, 2, 3) from at least one of the two particle fractions are transferred into the other of the two particle fractions or into a further particle fraction.

13. Particle manipulation method according to claim 12, wherein - the actuator device (30) has at least one electrode (31A, 31B) and exerts the actuating force on the particles (1, 2, 3) by means of negative dielectrophoresis.

14. Particle manipulation method according to any one of claims 10 to 13, wherein - the fluidic channel arrangement (10) following the flow direction (S) to the second actuator section (12B) contains at least one further analysis section (11C), - the fluidic channel arrangement (10) has at least one further reversing section (14, 15, 16) with adjacent legs, wherein the at least one further analysis section (11C) in one of the legs is arranged adjacent to the second analysis section (11A) in the other leg in a direction transverse to the flow direction (S), and - additionally, a radiation-based detection of at least one particle property of particles (1, 2, 3) in which at least one further analysis section (11C) with the detector device (20) is provided, wherein the at least one further analysis section (11C) is covered by the field of view (21) of the detector device (20).

15. Particle manipulation method according to claim 14, wherein - the fluidic channel arrangement (10) in the flow direction (S) following the further analysis section contains at least one further actuator section (12C), and - on at least one of the particles (1, 2, 3) in which at least one further actuator section (12C) a further actuator step, in particular comprising at least one of an exercise of a trigger action, a performance of a measurement and an exercise of an actuating force, is applied.

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