Apparatus and methods for separating particles in liquid, kits including the apparatus, and applications of the apparatus.

Abstract

The present invention relates to an apparatus, a method and a kit for separating particles of different sizes in a liquid. The present invention additionally relates to the use of the apparatus. The apparatus and the method involve the ability to bind particles to solid phase particles with different diameters in a liquid, the hydrodynamic diameter of the solid phase particles determines whether the particles can pass through the pores of the filter element, and the diameter of said pores can be changed in a controlled manner (e.g. the diameter can be increased or decreased). Thus, particles of equal size (e.g. B cells and T cells) in a liquid can be separated from each other with a high separation efficiency, and the particles can be separated simply, quickly and cheaply. A high yield can be produced and the particles can be provided in a liquid that can be applied for therapy.

Description

Technical Field

[0001] An apparatus, a method, and a kit for separating particles of different sizes in a liquid are provided. Use of the apparatus is additionally provided. The apparatus and method are based on the fact that particles in a liquid can bind to solid phase particles having different diameters, and the hydrodynamic diameter of the solid phase particles determines whether the particles can pass through pores of a filter element whose diameter can be altered in a targeted manner (e.g., increased or decreased). Therefore, particles of the same size in a liquid (e.g., B cells and T cells) can be separated from each other with high separation efficiency, and particle separation can be performed in a simple, rapid, and low-cost manner. High yield can also be achieved, and the particles can be provided in a liquid usable for therapy. Background Art

[0002] Numerous cell-based separation processes require the use of technical membranes for separating molecules or particles of different sizes from each other. Conventionally used membranes have predefined pore sizes or mesh sizes. Components whose size exceeds the pore size are blocked thereby, while smaller components can pass through the membrane. However, in many separation processes such as filtration of particles from blood, more than two different-sized particles (i.e., blood cells and / or endosomal vesicles of different sizes) need to be separated with high purity quality. Currently, expensive and complex multi-step processes or series connection of membranes with different pore sizes are required for this purpose.

[0003] Methods and apparatuses are known in the prior art that can separate specific types of particles in a liquid (e.g., specific cells from whole blood) from other particles in the liquid in a specific manner (see WO 2016 / 092025 A1). These methods and apparatuses are based on the specific binding of target particles (e.g., target cells) to solid-phase particles immobilized between two membranes of a membrane cartridge via low-affinity fab fragments. The target particles can bind to the modified solid-phase particles, and other particles (e.g., cells) having a diameter smaller than the pore size of the two membranes and / or not binding to the solid-phase particles are separated from the target particles.

[0004] This known method can be used to provide target cells in high purity and yield. However, due to the fixedly specified pore size (e.g., 45 μm) of the membrane used in this method and apparatus, and the specific affinity of the fab fragments for target cells, it is not possible to provide multiple hematopoietic fractions, each containing different types of particles, in a single step or with a single apparatus (membrane cartridge). In addition, only small amounts of blood samples are often available. It is often not possible to successfully perform the separation process after splitting this small sample across multiple membrane cartridges containing differently modified solid-phase particles. Therefore, there is a need for a method that allows the separation of a particle-containing liquid into multiple fractions, each containing a specific type of particle, in a single apparatus.

[0005] Prior art has shown that filter elements for separating unwanted components from a fluid flow can change the pore size of the filter element by applying a voltage to the filter element and / or by applying mechanical force to the filter element (see, for example, DE 10 2016 213 565 A1). The drawbacks of this filter element are that the change in pore size simultaneously affects the change in the cross-sectional surface of the pore (e.g., from a rectangular cross-section to a rhomboid cross-section, or from a circular cross-section to an elliptical cross-section), and that it is not possible to set the selectivity of the filter element for allowing particles of a specific shape to pass through with high precision.

[0006] Films containing or derived from dielectric elastomers are further known from the prior art (see, for example, DE 10 2012 016 375 A1). It is further known that these films can be structured through the effects of electromagnetic radiation (e.g., laser radiation) (see, for example, DE 10 2012 016 378 A1). [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] WO 2016 / 092025 A1 [Patent Document 2] DE 10 2016 213 565 A1 [Patent Document 3] DE 10 2012 016 375 A1 [Patent Document 4] DE 10 2012 016 378 A1 [Overview of the project] [Problems that the invention aims to solve]

[0008] Starting from this, the objective of the present invention was to provide an apparatus and method for separating particles in a liquid that does not have the drawbacks of the prior art. In particular, the apparatus and method should enable not only the separation of particles of different sizes in a liquid (e.g., living cells and / or endosomes of different sizes) from one another with high separation efficiency, but also the separation of particles of the same size (e.g., B cells and T cells) from one another. The separated particles should be provided in a simple, rapid, and inexpensive manner, and in high yield, even with a small amount of liquid. In addition, the particles should be provided in a liquid that can be used for therapeutic purposes. Further use of the apparatus should be provided. [Means for solving the problem]

[0009] This objective is achieved by an apparatus having the features of claim 1, by a kit having the features of claim 15, by a method having the features of claim 16, and by a use having the features of claim 18. Dependent claims provide further advantageous developments.

[0010] According to the present invention, an apparatus for separating particles in a liquid, a) A container for receiving liquid (suspension), b) Surface filter element having an upper surface and a lower surface Includes, The filter element has continuous pores with a specified pore size, The filter element is positioned within the container to divide it into an upper section in the direction of the upper surface of the filter element and a lower section in the direction of the lower surface of the container, so that liquid particles in the upper section can only move into the lower section once they pass through the filter element, and the upper section of the container has an opening for receiving liquid containing particles. In the apparatus, The upper compartment of the container contains solid-phase particles of at least one group having a specific hydrodynamic diameter and exposing at least one molecule on its surface that is suitable for specifically binding to the surface molecules of a first type of particle, The filter element comprises or consists of a material suitable for changing the pore size of its pores by applying a voltage to the filter element and / or by the action of mechanical force on the filter element. An apparatus characterized by the above is provided.

[0011] The term "continuous pores" refers to pores that extend downward from the top to the bottom of the filter element. The term "having a specified pore size" means that the pore sizes of all pores in the filter element differ from each other by less than 10 μm, preferably less than 5 μm. The term "solid-phase particles" also includes "gel particles."

[0012] Using the apparatus according to the present invention, it is possible to perform the separation of different particles in a liquid (or suspension) in a single apparatus. Furthermore, by the specific binding of specific types of particles in the liquid (e.g., types of blood cells and / or types of vesicles in the blood) to solid-phase particles of different sizes, it is also possible to separate different types of particles having substantially the same size (i.e., substantially the same hydrodynamic diameter) from one another. The apparatus according to the present invention therefore has the advantage of not only being able to separate particles of different sizes in a liquid (suspension) (e.g., living cells and / or endosomes of different sizes) from one another with high separation efficiency, but also being able to separate particles of the same size (e.g., B cells and T cells) from one another. The separation of particles of the same size is achieved by binding each type of particle to be separated to solid-phase particles that "confer" a predetermined particle size to each type of particle. In addition, this apparatus makes it possible to provide the separated particles in high yield, even with small amounts of liquid, in a simple, rapid, and inexpensive manner. This is due to the fact that the pore size of the filter element of the apparatus can be varied, and therefore it is not necessary to continuously separate the particles of the liquid into multiple different apparatuses, each containing filter elements with different, fixed pore sizes. The separated particles can then be supplied into a therapeutically usable liquid (for example, its original liquid, such as plasma).

[0013] The apparatus according to the present invention can be designed as a membrane cartridge.

[0014] The apparatus may be characterized in that the upper compartment of the container contains at least one solid phase particle of a second group having a second hydrodynamic diameter, the second hydrodynamic diameter being different from the first hydrodynamic diameter, and the at least one solid phase particle of the second group exposing molecules on its surface that are suitable for specifically binding to the surface molecules of the second type of particle. This embodiment has the advantage that not only can one first type of particle be separated from other particles in the liquid in a targeted manner, but the second type of particle can also be separated from the first type of particle and from other particles in the liquid.

[0015] Furthermore, in this apparatus, the upper compartment of the container comprises at least one third group of solid phase particles having a third hydrodynamic diameter, the third hydrodynamic diameter being different from the first and second hydrodynamic diameters, and the third group of solid phase particles may be characterized in that they expose on their surface molecules suitable for specifically binding to surface molecules of a third type of particles. The upper compartment preferably comprises fourth, fifth, sixth, seventh, eighth, ninth, and / or tenth groups of solid phase particles, each of which has a hydrodynamic diameter different from that of other groups of solid phase particles in the upper compartment, and each group of solid phase particles exposes on their surface molecules suitable for specifically binding to surface molecules of different types of particles respectively. The advantage of this embodiment is that at least three groups of particles in a liquid can be selectively separated from each other and from other particles in the liquid.

[0016] The at least one molecule exposed on the surface of the solid phase particle and suitable for specifically binding to surface molecules of a first type of particle may comprise or consist of a polypeptide chain, the polypeptide chain is preferably selected from the group consisting of antibodies, antibody fragments, and derivatives thereof, and particularly preferably selected from the group consisting of Fab fragments or derivatives thereof.

[0017] The molecule may further comprise or consist of a polynucleotide, and the polynucleotide is preferably selected from the group consisting of DNA, RNA, and derivatives thereof.

[0018] The molecule may further comprise or consist of an oligosaccharide.

[0019] Preferably, the molecule is reversibly bound to the surface of the solid phase particle via non-covalent interaction, and preferably the binding is releasable by means selected from the group consisting of changing the concentration of a substance, changing the temperature, changing the pH, and a combination of the foregoing.

[0020] It is further preferred that this molecule is suitable for specifically binding to surface molecules of particles of a type having a hydrodynamic diameter smaller than that of solid-phase particles, preferably up to 10% of the hydrodynamic diameter of the solid-phase particles, more preferably up to 8%, particularly preferably up to 6%, very particularly preferably up to 4%, especially up to 2% of the hydrodynamic diameter of the solid-phase particles.

[0021] The device may comprise at least two conductive layers connected to a voltage source, preferably to the voltage source of the device.

[0022] At least one of the at least two conductive layers may be arranged on the upper side of the filter element, or may be arranged on the lower side of the filter element. One of the two conductive layers is preferably arranged on the upper side of the filter element, and the other of the two conductive layers is arranged on the lower side of the filter element.

[0023] The filter membrane may represent an electrically insulating layer. An electrically insulating layer for the at least two conductive layers may thus be unnecessary.

[0024] At least one of the at least two conductive layers, and optionally at least two conductive layers, may further contact at least one electrically insulating layer arranged between the at least one conductive layer and the filter element, and optionally each of the at least two conductive layers contacts at least one electrically insulating layer arranged between the respective conductive layer and the filter element. In this embodiment, electrical short circuits or short arcs between the first and the further conductive layer can be prevented. The electrically insulating layer may be arranged only on one side of the filter element (i.e., the upper side or the lower side thereof), or the electrically insulating layer may be arranged on both sides of the filter element.

[0025] At least one of the at least two conductive layers, and optionally at least two conductive layers, may further be arranged in the peripheral region of the pores of the filter element. This arrangement can be designed in the form of dots around the pores of the filter element.

[0026] At least one of the at least two conductive layers, and optionally at least two conductive layers, may be further arranged around the entire perimeter of the pores of the filter element. In this case, the conductive layer has continuous pores having a specified pore diameter at the same point as the filter element.

[0027] At least one of the at least two conductive layers, optionally at least two of the conductive layers, may contain or be derived from a polymer, optionally a conductive polymer.

[0028] In addition, at least one of the at least two conductive layers, optionally at least two conductive layers, may contain conductive particles, preferably carbon particles, particularly preferably single-walled or multi-walled carbon nanotubes. The proportion of conductive particles is, optionally, in the range of 0.001 to 30 wt%, preferably 0.01 to 3 wt%, relative to the total weight of the conductive layers. An advantage of this embodiment is that the conductive layers are conductive even when they consist of (primarily) non-conductive polymers. An inherently conductive polymer without additional conductive additives (in the composite layer) would be a special case.

[0029] At least one of the at least two conductive layers, and optionally at least two conductive layers, may further contain or be composed of a metal, the metal preferably located on the surface of the conductive layer. If metal is present, it may exist in the form of particles.

[0030] Preferably, at least one of the at least two conductive layers, and optionally at least two conductive layers, are connected to the filter element or to an electrical insulating layer by friction lock and / or material bonding.

[0031] At least one of at least two conductive layers, optionally at least two conductive layers, can be applied to the filter element or conductive layer via a process selected from the group consisting of pad printing, doctor knife coating, screen printing, inkjet printing, spraying, atomizing, evaporation, and combinations thereof, optionally combined with a laser structuring process.

[0032] In a preferred embodiment, the apparatus has a voltage source conductively connected to at least two conductive layers of the apparatus, preferably connected to at least two conductive layers applied to the upper and / or lower sides of the filter element of the apparatus. The advantage here is that the pore size of the pores of the filter element can be changed by applying a voltage to the filter element.

[0033] In a more preferred embodiment, the apparatus has means suitable for changing the pore size of the filter element by the action of mechanical force on the filter element, which is selected from the group consisting of a stamp for applying pressure to the filter element, a pneumatic device for applying pressure to the filter element, a bimetallic wire for applying pressure to the filter element, a NiTiCu alloy for applying pressure to the filter element, and combinations thereof. The advantage here is that the pore size of the filter element can be changed by the action of mechanical force on the filter element.

[0034] This apparatus may have means suitable for applying an oscillating fluid flow, preferably a fluid flow that oscillates perpendicular to the upper surface of the filter element, to the upper surface of the filter element. The advantage here is that clogging of the filter element, which may occur during particle separation, is prevented.

[0035] The device may further include a control unit configured to control the voltage of a voltage source and / or the mechanical force on a filter element.

[0036] Control via a control unit is preferably performed such that the voltage and / or mechanical force on the filter element changes stepwise during the separation of particles in the liquid, and this variation is preferably performed automatically over time or manually by user input. The voltage variation is particularly a decrease in voltage, and the variation in the mechanical force on the filter element is particularly an increase in the mechanical force on the filter element. In this case, the objective is to widen the pore size accordingly.

[0037] The control via the control unit can be further configured to vary the pore size of the filter element's pores in the range of 10 to 200 μm, preferably in the range of 20 to 180 μm, particularly preferably in the range of 30 to 160 μm, and especially in the range of 40 to 120 μm.

[0038] Separately, control via a control unit can be performed so that the pore size of the filter element changes automatically over time, or manually by input by the user of the device, in steps to larger pore sizes, preferably in steps of 5 to 15 μm, particularly preferably in steps of 9 to 11 μm, and very particularly preferably from a pore size of 10 μm to a pore size of 200 μm, particularly from a pore size of 40 μm to 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, up to a pore size of 120 μm. An advantage of the DEA film as a filter element is that the pore size is not necessarily limited to a specific step, but can be continuously changed by voltage and / or mechanical force.

[0039] This device may have means suitable for moving a liquid containing particles through a filter element.

[0040] This means preferably includes or comprises a stamp that is liquid-tight and movable within a first compartment of the container. The advantage here is that the user can manually control the transport of the liquid through the filter element via the movement of the stamp.

[0041] This means may further include, or may be, a pump, particularly preferably a pump configured to move a liquid having particles bidirectionally through a filter element. The advantage here is that the user can manually control the transport of the liquid through the filter element via the pump operation settings, or it can be automatically controlled (via the control unit of the device).

[0042] The filter element may contain or be made of an electroactive material. The filter element may further contain or be made of a piezoelectric material. The filter element may further contain or be made of a dielectric material. The filter element may be a dielectric elastomer actuator (DEA). The advantage of these materials or DEAs is that the pore size of the filter element can be set by changing the voltage applied to the filter element, and the shape of the cross-sectional surface of the pore does not change substantially or at all with the variation in its diameter (for example, the cross-sectional shape remains circular). This allows for more precise setting of the selectivity of the filter element than other filter elements where the shape of the cross-sectional surface of the pore also changes with the change in pore size (for example, changing from rectangular to rhombic, or from circular to elliptical). The functional principle of DEA here is based on the Maxwell stress tensor formed between two mutually isolated, oppositely charged electrodes, similar to a capacitor. When a voltage is applied, a potential difference is created between the electrodes, resulting in mechanical stress in the dielectric. Maxwell stress provides equibiaxial, linear movement, resulting in a real actuator effect that brings about a change in the pore size of the filter element's pores.

[0043] The filter element preferably further contains or is made of an elastic material. Such a material allows for better setting of the pore size by the action of mechanical forces on the filter element. If the filter element contains an elastic material (e.g., an elastomer), it is also advantageous for setting the pore size via the application of voltage. This macroscopic or mesoscopic actuator effect may therefore also be influenced by the ability to continuously set the voltage. DEA often contains or is made of an incompressible elastomer. Consequently, the filter element may also contain or be made of an incompressible material.

[0044] In one preferred embodiment, the filter element comprises or consists of a polymer, preferably an elastomer, and more preferably a thermoplastic elastomer. The polymer is selected from the group consisting of silicone elastomers, liquid rubber elastomers, and combinations thereof.

[0045] The filter element can have a thickness ranging from ≤250 μm, preferably ≤200 μm, particularly preferably ≤150 μm, and especially ≤100 μm, from top to bottom. The advantage of a thin filter element is that the device can be provided in a lighter, more compact, and less expensive manner.

[0046] In a preferred embodiment, the filter element is pretensioned via equibiaxial pre-stretching, preferably in the range of 50% to 150%. Pretensioning can be achieved by connecting the filter element to a fixed frame of the apparatus. It is advantageous in pretensioning that the original pore size of the filter element's pores, i.e., the pore diameter of the filter element's pores in the untensioned (relaxed) state, can be made smaller than the pore size required to perform the method for separating particles in a liquid. This can reduce the manufacturing cost of the filter element.

[0047] In one preferred embodiment, the filter element has continuous pores having a substantially circular cross-section. This is advantageous because, if the solid-phase particles in the upper compartment of the apparatus are spherical (which is preferable), the openings provided through the pores can therefore be adapted in a predetermined manner to the spherical diameter of the solid-phase particles.

[0048] The filter element is preferably suited to varying the pore size of its continuous pores in the range of 10 to 200 μm, preferably 20 to 180 μm, particularly preferably 30 to 160 μm, and very particularly preferably 40 to 120 μm, by the action of voltage on the filter element and / or mechanical force on the filter element. This suitability is particularly such that the variation in pore size occurs isotropically in all directions along the cross-sectional surface of the pores.

[0049] The particles to be separated can be selected from a group consisting of vesicles and living cells, preferably from the group consisting of blood vesicles and living cells, and particularly preferably from the group consisting of endosomal vesicles, exosomal vesicles, platelets, red blood cells, white blood cells, and combinations thereof.

[0050] Solid-phase particles suitable for specifically binding to the surface molecules of a first type of particle to be separated may contain or consist of a polymer, the polymer preferably selected from the group consisting of plastics, agarose, and combinations thereof.

[0051] The solid phase particles can be solid phase spheres, and preferably have a substantially round shape (spherical). A substantially round shape is advantageous because the solid phase particles thus have substantially the same spatial extent (i.e., the same diameter) in all directions of expansion, and their suitability for passing through pores having a given pore diameter can therefore be more accurately predicted and defined.

[0052] The present invention includes an apparatus and a unit, and includes the following means, namely, Means suitable for applying a voltage to a filter element, preferably including or comprising a voltage source, A means suitable for applying an oscillating fluid flow, preferably a fluid flow that oscillates perpendicular to the upper surface of the filter element, to the upper surface of the filter element. Means suitable for moving a liquid containing particles through a filter element, preferably selected from the group consisting of a stamp, a conveying device, preferably a pump, more preferably a pump configured to move a liquid containing particles bidirectionally through a filter element, and combinations thereof, which are liquid-tight and movable within a first compartment of a container. A kit having at least one, preferably all, of the above is further provided in accordance with the present invention.

[0053] Further methods for separating particles in a liquid are provided. a) Providing a solid phase particle of at least one group having a specific hydrodynamic diameter and exposing at least one molecule on its surface that is suitable for specifically binding to the surface molecules of a first type of liquid particle, b) A step of incubating a liquid having solid particles from at least one group until the solid particles of at least one group specifically bind to the surface molecules of first type particles of the liquid, c) Providing a filter element comprising, or derived from, a material suitable for changing the pore size of its pores by applying a voltage to the filter element and / or by applying a mechanical force to the filter element, d) A step of setting the pore size of the filter element by applying a voltage to the filter element and / or acting a mechanical force on the filter element so that only particles up to a desired particle size can pass through the filter element, wherein the set particle size is smaller than the hydrodynamic diameter of at least one group of solid-phase particles, e) A step of moving the liquid through a filter element, f) A step of isolating the liquid, g) A step of increasing the pore size of the filter element by reducing the voltage force and / or increasing the mechanical force on the filter element, so that particles up to a desired, in this case larger particle size, can pass through the filter element, wherein the particles are preferably particles of a first type of particle bonded to a group of solid-phase particles, h) Optionally, a step of adding a liquid which preferably does not contain any particles to a liquid which has not passed through. i) A step of moving the liquid through a filter element, j) A step of isolating a liquid containing particles of the first type of particle, k) Optionally, repeat steps g) through j) until all the particles of the liquid are separated into separate liquids according to their size. Includes.

[0054] This method may be characterized by being performed using the apparatus according to the present invention.

[0055] This method preferably follows the next step, namely, i) A step of setting the pore size of the filter element of the apparatus by applying a voltage to the filter element and / or acting a mechanical force on the filter element so that only particles up to a desired particle size can pass through the filter element, wherein the set particle size is smaller than the hydrodynamic diameter of at least one group of solid-phase particles, ii) A step of filling the upper compartment of the apparatus container with a liquid containing particles of different sizes, iii) Incubating a liquid in the upper compartment of a container of an apparatus having at least one group of solid particles until at least one solid particle of the at least one group of solid particles specifically binds to the surface molecules of a first type of particle (e.g., a first type of blood particle), iv) A step of moving the liquid into the lower compartment of the container through the filter element of the device, v) The step of isolating the liquid containing the passed particles from the lower compartment of the apparatus container, vi) a step of increasing the pore size of the filter element of the apparatus by reducing the voltage force and / or increasing the mechanical force on the filter element so that particles up to a desired, in this case larger particle size, can pass through the filter element, wherein the particles are preferably particles of a first type of particle bonded to a group of solid-phase particles, vii) Optionally, the step of filling the upper compartment of the apparatus container with a liquid that preferably contains no particles, viii) A step of moving the liquid into the lower compartment of the container through the filter element of the device, ix) A step of isolating the liquid containing particles of the first type from the lower compartment of the apparatus container, x) Optionally, repeat steps vi) through ix) until all the particles of the liquid are separated into separate liquids according to their size. Includes.

[0056] Further use of the apparatus and / or kits according to the present invention for separating particles of different sizes present in a liquid is proposed. This use may preferably relate to the isolation of one or more hematopoietic fractions from blood for diagnostic purposes and / or for manufacturing blood products, particularly for providing hematopoietic fractions for generating cell therapeutics. This use may also relate to the isolation of bacterial cells from blood. This use may further preferably relate to the isolation of endosomes or exosomes vesicles from blood, serum, or biological suspensions for diagnostic purposes and / or for providing endosomal vesicles for manufacturing vaccines. Apart from this, this use may relate to the isolation of tissue cells from mixed tissue cell fractions. This use may further relate to the isolation of cells from mixed cell suspensions derived from a bioreactor, where these cells are preferably selected from the group consisting of plant cells, animal cells, human cells, bacterial cells, yeast cells, and combinations thereof.

[0057] The subject matter of the present invention will be described in more detail with reference to the following figures and examples, without the intention of limiting it to the specific embodiments shown herein. [Brief explanation of the drawing]

[0058] [Figure 1] This diagram schematically illustrates the setting of the pore size of filter element 1, which contains or is made of an electroactive material. When a high voltage (in the kV range) is applied to the filter element by a voltage source 3 through two conductive layers 4, 4' located on the side walls of the pore 2 and separated from each other by an insulating layer 5, the pore 2 of filter element 1 contracts, and the pore size of the pore 2 becomes relatively small. When the voltage of the voltage source 3 is reduced to zero, the pore 2 of filter element 1 expands to its maximum value. The pore size of the pore 2 of filter element 1 between high voltage (e.g., several kV) and zero voltage can be set continuously to a target. [Figure 2] This figure shows a macroscopic record of the expansion of individual pores 2 in a filter element 1 containing or made of an electroactive material in response to the applied voltage. Here, the filter element is a DEA film. It can be seen that the pore diameter of these pores 2 can be reduced from 4.6 mm at zero applied voltage to 4.35 mm at an applied voltage of 3 kV (an electric field is applied to the filter element). The achieved reduction in the diameter of the illustrated pores 2 in this case reaches approximately 250 μm. [Figure 3]This diagram schematically illustrates the production of three groups of solid particles 6, 6', and 6'' of mutually different sizes, where the solid particles 6, 6', and 6'' of each group are reversibly bound to specific binding molecules 7, 7', and 7'' on their surfaces, respectively. In the first step, three groups of solid particles 6, 6', and 6'' are provided, each having solid particles 6, 6', and 6'' with different hydrodynamic radii between the groups (Figure 3A). In the second step, specific chemical functionalization of the surfaces of the solid particles 6, 6', and 6'' occurs, exposing the specific binding molecules 7, 7', and 7'' on the surfaces of the solid particles 6, 6', and 6'' of each group, where the specific binding molecules 7, 7', and 7'' differ among the three groups (Figure 3B). In the third step, binding of liquid particles 8, 8', and 8'' (e.g., blood cells) containing particles 8, 8', and 8'' to be separated occurs to the solid particles 6, 6', and 6'' of each group. This binding occurs such that each of the three groups of solid-phase particles 6, 6', and 6'' binds only to specific particles 8, 8', and 8'' (for example, specific blood cells such as B cells or T cells). Particles 8, 8', and 8'' of the same size (such as B cells and T cells) also bind to the considerably larger solid-phase particles 6, 6', and 6'', and the latter determine the hydrodynamic radius of each of the resulting complexes, thus allowing them to separate from one another in this manner. The specific hydrodynamic radius that determines the separation characteristics of the same-sized particles 8, 8', and 8'' by the filter element is therefore "assigned" to each of the aforementioned particles 8, 8', and 8'' of the same size. [Figure 4]This diagram schematically shows the penetration of the pores 2 of filter element 1 by the respective solid particles 6, 6', and 6''. Solid particles 6, 6', and 6'' are bound to each type of particle to be separated (8, 8', and 8'') via specific binding molecules 7, 7', and 7'', respectively. Since these particles have considerably larger diameters than the particles to be separated (8, 8', and 8''), the possibility of particles 8, 8', and 8'' passing through the pores 2 of filter element 1 is determined by the solid particles 6, 6', and 6'' to which they are bound. When a high voltage is applied to filter element 1 (left diagram), the pore diameter of its pores 2 becomes relatively small. Particle 8 can then pass through the pores 2 bound to the relatively smallest solid particle 6. When a low voltage is applied to filter element 1 (center diagram), the pore diameter of its pores 2 becomes larger. Particle 8' can then pass through the pores 2 bound to the larger solid particle 6'. If no voltage is applied to filter element 1 (right figure), the pore diameter of its pore 2 will be at its largest value (maximum). As a result, only the particle 8'' bound to the largest solid particle 6'' can pass through pore 2. [Figure 5]This diagram schematically illustrates an apparatus according to the present invention for separating particles in a liquid, designed in the form of a cartridge. The apparatus includes a container 9 for receiving a liquid (suspension) and a planar filter element 1 having an upper side 10 and a lower side 11, the filter element 1 having continuous pores 2 having a specified pore diameter. The filter element 1 is positioned within the container 9 so as to divide the container 9 into an upper section 12 having an opening 14 in the direction of the upper side 10 of the filter element 1 and a lower section 13 in the direction of the lower side 11 of the filter element 1, so that liquid particles in the upper section 12 can only move into the lower section 13 once they pass through the filter element 1, the upper section 12 of the container 9 having an opening 14 for receiving a liquid containing particles. The apparatus is characterized in that the upper section 12 of the container 9 includes a group of at least one solid-phase particle 6, 6', 6'' having a specific hydrodynamic diameter and exposing at least one molecule 7, 7', 7'' (not shown separately) on its surface that is suitable for specifically binding to the surface molecules of a first type of particle 8, 8', 8'' (not shown separately). The filter element 1 includes or consists of a material suitable for changing the pore size of its pores 2 by applying a voltage to the filter element 1 and / or by the action of a mechanical force on the filter element 1. The lower part of Figure 5 shows how, when the pore size of the pores 2 of the filter element 1 is small, only the smaller binding complexes 6, 7, 8 can initially pass through the pores 2 of the filter element 1 and move from the upper section 12 of the container 9 into the lower section 13 of the container 9. If the pore diameter of the pores 2 in the filter element 1 is subsequently expanded by the application of voltage and / or by the action of mechanical force, then larger binding complexes 6', 7', and 8' can also pass through the pores 2 of the filter element and move from the upper compartment 12 of the container 9 into the lower compartment 13 of the container. [Modes for carrying out the invention]

[0059] Example 1 - Manufacturing suitable solid-phase particles The solid-phase particles used in the apparatus can be manufactured effectively, rapidly, adaptively, potentially with high scalability, and resource-efficiently in water-in-oil emulsions.

[0060] Here, for example, a water-based agarose is applied to an oil phase in a liquid phase, and the agarose is deformed into droplets or beads. Subsequently, the agarose can be polymerized, and then the beads can be washed and functionalized. This method produces solid-phase agarose particles with a polydisperse size distribution. The size distribution can be precisely set by varying manufacturing parameters such as the agarose-to-oil ratio or the viscosity of the oil.

[0061] It is advantageous if the variation in the size of the generated solid phase particles is less than 10 μm, i.e., if they are relatively uniformly dispersed. It is even more advantageous if multiple fractions of solid phase particles of different sizes are generated, and the difference in the average diameter of the solid phase particles in each fraction is preferably at least 10 μm.

[0062] For example, fractions are provided in which the size variation of solid phase particles within a fraction is less than 10 μm, and the average diameter of solid phase particles in the first fraction is 40 μm, 50 μm in the second fraction, 60 μm in the third fraction, 70 μm in the fourth fraction, 80 μm in the fifth fraction, 90 μm in the sixth fraction, 100 μm in the seventh fraction, 110 μm in the eighth fraction, and 120 μm in the ninth fraction.

[0063] Example 2 - Bonding a binding molecule to a solid phase particle Chemically covalently bonded Strep-Tactin® can be attached to solid-phase particles (e.g., agarose particles) (e.g., via a chemical bonding process) to bind specific molecules suitable for specifically binding to the surface molecules of the first type of particle. A chemically covalently bonded antibody fragment (e.g., a fab fragment) (e.g., via the microbiological production of a protein containing both an antibody fragment and a Strep-tag®) can be used as a binding molecule. When the modified solid-phase particles are combined with the modified antibody fragment in aqueous solution, both bind to each other via a non-covalent (and reversible) Strep-Tactin®-Strep-tag® bond, i.e., a binding complex is formed.

[0064] For example, if an antibody fragment is selected to bind only to T cells, the T cells can then bind to the solid-phase particles via binding to the fab fragment, which is immobilized via Strep-Tactin®-Strep-tag® binding through non-covalent interactions on the solid-phase particles. The non-covalent Strep-Tectin®-Strep-tag® binding is reversible and can be separated by setting a specific biotin concentration in an aqueous solution. In other words, the T cells bound to the solid-phase particles after separation can then be separated again from the solid-phase particles by adding biotin.

[0065] Example 3 - Fabrication of filter films with selective pore size The fabrication of films containing dielectric elastomers is known from the prior art (see, for example, DE 10 2012 016 375 A1). Using such films, filter elements can be fabricated, such as those used in the method and apparatus according to the present invention.

[0066] It is also known that such films can be structured through the effects of electromagnetic radiation (e.g., laser radiation) (see, for example, DE 10 2012 016 378 A1).

[0067] To produce a filter film suitable for the apparatus and method according to the present invention, an array of continuous pores having a specified pore size is introduced into a filter film containing, or derived from, a dielectric polymer, for example. "Specified" pore size means that the variation in pore size of all pores in the filter element is less than 10 μm, preferably less than 5 μm. Pores meeting these requirements can be inserted into the filter film, for example, by electromagnetic radiation from a laser.

[0068] Example 4 - Method for separating particles in a liquid In the first step, different fractions of solid-phase particles of different sizes (for example, as in Example 1) are generated.

[0069] In the second step, these are functionalized with different cell-specific binding molecules (for example, as in Example 2).

[0070] In the third step, a filter membrane for separating particles is provided (for example, according to Example 3).

[0071] In the fourth step, solid-phase particles of different sizes (e.g., agarose particles) come into contact with a liquid containing particles to be separated (e.g., blood cells and blood vesicles). At this point, certain types of particles (certain blood cells) specifically bind to solid-phase particles of a certain size, thereby assigning ("assigned") the size of the solid-phase particles to them.

[0072] In the fifth step, the particles (e.g., blood cells) bound to each solid-phase particle are separated stepwise through a filter membrane, starting with the smallest fraction of the solid-phase particle, and the pore size of the filter membrane is gradually increased for this purpose. [Explanation of Symbols]

[0073] 1 filter element 2. Pores of filter elements 3. Voltage source 4, 4' conductive layer 5, 5' insulating layer 6, 6', 6'' solid phase particles 7, 7', 7'' binding molecule 8, 8', 8'' Particles of the solution (suspension) to be separated

Claims

1. A device for separating particles in a liquid, a) A container for receiving a liquid (suspension), b) A surface filter element having an upper surface and a lower surface Equipped with, The filter element has continuous pores having a specified pore diameter, The filter element is positioned within the container such that it divides the container into an upper section in the direction of the upper surface of the filter element and a lower section in the direction of the lower surface of the filter element, and the liquid particles in the upper section can only move into the lower section once they pass through the filter element, and the upper section of the container has an opening for receiving liquid containing particles. The upper section of the container comprises solid-phase particles of at least one group having a specific first hydrodynamic diameter and exposing at least one molecule on its surface that is suitable for specifically binding to the surface molecules of a first type of particle. In the apparatus, The filter element comprises or consists of a material suitable for changing the diameter of its pores by applying a voltage to the filter element and / or by applying a mechanical force to the filter element. The filter element is suitable for changing the diameter of its continuous pores in the range of 10 to 200 μm by the action of a voltage on the filter element and / or by the action of a mechanical force on the filter element. The aforementioned device has a voltage source, The apparatus has at least two conductive layers electrically connected to the voltage source, The apparatus is characterized in that at least one of the two conductive layers comprises a polymer and conductive particles.

2. The apparatus according to claim 1, wherein the upper compartment of the container comprises at least one solid phase particle of a second group having a second hydrodynamic diameter, the second hydrodynamic diameter being different from the first hydrodynamic diameter, and the at least one solid phase particle of the second group exposing molecules on its surface that are suitable for specifically binding to surface molecules of a second type of particle.

3. The apparatus according to claim 2, wherein the upper compartment of the container comprises at least one solid phase particle of a third group having a third hydrodynamic diameter, the third hydrodynamic diameter being different from the first and second hydrodynamic diameters, and the solid phase particle of the third group exposing molecules on its surface that are suitable for specifically binding to surface molecules of a third type of particle.

4. The at least one molecule that is exposed on the surface of the solid-phase particles and is suitable for specifically binding to the surface molecules of the first type of particles is i) containing or consisting of polypeptide chains, and / or ii) containing or consisting of polynucleotides, and / or iii) containing or derived from oligosaccharides, and / or iv) Reversibly bonded to the surface of the solid phase particles via non-covalent interactions, and / or v) The apparatus according to any one of claims 1 to 3, characterized in that it is suitable for specifically binding to surface molecules of a type of particle having a hydrodynamic diameter smaller than that of the solid phase particle.

5. At least one of the at least two conductive layers is i.) Applied to the upper or lower side of the filter element, and / or ii.) Contacting and / or an at least one electrically insulating layer disposed between the at least one conductive layer and the filter element. iii.) Displaced in the peripheral region of the pores of the filter element, and / or iv.) A continuous pore having a specified diameter at the same point as the filter element, arranged around the pore of the filter element, and / or v. ) containing metal and / or vi.) The apparatus according to any one of claims 1 to 4, characterized in that it is connected to the filter element or an electrical insulating layer by friction lock and / or material bonding.

6. The apparatus according to any one of claims 1 to 5, wherein the apparatus has means suitable for changing the diameter of the pores of the filter element by applying a mechanical force to the filter element, the means being selected from the group consisting of a stamp for applying pressure to the filter element, a pneumatic device for applying pressure to the filter element, a bimetallic wire for applying pressure to the filter element, a NiTiCu alloy for applying pressure to the filter element, and combinations thereof.

7. The apparatus according to any one of claims 1 to 6, characterized in that it has means suitable for applying a fluid flow that vibrates in a direction perpendicular to the upper surface of the filter element to the upper surface of the filter element.

8. The apparatus has a control unit configured to control the voltage of the voltage source and / or the mechanical force on the filter element, thereby, i) The voltage and / or the mechanical force on the filter element changes stepwise during the separation of particles in the liquid, and / or ii) The pore diameter of the pores of the filter element varies in the range of 10 to 200 μm, and / or iii) The apparatus according to any one of claims 1 to 7, characterized in that the pore diameter of the pores of the filter element changes in steps to a larger pore diameter automatically over time or manually by input by the user of the apparatus.

9. The apparatus has means suitable for moving the liquid having the particles through the filter element, and the means is i.) Includes, or comprises, a stamp that is liquid-tight and movable within the first compartment of the container, and / or ii.) A pump comprising or comprising a pump configured to move the liquid having the particles bidirectionally through the filter element. The apparatus according to any one of claims 1 to 8, characterized in that

10. The aforementioned filter element is i) Electroactive, and / or ii) Piezoelectric and / or iii) Dielectric and / or iv) Elastic and / or v) Incompressible, and / or vi) It is an elastomer. The apparatus according to any one of claims 1 to 9, characterized in that it includes or consists of a material.

11. The aforementioned filter element is i) Having an extent of ≤250 μm in the direction from top to bottom, and / or ii) Pretensioned and / or iii) Having continuous pores with a substantially circular cross-section, and / or iv) The apparatus according to any one of claims 1 to 10, characterized in that it is suitable for changing the diameter of the continuous pores in the filter element in the range of 20 to 180 μm by the action of a voltage on the filter element and / or by the action of a mechanical force on the filter element.

12. The apparatus according to any one of claims 1 to 11, characterized in that the particles are selected from the group consisting of vesicles and living cells.

13. The solid phase particles are i) containing or consisting of polymers, and / or ii) It is a solid sphere. The apparatus according to any one of claims 1 to 12, characterized in that

14. i) The apparatus according to any one of claims 1 to 13, ii) The next means, namely, Means suitable for applying a fluid flow oscillating in a direction perpendicular to the upper surface of the filter element to the upper surface of the filter element, and Means suitable for moving the liquid having the particles through the filter element, selected from the group consisting of a stamp liquid-tight and movable within a first compartment of the container, a pump configured to move the liquid having the particles bidirectionally through the filter element, and combinations thereof. A unit having at least one of the following, A kit that includes the following:

15. A method for separating particles in a liquid, a) Providing a solid phase particle of at least one group having a specific hydrodynamic diameter and exposing at least one molecule on its surface that is suitable for specifically binding to the surface molecules of a first type of particle of the liquid; b) Incubating the liquid having the solid particles of the at least one group until the solid particles of the at least one group specifically bind to the surface molecules of the first type of particles of the liquid; c) Providing a filter element comprising, or comprising, a material suitable for changing the pore size of its pores by applying a voltage to the filter element and / or by applying a mechanical force to the filter element; d) A step of setting the pore size of the pores of the filter element by applying a voltage to the filter element and / or acting a mechanical force on the filter element so that only particles up to a desired particle size can pass through the filter element, wherein the set particle size is smaller than the hydrodynamic diameter of the solid phase particles of the at least one group, e) The step of moving the liquid through the filter element, f) The step of isolating the liquid, g) a step of increasing the pore diameter of the pores of the filter element by reducing the voltage force and / or increasing the mechanical force on the filter element, so that particles up to a desired, in this case larger particle size, can pass through the filter element, wherein the particles are preferably particles of the first type of particles bonded to the group of solid-phase particles, h) A step of moving the liquid through the filter element, i) A step of isolating the liquid containing particles of the first type of particles, Includes, The method described above is carried out using the apparatus described in any one of claims 1 to 13.

16. The above method proceeds to the next step, namely, i) A step of setting the pore size of the pores of the filter element of the apparatus by applying a voltage to the filter element and / or by applying a mechanical force to the filter element, so that only particles up to a desired particle size can pass through the filter element, wherein the set particle size is smaller than the hydrodynamic diameter of the solid phase particles of the at least one group; ii) A step of filling the upper compartment of the container of the apparatus with a liquid containing particles of different sizes, iii) Incubating the liquid in the upper compartment of the container of the apparatus having the solid particles of the at least one group until at least the solid particles of the at least one group specifically bind to the surface molecules of the first type of particles of the particles; iv) A step of moving the liquid into the lower compartment of the container through the filter element of the apparatus, v) The step of isolating the liquid having the particles that have passed through from the lower compartment of the container of the apparatus, vi) The step of increasing the pore size of the pores of the filter element of the apparatus by reducing the voltage force and / or increasing the mechanical force on the filter element, so that particles up to a desired, in this case larger particle size, can pass through the filter element; vii) A step of moving the liquid into the lower compartment of the container through the filter element of the apparatus, viiii) The step of isolating the liquid containing the first type of particles from the lower compartment of the container of the apparatus, A method for separating particles in a liquid according to claim 15, characterized by comprising:

17. Use of the apparatus and / or the kit according to any one of claims 1 to 13 for separating particles of different sizes present in a liquid.

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