DEVICE AND METHOD FOR SEPARATING PARTICLES IN A LIQUID, KIT CONTAINING THE DEVICE, AND APPLICATIONS OF THE DEVICE

Abstract

The invention relates to a device, a method, and a kit for separating particles of different sizes in a liquid. The invention additionally relates to applications of the device. The device and the method involve the capability of binding particles to solid phase particles with different diameters in a liquid, whereby the hydrodynamic diameter of the solid-phase particles determines whether the particles can pass through pores of a filter element, the diameter of said pores being modifiable 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) of a liquid can be separated from one another with a high degree of separation efficiency, wherein the particles can be separated simply, quickly, and inexpensively. High yields can be produced, and the particles can be provided in a therapeutically applicable liquid.

Claims

1-18. (canceled)

19. A device for separating particles in a liquid comprising a) a container for receiving the liquid; b) an areal filter element having an upper side surface and a lower side surface, wherein the filter element has continuous pores having a defined pore diameter; wherein the filter element is arranged in the container such that it divides the container into an upper compartment in the direction of the upper side surface of the filter element and into a lower compartment in the direction of the lower side surface of the filter element, so that particles of a liquid in the upper compartment can only move into the lower compartment if they pass through the filter element, with the upper compartment of the container having an opening for receiving the liquid containing particles, wherein the upper compartment of the container comprises at least one group of solid phase particles that have a specific hydrodynamic diameter and that expose at least one molecule at their surface that is suitable to specifically bind to a surface molecule of a first kind of particle; and the filter material comprises a material that is suitable to change, by application of an electrical voltage to the filter element and/or by the action of a mechanical force on the filter element, the diameter of its pores.

20. The device in accordance with claim 19, wherein the upper compartment of the container comprises at least one second group of solid phase particles having a second hydrodynamic diameter, with the second hydrodynamic diameter differing from the first hydrodynamic diameter, and with the at least one second group of solid phase particles exposing a molecule at their surface that is suitable to specifically bind to a surface molecule of the second group.

21. The device in accordance with claim 20, wherein the upper compartment of the container comprises at least one third group of solid phase particles having a third hydrodynamic diameter, with the third hydrodynamic diameter differing from the first and second hydrodynamic diameters, and with the third group of solid phase particles exposing a molecule at their surface that is suitable to bind to a surface molecule of a third kind of particle.

22. The device in accordance with claim 21, wherein the upper compartment further includes a fourth, fifth, sixth, seventh, eighth, ninth, and/or tenth group of solid phase particles each comprising a hydrodynamic diameter that differs from other groups of solid phase particles in the upper compartment, and with the solid phase particles of the respective groups exposing a molecule at their surface that is suitable to specifically bind to a surface molecule of a respective other kind of particle.

23. The device in accordance with claim 19, wherein the at least one molecule that is exposed at the surface of the solid phase particles and that is suitable to specifically bind to a surface molecule of a first kind of particle i) comprises a polypeptide chain; and/or ii) comprises a polynucleotide; and/or iii) comprises an oligosaccharide; and/or iv) is reversibly bound to the surface of the solid phase particles via non-covalent interactions; and/or v) is suitable to specifically bind to a surface molecule of a kind of particle that has a smaller hydrodynamic diameter than the solid phase particles.

24. The device in accordance with claim 23, wherein i) the polypeptide chain is selected from the group consisting of antibodies, antibody fragment, and derivatives thereof; and/or ii) the polynucleotide is selected from the group consisting of DNA, RNA, and derivatives thereof; and/or iii) is reversibly bound to the surface of the solid phase particles via non-covalent interactions such that the binding is releasable via a measure selected from the group consisting of changing a concentration of a substance, changing a temperature, changing the pH, and combinations thereof; and/or v) the smaller hydrodynamic diameter is a hydrodynamic diameter that amounts to a maximum of 10% of the hydrodynamic diameter of the solid phase particles.

25. The device in accordance with claim 19, wherein the device has at least two electrically conductive layers that are connected to an electrical voltage source, with at least one of the at least two electrically conductive layers, optionally the at least two electrically conductive layers i) are applied to the upper side or to the lower side of the filter element, with optionally one of the layers being applied to the upper side of the filter element and the other being applied to the lower side of the filter element; and/or ii) at least one electrical insulation layer being arranged between the at least one electrically conductive layer and the filter element, with the at least two electrically conductive layers, optionally each contacting at least one electrical insulation layer that is arranged between the at least two electrically conductive layers and the filter element; and/or iii) are arranged in a marginal region of the pores of the filter element; and/or iv) are fully arranged around the pores of the filter element and having continuous pores having a defined pore diameter at the same points as the filter element; and/or v) comprising a polymer, optionally an electrically conductive polymer; and/or vi) comprising electrically conductive particles, with the portion of the electrically conductive particles optionally being in the range from 0.001 to 30 wt %, relative to the total weight of the electrically conductive layer; and/or vii) comprising a metal; and/or viii) are connected to the filter element or to an electrically insulating layer with a friction-lock and/or a material bond; wherein at least one of the at least two electrically conductive layers, optionally the at least two electrically conductive layers, is/are applied to the filter element or to an electrically conductive layer via a process selected from the group consisting of pad printing, doctor knife coating, screen printing, inkjet printing, jetting, spraying, vaporization, and combinations thereof, optionally combined with a laser structuring process.

26. The device in accordance with claim 19, wherein the device has an electrical voltage source that is electrically conductively connected to at least two electrically conductive layers.

27. The device in accordance with one claim 19, wherein the device has a means that is suitable to change the pore diameter of its pores by the action of a mechanical force on the filter element, wherein the means is selected from the group consisting of a stamp for exerting a pressure on the filter element, a pneumatic device for exerting a pressure on the filter element, a bimetallic wire for exerting a pressure on the filter element, an NiTiCu alloy for exerting a pressure on the filter element, and combinations thereof.

28. The device in accordance with claim 19, wherein the device has a means that is suitable to apply an oscillating fluid flow to the upper side surface of the filter element.

29. The device in accordance with claim 19, which has a control unit that is configured to control an electrical voltage of a voltage source and/or a mechanical force on the filter element.

30. The device in accordance with claim 19, which has a means that is suitable to move the liquid having particles through the filter element.

31. The device in accordance with claim 19, wherein the filter element comprises a material that i) is electroactive; and/or ii) is piezoelectric; and/or iii) is dielectric; and/or iv) is elastic; and/or v) is incompressible; and/or vi) is a polymer.

32. The device in accordance with claim 19, wherein the filter element i) has an extent from the upper side in the direction of the lower side in the range from ?250 ?m; and/or ii) is pretensioned; and/or iii) has continuous pores that have a substantially round cross-section; and/or iv) is suitable to change the pore diameter of its continuous pores by the action of an electrical voltage on the filter element and/or by the action of a mechanical force on the filter element in a range from 10 to 200 ?m.

33. The device in accordance with claim 19, wherein the particles are selected from the group consisting of vesicles and biological cells.

34. The device in accordance with claim 19, wherein the solid phase particles i) comprise a polymer; and/or ii) are solid phase spheres.

35. A kit comprising i) a device in accordance with 19; and ii) a unit that has at least one of the following means: a means that is suitable to apply an electrical voltage to the filter element; a means that is suitable to apply an oscillating fluid flow; and a means that is suitable to move the liquid having the particles through the filter element.

36. A method for separating particles in a liquid comprising the steps of: a) providing at least one group of solid phase particles that have a specific hydrodynamic diameter and that expose at least one molecule at their surface that is suitable to specifically bind to a surface molecule of a first kind of particle of the particles of the liquid; and b) incubating the liquid having the at least one group of solid phase particles until the solid phase particles of the at least one group of solid phase particles have been specifically bound to a surface molecule of a first kind of particle of the particles of the liquid; c) providing a filter element that comprises a material that is suitable to change, by application of an electrical voltage to the filter element and/or by the action of a mechanical force on the filter element, the pore diameter of its pores; d) setting the pore diameter of the pores of the filter element via application of an electrical voltage to the filter element and/or the action of a mechanical force on the filter element so that only particles up to a desired particle diameter can pass through the filter element, with the set particle diameter being smaller than a hydrodynamic diameter of the at least one group of solid phase particles; e) moving the liquid through the filter element; f) isolating the liquid; g) increasing the pore diameter of the pores of the filter element by reducing the power of the electrical voltage and/or by increasing the mechanical force on the filter element so that particles up to a desired, now larger particle diameter can pass through the filter element; h) optionally adding a liquid that does not comprise any particles to the liquid having the particles that have not passed through; i) moving the liquid through the filter element; j) isolating the liquid that comprises the particles of the first kind of particle; k) optionally repeating steps g) to j) until all the particles of the liquid are present separated by their size in separate liquids.

37. The method for separating particles in a liquid in accordance with claim 36, wherein the separation is carried out in a device comprising a) a container for receiving the liquid; b) an areal filter element having an upper side surface and a lower side surface, wherein the filter element has continuous pores having a defined pore diameter; wherein the filter element is arranged in the container such that it divides the container into an upper compartment in the direction of the upper side surface of the filter element and into a lower compartment in the direction of the lower side surface of the filter element, so that particles of a liquid in the upper compartment can only move into the lower compartment if they pass through the filter element, with the upper compartment of the container having an opening for receiving the liquid containing particles, wherein the upper compartment of the container comprises at least one group of solid phase particles that have a specific hydrodynamic diameter and that expose at least one molecule at their surface that is suitable to specifically bind to a surface molecule of a first kind of particle; and the filter material comprises a material that is suitable to change, by application of an electrical voltage to the filter element and/or by the action of a mechanical force on the filter element, the diameter of its pores.

38. The method in accordance with claim 37, comprising: i) setting the pore diameter of the pores of the filter element of the device via application of an electrical voltage to the filter element and/or via the action of a mechanical force on the filter element so that only particles up to a desired particle diameter can pass through the filter element, with the set particle diameter being smaller than a hydrodynamic diameter of the at least one group of solid phase particles; ii) filling the upper compartment of the container of the device with a liquid that comprises particles having different sizes; iii) incubating the liquid in the upper compartment of the container of the device having the at least one group of solid phase particles until at least solid phase particles of the at least one group of solid phase particles have specifically bound to a surface molecule of a first kind of particle of the particles; iv) moving the liquid through the filter element of the device into the lower compartment of the container; v) isolating the liquid having the particles that have passed through from the lower compartment of the container of the device; vi) increasing the pore diameter of the pores of the filter element of the device by reducing the power of the electrical voltage and/or by increasing a mechanical force on the filter element so that particles up to a desired, now larger particle diameter can pass through the filter element; vii) optionally filling the upper compartment of the container of the device with a liquid that does not comprise any particles; viii) moving the liquid through the filter element of the device into the lower compartment of the container, ix) isolating the liquid that comprises the particles of the first kind of particles from the lower compartment of the container of the device; and x) optionally repeating steps vi) to ix) until all the particles of the liquid are present separated by their size in separate liquids.

Description

[0086] The subject matter in accordance with the invention will be explained in more detail with reference to the following Figures and examples without intending to restrict it to the specific embodiments shown here.

[0087] FIG. 1 schematically shows a setting of the pore size of a filter element 1 that comprises or consists of an electroactive material. If a high electrical voltage (in the kV range) is applied by an electrical voltage source 3 to the filter element via two electrically conductive layers 4, 4 that are located at the side wall of a pore 2 and are separated from one another by an isolation layer 5, the pores 2 of the filter element 1 contract and the pore diameter of the pores 2 is comparatively small. If the voltage of the electrical voltage source 3 is reduced to zero, the pores 2 of the filter element 1 expand to a maximum value. The pore diameter of the pores 2 of the filter element 1 between the high voltage (e.g., some kV) and the voltage value zero can be set in a targeted and continuous manner.

[0088] FIG. 2 shows a macroscopic recording of an expansion of an individual pore 2 of a filter element 1 that comprises or consists of an electroactive material in dependence on the applied electrical voltage. The filter element here is a DEA membrane. It can be recognized that the pore diameter of this pore 2 can be reduced from a diameter of 4.6 mm at an applied electrical voltage of zero to a pore diameter of 4.35 mm at an applied voltage of 3 kV (electrical field is applied to the filter element). The achieved reduction of the diameter of the pore 2 shown amounts to approximately 250 ?m in this case.

[0089] FIG. 3 schematically shows the manufacture of three groups having solid phase particles 6, 6, 6 of mutually different sizes, with the solid phase particles 6, 6, 6 of each group respectively having reversibly bound to a specific binding molecule 7, 7, 7 at the surface. In a first step, three groups of solid phase particle 6, 6, 6 are provided that each have solid phase particles 6, 61, 6 having a hydrodynamic radius differing among the groups (FIG. 3A). In a second step, a specific chemical functionalization of the surface of solid phase particles 6, 61, 6 takes place so that the solid phase particles 6, 61, 6 of each group expose a specific binding molecule 7, 7, 7 at their surface and the specific binding molecule 7, 7, 7 differs among the three groups (FIG. 3B). In a third step, a binding takes place of particles 8, 81, 8 (e.g., blood cells) of the liquid that comprises the particles 8, 81, 8 to be separated to the solid phase particles 6, 6, 6 of the respective groups. The binding takes place such that each of the three groups of solid phase particles 6, 61, 6 only binds specific particles 8, 8, 8 (e.g., specific blood cells such as B cells or T cells). Particles 8, 8, 8 of the same size (such as B cells and T cells) can also be separated from one another by this measure since they are bound to considerably larger solid phase particles 6, 6, 6 and the latter determine the respective hydrodynamic radius of the complexes produced. A respective specific hydrodynamic radius that determines the separation properties of the particles of the same size 8, 81, 8 by the filter element is thus respectively imparted to said particles 8, 8, 8 of the same size.

[0090] FIG. 4 schematically shows a penetration of a pore 2 of the filter element 1 by the respective solid body particles 6, 6, 6. Since the solid body particles 6, 6, 6 are each bound to a respective kind of particle of the particles 8, 8, 8 to be separated via a specific binding molecule 7, 7, 7 and have a considerably larger diameter than the particles 8, 8, 8 to be separated, the possibility of the particles 8, 8, 8 to be separated passing through the pores 2 of the filter element 1 is determined by the solid body particle 6, 6, 6 to which they are bound. If a high electrical voltage is applied to the filter element 1 (left image), the pore diameter of its pores 2 is relatively small. The particles 8 can now consequently pass through the pore 2 that are bound to the relatively smallest solid body particles 6. If a lower electrical voltage is applied to the filter element 1 (middle image), the pore diameter of its pores 2 becomes larger. The particles 8 can now consequently pass through the pore 2 that are bound to larger body particles 6. If no electrical voltage is applied to the filter element 1 (right image), the pore diameter of its pores 2 adopts the largest value (maximum value). Only the particles 8 can consequently pass through the pore 2 that are bound to the largest solid body particles 6.

[0091] FIG. 5 schematically shows a device in accordance with the invention for separating particles in a liquid that is here designed in the form of a cartridge. The device comprises a container 9 for receiving a liquid (suspension) and a planar filter element 1 having an upper side surface 10 and a lower side surface 11, with the filter element 1 having continuous pores 2 having a defined pore diameter. The filter element 1 is arranged in the container 9 such that it divides the container 9 into an upper compartment 12 having an opening 14 in the direction of the upper side surface 10 of the filter element 1 and into a lower compartment 13 in the direction of the lower side surface 11 of the filter element 1 so that particles of a liquid in the upper compartment 12 can only move into the lower compartment 13 if they pass through the filter element 1, with the upper compartment 12 of the container 9 having an opening 14 for receiving a liquid having particles. The device is characterized in that the upper compartment 12 of the container 9 comprises at least one group of solid phase particles 6, 6, 6 that have a specific hydrodynamic diameter and that expose at least one molecule 7, 7, 7 at their surface (not shown separately) that is suitable to specifically bind to a surface molecule of a first kind of particle 8, 8, 8 (not shown separately). The filter material 1 comprises or consists of a material that is suitable to change the pore diameter of its pores 2 by application of an electrical voltage to the filter element 1 and/or by the action of a mechanical force on the filter element 1. It is shown in the lower part of FIG. 5 how initially only the smaller binding complexes 6, 7, 8 can pass through the pores 2 of the filter element 1 in the case of a small pore diameter of the pores 2 of the filter element 1 and can move from the upper compartment 12 of the container 9 into the lower compartment 13 of the container 9. If the pore diameter of the pores 2 of the filter element 1 is subsequently enlarged by application of an electrical voltage and/or by the action of a mechanical force, the larger binding complexes 6, 7, 8 can now also pass through the pores 2 of the filter element and can move from the upper compartment 12 of the container 9 into the lower compartment 13 of the container.

EXAMPLE 1MANUFACTURE OF SUITABLE SOLID PHASE PARTICLES

[0092] The solid phase particles used in the device can be manufactured effectively, quickly adaptably, potentially high scalably, and resource savingly in water in oil emulsions.

[0093] Water based agarose is here, for example, applied into an oil phase in a liquid phase, with the agarose deforming into droplets or beads. After a subsequent polymerization of the agarose, the beads can be washed and functionalized. This method produces solid phase particles of agarose with a polydisperse size distribution. The size distribution can be precisely set by the variation of the manufacturing parameters such as the agarose to oil ratio or oil viscosity.

[0094] It is advantageous if the produced solid phase particles have a size variation of less than 10 ?m, that is are relatively homodisperse. It is furthermore advantageous if a plurality of different size fractions of solid phase particles are produced, with the difference of the mean diameters of the solid phase particles between the individual fractions amounting to preferably at least 10 ?m.

[0095] Fractions are, for example, provided in which the solid phase particles within a fraction have a size variation of less than 10 ?m and in which the mean diameter of the solid phase particles of a first fraction amounts to 40 ?m, of a second fraction to 50 ?m, of a third fraction to 60 ?m, of a fourth fraction to 70 ?m, of a fifth fraction to 80 ?m, of a sixth fraction to 90 ?m, of a seventh fraction to 100 ?m, of an eighth fraction to 110 ?m, and of a ninth fraction to 120 ?m.

EXAMPLE 2BINDING A BINDING MOLECULE TO THE SOLID PHASE PARTICLES

[0096] Chemically covalent Strep-Tactin? is, for example, bound (e.g., via a chemical coupling process) to the solid phase particles (e.g., agarose particles) to bind a specific molecule that is suitable to specifically bind to a surface molecule of a first kind of particle. An antibody fragment (e.g., fab fragment) that has chemically covalently bound (e.g., via microbiological production of a protein that comprises both the antibody fragment and the Step-Tag?) a Strep-Tag? can, for example, be used as a binding molecule. If the modified solid phase particles are now combined with the modified antibody fragments in a watery solution, both bind to one another via a noncovalent (and reversible) Strep-Tactin?-Strep-Tag? binding, i.e., a binding complex is produced.

[0097] If the antibody fragment is selected such that it, for example, only binds T cells, T cells can be found to the solid phase particles via the binding to the fab fragment that is in turn immobilized via the Strep-Tactin?-Strep-Tag? binding via noncovalent interactions on the solid phase particle. The noncovalent Strep-Tectin?-Strep-Tag? binding is reversible and can be separated by setting a specific biotin concentration in the aqueous solution. In other words, the T cells bound to the solid phase particles after the separation has taken place can in turn again be separated from the solid phase particles via the addition of biotin.

EXAMPLE 3MANUFACTURE OF A FILTER MEMBRANE HAVING A SELECTIVE PORE SIZE

[0098] The manufacture of a membrane that comprises a dielectric elastomer is known from the prior art (see e.g., DE 10 2012 016 375 A1). Such membranes can be used to manufacture a filter element such as is used in the method in accordance with the invention and in the device in accordance with the invention.

[0099] It is furthermore known that such membranes can be structured via the effect of electromagnetic radiation (e.g., of a laser) (see e.g., DE 10 2012 016 378 A1).

[0100] To manufacture a filter membrane suitable for the device in accordance with the invention and for the method in accordance with the invention, an array of continuous pores having a defined pore diameter is introduced, for example, into a filter membrane that comprises or consists of a dielectric polymer. Pores having a defined pore diameter means that the pore diameter of all the pores of the filter element varies by less than 10 ?m, preferably less than 5 ?m. Pores having these requirements can, for example, be inserted into the filter membrane via an electromagnetic radiation of a laser.

EXAMPLE 4METHOD FOR SEPARATING PARTICLES IN A LIQUID

[0101] In a first step, different fractions of solid phase particles of different sizes (e.g., in accordance with Example 1) are produced.

[0102] In a second step, they are respectively functionalized with the different cell specific binding molecules (e.g., in accordance with Example 2).

[0103] In a third step, a filter membrane for separating the particles is provided (e.g., in accordance with Example 3).

[0104] In a fourth step, the solid phase particles (e.g., agarose particles) of different sizes are contacted by a liquid that comprises the particles to be separated (e.g., blood cells and vesicles of blood). In this respect, specific kinds of particles (specific blood cells) specifically bind to solid phase particles of a specific size, whereby the size of the solid phase particles is placed on (imparted to) them.

[0105] In a fifth step, the particles bound to the respective solid phase particles (e.g., blood cells) are separated stepwise, starting with the smallest fraction of the solid phase particles, via the filter membrane, with the pore diameter of the filter membrane being increased stepwise for this purpose.

REFERENCE NUMERAL LIST

[0106] 1: filter element; [0107] 2: pore of the filter element [0108] 3: electrical voltage source [0109] 4, 4: electrically conductive layer [0110] 5, 5: isolation layer [0111] 6, 6, 6: solid phase particles [0112] 7, 7, 7: binding molecule [0113] 8,8,8: particles of the solution (suspension) to be separated