Device for fractionating suspension sample

Abstract

To provide a device for fractionating a suspension sample.SOLUTION: The present invention relates to a device for fractionating a suspension sample and to uses of such device. Furthermore, the present invention also relates to a method of fractionating a suspension sample into a liquid phase and a solid phase. The present invention also relates to a device and method for separating a sample comprising a solvent and two or more components having different molecular weights and being dissolved in the solvent, into the two or more components. The device may include at least one polymer matrix element.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a device for fractionating a suspension sample and to the use of such a device.Furthermore, the present invention also relates to a method for fractionating a suspension sample into a liquid phase and a solid phase.The present invention also relates to a device and a method for separating a sample comprising a solvent and two or more components having different molecular weights and dissolved in said solvent into said two or more components. [Background technology]

[0002] The separation of suspended samples into different phases has been the subject of intense research and practice for many years: clarification of beer or wine or skimming of milk are prime examples. Blood can be considered as a good example of a particulate fraction suspended in an aqueous liquid. Fractionation of whole blood into cellular and plasmatic components constitutes an important step in many bioanalytical workflows. Typically, this is achieved by centrifuging the sample in a corresponding sample collection vessel. After separating the cellular components from the liquid components by density in a centrifugal field or by coagulation followed by centrifugation, the plasma or serum is pipetted and further processed. During centrifugation, the sample vessel is moved around the rotation axis of the centrifuge. The effective centrifugal force causes accelerated sedimentation and solidification of the so-called blood cake or clot, which is in fact the cellular fraction of blood. The solidity of such a sedimented cellular fraction is decisive for the subsequent withdrawal of the plasma or serum. Such withdrawal is performed using a pipette with the centrifuge tube in an upright position. Contacting the cellular fraction with the tip of the pipette, which would cause mixing of the two phases, should be avoided. This is a standard procedure in laboratories, but requires the presence of a suitable laboratory centrifuge and suitable laboratory technicians [1]. In a further variant of this methodology, the sample tube is filled with a thixotropic gel that allows the separation of plasma and cellular fractions [2]. However, it is desirable to achieve fractionation without centrifugation. Different filter-based methods are described in the literature. Typically, a dedicated filter is used that retains the cellular components and allows the passage of the plasma liquid [3]. Since cells tend to block the filters, alternative approaches have been developed (e.g., by using membrane filters or hollow fiber membranes with sedimentation assisted filtration [4], sample dilution [5] or tangential flow and pumps [6]). A blood sample is pumped through hollow fibers with asymmetric pores. The plasma can penetrate the hollow fiber wall and be collected on the outside of the hollow fiber. Cells cannot penetrate the membrane and remain in the internal volume of the hollow fiber. Due to the tangential flow with respect to the membrane, clogging of the membrane holes is avoided.By passing the sample back and forth through the hollow fibers, the cell fraction can be enriched and / or the plasma fraction separated. However, the method requires a considerable effort in terms of the equipment used and is characterized by a somewhat significant dead volume that limits its use to samples of rather large volumes. Thus, small samples in the sub-milliliter range cannot be efficiently processed using this technique. A different method for plasma separation is based on the use of a soluble matrix that is placed in physical contact with the downstream surface of a filtration membrane. The soluble matrix is ​​used to passively draw the sample through the membrane by forces generated through the dissolution process of the matrix in the plasma [7]. However, this approach is only feasible if the matrix material does not interfere with downstream analytical processes. A similar approach shows the use of a solid monolith to filter blood [8]. Typically, in this approach, it is necessary to add a buffer solution to obtain a sample of plasma from the device. The drawback of this method is that the obtained sample is diluted and altered by the buffer used. Moreover, the process as all filter-based approaches in general are not efficient in terms of the volume of plasma obtained. Microfluidic-based tools have also been shown to be suitable for the purpose of plasma separation [9]. However, the need for complex microstructures and low plasma production yields have prevented this technology from becoming widely applied. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Tuck, MK, et al., Standard operating procedures for serum and plasma collection: early detection research network consensus statement standard operating procedure integration working group. J Proteome Res, 2009. 8(1): p. 113-7. [non-patent document 2] Smith, WC, Improved method for separating the cellular components of blood samples. 1989, Google Patents. [non-patent document 3] Pall David B, RENYUS, et al., Device and method for blood separation. | Device and method for separating blood. | Device and method for the separation of blood. | Device and method for blood separation Device and method for the separation of blood Device and Method for Separating Blood, GCNYUS Pall Corporation, Editor. 1991: EP. [non-patent document 4] Liu, C., et al., Membrane-based, sedimentation-assisted plasma separator for point-of-care applications. Analytical chemistry, 2013. 85(21): p. 10463-10470. [non-patent document 5] Nakayama, K. and K. Morimoto, Assessment of accuracy of immediate blood separation method: a novel blood analysis strategy. Environ Health Prev Med, 2011. 16(1): p. 1-5. [non-patent document 6] Hagihara Takeaki, OsOkJP and OsOkJP Aoki Satoshi, A compact plasma separator and an apparatus containing the same. | Compact plasma separator and device with such a separator. | Compact plasma separator and device containing a tel separator. | A compact plasma separator and an apparatus containing the same | Compact plasma separator and device containing a tel separator | Compact plasma separator and device with such a separator, CKTJP Asahi Medical Co. Ltd., editor. 1993: EP. [non-patent document 7] McNeely Michael Ryan, US, AUTOMATIC PLASMA SEPARATION AND METERING | AUTOMATIC PLASMA SEPARATION AND DOSAGE, USMMRUS McNeely Michael Ryan, Editor. 2017: WO. [non-patent document 8] Rueda Ivan, PCAUS, et al., PROCESSING BLOOD SAMPLES TO DETECT TARGET NUCLEIC ACIDS, CCAUSMINCUSMINC Monolythix Inc, Editor. 2017: US. [Non-Patent Document 9] Mukherjee, S., et al., Plasma separation from blood: the 'lab-on-a-chip' approach. Crit Rev Biomed Eng, 2009. 37(6): p. 517-29. Summary of the Invention [Problem to be solved by the invention]

[0004] Thus, there is a need for a simple tool to separate / fractionate a suspension sample into different phases, preferably a liquid and a solid phase.

[0005] A sample comprising a solvent and two components having different molecular weights and dissolved in the solvent. We also need a simple tool to separate the pull into its two components.

[0006] There is a further need for tools for phase separation over a wide range of sample volumes, and for tools for phase separation of suspension samples in the microliter range, i.e., where very small volumes of sample (e.g., less than 1 ml) are available. [Means for solving the problem]

[0007] In a first aspect, the present invention relates to a device configured for separating a sample comprising a solvent and two components with different molecular weights and dissolved in said solvent into said two components, or for fractionating a suspension sample into a liquid phase and a solid phase, said device comprising at least one polymeric matrix element, said polymeric matrix element comprising a porous polymer scaffold and an interstitial pore space within said polymeric scaffold, said porous polymeric scaffold being composed of a polymer responsive to a change in at least one external condition to which said polymeric matrix element is exposed during use of said device, such as pH, temperature, salt conditions, the presence or absence of a chemical. In one embodiment, said at least one polymeric matrix element alternatively has the ability to reversibly adopt an expanded and contracted state, where the reversible adaptation of the expanded or contracted state occurs in response to a change in at least one external condition to which said polymeric matrix element is exposed during use of said device. Preferably, said at least one polymeric matrix element is Repeatedly, It has the ability to reversibly assume such expanded and contracted states.

[0008] In one embodiment, the device further comprises at least one sample vessel, the at least one sample vessel comprising or configured to comprise the at least one polymer matrix element, the at least one sample vessel being capable of receiving a sample comprising a solvent and two components having different molecular weights and dissolved in the solvent, or a suspension sample. The sample vessel, as used herein, is a vessel in which the sample to be separated or fractionated into different components is provided or introduced prior to separation or fractionation.

[0009] In one embodiment, the at least one polymeric matrix element is either a macroscopically distinct particle, preferably in the shape of a bead, sphere, nodule, ball, cube, cuboid, block, ellipsoid, or irregularly shaped body, or the at least one polymeric matrix element is a macroscopic body or layer immobilized on the surface of a substrate within the device.

[0010] In one embodiment, the at least one polymeric matrix element is detachable or removable from the device or configured to be detachable or removable, preferably wherein the at least one polymeric matrix element is a disposable, single-use polymeric matrix element.

[0011] In one embodiment, the device further comprises means for effecting a change in at least one external condition to which the polymer matrix element is exposed during use of the device, wherein preferably said means for effecting a change in at least one external condition is a heating and / or cooling means.

[0012] In one embodiment, said means, preferably said heating and / or cooling means, are configured to expose said at least one polymer matrix element to at least one change of external condition, wherein preferably said means is part of said device and / or is integrated into said device and / or is in contact with said suspension sample. The suspension sample may be adapted to be in contact with the sample.

[0013] In one embodiment, the device further comprises a carrier tool configured to allow handling of the at least one polymeric matrix element, the carrier tool acting as a substrate having a surface on which the at least one polymeric matrix element is immobilized, the carrier tool being dimensioned such that it can be immersed in a sample comprising a solvent and two components having different molecular weights and dissolved in the solvent, or in a suspension sample, and such that it can contact the at least one polymeric matrix element with the sample, preferably the suspension sample.

[0014] In one embodiment, the carrier tool is configured to effect a change in at least one external condition to which the polymer matrix element is exposed during use of the device, such as pH, temperature, salt conditions, presence or absence of chemicals, wherein preferably the carrier tool is configured to effect a change in temperature.

[0015] In one embodiment, the carrier tool comprises a heating and / or cooling means for heating and / or cooling the at least one polymer matrix element or the carrier tool is connected or in contact with a heating and / or cooling means for heating and / or cooling the at least one polymer matrix element, wherein preferably the heating and / or cooling means is a heating and / or cooling means as defined above.

[0016] In one embodiment, the carrier tool is a disposable, single use carrier tool and is configured to be releasably connected to, releasably contacted by or releasably included with the heating and / or cooling means, preferably wherein the carrier tool is configured as a pen, stick, rod, cylinder, bar, rod, baton or member, wherein the carrier tool has an interface that releasably connects to, releasably contacts or releasably includes the heating and / or cooling means.

[0017] In one embodiment, the device further comprises at least one liquid phase collection vessel, which, if present, is configured to capture the polymer matrix elements and / or the carrier tool with the polymer matrix elements immobilized on the carrier tool. It should be noted that such liquid phase collection vessel is a vessel different from the sample vessel. The liquid phase collection vessel is typically a vessel to which the polymer matrix elements and / or the carrier tool with the polymer matrix elements immobilized on the carrier tool, if present, are transferred after the polymer matrix elements have been exposed to a suspension sample and after capturing a liquid phase from such suspension sample. In effect, the liquid phase collection vessel serves the purpose of collecting the liquid phase discharged from the polymer matrix elements.

[0018] In one embodiment, the liquid phase collection vessel comprises a means for taking up a defined volume of the liquid phase and / or a means for measuring the volume of the liquid phase.

[0019] It should be noted that after the polymeric matrix element takes up the liquid phase from a suspension sample, some portion of the solid phase from the suspension may become adhered or attached to the polymeric matrix element, and therefore it may be necessary to remove such solid phase from the polymeric matrix element, preferably from the outside of the polymeric matrix element.

[0020] In one embodiment, for removing the solid phase from the polymeric matrix element, in particular when a part of the solid phase is attached or adhered to the polymeric matrix element, a) the sample vessel and / or, if present, the carrier tool, and / or, if present, the liquid phase collection vessel, comprise a means for mechanically removing the solid phase from the polymeric matrix element, such as an orifice, a constriction, a sliding ring, a lip, an edge or an opening, the internal dimensions of which match the external dimensions of the polymeric matrix element; or b) the sample vessel and / or, if present, the carrier tool, and / or, If present, the liquid phase collection vessel comprises a substance having an affinity for the solid phase, to which the solid phase preferably adheres when in or comes into contact with the substance; or c) the sample vessel and / or, if present, the carrier tool and / or, if present, the liquid phase collection vessel comprise a means for washing the solid phase from the polymeric matrix elements, e.g. a jet ejecting a liquid that washes the solid phase from the polymeric matrix elements; or d) the device further comprises a separation tool, e.g. a spatula, blade, scraper, for removing the solid phase from the polymeric matrix elements.

[0021] In one embodiment, the device further comprises means for further processing and / or analyzing the liquid phase, where preferably, the means for further processing and / or analyzing the liquid phase comprises a spectrophotometer and / or means for adding one or several chemical agents to the liquid phase.

[0022] In one embodiment, the sample comprising a solvent and two components having different molecular weights and dissolved in the solvent is an aqueous sample comprising a macromolecular component, such as a protein or peptide or nucleic acid, as one of the two components, and a low molecular component, such as a dye or salt, as the other of the two components. In one embodiment, the suspension sample is a whole blood sample, the liquid phase of the whole blood sample is plasma, and the solid phase of the whole blood sample is or comprises blood cells of the whole blood sample.

[0023] In one embodiment, the polymer responsive to changes in at least one external condition to which the polymer matrix element is exposed during use of the device is preferably poly(N-isopropylacrylamide) (pNIPAM), poly(N-isopropylacrylamide / N,N'-methylenebis(acrylamide), poly(N-isopropylacrylamide)-co-(acrylic acid), poly(N-isopropylacrylamide)-co-(acrylic acid)-co-(poly(ethylene glycol) methyl ether acrylate, poly(N-isopropylacrylamide)-co-(poly(ethylene glycol) diacrylate, poly(N-isopropylacrylamide)-co-(poly(ethylene glycol) methyl ether acrylate, poly[2-(dimethylamino)ethyl methacrylate]ethyl meth ... The thermoresponsive polymer is a thermoresponsive polymer having a lower critical solution temperature (LCST polymer) selected from poly(N-acryloylglycinamide) (PNAGA), poly(allylamine)-co-poly(allylurea) and its derivatives, poly(methacrylamide), poly(N-acryloylaspartamide), poly(N-methacryloylglutamine amide), poly(acrylamide)-co-(acrylonitrile), poly(sulfobetaine), poly(phosphorylcholine).

[0024] In one embodiment, the responsive polymer, in particular the thermoresponsive polymer, is preferably are crosslinked either by at least one crosslinking reagent which crosslinks and interconnects between different polymer chains within the polymer, or by providing a substrate within the device, in particular a substantially flat or curved substrate, adjacent to the polymer matrix element and by interfacially crosslinking different polymer chains of the polymer to the substrate, in particular to the surface of the substrate.

[0025] In one embodiment, the at least one polymeric matrix element has the ability to reversibly, preferably repeatedly reversibly, assume expanded and contracted states, and preferably, in the expanded state, the interstitial pore space has a volume that is at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80% and even more preferably at least 90% of the total volume of the polymeric matrix element when it is in the expanded state.

[0026] In one embodiment, the interstitial pore space is not accessible to the solid phase of the suspension sample, but is accessible to the liquid phase of the suspension sample, and is preferably dimensioned to accommodate at least more than 50% of the liquid phase of the suspension sample, more preferably more than 75%, and even more preferably more than 90% of the liquid phase of the suspension sample.

[0027] In one embodiment the interstitial pore space comprises pores, the average diameter of the pores being <5 μm, preferably <3 μm, more preferably <1 μm.

[0028] In one embodiment, the device is dimensioned to fractionate a suspension sample having a volume in the range of 10 μl to 500 μl, wherein the interstitial pore space has a volume that captures at least 50%, preferably at least 75%, more preferably at least 90% and even more preferably at least 95% of the suspension sample.

[0029] In one embodiment, the suspension sample has a volume of 10 μl to 200 μl, preferably 50 μl to 200 μl, more preferably 100 μl to 200 μl.

[0030] In a further aspect, the present invention provides a method for fractionating a suspension sample into a liquid phase and a solid phase, the method comprising: a) providing, in any order, but separately from each other, a device according to the invention (as defined above) and a suspension sample, wherein said at least one polymeric matrix element of said device is in a dry state or said at least one polymeric matrix element of said device is provided under conditions in which said responsive polymer is in a contracted state but said at least one polymeric matrix element of said device is in a wet state; b) exposing the at least one polymeric matrix element of the device to the suspension sample under external conditions that allow the polymeric matrix element to reversibly assume an expanded state, thus allowing the liquid phase in the suspension sample to enter the interstitial pore spaces of the at least one polymeric matrix element, while simultaneously excluding the solid phase in the suspension sample from the interstitial pore spaces; c) removing said at least one polymeric matrix element from said sample, thereby removing said liquid phase contained within said interstitial pore space from said sample, thereby separating said liquid phase from a solid phase in said sample.

[0031] In one embodiment, in step c), the at least one polymer matrix element, if present, is removed from the sample vessel (as further defined above) and, if present, transferred to a liquid phase collection vessel (as further defined above).

[0032] In one embodiment, the method further comprises: d) changing the external conditions to which the polymer matrix elements are exposed to conditions that allow the polymer matrix elements to reversibly assume a contracted state, thereby releasing the liquid phase from the polymer matrix elements.

[0033] In one embodiment, the polymer responsive to a change in at least one external condition is a thermoresponsive polymer (as further defined above), and the external condition is temperature.

[0034] In one embodiment, the suspension sample is a whole blood sample, the liquid phase of the whole blood sample is plasma and the solid phase of the whole blood sample is or comprises blood cells of the whole blood sample, preferably a majority of whole blood cells, more preferably whole blood cells of the whole blood sample.

[0035] In a further aspect, the present invention also relates to the use of a device (as defined above) or of a polymer (as defined above) responsive to changes in at least one external condition, such as pH, temperature, salt conditions, presence or absence of a chemical, for fractionating a suspension sample into liquid and solid phases, preferably for fractionating a whole blood sample into plasma and blood cells, wherein preferably said use comprises steps a) to c) (as defined above) and optionally step d) (also as defined above).

[0036] In a further aspect, the present invention provides a method for separating a sample comprising a solvent and two components having different molecular weights and dissolved in said solvent into said two components, said method comprising: a) providing, in any order, but separately from each other, a device according to the invention (as defined above) and a sample (hereinafter in this embodiment "sample") comprising a solvent and two components having different molecular weights and dissolved in said solvent, wherein said at least one polymer matrix element of said device is in a dry state or said at least one polymer matrix element of said device is provided under conditions in which said responsive polymer is in a contracted state but said at least one polymer matrix element of said device is in a wet state; b) exposing the at least one polymeric matrix element of the device to the sample under external conditions that allow the polymeric matrix element to reversibly assume an expanded state, thus allowing components that dissolve in such solvent and have a low molecular weight (and thus a size small enough to fit into the pores) to enter the interstitial pore spaces of the at least one polymeric matrix element, while simultaneously excluding other components that dissolve in such solvent and have a high molecular weight (and thus a size too large to fit into the pores) from the interstitial pore spaces; c) removing said at least one polymer matrix element from said sample, thereby removing said solvent and said small molecular components dissolved in said solvent and contained within said interstitial pore space from said sample, thereby separating said small molecular components dissolved in said solvent from macromolecular compounds in said sample.

[0037] In one embodiment, in step c), the at least one polymer matrix element, if present, is removed from the sample vessel (as further defined above) and, if present, transferred to a liquid phase collection vessel (as further defined above).

[0038] In one embodiment, the method further comprises: d) changing the external conditions to which the polymer matrix elements are exposed to conditions that allow the polymer matrix elements to reversibly assume a contracted state, thereby releasing the solvent and the low molecular weight components dissolved in the solvent from the polymer matrix elements.

[0039] In one embodiment, the polymer responsive to a change in at least one external condition is a thermoresponsive polymer (as further defined above), and the external condition is temperature.

[0040] In one embodiment, the sample, which includes a solvent and two components with different molecular weights and dissolved in the solvent, is an aqueous solution of biomolecules (e.g., proteins or nucleic acids) that further includes other dissolved components in an aqueous solvent (e.g., buffers, and / or salts, and / or low molecular weight components (e.g., dyes, etc.)). The sample can also be a preparation of large macromolecular biomolecular complexes (e.g., protein complexes, cell organelles, ribosomes, etc.).

[0041] In a further aspect, the present invention also relates to the use of a device (as defined above) or of a polymer (as defined above) responsive to a change in at least one external condition, such as pH, temperature, salt conditions, presence or absence of a chemical, for separating a sample (as defined above) comprising a solvent and two components having different molecular weights and dissolved in said solvent into said two components, wherein preferably said use comprises steps a) to c) (as defined above) and optionally step d) (also as defined above).

[0042] The inventors have invented a device configured for fractionating a suspension sample into a liquid phase and a solid phase, said device utilizing a polymer that is responsive to changes in at least one external condition to which said polymer is exposed.

[0043] The device can thus act as a filter to separate a solid phase from a liquid phase.

[0044] Similarly, in certain embodiments, the device can also be used to separate two or more components that are dissolved in a solvent within a sample (and thus can no longer be distinguished by their different phase states ("solid" vs. "liquid")) according to their different sizes, which differ only by their molecular weight. Thus, a device according to such an embodiment can act as a molecular sieve and / or it can be used to enrich dissolved components of a defined molecular weight (e.g., as in dialysis). Thus, the methodology of the present invention ranges from separating solid particles suspended in an aqueous medium (e.g., of a size in the μm range) from a liquid phase within such an aqueous medium, to a size separation according to the molecular weight of two components that have different molecular weights and are dissolved in the liquid phase, one of which has a high molecular weight (e.g., a biomolecule, such as a protein, peptide, nucleic acid, biomolecule complex) and is therefore of a size in the nm or sub-nm range, and the other dissolved component is a low molecular weight component (e.g., a salt or buffer, or other low molecular weight component, such as a dye).

[0045] Thus, embodiments according to the invention relate to a filtration action to separate a solid phase from a liquid phase, while other embodiments relate to a molecular sieve action to separate two dissolved components according to their molecular weight.

[0046] In some embodiments directed to molecular sieving, the separation achieved may not be complete, and in these embodiments, the devices and methods according to the invention include: It is directed to the purpose of enriching one of several, e.g. two, components over each other, where these components have different molecular weights and are (initially) dissolved in a solvent and a sample comprising the two (or more) components (different in their respective molecular weights).

[0047] The term "two components" as used herein should not be interpreted as being limited to the presence of only two components (of different molecular weights). Rather, it is intended to allow the presence of additional components with different molecular weights, which can also be separated using the device and method according to the present invention. In these cases involving more than two components, the device and method according to the present invention may be used repeatedly, using different polymer matrix elements, the interstitial pore space, in particular the pore size, of the different polymer matrix elements being adapted to the respective molecular weights of the components to be separated. The inventors expect that the average pore size or average pore diameter of the respective polymer matrix elements will define the molecular weight boundary or molecular weight cutoff at which separation into different components occurs.

[0048] The polymer used in any of the embodiments according to the present invention is a polymer that is responsive to a change in at least one external condition to which the polymer is exposed. Typically, the "response" to a change in at least one external condition is a change in the volume and / or phase of the polymer. Such polymers are sometimes referred to herein as "responsive polymers". More specifically, the device comprises at least one polymer matrix element that comprises a porous polymer scaffold and an interstitial pore space within the polymer scaffold, where the porous polymer scaffold is composed of a polymer that is responsive to a change in at least one external condition to which the polymer matrix element is exposed during use of the device. Representative examples of such external conditions are pH, temperature, salt conditions, and / or the presence or absence of a particular chemical. In an embodiment of the present invention, the polymer matrix element or a plurality of such polymer matrix elements of the device is contacted with a suspension sample. Taking temperature as an example of an external condition, the polymer matrix element is composed of a thermoresponsive polymer that exhibits a thermoresponsive phase change behavior when a critical temperature is reached. In this case, such a responsive polymer may be a thermoresponsive polymer, and may have a lower critical solution temperature (LCST) polymer or an upper critical solution temperature (UCST) polymer. When a lower critical solution temperature (LCST) polymer is below its lower critical solution temperature (LCST), the corresponding polymer matrix element is very porous and shrinks when the polymer matrix element is heated above the LCST. When a polymer matrix element below the LCST was previously in an aqueous environment, the respective interstitial pore spaces within the polymer matrix element are filled with liquid, whereas upon heating to reach and exceed the lower critical solution temperature, the polymer matrix element shrinks and any liquid contained in the interstitial pore spaces is expelled. Polymers that are responsive to changes in at least one external condition (e.g., thermoresponsive polymers) have been studied and are known [10-12]. To the best of the inventors' knowledge, the use of such responsive polymers to fractionate suspension samples has not been described before.

[0049] In one embodiment, the device according to the present invention further comprises at least one sample vessel intended to receive a suspension sample to be fractionated or a sample comprising a solvent and two components with different molecular weights and dissolved in said solvent. The sample vessel comprises or is configured to comprise said at least one polymer matrix element. It should be noted that the device according to the present invention may also comprise more than one polymer matrix element, for example a plurality of polymer matrix elements.

[0050] In one embodiment, the polymer matrix elements are macroscopically distinct particles, e.g. , beads, spheres, blobs, balls, cubes, cuboids, blocks, ellipsoids, or preferably irregularly shaped bodies dimensioned to fit into the device and, preferably, into the sample vessel, if present. In another embodiment, the at least one polymeric matrix element is a macroscopic body, portion or layer immobilized on a surface of a substrate within the device. In this embodiment, the surface of the substrate on which the polymeric matrix element is immobilized can be any suitable surface of a substrate located within the device. By way of example, if the device comprises a sample vessel, a portion of the inner surface of the sample vessel can have the at least one polymeric matrix element immobilized on a portion of the inner surface. The immobilization of the at least one polymeric matrix element on such surfaces occurs by known means (e.g., by suitable covalent bonding / crosslinking chemistry). The term "macroscopic particle" or "macroscopic body, part or layer" refers to a particle, body, part or layer that can be recognized and observed using the naked eye and does not require specific optical means (e.g., a microscope) to observe such a particle. The term "solitary" in this context refers to the fact that the particle can be identified as a proper particle that defines a boundary and is separate from its surroundings.

[0051] In one embodiment, the at least one polymeric matrix element is detachable or removable from the device. This is because the polymeric matrix element acts as a reservoir volume into which the liquid phase of the suspension sample is captured. Once the liquid or liquid phase of the suspension sample is captured into the at least one polymeric matrix element, separation from other phases of the suspension sample can be easily achieved by removing the at least one polymeric matrix element from the device. In many cases, once the polymeric matrix element captures the liquid phase from the suspension sample and is removed from the device, and the liquid phase is expelled from the polymeric matrix sample by appropriate change of conditions, the polymeric matrix element can be discarded, having served its purpose. Thus, in a preferred embodiment, the at least one polymeric matrix element is a disposable, single-use polymeric matrix element. This may be particularly relevant when the device is to be used for separation / fractionation of suspension samples under sterile conditions.

[0052] In one embodiment, the device further comprises a means for effecting a change in at least one further external condition to which the polymer matrix elements are exposed during use of the device. Such means for effecting a change in at least one external condition may take a number of different forms. For example, the means may be a reservoir of a pH adjusting agent, allowing the addition of such an agent to the suspension sample. In another embodiment, the means may be a reservoir containing a certain buffer, salt solution or other chemical, allowing a defined amount of such buffer, salt solution or other chemical to be added to the suspension sample. In yet another embodiment, such means for effecting a change in at least one external condition is a heating and / or cooling means. Such heating and / or cooling means are particularly preferred when the responsive polymer is a thermoresponsive polymer. In one embodiment, such heating and / or cooling means may be or may include a heater, for example in the form of a heated filament, alone or in conjunction with a suitable cooling means (for example a fan or a tube through which a cooling liquid can flow). In another embodiment, the heating and / or cooling means may be one or several Peltier elements that may operate in either heating or cooling mode. In yet another embodiment, the heating and / or cooling means may only comprise heating means, where cooling is achieved by convective heat transfer.

[0053] In one embodiment, the means for bringing about a change in at least one external condition, preferably said heating and / or cooling means, causes said at least polymer matrix elements to undergo at least The means, preferably the heating and / or cooling means, is configured to be exposed to a change in one external condition. In one embodiment, the means, preferably the heating and / or cooling means, forms part of the device for fractionation, or they are integrated into the device, and / or they are configured to come into contact with or be connected to the suspension sample. By way of example, when the means is a heating and / or cooling means, and when the device comprises a container, the heating and / or cooling means can be configured to surround the container, thereby influencing, controlling and modifying, if necessary, the temperature of the suspension sample and the temperature of the at least one polymer matrix element that comes into contact with the suspension sample.

[0054] In one embodiment, the device configured for fractionating a suspension sample further comprises a carrier tool configured to allow handling of the at least one polymeric matrix element. Such a carrier tool acts as a substrate having a surface on which the at least one polymeric matrix element can be immobilized. The carrier tool is dimensioned to be immersed in a suspension sample and to bring the at least one polymeric matrix element into contact with the suspension sample. In this embodiment, the carrier tool serves the purpose of handling the at least one polymeric matrix element in an easy manner in that it allows the at least one polymeric matrix element immobilized on the carrier tool to be exposed to the suspension sample and, if necessary and desired, to be removed from the suspension sample. Such a carrier tool can be configured in multiple ways. For example, it can be a rod, stick, etc., that allows the polymeric matrix element to be immersed in the suspension sample. Furthermore, in a preferred embodiment, the carrier tool itself can be configured to allow the polymeric matrix element to effect a change in at least one external condition to which it is exposed during use of the device. In a preferred embodiment, said carrier tool is configured to effect a change in temperature, preferably comprising or being connected to or in contact with heating and / or cooling means forming part of said device. In a preferred embodiment, such heating and / or cooling means is said heating and / or cooling means (as further defined above). In one embodiment, said carrier tool is a disposable, single-use carrier tool, configured to be detachably connected to, detachably contacted to or detachably comprising said heating and / or cooling means.It is preferred that the carrier tool is configured as a pen, stick, rod, cylinder, bar, rod, baton, or member, where the carrier tool has an interface that is detachably connected to, detachably contacted with, or detachably includes the heating and / or cooling means. In one specific embodiment, the carrier tool is configured as a hollow cylinder into which the heating and / or cooling means is inserted. In this embodiment, activation of the heating and / or cooling means changes the temperature of the carrier tool, and thus of any at least one polymer matrix element immobilized thereon.

[0055] In one embodiment, the device further comprises at least one liquid phase collection vessel configured to capture the polymer matrix element and / or, if present, the carrier tool on which the polymer matrix element is immobilized.

[0056] In one embodiment, the liquid phase collection vessel serves as a reservoir and / or storage module for the liquid phase. In one embodiment, the liquid phase collection vessel is dimensioned to receive at least 50%, preferably at least 75%, more preferably at least 90% and even more preferably at least 95% of the liquid phase of the suspension sample. In one embodiment, the total volume of said liquid phase collection vessel does not exceed 5 ml, preferably 2 ml, more preferably 1 ml and even more preferably 0.5 ml.

[0057] In one embodiment, during use of the device, the polymeric matrix element and / or, if present, the carrier tool on which the polymeric matrix element is immobilized, are transferred from the sample vessel to the liquid phase collection vessel under conditions that allow the polymeric matrix element to take up at least a portion of the liquid phase of the suspension sample after contacting the polymeric matrix element with the suspension sample. In such an embodiment, it is preferred that, after transferring the polymeric matrix element containing the liquid phase to the liquid phase collection vessel, conditions are applied in which at least a portion of the liquid phase contained in the polymeric matrix element is released from the polymeric matrix element into the liquid phase collection vessel.

[0058] In one embodiment, the liquid phase collection vessel serves as a reservoir and / or storage device for the liquid phase.

[0059] In one embodiment, the device for fractionating a suspension sample further comprises a means for further processing and / or analyzing the liquid phase, where preferably the means for further processing and / or analyzing the liquid phase comprises a spectrophotometer and / or a means for adding a chemical agent to the liquid phase. Such a chemical agent may be used to further modify the liquid phase. For example, a spectrophotometer may be used to detect the presence or absence of one or several analytes, for which the addition of a chemical reagent may be necessary to enable such detection. For example, such a chemical reagent may be one or several dyes, chromophores, antibodies, or other suitable detection agents.

[0060] In one embodiment, the liquid phase collection vessel comprises a means for taking up a defined volume of the liquid phase and / or a means for measuring the volume of the liquid phase of the suspension sample.

[0061] In one embodiment, a) the sample vessel and / or the carrier tool, if present, and / or the liquid phase collection vessel, if present, comprises a means for mechanically removing the solid phase from the polymeric matrix element, such as an orifice, a constriction, a sliding ring, a lip, an edge or an opening, the internal dimensions of which match the external dimensions of the polymeric matrix element; or b) the sample vessel and / or the carrier tool, if present, and / or the liquid phase collection vessel, if present, comprises a material having an affinity for the solid phase, which is capable of removing the solid phase from the polymeric matrix element. The solid phase preferably comprises a substance to which it adheres when in contact or comes into contact with the substance; or c) the sample vessel and / or, if present, the carrier tool and / or, if present, the liquid phase collection vessel comprise a means for washing the solid phase from the polymeric matrix element (e.g. a jet ejecting a liquid which washes the solid phase from the polymeric matrix element); or d) the device further comprises a separation tool (e.g. a spatula, blade, scraper) for removing the solid phase from the polymeric matrix element.

[0062] The device according to the invention is particularly useful in the context of fractionating whole blood into plasma and blood cells as liquid and solid phases, respectively. Thus, in one embodiment, the suspension sample is a whole blood sample and the device is configured for fractionating whole blood into plasma and a solid phase comprising blood cells. In one embodiment, the plasma is platelet enriched. In another embodiment, the plasma is devoid of platelets. In one embodiment, the polymer responsive to a change in at least one external condition to which the polymer matrix element is exposed is a thermoresponsive polymer. The thermoresponsive polymer is a thermoresponsive polymer. Such a thermoresponsive polymer is either a thermoresponsive polymer with a lower critical solution temperature (LCST) or a thermoresponsive polymer with an upper critical solution temperature (UCST). The term "thermoresponsive polymer" refers to a polymer whose physical qualities, in particular its affinity for water, depend on the external temperature to which the polymer is exposed. In one embodiment, the thermoresponsive polymer according to the invention shows a change in affinity for water with temperature. For example, a thermoresponsive polymer with a lower critical solution temperature (LCST) is in a hydrated and expanded state at a temperature below such LCST. At a temperature above the LCST, the polymer is in a contracted and non-hydrated state. By cycling the at least one polymer matrix element between the two states, it is possible to repeatedly (if desired) and reversibly accommodate liquid in the polymer matrix element and then expel the liquid from the polymer matrix element again if and when desired. When such liquid is taken up into the polymer matrix element and into the interstitial pore space within the polymer matrix element, the solid particles in the suspension sample remain outside the polymer matrix element. Thus, separation of the liquid phase within the polymer matrix element and the solid phase outside the polymer matrix element is achieved. When the polymer matrix element is in a swollen state, the solvent / liquid has access to all or substantially all of the pores, or a defined percentage of the pores, whereas the solid phase (e.g., blood cells) does not have access to the pores. In such a swollen state, the polymer matrix element can be removed and separated from the blood cells, thus achieving fractionation into different phases.

[0063] In one embodiment, the responsive polymer, in particular the thermoresponsive polymer, is crosslinked, either by at least one crosslinking reagent, which preferably crosslinks and interconnects between different polymer chains within the polymer, or by providing a substrate, in particular a substantially flat or curved substrate, in the device adjacent to the polymer matrix element, and by interfacially crosslinking different polymer chains of the polymer to the substrate, in particular the surface of the substrate. In one embodiment, particularly in this context, the surface of the substrate to which the responsive polymer is crosslinked (or immobilized) is the surface of the at least one sample vessel, if present, or the surface of the carrier tool, if present.

[0064] In an embodiment according to the invention, the at least one polymeric matrix element has the ability to reversibly assume an expanded and contracted state due to the presence of the responsive polymer. Preferably, in the expanded state, the interstitial pore space has a volume that is at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80% and even more preferably at least 90% of the total volume of the polymeric matrix element when the polymeric matrix element is in the expanded state.

[0065] Moreover, in one embodiment, the pore space is not accessible to the solid phase of the suspension sample, but is accessible to the liquid phase of the suspension sample. Taking whole blood as an example, the pore space is accessible to the plasma in the blood, but not to the blood cells, preferably red blood cells and white blood cells. The pore space may be accessible to platelets, depending on whether a platelet-rich plasma is desired. The pore space may be appropriately adjusted to have pores, the average diameter of the pores allowing the exclusion of the desired solid phase and the inclusion / incorporation of the desired liquid phase into the pore space. For example, in one embodiment, the pore space may have pores, the average diameter of the pores being <5 μm, preferably <3 μm, more preferably <1 μm. When the device is used to effect size separation of two dissolved components differing from each other by their respective molecular weights and / or to enrich one of these two components, the pore space may be appropriately adjusted to have pores, the average diameter of the pores being <5 μm, preferably <3 μm, more preferably <1 μm. , pores whose average diameter is well below the μm range, for example 0.1 nm to 500 nm, preferably 1 nm to 100 nm. The size of the pores is chosen to suit the intended purpose of the device.

[0066] In one embodiment, the interstitial pore space is inaccessible to the solid phase of the suspension sample but is accessible to the liquid phase of the suspension sample. Preferably, the interstitial pore space is dimensioned to accommodate at least more than 50% of the liquid phase of the suspension sample, more preferably more than 75% and even more preferably more than 90% of the liquid phase of the suspension sample.

[0067] In one embodiment, the device is particularly suitable and configured for fractionating suspension samples having a volume <1 ml. In one embodiment, the device according to the invention is dimensioned to fractionate suspension samples having a volume in the range of 10 μl to 500 μl, and the interstitial space of the at least one polymeric matrix element of the device has a volume that captures at least 50%, preferably at least 75%, more preferably at least 90% and even more preferably at least 95% of the suspension sample. Thus, in one embodiment, the interstitial space of the polymeric matrix element has a volume that is at least 50% of the suspension sample volume of 10 μl to 500 μl. That is, the volume of the interstitial space is at least 5 μl to at least 200 μl to 250 μl; preferably, the interstitial space has a volume that is at least 75% of the suspension sample volume of 10 μl to 500 μl. That is, the interstitial space has a volume of at least 7.5 μl to at least 375 μl; more preferably, the interstitial space has a volume that is at least 90% of the suspension sample volume of 10 μl to 500 μl. That is, the interstitial space has a volume of at least 9 μl to at least 450 μl; and even more preferably, the interstitial space has a volume that is at least 95% of the suspension sample volume of 10 μl to 500 μl. That is, the interstitial space has a volume of at least 9.5 μl to at least 475 μl. In this context, in one embodiment, the total volume of the interstitial spaces of the polymer matrix elements does not exceed the volume of the suspension sample. Thus, by way of example, if the volume of the suspension sample is 500 μl, the volume of the interstitial spaces is preferably 500 μl or less. In one embodiment, the volume of the pore space is 250 μl or 375 μl or 450 μl or 475 μl. In another exemplary embodiment, when the suspension sample has a volume in the range of 10 μl to 200 μl, the volume of the pore space is in the range of 5 μl to 200 μl.In another embodiment, when the suspension sample has a volume of 50 μl to 200 μl, the volume of the interstitial pore space is in the range of 25 μl to 200 μl.In yet another embodiment, when the suspension sample has a volume in the range of 100 μl to 200 μl, the volume of the interstitial pore space is in the range of 50 μl to 200 μl.

[0068] In a further aspect, the present invention also provides a method for fractionating a suspension sample into a liquid phase and a solid phase, said method comprising: a) providing, in any order, but separately from each other, a device according to the invention (as defined above) and a suspension sample, wherein said at least one polymeric matrix element of said device is in a dry state or said at least one polymeric matrix element of said device is provided under conditions in which said responsive polymer is in a contracted state but said at least one polymeric matrix element of said device is in a wet state; b) allowing the at least one polymeric matrix element of the device to reversibly assume an expanded state such that the liquid phase in the suspension sample enters the interstitial pore space of the at least one polymeric matrix element. exposing said suspension sample to external conditions which permit the suspension sample to be simultaneously displaced from said interstitial pore space by a solid phase in said suspension sample; c) removing the at least one polymer matrix element from the sample, thereby removing the liquid phase contained in the interstitial pore space from the sample, thereby separating the liquid phase from the solid phase in the sample.

[0069] In one embodiment, during use of the device in the fractionation method, in step c) the polymeric matrix elements and / or, if present, the carrier tool on which they are immobilized are transferred from the sample vessel to the liquid phase collection vessel after contacting the polymeric matrix elements with the suspension sample under conditions allowing the polymeric matrix elements to take up at least a portion of the liquid phase of the suspension sample. In such an embodiment, it is preferred that, after transferring the polymeric matrix elements comprising the liquid phase to the liquid phase collection vessel, conditions are applied under which at least a portion of the liquid phase contained in the polymeric matrix elements is released from the polymeric matrix elements into the liquid phase collection vessel.

[0070] In one embodiment, the liquid phase collection vessel serves as a reservoir and / or storage device for the liquid phase.

[0071] In one embodiment of the method, the method further comprises: d) changing the external conditions to which the polymer matrix elements are exposed to conditions that allow the polymer matrix elements to reversibly assume a contracted state, thereby releasing the liquid phase from the polymer matrix elements.

[0072] It should be noted that in certain embodiments of the method according to the invention, in step a), the device according to the invention (and thus the polymeric matrix elements contained therein) can be provided in a wet but contracted state instead of dry, i.e. the polymeric matrix elements are provided under external conditions that are not dry but in which the responsive polymer is in a contracted state but may be wet. For example, there may be a certain amount of buffer (or other liquid) or a layer of buffer (or other liquid) covering the polymeric matrix elements. In this embodiment, subsequently, in step b), the at least one polymeric matrix element is exposed to the suspension sample under external conditions that allow the polymeric matrix element to adopt a reversibly expanded state and thus allow the liquid phase in the suspension sample to enter the interstitial pore space of the at least one polymeric matrix element (as in the embodiment in which the polymeric matrix element is provided in a dry state). Subsequent step c) and subsequent optional step d) are as defined above.

[0073] In a preferred embodiment according to the present invention, the polymer that is responsive to a change in at least one external condition (i.e., a "responsive polymer") is a thermoresponsive polymer (as further defined above), said external condition being temperature.

[0074] In a preferred embodiment of the method according to the invention, the suspension sample is a whole blood sample, the liquid phase of the whole blood sample is plasma and the solid phase of the whole blood sample is or comprises the blood cells of the whole blood sample. In a preferred embodiment, the solid phase of the whole blood sample is or comprises the majority of the whole blood cells, even more preferably the whole blood cells of the whole blood sample.

[0075] In a still further aspect, the present invention also relates to a detector according to the present invention for fractionating a suspension sample into liquid and solid phases, preferably for fractionating a whole blood sample into plasma and blood cells. The use of a polymer (as further defined above) that is responsive to a change in a device (as defined above) or to at least one external condition, such as pH, temperature, solid conditions, presence or absence of a chemical, wherein preferably said use relates to a use comprising steps a) to c) (as further defined above) and optionally step d) (also as further defined above).

[0076] Without wishing to be bound by any theory, the inventors have found that the use of a porous polymer scaffold and a responsive polymer forming interstitial pore space within the porous polymer scaffold, which together constitute a polymer matrix element, allows for efficient fractionation of a suspension sample into liquid and solid phases. This is particularly true when the suspension sample volume is small, i.e., <1 ml. In a preferred embodiment according to the invention, the sample size used is well below 1 ml, for example <200 μl, preferably <150 μl. For example, in one embodiment, a representative blood sample used according to the invention is a sample obtained by capillary tube, the volume of such a sample typically does not exceed 140 μl (i.e., <140 μl), and the representative amount of plasma from such a sample is <80 μl. The device according to the invention is extremely easy to use and can be easily adapted to different suspension sample types. A particularly preferred suspension sample type is whole blood, and devices according to the invention allow multiple samples to be processed within a short period of time due to the preferred disposability of the individual polymer matrix elements used within certain embodiments of the device, where the disposable polymer matrix elements are detachable or removable from the device. It is also contemplated that a device for fractionating suspension samples according to the invention may include more than one polymer matrix element, for example, 2, 3, 4, 5, 6, 10, 20, 30, 40, 50, ... 100, 10,000, etc. polymer matrix elements.

[0077] The device may be a portable device, or may be configured as a bench-top device or as a large-scale workstation device that allows many samples to be processed simultaneously. In the latter case, the device may further be configured to include a suitable control unit that provides the movement of each polymer matrix element, a robotic arm, and a suitable electronic control circuit that coordinates the control unit that can provide the addition of a sample, the adaptation of external conditions (e.g., temperature that provides for the incorporation of a liquid phase into the polymer matrix element), the removal of the polymer matrix element from the sample, and the change of external conditions (e.g., temperature) to switch the polymer matrix element between an expanded and a contracted state. [Brief description of the drawings]

[0078] The present invention is further illustrated by the drawings. [Figure 1] FIG. 1 shows one embodiment of a device according to the present invention that includes a polymer matrix element contained within a sample vessel that is used to fractionate a blood sample.

[0079] [Figure 2-1] Figure 2 (A)-C) show photographs of fractionation performed according to the principle shown in Figure 1, and (D) shows the results of a cytometric analysis performed on a sample fractionated according to the invention. [Figure 2-2] Figure 2 (A)-C) show photographs of fractionation performed according to the principle shown in Figure 1, and (D) shows the results of a cytometric analysis performed on a sample fractionated according to the invention.

[0080] [Diagram 3] FIG. 3 shows another embodiment of a device according to the invention, in which the polymer matrix elements are immobilized on a carrier tool configured to be detachably connected to heating and / or cooling means.

[0081] [Figure 4]FIG. 4 illustrates one embodiment of a carrier tool having polymer matrix elements immobilized on a surface of the carrier tool.

[0082] [Figure 5-1] FIG. 5 shows a prototype of an embodiment of a portable instrument including heating and / or cooling means, (A)=exploded view, and B)=assembled device, and (C) an embodiment of a device for fractionation including a disposable carrier tool with immobilized polymer matrix elements and heating and / or cooling means according to the present invention. [Figure 5-2] FIG. 5 shows a prototype of an embodiment of a portable instrument including heating and / or cooling means, (A)=exploded view, and B)=assembled device, and (C) an embodiment of a device for fractionation including a disposable carrier tool with immobilized polymer matrix elements and heating and / or cooling means according to the present invention.

[0083] [Figure 6-1] 6(A)-F) show the results of the plasma separation process of a whole blood sample using the plasma separation device as illustrated in FIG. [Figure 6-2] 6(A)-F) show the results of the plasma separation process of a whole blood sample using the plasma separation device as illustrated in FIG. [Figure 6-3] 6(A)-F) show the results of the plasma separation process of a whole blood sample using the plasma separation device as illustrated in FIG.

[0084] [Figure 7] FIG. 7 shows the fluorescence emission spectra of stock solutions of carboxy-fluorescein and R-phycoerythrin (R-PE), of the same stock solutions after exposure to two different polymeric matrix elements at room temperature (during which the exposed portions of the stock solutions may enter the interstitial pore spaces of the polymeric matrix elements), and of the liquid expelled from two different polymeric matrix elements after they were heated to 40° C. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0085] More specifically, FIG. 1 shows a sample vessel with a polymeric matrix element in a dry state. After adding a sample to the sample vessel, the polymeric matrix element is wetted and liquid is taken up by the polymeric matrix element, whereas the solid phase within the sample remains outside the polymeric matrix element. Upon contact with the liquid, the polymeric matrix element swells considerably, increasing its volume by approximately 10× magnification. The remaining sample that is not incorporated into the polymeric matrix element is more or less a solid phase or an enriched sample of the suspended members, e.g., cells. In contrast, the polymeric matrix element itself contains the liquid phase of the sample, without the excluded suspended solid members. In the case of a blood sample, the incorporated liquid phase is plasma. Depending on the size of the pores in the polymeric matrix element, this can be plasma with or without platelets. In a further step, the phase with the enriched solid phase can be removed from the sample vessel, or the liquid phase can be removed by removing the polymeric matrix element from the sample vessel. The polymer matrix element is then exposed to conditions that cause the polymer to shrink again, thus reducing the pore size of the polymer matrix element and thus displacing liquid from the polymer matrix element.For example, if the polymer is an LCST polymer, such shrinkage can be achieved by increasing the temperature to a temperature above the LCST.The liquid phase thus released, for example the plasma released, can be collected from the container.

[0086] Figure 2 shows the results of a fractionation carried out according to the principle outlined for Figure 1. More specifically, four different polymer matrix elements were generated as macroscopically independent particles and exposed to whole blood (A), from which they were removed after a defined incubation and placed in a separate additional container (B). They were then exposed to conditions that allowed the liquid phase (plasma) to be released from the polymer matrix elements (C). Figure 2 D) shows the results of a cell counting analysis of the different samples processed in Figures 2 A)-C), as well as of whole blood and centrifuged plasma.

[0087] FIG. 3 shows another embodiment of the device according to the invention and its use. In this embodiment, a sample vessel containing a suspension sample is provided and a polymer matrix element attached to a carrier tool is introduced into the suspension sample. The carrier tool is preferably thermally conductive, so that the temperature to which the polymer matrix element is exposed can be easily changed by connecting the carrier tool to heating and / or cooling means. Upon contacting the polymer matrix element with the suspension sample, the polymer matrix element takes up the liquid and excludes the suspended solid phase, e.g. cells. Again, taking blood as an example, plasma is collected in the polymer matrix element until the capacity of the pores in the interstitial pore space is reached. Thereafter, the polymer matrix element is then removed from the sample vessel and transferred to a further vessel, i.e. a vessel for collecting the liquid phase (hereinafter sometimes also referred to as "liquid phase collection vessel"), where again the external conditions are changed to shift the polymer matrix element from an expanded to a contracted state, thus releasing the liquid incorporated therein. When the responsive polymer of the polymer matrix element is an LCST polymer, the change in external condition is an increase in temperature above the LCST.

[0088] FIG. 4 illustrates an embodiment of a carrier tool with polymer matrix elements immobilized on the surface of the carrier tool in accordance with the present invention.

[0089] 5 shows an embodiment of a portable instrument forming part of a device according to the invention, comprising heating and / or cooling means (here: a heating rod) configured to connect with polymer matrix elements immobilized on a disposable carrier tool. Panel A shows an exploded view of the portable instrument, panel B shows the portable instrument in assembled form; panel C) shows an embodiment of a device for fractionation according to the invention, comprising a disposable carrier tool with immobilized polymer matrix elements and heating and / or cooling means (here: a heating rod).

[0090] Figure 6 shows the results of a plasma separation process using a plasma separation device as illustrated in Figure 5. Panels A) to F) show different stages of the separation process: Panel A) shows an embodiment of a sample vessel containing whole blood; Panel B) shows a disposable carrier tool (as shown in Figure 4) on which a polymer matrix element is immobilized, inserted into the sample vessel; Panel C) shows the handheld device of Figure 5 connected with the disposable carrier tool by the heating and / or cooling means (heating rod); Panel D) shows the combination of the handheld device of Figure 5 with the disposable carrier tool after it has been withdrawn from the sample vessel; Panel e) shows the combination of the handheld device of Figure 5 with the disposable carrier tool after it has been inserted into a new vessel ("liquid-phase collection vessel"); and Panel F) shows both the (first) sample vessel and the liquid-phase collection vessel after the combination of the handheld device of Figure 5 with the disposable carrier tool has been withdrawn.

[0091] FIG. 7 shows stock solutions of carboxy-fluorescein and R-phycoerythrin (R-PE) exposed to two different polymer matrix elements at room temperature during which the exposed portion of the stock solution can enter the interstitial pore space of the polymer matrix elements. Fluorescence emission spectra of the same stock solution later, as well as of the liquid expelled from the two different polymeric matrix elements after they had been heated to 40° C. The removal of R-phycoerythrin from the liquid that enters the polymeric matrix elements (and the corresponding enrichment in the liquid "supernatant" that cannot enter the polymeric matrix elements) can be seen.

[0092] Reference is further made to the following examples, which are offered to illustrate, but not to limit, the invention. EXAMPLES

[0093] Example 1 Embodiments Describing the Preparation of Polymer Matrix Elements in a Sample Container and Their Application for Plasma Separation Four different formulations of LCST polymer matrix were prepared and contained in the sample vessel.

[0094] The protocol applied was as follows: First, a solution containing N-isopropylacrylamide (NIPAM) and N,N'-methylenebisacrylamide (BIS) monomers and ammonium persulfate (APS) was prepared (premixed NBA). [Table 1]

[0095] To evaluate the effect of different additives on the above formulation, the following variations were prepared: [Table 2]

[0096] Approximately 100 μL plugs (i.e., "visible individual particles") were thus formed in 0.5 mL microreaction vials by mixing the above liquid with 2 μL of 5% TEMED (degassed) under argon, after which polymerization was allowed to occur at room temperature for 30 min.

[0097] The vial was placed on a heat block at a temperature of 50° C. The shrinkage of the polymer was easily observed. The expelled liquid was collected with a micropipette and discarded. The plug was removed from the micro reaction vial and transferred to a 2 ml reaction tube. 500 μl of PBS buffer was added to the tube, followed by incubation at 20° C. for 1 hour to allow the polymer to swell again. The excess PBS was removed and the tube was placed in a 2 ml reaction tube. The polymer was allowed to shrink by incubating at 60° C. for 15 minutes. The drained liquid was discarded. This washing procedure was repeated one more time.

[0098] A 150 μL sample of whole blood, freshly collected in an EDTA-coated vacutainer tube, was added to each vial containing the shrunk polymer as shown in FIG. 2A) and allowed to sit at room temperature for 30 minutes.

[0099] After 30 minutes, the samples were removed from the vials with swollen plugs. Figure 2B) shows the four vials with the plugs. It is clear that different matrices took up different amounts of liquid. Furthermore, different amounts of whole blood stuck to the plugs depending on their formulation.

[0100] The vials with the plugs were then placed on a heat block at 40° C. for 1 minute. The polymer contracted and liquid was expelled from the matrix material. The expelled liquid was collected in a separate vial. As shown in FIG. 2C), different amounts of liquid were expelled at the defined temperatures, as well as different coloration levels of the samples due to the trapped whole blood can be observed.

[0101] The collected samples were analyzed on a laboratory hematology analyzer (Sysmex) and data were compared against cell counts obtained for whole blood ("WBC" = white blood cells; "RBC" = red blood cells; and "PLT" = platelets) and plasma generated by centrifugation of 250 μl samples at 1,500 g for 1 min. Data for two aliquots of one sample of whole blood ("White Blood Cells #1" and "White Blood Cells #2"), two plasma samples generated by centrifugation from the same sample of whole blood ("Centrifuged Plasma #1" and "Centrifuged Plasma #2"), and data for fluids collected from different formulations of LCST polymer matrices ("Matrix 1", "Matrix 2", "Matrix 3", "Matrix 4") are summarized in Figure 2D.

[0102] The separation effect achieved by application of the matrices is clearly visible. Moreover, a simple approach to the optimization of the separation effect by adding different reagents to the polymer preparation is presented. Matrices 1 and 2 appear to be the most efficient ones in separating blood cells from the liquid components of the blood sample.

[0103] Example 2 Polymer matrix elements immobilized on a substrate An aluminum shell was designed as part of a disposable for plasma separation. The aluminum shell functions as a carrier tool that acts as a substrate with a surface on which polymer matrix elements are immobilized. The shell surface was roughened with sandpaper to allow the polymer matrix elements to better adhere to the surface.

[0104] The formulation of the LCST polymer matrix as the polymer matrix component was prepared as follows: First, a solution containing N-isopropylacrylamide (NIPAM) and N,N′-methylenebisacrylamide (BIS) monomers and ammonium sulfate (APS) was prepared (premix NBA). [Table 3]

[0105] To this mix, PEG methyl ether acrylate and water were added as follows: [Table 4]

[0106] A 194 μl aliquot of this solution was added to a Sarsted Microvette sampling vessel, the solution in the vessel was degassed and saturated with argon in four push-pull cycles.

[0107] These aliquots were mixed with 4 μl of 5% TEMED (degassed) under argon and the aluminum shell was immersed in the solution. After an incubation period of 30 minutes, the aluminum shell was exposed to a heat source, causing the polymer to shrink onto the shell. The shell and polymer were removed from the vial and transferred to a new vial containing PBS wash buffer. The polymer was allowed to reswell in the wash buffer for 1 hour. The wash step was repeated twice.

[0108] This disposable device is shown in FIG. 4 and was subsequently used as a carrier tool according to the exemplary process described in Example 3.

[0109] Example 3 Description of the prototype as developed by Blink AG As a possible product based on the present invention, a portable separation device has been developed. Such a product may be particularly useful in situations that allow diagnostic applications at the point of care. Many diagnostic tests require plasma as a sample matrix. However, in many environments, there are no suitable means available to generate plasma from whole blood, especially from capillary blood samples that can otherwise be easily obtained by common finger prick techniques. Thus, the inventors describe a pen-like portable battery-operated instrument in combination with a disposable device.

[0110] The instrument consists of the basic modules shown in Figures 5A) (exploded view) and 5B) (assembled device) that are to be used with a disposable carrier tool as shown in Figure 4. The combination of the handheld device and disposable carrier tool of Figures 5A) and B) is one embodiment of a device configured for fractionation according to the present invention. This is shown in Figure 5C.

[0111] The portable devices of Figures 5A) and 5B) are designed to heat a metal rod to a predefined temperature (above the LCST of the filter polymer). The control circuitry is housed on a separate electronic board that includes a power management circuit connected to an AA battery housed within the device body, an LED for reporting the device's status, and a button for activating the device. The control circuitry is connected to a heating rod ("heating and / or cooling means") that has dimensions that allow it to be combined with and introduced into a special "disposable carrier tool" (e.g., as shown in Figure 4) that has an LCST polymer coating that acts as a polymer matrix element immobilized on the surface of the carrier tool.

[0112] The "disposable carrier tool"-device comprises a plastic part with a recess that securely accommodates the heating rod of the device. On its outer surface, the disposable carrier tool is coated with a LCST polymer as outlined in Example 2 and shown in FIG.

[0113] The disposable carrier tool is designed to fit into a sample vessel, for example a standard Microvette sample vessel containing the required volume of collected whole blood (for example 100 μL). When the disposable carrier tool is introduced into the sample vessel, the plasma coats the polymer and occupies its inner space, whereas any specific substances contained in the sample remain outside the polymer matrix. After a sufficient incubation time, the disposable carrier tool is brought into contact with the portable instrument of FIGS. 5A) and 5B) and its heating and / or cooling means, i.e. the heating rod, by introducing the heating rod of the instrument into a recess of the disposable carrier tool. The disposable carrier tool is attached to the rod by pressing the rod firmly into the disposable carrier tool. The instrument with the attached disposable carrier tool is then removed from the sample vessel and the disposable carrier tool is introduced into another vessel, i.e. an empty vessel ("liquid-phase collection vessel"). The portable instrument is activated by pressing a button. A green (or any other suitable color) LED glows until a predefined temperature above the LCST is reached. The LED now glows green all the time. The polymer contracts within the disposable carrier tool, releasing the captured plasma into the liquid phase collection container. The disposable carrier tool is extracted from the liquid phase collection container, removed from the portable device of Figures 5A and 5B, and safely discarded. The portable device can now be used for a new separation task. The device is switched off by pressing the button once. Any faults are indicated by a red light.

[0114] The whole workflow is illustrated in Figures 6A)-F), where Figure 6A) shows a standard sample vessel containing a suspension sample (in this case whole blood); Figure 6B) shows the disposable carrier tool of Figure 4 (with a polymer matrix element immobilized on the carrier tool) inserted into the sample vessel such that the polymer matrix element becomes exposed to the suspension sample (i.e. in this case the whole blood); Figure 6C) shows the disposable carrier tool connected with the heating and / or cooling means (in this case a heating rod) of the handheld device of Figure 5); Figure 6D) shows the combination of the disposable carrier tool and handheld device being withdrawn from the sample vessel after exposure to the suspension sample for a defined period of time and after the liquid phase has been bound by or incorporated into the polymer matrix element; Figure 6E) shows the same combination (disposable carrier tool and handheld device) being introduced into a further empty vessel, i.e. a liquid phase collection vessel, where the heating and The heating and / or cooling means (in this case a heating rod) is actuated to heat above the LCST of the polymer matrix element, thereby expelling the liquid phase bound by the polymer matrix element into the liquid phase collection vessel; FIG. 6E) shows (on the left) the (first) sample vessel in which whole blood was originally held and exposed to the polymer matrix element, and (on the right) the (further) liquid phase collection vessel into which the liquid phase has been expelled after the heating rod has been heated above the LCST of the polymer matrix element.

[0115] Example 4 Enrichment and depletion of soluble fluorescent compounds of different sizes The invented methodology was applied to enrich soluble molecules in solution in small volumes of liquid.

[0116] Polymer plugs (i.e., "polymer matrix elements") prepared according to the methodology described in Example #1 were used to vary the composition of a stock solution containing 1.62 μmol / L of the small organic dye 5-carboxyfluorescein (M=376.32 g / mol; C0537 SIGMA-ALDRICH) ("5CF") and 0.4 nmol / L of the polymeric fluorescent protein R-phycoerythrin (M=250 kDa, Thermo Fisher P801) ("R-PE"). Fluorescence spectra of the stock solutions were collected using a Lambda 50 Fluorescence Spectrometer (Perkin Elmer). The spectra are shown as solid black lines in FIG. 7 (emission peak at 570 nm for R-PE).

[0117] Two different plugs of LCST polymer matrix prepared as outlined in Example #1, composed of PNIPAM / PEG acrylate and PNIPAM, were used to investigate the effect of the matrix on the composition of the liquid. The vial containing the plug was placed in a 40° C. heat block for 1 minute. The expelled liquid was collected with a micropipette and discarded. 150 μL of the stock solution containing 5-carboxyfluorescein and R-phycoerythrin was added to the vial and allowed to sit at room temperature for 5 minutes. The supernatant was then collected and fluorescence spectra were obtained for both samples ("supernatants"). The vial with the plug was placed in a 40° C. heat block for 1 minute and the expelled liquid was collected with a micropipette. Fluorescence spectra were obtained for each of the expelled liquid samples ("Matrix 1" (open circles) and "Matrix 4" (open diamonds)) and compared against the spectrum of the stock solution (shown as solid black line) and against the supernatants (Matrix 1 (closed circles) and Matrix 4 (closed diamonds)). Normalized spectra of the two different matrix preparations and the stock solutions are shown in FIG. 7. Both polymeric matrix elements increased the R-phycoerythrin concentration in the respective supernatants by excluding R-phycoerythrin from the interstitial pore spaces of the polymeric matrix elements, thereby removing R-phycoerythrin from the liquid that enters the interstitial pore spaces of the polymeric matrix elements. It can be seen that R-phycoerythrin can be enriched in the matrix (FIG. 7: "Supernatant Matrix 1" (filled circles) and "Supernatant Matrix 4" (filled diamonds)) and that R-phycoerythrin is subsequently released again when the polymer matrix element is heated above its LCST (FIG. 7: "Expelled Liquid Matrix 1" (open circles) and "Expelled Liquid Matrix 4" (open diamonds)). Thus, the polymer matrix elements according to the invention can be successfully used to separate and / or enrich / remove, respectively, two dissolved components of different molecular weight. It is also clear from the data that the different matrix preparations show different abilities to enrich or remove certain reagents from solution. References: [ka] For example, the present invention provides the following: (Item 1) A device configured for separating a sample comprising a solvent and two components having different molecular weights and dissolved in the solvent into the two components, or for fractionating a suspension sample into a liquid phase and a solid phase, the device comprising at least one polymeric matrix element, the polymeric matrix element comprising a porous polymer scaffold and interstitial pore space within the polymeric scaffold, the porous polymer scaffold being composed of a polymer that is responsive to changes in at least one external condition to which the polymeric matrix element is exposed during use, such as pH, temperature, salt conditions, the presence or absence of a chemical. (Item 2) The method further includes at least one sample vessel, the at least one sample vessel being 2. The device according to claim 1, comprising or configured to comprise at least one polymer matrix element, wherein the at least one sample vessel is capable of receiving a sample comprising a solvent and two components having different molecular weights and dissolved in the solvent, or a suspension sample. (Item 3) The device of any of the preceding items, wherein the at least one polymeric matrix element is either a macroscopically distinct particle, preferably in the shape of a bead, sphere, nodule, ball, cube, cuboid, block, ellipsoid, or irregularly shaped body, or the at least one polymeric matrix element is a macroscopic body or layer immobilized on the surface of a substrate within the device. (Item 4) The device described in any of the preceding items, wherein the at least one polymeric matrix element is detachable or removable from the device, preferably, the at least one polymeric matrix element is a disposable, single-use polymeric matrix element. (Item 5) The device according to any of the preceding items, further comprising a means for effecting a change in at least one external condition to which the polymer matrix element is exposed during use of the device, preferably wherein the means for effecting a change in at least one external condition is a heating and / or cooling means. (Item 6) 6. The device according to item 5, wherein said means, preferably said heating and / or cooling means, are configured to expose said at least one polymer matrix element to at least one change of external condition, wherein preferably said means is part of said device and / or is integrated into said device and / or is configured to come into contact with or be connected to said suspension sample. (Item 7) The device described in any of the preceding items, further comprising a carrier tool configured to allow handling of the at least one polymeric matrix element, the carrier tool acting as a substrate having a surface on which the at least one polymeric matrix element is immobilized, the carrier tool being dimensioned such that it can be immersed in a sample comprising a solvent and two components having different molecular weights and dissolved in the solvent, or in a suspension sample, and such that the at least one polymeric matrix element can be contacted with the sample, preferably the suspension sample. (Item 8) 8. The device of claim 7, wherein the carrier tool is configured to effect a change in at least one external condition to which the polymer matrix element is exposed during use of the device, such as pH, temperature, salt conditions, presence or absence of a chemical, and wherein preferably the carrier tool is configured to effect a change in temperature. (Item 9) 9. The device according to item 8, wherein the carrier tool comprises a heating and / or cooling means for heating and / or cooling the at least one polymer matrix element or the carrier tool is connected or in contact with a heating and / or cooling means for heating and / or cooling the at least one polymer matrix element, preferably wherein the heating and / or cooling means is a heating and / or cooling means according to item 5 or 6. (Item 10) The carrier tool is a disposable, single-use carrier tool and is configured to be releasably connected to, releasably contacted with, or releasably include, the heating and / or cooling means, wherein preferably, the carrier tool is 10. The device according to any of items 7 to 9, wherein the carrier tool is configured as a carrier tool, stick, rod, cylinder, bar, stick, baton or member, wherein the carrier tool has an interface that detachably connects to, detachably contacts or detachably includes said heating and / or cooling means. (Item 11) The device of any of the preceding items, further comprising at least one liquid phase collection vessel configured to capture the polymer matrix element and / or, if present, the carrier tool on which the polymer matrix element is immobilized. (Item 12) a) the sample vessel and / or, if present, the carrier tool, and / or, if present, the liquid phase collection vessel, comprises a means for mechanically removing the solid phase from the polymer matrix element, such as an orifice, a constriction, a sliding ring, a lip, an edge or an opening, the internal dimensions of which match the external dimensions of the polymer matrix element; or b) the sample vessel and / or, if present, the carrier tool, and / or, if present, the liquid phase collection vessel, comprises a substance that has an affinity for the solid phase, for which the solid phase is 12. The device according to any of items 2 to 11, wherein the solid phase is preferably adhered when in or comes into contact with the substance; or c) the sample vessel and / or, if present, the carrier tool and / or, if present, the liquid phase collection vessel comprises a means for washing the solid phase from the polymeric matrix elements, e.g. a jet ejecting a liquid which washes the solid phase from the polymeric matrix elements; or d) the device further comprises a separation tool, e.g. a spatula, blade, scraper, for removing the solid phase from the polymeric matrix elements. (Item 13) The device according to any of the preceding items, wherein the sample comprising a solvent and two components having different molecular weights and dissolved in the solvent is an aqueous sample comprising a macromolecular component, such as a protein or peptide or nucleic acid, as one of the two components, and a small molecular component, such as a dye or salt, as the other of the two components, or the suspension sample is a whole blood sample, wherein the liquid phase of the whole blood sample is plasma and the solid phase of the whole blood sample is or comprises blood cells of the whole blood sample. (Item 14) The polymer responsive to changes in at least one external condition to which the polymer matrix element is exposed during use of the device is preferably selected from the group consisting of poly(N-isopropylacrylamide) (pNIPAM), poly(N-isopropylacrylamide / N,N'-methylenebis(acrylamide), poly(N-isopropylacrylamide)-co-(acrylic acid), poly(N-isopropylacrylamide)-co-(acrylic acid)-co-(poly(ethylene glycol) methyl ether acrylate, poly(N-isopropylacrylamide)-co-(poly(ethylene glycol) diacrylate, poly(N-isopropylacrylamide)-co-(poly(ethylene glycol) methyl ether acrylate, poly[2-(dimethylamino)ethyl methacrylate] (pDMAEMA), hydrochloride, ethyl methacrylate ... The thermo-responsive polymer is a thermo-responsive polymer having a lower critical solution temperature (LCST polymer) selected from poly(N-acryloylglycinamide) (PNAGA), poly(allylamine)-co-poly(allylurea) and its derivatives, poly(methacrylamide), poly(N-acryloylaspartamide), poly(N-methacryloylglutamine amide), poly(acrylamide)-co-(acrylonitrile), poly(sulfobetaine), poly(phosphorylcholine), poly(phosphorylcholine), poly(N-acryloylglycinamide)-co-(N-acryloylaspartamide), poly(N-methacryloylglutamine amide), poly(acrylamide)-co-(acrylonitrile), poly(sulfobetaine), poly(phosphorylcholine), poly(phosphorylcholine), poly(N-acryloylglycinamide)-co-(N-acryloylaspartamide), poly(N-methacryloylglutamine amide), poly(acrylamide)-co-(acrylonitrile), poly(sulfobetaine), poly(phosphorylcholine), poly(phosphorylcholine), poly(N-acryloylglycinamide)-co-(N-acryloylaspartamide), poly(phosphorylcholine ... The device according to any of the preceding items, wherein the responsive polymer, in particular the thermoresponsive polymer, is preferably crosslinked either by at least one crosslinking reagent which crosslinks and interconnects between different polymer chains within the polymer or by providing a substrate within the device, in particular a substantially flat or curved substrate, adjacent to the polymer matrix element and by interfacially crosslinking different polymer chains of the polymer to the substrate, in particular to the surface of the substrate. (Item 15) 2. A device according to any of the preceding items, wherein the interstitial pore space comprises pores, the average diameter of the pores being <5 μm, preferably <3 μm, more preferably <1 μm. (Item 16) A device according to any of the preceding items, wherein the device is dimensioned to fractionate a suspension sample having a volume in the range of 10 μl to 500 μl, and the interstitial pore space has a volume to capture at least 50%, preferably at least 75%, more preferably at least 90%, and even more preferably at least 95% of the suspension sample, and wherein preferably the suspension sample has a volume of 10 μl to 200 μl, preferably 50 μl to 200 μl, more preferably 100 μl to 200 μl. (Item 17) 1. A method for fractionating a suspension sample into a liquid phase and a solid phase, the method comprising: a) providing, in any order but separately from each other, a device according to any of items 1 to 16 and a suspension sample, wherein the at least one polymer matrix element of the device is in a dry state or the at least one polymer matrix element of the device is provided under conditions in which the responsive polymer is in a contracted state but the at least one polymer matrix element of the device is in a wet state; b) exposing the at least one polymeric matrix element of the device to the suspension sample under external conditions that allow the polymeric matrix element to reversibly assume an expanded state, thus allowing the liquid phase in the suspension sample to enter the interstitial pore spaces of the at least one polymeric matrix element, while simultaneously excluding the solid phase in the suspension sample from the interstitial pore spaces; c) removing said at least one polymer matrix element from said sample, thereby removing said liquid phase contained within said interstitial pore space from said sample, thereby separating said liquid phase from a solid phase in said sample; The method includes: (Item 18) 18. The method according to item 17, wherein in step c) the at least one polymer matrix element, if present, is removed from a sample vessel as defined in item 2 and, if present, is transferred to a liquid phase collection vessel as defined in item 11. (Item 19) The method comprises: d) changing the external conditions to which the polymer matrix elements are exposed to conditions that allow the polymer matrix elements to reversibly adopt a contracted state, thereby releasing the liquid phase from the polymer matrix elements; 19. The method according to any one of items 17 to 18, further comprising: (Item 20) Use of a device as defined in any of items 1 to 16 or of a polymer responsive to a change in at least one external condition, such as pH, temperature, salt conditions, presence or absence of a chemical, as defined in any of items 1 to 14 for fractionating a suspension sample into liquid and solid phases, preferably for fractionating a whole blood sample into plasma and blood cells, wherein preferably said use comprises steps a) to c) as defined in any of items 17 to 18 and, optionally, step d) as defined in item 19.

Claims

[Claim 1] The invention described in the specification.

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