Device for fractionating a suspension sample

Abstract

The present invention relates to a device for fractionating a suspension sample and 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 the solvent into said two or more components. The device may comprise at least one polymer matrix element.

Description

[Technology 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 method for separating a sample containing a solvent and two or more components having different molecular weights and dissolved in the solvent into those two or more components. [Background technology]

[0002] Separating suspended solid samples into different phases has been a subject of intense research and practice for many years. Clarification of beer or wine, or skimming milk, are prime examples. Blood can be considered a good example of a particulate fraction suspended in an aqueous liquid. The fractionation of whole blood into cellular and plasma components constitutes a crucial step in many bioanalytical workflows. Typically, this is achieved by centrifugation of the sample in a corresponding sample collection container. After separating the cellular components from the liquid components by density in the centrifugal field or by centrifugation following coagulation, the plasma or serum is pipetted and further processed. During centrifugation, the sample container is moved around the axis of rotation of the centrifuge. Effective centrifugal force causes accelerated sedimentation and solidification of the so-called blood cake, which is effectively the cellular fraction of the blood. The solidity of such a settled cellular fraction has a decisive effect on the subsequent withdrawal of the plasma or serum. Such withdrawal is performed using a pipette with the centrifugation tube in an upright position. Contact with the cellular fraction at the tip of the pipette, which is likely to cause mixing of the two phases, should be avoided. This is a standard procedure in the laboratory but requires the presence of a suitable laboratory centrifuge and a qualified laboratory technician [1]. In a further variation of this methodology, there are sample tubes filled with thixotropic gel that allow for the separation of plasma and cell fractions [2]. However, achieving fractionation without centrifugation is desirable. Different filter-based methods have been described in the literature. Typically, dedicated filters are used to retain cellular components and allow the passage of plasma fluid [3]. Since cells tend to clog the above filters, alternative approaches have been developed (e.g., by sedimentation-assisted filtration [4], sample dilution [5], or by using membrane filters or hollow fiber membranes with tangential flow and pumps [6]). A blood sample is pumped through hollow fibers having asymmetric pores. The plasma can penetrate the walls of the hollow fibers and be collected outside the hollow fibers. Cells cannot permeate the membrane and remain within the internal volume of the hollow fibers. Tangential flow across the membrane prevents clogging of the membrane pores.By passing the above sample back and forth through the above hollow fiber, the above cell fraction can be enriched and / or the plasma fraction can be separated. However, the above method requires considerable effort with respect to the device used and is characterized by a rather significant dead volume that limits its use to fairly large volume samples. Thus, small samples in the range of less than a milliliter cannot be efficiently processed using this technique. Different methods for plasma separation are based on the use of a soluble matrix that is placed in physical contact with the downstream surface of the filtration membrane. The above soluble matrix is used to passively draw in the above sample through the above membrane by the force generated through the dissolution process of the above matrix in plasma [7]. However, this approach is only feasible if the above matrix material does not interfere with the downstream analysis process. A similar approach involves the use of a solid monolith for filtering blood [8]. Typically, in this approach, it is necessary to add a certain buffer to obtain a plasma sample from the above device. The drawback of this method is that the resulting sample is diluted and modified by the buffer used. Furthermore, the process as a whole for all filter-based approaches is not efficient with respect to the volume of plasma obtained. Microfluidics-based tools have also been shown to be suitable for plasma separation purposes [9]. However, the need for complex microstructures and the low plasma production yield have prevented this technology from being widely applied.

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

[0004] Therefore, a simple tool is needed to separate / fractionate a suspended sample into different phases, preferably a liquid phase and a solid phase.

[0005] A simple tool is also needed to separate a sample containing a solvent and two components having different molecular weights and dissolved in the solvent into those two components.

[0006] There is a greater need for tools for phase separation across a wide range of sample volumes. Specifically, there is a greater need for tools for phase separation of suspended samples when samples in the microliter range, i.e., very small volumes (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 containing a solvent and two components having different molecular weights and dissolved in the solvent into two constituent elements, or for fractionating a suspension sample into a liquid phase and a solid phase, wherein the device comprises at least one polymer matrix element, the polymer matrix element comprising a porous polymer scaffold and interstitial pore spaces within the polymer scaffold, the porous polymer scaffold being a polymer that responds to changes in at least one external condition to which the device is exposed during use, e.g., pH, temperature, salt conditions, presence or absence of a chemical substance. In one embodiment, the at least one polymer matrix element has the ability to selectively (alternatively) reversibly assume an expanded state and a contracted state, where the reversible adaptation of the expanded or contracted state occurs in response to changes in at least one external condition to which the polymer matrix element is exposed during use of the device. Preferably, the at least one polymer matrix element is To repeat, It has the ability to reversibly switch between such expanded and contracted states.

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

[0009] In one embodiment, the at least one polymer matrix element is either a visible independent particle, preferably in the shape of a bead, sphere, lump, ball, cube, rectangular prism, block, ellipsoid, or irregularly shaped body, or the at least one polymer matrix element is a visible body or layer fixed on the surface of the substrate within the device.

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

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

[0012] In one embodiment, the means, preferably the heating and / or cooling means, are configured to expose the at least one polymer matrix element to at least one change in external conditions, where preferably the means are part of the device and / or incorporated into the device and / or come into contact with or connect to the suspension sample.

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

[0014] In one embodiment, the carrier tool is configured to cause 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, or the presence or absence of a chemical substance, where preferably the carrier tool is configured to cause a change in temperature.

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

[0016] In one embodiment, the carrier tool is a disposable, single-use carrier tool configured to be detachably connected to, detachably contacted with, or detachably included in, the heating and / or cooling means, 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 is detachably connected to, detachably contacted with, or detachably included in, the heating and / or cooling means.

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

[0018] In one embodiment, the liquid phase collection container includes means for collecting a specified volume of the liquid phase and / or means for measuring the volume of the liquid phase.

[0019] It should be noted that after the polymer matrix element has taken up the liquid phase from the suspension sample, some portion of the solid phase from the suspension may adhere to or attach to the polymer matrix element. Therefore, it may be necessary to remove such solid phase from the polymer matrix element, preferably from the outside of the polymer matrix element.

[0020] In one embodiment, in order to remove the solid phase from the polymer matrix element, particularly when a part of the solid phase is attached or adhered to the polymer matrix element, a) the sample container and / or, if present, the carrier tool, and / or, if present, the liquid phase collection container includes means for mechanically removing the solid phase from the polymer matrix element, such as an orifice, constriction, sliding ring, lip, edge or opening, and the internal dimension of the means matches the outer dimension of the polymer matrix element; or b) the sample container and / or, if present, the carrier tool, and / or, if present, the liquid phase collection container includes a substance having an affinity for the solid phase, and the solid phase preferably adheres to the substance when in contact with or about to come into contact with the substance; or c) the sample container and / or, if present, the carrier tool, and / or, if present, the liquid phase collection container includes means for flushing the solid phase from the polymer matrix element, such as a jet for discharging a liquid for flushing the solid phase from the polymer matrix element; or d) the device further includes a separation tool for removing the solid phase from the polymer matrix element, such as a spatula, blade, scraper.

[0021] In one embodiment, the device further includes means for further processing and / or analyzing the liquid phase, where preferably the means for further processing and / or analyzing the liquid phase includes 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 high molecular weight component, such as a protein, peptide, or nucleic acid, as one of the two components, and a low molecular weight 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 matrix element, the polymer responsive to changes in at least one external condition to which the device is exposed during use, 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] (pDMAEMA), hydroxypropyl cellulose, poly(vinyl caprolactam) (P(VCL)), and polyvinyl methyl ether, a thermoresponsive polymer (LCST polymer) having a lower critical solution temperature selected therefrom, or the thermoresponsive polymer is preferably poly(N-acryloylglycinamide) (PNAGA), poly(allylamine)-co-poly(allylurea) and its derivatives, poly(methacrylamide), poly(N-acryloylasparagine amide), poly(N-methacryloylglutamine amide), poly(acrylamide)-co-(acrylonitrile), poly(sulfobetaine), poly(phosphorylcholine), a thermoresponsive polymer having an upper critical solution temperature (UCST) selected therefrom, any thermoresponsive polymer.

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

[0025] In one embodiment, the at least one polymer matrix element has the ability to reversibly, preferably repeatedly, switch between an expanded state and a contracted state, wherein, preferably, in the expanded state, the pore space has a volume of 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 polymer matrix element when the polymer matrix element is in the expanded state.

[0026] 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, and is preferably sized to accommodate at least 50% of the liquid phase of the suspension sample, more preferably more than 75%, and even more preferably more than 90%.

[0027] In one embodiment, the pore space has holes, and the average diameter of the holes is <5 μm, preferably <3 μm, and more preferably <1 μm.

[0028] In one embodiment, the device is sized to fractionate a suspension sample having a volume in the range of 10 μl to 500 μl, where the pore space has a volume that can accommodate 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, and more preferably 100 μl to 200 μl.

[0030] In a further aspect, the present invention relates to a method for fractionating a suspension sample into a liquid phase and a solid phase, wherein the above method is a) A step of providing, in any order but separately from one another, a device according to the present invention (as defined above) and a suspension sample, wherein the device is provided under conditions in which 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 in a responsive polymer contracted state, but the at least one polymer matrix element of the device is in a wet state; b) Exposing the at least one polymer matrix element of the device to the suspension sample under external conditions that allow the polymer matrix element to reversibly enter an expanded state, thereby allowing the liquid phase in the suspension sample to enter the pore space of the at least one polymer matrix element, while simultaneously removing the solid phase in the suspension sample from the pore space; c) The present invention relates to a method comprising the step of removing at least one polymer matrix element from the sample, thereby removing the liquid phase contained in the pore space from the sample, and thereby separating the liquid phase from the solid phase in the sample.

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

[0032] In one embodiment, the above method is d) The process further includes changing the external conditions to which the polymer matrix element is exposed to conditions that allow the polymer matrix element to reversibly enter a contracted state, thereby releasing the liquid phase from the polymer matrix element.

[0033] In one embodiment, the polymer responsive to a change in at least one external condition is a thermoresponsive polymer (as further defined above), where 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 the blood cells of the whole blood sample, preferably the majority of the whole blood cells, more preferably the whole blood cells of the whole blood sample, or comprising these.

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

[0036] In a further aspect, the present invention relates to a method for separating a sample comprising a solvent and two components having different molecular weights and dissolved in the solvent into the two components, wherein the method is a) A step of providing, in any order but separately from one another, a device according to the present invention (as defined above), and a sample (hereinafter referred to herein as the "sample") comprising a solvent and two components having different molecular weights and dissolved in the solvent, wherein the device is provided under conditions in which at least one polymer matrix element of the device is in a dry state, or in which the responsive polymer is in a shrunk state but the at least one polymer matrix element of the device is in a wet state; b) Exposing the sample to the at least one polymer matrix element of the device under external conditions that allow the polymer matrix element to reversibly assume an expanded state, and thus allow components that dissolve in such a solvent (typically water or an aqueous solution) and have a low molecular weight (and therefore a size small enough to fit into the pores) to enter the pore space of the at least one polymer matrix element, while simultaneously dissolving in such a solvent and removing other components that have a high molecular weight (and therefore a size too large to fit into the pores) from the pore space; c) A method comprising the step of removing at least one polymer matrix element from the sample, thereby removing the solvent and the low molecular weight components dissolved in the solvent and contained in the pore spaces from the sample, thereby separating the low molecular weight components dissolved in the solvent from the polymer compounds in the sample.

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

[0038] In one embodiment, the above method is d) The process further includes changing the external conditions to which the polymer matrix element is exposed to conditions that allow the polymer matrix element to reversibly assume a contracted state, thereby releasing the solvent and the low molecular weight components dissolved in the solvent from the polymer matrix element.

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

[0040] In one embodiment, the sample comprising a solvent and two components having different molecular weights and dissolved in the solvent is an aqueous solution of a biomolecule (e.g., protein or nucleic acid) further comprising other dissolved components in the aqueous solvent (e.g., buffer, and / or salt, and / or low molecular weight components (e.g., dyes)). The sample may also be a preparation of a large macromolecular biomolecular complex (e.g., protein complex, cell organelle, ribosome, etc.).

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

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

[0043] The above device can therefore act as a filter for separating the solid phase from the liquid phase.

[0044] Similarly, in certain embodiments, the device may also be used to separate two or more components that are dissolved in a solvent in a sample (and therefore no longer distinguishable by their different phase states ("solid" vs. "liquid")) and differ only by their molecular weight according to their different sizes. Thus, a device according to such embodiments may act as a molecular sieve, and / or it may 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 the liquid phase in such an aqueous medium to size separation according to the molecular weight of two components having different molecular weights and dissolved in the liquid phase (one of the components having a high molecular weight (e.g., biomolecules, e.g., proteins, peptides, nucleic acids, biomolecular complexes) and therefore of a size in the nm range or less, and the other dissolved component being a low molecular weight component (e.g., a salt or buffer, or other low molecular weight components, e.g., dyes)).

[0045] Therefore, embodiments according to the present invention relate to a filtration action for separating a solid phase from a liquid phase. Other embodiments relate to a molecular sieving action for separating two dissolved components according to their molecular weight.

[0046] In some embodiments aimed at molecular sieving, the separation achieved does not have to be perfect, and in these embodiments, the devices and methods according to the present invention relate to the objective of enriching one of several components, for example, two, in proportion to each other. Here, these components have different molecular weights and are dissolved (first) in a solvent and a sample containing two (or more) components (each differing in molecular weight).

[0047] The term “two components” as used herein should not be construed as being limited to the presence of only two components (of different molecular weights). Rather, it is intended to allow for the presence of further components having different molecular weights, and such further components may also be separated using the devices and methods according to the present invention. In these cases involving more than two components, the devices and methods according to the present invention may be used repeatedly with different polymer matrix elements, the pore space between the different polymer matrix elements, particularly the pore size, is adapted to the respective molecular weights of the components to be separated. The inventors anticipate that the average pore size or average pore diameter of each of the polymer matrix elements will define a molecular weight boundary or molecular weight cutoff at which separation to different components occurs.

[0048] The polymer used in any embodiment of the present invention is a polymer that is responsive to changes 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 including a porous polymer scaffold and pore spaces within the polymer scaffold, wherein the porous polymer scaffold is composed of a polymer to which the polymer matrix element is responsive to changes in at least one external condition to which the device is exposed during use. Typical examples of such external conditions are pH, temperature, salt conditions, and / or the presence or absence of a particular chemical substance. In embodiments of the present invention, the polymer matrix element of the device, or a plurality of such polymer matrix elements, is brought into contact with a suspended sample. Taking temperature as an example of an external condition, the polymer matrix element is composed of a thermoresponsive polymer that exhibits thermally responsive phase change behavior when it reaches a critical temperature. 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. If a lower critical solution temperature (LCST) polymer is below its lower critical solution temperature (LCST), the corresponding polymer matrix element is highly porous and shrinks when heated above the LCST. If a polymer matrix element below the LCST is previously in an aqueous environment, each pore space within the polymer matrix element is filled with liquid, whereas when heated to and above the lower critical solution temperature, the polymer matrix element shrinks and any liquid contained in the pore space 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 our knowledge, the use of such responsive polymers for fractionating suspended samples has not been previously described.

[0049] In one embodiment, a device according to the present invention further includes at least one sample container intended to receive a suspension sample or solvent to be fractionated and a sample comprising two components having different molecular weights and dissolved in the solvent. The sample container comprises or is configured to comprise the at least one polymer matrix element. It should be noted that a 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 element is a visible independent particle, such as a bead, sphere, lump, ball, cube, rectangular prism, block, ellipsoid, or preferably an irregularly shaped body that is sized to fit into the device and, if present, into the sample container. In another embodiment, the at least one polymer matrix element is a visible body, portion, or layer that is immobilized on the surface of a substrate within the device. In this embodiment, the surface of the substrate to which the polymer matrix element is immobilized can be any suitable surface of the substrate located within the device. For example, if the device includes a sample container, a portion of the inner surface of the sample container may have the at least one polymer matrix element immobilized on that portion of the inner surface. Immobilization of the at least one polymer matrix element on such a surface occurs by known means (e.g., by a suitable covalent / crosslinking chemical phenomenon). The terms “visible particle” or “visible body, part, or layer” refer to a particle, body, part, or layer that can be recognized and observed using the naked eye, and that does not require specific optical means (e.g., a microscope) to observe such a particle. In this context, the term “solitary” refers to the fact that the above particle can be identified as a suitable particle that defines a boundary and is separated from its surroundings.

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

[0052] In one embodiment, the device further includes means to cause the polymer matrix elements to undergo a change in at least one further external condition to which they are exposed during use of the device. Such means to cause a change in at least one external condition can take several different forms. For example, the means may be a reservoir for such a pH adjusting agent, which allows the addition of the pH adjusting agent to the suspension sample. In another embodiment, the means may be a reservoir containing some kind of buffer, salt solution or other chemical, which allows a specified amount of such buffer, salt solution or other chemical to be added to the suspension sample. In yet another embodiment, such means to cause a change in at least one external condition is a heating and / or cooling means. Such a heating and / or cooling means is particularly preferred when the responsive polymer is a thermoresponsive polymer. In one embodiment, such a heating and / or cooling means may be a heater, for example, in the form of a heating filament, either alone or connected to a suitable cooling means (e.g., a fan or a tube through which a coolant can flow), or may include such a heater. In another embodiment, the heating and / or cooling means may be one or more Peltier elements that can operate in either a heating mode or a cooling mode. In yet another embodiment, the heating and / or cooling means may consist only of a heating means, where cooling is achieved by convective heat transfer.

[0053] In one embodiment, means, preferably the heating and / or cooling means, that bring about at least one change in external conditions are configured to expose the at least polymer matrix elements to the change in at least one external condition. In one embodiment, the means, preferably the heating and / or cooling means, form part of the device for fractionation, or they are incorporated into the device, and / or they are configured to come into contact with the suspension sample or to be connected to the suspension sample. For example, if the means is a heating and / or cooling means, and the device includes a container, the heating and / or cooling means may be configured to surround the container and thereby, if necessary, influence, control, and modify 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, a device configured for fractionating a suspension sample further includes a carrier tool configured to enable handling of the at least one polymer matrix element. Such a carrier tool acts as a substrate having a surface to which the at least one polymer matrix element can be immobilized. The carrier tool is sized to be immersed in the suspension sample and to bring the at least one polymer matrix element into contact with the suspension sample. In this embodiment, the carrier tool serves the purpose of handling the at least one polymer matrix element in an easy manner, in that it enables exposure of the at least one polymer matrix element immobilized on the carrier tool to the suspension sample and, if necessary and desirable, removal of the at least one polymer matrix element from the suspension sample. Such a carrier tool can be configured in several ways. For example, it may be a rod, a stick, etc., that enables immersion of the polymer matrix element in the suspension sample. Furthermore, in a preferred embodiment, the carrier tool itself may be configured to cause a change in at least one external condition to which the polymer matrix element is exposed during use of the device. In a preferred embodiment, the carrier tool is configured to produce a temperature change and preferably includes heating and / or cooling means, or is connected to or in contact with heating and / or cooling means that form part of the device. In a preferred embodiment, such heating and / or cooling means is the heating and / or cooling means (as further defined above). In one embodiment, the carrier tool is a disposable, single-use carrier tool and is configured to be detachably connected to, detachably in contact with, or detachably included in the heating and / or cooling means.The carrier tool may be configured as a pen, stick, rod, cylinder, bar, rod, baton, or component, and it is preferable that 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, the operation of the heating and / or cooling means changes the temperature of the carrier tool and, therefore, the temperature of any at least one polymer matrix element fixed thereon.

[0055] In one embodiment, the device further includes at least one liquid phase collection container, the at least one liquid phase collection container configured to collect the polymer matrix element and / or, if present, the polymer matrix element immobilized on the carrier tool.

[0056] In one embodiment, the liquid phase collection container acts as a reservoir and / or storage module for the liquid phase. In one embodiment, the liquid phase collection container is sized to accept 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 the liquid phase collection container 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 carrier tool on which the polymer matrix element and / or, if present, the polymer matrix element is immobilized is transferred from the sample container to the liquid phase collection container under conditions that allow the polymer matrix element to take up at least a portion of the liquid phase of the suspension sample after the polymer matrix element has come into contact with the suspension sample. In such embodiments, it is preferable that, after the polymer matrix element containing the liquid phase is transferred to the liquid phase collection container, conditions are applied that allow at least a portion of the liquid phase contained in the polymer matrix element to be released from the polymer matrix element to the liquid phase collection container.

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

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

[0060] In one embodiment, the liquid phase collection container includes means for collecting a specified volume of the liquid phase and / or means for measuring the volume of the liquid phase of the suspension sample.

[0061] In one embodiment, a) the sample container and / or the carrier tool, if present, and / or the liquid phase collection container, include means for mechanically removing the solid phase from the polymer matrix element (e.g., an orifice, a constriction, a sliding ring, a lip, a rim, or an opening (whose internal dimensions match the external dimensions of the polymer matrix element)); or b) the sample container and / or the carrier tool, and / or the liquid phase collection container, if present, include a substance having affinity for the solid phase, and the The device further comprises a separation tool for removing the solid phase from the polymer matrix elements (e.g., a spatula, blade, scraper).

[0062] Devices according to the present invention are particularly useful in situations where whole blood is fractionated into plasma and blood cells as a liquid phase and a solid phase, respectively. Thus, in one embodiment, the suspension sample is a whole blood sample, and the device is configured to fractionate the whole blood into a solid phase containing plasma and blood cells. In one embodiment, the plasma is platelet-enriched. In another embodiment, the plasma is platelet-deficient. In one embodiment, the polymer responsive to changes in at least one external condition to which the polymer matrix element is exposed is a thermoresponsive polymer. Such a thermoresponsive polymer is either a thermoresponsive polymer having a lower critical solution temperature (LCST) or a thermoresponsive polymer having an upper critical solution temperature (UCST). The term “thermoresponsive polymer” means a polymer whose physical properties, 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 present invention exhibits a change in affinity for water with temperature. For example, a thermoresponsive polymer having a lower critical solution temperature (LCST) is hydrated and expanded at temperatures below such LCST. At temperatures above the LCST, the polymer is in a contracted, unhydrated state. By circulating the at least one polymer matrix element between the two states, it is possible to repeatedly (preferably) and reversibly contain the liquid in the polymer matrix element, and then, if desired and as needed, discharge the liquid back from the polymer matrix element. When such liquid is incorporated into the polymer matrix element, into the pore spaces within the polymer matrix element, the solid particles in the suspension sample remain outside such 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 the expanded state, the solvent / liquid has access to all or substantially all of the pores, or a specified proportion of the pores, whereas the solid phase (e.g., blood cells) does not have access to the pores.In this swollen state, the polymer matrix elements can be removed from 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, preferably by at least one crosslinking reagent that crosslinks and interconnects different polymer chains within the polymer, or by providing a substrate in the device, in particular a substantially flat or curved substrate, adjacent to the polymer matrix elements, and by interfacial crosslinking of 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, if present, the surface of the at least one sample container, or, if present, the surface of the carrier tool.

[0064] In embodiments according to the present invention, the at least one polymer matrix element has the ability to reversibly enter an expanded state and a contracted state due to the presence of the responsive polymer. Preferably, in the expanded state, the pore space has a volume of 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 polymer matrix element when the polymer matrix element is in the expanded state.

[0065] Furthermore, 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. Depending on whether platelet-enriched plasma is desired, the pore space may be accessible to platelets. The pore space may be appropriately tuned to have pores, the average diameter of which allows for the exclusion of a desired solid phase and the inclusion / incorporation of a desired liquid phase into the pore space. For example, in one embodiment, the pore space may have pores, the average diameter of which is <5 μm, preferably <3 μm, more preferably <1 μm. If the above device is used to provide size separation of two dissolved components that differ from each other by their respective molecular weights, and / or to enrich one of these two components, the pore space may have pores, the average diameter of which 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 selected to suit the intended purpose of the above device.

[0066] 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. Preferably, the pore space is sized to accommodate at least 50% of the liquid phase of the suspension sample, more preferably 75%, and even more preferably 90%.

[0067] In one embodiment, the device is particularly suitable and configured for fractionating a suspension sample having a volume <1 ml. In one embodiment, the device according to the present invention is sized to fractionate a suspension sample having a volume in the range of 10 μl to 500 μl, and the pore space of the at least one polymer matrix element of the device has a volume that takes up 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 pore space of the polymer 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 pore space is at least 5 μl to at least 200 μl to 250 μl; preferably, the pore space has a volume that is at least 75% of the suspension sample volume of 10 μl to 500 μl. That is, the pore space has a volume of at least 7.5 μl to at least 375 μl; more preferably, the pore space has a volume of at least 90% of the suspension sample volume of 10 μl to 500 μl. That is, the pore space has a volume of at least 9 μl to at least 450 μl; and even more preferably, the pore space has a volume of at least 95% of the suspension sample volume of 10 μl to 500 μl. That is, the pore space has a volume of at least 9.5 μl to at least 475 μl. In this situation, in one embodiment, the total volume of the pore space of the polymer matrix element does not exceed the volume of the suspension sample. Therefore, for example, if the volume of the suspension sample is 500 μl, the volume of the pore space is preferably 500 μl or less. In one embodiment, the volume of the pore space is 250 μl, 375 μl, 450 μl, or 475 μl. In another exemplary embodiment, if 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, if the suspension sample has a volume of 50 μl to 200 μl, the volume of the pore space is in the range of 25 μl to 200 μl. In yet another embodiment, if the suspension sample has a volume in the range of 100 μl to 200 μl, the volume of the 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, wherein the method is: a) A step of providing, in any order but separately from one another, a device according to the present invention (as defined above) and a suspension sample, wherein the device is provided under conditions in which 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 in a responsive polymer contracted state, but the at least one polymer matrix element of the device is in a wet state; b) Exposing the at least one polymer matrix element of the device to the suspension sample under external conditions that allow the polymer matrix element to reversibly enter an expanded state, thereby allowing the liquid phase in the suspension sample to enter the pore space of the at least one polymer matrix element, while simultaneously removing the solid phase in the suspension sample from the pore space; c) A method comprising the step of removing at least one of the above polymer matrix elements from the sample, thereby removing the liquid phase contained in the 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 described above, in step c), the polymer matrix element and / or, if present, the carrier tool on which the polymer matrix element is immobilized is transferred from the sample container to the liquid phase collection container after the polymer matrix element has come into contact with the suspension sample under conditions that allow the polymer matrix element to take up at least a portion of the liquid phase of the suspension sample. In such embodiments, it is preferable that after the polymer matrix element containing the liquid phase is transferred to the liquid phase collection container, conditions are applied such that at least a portion of the liquid phase contained in the polymer matrix element is released from the polymer matrix element into the liquid phase collection container.

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

[0071] In one embodiment of the above method, the method is d) The process may further include changing the external conditions to which the polymer matrix element is exposed to conditions that allow the polymer matrix element to reversibly enter a contracted state, thereby releasing the liquid phase from the polymer matrix element.

[0072] It should be noted that in certain embodiments of the method according to the present invention, in step a), the device according to the present invention (and therefore the polymer matrix elements contained within the device) may be provided in a wet but shrunk state instead of a dry state; that is, the polymer matrix elements are provided under external conditions that are not dry, but the responsive polymer is in a shrunk state, although they 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 polymer matrix elements. In this embodiment, in step b), the at least one polymer matrix element is exposed to the suspension sample under external conditions that allow the polymer matrix element to take on a reversibly expanded state, and therefore allow the liquid phase in the suspension sample to enter the pore spaces of the at least one polymer matrix element (as in embodiments where the polymer matrix element is provided in a dry state). Subsequent steps c) and the subsequent optional step d) are as previously defined.

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

[0074] In a preferred embodiment of the method according to the present 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 the blood cells of the whole blood sample or contains the blood cells of the whole blood sample. In a preferred embodiment, the solid phase of the whole blood sample is the majority of the whole blood cells, more preferably the whole blood cells of the whole blood sample or contains them.

[0075] In further aspects, the present invention also relates to the use of a device according to the present invention (as defined above) for fractionating a suspension sample into liquid and solid phases, preferably for fractionating a whole blood sample into plasma and blood cells, or to the use of a polymer (as further defined above) that is responsive to at least one external condition, e.g., pH, temperature, solid conditions, or the presence or absence of a chemical substance, wherein the use preferably comprises steps a) to c) (as further defined above) and optionally step d) (likewise as further defined above).

[0076] While we do not wish to be constrained by any theory, the inventors have found that the use of a porous polymer scaffold and a responsive polymer that forms pore spaces within the porous polymer scaffold, together, constitutes a polymer matrix element, enabling efficient fractionation of the suspended sample into liquid and solid phases. This is especially true when the suspended sample volume is small, i.e., <1 ml. In preferred embodiments according to the present invention, the sample size used is well below 1 ml, for example, <200 μl, preferably <150 μl. For example, in one embodiment, a typical blood sample used according to the present invention is a sample obtained by capillary tube, the volume of such a sample is typically not exceeding 140 μl (i.e., <140 μl), and from such a sample, a typical amount of plasma is <80 μl. The device according to the present invention is extremely easy to use and can be readily adapted to different suspended sample types. A particularly preferred suspension sample type is whole blood, and the device according to the present invention allows for the processing of multiple samples in a short period of time due to the preferred disposability of the individual polymer matrix elements used in certain embodiments of the device, which are detachable or removable from the device. It is also conceivable that a single device for fractionating a suspension sample according to the present invention may contain more than one polymer matrix element, for example, 2, 3, 4, 5, 6, 10, 20, 30, 40, 50, ... 100, 10,000 or more polymer matrix elements.

[0077] The above device may be a portable device, a benchtop device, or a large-scale workstation device capable of processing many samples simultaneously. In the latter case, the device may further include a suitable control unit, a robotic arm, and a suitable electronic control circuit that can bring about the movement of each polymer matrix element, as well as the control unit, which can bring about the addition of a sample, the adaptation of external conditions (e.g., temperature that brings about the incorporation of the liquid phase into the polymer matrix element), the removal of the polymer matrix element from the sample, and the change in external conditions (e.g., temperature) in order to switch the polymer matrix element between an expanded and a contracted state. [Brief explanation of the drawing]

[0078] Furthermore, the present invention is illustrated by drawings. [Figure 1] Figure 1 shows one embodiment of a device according to the present invention, which includes a polymer matrix element contained within a sample container used for fractionating a blood sample.

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

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

[0081] [Figure 4] Figure 4 shows one embodiment of a carrier tool in which polymer matrix elements are immobilized on the surface of the carrier tool.

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

[0083] [Figure 6-1] Figures 6(A)–F)) show the results of the plasma separation process of whole blood samples using the plasma separation device illustrated in Figure 5. [Figure 6-2] Figures 6(A)–F)) show the results of the plasma separation process of whole blood samples using the plasma separation device illustrated in Figure 5. [Figure 6-3] Figures 6(A)–F)) show the results of the plasma separation process of whole blood samples using the plasma separation device illustrated in Figure 5.

[0084] [Figure 7] Figure 7 shows the fluorescence emission spectra of the stock solutions of carboxyfluorescein and R-phycoerythrin (R-PE) after exposure to two different polymer matrix elements at room temperature (during which the exposed portion of the stock solution may enter the interporal spaces of the polymer matrix elements), and after the two different polymer matrix elements are heated to 40°C and the liquids discharged from them. [Modes for carrying out the invention]

[0085] More specifically, Figure 1 shows a sample container with a polymer matrix element in a dry state. After the sample is added to the sample container, the polymer matrix element is moistened, and the liquid is incorporated by the polymer matrix element, while the solid phase within the sample remains outside the polymer matrix element. Upon contact with the liquid, the polymer matrix element swells considerably, increasing its volume by approximately 10 × 10⁻¹. The residual sample not incorporated into the polymer matrix element is more or less a solid phase or the suspended members, e.g., a cell-enriched sample. In contrast, the polymer matrix element itself contains the liquid phase of the sample, without any 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 polymer matrix element, this may be platelet-containing or platelet-free plasma. In a further step, the phase having the enriched solid phase may be removed from the sample container, or the liquid phase may be removed by removing the polymer matrix element from the sample container. Subsequently, the polymer matrix element is exposed to conditions that cause the polymer to shrink again, thereby reducing the pore size of the polymer matrix element and thus moving the liquid away from the polymer matrix element. For example, if the polymer is an LCST polymer, such shrinkage can be achieved by raising the temperature to a temperature above that of the LCST. The liquid phase thus released, for example, released plasma, can be collected from the container.

[0086] Figure 2 shows the results of fractionation performed according to the principles outlined in Figure 1. More specifically, four different polymer matrix elements were generated as visible independent particles, exposed to whole blood (A), and after a prescribed incubation, the particles were removed and placed in separate containers (B). These 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 cell counting analysis of the different samples processed in Figures 2 A) to C), as well as of whole blood and centrifuged plasma.

[0087] Figure 3 shows another embodiment of a device according to the present invention and its use. In this embodiment, a sample container containing a suspended sample is provided, and a polymer matrix element attached to a carrier tool is introduced into the suspended sample. The carrier tool is preferably thermally conductive, and therefore the temperature to which the polymer matrix element is exposed can be easily changed by connecting the carrier tool to a heating and / or cooling means. When the polymer matrix element is brought into contact with the suspended sample, the polymer matrix element takes in the liquid and excludes the suspended solid phase, such as cells. To reiterate, taking blood as an example, plasma is collected in the polymer matrix element until it reaches the capacity of the pores in the interstitial space. The polymer matrix element is then removed from the sample container and transferred to a further container, i.e., a container for collecting the liquid phase (hereinafter sometimes referred to as the “liquid phase collection container”), and here again, the external conditions are changed to shift the polymer matrix element from an expanded state to a contracted state, and therefore release the liquid incorporated into the polymer matrix element. If the responsive polymer of the polymer matrix element described above is an LCST polymer, then the change in external conditions is a temperature increase exceeding that of the LCST.

[0088] Figure 4 shows an embodiment of a carrier tool in which polymer matrix elements are immobilized on the surface of the carrier tool according to the present invention.

[0089] Figure 5 shows an embodiment of a portable device forming part of a device according to the present invention, which includes a polymer matrix element immobilized on a disposable carrier tool and a heating and / or cooling means (in this case, a heating rod) configured to connect to it. Panel A shows an exploded view of the portable device, and Panel B shows the portable device in its assembled form; Panel C) shows an embodiment of a device for fractionation according to the present invention, which includes a disposable carrier tool having an immobilized polymer matrix element and a heating and / or cooling means (here: 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 container containing whole blood; Panel B) shows a disposable carrier tool (as shown in Figure 4) on which polymer matrix elements are immobilized on a disposable carrier tool inserted into the sample container; Panel C) shows the portable device of Figure 5 connected to the disposable carrier tool by the heating and / or cooling means (heating rod); Panel D) shows the portable device of Figure 5 combined with the disposable carrier tool after it has been withdrawn from the sample container; Panel e) shows the portable device of Figure 5 combined with the disposable carrier tool after it has been inserted into a new container ("liquid phase collection container"); and Panel F) shows both the (first) sample container and the liquid phase collection container after the portable device of Figure 5 combined with the disposable carrier tool has been withdrawn.

[0091] Figure 7 shows the fluorescence emission spectra of the same stock solutions of carboxyfluorescein and R-phycoerythrin (R-PE) after exposure to two different polymer matrix elements at room temperature (during which the exposed portion of the stock solution may enter the interporal spaces of the polymer matrix elements), as well as the liquids discharged from these two different polymer matrix elements after they were heated to 40°C. Removal of R-phycoerythrin from the liquids entering the polymer matrix elements (and corresponding enrichment in the "supernatant" liquids that cannot enter the polymer matrix elements) can be observed.

[0092] Furthermore, the following examples are referenced. These examples are given to illustrate the present invention and not to limit it. [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 the LCST polymer matrix were prepared and placed in sample containers.

[0094] The protocol applied was as follows: First, a solution containing N-isopropylacrylamide (NIPAM), N,N'-methylenebisacrylamide (BIS) monomer, and ammonium persulfate (APS) was prepared (premix 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 of plugs (i.e., "visible independent particles") were formed in a 0.5 mL microreaction vial by mixing the above liquid with 2 μL of 5% TEMED (degassed) under argon. Polymerization was then allowed to occur at room temperature for 30 minutes.

[0097] The vial was placed on a heat block at 50°C. Shrinkage of the polymer was easily observed. The discharged liquid was collected with a micropipette and discarded. The plug was removed from the microreaction vial and transferred to a 2 ml reaction tube. 500 μl of PBS buffer was added to the tube, and then incubated at 20°C for 1 hour to swell the polymer again. Excess PBS was removed, and the tube was incubated at 60°C for 15 minutes to shrink the polymer. The discharged liquid was discarded. This washing procedure was repeated one more time.

[0098] Fresh 150 μL whole blood samples collected in EDTA-coated vacuum tubes were added to vials containing the shrunk polymer as shown in Figure 2A), and allowed to stand at room temperature for 30 minutes.

[0099] After 30 minutes, the above samples were removed from the vials containing the swollen plugs. Figure 2B) shows the four vials with the plugs. It is clear that different matrices absorbed different amounts of fluid. Furthermore, different amounts of whole blood adhered to the plugs, depending on their composition.

[0100] Next, the vial with the plug was placed on a heat block at 40°C for 1 minute. When the polymer shrunk, the liquid was discharged from the matrix material. The discharged liquid was collected in a separate vial. As shown in Figure 2C), different amounts of liquid were discharged at the specified temperature, and similarly, different levels of coloration of the sample may be observed due to the captured whole blood.

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

[0102] The separation effect achieved by applying the above matrix is ​​clearly evident. Furthermore, a simple approach to optimizing the separation effect by adding different reagents to the polymer preparation is shown. Matrices 1 and 2 appear to be the most efficient in separating blood cells from the liquid components of a blood sample.

[0103] Example 2 Polymer matrix elements immobilized on a substrate An aluminum shell was designed as part of a disposable component for plasma separation. The aluminum shell functions as a carrier tool, acting as a substrate with a surface to which polymer matrix elements are immobilized. The shell surface was roughened with sandpaper to improve adhesion of the polymer matrix elements to the surface.

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

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

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

[0107] These aliquots were mixed with 4 μl of 5% TEMED (degassed) under argon, and the aluminum shells were immersed in the solution. After a 30-minute incubation period, the aluminum shells were brought into contact with a heat source to shrink the polymer on the shells. The shells and polymers were removed from the vial and transferred to a new vial containing PBS washing buffer. The polymers were re-swelled in the washing buffer for 1 hour. The washing process was repeated twice.

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

[0109] Example 3 Description of the prototype developed by Blink AG Based on the present invention, we have developed a portable isolation device as a conceivable product. Such a product may be particularly useful in situations that enable point-of-care diagnostic applications. Many diagnostic tests require plasma as a sample matrix. However, in many environments, there is no suitable means available to generate plasma from whole blood, particularly from capillary blood samples that can be easily obtained by other means, such as the common fingertip puncture method. Therefore, we describe a pen-like, portable, battery-powered device in combination with a disposable device.

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

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

[0112] The “disposable carrier tool” device described above includes a plastic portion with a recess for securely housing the heating rod of the device. On its outer surface, the disposable carrier tool is coated with an LCST polymer as outlined in Example 2 and shown in Figure 4.

[0113] The disposable carrier tool described above is designed to fit into a standard Microvette sample container, for example, a sample container containing the required amount of collected whole blood (e.g., 100 μL). When the disposable carrier tool is introduced into the sample container, the plasma coats the polymer and occupies its inner space, while 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 device shown in Figures 5A) and 5B), as well as their heating and / or cooling means, i.e., the heating rod, by introducing the heating rod of the device into the recess of the disposable carrier tool. The disposable carrier tool is attached to the rod by firmly pushing the rod into the disposable carrier tool. The device with the disposable carrier tool attached is then removed from the sample container, and the disposable carrier tool is introduced into another container, i.e., an empty container ("liquid phase collection container"). The portable device is activated by pressing a button. A green (or any other appropriate color) LED lights up until a predetermined temperature above the LCST is reached. The LED remains green at this point. The polymer contracts within the disposable carrier tool, releasing the captured plasma into the liquid-phase collection container. The disposable carrier tool is removed from the liquid-phase collection container and taken out of the portable device shown in Figures 5A and 5B for safe disposal. The portable device can now be used for a new separation task. The device is switched off by pressing the button once. Any malfunction is indicated by a red light.

[0114] The overall workflow is shown in Figures 6A) to F), where Figure 6A) shows a standard sample container containing a suspension sample (in this case, whole blood); Figure 6B) shows the disposable carrier tool of Figure 4 (with the polymer matrix elements immobilized on the carrier tool) inserted into the sample container so that the polymer matrix elements are exposed to the suspension sample (i.e., in this case, the whole blood); Figure 6C) shows the disposable carrier tool connected to a heating and / or cooling means (in this case, a heating rod) of the portable device of Figure 5); Figure 6D) shows the disposable carrier tool after exposure to the suspension sample for a specified period and after the liquid phase has been bonded to or incorporated into the polymer matrix elements. Figure 6E) shows the combination of the rear tool and the portable device withdrawn from the sample container; Figure 6E) shows the same combination (disposable carrier tool and portable device) introduced into a further empty container, i.e., a liquid phase collection container, where the heating and / or cooling means (in this case, a heating rod) is operated to heat above the LCST of the polymer matrix element, thereby releasing the liquid phase bound by the polymer matrix element into the liquid phase collection container; Figure 6E) shows (on the left) the (first) sample container in which the whole blood was originally held and exposed to the polymer matrix element, and (on the right) the (further) liquid phase collection container into which the liquid phase has been released after the heating rod has been heated above the LCST of the polymer matrix element.

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

[0116] Polymer plugs (i.e., "polymer matrix elements") prepared according to the methodology described in Example #1 were used to alter 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 high molecular weight fluorescent protein R-phycoerythrin (M=250 kDa, Thermo Fisher P801) ("R-PE"). The 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 Figure 7 (the emission peak for R-PE is at 570 nm).

[0117] Two different plugs of LCST polymer matrix, prepared as outlined in Example #1, consisting of PNIPAM / PEG acrylate and PNIPAM, were used to investigate the effect of the matrix on the composition of the above liquid. Vials containing the above plugs were placed on a 40°C heat block for 1 minute. The discharged liquid was collected with a micropipette and discarded. 150 μL of the above stock solution containing 5-carboxyfluorescein and R-phycoerythrin was added to the vial and allowed to stand at room temperature for 5 minutes. The supernatant was then collected and fluorescence spectra were obtained for both samples ("supernatant"). Vials containing the above plugs were placed on a 40°C heat block for 1 minute, and the discharged liquid was collected with a micropipette. Fluorescence spectra were obtained for the drained liquid samples ("Matrix 1" (white circles) and "Matrix 4" (white diamonds)) and compared to the spectra of the stock solution (shown by the solid black lines) and the supernatants (Matrix 1 (black circles) and Matrix 4 (black diamonds)). The normalized spectra of the two different matrix preparations and the stock solution are shown in Figure 7. Both polymer matrix elements remove R-phycoerythrin from the interpore spaces of the polymer matrix elements, thereby removing R-phycoerythrin from the liquid entering the interpore spaces of the polymer matrix elements, thus removing R-phycoerythrin from the respective supernatants. It can be observed that phosphorus can be enriched (Figure 7: “Supernatant Matrix 1” (black circle) and “Supernatant Matrix 4” (black diamond)), and when the polymer matrix elements are heated above their LCST, R-phycoerythrin is subsequently released again (Figure 7: “Discharged Liquid Matrix 1” (white circle) and “Discharged Liquid Matrix 4” (white diamond)). Thus, polymer matrix elements according to the present invention can be successfully used to separate and / or enrich / remove two dissolved components of different molecular weights, respectively. It is also evident from the data that the different matrix preparations exhibit different abilities to enrich or remove certain reagents from solution. References: [Formula 1]

Claims

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 device is exposed during use, such as pH, temperature, salt conditions, or the presence or absence of a chemical.

2. 10. The device of claim 1, further comprising at least one sample vessel containing or configured to contain the at least one polymer matrix element, the at least one sample vessel being capable of receiving a sample containing a solvent and two components having different molecular weights and dissolved in the solvent, or a suspension sample.

3. 10. A device according to any of the preceding claims, wherein the at least one polymeric matrix element is either a macroscopically visible discrete 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.

4. 10. The device of any of the preceding claims, 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.

5. 10. A device according to any of the preceding claims, wherein 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 the means for effecting a change in at least one external condition is a heating and / or cooling means.

6. 6. The device of claim 5, wherein said means, preferably said heating and / or cooling means, is configured to expose said at least one polymer matrix element to a change in at least one external condition, wherein preferably said means is part of said device and / or is incorporated into said device and / or is configured to come into contact with or be connected to said suspension sample.

7. 10. A device according to any of the preceding claims, 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 it can bring the at least one polymeric matrix element into contact with the sample, preferably the suspension sample.

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 chemicals, wherein preferably the carrier tool is configured to effect a change in temperature.

9. 9. The device according to claim 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 to 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 according to claim 5 or 6.

10. 10. The device of any of claims 7 to 9, wherein the carrier tool is a disposable, single-use carrier tool and is configured to be detachably connected to, detachably contacted by, or detachably include, the heating and / or cooling means, preferably wherein the carrier tool is configured as a pen, stick, rod, cylinder, bar, rod, baton, or member, and wherein the carrier tool has an interface that detachably connects to, detachably contacts, or detachably includes, the heating and / or cooling means.

11. 10. A device as described in any of the preceding claims, further comprising at least one liquid phase collection container configured to capture the polymer matrix element and / or, if present, the carrier tool on which the polymer matrix element is immobilized.

12. a) the sample vessel and / or, if present, the carrier tool, and / or, if present, the liquid phase collection vessel, comprise means for mechanically removing the solid phase from the polymer matrix element, such as an orifice, a constriction, a sliding ring, a lip, a rim 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, comprise a substance that has an affinity for the solid phase, for which the solid phase is , preferably adheres when in 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 means for washing the solid phase from the polymer matrix element, for example a jet for ejecting a liquid that washes the solid phase from the polymer matrix element; or d) the device according to any one of claims 2 to 11, further comprising a separation tool for removing the solid phase from the polymer matrix element, for example a spatula, blade, scraper.

13. A device according to any of the preceding claims, 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, 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 wherein 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.

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 a thermoresponsive polymer that is a thermoresponsive polymer having a lower critical solution temperature (LCST polymer) selected from 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), hydroxypropyl cellulose, poly(vinyl caprolactam) (P(VCL)), and polyvinyl methyl ether; or the thermoresponsive polymer 10. The device according to any of the preceding claims, wherein the polymer is preferably a thermo-responsive polymer having an upper critical solution temperature (UCST) selected from poly(N-acryloylglycinamide) (PNAGA), poly(allylamine)-co-poly(allylurea) and derivatives thereof, poly(methacrylamide), poly(N-acryloylaspartamide), poly(N-methacryloylglutamine amide), poly(acrylamide)-co-(acrylonitrile), poly(sulfobetaine), poly(phosphorylcholine), wherein more preferably said responsive polymer, in particular said thermo-responsive polymer, is crosslinked, preferably by either by at least one crosslinking reagent crosslinking and interconnecting between different polymer chains within said polymer, or by providing a substrate, in particular a substantially flat or curved substrate, within the device adjacent to the polymer matrix element and by interfacially crosslinking different polymer chains of said polymer to the substrate, in particular to the surface of the substrate.

15. 10. A device according to any of the preceding claims, wherein the interstitial pore space comprises pores, the average diameter of the pores being <5 μm, preferably <3 μm, more preferably <1 μm.

16. A device according to any of the preceding claims, 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 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, 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.

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 claims 1 to 16 and a suspension sample, wherein the at least one polymeric matrix element of the device is in a dry state or the at least one polymeric matrix element of the device is provided under conditions in which the responsive polymer is in a contracted state but the at least one polymeric 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 polymeric matrix element from said sample, thereby removing said liquid phase contained in said interstitial pore space from said sample, thereby separating said liquid phase from a solid phase in said sample; A method that encompasses

18. 18. The method of claim 17, wherein in step c) the at least one polymer matrix element, if present, is removed from a sample vessel as defined in claim 2 and transferred to a liquid phase collection vessel as defined in claim 11, if present.

19. The method comprises: d) changing the external conditions to which the polymeric matrix elements are exposed to conditions that allow the polymeric matrix elements to reversibly assume a contracted state, thereby releasing the liquid phase from the polymeric matrix elements; 19. The method of any one of claims 17 to 18, further comprising:

20. 16. Use of a device as defined in any of claims 1 to 16 or of a polymer responsive to changes in at least one external condition, such as pH, temperature, salt conditions, presence or absence of a chemical, as defined in any of claims 1 to 14 for fractionating a suspension sample into a liquid phase and a solid phase, 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 claims 17 to 18 and, optionally, step d) as defined in claim 19.

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