CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of U.S. Provisional Patent Application No. 63/380,341, entitled "Separation Media and Methods of Use," filed October 20, 2022, the disclosure of which is incorporated by reference herein in its entirety.

TECHNICAL FIELD

[0002] The present disclosure is directed to separation media, their components, and methods of using such media. More particularly, the present disclosure is related to separation media having one or more layers for separating serum and/or plasma from whole blood without the need for centrifugation.

BACKGROUND

[0003] Separating serum from whole blood has traditionally been performed through centrifugation, filtration, and migration processes. Centrifugation separates serum from blood cell components based on density, while filtration separates serum from blood cell components based on size. In contrast, migration separation, or lateral separation, separates serum from blood cells based on capabilities of various blood components to move within a porous matrix. [0004] Although fiber-based whole blood separation media are available on the market, there exists a need for separation media capable of separating serum and/or plasma from whole blood in a faster, more efficient, more user-friendly manner, with lower protein binding and high serum and/or plasma transfer efficiency. In particular, there exists a need for separation media capable of separating serum and/or plasma from whole blood without the need for centrifugation.

SUMMARY

[0005] The instant disclosure is directed to separation media, their components, and methods of using such media. In an embodiment, a separation medium may comprise a first layer and a second layer, wherein the first layer is fixedly coupled to the second layer. In some embodiments, the first layer may comprise a porous fiber matrix, and the second layer may comprise a polymer membrane. In certain embodiments, the second layer may have a property, such as a density, a pore size, a pore volume, a thickness, or a combination thereof, that varies in a direction, such as a length direction, a width direction, a height direction, or a combination thereof. In some embodiments, the first layer may be fixedly coupled to the second layer by an adhesive, a thermal bond, or a combination thereof.

[0006] In an embodiment, a method of using such a separation medium may comprise exposing a first end of the separation medium to a fluid comprising whole blood, and drawing the fluid from the first end of the separation medium toward a second end of the separation medium. In certain embodiments, the step of drawing may be accomplished via capillary action. In some embodiments, the step of drawing may thereby capture a first component of the whole blood in a first portion of the separation medium, and may thereby also separate the first component of the whole blood from a second component of the whole blood. In certain embodiments, the first component of the whole blood may comprise red blood cells, white blood cells, platelets, or a combination thereof. In some embodiments, the second component of the whole blood may comprise serum or plasma. In an embodiment, the method does not include centrifugation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIG. 1 is an optical microscopy image of an embodiment of a first layer comprising a porous fiber matrix in accordance with the present disclosure.

[0008] FIG. 2 is a photo of an embodiment of a first layer comprising a porous fiber matrix in accordance with the present disclosure.

[0009] FIG. 3 is a photo of an embodiment of a second layer comprising a polymer membrane in accordance with the present disclosure.

[0010] FIG. 4 is a schematic representation of a side view of a separation medium in accordance with the present disclosure.

[0011] FIG. 5A is a top view photo of an embodiment of a separation medium in accordance with the present disclosure.

[0012] FIG. 5B is an alternative view photo of the embodiment of the separation medium shown in FIG. 5A in accordance with the present disclosure.

[0013] FIG. 5C is a side view photo of the embodiment of the separation medium shown in FIG. 5A in accordance with the present disclosure.

[0014] FIG. 6A is a photo of an embodiment of a method of using a separation medium for about 2 minutes in accordance with the present disclosure.

[0015] FIG. 6B is a photo of the embodiment of the method of using the separation medium shown in FIG. 6A for about 5 minutes in accordance with the present disclosure. [0016] FIG. 7 is a photo of a comparison of three samples exposed to a fluid comprising whole blood, with Sample 3 being an embodiment of a separation medium comprising a first layer and a second layer in accordance with the present disclosure.

[0017] FIG. 8 is a schematic illustration of a process used to prepare separation media in accordance with the present disclosure.

[0018] FIG. 9 is a photo of separation media undergoing dip treatment with a treatment formulation in accordance with the present disclosure.

[0019] FIG. 10 is a photo of dip-treated separation media on a sieve tray in accordance with the present disclosure.

[0020] FIG. 11 is a pore diameter distribution graph for a separation medium in accordance with the present disclosure.

[0021] FIG. 12A is an optical microscopy image of a top layer having small fiber diameters in accordance with the present disclosure.

[0022] FIG. 12B is an optical microscopy image of a top layer having large fiber diameters in accordance with the present disclosure.

[0023] FIG. 13 A is a scanning electron microscopy (SEM) image of a matte side of a second layer comprising a polymer membrane in accordance with the present disclosure.

[0024] FIG. 13B is an SEM image of a shiny side of a second layer comprising a polymer membrane in accordance with the present disclosure.

[0025] FIG. 14 shows an individual data plot for pore diameters of both matte and shiny sides of second layers comprising polymer membranes in accordance with the present disclosure. [0026] FIG. 15A is a photo of severe blood pooling on a second layer tested alone.

[0027] FIG. 15B is a photo of severe blood pooling on another second layer tested alone.

[0028] FIG. 15C is a photo showing plasma separation on a first layer tested alone.

[0029] FIG. 16A is a schematic representation of the boundary line definitions (i.e., intersections/cut lines) for a separation medium cut in accordance with the present disclosure. [0030] FIG. 16B is a photo of an embodiment of a separation medium before the cuts illustrated in FIG. 16A were made.

[0031] FIG. 16C is a photo of the embodiment of the separation medium of FIG. 16B, after the cuts illustrated in FIG. 16A were made using a razor blade.

[0032] FIG. 16D is a photo showing how an exemplary section the embodiment of the separation medium illustrated in FIG. 16A was measured after being cut.

[0033] FIG. 17 is a photo of a top view of a test strip 30 seconds after exposure to whole blood in accordance with the present disclosure. [0034] FIG. 18A is a photo of the bottom view of an embodiment of a separation medium (with the second layer shown) after plasma separation from whole bovine blood in accordance with the present disclosure.

[0035] FIG. 18B is a photo of the bottom view of another embodiment of a separation medium with the second layer shown) after plasma separation from whole bovine blood in accordance with the present disclosure.

[0036] FIG. 18C is a photo of the bottom view of yet another embodiment of a separation medium with the second layer shown) after plasma separation from whole bovine blood in accordance with the present disclosure.

[0037] FIG. 18D is a photo of the bottom view of still another embodiment of a separation medium with the second layer shown) after plasma separation from whole bovine blood in accordance with the present disclosure.

[0038] FIG. 19 is a photo of two embodiments of separation media (top and bottom of photo) after plasma separation from whole human blood in accordance with the present disclosure.

[0039] FIG. 20 is a photo of a drop of whole blood completely absorbed by the first layer of a separation medium by the fourth second of testing in accordance with the present disclosure.

[0040] FIG. 21 is a photo of a second layer of a test medium, showing that by the eleventh second of testing, plasma had begun to separate sufficiently from 10 pL of whole blood in accordance with the present disclosure.

[0041] FIG. 22A is a photo of an embodiment of a separation media test trip cut to 0.4mm thickness x 5mm width x 20mm length in accordance with the present disclosure.

[0042] FIG. 22B is a photo of an embodiment of a plasma transfer membrane cut to 0.17mm thickness x 5mm width x 30mm length in accordance with the present disclosure.

[0043] FIG. 23 A is a photo of an embodiment of a separation medium test strip (left) coupled with an embodiment of a plasma transfer membrane (right) in accordance with the present disclosure.

[0044] FIG. 23B is a photo showing that the separation medium test strip (left) and plasma transfer membrane (right) of FIG. 23 A were assembled with an overlap.

[0045] FIG. 23C is a photo showing the components of FIG. 23B, with a 50g calibrated weight placed on top of the components at the point of overlap.

[0046] FIG. 24 is a photo of an embodiment of a plasma transfer membrane fully saturated with plasma in accordance with the present disclosure.

[0047] FIG. 25A is a photo of a separation medium test strip (left) and a plasma transfer membrane (right) in ambient fluorescent light in accordance with the present disclosure. [0048] FIG. 25B is a photo of the separation medium test strip (left) and plasma transfer membrane (right) of FIG. 25 A under UV light for easier visualization in accordance with the present disclosure.

[0049] FIG. 26A is a photo showing the plasma transferred onto an embodiment of a plasma transfer membrane from 10 pL bovine whole blood in accordance with the present disclosure. [0050] FIG. 26B is a photo of an embodiment of a separation medium test strip (bottom) and plasma transfer membrane (top) after the separation of 20pL of human whole blood in accordance with the present disclosure.

[0051] FIG. 26C is a photo of the length of the plasma-soaked section of a plasma transfer membrane measured in accordance with the present disclosure.

[0052] FIG. 27A is a schematic illustration of the serial dilution preparation used for UV-Vis absorbance analysis in accordance with the present disclosure.

[0053] FIG. 27B is a photo of a serial dilution from whole bovine blood, 30-40% het, completed according to the schematic illustration in FIG. 27A.

[0054] FIG. 27C is a photo of a serial dilution from pure bovine plasma completed according to the schematic illustration in FIG. 27A.

[0055] FIG. 28 is a photo of (left) the plasma-containing section and red blood cell-containing section of an embodiment of a separation medium after being cut, and (right) the plasmacontaining section and the red blood cell -containing section after being further cut into small pieces and inserted into a separate ImL low protein binding microcentrifuge tube in accordance with the present disclosure.

[0056] FIG. 29A is a UV-Vis spectrum of whole bovine blood, 30-40% het in accordance with the present disclosure.

[0057] FIG. 29B is a UV-Vis spectrum of whole bovine blood, 30-40% het after first dilution in accordance with the present disclosure.

[0058] FIG. 29C is a UV-Vis spectrum of whole bovine blood, 30-40% het after second dilution in accordance with the present disclosure.

[0059] FIG. 29D shows the four separate UV-Vis spectra of whole blood and 3 of the serial dilutions, where the labels DI, D2 and D3 represent serial dilution 1, serial dilution 2, and serial dilution 3, respectively, in accordance with the present disclosure.

[0060] FIG. 30A shows the UV-Vis spectrum of pure bovine plasma in K2EDTA in accordance with the present disclosure.

[0061] FIG 30B shows the UV-Vis spectra of pure plasma and all dilutions overlayed in accordance with the present disclosure. [0062] FIG. 31 A is a UV-Vis spectra overlay of the red blood cell -containing section (marked “red section elution”) and the plasma-containing section (marked “yellow section elution”) of an embodiment of separation media after separation of human whole blood in accordance with the present disclosure.

[0063] FIG. 3 IB is a UV-Vis spectra overlay of each plasma-containing section (marked “yellow section”) (N=5) in accordance with the present disclosure.

[0064] FIG. 31C is a UV-Vis spectra overlay of each red blood cell-containing section (marked “red section”) (N=5) in accordance with the present disclosure.

[0065] FIG. 32A is a photo showing a test strip assembly in accordance with the present disclosure.

[0066] FIG. 32B is a photo showing a low protein binding microcentrifuge tube containing a portion of a plasma transfer membrane as well as PBS buffer in accordance with the present disclosure.

[0067] FIG. 33 is a standards calibration curve of IgG constructed by linear regression using six standards of known concentrations in accordance with the present disclosure.

[0068] FIG. 34 is a representative UV-Vis curve of an elution sample with IgG present in accordance with the present disclosure.

[0069] FIG. 35 is a boxplot graph comparing the IgG recovery from the separation medium described herein (marked “Plasma Separation Product”), the positive control polyamide test strip (marked “Positive Control”), and a third-party product (marked “Competitor’s Product”), in accordance with the present disclosure.

[0070] FIG. 36A is a photo of the competitor products (1, 2, and 3) and an embodiment of a separation medium described herein (bottom), 2 minutes and 38 seconds after application of 10 pL of human whole blood, het 40-50% to the left-most side of each product in accordance with the present disclosure.

[0071] FIG. 36B is a photo of the samples of FIG. 36A, imaged under UV light for improved visibility.

DETAILED DESCRIPTION

[0072] This disclosure is not limited to the particular systems, devices, media, and methods described, as these may vary. The terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the disclosure. [0073] The following terms shall have, for the purposes of this application, the respective meanings set forth below. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Nothing in this disclosure is to be construed as an admission that the embodiments described in this disclosure are not entitled to antedate such disclosure by virtue of prior invention.

[0074] As used herein, the singular forms “a,” “an,” and “the” include plural references, unless the context clearly dictates otherwise. Thus, for example, reference to a “fiber” is a reference to one or more fibers and equivalents thereof known to those skilled in the art, and so forth.

[0075] As used herein, the term “consists of’ or “consisting of’ means that the device, medium, or method includes only the elements, steps, or ingredients specifically recited in the particular claimed embodiment or claim.

[0076] In embodiments or claims where the term “comprising” is used as the transition phrase, such embodiments can also be envisioned with replacement of the term “comprising” with the terms “consisting of’ or “consisting essentially of.”

[0077] As used herein, the term “fluid” means a liquid. Fluids include, but are not limited to, water, aqueous fluids, and bodily fluids such as urine, blood, drainage fluid from a wound, saliva, and combinations thereof.

[0078] As discussed herein separation media capable of separating serum and/or plasma from whole blood in a faster, more efficient, more user-friendly manner, with lower protein binding and high serum and/or plasma transfer efficiency, would be advantageous. In particular, there exists a need for separation media capable of separating serum and/or plasma from whole blood without the need for centrifugation.

[0079] Such a separation medium could be used, for example, for rapid sample intake, with fast absorbency and minimal blood pooling. Such a separation medium would also reduce or eliminate the need for any post-treatments to achieve plasma separation, and would reduce hemolysis and red blood cell leakage, leading to increased assay sensitivity. Such a separation medium could also include a customizable bed volume to accommodate a range of sample volumes and would comprise robust, readily available materials for efficient manufacturing. Such a separation medium could provide lower protein binding (i.e., fewer proteins bound to the separation medium), thereby enabling the separation media to be utilized for the separation of an antibody, enzyme, antigen, biomarker or any other protein present in whole blood, and thus making it suitable for a range of diagnostic tests. Such a separation medium could also facilitate high serum and/or plasma transfer efficiency onto another medium, making it optimal for diagnostic tests. [0080] In some embodiments, the separation medium may comprise the first layer, the second layer, or a combination thereof, as described herein. In an embodiment, a separation medium may comprise a first layer and a second layer.

[0081] In some embodiments, the first layer may comprise a porous fiber matrix. In certain embodiments, the porous fiber matrix may comprise one or more fibers. In some embodiments, the fibers may have an average diameter from about 3 pm to about 40 pm. The fibers may have an average diameter of, for example, about 3 pm, about 4 pm, about 5 pm, about 6 pm, about 7 pm, about 8 pm, about 9 pm, about 10 pm, about 11 pm, about 12 pm, about 13 pm, about 14 pm, about 15 pm, about 16 pm, about 17 pm, about 18 pm, about 19 pm, about 20 pm, about 21 pm, about 22 pm, about 23 pm, about 24 pm, about 25 pm, about 26 pm, about 27 pm, about 28 pm, about 29 pm, about 30 pm, about 31 pm, about 32 pm, about 33 pm, about 34 pm, about 35 pm, about 36 pm, about 37 pm, about 38 pm, about 39 pm, about 40 pm, or any range between any two of these values, including endpoints.

[0082] In an embodiment, the fibers of the first layer may be bound fibers. In some embodiments, the fibers may comprise one or more polymers. In an embodiment, the polymers may comprise one or more thermoplastic polymers. The polymers may include, for example, hydrocarbon resins, polyethylene, polypropylene, polyvinyl chloride, polyester, polyethylene glycol, propylene, polyamides, ethylene terephthalate, butylene terephthalate, ethylene, nylon, derivatives thereof, co-polymers thereof, or combinations thereof.

[0083] In certain embodiments, the porous fiber matrix may comprise bicomponent fibers. In some embodiments, the bicomponent fibers may have a core-sheath configuration. In an embodiment, the core-sheath configuration may comprise a core material selected from the group consisting of polyester, polypropylene, and combinations thereof. In an embodiment, the core-sheath configuration may further comprise a sheath material comprising polyester, nylon, polyolefins, and combinations thereof.

[0084] In some embodiments, the first layer may have a thickness from about 200 pm to about 2,000 pm. The first layer may have a thickness of, for example, about 200 pm, about 250 pm, about 300 pm, about 350 pm, about 400 pm, about 450 pm, about 500 pm, about 550 pm, about 600 pm, about 650 pm, about 700 pm, about 750 pm, about 800 pm, about 850 pm, about 900 pm, about 950 pm, about 1,000 pm, about 1,050 pm, about 1,100 pm, about 1,150 pm, about 1,200 pm, about 1,250 pm, about 1,300 pm, about 1,350 pm, about 1,400 pm, about 1,450 pm, about 1,500 pm, about 1,550 pm, about 1,600 pm, about 1,650 pm, about 1,700 pm, about 1,750 pm, about 1,800 pm, about 1,850 pm, about 1,900 pm, about 1,950 pm, about 2,000 pm, or any range between any two of these values, including endpoints. In an embodiment, the first layer may have a thickness of about 500 pm.

[0085] In some embodiments, the porous fiber matrix described herein may have a minimum pore size (i.e., pore diameter) from about 1 pm to about 40 pm. The porous fiber matrix may have a minimum pore size of, for example, about 1 pm, about 2 pm, about 3 pm, about 4 pm, about 5 pm, about 6 pm, about 7 pm, about 8 pm, about 9 pm, about 10 pm, about 11 pm, about 12 pm, about 13 pm, about 14 pm, about 15 pm, about 16 pm, about 17 pm, about 18 pm, about 19 pm, about 20 pm, about 21 pm, about 22 pm, about 23 pm, about 24 pm, about 25 pm, about 26 pm, about 27 pm, about 28 pm, about 29 pm, about 30 pm, about 31 pm, about 32 pm, about 33 pm, about 34 pm, about 35 pm, about 36 pm, about 37 pm, about 38 pm, about 39 pm, about 40 pm, or any range between any two of these values, including endpoints. In an embodiment, the minimum pore size may be measured using porosimetry testing.

[0086] In some embodiments, the porous fiber matrix described herein may have a maximum pore size (i.e., pore diameter) from about 15 pm to about 200 pm. The porous fiber matrix may have a maximum pore size of, for example, about 15 pm, about 20 pm, about 25 pm, about 30 pm, about 35 pm, about 40 pm, about 45 pm, about 50 pm, about 55 pm, about 60 pm, about 65 pm, about 70 pm, about 75 pm, about 80 pm, about 85 pm, about 90 pm, about 95 pm, about 100 pm, about 105 pm, about 110 pm, about 115 pm, about 120 pm, about 125 pm, about 130 pm, about 135 pm, about 140 pm, about 145 pm, about 150 pm, about 155 pm, about 160 pm, about 165 pm, about 170 pm, about 175 pm, about 180 pm, about 185 pm, about 190 pm, about 195 pm, about 200 pm, or any range between any two of these values, including endpoints. In an embodiment, the maximum pore size may be measured using porosimetry testing.

[0087] In certain embodiments, the porous fiber matrix described herein may have a porosity from about 40% to about 90%. The porosity may be, for example, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or any range between any two of these values, including endpoints. In an embodiment, the porosity may be a theoretical (e.g., calculated or approximated) porosity based on the specific gravity of the polymer or polymers that comprise the porous fiber matrix.

[0088] In some embodiments, the porous fiber matrix described herein may have theoretical (e.g., calculated or approximated) void volume from about 30% to about 90%. The porosity may be, for example, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or any range between any two of these values, including endpoints.

[0089] In an embodiment, the first layer may have a property that varies in a direction, as described herein. In some embodiments, the property of the first layer that may vary in a direction may be, for example, a density, a pore size, a pore volume, a fiber diameter, or a combination thereof. The direction of variation of the property of the first layer may be, for example, a length direction (i.e., the property varies laterally along the first layer), a width direction (i.e., the property varies along the width of the first layer), a height direction (i.e., the property varies vertically along the first layer), or a combination thereof.

[0090] In some embodiments, the first layer may further comprise an agglomeration medium. In an embodiment, the agglomeration medium may comprise a red blood cell agglomeration medium. In some embodiments, the agglomeration medium may comprise polyelectrolytes, polylysines, lectin, chitosan, water-soluble polymers, or any combination thereof.

[0091] In certain embodiments, the first layer may further comprise a blood cell-stabilizing medium selected from the group consisting of polyols, polyelectrolytes, non-ionic surfactants (non-limiting examples include PLURONIC, PLURONIC F-68, Tween, and the like), water- soluble polymers, and combinations thereof.

[0092] FIG. 1 is an optical microscopy image of an embodiment of a first layer comprising a porous fiber matrix in accordance with the present disclosure. FIG. 1 includes a 50 pm scale bar in the lower right corner. FIG. 2 is a photo of an embodiment of a first layer comprising a porous fiber matrix in accordance with the present disclosure. The porous fiber matrix of FIG. 2 comprises a flat sheet.

[0093] Table 1 details certain characteristics of sample embodiments of a porous fiber matrix as described herein:

Table 1. [0094] In some embodiments, the second layer of the separation medium may comprise a polymer membrane. In some embodiments, the polymer membrane may comprise a polymer. The polymer may be, for example, polysulfone, derivatives thereof, copolymers thereof, or a combination thereof. In an embodiment, the polymer membrane comprises polysulfone. In some embodiments, the second layer may further comprise at least one hydrophilic additive.

[0095] In some embodiments, the second layer may have a thickness from about 90 pm to about 375 pm. The second layer may have a thickness of, for example, about 90 pm, about 100 pm, about 125 pm, about 150 pm, about 175 pm, about 200 pm, about 225 pm, about 250 pm, about 275 pm, about 300 pm, about 325 pm, about 350 pm, about 375 pm, or any range between any two of these values, including endpoints. In an embodiment, the second layer may have a thickness of about 150 pm.

[0096] In some embodiments, the second layer may have an average pore size (i.e., pore diameter) from about 0.03 pm to about 2 pm. second layer may have an average pore size of, for example, about 0.03 pm, about 0.05 pm, about 0.1 pm, about 0.2 pm, about 0.3 pm, about 0.4 pm, about 0.5 pm, about 0.6 pm, about 0.7 pm, about 0.8 pm, about 0.9 pm, about 1 pm, about 1.1 pm, about 1.2 pm, about 1.3 pm, about 1.4 pm, about 1.5 pm, about 1.6 pm, about 1.7 pm, about 1.8 pm, about 1.9 pm, about 2 pm, or any range between any two of these values, including endpoints.

[0097] In certain embodiments, the second layer may have a density from about 0.08 g/cc to about 0.2 g/cc. The second layer may have a density of, for example, about 0.08 g/cc, about 0.09 g/cc, about 0.1 g/cc, about 0.11 g/cc, about 0.12 g/cc, about 0.13 g/cc, about 0.14 g/cc, about 0.15 g/cc, about 0.16 g/cc, about 0.17 g/cc, about 0.18 g/cc, about 0.19 g/cc, about 0.2 g/cc, or any range between any two of these values, including endpoints.

[0098] In an embodiment, the second layer may have a property that varies in a direction, as described herein. This variation of one or more properties in one or more directions, as described herein, may be referred to as “asymmetric,” such that the second layer having a property that varies in a direction as described herein may be referred to as an “asymmetric” polymer membrane. In some embodiments, the property of the second layer that may vary in a direction may be, for example, a density, a pore size, a pore volume, a thickness, or a combination thereof. The direction of variation of the property of the second layer may be, for example, a length direction (i.e., the property varies laterally along the second layer), a width direction (i.e., the property varies along the width of the second layer), a height direction (i.e., the property varies vertically along the second layer), or a combination thereof. [0099] FIG. 3 is a photo of an embodiment of a second layer comprising a polymer membrane in accordance with the present disclosure. In the photo, the second layer is secured to a surface using paper clips, which are not a component of the embodiment of the separation medium.

[0100] In an embodiment, the first layer of the separation medium may be fixedly coupled to the second layer. In some embodiments, the first layer may be fixedly coupled to the second layer by an adhesive, a thermal bond, or a combination thereof. In an embodiment, the adhesive may comprise a low heat activated polymeric web adhesive. In an embodiment, the first layer may be fixedly coupled to the second layer via thermal bonding. In an embodiment, the first layer may be fixedly coupled to the second layer via “point bonding.”

[0101] FIG. 4 is a schematic representation of a side view of a separation medium in accordance with the present disclosure. In FIG. 4, the separation medium 400 comprises a first layer 410 comprising a porous fiber matrix, and a second layer 420 comprising a polymer membrane, as described herein. The first layer 410 has a property, such as a density, a pore size, a pore volume, a fiber diameter, or a combination thereof, that varies in a direction, such as a length direction (i.e., the property varies laterally along the first layer 410), a width direction (i.e., the property varies along the width of the first layer 410), or a height direction (i.e., the property varies vertically along the first layer 410), as described herein. In addition, the second layer 420 has a property, such as a density, a pore size, a pore volume, a fiber diameter, or a combination thereof, that varies in a direction, such as a length direction (i.e., the property varies laterally along the second layer 420), a width direction (i.e., the property varies along the width of the second layer 420), or a height direction (i.e., the property varies vertically along the second layer 420), as described herein. In the embodiment shown in FIG. 4, the first layer 410 of the separation medium 400 is fixedly coupled to the second layer 420 by a component 430 that comprises an adhesive, a thermal bond, or a combination thereof. In the embodiment shown in FIG. 4, the first layer 410 has a thickness of about 500 pm, and the second layer 420 has a thickness of about 150 pm. In the separation medium embodiment shown in FIG. 4, the first layer 410 has a length smaller than that of the second layer 420, thereby creating a separation medium 400 having a non-uniform or “staggered” configuration. [0102] In an embodiment, a separation medium as described herein may have an input volume from about 30 pL to about 40 pL. In certain embodiments, the input volume may be customized for a range of sample volumes, for example by varying one or more dimensions of the first layer, varying one or more dimensions of the second layer, or a combination thereof.

[0103] In some embodiments, a separation medium as described herein may have a separation time (e.g., a plasma or serum separation time) from about 20 seconds to about 90 seconds. The separation time may be, for example, about 20 seconds, about 30 seconds, about 40 seconds, about 50 seconds, about 60 seconds, about 70 seconds, about 80 seconds, about 90 seconds, or any range between any two of these values, including endpoints. In some embodiments, the separation media described herein may result in no visible hemolysis during or after the separation time. In certain embodiments, the separation media described herein may result in no visible leakage of blood cells (e.g., red blood cells) during the separation time. In an embodiment, the separation medium described herein may absorb whole blood into the top layer within about 1 second to about 2 seconds. In an embodiment, the separation medium described herein may begin separating the plasma from the whole blood within about 10 seconds.

[0104] In certain embodiments, a separation medium as described herein may have a recovery volume (e.g., a plasma recovery volume or a serum recovery volume) of greater than 50%. The recovery volume may be, for example, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, or any range between any two of these values, including endpoints.

[0105] In some embodiments, a separation medium as described herein may have a plasma separation efficiency ranging from about 55% to about 95%. The separation efficiency may be, for example, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or any range between any two of these values, including endpoints.

[0106] In some embodiments, a separation medium as described herein may have a capacity to hold a whole blood volume from about 10 pL to about 200 pL. The separation medium may have the capacity to hold a whole blood volume of, for example, about 10 pL, about 20 pL, about 30 pL, about 40 pL, about 50 pL, about 60 pL, about 70 pL, about 80 pL, about 90 pL, about 100 pL, about 110 pL, about 120 pL, about 130 pL, about 140 pL, about 150 pL, about 160 pL, about 170 pL, about 180 pL, about 190 pL, about 200 pL, or any range between any two of these values, including endpoints.

[0107] FIG. 5A is a top view photo of an embodiment of a separation medium in accordance with the present disclosure. In FIG. 5A, the separation medium 500 comprises a first layer 510 comprising a porous fiber matrix, and a second layer 520 comprising a polymer membrane, as described herein. The first layer 510 has a property, such as a density, a pore size, a pore volume, a fiber diameter, or a combination thereof, that varies in a direction, such as a length direction (i.e., the property varies laterally along the first layer 510), a width direction (i.e., the property varies along the width of the first layer 510), or a height direction (i.e., the property varies vertically along the first layer 510), as described herein. In addition, the second layer 520 has a property, such as a density, a pore size, a pore volume, a fiber diameter, or a combination thereof, that varies in a direction, such as a length direction (i.e., the property varies laterally along the second layer 520), a width direction (i.e., the property varies along the width of the second layer 520), or a height direction (i.e., the property varies vertically along the second layer 520), as described herein. FIG. 5B is an alternative view photo of the embodiment of the separation medium shown in FIG. 5A in accordance with the present disclosure. FIG. 5C is a side view photo of the embodiment of the separation medium shown in FIG. 5 A in accordance with the present disclosure.

[0108] In some embodiments, the separation medium described herein may be configured to draw a fluid from a first end of the separation medium toward a second end of the separation medium. In an embodiment, the fluid may be drawn from the first end toward the second end via capillary action. In certain embodiments, the fluid may comprise whole blood. In some embodiments, the separation medium may be configured to draw the whole blood from the first end of the separation medium to the second end of the separation medium, thereby: capturing a first component of the whole blood in a first portion of the separation medium; optionally, capturing a second component of the whole blood in a second portion of the separation medium; and separating the first component of the whole blood from the second component of the whole blood. In some embodiments, the first component of the whole blood may comprise a red blood cell, a white blood cell, a platelet, or a combination thereof. In certain embodiments, the second component of the whole blood may comprise serum or plasma.

[0109] In an embodiment, a method of using a separation medium as described herein may comprise exposing a first end of the separation medium to a fluid comprising whole blood. In some embodiments, the method may further comprise drawing the fluid from the first end of the separation medium toward a second end of the separation medium. In certain embodiments, the fluid may be drawn via capillary action, as described herein.

[0110] In some embodiments, drawing the fluid may thereby capture a first component of the whole blood in a first portion of the separation medium, as described herein. In some embodiments, drawing the fluid may thereby also separate the first component of the whole blood from a second component of the whole blood, as described herein. In certain embodiments, the first component of the whole blood may comprise red blood cells, white blood cells, platelets, or a combination thereof, as described herein. In some embodiments, the second component of the whole blood may comprise serum or plasma. In an embodiment, the method does not include centrifugation. In certain embodiments, the method does not lead to red blood cell breakthrough, hemolysis, or a combination thereof, in the sample collected and separated.

[OHl] In some embodiments, a device may comprise an embodiment of a separation medium as described herein. In an embodiment, the device may further comprise a sample well. In certain embodiments, the sample well is configured to receive a sample of whole blood. The sample of whole blood may comprise one drop or several drops.

[0112] In certain embodiments, the device may be configured to be used in a test that requires the separation of plasma, serum, or a combination thereof from whole blood. The test may include, for example, an alanine aminotransferase (ALT) test, an aspartate aminotransferase (AST) test, a blood urea nitrogen (BUN) test, a C-reactive protein (CRP) test, a creatinine test, a direct low density lipoprotein (LDL) test, a lipid panel test, a vitamin D test, a thyroid stimulating hormone (TSH) test, a serum nicotine test, a cortisol test, a malaria test, a dengue test, a human immunodeficiency virus (HIV) test, a hepatitis C virus (HCV) test, and combinations thereof. In an embodiment, the device is a lateral flow assay device. In some embodiments, the device may be configured for use as a rapid diagnostic test (RDT) device. Such RDT devices may be used to test a subject for, for example, HbsAg, dengue, and malaria, and the like. In certain embodiments, the device may be configured for use at a point of care, such as a hospital or healthcare provider’s office.

[0113] Embodiment 1 : A separation medium comprising: a first layer comprising a porous fiber matrix; and a second layer comprising a polymer membrane, the second layer having a property that varies in a direction; wherein the first layer is fixedly coupled to the second layer. [0114] Embodiment 2: The separation medium of embodiment 1, wherein the porous fibrous matrix comprises bicomponent fibers.

[0115] Embodiment 3: The separation medium of embodiment 2, wherein the bicomponent fibers have a core-sheath configuration.

[0116] Embodiment 4: The separation medium of embodiment 3, wherein the core-sheath configuration comprises: a core material selected from the group consisting of polyester, polypropylene, polybutylene ester, and combinations thereof; and a sheath material comprising polyester.

[0117] Embodiment 5: The separation medium of any of embodiments 1-4, wherein the first layer has a thickness from about 200 pm to about 2,000 pm.

[0118] Embodiment 6: The separation medium of any of embodiments 1-5, wherein the porous fiber matrix comprises a plurality of fibers having an average diameter from about 3 pm to about 40 pm. [0119] Embodiment 7: The separation medium of any of embodiments 1-6, wherein the porous fiber matrix comprises a plurality of fibers, the plurality of fibers comprising a polymer selected from the group consisting of polyethylene, polypropylene, polyvinyl chloride, polyester, polyethylene glycol, propylene, polyamides, ethylene terephthalate, butylene terephthalate, ethylene, nylon, derivatives thereof, copolymers thereof, and combinations thereof.

[0120] Embodiment 8: The separation medium of any of embodiments 1-7, wherein the porous fiber matrix has a minimum pore size from about 1 pm to about 40 pm.

[0121] Embodiment 9: The separation medium of any of embodiments 1-8, wherein the porous fiber matrix has a maximum pore size from about 15 pm to about 115 pm.

[0122] Embodiment 10: The separation medium of any of embodiments 1-9, wherein the porous fiber matrix has a porosity from about 50% to about 90%.

[0123] Embodiment 11 : The separation medium of any of embodiments 1-10, wherein the first layer further comprises an agglomeration medium selected from the group consisting of polyelectrolytes, polylysines, lectin, chitosan, water-soluble polymers, and combinations thereof.

[0124] Embodiment 12: The separation medium of any of embodiments 1-11, wherein the first layer further comprises a blood cell-stabilizing medium selected from the group consisting of polyols, polyelectrolytes, non-ionic surfactants, water-soluble polymers, and combinations thereof.

[0125] Embodiment 13: The separation medium of any of embodiments 1-12, wherein the polymer membrane comprises a polymer selected from the group consisting of polysulfone, derivatives thereof, and copolymers thereof.

[0126] Embodiment 14: The separation medium of any of embodiments 1-13, wherein the second layer further comprises at least one hydrophilic additive.

[0127] Embodiment 15: The separation medium of any of embodiments 1-14, wherein the second layer has a thickness from about 90 pm to about 300 pm.

[0128] Embodiment 16: The separation medium of any of embodiments 1-15, wherein the second layer has an average pore size from about 0.03 pm to about 2 pm.

[0129] Embodiment 17: The separation medium of any of embodiments 1-16, wherein the second layer has a density from about 0.08 g/cc to about 0.2 g/cc.

[0130] Embodiment 18: The separation medium of any of embodiments 1-17, wherein the property is selected from the group consisting of a density, a pore size, a pore volume, a thickness, and combinations thereof. [0131] Embodiment 19: The separation medium of any of embodiments 1-18, wherein the direction is selected from the group consisting of a length direction, a width direction, a height direction, and combinations thereof.

[0132] Embodiment 20: The separation medium of any of embodiments 1-19, wherein the first layer has a second property that varies in a second direction.

[0133] Embodiment 21 : The separation medium of embodiment 20, wherein the second property is selected from the group consisting of a density, a pore size, a pore volume, a fiber diameter, and combinations thereof.

[0134] Embodiment 22: The separation medium of embodiment 20 or 21, wherein the second direction is selected from the group consisting of a length direction, a width direction, a height direction, and combinations thereof.

[0135] Embodiment 23: The separation medium of any of embodiments 1-22, wherein the first layer is fixedly coupled to the second layer by an adhesive, a thermal bond, or a combination thereof.

[0136] Embodiment 24: The separation medium of embodiment 23, wherein the adhesive comprises a low heat activated polymeric web adhesive.

[0137] Embodiment 25: The separation medium of any of embodiments 1-24, wherein the separation medium is configured to draw a fluid from a first end of the separation medium toward a second end of the separation medium via capillary action.

[0138] Embodiment 26: The separation medium of embodiment 25, wherein the fluid comprises whole blood, and wherein the separation medium is configured to draw the whole blood from the first end of the separation medium to the second end of the separation medium, thereby capturing a first component of the whole blood in a first portion of the separation medium and separating the first component of the whole blood from a second component of the whole blood.

[0139] Embodiment 27: The separation medium of embodiment 26, wherein the first component of the whole blood is selected from the group consisting of a red blood cell, a white blood cell, a platelet, and a combination thereof.

[0140] Embodiment 28: The separation medium of embodiment 26 or 27, wherein the second component of the whole blood is selected from the group consisting of serum and plasma.

[0141] Embodiment 29: A method of using the separation medium of any of embodiments 1- 28, the method comprising: exposing a first end of the separation medium to a fluid comprising whole blood; and drawing the fluid from the first end of the separation medium toward a second end of the separation medium via capillary action, thereby: capturing a first component of the whole blood in a first portion of the separation medium, and separating the first component of the whole blood from a second component of the whole blood.

[0142] Embodiment 30: The method of embodiment 29, wherein the first component of the whole blood is selected from the group consisting of a red blood cell, a white blood cell, a platelet, and a combination thereof.

[0143] Embodiment 31 : The method of embodiment 29 or 30, wherein the second component of the whole blood is selected from the group consisting of serum and plasma.

[0144] Embodiment 32: The method of any of embodiments 29-31, wherein the method does not include centrifugation.

[0145] Embodiment 33: A device comprising the separation medium of any of embodiments 1-28, and a sample well.

[0146] Embodiment 34: The device of embodiment 33, wherein the sample well is configured to receive a sample of whole blood.

[0147] Embodiment 35: The device of embodiment 33 or 34, wherein the device is configured to be used in a test that requires the separation of plasma, serum, or a combination thereof from whole blood.

[0148] Embodiment 36: The device of embodiment 35, wherein the test is selected from the group consisting of an alanine aminotransferase (ALT) test, an aspartate aminotransferase (AST) test, a blood urea nitrogen (BUN) test, a C-reactive protein (CRP) test, a creatinine test, a direct low density lipoprotein (LDL) test, a lipid panel test, a vitamin D test, a thyroid stimulating hormone (TSH) test, a serum nicotine test, a cortisol test, a malaria test, a dengue test, a human immunodeficiency virus (HIV) test, a hepatitis C virus (HCV) test, and combinations thereof.

[0149] Embodiment 37: The device of any of embodiments 33-36, wherein the device is a lateral flow assay device.

EXAMPLES

Example 1 : Method of using a separation medium

[0150] FIG. 6A is a photo of an embodiment of a method of using a separation medium for about 2 minutes, as described herein. In FIG. 6A, a first end 640A of a first separation medium 600A was exposed to a first sample of fluid comprising whole blood (40 pL of bovine blood in EDTA), and the fluid was drawn via capillary action from the first end 640A toward a second end 650A of the first separation medium 600A, as described herein. By drawing the fluid via capillary action from the first end 640A toward the second end 650A, a first component of the whole blood was captured in a first portion 660A of the first separation medium 600A, and a second component of the whole blood was captured in a second portion 670A of the separation medium 600A. Accordingly, the first component of the whole blood was separated from the second component of the whole blood.

[0151] Also in FIG. 6A, a first end 640B of a second separation medium 600B was exposed to a second sample of fluid comprising whole blood (40 pL of bovine blood in EDTA), and the fluid was drawn via capillary action from the first end 640B toward a second end 650B of the second separation medium 600B, as described herein. By drawing the fluid via capillary action from the first end 640B toward the second end 650B, a first component of the whole blood was captured in a first portion 660B of the second separation medium 600B, and a second component of the whole blood was captured in a second portion 670B of the second separation medium 600B. Accordingly, the first component of the whole blood was separated from the second component of the whole blood.

[0152] In FIG. 6A, the first component of the whole blood in each sample comprised red blood cells, white blood cells, platelets, or a combination thereof, as described herein; and the second component of the whole blood in each sample comprised serum or plasma. In FIG. 6A, the method was carried out on the first separation medium 600A and the second separation medium 600B for 2 minutes and 14 seconds (i.e., about 2 minutes), as shown on the digital timer on the left-hand side of the photo. In FIG. 6B, the method was carried out on the first separation medium 600A and the second separation medium 600B for 4 minutes and 41 seconds (i.e., about 5 minutes), as shown on the digital timer on the left-hand side of the photo.

Example 2: Performance of a separation medium compared to traditional media

[0153] FIG. 7 is a photo of a comparison of three samples exposed to a fluid comprising whole blood. The top row shows Sample 1, which comprised commercially available untreated asymmetrical polysulfone (5mm width x 35mm length), exposed to a fluid comprising whole blood (30 pL of bovine blood having a hematocrit (HCT) of about 40%), after 20 seconds (left), 120 seconds (center), and 180 seconds (right). The middle row of FIG. 7 shows Sample 2, which comprised commercially available treated glass fiber (5mm width x 35mm length), exposed to a fluid comprising whole blood (30 pL of bovine blood having a hematocrit (HCT) of about 40%), after 20 seconds (left), 120 seconds (center), and 180 seconds (right). The bottom row of FIG. 7 shows Sample 3, which was an embodiment of a separation medium (5mm width x 35mm length) comprising a first layer and a second layer as described herein, exposed to a fluid comprising whole blood (30 pL of bovine blood having a hematocrit (HCT) of about 40%), after 20 seconds (left), 120 seconds (center), and 180 seconds (right).

[0154] The results of this experiment demonstrated that the separation media described herein could be used for rapid sample intake, with fast absorbency and minimal blood pooling. This experiment also demonstrated that the separation media described herein may reduce or eliminate the need for any post-treatments to achieve plasma separation, and may reduce hemolysis and red blood cell leakage, leading to increased assay sensitivity.

Example 3 : Material Construction and Characterization

Manufacturing process

[0155] In an experiment, a first layer comprising a porous meltblown fiber matrix was layered with a second layer comprising an asymmetric polymer membrane, with an adhesive layer between the first and second layers. The layers were laminated together to form a separation medium as described herein. The lamination was accomplished by using a low heatcompression process where heat was applied to the first and second layers along with compression. FIG. 8 is a schematic illustration of the process used to prepare the separation media used in this experiment. The first layer 810 comprised a porous meltblown fiber matrix having a thickness ranging from 200 to 800 pm. The second layer 820 comprised an asymmetric polymer membrane having a thickness of about 150 pm. The adhesive layer 830 comprised a thermally activated, low melting point PET adhesive. The adhesive layer 830 was positioned between the first layer 810 and the second layer 820, and the first layer 810, the second layer 820, and the adhesive layer 830 were laminated (denoted by arrow 840) using a low heat-compression process where heat and compression were applied to the first layer 810 and the second layer 820. The process resulted in a separation medium 850 comprising the first layer 810 and the second layer 820. The first layer 810 and the second layer 820 are not easily separable (i.e., not separable without damaging at least one of the first layer 810 and the second layer 820) in the separation medium 850. The adhesive layer 830 is not shown as a distinct layer in the separation medium 850. In the separation medium embodiment shown in FIG. 8, the first layer 810 has a length that is approximately equal to that of the second layer 820, thereby creating a separation medium 850 having a “uniform” configuration.

[0156] The separation medium was then treated with a treatment formulation to produce the final separation medium as described herein. To prepare the treatment formulation, 1% (w/v) solution of Ficoll was prepared by mixing approx. 0.5g of Ficoll in 50 mL DI water. Next, 1% (w/v) solution of Polyvinylpyrrolidone (PVP) by prepared by mixing approx. 0.5g of PVP (1,300,000 g/mol) in 50 mL DI water. Next, using a magnetic stir bar and a stir plate, a formulation was prepared using the following ratio: Alsever’s solution: 1% Ficoll: 1% PVP: 10% PLURONIC F-68 (a polyoxyethylene-polyoxypropylene block copolymer) = 3: 6: 2: 0.5. Each separation medium was cut into test articles with dimensions of 5mm W x 100mm L using a paper cutter. Next, the physical dimensions (thickness, width, length) and weight of each strip were measured using a slide gauge and a scale, respectively, and recorded. Each strip was placed in a labeled plastic compartment box with 12 dividers. Next, approx. 2 mL of the treatment formulation was poured into each compartment and left to sit for 20 min. Each strip was fully immersed in the treatment formulation. FIG. 9 is a photo of the experimental separation media undergoing dip treatment with the treatment formulation as described herein. [0157] After 20 min, each strip was carefully removed using tweezers, and then placed on a sieve tray, ensuring that none of the test strips contacted each other. FIG. 10 is a photo of the dip-treated separation media placed on a sieve tray to be dried. The sieve tray was placed on the top rack of a pre-heated oven with the temperature set to 50 °C. After 15 min, the sieve tray was removed using heat-resistant gloves, and the sieve tray was let to sit until it reached room temperature. The physical dimensions (length, width, thickness) and weight of each finally formed separation medium as described herein (sometimes referred to as a “strip”) were remeasured and recorded. In large-scale manufacturing, this dip treatment process could be used, or an alternative vacuum treatment process, or an alternative in-line/continuous treatment process at negative pressure or high pressure, could also be used.

Characterization

[0158] Pore diameter distribution and pore diameters of all samples were analyzed in a porosimetry instrument while the fiber diameters were measured and analyzed via optical microscopy. The physical dimensions such as the length, the width and the thickness of samples were gathered utilizing a slide gage. The weights of samples were obtained from a scale.

[0159] Characterization of the first layer:

[0160] The porosity of a first layer comprising a polymeric porous media with a tortuous pore structure as described herein is a function of the specific gravities of the polymers used and its weight. It is computed using Equations 1 and 2 below: dry weight of sample (g)

Density of sample = Equation 1 bulk volume of sample (cc) density of sample(g/cc)

Porosity of sample = 1 — Equation 2 specific gravity of polymers (g/cc)

[0161] where, specific gravity of polymers used in the present study ranged from 0.9 - 1.38 g/cc.

Table 2: Physical dimensions and theoretical pore volume of a variety of first layers comprising porous media tested in the present investigation (N=10/sample)

[0162] Pore diameter data were gathered using a porosimetry instrument and standardized test methods, such as ASTM D4404-01 or ASTM UOP578-02, may be followed to conduct pore diameter distribution testing. Table 3 below is a list of samples tested in the present investigation.

Table 3: Smallest, average, and largest pore diameter detected of a variety of samples tested during the present investigation (N=3/each)

[0163] Porous media such as the top layer described herein comprises a pore diameter distribution, which is common for media manufactured via nonwoven technologies. FIG. 11 is a pore diameter distribution graph for sample 18943 tested in this experiment.

[0164] Optical microscopy was conducted on the first layer of the separation medium embodiment reported herein, and the measured fiber diameters from a range of first layers tested during the investigation for the optimal plasma separation media are reported in table 4 (N=20).

Table 4: Fiber diameter of a list of samples tested during the present investigation (N=20/sample).

[0165] The measured fiber diameter in an exemplary first layer (sample ID 18943) tested falls under the “medium fiber diameter” range of the manufacturing process. Sample ID 18943, when laminated with the second layer, resulted in optimal plasma separation efficiencies with very high plasma yield from 10 pL of whole animal and human blood. These results are discussed in greater detail herein.

[0166] FIGS. 1 and 12A-12B are optical microscopy images of cross sections of top layers in the separation medium embodiments formed in this example. FIG. 12A demonstrates the presence of small fiber diameters in a top layer; FIG. 1 demonstrates the presence of medium fiber diameters in a top layer; and FIG. 12B demonstrates presence of achieve large fiber diameters in a top layer.

[0167] Characterization of the second layer:

[0168] A range of second layers comprising polysulfone membranes were tested in the present investigation, and the second layer selected to manufacture the separation media described in this experiment had a thickness of 360 ± 30 pm measured via a non-contact gage (laser beam method).

[0169] The pore diameter distribution for the second layer was analyzed via scanning electron microscopy (SEM). An asymmetric polysulfone membrane a “matte” side and a “shiny” side. FIG. 13 A is an SEM image of a matte side of an asymmetric poly sulfone membrane, and FIG. 13B is an SEM image of a shiny side of an asymmetric polysulfone membrane. As shown in FIG. 13 A, the matte side of the asymmetric polysulfone membranes used in this experiment had a more open pore structure at 12.11 ± 9.02 pm (N=12). As shown in FIG. 13 A, the shiny side of the asymmetric polysulfone membranes used in this experiment had a smaller pore diameter, at 1.04 ± 0.48 pm (N=12). FIG. 14 shows the individual data plot for the pore diameter of both matte and shiny sides of the second layers comprising polymer membranes used in this experiment.

[0170] In the present example, the matte side of the second layer was laminated to the first layer. To ensure that blood wicks passively from the first layer to the second layer, the pore diameter distribution in each layer in the embodiment was constructed to be sequentially arranged from large to small as the fluid travels from the first layer to the second layer.

Results

[0171] An important result from the present investigation was that none of the layers in the present embodiment (i.e., the first or second layers) individually were able to achieve plasma separation with high quality in 1 - 5 min time span. FIGS. 15A-15B are each photos demonstrating that blood separation testing yielded severe blood pooling on second layers alone, and FIG. 15C is a photo demonstrating that blood separation testing yielded no plasma separation on a first layer alone. In contrast, the porous separation media comprising first and second layers fixedly coupled to one another achieved quality plasma separation in a time period between about 10 seconds and about 12 minutes, further demonstrating the synergistic effect of the components of the porous separation media described herein and tested in this investigation.

Example 4: Plasma separation efficiency, separation time, and plasma yield

Methods

[0172] The separation media prepared as described in Example 3 were tested to determine plasma yield, plasma separation efficiency, plasma separation speed, plasma separation consistency, and plasma quality. Whole bovine blood was tested. The whole bovine blood as received contained Alsever’s solution (9.5 g/L Na-Citrate, 21 g/L Dextrose, 4.25 g/L NaCl, and 10 ml/L of 5.5% Citric Acid solution) at a 50:50 ratio of whole blood: Alsever’s Solution.

[0173] Before each experiment, the bovine blood bottle was removed from a refrigerator and placed in a chemical fume hood for approximately 1 hour in order for the blood to reach room temperature gradually. It was important to never heat the bottle for quicker thawing, which would lead to blood hemolysis.

[0174] A test strip was placed on a plexiglass stage, and 10 pL of whole blood was dispensed directly onto one end of the test strip using a pipettor. The timer was started as soon as blood was dispensed onto the test strip. Blood would begin to flow laterally (in the x-direction), and as it did, photos and observations were captured. The time taken by the embodiment to completely absorb the dispensed blood was recorded. Photos were taken when the blood drop was completely absorbed to the test strip, and at 1-2 min intervals afterwards. After 5 min, the timer was stopped, and the test strip was flipped so that the bottom of the test strip was now facing upwards. Using a razor blade and cut-resistant gloves, the test strip was cut at the intersection between the red blood cell -containing portion (appearing red) and the plasmacontaining portion (appearing yellow). Next, the test strip was cut at the intersection between the plasma-containing portion (appearing yellow) and the fluid-free portion (appearing white). Next, the length and the weights of the red color section and the yellow color section were measured individually using a ruler and a scale, respectively. FIG. 16A is a schematic representation of the boundary line definitions (i.e., intersections/cut lines) for a separation medium (i.e., test strip) 1600. Each separation medium 1600 was cut at the first intersection 1610 between the red blood cell-containing section 1620 and the plasma-containing section 1630. Each separation medium 1600 was also cut at the second intersection 1640 between the plasma-containing section 1630 and the fluid-free section 1650. FIG. 16B is a photo of an exemplary separation medium before the cuts illustrated in FIG. 16A were made. FIG. 16C is a photo of the exemplary separation medium of FIG. 16B after the cuts illustrated in FIG. 16A were made using a razor blade. FIG. 16D is a photo showing how an exemplary section (i.e., 1650) illustrated in FIG. 16A was measured after being cut.

[0175] The recorded lengths and weights of each cut section were used to calculate the plasma separation efficiency using the following equations: dry weight of test strip (g)

Density of test strip = Equation 3 bulk volume of test strip (cc) density of test article (g/cc)

Porosity of test strip = 1 Equation 4 specific gravity of polymers (g/cc)

[0176] where the specific gravity of polymers was 1.044 g/cc.

Bulk volume of plasma section = thickness X width X length (mm ³ ) Equation 5

Plasma Yield = porosity x bulk volume of plasma section x 80% (p.L) Equation 6

[0177] where 80% is the theoretical factor accounting for open pores.

Plasma Separation Efficiency = ^p ^ ^{asma yield} G ^L ) Equation 7 blood input volume ( .L)

[0178] The first assumption was made in Equation 4. Because a majority (i.e., > about 90%) of each separation medium comprises the first layer, the average of the specific gravities of the polymers in the porous pad was used to compute the porosity of the embodiment as a whole. The second assumption made was in Equation 6 to ensure that the plasma yield was not overestimated. In porous products with a tortuous pathway like the top layer of the separation media described herein, there often are closed pores within the pore structure. Because these closed pores are not able to absorb and hold any fluids, it is important to estimate the pore volume of the open pores. Generally, about 80% - 90% of a porous product with a tortuous pathway contains open pores. Therefore, the lower end of this range was used as the factor to account for the true pore volume.

[0179] Finally, plasma separation performance testing was conducted using human blood at hematocrit (het) of approximately 50-60% as received from the blood supplier. One of the major pain-points in blood cell separation without centrifugation is higher hematocrit levels, and the separation media described herein were challenged against higher hematocrit blood samples in order to validate a variety of applications such as blood tests in pregnant women, newborns, and other patient groups with higher hematocrit levels. All the plasma and blood samples for this study were ethically obtained and procured through Lampire Biological Laboratories (PA).

Results and Discussion

[0180] The plasma yield from whole bovine blood obtained ranged from 55% - 95% with an average of 71% and standard deviation of 15% (N=10). Table 5 below is representation of the plasma yield and efficiency calculation.

Table 5: Consolidated results of density, porosities, plasma yield and plasma separation efficiencies of 10 independent test strips.

[0181] FIG. 17 is a photo of a top view of a test strip 30 seconds after exposure to whole blood. The clear-colored section 1710 is completely saturated with liquid plasma. The assumption made in Equation 6 above would only hold if the test strip was completely saturated in plasma, which is demonstrated in FIG. 17.

[0182] The plasma separation from whole blood was observed from a bottom view (i.e., from observing the second layer of the separation medium) in the experiments described herein. FIGS. 18A-18D are photos of the bottom view of a separation medium (with the second layer shown) after plasma separation from whole bovine blood as described herein. Collectively, the whole bovine blood used in the samples shown in FIGS. 18A-18D had 30-40% het. In each of FIGS. 18A-18D, the plasma-containing section of each test strip (center) contains plasma separated from whole blood. It is visually clear how the plasma-containing section of each strip (center) is longer than the red blood cell-containing section (left), suggesting one could expect to collect plasma at greater than 60 - 70% efficiency. These separation media were also tested after they were aged under room conditions for 1 - 2 months, and the separation efficiencies fell within 55 - 95%, indicating that the separation media described herein exhibit no aging phenomena.

[0183] The plasma yield obtained from whole human blood using the separation media described herein was 52 ± 7% (N=10). FIG. 19 is a photo of two test strips (top and bottom) showing examples of plasma separation efficiency of human blood using the materials and methods described herein. [0184] When 10 pL of whole blood (whether bovine or human) was pipetted on top of the left end of a test strip, the whole blood was absorbed by the top layer of the separation medium (i.e., test strip) within 1 - 3 seconds. Photo capture by humans lagged behind this fast phenomenon. FIG. 20 is a photo of a drop of whole blood completely absorbed by the first layer of a separation medium as described herein by the fourth second of testing (as indicated by the timer on the left of the photo). Similarly, capturing the true plasma separation time for each tested separation medium suffered human limitations due to the fast nature with which plasma separation occurred in the test strips. FIG. 21 is a photo of a second layer of a test medium, showing that by the eleventh second of testing (as indicated by the timer on the left of the photo), plasma had begun to separate sufficiently from 10 pL of whole blood. Across the samples tested, plasma separation began at approximately 10 seconds with both bovine and human whole blood.

Example 5: Plasma Transfer Efficiency

Methods

[0185] Separation media prepared as described in Example 3 were used for plasma transfer efficiency testing. The separation media test trips were cut to 0.4mm thickness x 5mm width x 20mm length test strips as shown in FIG. 22A, while plasma transfer membranes were cut to dimensions of 0.17mm thickness ^x 5mm width x 30mm length as shown in FIG. 22B.

[0186] FIG. 23 A shows that each separation medium test strip (left) was coupled with a plasma transfer membrane (right). FIG. 23B shows that the separation medium test strip (left) and plasma transfer membrane (right) of FIG. 23A were assembled with an overlap. It was important to orient the two strips so that the second layer of the separation medium test strip was in contact with the dull side of the plasma transfer membrane. This is because poly sulfone membranes have asymmetric pore size gradients, and hence the directionality of the pore structure is important in the assembly. Afterward the components were assembled with the overlap as shown in FIG. 23B, a 50g calibrated weight was placed on top of the components at the point of overlap, as shown in FIG. 23 C, to ensure sufficient contact and compression at the site of the overlap so that the plasma could passively flow onto the membrane. Some level of compression was compulsory for plasma wicking to occur.

[0187] Next, the test was initiated by placing/dropping 10 pL or 20 pL of whole blood on each separation medium test strip. After 5 min, the timer was stopped, and the separation medium test strip and plasma transfer membrane were removed from the assembly. Because the plasma transfer membrane was fully saturated with plasma, using the measured dimensions of the membrane where the plasma was collected, the plasma volume transferred onto the membrane was calculated using Equation 8 below:

Plasma volume transferred = thickness x width x length (mm ³ ) Equation 8

[0188] where thickness, width and length are the dimensions of the membrane section where there was visual indication of plasma.

Plasma Transfer Efficiency = P^ vo _l umetransfer _r .d (mm») _x j _fl0 Equation 9

^J input blood volume (pL)

Results and Discussion

[0189] Once the separation medium test strip was removed, each plasma transfer membrane clearly showed that plasma was transferred without signs of red blood cell breakthrough or hemolysis. FIG. 24 shows a plasma transfer membrane fully saturated with plasma 2410.

[0190] To clearly visualize plasma transfer with no indications of hemolysis, photos were obtained under UV light, which demonstrated that the plasma transferred passively to the membrane did not contain any red blood cells. If there were red blood cells, the membrane section would have shown some spots or streaks of dark colored regions. FIG. 25A is a photo of a separation medium test strip (left) and plasma transfer membrane (right) in ambient fluorescent light. The plasma transfer membrane of FIG. 25A is fully saturated with plasma 2510. FIG. 25B is a photo of the separation medium test strip (left) and plasma transfer membrane (right) of FIG. 25A under UV light for easier visualization of the saturation with plasma 2510. In the examples shown in FIGS. 24 and 25A-25B, plasma passively transferred onto a plasma transfer membrane from a 20 pL human whole blood sample with a 50-60% het. [0191] Plasma transfer efficiencies were computed using the methods and Equations 8 and 9 described above. FIG. 26 A is a photo showing the plasma transferred onto a plasma transfer membrane from 10 pL bovine whole blood. FIG. 26B shows a separation medium test strip (bottom) and plasma transfer membrane (top) after the separation of 20pL of human whole blood. FIG. 26B also shows the method by which the length of the plasma-soaked section of the plasma transfer membrane (top) was measured. For example, in FIG. 26C, the length of the plasma-soaked section of the plasma transfer membrane measured was 19mm. The thickness and width were measured at 0.17mm and 5mm, respectively. Therefore, the plasma volume transferred was 0.17mm x 5mm x 19mm = 16.15mm ³ . The input human whole blood volume was 20 pL, and therefore, the plasma transfer efficiency is 16.15/20 = 81%. The average plasma transfer efficiency of human whole blood was 86 ± 4% (N=5). The coefficient of variation (CV) was 5% while the min and max were 81% and 89%, respectively.

Example 6: Plasma Quality and Levels of Hemolysis

Methods

[0192] In order to determine whether the plasma separated by separation media and methods described herein contained red blood cells or hemolyzed red blood cells, an elution method and UV-Vis spectroscopy detection method were developed. A Nanodrop 2000 (ThermoFisher Scientific) spectrophotometer was used to conduct the UV-Vis testing to determine plasma quality and levels of hemolysis and red cell breakthrough.

[0193] Prior to all testing, the Nanodrop 2000 instrument was calibrated using a vial of CF-1 calibration fluid (aqueous potassium dichromate; K^C O?). 10 aliquots of 1 pL of the CF-1 calibration fluid was used in 10 independent runs to complete the calibration process successfully.

[0194] Literature suggests that 420nm and 580nm are well established absorbance peaks for whole blood and the absorbance peak for plasma is around 540nm; studies have shown that the absorbance wavelength maxima /max shifts depending on the varying levels of hematocrit (het) in blood samples. See Roggan, Andre, et al. "Optical properties of circulating human blood in the wavelength range 400-2500nm." Journal of biomedical optics 4.1 (1999): 36-46, which is incorporated herein by reference.

[0195] Therefore, in order to determine the absorbance wavelength maxima max) of pure plasma and whole blood, UV-Vis spectra were obtained in the range of 200nm - 750nm using samples from 2 distinct sample groups. Pure plasma was obtained by adding K2EDTA to whole bovine blood and centrifuging the blood sample at 5000 rpm for 5 min. The top layer was syphoned off carefully without disturbing the buffy coat or the sedimented red blood cell layer at the very bottom. All the plasma and blood samples for this study were ethically obtained and procured through Lampire Biological Laboratories (PA).

[0196] (i) 1 :5 serial dilutions in PBS buffer (280mMNaCl, 20mM phosphate buffer, 6mM

KC1 at pH 7.4) were prepared from (i) pure bovine plasma, and (ii) whole bovine blood. The dilution equation indicated by Equation 10 below was used to determine the successive aliquots needed to prepare the serial standards solutions using the pure sample.

Mi X V _X = M ₂ X V ₂ Equation 10 [0197] where Mi is the concentration of the original solution, M2 is the concentration of the dilution, Vi is the volume of the original solution, and V2 is the volume of the dilution.

[0198] FIG. 27A is a schematic illustration of the serial dilution preparation used for UV-Vis absorbance analysis. FIG. 27B is a photo of a serial dilution from whole bovine blood, 30-40% het, completed according to the schematic illustration in FIG. 27A. The color change as the dilution progresses is visible to the naked eye. FIG. 27C is a photo of a serial dilution from pure bovine plasma completed according to the schematic illustration in FIG. 27A. The color change as the dilution progresses is visible to the naked eye.

[0199] Next, UV-Vis spectra were generated at the 1mm absorbance values using triplicates of each dilution level. Prior to testing, absorbance measurements for a blank solution were read by loading a 1 pL sample of the PBS buffer used in the serial dilutions.

[0200] After gathering UV-Vis absorbance spectra of all original samples and dilutions, the absorbance wavelength maxima Umax) of all the spectra were analyzed. Pure plasma and the whole blood were expected to have at least one distinctive difference in k _ma x.

[0201] This test method was used to detect whether the separation medium described herein causes any hemolysis and whether the plasma collected in the downstream end of the separation medium contains red blood cells (RBC) or RBC debris such as heme protein, indicating hemolysis during the separation process.

[0202] First, 5mm wide x 30mm length strips were cut and treated with a treatment formulation as described in Example 3. Next, 10 pL of whole bovine blood in K2EDTA anti -coagulant was dispensed onto the separation medium test strip. After 5 min, the plasma-containing section and red blood cell-containing section were each cut using a razor blade as described in Example 4 above, and as shown in FIG. 28 (left). The plasma-containing section and red blood cellcontaining section were then each chopped into several small pieces and inserted into a separate ImL low protein binding microcentrifuge tube, as shown in FIG. 28 (right). 80 pL of PBS buffer was pipetted into the centrifuge tube, and was allowed to sit at room temperature for 10 min. The contents in each centrifuge tube were analyzed using UV-Vis spectroscopy. The absorbance from 200nm - 750nm was read to determine the distinct peaks exhibited by the two separate elution liquids. Prior to UV-Vis testing with the test sample, the instrument was calibrated using 1 pL of a blank solution, where the blank solution was prepared by incubating a piece of the separation medium in PBS buffer for 10 min at room temperature. From the UV- Vis spectra, the absorbance wavelengths (X), which were distinctively observed in blood but not present in plasma, were used to determine whether the plasma separation section in the separation medium contained any RBC or RBC debris. Results and Discussion

[0203] Whole blood as well as the first dilution yielded UV-Vis spectra with distinctive peaks. FIG. 29A is a UV-Vis spectrum of whole bovine blood, 30-40% het. FIG. 29B is a UV-Vis spectrum of whole bovine blood, 30-40% het after first dilution. FIG. 29C is a UV-Vis spectrum of whole bovine blood, 30-40% het after second dilution, indicating higher noise levels. FIG. 29D shows the four separate UV-Vis spectra of whole blood and 3 of the serial dilutions, where the labels DI, D2 and D3 represent serial dilution 1, serial dilution 2, and serial dilution 3, respectively. The UV-Vis spectra run from 200nm - 750nm indicated several distinctive peaks for whole blood as illustrated in FIGS. 29A-29D. These peaks are at 200nm and 280nm (proteins present in whole blood), 350nm, 417nm, 540nm and 580nm.

[0204] In whole blood, the primary proteins contributing to the 220nm and 280nm are likely to be albumin, globulins, fibrinogen and other proteins. Hemoglobin is also a protein; however, this protein, being responsible for the red color of blood, absorbs light in the visible range (540nm - 750nm) and is not a significant contributor to absorption at 200nm. The absorbance peaks began to experience high noise levels by the second dilution as indicated in FIG. 29C, indicating that this perhaps is the lowest detection concentration limit for the test method.

[0205] The observed wavelengths of the pure plasma were at 230nm and 280nm, owing to the proteins present in the plasma. Proteins contain peptide bonds which strongly absorb UV light in the wavelength range of 190nm - 230nm. This region historically has been attributed to interference as well. However, no peak was observed in the 300nm - 750nm wavelength. FIG. 30A shows the UV-Vis spectrum of pure bovine plasma in K2EDTA. FIG 30B shows the UV- Vis spectra of pure plasma and all dilutions overlayed.

[0206] Whole blood and all its dilutions showed peaks at 417nm, 540nm, and 580nm, which were not present in pure plasma and its dilutions. Therefore, 417nm, 540nm, and 580nm were chosen as the absorbance peaks to look for RBC contamination in the plasma separation sections of the separation media.

[0207] The PBS buffer elution from each separated centrifuge tube resulted in two UV-Vis spectra which indicates a peak at 200nm in both samples, owing to the proteins present in blood and plasma. However, the red blood cell -containing section also indicated absorbance at 417nm which did not show up in the plasma-containing section. FIG. 31 A is a UV-Vis spectra overlay of the red blood cell -containing section (marked “red section elution”) and the plasmacontaining section (marked “yellow section elution”) of the separation media after separation of human whole blood. FIG. 3 IB is a UV-Vis spectra overlay of each plasma-containing section (marked “yellow section”) (N=5). FIG. 31C is a UV-Vis spectra overlay of each red blood cell-containing section (marked “red section”) (N=5). These spectra indicate that the plasma-containing section does not have any heme protein, RBC or any other debris that can be attributed to the red color in whole blood. These observations were made from 5 independent plasma separation trials.

[0208] Therefore, in conclusion, the plasma separated by separation media described herein is of high quality and is suitable for lateral flow assay (LFA) and point of care (POC) systems where chromogenic reading is the method of downstream detection.

Example 7: Recovery of Anti -Human IgG

Methods

[0209] In order to determine whether separation medium described herein causes non-specific binding of relevant biomarkers, anti-human IgG was used. IgG is a type of antibody present in circulating human blood that is responsible for controlling a variety of infections or illnesses. Given the clinical relevance, anti-human IgG antibody was utilized as the model analyte to study the levels of non-specific binding. The level of non-specific binding of IgG was assessed by determining the recovery% of IgG from the separation medium.

[0210] The anti-human IgG (Fc specific) antibody produced in rabbits was stored at -20°C, and prior to each experimentation day, the vial was brought to room temperature by leaving the vial in a chemical hood for approx. 2 hours. A Nanodrop 2000 (ThermoFisher Scientific) spectrophotometer was used to conduct the UV-Vis testing to determine the levels of recovery of human IgG.

[0211] Prior to testing, the Nanodrop 2000 instrument was calibrated using a vial of CF-1 calibration fluid (aqueous potassium dichromate; K^C O?). 10 aliquots of 1 pL of the CF-1 calibration fluid was used in 10 independent runs to complete the calibration process successfully.

[0212] In order to validate the UV-Vis spectroscopy test method, a calibration curve was generated using five concentration values of the anti -human IgG (Fc specific) antibody in triplicate. The newly developed method’s accuracy, sensitivity, and linearity were assessed according to the ICH Harmonized Tripartite Guidelines. See Choudhari VP, Parekar SR, Chate SG, Bharande PD, Kuchekar BS. Development and validation of UV-Visible spectrophotometric baseline manipulation methodology for simultaneous analysis of drotraverine and etoricoxib in pharmaceutical dosage forms. Pharm Methods. (2011) Oct;2(4):247-52. doi: 10.4103/2229-4708.93395; PMID: 23781465; PMCID: PMC3658078, which is incorporated herein by reference.

[0213] First, the concentration of the anti-human antibody as received was measured using the Protein A280, IgG method in-built in the NanoDrop 2000 Spectrophotometer. The measured protein concentration was 31.81 mg/mL (N=3).

[0214] Next, 12.5 pL of IgG as received was dissolved in 37.5 pL of PBS buffer (280mM NaCl, 20mM phosphate buffer, 6mM KC1 at pH 7.4) to achieve a 50 pL solution at of 8mg/mL. This was used as the stock solution. The dilution equation indicated in Equation 10 above was used to determine the successive aliquots needed to prepare the successive standard solutions using the stock solution. The dilutions yielded 2 mg/mL, 0.5 mg/mL, 1 mg/mL and 0.03 mg/mL standard solutions respectively. Next, each standard solution was run in the Nanodrop 2000 instrument in triplicates to obtain a total of 15 determinations in the specified range. Prior to testing, absorbance measurements for a blank solution was taken by loading a 1 pL sample of the PBS buffer used in the serial dilutions. The x-axis of the regression graph was the nominal concentration, while the y-axis was the measured concentration.

[0215] The precision of an analytical procedure is the closeness of agreement between a series of measurements obtained by multiple sampling of a homogenous sample. This was assessed by the coefficient of variation (%CV), which was calculated by Equation 11 below:

_{Q/ r} , _T > standard deviation of the triplicates > _ / 0 Ci V — _ , . .. X J. u u Equation 11 mean of the triplicates

[0216] The accuracy of the method was assessed by the %recovery from the theoretical concentration, which was calculated by Equation 12 below: observed concentration

% recovery = - _{: :} — x 100 Equation 12 nominal concentration

[0217] Linearity is the procedure’s ability to obtain test results which are directly proportional to the concentration of the standard (anti-human IgG antibody) and was assessed by fitting the data into a linear regression numerical model.

[0218] The limit of detection (LOD) of a method is the lowest amount of analyte which can be detected by the method reliably and was calculated according to Equation 13 below: LOD = ^{3,3 X g} Equation 13 slope ¹

[0219] Limit of quantitation (LOQ) is the lowest amount of analyte which can be quantitatively determined with suitable precision and accuracy and was determined by Equation 14 below: Equation 14

[0220] In both the LOD and LOQ equations, slope = slope of the calibration regression line, o is the standard deviation of the y-intercepts of the regression line calculated using Equation 15 below. The standard error of the regression line (SE) was computed using the Data Analysis tool in MS Excel. N = number of standards used to develop the calibration curve. For this study, N=6. Equation 15

[0221] Linearity, LOD, and LOQ were calculated according to the ICH Harmonized Tripartite Guidelines as discussed above.

[0222] The test method developed and validated as indicated above was used to detect whether the present invention causes any non-specific binding of IgG.

[0223] First, 5mm wide x 30mm length strips of a separation medium prepared as described in Example 3 were cut and treated with a treatment formulation as described in Example 3. The test strips were laid on a nitrocellulose membrane as shown in FIG. 32A. 30 pL of 12 mg/mL anti-human antibody dissolved in PBS was placed on the separation medium, which was overlapped with a plasma transfer membrane comprising nitrocellulose at the bottom. FIG. 32A is a photo showing the resulting test strip assembly. Nitrocellulose membrane was used as the plasma transfer membrane here in order to mimic a real lateral flow assay (LFA) test.

[0224] Next, 20 pL of anti-human IgG antibody solution (12mg/mL) was dispensed onto the separation medium. After 5 min, the plasma transfer membrane was removed, and the membrane piece, which had liquid absorbed onto it, was cut using a razor blade. It was then inserted into a low protein binding microcentrifuge tube containing 50 pL of PBS buffer (280mM NaCl, 20mM phosphate buffer, 6mM KC1, pH = 7.4) and sat at room temperature for 10 min as shown in FIG. 32B. The elution in the centrifuge tube was analyzed using UV-Vis spectroscopy, i.e., the absorbance at 280nm was read to determine the amount of IgG eluted from the nitrocellulose membrane piece. Prior to UV-Vis testing with the test sample, the instrument was calibrated using 1 pL of a blank solution, where the blank solution was prepared by extracting a poly sulfone membrane piece using PBS buffer. The eluted samples from each centrifuge tube were analyzed in triplicates to obtain 15 determinations. weight (mg) = M ₂ @ x V ₂ (mL) Equation 16 n,

% _T j mg oflgG used „„ IgG recovered = - - - * — - - - x 100 Equation 17

° mg of IgG on nitrocellulose membrane

[0225] The positive control used in the study was a polyamide test strip. The same test method, elution method, and equation were used in order to conduct UV-Vis experimentation. Polyamides such as Nylon 6,6 and Nylon 11 are known to cause non-specific binding of charged molecules such as proteins, antibodies, enzymes, DNA and RNA molecules. See Senkovenko, A.M., Moysenovich, A.M., Maslakova, A.A. Measurements of IgG Antibodies Adsorption onto Electrospun Nylon-6 Membranes. BIOPHYSICS 67, 440-444 (2022); Leda R Castilho, Wolf-Dieter Deckwer, F. Birger Anspach. Influence of matrix activation and polymer coating on the purification of human IgG with protein A affinity membranes, Journal of Membrane Science, Volume 172, Issues 1-2, pages 269-277 (2000); each of which is incorporated by reference herein. Therefore, polyamide was used as the positive control for this study.

Results and Discussion

[0226] FIG. 33 is a standards calibration curve of IgG constructed by linear regression using six standards of known concentrations. Error bars represent ±1 SD from triplicate runs per each standard (n=18 determinations). The coefficient of determination (r ² ) of the calibration curve was 0.9988 indicating that the regression equation is capable of approximating the real data very well. The slope of the linear regression equation was 0.92 while the standard error (SE) computed by the regression model was 0.05151. These values along with the eqn. 2.4 - 2.6 were used to compute the LOD and LOQ values.

Table 6: Regression statistics of the nominal vs. measured concentrations of IgG standards. session Statistics

Multiple R 0.99988284 R Square 0.99976568 Adjusted R Square 0.9997071

[0227] The accuracy and precision of the developed method are evaluated from the %recovery and the %CV respectively, shown in Table 6 above. The %CV for five of the six concentration levels were well below 11%, and the %recovery for all levels ranged from 92% - 100%. The detection limit (LOD) and quantitation limit (LOQ) determined as per the ICH Harmonized Tripartite Guidelines were 0.45 mg/mL and 1.37 mg/mL respectively.

Table 7: Recovery analysis and system suitability parameter - UV-Vis Spectrophotometry (n=18 determinations)

[0228] The elution method described above was used to analyze a sample of only plasma transfer membrane (comprising nitrocellulose) immersed in 50 pL of PBS buffer. However, there was no absorbance at 280nm, indicating that the native plasma transfer membrane did not have any proteins on it, which would have caused any interferences in the elution study.

[0229] The measured concentration of the IgG stock solution used for the elution testing was 11.611 mg/mL, and the volume of stock solution used for testing was 20pL. Therefore, the amount of IgG antibodies used in each test was computed at 0.23 mg. FIG. 34 is a representative UV-Vis curve of an elution sample with IgG present in it (i.e., a spectrograph of IgG eluted from the nitrocellulose plasma transfer membrane after the experimentation was conducted). [0230] The experimentation conducted to determine the recovery of IgG from the separation medium yielded concentration values which were in the range of 1 - 3 mg/mL, which were well above the LOD and LOQ of the analytical method developed and reported here. The detected concentrations of IgG during experimentation were also well within the range of concentrations in the calibration curve which had lower %CV as shown in Table 6 and were well within the linear range of the analytical method as well.

[0231] The amount of IgG recovered from the nitrocellulose plasma transfer membrane was 44 ±4%, indicating that even without a chase buffer, the separation medium does not result in significant loss of IgG during an experiment. This was compared to the positive control polyamide test strip. The (+) control resulted in IgG recovery of only 10 ±4%, indicating significant loss of IgG. In order to benchmark the separation medium described herein with products in the market, another competitor product currently used in the market for plasma separation was obtained and tested using the same elution and UV-Vis test method. The results indicated that the competitor’s material resulted in 33±2% IgG recovery, which was 10% lower than that of the separation medium described herein. FIG. 35 shows boxplot graphs comparing the IgG recovery from the separation medium described herein (“plasma separation product”), the positive control polyamide test strip (“positive control”), and the commercially available competitor product (“competitor’s product”). In order to determine whether the observed difference between the two means of the two populations, a student’s t-test assuming unequal variances was conducted. The difference between the two means was statistically significant with a p<0.05. The separation medium and methods described herein thus recover a clinically relevant biomarker (anti-human IgG antibody) at approx. 45% without the usage of a chase buffer. With a chase buffer, the recovery percentage is expected to be even higher.

[0232] Because the separation medium described herein does not cause significant nonspecific binding of a clinically relevant biomarker such as IgG, this product could be used in the development of rapid diagnostic products such as LFA (lateral flow assays) or POC (point of care) devices used to detect proteins such as enzymes, hormones, and/or antibodies as well as a variety of antigens with a protein coating on their surface.

Example 8: Comparison to Commercially Available Products

Methods

[0233] Additional testing of the separation medium described herein, alongside three commercially available competitor products that are marketed as suitable for blood volumes as low as 10 pL, demonstrated that the separation medium described herein separates plasma at high yields and is suitable for diagnostic applications. 10 pL of human whole blood, het 40- 50%, was used to test each product.

[0234] FIG. 36A is a photo of the competitor products 3610, 3620, and 3630, and an embodiment of a separation medium 3640 as described herein, 2 minutes and 38 seconds after application of 10 pL of human whole blood, het 40-50% to the left-most side of each product. Plasma separation is evident from the plasma-containing section 3640a of the separation medium 3640.

[0235] FIG. 36B is a photo of the competitor products 3610, 3620, and 3630, and the separation medium 3640 of FIG. 36A, imaged under UV light for improved visibility. Again, plasma separation is evident from the plasma-containing section 3640a of the separation medium 3640.

[0236] In the above detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be used, and other changes may be made, without departing from the spirit or scope of the subj ect matter presented herein. It will be readily understood that various features of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.

[0237] The present disclosure is not to be limited in terms of the particular embodiments described in this application, which are intended as illustrations of various features. Many modifications and variations can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this disclosure is not limited to particular methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0238] With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity.

[0239] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (for example, bodies of the appended claims) are generally intended as “open” terms (for example, the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” et cetera). While various compositions, methods, and media are described in terms of “comprising” various components or steps (interpreted as meaning “including, but not limited to”), the compositions, methods, and media can also “consist essentially of’ or “consist of’ the various components and steps, and such terminology should be interpreted as defining essentially closed-member groups. It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present.

[0240] For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles "a" or "an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an" (for example, “a” and/or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations.

[0241] In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (for example, the bare recitation of "two recitations," without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, et cetera” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (for example, “a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, et cetera). In those instances where a convention analogous to “at least one of A, B, or C, et cetera” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (for example, “a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, et cetera). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.” [0242] In addition, where features of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0243] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, et cetera. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, et cetera. As will also be understood by one skilled in the art all language such as “up to,” “at least,” and the like include the number recited and refer to ranges that can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 fibers refers to groups having 1, 2, or 3 fibers. Similarly, a group having 1-5 fibers refers to groups having 1, 2, 3, 4, or 5 fibers, and so forth.

[0244] The term “about,” as used herein, refers to variations in a numerical quantity that can occur, for example, through measuring or handling procedures in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of compositions or reagents; and the like. Typically, the term “about” as used herein means greater or lesser than the value or range of values stated by 1/10 of the stated values, e.g., ±10%. The term “about” also refers to variations that would be recognized by one skilled in the art as being equivalent so long as such variations do not encompass known values practiced by the prior art. Each value or range of values preceded by the term “about” is also intended to encompass the embodiment of the stated absolute value or range of values. Whether or not modified by the term “about,” quantitative values recited in the claims include equivalents to the recited values, e.g., variations in the numerical quantity of such values that can occur, but would be recognized to be equivalents by a person skilled in the art. [0245] Various of the above-disclosed and other features and functions, or alternatives thereof, may be combined into many other different systems or applications. Various presently unforeseen or unanticipated alternatives, modifications, variations or improvements therein may be subsequently made by those skilled in the art, each of which is also intended to be encompassed by the disclosed embodiments.