Method and kit for removing particles from a fluid

Abstract

The present disclosure provides methods and kits for removing particles from a fluid. The method for removing red blood cells includes obtaining a device having a microstructured substrate with microstructures extending across a first surface, at least a portion of an outer surface of the microstructures configured to allow capillary action. The device also includes a cover positioned a selected distance from the top of the first surface of the microstructured substrate and at least one sidewall attaching the cover to the first surface of the microstructured substrate along the periphery of the first surface of the microstructured substrate. The method further includes filling the device with a volume of blood through the first aperture by capillary action, waiting a sufficient time for at least a portion of the red blood cells to settle within the first open volume of the microstructures, and fluidly coupling the device with a collection article, thereby allowing a portion of the initial volume of blood from which at least a portion of the red blood cells were drawn into the first open volume of the microstructures to flow out of the device. Kits containing the device and the collection article are also included.

Description

[Technical Field]

[0001] Identification and quantification of biomarkers in blood often requires the removal of red blood cells (RBCs) prior to analysis. Red blood cells are present in whole blood at concentrations ranging from 35% to 50%, reported as the hematocrit level. This high concentration can interfere with biomarker assays. The level of interference varies across the physiological hematocrit range, resulting in variability in reported biomarker concentrations. Interference in optical detection assays (e.g., ELISA) can be caused by the light scattering effect and absorbance of hemoglobin in the visible spectrum. RBCs can also interfere with electrochemical detection assays, such as those commonly utilized in glucose test strips. Chemical species, such as oxygen present in RBCs, can interfere with redox reactions.

[0002] In centralized hospital or clinical laboratories, red blood cell separation is performed by centrifugation. The separation process requires a large volume (e.g., milliliters) of blood drawn from a vein into a test tube. During centrifugation, the red blood cells are packed to the bottom of the tube, leaving the remaining blood (e.g., cell-free plasma) accessible in the upper layer for further analysis. In these centralized settings, biomarker detection is then typically performed on large, complex analyzers capable of automated liquid handling and accessible for frequent verification of assay performance through calibration.

[0003] Point-of-care hematology analyzers used outside of central laboratories also require the removal of red blood cells before analysis. In situations where rapid results are required, access to intravenous blood volume and tabletop centrifugation are often unavailable or too time-consuming. A finger prick collects a blood volume of approximately 5 microliters. Glucose test strips typically accept blood volumes of less than 1 microliter and are subject to the above-mentioned interferences. Removal of red blood cells from these small volumes remains a challenge. Summary of the Invention

[0004] In a first aspect, a method for separating red blood cells from blood is provided. The method includes obtaining a device including a microstructured substrate with a plurality of microstructures extending across a first surface of the microstructured substrate. At least a portion of the outer surface of the plurality of microstructures is configured to enable capillary action. The device also includes a cover positioned a selected distance from a top of the first surface of the microstructured substrate and at least one sidewall attaching the cover to the first surface of the microstructured substrate along a periphery of the first surface of the microstructured substrate. The device further includes a first aperture defined by at least one of the microstructured substrate or the cover, and a second aperture defined by at least one of the microstructured substrate or the cover. The first surface of the microstructured substrate, together with the at least one sidewall, defines a first open volume that is the sum of the open spaces between the plurality of microstructures from the bottom to the top of each microstructure, and the cover, together with the top of the first surface of the microstructured substrate and the at least one sidewall, defines a second open volume adjacent to the first open volume, wherein the first open volume has a percentage of the 100% open volume that is greater than the volume percentage of red blood cells present in the blood, where the combined first and second open volumes represent 100% open volume. The method further includes filling the device with a volume of blood through the first aperture by capillary action and waiting a sufficient time for at least a portion of the red blood cells to settle within the first open volumes of the plurality of microstructures. Additionally, the method includes fluidly connecting the device to the collection article at either the first aperture or the second aperture, thereby causing at least 10% of the initial volume of blood from which at least a portion of the red blood cells have been drawn into the first open volume of the plurality of microstructures to flow out of the device and onto the collection article by capillary action.

[0005] In a second aspect, a method for separating solid particles from a fluid is provided. The method includes obtaining a device including a microstructured substrate with a plurality of microstructures extending across a first surface of the microstructured substrate. At least a portion of the outer surface of the plurality of microstructures is configured to enable capillary action. The device also includes a cover positioned a selected distance from a top of the first surface of the microstructured substrate and at least one sidewall portion attaching the cover to the first surface of the microstructured substrate along a periphery of the first surface of the microstructured substrate. The device further includes a first aperture defined by at least one of the microstructured substrate or the cover, and a second aperture defined by at least one of the microstructured substrate or the cover. The first surface of the microstructured substrate, together with at least one sidewall, defines a first open volume that is the sum of the open spaces between the plurality of microstructures from the bottom to the top of each microstructure, and the cover, together with the top of the first surface of the microstructured substrate and the at least one sidewall, defines a second open volume adjacent to the first open volume. The first open volume has a greater volume percentage of the 100% open volume than the volume percentage of particles present in the fluid, where the combined first and second open volumes equal 100% open volume. The method further includes filling the device with a volume of fluid through the first aperture by capillary action and waiting a sufficient time for at least a portion of the particles to settle into the first open volumes of the plurality of microstructures. Additionally, the method includes fluidly connecting the device to the collection article at either the first aperture or the second aperture, thereby causing at least 10% of the initial volume of blood from which at least a portion of the red blood cells have been drawn into the first open volume of the plurality of microstructures to flow out of the device and onto the collection article by capillary action.

[0006] In a third aspect, a kit is provided. The kit includes a device and a collection article. The device includes a microstructured substrate having a plurality of microstructures extending across a first surface of the microstructured substrate. The microstructures cover at least 90% of the first surface of the microstructured substrate, and at least a portion of the outer surface of the plurality of microstructures is configured to enable capillary action. The device also includes a cover positioned a selected distance from the top of the first surface of the microstructured substrate and at least one sidewall attaching the cover to the first surface of the microstructured substrate along the periphery of the first surface of the microstructured substrate. The device further includes a first aperture defined by at least one of the microstructured substrate or the cover, and a second aperture defined by at least one of the microstructured substrate or the cover. The first surface of the microstructured substrate, together with the at least one sidewall, defines a first open volume that is the sum of the open spaces located between the plurality of microstructures from the bottom to the top of each microstructure, and the cover, together with the top of the first surface of the microstructured substrate and the at least one sidewall, defines a second open volume located adjacent to the first open volume. The collection article has a surface that exhibits an advancing contact angle with water of less than 90 degrees.

[0007] It has been discovered that devices, kits, and methods according to at least certain embodiments of the present disclosure can provide for the removal of solid particles (e.g., red blood cells) from very small volumes (e.g., microliters) of fluid (e.g., blood).

[0008] The above summary of the present disclosure is not intended to describe each embodiment or every implementation disclosed in the present disclosure. The following description more particularly exemplifies exemplary embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list. [Brief explanation of the drawings]

[0009] [Figure 1]1 is a generalized flowchart of an exemplary method. [Figure 2A] FIG. 1 is a perspective view of a generalized schematic diagram of an exemplary device. [Figure 2B] FIG. 1 is a perspective view of a generalized schematic diagram of a microstructured substrate for use in an exemplary device. [Figure 3A] 1 is a scanning electron microscope (SEM) image of a cross section of a portion of an exemplary device, marked up to illustrate portions of an exemplary method. [Figure 3B] 3B is a cross-sectional SEM image of a portion of the exemplary device and exemplary collection item of FIG. 3A, marked up to show different parts of the exemplary method. [Figure 4A] 1 is a schematic cross-sectional view of a microstructured substrate having a plurality of ribs alternating with channels. [Figure 4B] FIG. 1 is a schematic perspective view of a microstructured substrate having a plurality of ribs alternating with channels, the ribs including caps on their top surfaces. [Figure 5A] FIG. 1 is a schematic cross-sectional view of a microstructured substrate having a linear array of prisms. [Figure 5B] 1 is an SEM image of a cross section of a portion of an exemplary device. [Figure 5C] FIG. 1 is a schematic perspective view of a portion of a microstructured substrate having an array of peak structures and adjacent valleys in a particular orientation. [Figure 5D] FIG. 1 is a schematic perspective view of a portion of a microstructured substrate having an array of peak structures and adjacent valleys in another particular orientation. [Figure 5E] 1 is a schematic cross-sectional view of a microstructured substrate having a faceted structure. [Figure 6A] 1 is a schematic cross-sectional view of a microstructured substrate having a two-dimensional array of protrusions. [Figure 6B] FIG. 10 is a top view of four representative engineered micropatterned regions for two-dimensional arrays of protrusions. [Figure 7A] FIG. 1 is a schematic cross-sectional view of a microstructured substrate having a plurality of cavities extending between two major surfaces. [Figure 7B] FIG. 1 is a generalized schematic exploded view of a microstructured substrate having a plurality of cavities extending between two major surfaces. [Figure 8A] FIG. 1 is an exploded and generalized schematic diagram of the device of Example 1. [Figure 8B] FIG. 1 is a generalized schematic top view of the device of Example 1. [Figure 8C] FIG. 1 is a generalized schematic top view of two components used to attach a pump secured to the device of Example 1. [Figure 8D] FIG. 1 is a generalized schematic perspective view of the device of Example 1 adapted to be attached to a pump. [Figure 9] 1 is a generalized flowchart of another exemplary method. [Figure 10A] FIG. 1 is a schematic perspective view of a portion of a microstructured substrate having an array of fluidly connected wells. [Figure 10B] FIG. 1 is a schematic top view of a portion of a microstructured substrate having an array of fluidly connected wells having circular geometries. [Figure 11A] 1 is a generalized schematic diagram of an exemplary collectible item. [Figure 11B] FIG. 1 is a generalized schematic diagram of another exemplary collectible item. [Figure 12] 1 is a schematic cross-sectional view of a further exemplary collection article. [Figure 13] FIG. 1 is a schematic cross-sectional view of a microstructured substrate having an array of generally upright stems. DETAILED DESCRIPTION OF THE INVENTION

[0010] The above-identified figures set forth several embodiments of the present disclosure; however, as noted in the description, other embodiments are also contemplated. The drawings are not necessarily drawn to scale. In all cases, this disclosure presents the invention by way of representation and not limitation.

[0011] As used herein, the term "microreplication" refers to the production of microstructured surfaces by a process in which the structured surface features retain individual feature fidelity during fabrication.

[0012] As used herein, the term "microstructure" encompasses both structures (i.e., features) that protrude above a major surface of the substrate and structures that are recessed below the major surface of the substrate. Combinations of protruding and recessed features are contemplated. Microstructure further means that the structure is a predetermined molded structure (e.g., a molded structure such as may be obtained by molding a polymeric thermoplastic against a tooling surface that bears a negative of the microstructure desired to be provided on the first major surface of the substrate) having dimensions ranging from about 5 micrometers to about 3000 micrometers in at least two orthogonal directions. One of these orthogonal directions may often be perpendicular to the plane of the substrate (e.g., along the z-axis), and thus this dimension may include, for example, the height of a protrusion or the depth of a recess.

[0013] As used herein, the term "capillary action" refers to the flow of a fluid without the assistance of an external force (e.g., pressure, gravity, vacuum, etc.). Capillary action often occurs for aqueous fluids in contact with hydrophilic surfaces. Aqueous fluids contain 50% or more water by volume.

[0014] As used herein, the term "hydrophilic" refers to a surface that is wetted by aqueous solutions, and does not describe whether the material absorbs the aqueous solution. "Wetting" means that the surface exhibits spontaneous wicking when contacted with aqueous fluids. "Spontaneous" means that it occurs without an external force. In some embodiments, a hydrophilic surface exhibits an advancing (maximum) water contact angle of less than 90°, preferably 45° or less.

[0015] As used herein, the term "hydrophobic" refers to a surface that lacks spontaneous wicking when contacted with aqueous fluids. In some embodiments, a hydrophobic surface exhibits an advancing water contact angle of 70° or greater, preferably 90° or greater.

[0016] As used herein, "curing" means solidifying or partially solidifying a composition by any mechanism, e.g., heat, light, radiation, electron beam, microwave, chemical reaction, or a combination thereof. As used herein, the term "solidifiable" refers to a material that can be hardened or solidified by, for example, heating to remove solvent, heating to cause polymerization, chemical crosslinking, radiation-induced polymerization or crosslinking, etc. As used herein, "cured" refers to a material or composition that has been hardened or partially solidified (e.g., polymerized or crosslinked) by curing.

[0017] As used herein, a polymer "film" is a polymer material in the form of a generally flat sheet that is flexible and strong enough to be processed in a roll-to-roll manner. Roll-to-roll refers to a process in which the material is wound onto or unwound from a support and further processed in some way. Examples of further processing include coating, slitting, blanking, and exposure to radiation. Polymer films can be produced in a variety of thicknesses, generally ranging from about 5 micrometers to 1000 micrometers.

[0018] As used herein, "solid" refers to a state of matter that is neither a liquid nor a gas, and a solid has a stable three-dimensional form.

[0019] As used herein, "fluid" refers to a composition that contains a liquid (i.e., a state of matter that is not a solid or a gas), and includes solutions, suspensions, and emulsions.

[0020] As used herein, "outer surface" with respect to a microstructure refers to the outermost surface of the microstructure.

[0021] As used herein, "thermoplastic" refers to a polymer that flows when heated sufficiently above its glass transition temperature and becomes solid when cooled. In contrast, "thermoset" refers to a polymer that becomes permanently hardened upon curing and does not flow when subsequently heated. Thermoset polymers are typically crosslinked polymers.

[0022] As used herein, the "glass transition temperature" (T g The term T refers to the transition of a polymer from a glassy to a rubbery state and can be measured using differential scanning calorimetry (DSC), for example, at a heating rate of 10°C per minute in a nitrogen stream. g When a reference is made to a T, it refers to the T of the homopolymer of that monomer. g The homopolymer is T g The molecular weight must be high enough so that the T g It is generally recognized that the T increases with increasing molecular weight up to a critical value. Homopolymers also contain moisture, residual monomers, solvents, and g It is also understood that the DSC is substantially free of other contaminants that may affect the performance of the polymer. A suitable DSC method and analysis mode is as described in Matsumoto, A. et al., J. Polym. Sci. A., Polym. Chem. 1993, 31, 2531-2539.

[0023] As used herein, "transparent" refers to a material (e.g., a layer) that has at least 50% transmittance, 70% transmittance, or optionally greater than 90% transmittance over at least the 400 nanometer (nm) to 700 nm portion of the visible light spectrum.

[0024] The words "preferred" and "preferably" refer to embodiments of the present disclosure that may offer certain benefits, under particular circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, or is intended to exclude other embodiments from the scope of the present disclosure.

[0025] In this application, terms such as "a," "an," and "the" are not intended to refer only to a singular entity, but include a general class of which a particular example may be used for illustration. The terms "a," "an," and "the" are used interchangeably with the term "at least one." The phrases "at least one of" and "including at least one of" following a list refer to any one of the items in the list, and any combination of two or more items in the list.

[0026] As used herein, the term "or" is generally used in its ordinary sense, including "and / or," unless the context clearly dictates otherwise. The term "and / or" refers to one or all of the listed elements or a combination of any two or more of the listed elements.

[0027] Also, all numbers herein are intended to be modified by the term "about," and preferably by the term "exactly." When used herein in connection with a measured quantity, the term "about" refers to the variation in the measured quantity that would be expected by one of ordinary skill in the art making the measurement and exercising a level of care commensurate with the purpose of the measurement and the precision of the measuring device used.

[0028] The term "generally," when used herein as a modifier to a characteristic or attribute, unless specifically defined otherwise, means that the characteristic or attribute is readily recognizable by one of ordinary skill in the art, but does not require absolute precision or perfect agreement (e.g., within + / - 20% for quantifiable characteristics). The term "substantially," unless specifically defined otherwise, means a high degree of approximation (e.g., within + / - 10% for quantifiable characteristics), but again does not require absolute precision or perfect agreement. Terms such as same, equal, uniform, constant, exactly, etc., are understood to be within normal tolerances or measurement errors applicable to the particular circumstances, rather than requiring absolute precision or perfect agreement.

[0029] Point-of-care biomarker analysis requires simple and efficient separation of red blood cells from microliter volumes of blood without hemolysis, dilution, or significant loss of blood to dead space.

[0030] In a first aspect, the present disclosure provides a method for separating red blood cells from blood, the method comprising: a) 1) a microstructured substrate comprising a plurality of microstructures extending across a first surface of the microstructured substrate, at least a portion of an outer surface of the plurality of microstructures configured to allow capillary action; 2) a cover positioned a selected distance from the top of the first surface of the microstructured substrate; 3) at least one sidewall attaching the cover to the first surface of the microstructured substrate along the periphery of the first surface of the microstructured substrate; 4) a first aperture defined by at least one of the microstructured substrate or the cover; and 5) a second aperture defined by at least one of the microstructured substrate or the cover; and Equipped with obtaining a device wherein a first surface of the microstructured substrate, together with at least one sidewall, defines a first open volume that is the sum of the open spaces located between a plurality of microstructures from the bottom to the top of each microstructure; and a cover, together with the top of the first surface of the microstructured substrate and the at least one sidewall, defines a second open volume located adjacent to the first open volume, wherein the first open volume has a percentage of 100% open volume that is greater than the volume percentage of red blood cells present in blood, where the combined total of the first open volume and the second open volume is 100% open volume; b) filling the device with a volume of blood through the first aperture by capillary action; c) waiting a sufficient time for at least a portion of the red blood cells to settle within the first open volume of the plurality of microstructures; d) fluidly coupling the device with the collection article at either the first aperture or the second aperture, thereby causing at least 10% of the initial volume of blood, with at least a portion of the red blood cells drawn into the first open volume of the plurality of microstructures of the device, to flow out of the device and onto the collection article by capillary action; Includes:

[0031] Referring to FIG. 1 , the method includes obtaining a device (the device is as described above), 110, filling the device with a volume of blood through a first aperture by capillary action, 120, waiting a time sufficient for at least a portion of the red blood cells to settle within the first open volume of the plurality of microstructures, 130, and fluidly connecting the device to a collection article at either the first aperture or the second aperture, thereby causing at least 10% of the initial volume of blood from which at least a portion of the red blood cells have been drawn into the first open volume of the plurality of microstructures of the device to flow out of the device onto the collection article by capillary action, 140. In some cases, the waiting time is sufficient for at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even at least 95% of the red blood cells to settle within the first open volume of the plurality of microstructures.

[0032] 2 and 3A, an exemplary device 200 is shown. The device 200 comprises a microstructured substrate 210 comprising a plurality of microstructures 230 extending across a first surface 202 of the microstructured substrate 210. At least a portion of an outer surface 232 of the plurality of microstructures 230 is configured to enable capillary action. The device 200 also includes a cover 220 positioned a selected distance D from the top of the first surface 221 of the microstructured substrate 210 and at least one sidewall 240 attaching the cover 220 to the first surface 202 of the microstructured substrate 210 along a perimeter P of the first surface of the microstructured substrate 210. The perimeter P collectively comprises each of the sides Pa, Pb, Pc, Pd, ...Pn of a particular device. The device 200 shown in FIG. 2A has four sides Pa, Pb, Pc, and Pd, and therefore the perimeter P includes each of these four sides. Other shapes of devices having numbers of sides other than four are contemplated.

[0033] Optionally, at least one sidewall 240 may include an adhesive layer disposed between the cover 220 and the microstructured substrate 210 (e.g., to attach the cover to the substrate), such as the double-sided tape used in Preparative Example 6 below. This may be particularly useful when the microstructured substrate is a microstructured film. Suitable materials for the adhesive layer include, for example, pressure-sensitive adhesives. The adhesive layer can be prepared by coating a film of adhesive containing an adhesive polymer. Preferably, the adhesive comprises an adhesive polymer and a crosslinker. As used herein, the term "adhesive polymer" refers to a polymer that exhibits adhesive properties at ambient temperatures (e.g., 20°C to 25°C). The adhesive polymer may be, for example, an acrylic polymer, polyurethane, polyolefin, or polyester. In selected embodiments, the adhesive layer comprises a double-sided coated adhesive film. Some suitable commercially available double-coated adhesive films are sold by 3M Company (St. Paul, MN) under the trade names 3M Medical Silicone Tape 2477P, and 3M Medical Tape 1509, 1510, 1513, 1522, 9874, and 9877.

[0034] Additionally, the device includes a first aperture 250 defined by at least one of the microstructured substrate 210 or the cover 220, and a second aperture 260 defined by at least one of the microstructured substrate 210 or the cover 220. The device shown in Figure 2A includes a first aperture 250 defined by the cover 220, and a second aperture 260 defined by both the microstructured substrate 210 and the cover 220.

[0035] In selected embodiments, the sidewalls, microstructured substrate, and cover are hermetically sealed to one another at their contact points (e.g., seams), which minimizes leakage of the fluid sample from any seams between the three (e.g., fluid may enter or exit primarily or exclusively through the first aperture and / or the second aperture). In some cases, the microstructured substrate itself includes sidewalls within its structure, such that at least one sidewall is part of the microstructured substrate.

[0036] Suitable materials for use as the cover include, but are not limited to, polyolefins (e.g., high-density polyethylene (HDPE), medium-density polyethylene (MDPE), or low-density polyethylene (LDPE)), polyesters, polyamides, poly(vinyl chloride), polyetheresters, polyimides, polyesteramides, polyacrylates, polyvinyl acetate, or hydrolyzed derivatives of polyvinyl acetate. In certain embodiments, polyolefins are preferred due to their excellent physical properties, ease of processing, and typically low cost. Polyolefins are also generally strong, durable, and retain their shape well, making them easy to handle after the article is formed. In selected embodiments, the film layer comprises the polyester polyethylene terephthalate (PET). One suitable commercially available PET is a 5-mil (127 micrometer) thick PET sheet sold under the trade name "MELINEX 454" by Tekra (New Berlin, WI). A suitable commercially available LDPE is sold under the trade name "DOW 955I LDPE" by The Dow Chemical Company (Midland, MI). Additionally, various additives may be included in the cover layer, such as surface energy modifiers (eg, surfactants and hydrophilic polymers), plasticizers, antioxidants, pigments, release agents, antistatic agents, and the like.

[0037] The first surface 202 of the microstructured substrate 210, together with at least one sidewall 240, defines a first open volume 270, which is the sum of the open spaces located between the plurality of microstructures 230 from the bottom 236 to the top 221 of each microstructure 230. (For simplicity, the arrows in 270 only point to the portion of the first open volume between two adjacent microstructures 230.) The cover 220, together with the top 221 and at least one sidewall 240 of the first surface 202 of the microstructured substrate 210, defines a second open volume 280 adjacent to the first open volume 270, with the first open volume having a percentage of the 100% open volume greater than the volume percentage of red blood cells present in the blood, where the combined first and second open volumes are 100% open volume. To calibrate the device to blood containing a certain volume percentage of particles (e.g., solid particles) such as red blood cells, it can be concluded that the first open volume must have a larger percentage of that 100% open volume than the volume percentage of particles present in the fluid, considering that the first open volume and the second open volume together total 100% open volume. For example, if the volume percentage of particles is 20% of the total volume of the fluid, the preferred ratio of the first open volume to the second open volume is greater than 1:4 (e.g., 1.1:4). If the volume percentage of particles is 75% of the total volume of the fluid, the preferred ratio of the first open volume to the second open volume is greater than 3:1 (e.g., 3.1:1).

[0038] In some cases, the ratio of first open volume 270 to second open volume 280 is 1:1 or greater, 1.1:1, 1.2:1, 1.3:1, or 1.4:1 or greater, and is up to 2.0:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, or up to 1.5:1. Bracket 272 indicates the height of first open volume 270, and bracket 282 indicates the height of second open volume 280 and indicates that in this device 220, second volume 280 is stacked directly on top of first open volume 270 when device 220 is positioned as shown by the z and y axes. It should be noted that the device 200 in Figures 3A and 3B is not to scale where the ratio of first open volume 270 to second open volume 280 is 1:1 or greater, and is shown to aid in describing the devices and methods provided herein.

[0039] If desired, it may also be useful to select a particular relationship (e.g., ratio) between the average height of the plurality of microstructures and the average width of the pitch between each of the plurality of microstructures. Preferably, the first open volume is large enough so that, when all red blood cells have settled, no red blood cells protrude above the top of the microstructures.

[0040] As described above, at least a portion of the outer surface of the plurality of microstructures is configured to enable capillary action. Capillary action is known in the art to refer to fluid flow without the assistance of external forces, typically for aqueous fluids (having 50% or more water by volume) in contact with a hydrophilic surface. Thus, when blood is introduced into the first aperture of the device, the blood spontaneously transports along the outer surface of the microstructure, thereby spreading within the area of ​​the microstructure. Two general factors affect the ability of a microstructure to spontaneously transport fluid are (i) the structure or topography of the surface (e.g., capillary, cavity shape) and (ii) the properties of the surface (e.g., surface energy). To achieve a desired amount of fluid transport capacity, designers may adjust the structure or topography of the substrate layer and / or the surface energy of the capillary microstructured surface. To achieve wicking, the surface of the capillary microstructure must be "wettable" by the transported liquid (e.g., a substance in a liquid state). Optionally, the wettability of a solid surface by a liquid is characterized by the contact angle that a liquid makes with the solid surface after being deposited on a horizontally positioned surface and allowed to settle thereon. This angle is sometimes referred to as the "static equilibrium contact angle," or simply the "advancing contact angle" herein. In some cases, a material is considered hydrophilic if it has an advancing contact angle of less than 90 degrees, while a hydrophilic surface exhibits an advancing (maximum) water contact angle of less than 90 degrees, preferably 45 degrees or less.

[0041] A sufficient portion of the exterior surface of the microstructures must be hydrophilic so that when fluid is introduced through the first aperture, the microstructured substrate can undergo capillary action to transport the fluid (e.g., blood) throughout the device, allowing solid particles to access the open volume between the microstructures and settle out of the bulk of the fluid. In some cases, 50% or more of the area of ​​the exterior surface of the microstructures can undergo capillary action, and 60%, 70%, 80%, 90%, or 95% or more of the exterior surface of the microstructures can undergo capillary action. In selected embodiments, at least a portion of the major surface 223 of the cover 220 facing the microstructured substrate 230 is hydrophilic to aid in capillary action of the fluid within the device 200. The hydrophilicity of the exterior surface of the microstructures and / or the major surface of the cover, according to any device described herein, can be achieved by one or more of material selection, additives included in the material, or surface treatment. In some embodiments, the microstructures have an exterior surface that includes a surfactant, a surface treatment, a hydrophilic polymer, a flocculating agent, or any combination thereof. Suitable surfactants include, but are not limited to, C8-C18 alkane sulfonates, C8-C18 secondary alkane sulfonates, alkyl benzene sulfonates, C8-C18 alkyl sulfates, alkyl ether sulfates, sodium laureth 4 sulfate, sodium laureth 8 sulfate, dioctyl sulfosuccinate, sodium salt, lauroyl lacrylate, stearoyl lactylate, or any combination thereof. The one or more surfactants can be applied by conventional methods, for example, by spreading a coating of the surfactant on the microstructured surface and allowing the coating to dry. Suitable surface treatments include hydrophilic coatings comprising plasma-deposited silicon / oxygen materials and / or diamond-like glass (DLG) materials. Plasma deposition of silicon / oxygen materials and DLG materials is described, for example, in PCT Publication WO 2007 / 075665 (Somasiri et al.).Further examples of suitable DLG materials are disclosed in U.S. Patent Nos. 6,696,157 (David et al.), 6,881,538 (Haddad et al.), and 8,664,323 (Iyer et al.). Suitable hydrophilic polymers include, but are not limited to, polyesters, polyamides, polyurethanes, poly(vinyl alcohols), poly(alkylene glycols), poly(alkylene oxides), poly(vinylpyrrolidones), rubber elastomers, or any combination thereof.

[0042] It is known to use certain ionic polymers, particularly cationic polymers, for the aggregation of cells and / or cell debris and for the precipitation of proteins. When a flocculant is used, a device with larger microstructures (e.g., height and / or depth, pitch between adjacent microstructures, etc.) may be used than when a flocculant is not used. This is because particles tend to agglomerate and have larger sizes when aggregated, so more space may be required to retain the particles within the microstructure. In contrast, devices with larger microstructures may be less effective at retaining particles (e.g., red blood cells) separated from unagglomerated fluid (e.g., blood). Therefore, the microstructured surface of the device may be selected in part based on the expected size of the particles or aggregated particles.

[0043] Polymers used as aggregating agents can be unmodified (e.g., polyethyleneimine) or modified (e.g., guanylated polyethyleneimine). In some embodiments, suitable aggregating agents are hydrophilic and non-hemolytic (i.e., do not lyse red blood cells). Some suitable aggregating agents are described in detail in U.S. Pat. Nos. 8,435,776 (Rasmussen et al.) and 10,005,814 (Rasmussen et al.), which are incorporated herein by reference in their entireties. In certain cases, the aggregating agent comprises an unmodified or modified (e.g., functionalized) amino polymer. Suitable amino polymers can be selected from the group consisting of, for example, polyethyleneimine, polylysine, polyaminoamide, polyallylamine, polyvinylamine, polydimethylamine-epichlorohydrin-ethylenediamine, polydiallyldimethylammonium chloride, cationic polyacrylamide (CPAM), polyaminosiloxane, and dendrimers formed from polyamindoamine (PAMAM) and polypropyleneimine. Suitable modified amino polymers can be prepared by functionalizing one or more amino polymers selected from the group consisting of polyethyleneimine, polylysine, polyaminoamide, polyallylamine, polyvinylamine, polydimethylamine-epichlorohydrin-ethylenediamine, polydiallyldimethylammonium chloride, CPAM, polyaminosiloxane, and dendrimers formed from PAMAM and polypropyleneimine. For example, functionalization can include reacting the amino polymer with an alkylating agent, an acylating agent, or a guanylating agent. In selected embodiments, the flocculating agent comprises a modified or unmodified polyethyleneimine polymer.

[0044] In some embodiments, suitable agglomerating agents include modified or unmodified materials selected from the group consisting of gelatin, collagen, fibrinogen, dextran, hydroxyethyl starch (HES), pentastarch, polyvinylpyrrolidone (PVP), and polyethylene glycol (PEG). In selected embodiments, the agglomerating agent includes modified or unmodified gelatin.

[0045] The one or more flocculants can be applied by conventional methods, for example, by applying a coating of the flocculant onto the surface of the microstructures and allowing the coating to dry.

[0046] In some cases, the agglutinating agent has a weight average molecular weight (Mw) of 5,000 grams per mole (g / mol) or more, 10,000 g / mol, 20,000 g / mol, 30,000 g / mol, 40,000 g / mol, 50,000 g / mol, 60,000 g / mol, 70,000 g / mol, 80,000 g / mol, 90,000 g / mol, 100,000 g / mol, 110,000 g / mol, 120,000 g / mol, 130,000 g / mol, 140,000 g / mol, or 150,000 g / mol or more, and up to 500,000 g / mol, as measured by gel permeation chromatography. Sometimes, a high molecular weight, for example, 50,000 g / mol or more, can be useful for agglutinating particles (e.g., red blood cells).

[0047] Advantageously, at least a portion of the agglutinating agent tends to dissolve, disperse, or a combination thereof in the fluid (e.g., blood) after the device is filled with a volume of fluid. In some cases, the amount of agglutinating agent used is greater than or equal to 0.01 micrograms per milliliter of fluid (μg / mL), 0.1 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, 25 μg / mL, 50 μg / mL, 75 μg / mL, 100 μg / mL, 150 μg / mL, 250 μg / mL, 500 μg / mL, 750 μg / mL The flocculant concentration in a volume of fluid sample (e.g., blood) is designed to be greater than or equal to 1000 μg / mL, 1500 μg / mL, and less than or equal to 5000 μg / mL, 4000 μg / mL, 3000 μg / mL, 2000 μg / mL, 1000 μg / mL, 500 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL, 10 μg / mL, or 2 μg / mL. Stated another way, in some embodiments, the flocculant is present in a volume of fluid (e.g., blood) in an amount between 0.01 μg / mL and 5000 μg / mL. The amount of flocculant when in the form of a dry coating on the surface of the device will vary based on the molecular weight (Mw) of the particular flocculant.

[0048] 3A and 3B also incorporate an illustration on an SEM image of the device 200 to provide a concept of how the device is typically used. For example, after blood 295 fills the device 200 using capillary action, red blood cells 290 begin to settle between the microstructures 230 and into the first open volume 270. In FIG. 3A, only three red blood cells 290 are depicted as being located within the first open volume 270. Referring to FIG. 3B, the device 200 is depicted after waiting a sufficient time for all of the red blood cells 290 to settle within the first open volume 270 of the plurality of microstructures 230. In many cases, the red blood cells settle due to gravity alone. A volume of blood 295, from which the red blood cells have been removed, is present in the second open volume 280. In use, when the device is fluidly connected to the collection article 100 at either the first aperture (not shown) or the second aperture 260, some amount of the initial volume of blood, from which at least a portion of the red blood cells have been drawn into the first open volume 270 of the plurality of microstructures 230, flows out of the device 200 onto the collection article 100 by capillary action.

[0049] The collection article 100 is not particularly limited as long as it is capable of capillary action. Numerous types of collection articles are described below. The collection article 100 depicted in FIG. 3B comprises a microstructured substrate 104 comprising a plurality of microstructures 102 extending across a first surface 101 of the microstructured substrate 104. Each of the microstructures 102 has a top surface 103. The collection article 100 is positioned such that the top surfaces 103 of the plurality of microstructures 102 are located closer to the top surfaces 221 of the plurality of microstructures 230 of the device 200 than to the second, opposite surface 211 of the microstructured substrate 210 of the device 200. In other words, the positions of the microstructures 102 of the collection article 100 are inverted relative to the positions of the microstructures 230 of the device 200. Typically, fluids tend to flow preferentially to dry surfaces configured for capillary flow, so blood 295 can be caused to flow out of second volume 280 of device 200 by abutting edge 105 of collection device 100 against aperture (250 or 260) of device 200 such that edge 105 contacts blood 295. If desired, blood can then be removed from the collection article via wicking (e.g., into a test container for analysis, etc.).

[0050] Optionally, the method further includes passing the fluid (e.g., blood) through a filter before entering the device, after exiting the device, or both. For example, filtering the fluid can be useful for removing at least one undesirable component from the fluid. Suitable filters include, but are not limited to, nonwoven filters, woven filters, membrane filters, paper filters, and sponge filters. Exemplary suitable filters include, for example, glass fiber filters and asymmetric polysulfone / polyethersulfone filters (e.g., VIVID Plasma Separation membranes commercially available from Pall Corporation (Port Washington, NY), or the Cobetter OneStep Plasma Separation Membrane or Cobetter RB series, both commercially available from Cobetter Filtration Equipment Co., Ltd. (Hangzhou, China)).

[0051] In some cases, the method further includes adding an agglutinating agent to the volume of fluid (e.g., blood) prior to filling the device with the volume of fluid (e.g., blood). The agglutinating agent may be as described in detail above, including at a concentration present in the volume of fluid (e.g., an amount of 0.01 micrograms to 5000 micrograms per mL of fluid).

[0052] Red blood cells tend to comprise 35% to 50% of the total volume of whole blood. A ratio of 1:1 or greater between the first and second open volumes provides sufficient space between the microstructures in the first open volume to retain up to all red blood cells in an undiluted whole blood sample, while leaving the second open volume free for blood containing few to no red blood cells. Thus, when the device is contacted with a collection article after sedimentation, the blood (and possibly plasma) from which the red blood cells have been removed preferentially exits the device because it is located closer to the aperture. In contrast, sedimented red blood cells tend to be retained between the microstructures in the first open volume and are less likely to be removed from the device by contact with the collection article. In some cases, the ratio of the first and second open volumes is selected to minimize the volume of blood that may settle into the first open volume along with the red blood cells, while maximizing the volume of blood (from which the red blood cells have been removed) that can be flowed out of the device for analysis. By selecting a ratio of the first open volume to the second open volume of 1:1 or greater, dilution of the whole blood is typically not required for effective separation of red blood cells from the blood, and thus the blood can be used in undiluted form. However, with diluted blood, smaller ratios can be used, such as 0.9:1 or less, 0.8:1, 0.7:1, 0.6:1, or even 0.5:1 or less.

[0053] Advantageously, devices according to at least certain embodiments of the present disclosure effectively separate (at least a portion of) red blood cells from small volumes of blood while minimizing retention of analyzable blood within the device. This is in contrast to devices that use flow streams or that result in dead volume losses. Examples of red blood cell separation devices that require flowing blood include U.S. Patent Application Publication No. 2008 / 0135502 (Pyo et al.), U.S. Patent No. 11,262,347 (Yun et al.), U.S. Patent No. 10,518,196 (Puleo et al.), Korean Patent Application Publication No. 2010 / 0048507 (Chun et al.), and Taiwanese Patent No. I338134 (Chou et al.).

[0054] Preferably, at least 10%, 15%, 20%, 25%, 30%, 35%, or even at least 40% of the red blood cell-depleted blood flows out of the device upon contact with the collection article. The greater the percentage, the more efficient the separation method using a device according to the present disclosure and the less analyzable sample volume is lost to the device.

[0055] Devices according to at least certain embodiments of the present disclosure are suitable for use in removing solid particles (e.g., red blood cells) from fluids (e.g., blood) when the sample volume is 120 microliters or less, 110 microliters, 100 microliters, 90 microliters, 80 microliters, 70 microliters, 60 microliters, 50 microliters, 40 microliters, 30 microliters, 20 microliters, 10 microliters, or even 5 microliters or less, and 1 microliter or more, 2 microliters, 3 microliters, 4 microliters, 5 microliters, 6 microliters, 7 microliters, 8 microliters, 9 microliters, 10 microliters, 12 microliters, 15 microliters, 25 microliters, 35 microliters, or 45 microliters or more. For example, the sum of the first open volume and the second open volume of the device can be between 5 microliters and 120 microliters.

[0056] In some cases, the first aperture is defined by the cover of the device, allowing a volume of fluid (e.g., blood) to be introduced into the device using gravity and / or wicking of the fluid into the aperture. In other cases, the first aperture is defined by the microstructured substrate. For example, referring to FIG. 2B , the first aperture 250 can be provided as a reservoir configured to hold at least a certain minimum volume of fluid. The reservoir can be defined by a portion of the microstructured substrate 210 adjacent one end 237 of the microstructure 230. Similarly, the second aperture 260 can be a reservoir configured to hold fluid as it exits the microstructured surface 202 of the microstructured substrate 210. Often, it is advantageous for the second aperture to be located a distance away from the first aperture to facilitate the evacuation of displacement gas (typically air) from the device as fluid is deposited into the device through the first aperture. Optionally, a positive pressure can be provided by a pump to deposit a volume of blood into the first aperture. Optionally, a volume of blood can be wicked into the device through the first aperture. In some cases, it may be preferable for blood 295 to exit through the second aperture 260.

[0057] Louver structure 3A and 3B, the microstructure 230 of certain embodiments of the device comprises a "louver structure" with ribs separated by channels. This configuration is also shown in FIG. 4A, where the microstructure 230 comprises a plurality of ribs 230 alternating with channels 201 extending across the first surface 202 of the microstructured substrate 210, each of the ribs 230 comprising sidewalls 233 and 234 and a top surface 221, and each of the channels 201 comprising a bottom surface 205.

[0058] More specifically, the depicted microstructured substrate 210 comprises a plurality of channels 201a-201d on a base layer 213. As shown in FIG. 4A, a continuous land layer "L" may exist between the bottoms 205 of the channels and the top surface 202 of the base layer 213. Alternatively, the channels 201 may extend across the entire microstructured substrate 210. In some cases (as shown in FIG. 4B), the bottoms 205 of the channels may coincide with the top surface 202 of the base layer 213. In an exemplary embodiment, the base layer 213 is a preformed film comprising a different organic polymeric material than the ribs 230.

[0059] The height and width of rib (e.g., protrusion) 230 is defined by adjacent channels (e.g., 201a and 201b). Rib 230 can be defined by a top surface 221, a bottom surface 231, and sidewalls 233 and 234 that join top surface 221 to bottom surface 231. Sidewalls 233 and 234 can be parallel to one another. More typically, the sidewalls have a wall angle.

[0060] The ribs 230 can be defined by a width "W." Often, the ribs 230 have a width parallel to the first surface of the microstructured substrate and a height perpendicular to the first surface of the microstructured substrate. Excluding the land areas "L," the ribs 230 typically have nominally the same height as the channels 201. In typical embodiments, the height "H" of the channels 201 and / or ribs 230 is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 micrometers. In some embodiments, the height is no greater than 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, or 100 micrometers. In some embodiments, the height of the channels 201 and / or ribs 230 is in the range of 50 micrometers to 250 micrometers. The microstructured substrate typically comprises a plurality of ribs 230 having nominally the same height and width. In some embodiments, the ribs 230 have a height "H," a maximum width "W" at their widest point, and an aspect ratio H / W of at least 1.5. In some embodiments, H / W is at least 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.5, 4.0, 4.5, or 5.0. In other embodiments, the aspect ratio of the ribs is at least 6, 7, 8, 9, or 10. In other embodiments, the aspect ratio of the ribs is at least 15, 20, 25, 30, 35, 40, 45, or 50.

[0061] The channels 201 have a height "H" defined by the distance between the bottom surface 205 and the top surface 221, with such top and bottom surfaces typically parallel to the top surface 202 of the base layer 213. The channels 201 have a maximum width "W" and are spaced apart along the microstructured surface 202 by a pitch "P." The width "W" of a channel at its base (i.e., adjacent the bottom surface 205) is typically nominally the same as the width of the channel adjacent the top surface 221. However, if the width of a channel at its base differs from its width adjacent the top surface, the width is defined by the maximum width. The maximum widths of multiple channels can be averaged over a region of interest, such as the region for calculating the first open volume. A microstructured substrate may comprise multiple channels having nominally the same height and width. In typical embodiments, the channels generally have widths of 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 micrometer or less. In some embodiments, the channels generally have widths of no more than 900, 800, 700, 600, or 500 micrometers, hi some embodiments, the channels have widths of at least 50, 60, 70, 80, 90, or 100 micrometers.

[0062] In some embodiments, the wall angle θ is greater than 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 degrees. In some embodiments, the wall angle is less than or equal to 110, 109, 108, 107, 106, 105, 104, 103, 102, 101, 100, 99, 98, 97, 96, or 95 degrees. In some embodiments, the wall angle approaches 90 degrees. When the wall angle is 90 degrees, the angle between the channel 201 and the top surface 221 is also 90 degrees. Depending on the wall angle, the rib can have a rectangular or trapezoidal cross-section. In some embodiments, the sidewalls can be described as including first and second sidewalls, with the first sidewall having a wall angle with a line parallel to the first surface of the microstructured substrate between 0 degrees and +10 degrees or between 0 degrees and -10 degrees relative to the bottom surface of the microstructured substrate.

[0063] In some embodiments, the ribs 230 have a pitch "P" of at least 10 micrometers. The pitch is the distance between the beginning of a first rib and the beginning of a second rib, as depicted in FIG. 4A. The pitch can be at least 15, 20, 25, 30, 35, 40, 45, or 50, 60, or 70 micrometers. The pitch is generally 1 mm or less. The pitch is typically 900, 800, 700, 600, or 500 micrometers or less. In some embodiments, the pitch is typically 550, 500, 450, 400, 350, 300, 250, or 200 micrometers or less. In some embodiments, the pitch is 175, 150, 100 micrometers or less. In typical embodiments, the ribs are uniformly spaced and have a single pitch. Alternatively, the ribs can be spaced such that the pitch between adjacent ribs is not the same. In this latter embodiment, at least some, typically a majority (at least 50, 60, 70, 80, 90% or more of all ribs) have the above pitch. The pitch of the channels is within the same ranges as described for the ribs. Optionally, the channels have an average pitch of 10 micrometers to 200 micrometers. The pitch and height of the ribs can be important to facilitate coating of the ribs with the coating. If the ribs are spaced too closely together, it can be difficult to uniformly coat the sidewalls. If the ribs are spaced too far apart, the coating may not be effective in providing its intended function.

[0064] A variation of the louver structure of Figure 4A is depicted in Figure 4B, where in some cases, the top surface 221 of each rib 230 is the apex of a cap 235 disposed on the sidewalls 233, 234, the cap 235 having a width ("CW") greater than the width ("WW") between the opposing sidewalls 233, 234. Without wishing to be bound by theory, it is believed that the presence of caps on the ribs (e.g., undercut features) may help retain particles in the channels as they settle into the device.

[0065] Louver structures can be prepared by any suitable method. In one embodiment, a structure, such as the microstructured substrate 210 shown in FIG. 4A, can be prepared by a method including: (a) preparing a polymerizable composition; (b) depositing the polymerizable composition onto a negative microstructured molding surface (e.g., a tool) of a master in an amount just sufficient to fill the cavities of the master; (c) filling the cavities by transferring a bead of the polymerizable composition between the master and a base layer (e.g., a preformed film), at least one of which is flexible; and (d) curing the composition. Deposition temperatures can range from ambient to about 180°F (82°C). The master can be a metal, such as nickel, chromium, or nickel-plated copper or brass, or a thermoplastic material that is stable under polymerization conditions and has a surface energy that allows the polymerized material to be cleanly removed from the master. If the base layer is a preformed film, one or more of the surfaces of the film may be optionally primed or otherwise treated to promote adhesion with the microstructured organic material.

[0066] In one embodiment, a structure, e.g., the microstructured substrate 210 shown in Figure 4B with caps on the ribs, can be prepared by known methods for creating undercut features (e.g., by partially disassembling a multi-part mold after molding to open each of the cavities and allow for easy removal of the features, or by first molding straight ribs without undercuts and then forming caps on their stems in a separate molding step after demolding). Alternatively, such a microstructured substrate may be made according to the disclosure of PCT Publication WO 2015 / 041844 (Rule et al.), in which a polyolefin resin is deposited into a mold cavity to form a first layer including a plurality of undercut features on and extending from a unitary backing, and the first layer is demolded from the mold cavity at a rate of at least 150 millimeters per minute (mm / min).

[0067] The polymerizable resin can include a combination of first and second polymerizable components selected from (meth)acrylate monomers, (meth)acrylate oligomers, and mixtures thereof. As used herein, "monomer" or "oligomer" refers to any substance that can be converted into a polymer. The term "(meth)acrylate" refers to both acrylate and methacrylate compounds. In some cases, the polymerizable composition can include (meth)acrylated urethane oligomers, (meth)acrylated epoxy oligomers, (meth)acrylated polyester oligomers, (meth)acrylated phenolic oligomers, (meth)acrylated acrylic oligomers, and mixtures thereof.

[0068] The polymerizable resin may be a radiation-curable polymer resin, such as a UV-curable resin. In some cases, polymerizable resin compositions useful in the microstructured substrates of the present disclosure may include those described in U.S. Pat. No. 8,012,567 (Gaides et al.).

[0069] The chemical composition and thickness of the base layer can depend on the end use of the microstructured substrate. In a typical embodiment, the thickness of the base layer can be at least about 0.025 millimeters (mm), and can be from about 0.05 mm to about 0.25 mm. Useful base layer materials include, for example, styrene-acrylonitrile, cellulose acetate butyrate, cellulose acetate propionate, cellulose triacetate, polyethersulfone, polymethyl methacrylate, polyurethane, polyester, polycarbonate, polyvinyl chloride, polystyrene, polyethylene naphthalate, copolymers or blends based on naphthalenedicarboxylic acid, polyolefin-based materials such as cast or stretched films of polyethylene, polypropylene, and polycycloolefin, polyimide, and glass. If desired, the base layer can contain mixtures or combinations of these materials. In some embodiments, the base layer can be multilayered or contain dispersed components suspended or dispersed in a continuous phase.

[0070] Examples of base layer materials include polyethylene terephthalate (PET) and polycarbonate (PC). An example of a useful PET film is photo-grade polyethylene terephthalate available from DuPont Films (Wilmington, Del.) under the trade name "Melinex 618." Examples of optical-grade polycarbonate films include LEXAN polycarbonate film 8010 available from GE Polymershapes (Seattle, WA) and Panlite 1151 available from Teijin Kasei (Alpharetta, GA).

[0071] Alternatively, the microstructured substrate 210 can be prepared by melt extrusion, i.e., casting a fluid resin composition onto a master negative microstructured molding surface (e.g., a tool) and allowing the composition to solidify. In this embodiment, the ribs 230 are interconnected in a continuous layer to the base layer 213. The individual ribs and the connections between them generally comprise the same thermoplastic material. The thickness of the land layer (i.e., excluding the portion resulting from the replicated microstructure) is typically 0.001 inches to 0.100 inches, preferably 0.003 inches to 0.010 inches. Resin compositions suitable for melt extrusion are dimensionally stable, durable, weather-resistant, and transparent materials that can be easily molded into the desired shape. Examples of suitable materials include acrylics, such as Plexiglas brand resins manufactured by Rohm and Haas Company (Philadelphia, PA); polycarbonates; reactive materials, such as thermosetting acrylates and epoxy acrylates; polyethylene-based ionomers, such as those sold under the SURLYN brand name by Dow Chemical (Midland, MI) EI Dupont de Nemours and Co., Inc.; (poly)ethylene-co-acrylic acid; polyesters; polyurethanes; and cellulose acetate butyrate.

[0072] In yet another embodiment, the master negative microstructured molding surface (eg, tool) can be used as an embossing tool, as described, for example, in US Pat. No. 4,601,861 (Pricone).

[0073] Further details regarding microstructured substrates having such louvered structures and methods for forming them are described in WO 2019 / 118685 (Schmidt et al.) and WO 2020 / 026139 (Schmidt et al.), each of which is incorporated herein by reference.

[0074] Prism Structure FIG. 5A illustrates an alternative microstructured substrate 309 comprising a linear array of regular prisms 320. Each prism has a first facet 321 and a second facet 322. The prisms are typically formed on a base member 310 (e.g., a preformed polymer film) having a first plane 331 on which the prisms are formed and a second surface 332 that is substantially flat or planar and opposite the first surface. In some embodiments, the prisms are right-angle prisms. By right-angle prism, we mean that the apex angle θ, 340, is typically approximately 90°. However, this angle may range from 5° to 90°, or even from 20° to 80°. In selected embodiments, the microstructures comprise linear prisms having an apex angle of 90° or less. In some embodiments, the apex angle of the peak structures is typically twice the wall angle, particularly when the facets of the peak structures are interconnected by valleys between the peak structures. Thus, the apex angle is typically greater than 5 degrees, more typically at least 25, 30, 35, 40, 45, 50, 55, or 60 degrees. The apex angle of the peak structures is typically less than 90 degrees, more typically less than 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, or 35 degrees. Optionally, there is a gap between adjacent peak structures 320, for example, in or parallel to the first plane 331.

[0075] These apexes may be sharp (as shown), rounded, or truncated. In some cases, using sharp or rounded apexes is advantageous because particles are less likely to settle on the apex than on truncated (e.g., flat) apexes. Preferably, the radius of the prism apex is smaller than the radius of the particle (e.g., red blood cell). The spacing between (e.g., prism) peaks may be characterized as the pitch ("P"). In this embodiment, the pitch is also equal to the maximum width of the valley. Thus, the pitch, as noted above, ranges from greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 microns, up to 250 microns. The length ("L") of the prismatic microstructure is typically the largest dimension and can span the dimensions of the microstructured surface, film, or article. The prism facets need not be identical, and the prisms may be angled relative to one another. The facets of adjacent peak structures are typically connected at the bottom of the valley, i.e., adjacent to the planar base layer. The facets of the peak structures form a continuous surface in the same direction. For example, in Figure 5A, facets 321 and 322 of the prismatic peak structures are continuous along the length (L) of the microstructure, i.e., the y-direction.

[0076] Optionally, a continuous land layer 360 can be present between the bottom of the channel or valley and the top surface 331 of the (e.g., flat) base member 310. In some embodiments, for example, when the microstructured surface is prepared from casting and curing a polymerizable resin composition, the thickness of the land layer typically ranges from at least 0.5, 1, 2, 3, 4, or 5 microns to up to 50 microns. In some embodiments, the thickness of the land layer is no greater than 45, 40, 35, 30, 25, 20, 15, or 10 microns.

[0077] 5B, a cross-sectional SEM image of a portion of an exemplary device of Preparative Example 12 having prismatic microstructures is provided. First surface 302 of microstructured substrate 325, together with at least one sidewall 340, defines a first open volume 370 that is the sum of the open spaces located between multiple microstructures 320 from the bottom 336 to the top 327 of each microstructure 320. (For simplicity, the arrow in 370 only points to the portion of the first open volume between two adjacent microstructures 320.) The cover 352, together with the top 327 and at least one sidewall 340 of the first surface 302 of the microstructured substrate 325, defines a second open volume 380 located adjacent to the first open volume 370, and the first open volume has a percentage of the 100% open volume that is greater than the volume percentage of red blood cells present in the blood, where the combined total of the first open volume and the second open volume is 100% open volume.

[0078] In selected embodiments, the microstructure comprises an array of peak structures and adjacent valleys, the valleys having a maximum width in the range of 10 microns to 250 microns, and the peak structures having an apex angle of greater than 5 degrees up to 90 degrees.

[0079] The orientation of the linear array of peak structures and adjacent valleys extending across the first surface of the microstructured substrate is not particularly limited, and they may be disposed at an angle between 0 and 90 degrees relative to the flow direction of the device. More specifically, the peak structures and adjacent valleys may be disposed at an angle of 0 degrees or greater, 5 degrees, 10 degrees, 15 degrees, 20 degrees, 25 degrees, 30 degrees, 35 degrees, 40 degrees, 45 degrees, 50 degrees, 55 degrees, 60 degrees, 65 degrees, 70 degrees, or 75 degrees or greater, and 90 degrees, 85 degrees, 80 degrees, 75 degrees, 70 degrees, 65 degrees, 60 degrees, 55 degrees, 50 degrees, 45 degrees, 40 degrees, 35 degrees, 30 degrees, 25 degrees, 20 degrees, or 15 degrees or less. For example, in device 300 of FIG. 5B, peak structures 320 and valleys 301 are disposed at an angle of 0 degrees relative to the flow direction of device 300 (e.g., in the same direction as the flow). On the microstructured substrate 325 of Figure 5C, the peak structures 322 and valleys 301 are arranged at a 90 degree angle (e.g., perpendicular) relative to the flow direction ("F") of a device including the microstructured substrate 325. On the microstructured substrate 325 of Figure 5D, the peak structures 320 and valleys 301 are arranged at a 45 degree angle relative to the flow direction ("F") of a device including the microstructured substrate 325. By selecting an arrangement orientation greater than 0 degrees (and, for example, up to 90 degrees), particles have a greater opportunity to come into contact with two or more microstructures in the direction of fluid flow into the device, potentially increasing the likelihood of being trapped within the microstructures.

[0080] Further details about microstructured substrates having such peak structure arrays and methods for forming them are described in U.S. Patent Application Publication No. 2021 / 0187819 (Connell et al.), which is incorporated herein by reference.

[0081] Facet Structure Similar to the prismatic structure, Figure 5E illustrates an alternative microstructured substrate 700 having a faceted structure. More specifically, Figure 5E illustrates a microstructured substrate 700 defining a bottom surface 705, a top surface 720, a first sidewall portion 732, and a facet 733. Stated another way, the microstructure comprises a facet 733 and a sidewall 732 that meets the facet 733 at a ridge 720 of the microstructure. The facet 733 and the sidewall 732 typically define an oblique angle therebetween.

[0082] In selected embodiments, the microstructures comprise facets and sidewalls that meet the facets at ridges of the microstructures, the facets and sidewalls defining an oblique angle therebetween.

[0083] Further details regarding microstructured substrates having such faceted structures and methods for forming them are described in WO 2020 / 250180 (Kenney et al.), which is incorporated herein by reference.

[0084] Protrusion array structure FIG. 6A illustrates another alternative microstructured substrate 400 having a protrusion array structure. More specifically, FIG. 6A is a schematic side view of a microstructured substrate 400 having a two-dimensional (x-axis and y-axis) array of protrusions 410 arranged across a first surface 420. Each of the protrusions 410 comprises a base 412, an apex 414, and one or more sides 416, 418 connecting the apex to the base. Optionally, each of the protrusions 410 is a spaced-apart post. For example, FIG. 6B is a top view of four representative engineered micropatterned regions for a two-dimensional array of protrusions, including spaced-apart posts 410 present in all but the bottom right image. Some microstructured surfaces may comprise protrusions with a range of aspect ratio values, such as an array of protrusions having a constant height and variable widths. In such cases, the surface is typically characterized by a maximum aspect ratio value.

[0085] In selected embodiments, the microstructure comprises a two-dimensional (x-axis and y-axis) array of protrusions arranged across the first surface of the microstructured substrate, each of the protrusions comprising a base, a top, and one or more sides connecting the top to the base.

[0086] Further details about microstructured substrates having such protrusion arrays and methods for forming them are described in WO 2020 / 097319 (Wolk et al.), which is incorporated herein by reference.

[0087] Cavity Array Structure 7A shows a further alternative microstructured substrate 500a having a cavity array structure. A "cavity array" is defined as a cavity array having at least about 100 cavities per cm. 2 , preferably at least about 10 / mm 2The cavity has a three-dimensional structure with dimensions such as openings having a diameter in the range of about 5 micrometers to 250 micrometers and a depth in the range of about 2 micrometers to 250 micrometers. The array may be any regular array, such as a close-packed array or a rectangular array, or the cavities may be randomly distributed. More specifically, FIG. 7A is a schematic cross-sectional view of a microstructured substrate 500a having a plurality of cavities 522 extending between a first major surface 514 and a second major surface 516. The microstructured substrate 500a comprises a microstructured layer 510 having a first major surface 514 and a second major surface 516, the microstructure comprising a plurality of cavities 522 extending between the first major surface 514 and the second major surface 516. Each cavity comprises a first opening 524, a second opening 528, and at least one sidewall 526 extending between the first opening 524 and the second opening 528. Each of the sidewall(s) 526 forms a sidewall angle θ with a line 515 normal to the first major surface 514 of the microstructured layer 510. Each of the cavities 522 further comprises a depth “D,” which is the perpendicular distance between the first aperture 524 and the second aperture 528. Optionally, the microstructured substrate 500a further comprises either an adhesive layer 540, a first substrate layer 530, or a second substrate layer 550.

[0088] 7B is a generalized schematic top perspective exploded view of a microstructured substrate 500b having a plurality of cavities 522 extending between two major surfaces. The microstructured substrate 500b includes a microstructured layer 510 having a first major surface 514 and an opposing second major surface 516. The first major surface 514 includes an array of discrete cavities 522. In one particular embodiment, each of the cavities 522 includes a cross-section parallel to the first major surface 514 that may be circular, elliptical, or polygonal. The cross-sections optionally decrease in size in the direction from the first major surface 514 to the second major surface 516. This embodiment of the microstructured substrate 500b further includes a (e.g., flexible) substrate 530 coupled to the second major surface 516 of the microstructured layer 510.

[0089] In selected embodiments, the microstructured substrate comprises a microstructured layer having first and second major surfaces, the microstructure comprising a plurality of cavities extending between the first and second major surfaces, each cavity comprising a first opening, a second opening, and at least one sidewall extending between the first opening and the second opening.

[0090] Further details regarding microstructured substrates with such cavity arrays and methods for forming them are described in US Pat. No. 9,329,311 (Halverson et al.), which is incorporated herein by reference.

[0091] Connected well structure 10A-10B show yet a further alternative microstructured substrate 1025, in which the microstructures comprise an array of fluidly connected wells, at least some of which are fluidly connected to at least two adjacent wells, each connected via a vent. For example, FIG. 10A is a schematic perspective view of a portion of a microstructured substrate 1025 having an array of fluidly connected wells 1077 connected to each other by vents 1087.

[0092] Figure 10B is a schematic top view of a portion of the microstructured substrate 1025 of Figure 10A having an array of fluidly connected wells 1077 having circular shapes connected to adjacent wells 1077 by vents 1087. In the particular configuration shown in Figures 10A-10B, wells 1077 in the center of the microstructured substrate 1025 (e.g., with at least one other well located between it and the periphery of the microstructured substrate) are each connected to four other wells 1077, with each connection being via a vent 1087. There are also wells 1077 located adjacent the periphery 1095, which are connected to one or two adjacent wells 1077 via vents 1087. For example, referring to Figure 10A, well 1077c is at a corner of the array and is attached to only one other well 1077d via vent 1087a. Similarly, a well can be attached to three other adjacent wells via vents, or to four, five, six, seven, eight, nine, ten, eleven, or twelve other adjacent wells via vents.

[0093] The shape of the wells is not particularly limited and can include curved, polygonal, irregular, or a combination thereof. In some embodiments, the wells comprise a circle, a triangle, a rectangle, an ellipse, or a combination thereof. When the wells comprise a shape with corners, the vents are optionally located at the corners (e.g., to reduce the possibility of trapping air bubbles at the corners).

[0094] Typically, each well has an open volume large enough to hold at least one sedimenting particle (e.g., red blood cells), for example, 100 femtoliters or more, 250 femtoliters, 500 femtoliters, 750 femtoliters, 1 picoliter, 100 picoliters, 250 picoliters, 500 picoliters, 750 picoliters, 1 nanoliter, 100 nanoliters, 200 nanoliters, 300 nanoliters, 400 nanoliters, 500 nanoliters, 600 nanoliters, The wells have an open volume of 700 nanoliters, 800 nanoliters, or 900 nanoliters or more and 1 microliter or less, 900 nanoliters, 800 nanoliters, 700 nanoliters, 600 nanoliters, 500 nanoliters, 400 nanoliters, 300 nanoliters, 200 nanoliters, 100 nanoliters, 1 nanoliter, 750 picoliters, 500 picoliters, 250 picoliters, 1 picoliter, 750 femtoliters, or 500 femtoliters or less. Stated another way, in some cases, each well has an open volume in the range of 100 femtoliters to 1 microliter or 500 femtoliters to 0.1 microliters.

[0095] Some typical dimensions for each well include a depth (i.e., the distance between the top surface 1027 of the microstructured substrate 2025 and the bottom surface 1029 of the well, as shown in FIG. 10A ) of 50 microliters or more, 75 microliters, 100 microliters, 125 microliters, 150 microliters, 175 microliters, 200 microliters, 225 microliters, 250 microliters, 275 microliters, 300 microliters, 325 microliters, or 350 microliters or more, and 500 microliters or less, 475 microliters, 450 microliters, 425 microliters, 400 microliters, 375 microliters, 350 microliters, 325 microliters, 300 microliters, 275 microliters, 250 microliters, 225 microliters, 200 microliters, 175 microliters, or 150 microliters or less. This same range of distances is also applicable to the diameter of the well. The diameter is the longest line passing through the center point of the shape.

[0096] Preferably, at least some of the vents between wells are located at the same depth as the bottom of the adjacent wells. This helps encourage air bubbles to exit the wells without becoming trapped near the bottom of the wells. In some embodiments, the vents have a total depth equal to the total depth of the adjacent wells, although this is not required. When the microstructured substrate is formed using a tool, creating vents that have the same depth as the adjacent wells tends to be more practical than creating vents that only connect the lower parts of two wells.

[0097] 10A , in some embodiments, the microstructured substrate 1025 further includes at least one sidewall 1097 disposed along the perimeter 1095 of the first surface 1027 of the microstructured substrate 1025. The one or more sidewalls 1097 have a height (“H”) that extends beyond the top surface of the plurality of microstructures by between 50 micrometers and 250 micrometers.

[0098] The interconnected well structure can be fabricated using a multiphoton exposure system, such as that described in U.S. Pat. No. 8,605,256 (DeVoe et al.), to create a patterned tool. Such a structured polymer tool is then typically plated with nickel to create a metallized tool. The nickel-plated tool is then used to create an impression-molded specimen using a press, such as a Carver Press (Carver, Wabash, IN), and resin (e.g., polypropylene resin). The press platens are heated (e.g., to 170°C), and the tool and resin are then pressed together at high force (e.g., 1000 pound force) for several minutes (e.g., 5-10 minutes). After cooling (e.g., until the platen temperature reaches 80°C), the pressure is released and the molded specimen can be removed. Using such a method, a microstructured substrate can be provided in the form of a microstructured film.

[0099] Further details regarding microstructured substrates with such connected wells and methods for forming them are described in commonly owned patent application Ser. No. 63 / 425,473, which is incorporated herein by reference.

[0100] Stem Web Structure 13 illustrates a further alternative microstructured substrate 1320 having a stem web structure. In such an embodiment, the microstructures 1326 comprise an array of upstanding stems 1326 extending across the first surface 1324 of the microstructured substrate 1320. The microstructures 1326 are generally upstanding stems of various shapes. By "substantially upstanding," it is meant that the stems project (e.g., in a planar direction) from the first surface 1324. The stems 1326 may project upward from the surface 1324 at a generally normal angle, or the stems 1326 may project at an angle away from the surface 1324. The stems may also be irregularly shaped so that they do not project at any one uniform angle.

[0101] The microstructured substrate 1320 includes a backing layer 1321 having a first surface 1324 including an array of generally upright stems 1326. The stems 1326 may be arranged in a regular or irregular array. Various stem patterns, such as hexagonal, diagonal, or sinusoidal, may be used. The stems 1326 may be at least partially composed of an elastomeric material. In some cases, the entire exterior surface of the stems 1326 is an elastomeric material. In the embodiment of FIG. 13, the backing layer 1321 is integrally formed with the stems 1326. The combination of the backing layer 1321 and the stems 1326 is sometimes referred to as a stem web. While the illustrated embodiment shows the stems 1326 as generally cylindrical, the sides of the stems 1326 typically have a slight taper 1335 to facilitate removal from a mold. As shown, the taper 1335 is inward from the base 1302 to the tip 1304 of the stem 1326. It is expressly contemplated that the stem may be configured to have an outward taper from the base to the tip of the stem. Various non-cylindrical shapes may also be utilized, such as a truncated cone or pyramid, a rectangle, a hemisphere, a square, a hexagon, an octagon, a gumdrop, etc.

[0102] The backing layer 1321 from which the stem 1326 directly extends is typically about 0.05 millimeters to about 0.5 millimeters (0.002 inches to 0.02 inches) thick. Accordingly, additional backing layer(s) 1322 are optionally applied to the second surface 1325 to reinforce the backing layer 1321 and form a multi-layered base or backing structure. As used herein, "backing" or "base" layer is used to refer to the collective backing or base structure. Such a structure may be single-layered or multi-layered (e.g., as shown in FIG. 13 ) with one or more layers supporting the generally upright stem 1326, although typically at most one of these layers 1321 is integrally formed with the stem 1326.

[0103] The stems typically have a height 1328 ranging from about 0.2 mm to about 3 mm, preferably from about 0.2 mm to about 1.5 mm. The separation or gap 1330 between adjacent stems 1326 generally ranges from about 0.25 mm to about 2.5 mm, more typically from about 0.4 mm to about 1.0 mm. This separation gap creates a percentage of free volume, which is the volume within the stem web that is not occupied by stems. The percentage of free volume is typically 60% to 98% of the stem web, more typically 85% to 95%. The stems 1326 have a maximum cross-sectional dimension 1329 ranging from about 0.076 mm to about 0.76 mm. The stems 1326 are arranged on the backing at a density of at least 15.5 per square centimeter (100 per square inch), more typically at least 50 per square centimeter. The stem density is generally at most about 1500 / cm², more typically at most about 500 / cm². The stem has an aspect ratio of at least 1.25, preferably at least 1.5, and most preferably at least 2.0. The aspect ratio refers to the ratio of the height of the stem to the maximum cross-sectional dimension. For stems with a circular cross-section, the maximum cross-sectional dimension is the diameter of the stem.

[0104] Suitable elastomeric stem materials include elastomer classes such as anionic triblock copolymers, polyolefin-based thermoplastic elastomers, halogen-containing polyolefin-based thermoplastic elastomers, dynamically vulcanized elastomeric-thermoplastic blend-based thermoplastic elastomers, thermoplastic polyetherester or polyester-based elastomers, polyamide or polyimide-based thermoplastic elastomers, ionomeric thermoplastic elastomers, hydrogenated block copolymers in thermoplastic elastomeric interpenetrating polymer networks, carbocationically polymerized thermoplastic elastomers, polymer blends containing styrene / hydrogenated butadiene block copolymers, and polyacrylate-based thermoplastic elastomers. Some specific examples of elastomers are natural rubber, butyl rubber, EPDM rubber, silicone rubber, e.g., polydimethylsiloxane, polyisoprene, polybutadiene, polyurethane, ethylene / propylene / diene terpolymer elastomers, chloroprene rubber, styrene-butadiene copolymers (random or block), styrene-isoprene copolymers (random or block), acrylonitrile-butadiene copolymers, mixtures thereof, and copolymers thereof. The block copolymers may be linear, radial, or star-shaped in configuration, and may be diblock (AB) or triblock (ABA) copolymers or mixtures thereof. Blends of these elastomers with each other or with modified non-elastomers are also contemplated. Commercially available elastomers include block polymers (e.g., polystyrene materials containing elastomeric segments) available under the KRATON™ trademark from KRATON Polymers Company, Houston, Texas.

[0105] Any of a number of conventional additives may also be added to elastomeric resinous materials such as those described above, including, for example, plasticizers, tackifiers, fillers, antioxidants, UV absorbers, hindered amine light stabilizers (HALS), dyes or pigments, opacifiers, and the like.

[0106] Suitable backing layer materials include thermoplastic polyurethanes, polyvinyl chloride, polyamides, polyimides, polyolefins (e.g., polyethylene and polypropylene), polyesters (e.g., polyethylene terephthalate), polystyrene, nylon, acetal, block polymers (e.g., polystyrene materials containing elastomeric segments available under the KRATON™ name from KRATON Polymers Company, Houston, Texas), polycarbonates, thermoplastic elastomers (e.g., polyolefin, polyester, or nylon types), and copolymers and blends thereof. In some cases, the entire stem web is formed from one or more thermoplastic materials, such as those listed above. The thermoplastic materials may also contain additives, including, but not limited to, fillers, fibers, antistatic agents, lubricants, wetting agents, blowing agents, surfactants, pigments, dyes, coupling agents, plasticizers, suspending agents, hydrophilic / hydrophobic additives, adhesives, and the like.

[0107] Further details regarding microstructured substrates having such stem webs and methods for forming them are described in WO 2009 / 020811 (Tuman et al.), which is incorporated herein by reference.

[0108] Collectibles As mentioned above, the collection article is not particularly limited as long as it allows for capillary action. Therefore, suitable collection articles include, for example, films, membranes, nonwoven fibrous sheets, woven fibrous sheets, foams, etc. The collection article may include one or more cavities and / or a microstructured surface for retaining fluid (e.g., blood) from which at least a portion of the particles (e.g., red blood cells) have been removed within the device. Materials described in detail above with respect to the outer surface of the microstructure of the device that provide a configuration that allows for capillary action are also suitable for the collection article. For example, a material that forms at least a portion of a surface that exhibits an advancing contact angle with water of less than 90 degrees.

[0109] In some cases, the collection items may be a) a first polymer layer having a substantially planar first major surface and an opposite second major surface; b) a second polymer layer bonded to the first polymer layer, the second polymer layer having a first major surface and a second major surface, the first major surface of the second polymer layer conforming to the second major surface of the first polymer layer, the second major surface of the second polymer layer defining a cavity with at least one wall, the second polymer layer having a channel connecting the cavity to at least one edge of the second polymer layer or the first major surface of the first polymer layer, and a surface of the cavity exhibiting an advancing contact angle with water of less than 90 degrees; Equipped with.

[0110] 11A-11B, a generalized schematic diagram of such a collection article 1100 is provided. One suitable collection article 1100 comprises a first polymer layer 1110 having a substantially planar first major surface 1111 and an opposite second major surface 1113, and a second polymer layer 1120 bonded to the first polymer layer 1110 and having a first major surface 1121 and a second major surface 1123, wherein the first major surface 1121 of the second polymer layer 1120 conforms to the second major surface 1113 of the first polymer layer 1110 and the second major surface 1123 of the second polymer layer 1120 is at least and a second polymer layer 1120 defining a cavity 1150 with at least one wall 1154, wherein the second polymer layer 1120 has a channel 1160 connecting the cavity 1150 to at least one edge 1125 of the second polymer layer 1120 (as shown in FIG. 11A ) or the second polymer layer 1120 has a channel 1160 connecting the cavity 1150 to a first major surface 1111 of the first polymer layer 1110 (as shown in FIG. 11B ). A surface 1152 of the cavity 1150 exhibits an advancing contact angle with water of less than 90 degrees.

[0111] In the embodiment shown in FIG. 11A , the channel 1160 also connects the cavity 1150 to the second edge of the second polymer layer 1120. In this embodiment, the channel 1160 is deep enough to extend through the second polymer layer into the first polymer layer 1110, and therefore the channel 1160 also connects the cavity 1150 to the second edge 1117 of the first polymer layer 1110. Also in the embodiment of FIG. 1A , optionally, the cavity 1150 has a first volume 1151, and the channel 1160 in the second polymer layer 1120 has a second volume 1161, the second volume 1161 being 0.1% to 10% of the first volume 1151. The first polymer layer of the embodiment of FIG. 11A further includes a fiducial mark 1119, i.e., a diamond-shaped mark located on the first major surface 1111 of the first polymer layer 1110.

[0112] 11B, the channel 1160 has a generally cylindrical shape and can be formed, for example, using laser drilling, through each of the first polymer layer 1110 and the second polymer layer 1120. Also in the embodiment of FIG. 11B, optionally, the cavity 1150 has a first volume 1151 and the channel 1160 in the second polymer layer 1120 has a second volume 1161, where the second volume 1161 is 0.001% to 0.1% of the first volume 1151.

[0113] Further details about collection articles having such structures and methods for forming them are described in PCT Publication No. WO 2020 / 261086 (Halverson et al.), which is incorporated herein by reference.

[0114] 12 is a schematic cross-sectional view of a further exemplary collection article 1200, including a fluid control film 1201. The fluid control film 1201 has primary channels 1230 and secondary channels 1231 defined by primary ridges 1220 and secondary ridges 1221, with the channels 1230, 1231 and ridges 1220, 1221 extending along a channel axis that is at an angle to the longitudinal axis, e.g., the x-axis, of the fluid control film 1201. Each primary channel 1230 is defined by a pair of primary ridges 1220 (first and second) on either side of the primary channel 1230. The primary ridges 1220 have a height hp measured from the bottom surface 1230a of the channel 1230 to the top surface 1220a of the ridge 1220.

[0115] The collection article 1200 has a thickness tv measured from the bottom surface 1201a of the collection article 1200 to the bottom surface of the channel 1230a. The thickness tv can be selected to allow droplets to wick into the collection article 1200 while still maintaining a robust structure. In some embodiments, the thickness tv of the fluid control layer is less than about 75 micrometers thick, or between about 20 micrometers and about 200 micrometers thick.

[0116] In some embodiments, the microstructure is disposed within the primary channel 1230. In some embodiments, the microstructure comprises secondary channels 1231 disposed between the first and second primary ridges 1220 of the primary channel 1230. Each of the secondary channels 1231 is associated with at least one secondary ridge 1221. A secondary channel 1231 may be located between a pair of secondary ridges 1221 or between a secondary ridge 1221 and a primary ridge 1220. Stated another way, in some cases, the microstructure of the collection article comprises a plurality of ribs 1220 alternating with channels 1230, each channel 1230 comprising at least one secondary channel 1231.

[0117] Further details about collection articles with such fluid control structures and methods of forming them are described in PCT Publication No. WO 2015 / 164632 (Halverson et al.), which is incorporated herein by reference.

[0118] In some embodiments, the collection article comprises the same microstructure as the device. More specifically, the collection article may comprise any of the microstructured substrates depicted in Figures 4A-7B or 10A-10B and described in detail above. In some cases, the microstructure of the device has a different size, a different shape, or both, than the microstructure of the collection article.

[0119] In a second aspect, the present disclosure provides a method for separating solid particles from a fluid, the method comprising: a) 1) a microstructured substrate comprising a plurality of microstructures extending across a first surface of the microstructured substrate, at least a portion of an outer surface of the plurality of microstructures configured to allow capillary action; 2) a cover positioned a selected distance from the top of the first surface of the microstructured substrate; 3) at least one sidewall attaching the cover to the first surface of the microstructured substrate along the periphery of the first surface of the microstructured substrate; 4) a first aperture defined by at least one of the microstructured substrate or the cover; and 5) a second aperture defined by at least one of the microstructured substrate or the cover; and Equipped with obtaining a device wherein a first surface of the microstructured substrate, together with at least one sidewall, defines a first open volume that is the sum of the open spaces located between a plurality of microstructures from the bottom to the top of each microstructure; and a cover, together with the top of the first surface of the microstructured substrate and the at least one sidewall, defines a second open volume located adjacent to the first open volume, wherein the first open volume has a greater volume percentage of 100% open volume than the volume percentage of particles present in the fluid, where the combined first and second open volumes are 100% open volume; b) filling the device with a volume of fluid through the first aperture by capillary action; c) waiting a sufficient time for at least a portion of the particles to settle within the first open volume of the plurality of microstructures; d) fluidly coupling the device with a collection article at either the first aperture or the second aperture, thereby causing at least 10% of the initial volume of fluid that has drawn at least a portion of the particles into the first open volume of the plurality of microstructures to flow out of the device and onto the collection article by capillary action; Includes:

[0120] 9 , the method includes obtaining a device (the device is as described above) 910, filling the device with a volume of fluid through a first aperture by capillary action 920, waiting a sufficient time for at least a portion of the particles to settle into the first open volume of the plurality of microstructures 930, and fluidly connecting the device to a collection article at either the first aperture or the second aperture, thereby causing at least 10% of the initial volume of fluid from which at least a portion of the particles were drawn into the first open volume of the plurality of microstructures to flow out of the device onto the collection article by capillary action 940. The respective features, materials, structures, etc. of the device and collection article for the method of the second aspect may be according to any of the embodiments of the device and collection article detailed above with respect to the first aspect. Similarly, the method of the second aspect may be according to any of the embodiments of the method of the first aspect detailed above.

[0121] In a third aspect, a kit is provided. The kit includes a device and a collection item, the device comprising: 1) a microstructured substrate comprising a plurality of microstructures extending across a first surface of the microstructured substrate, the microstructures covering at least 90% of the first surface of the microstructured substrate, and at least a portion of an outer surface of the plurality of microstructures configured to enable capillary action; 2) a cover positioned a selected distance from the top of the first surface of the microstructured substrate; 3) at least one sidewall attaching the cover to the first surface of the microstructured substrate along the periphery of the first surface of the microstructured substrate; 4) a first aperture defined by at least one of the microstructured substrate or the cover; and 5) a second aperture defined by at least one of the microstructured substrate or the cover; and Equipped with the first surface of the microstructured substrate together with the at least one sidewall defines a first open volume that is the sum of the open spaces located between the plurality of microstructures from the bottom to the top of each microstructure, and the cover together with the top of the first surface of the microstructured substrate and the at least one sidewall defines a second open volume located adjacent to the first open volume; The collection article has a surface that exhibits an advancing contact angle with water of less than 90 degrees.

[0122] The kit may contain any number of each of the devices and collection items. In some cases, the kit contains the same number of each of the devices and collection items. The features, materials, structure, etc. of each of the devices and collection items for the kit may be according to any of the embodiments of the devices and collection items detailed above with respect to the first aspect.

[0123] Illustrative Embodiments In a first embodiment, the present disclosure provides a method for separating red blood cells from blood. The method includes obtaining a device including a microstructured substrate comprising a plurality of microstructures extending across a first surface of the microstructured substrate, at least a portion of an outer surface of the plurality of microstructures configured to enable capillary action, a cover positioned a selected distance from a top of the first surface of the microstructured substrate, at least one sidewall attaching the cover to the first surface of the microstructured substrate along a periphery of the first surface of the microstructured substrate, a first aperture defined by at least one of the microstructured substrate or the cover, and a second aperture defined by at least one of the microstructured substrate or the cover. The first surface of the microstructured substrate, together with the at least one sidewall, defines a first open volume that is the sum of the open spaces between the plurality of microstructures from the bottom to the top of each microstructure, and the cover, together with the top of the first surface of the microstructured substrate and the at least one sidewall, defines a second open volume adjacent to the first open volume, wherein the first open volume has a percentage of the 100% open volume that is greater than the volume percentage of red blood cells present in the blood, where the combined first and second open volumes represent 100% open volume. The method further includes filling the device with a volume of blood through the first aperture by capillary action and waiting a sufficient time for at least a portion of the red blood cells to settle within the first open volumes of the plurality of microstructures. Additionally, the method includes fluidly connecting the device to the collection article at either the first aperture or the second aperture, thereby causing at least 10% of the initial volume of blood from which at least a portion of the red blood cells have been drawn into the first open volume of the plurality of microstructures to flow out of the device and onto the collection article by capillary action.

[0124] In a second embodiment, the present disclosure provides a method according to the first embodiment, wherein the collection article has a surface that exhibits an advancing contact angle with water of less than 90 degrees.

[0125] In a third embodiment, the present disclosure provides a method according to the first or second embodiment, wherein the collection article comprises: a) a first polymer layer having a substantially planar first major surface and an opposite second major surface; and b) a second polymer layer bonded to the first polymer layer and having a first major surface and a second major surface, wherein the first major surface of the second polymer layer conforms to the second major surface of the first polymer layer and the second major surface of the second polymer layer defines a cavity with at least one wall, the second polymer layer having a channel connecting the cavity to at least one edge of the second polymer layer or the first major surface of the first polymer layer, and wherein a surface of the cavity exhibits an advancing contact angle with water of less than 90 degrees.

[0126] In a fourth embodiment, the present disclosure provides a method according to the first or second embodiment, wherein the collection article comprises a microstructured substrate comprising a plurality of microstructures extending across a first surface of the microstructured substrate, at least a portion of an outer surface of the plurality of microstructures having a surface that exhibits an advancing contact angle with water of less than 90 degrees, and the collection article is positioned such that the plurality of microstructures is located closer to the plurality of microstructures of the device than to a second, opposite surface of the microstructured substrate of the device.

[0127] In a fifth embodiment, the present disclosure provides a method according to any of the first to fourth embodiments, wherein at least one sidewall is part of a microstructured substrate of the device.

[0128] In a sixth embodiment, the present disclosure provides a method according to any of the first to fifth embodiments, wherein at least 15%, at least 20%, or at least 30% of the blood flows out of the device upon contact with the collection article.

[0129] In a seventh embodiment, the present disclosure provides a method according to any of the first to sixth embodiments, wherein the time is sufficient for at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90% of the red blood cells to settle within the first open volume of the plurality of microstructures of the device.

[0130] In an eighth embodiment, the present disclosure provides a method according to any of the first to seventh embodiments, wherein the microstructured substrate of at least one of the device or collection article is a microstructured film.

[0131] In a ninth embodiment, the present disclosure provides a method according to any of the first to eighth embodiments, wherein the cavity of the collection device or at least a portion of the exterior surface of the plurality of microstructures of at least one of the device or collection article comprises a surfactant, a surface treatment, a hydrophilic polymer, a flocculating agent, or any combination thereof.

[0132] In a tenth embodiment, the present disclosure provides a method according to the ninth embodiment, wherein the agglutinating agent is hydrophilic and non-hemolytic.

[0133] In an eleventh embodiment, the present disclosure provides a method according to the ninth or tenth embodiment, wherein the flocculating agent comprises a modified or unmodified amino polymer selected from the group consisting of polyethyleneimine, polylysine, polyaminoamide, polyallylamine, polyvinylamine, polydimethylamine-epichlorohydrin-ethylenediamine, polydiallyldimethylammonium chloride, cationic polyacrylamide (CPAM), polyaminosiloxane, and dendrimers formed from polyaminated amine (PAMAM) and polypropyleneimine.

[0134] In a twelfth embodiment, the present disclosure provides a method according to any of the ninth to eleventh embodiments, wherein the flocculant comprises a modified or unmodified polyethyleneimine polymer.

[0135] In a thirteenth embodiment, the present disclosure provides a method according to any of the ninth to twelfth embodiments, wherein at least a portion of the agglutinating agent dissolves, disperses, or a combination thereof in the blood after filling the device with a volume of blood.

[0136] In a fourteenth embodiment, the present disclosure provides a method according to the thirteenth embodiment, wherein the agglutinating agent is present in the volume of blood in an amount between 0.01 micrograms and 5000 micrograms per milliliter of blood.

[0137] In a fifteenth embodiment, the present disclosure provides a method according to any of the first to fourteenth embodiments, further comprising removing the blood from which at least a portion of the red blood cells have been drawn from the collection article via wicking.

[0138] In a sixteenth embodiment, the present disclosure provides a method according to any of the first to fifteenth embodiments, wherein the microstructure of the device has a different size, a different shape, or both, than the microstructure of the collection article.

[0139] In a seventeenth embodiment, the present disclosure provides a method according to any of the first to sixteenth embodiments, wherein the first aperture is defined by a cover.

[0140] In an eighteenth embodiment, the present disclosure provides a method according to any of the first to seventeenth embodiments, wherein the second aperture is defined by the microstructured substrate and cover of the device.

[0141] In a nineteenth embodiment, the present disclosure provides a method according to any of the first to eighteenth embodiments, wherein the blood is undiluted.

[0142] In a twentieth embodiment, the present disclosure provides a method according to any of the first to nineteenth embodiments, wherein the red blood cells are allowed to settle by gravity alone.

[0143] In a twenty-first embodiment, the present disclosure provides a method according to any of the first to twentieth embodiments, wherein the device further comprises an adhesive layer disposed between the cover and the microstructured substrate.

[0144] In a twenty-second embodiment, the present disclosure provides a method according to any of the first to twenty-first embodiments, wherein the microstructure of at least one of the device or collection article comprises a plurality of ribs alternating with channels extending across the first surface of the microstructured substrate, each of the ribs having a sidewall and a top surface, and each of the channels having a bottom surface.

[0145] In a 23rd embodiment, the present disclosure provides a method according to any of the 1st to 22nd embodiments, wherein the top surface of each rib is the top of a cap disposed on the side wall, and the cap has a width greater than the width between the side walls on both sides.

[0146] In a twenty-fourth embodiment, the present disclosure provides a method according to the twenty-second embodiment, wherein the microstructure of the collection article comprises a plurality of ribs alternating with channels, each channel comprising at least one secondary channel.

[0147] In a twenty-fifth embodiment, the present disclosure provides a method according to any of the first to twenty-first embodiments, wherein the microstructure of at least one of the device or collection article comprises an array of peak structures and adjacent valleys, the valleys having a maximum width in the range of 10 microns to 250 microns, and the peak structures having an apex angle of greater than 5 degrees up to 90 degrees.

[0148] In a twenty-sixth embodiment, the present disclosure provides a method according to the twenty-fifth embodiment, wherein the array of peak structures and adjacent valleys extending across the first surface of the microstructured substrate is arranged at an angle between 0 degrees and 90 degrees relative to the flow direction of the device.

[0149] In a 27th embodiment, the present disclosure provides a method according to the 25th embodiment or the 26th embodiment, wherein the array of peak structures and adjacent valleys of at least one of the device or collection article further comprises gaps between adjacent peak structures.

[0150] In a twenty-eighth embodiment, the present disclosure provides a method according to any of the first to twenty-first embodiments, wherein the microstructure of at least one of the device or collection article comprises a two-dimensional (x-axis and y-axis) array of protrusions arranged across the first surface of the microstructured substrate, each of the protrusions comprising a base, an apex, and one or more sides connecting the apex to the base.

[0151] In a twenty-ninth embodiment, the present disclosure provides a method according to any of the first to twenty-first embodiments, wherein the microstructured substrate of at least one of the device or collection article comprises a microstructured layer having first and second major surfaces, the microstructure comprising a plurality of cavities extending between the first and second major surfaces, each cavity comprising a first opening, a second opening, and at least one sidewall extending between the first opening and the second opening.

[0152] In a thirtieth embodiment, the present disclosure provides a method according to any of the first to twenty-first embodiments, wherein at least one microstructure of the device or collection article comprises a facet and a sidewall that meets the facet at a ridge of the microstructure, the facet and the sidewall defining an oblique angle therebetween.

[0153] In a thirty-first embodiment, the present disclosure provides a method according to any of the first to twenty-first embodiments, wherein at least one microstructure of the device or collection article comprises an array of fluidly connected wells, at least some of the wells being fluidly connected to at least two adjacent wells, each connected via a vent.

[0154] In a thirty-second embodiment, the present disclosure provides a method according to any of the first to twenty-first embodiments, wherein the microstructure comprises an array of upstanding stems extending across the first surface of the microstructured substrate.

[0155] In a thirty-third embodiment, the present disclosure provides a method according to any of the first to thirty-first embodiments, wherein the volume of blood filled through the first aperture is up to 100 microliters of blood.

[0156] In a thirty-fourth embodiment, the present disclosure provides a method according to any of the first to thirty-third embodiments, wherein the ratio of the first open volume to the second open volume is greater than 1:1.

[0157] In a thirty-fifth embodiment, the present disclosure provides a method according to any of the first to thirty-fourth embodiments, further comprising passing the blood through a filter before entering the device, passing the blood with at least a portion of the red blood cells drawn into a first open volume of the plurality of microstructures after exiting the device, or both.

[0158] In a thirty-sixth embodiment, the present disclosure provides a method according to any of the first to thirty-fifth embodiments, further comprising adding an agglutinating agent to the volume of blood before filling the device with the volume of blood.

[0159] In a thirty-seventh embodiment, the present disclosure provides a method according to the thirty-sixth embodiment, wherein the agglutinating agent is added and is present in an amount of 0.01 micrograms to 5000 micrograms per mL of blood.

[0160] In a thirty-eighth embodiment, the present disclosure provides a method for separating solid particles from a fluid. The method includes obtaining a device including a microstructured substrate with a plurality of microstructures extending across a first surface of the microstructured substrate. At least a portion of an outer surface of the plurality of microstructures is configured to enable capillary action. The device also includes a cover positioned a selected distance from a top of the first surface of the microstructured substrate and at least one sidewall portion attaching the cover to the first surface of the microstructured substrate along a periphery of the first surface of the microstructured substrate. The device further includes a first aperture defined by at least one of the microstructured substrate or the cover, and a second aperture defined by at least one of the microstructured substrate or the cover. The first surface of the microstructured substrate, together with at least one sidewall, defines a first open volume that is the sum of the open spaces between the plurality of microstructures from the bottom to the top of each microstructure, and the cover, together with the top of the first surface of the microstructured substrate and the at least one sidewall, defines a second open volume adjacent to the first open volume. The first open volume has a greater volume percentage of the 100% open volume than the volume percentage of particles present in the fluid, where the combined first and second open volumes equal 100% open volume. The method further includes filling the device with a volume of fluid through the first aperture by capillary action and waiting a sufficient time for at least a portion of the particles to settle into the first open volumes of the plurality of microstructures. Additionally, the method includes fluidly connecting the device to the collection article at either the first aperture or the second aperture, thereby causing at least 10% of the initial volume of blood from which at least a portion of the red blood cells have been drawn into the first open volume of the plurality of microstructures to flow out of the device and onto the collection article by capillary action.

[0161] In a thirty-ninth embodiment, the present disclosure provides a kit. The kit includes a device and a collection article. The device includes a microstructured substrate with a plurality of microstructures extending across a first surface of the microstructured substrate. The microstructures cover at least 90% of the first surface of the microstructured substrate, and at least a portion of the outer surface of the plurality of microstructures is configured to enable capillary action. The device also includes a cover positioned a selected distance from a top of the first surface of the microstructured substrate and at least one sidewall attaching the cover to the first surface of the microstructured substrate along a periphery of the first surface of the microstructured substrate. Furthermore, the device includes a first aperture defined by at least one of the microstructured substrate or the cover, and a second aperture defined by at least one of the microstructured substrate or the cover. The first surface of the microstructured substrate, together with the at least one sidewall, defines a first open volume that is the sum of the open spaces located between the plurality of microstructures from the bottom to the top of each microstructure, and the cover, together with the top of the first surface of the microstructured substrate and the at least one sidewall, defines a second open volume located adjacent to the first open volume. The collection article has a surface that exhibits an advancing contact angle with water of less than 90 degrees.

[0162] In a fortieth embodiment, the present disclosure provides a kit according to the thirty-ninth embodiment, wherein the collection article comprises: a) a first polymer layer having a substantially planar first major surface and an opposite second major surface; and b) a second polymer layer bonded to the first polymer layer and having a first major surface and a second major surface, wherein the first major surface of the second polymer layer conforms to the second major surface of the first polymer layer and the second major surface of the second polymer layer defines a cavity with at least one wall, the second polymer layer having a channel connecting the cavity to at least one edge of the second polymer layer or the first major surface of the first polymer layer, and the surface of the cavity exhibits an advancing contact angle with water of less than 90 degrees.

[0163] In a forty-first embodiment, the present disclosure provides a kit according to the thirty-ninth embodiment or the fortieth embodiment, wherein the collection article comprises a microstructured substrate comprising a plurality of microstructures extending across a first surface of the microstructured substrate, and at least a portion of an outer surface of the plurality of microstructures has a surface that exhibits an advancing contact angle with water of less than 90 degrees.

[0164] In a forty-second embodiment, the present disclosure provides a kit according to the forty-first embodiment, wherein the microstructured substrate of at least one of the devices or collection articles is a microstructured film.

[0165] In a forty-third embodiment, the present disclosure provides a kit according to any of the thirty-ninth to forty-second embodiments, wherein the cavity of the collection device or at least a portion of the outer surface of the plurality of microstructures of at least one of the devices or collection articles comprises a surfactant, a surface treatment agent, a hydrophilic polymer, a flocculating agent, or any combination thereof.

[0166] In a forty-fourth embodiment, the present disclosure provides a kit according to the forty-third embodiment, wherein the agglutinating agent on at least a portion of the plurality of microstructures of the device is hydrophilic and non-hemolytic.

[0167] In a forty-fifth embodiment, the present disclosure provides a method according to the forty-third or forty-fourth embodiment, wherein the flocculating agent on at least a portion of the plurality of microstructures of the device comprises a modified or unmodified amino polymer selected from the group consisting of polyethyleneimine, polylysine, polyaminoamide, polyallylamine, polyvinylamine, polydimethylamine-epichlorohydrin-ethylenediamine, polydiallyldimethylammonium chloride, cationic polyacrylamide (CPAM), polyaminosiloxane, and dendrimers formed from polyaminatedamine (PAMAM) and polypropyleneimine.

[0168] In a 46th embodiment, the present disclosure provides a method according to any of the 43rd to 45th embodiments, wherein the flocculating agent on at least a portion of the plurality of microstructures of the device comprises a modified or unmodified polyethyleneimine polymer.

[0169] In a forty-seventh embodiment, the present disclosure provides a kit according to any of the forty-first to forty-sixth embodiments, wherein the microstructure of the device has a different size, a different shape, or both, than the microstructure of the collection item.

[0170] In a forty-eighth embodiment, the present disclosure provides a kit according to any of the thirty-ninth to forty-seventh embodiments, wherein at least one sidewall is part of a microstructured substrate of the device.

[0171] In a forty-ninth embodiment, the present disclosure provides a kit according to any of the thirty-ninth to forty-eighth embodiments, wherein the first aperture is defined by a cover.

[0172] In a 50th embodiment, the present disclosure provides a kit according to any of the 39th to 49th embodiments, wherein the second aperture is defined by the microstructured substrate and cover of the device.

[0173] In a 51st embodiment, the present disclosure provides a kit according to any of the 39th to 50th embodiments, wherein the device further comprises an adhesive layer disposed between the cover and the microstructured substrate.

[0174] In a 52nd embodiment, the present disclosure provides a kit according to any of the 39th to 51st embodiments, wherein the microstructure of at least one of the devices or collection articles comprises a plurality of ribs alternating with channels extending across the first surface of the microstructured substrate, each of the ribs having a sidewall and a top surface, and each of the channels having a bottom surface.

[0175] In a 53rd embodiment, the present disclosure provides a kit according to the 52nd embodiment, in which the top surface of each rib is the top of a cap placed on the side wall, and the cap has a width greater than the width between the side walls on both sides.

[0176] In a fifty-fourth embodiment, the present disclosure provides a kit according to the fifty-second embodiment, wherein the microstructure of the collection article comprises a plurality of ribs alternating with channels, each channel comprising at least one secondary channel.

[0177] In a 55th embodiment, the present disclosure provides a kit according to any of the 39th to 51st embodiments, wherein the microstructure of at least one of the device or collection article comprises an array of peak structures and adjacent valleys, the valleys having a maximum width in the range of 10 microns to 250 microns, and the peak structures having an apex angle of more than 5 degrees up to 90 degrees.

[0178] In a 56th embodiment, the present disclosure provides a kit according to the 55th embodiment, wherein the array of peak structures and adjacent valleys extending across the first surface of the microstructured substrate are arranged at an angle between 0 degrees and 90 degrees relative to the flow direction of the device.

[0179] In a 57th embodiment, the present disclosure provides a kit according to the 55th embodiment or the 56th embodiment, wherein the array of peak structures and adjacent valleys of at least one of the devices or collection articles further comprises gaps between adjacent peak structures.

[0180] In a 58th embodiment, the present disclosure provides a kit according to any of the 39th to 51st embodiments, wherein the microstructure of at least one of the devices or collection articles comprises a two-dimensional (x-axis and y-axis) array of protrusions arranged across the first surface of the microstructured substrate, each of the protrusions comprising a base, an apex, and one or more sides connecting the apex to the base.

[0181] In a fifty-ninth embodiment, the present disclosure provides a kit according to any of the thirty-ninth to fifty-first embodiments, wherein the microstructured substrate of at least one of the device or collection article comprises a microstructured layer having first and second major surfaces, the microstructure comprising a plurality of cavities extending between the first and second major surfaces, each cavity comprising a first opening, a second opening, and at least one sidewall extending between the first opening and the second opening.

[0182] In a 60th embodiment, the present disclosure provides a kit according to any of the 39th to 51st embodiments, wherein the microstructure of at least one of the device or collection article comprises a facet and a sidewall that meets the facet at a ridge of the microstructure, the facet and the sidewall defining an oblique angle therebetween.

[0183] In a 61st embodiment, the present disclosure provides a kit according to any of the 39th to 51st embodiments, wherein at least one microstructure of the device or collection article comprises an array of interconnected wells, at least 80% of the wells being fluidly connected to at least two adjacent wells, each connected via a vent.

[0184] In a 62nd embodiment, the present disclosure provides a method according to any of the 39th to 51st embodiments, wherein the microstructure comprises an array of upstanding stems extending across the first surface of the microstructured substrate.

[0185] In a 63rd embodiment, the present disclosure provides a kit according to any of the 39th to 62nd embodiments, wherein the ratio of the first open volume to the second open volume is greater than 1:1.

[0186] In a 64th embodiment, the present disclosure provides a kit according to any of the 39th to 63rd embodiments, further comprising at least one filter.

[0187] In a 65th embodiment, the present disclosure provides a method according to any of the 43rd to 46th embodiments, wherein the aggregating agent on at least a portion of the plurality of microstructures of the device comprises a modified or unmodified material selected from the group consisting of gelatin, collagen, fibrinogen, dextran, hydroxyethyl starch (HES), pentastarch, polyvinylpyrrolidone (PVP), and polyethylene glycol (PEG).

[0188] In a 66th embodiment, the present disclosure provides a method according to any of the 43rd to 47th embodiments or the 65th embodiment, wherein the aggregating agent on at least a portion of the plurality of microstructures of the device comprises modified or unmodified gelatin. [Example]

[0189] The objects and advantages of the present disclosure are further illustrated by the following examples, although the particular materials and amounts thereof recited in these examples, as well as other conditions and details, should not be construed to unduly limit the present disclosure. Unless otherwise specified, all parts, percentages, ratios, etc. in the examples and the remainder of the specification are by weight. The Table of Materials (below) lists the materials used in the examples and their suppliers.

[0190] material [Table 1]

[0191] Preparative Examples 1-4. Procedures for Preparing Prismatically Structured Microstructured Films A UV-curable resin was prepared from PHOTOMER 6210 aliphatic urethane diacrylate oligomer (75 parts), SR238 1,6-hexanediol diacrylate (25 parts), and LUCIRIN TPO photoinitiator (0.5%). The components were blended in a high-speed mixer, heated in an oven at approximately 70°C for 24 hours, and then cooled to room temperature. A copper button was used as a template to prepare a linear prism film. Both the button and the compounded resin were heated in an oven at approximately 70°C for 15 minutes. The warmed resin was applied to the center of the warmed button using a transfer pipette. A piece of MELINEX 618 PET support film (DuPont Teijin Films, Chester, VA) was placed on top of the applied resin, followed by a glass plate. The primed side of the PET film was positioned in contact with the resin. The glass plate was held in place with hand pressure until the resin completely covered the surface of the button. The glass plate was carefully removed, and if any air bubbles were present, they were removed using a rubber hand roller.

[0192] The samples were cured with UV light by passing them twice through a UV processor (model QC 120233AN equipped with two Hg vapor lamps, obtained from RPC Industries, Plainfield, IL) at a speed of 15.2 meters / min under a nitrogen atmosphere. The cured microstructured film with the array pattern shown in Figure 5A was removed from the copper template by gently pulling it apart at a 90° angle. A release liner-backed adhesive layer (8 mil thick, obtained from 3M Corporation as 3M 8188 Optically Clear Adhesive) was applied to the backside (i.e., non-microstructured surface) of the microstructured film as needed using a hand roller. The characteristics of the prepared linear prism microstructured films are reported in Table 1 as Preparative Examples 1-4.

[0193] A surfactant coating was applied by cutting the microstructured film into 2.54 cm x 7.62 cm pieces, immersing each piece in a 0.1 weight percent aqueous solution of IPEGAL-CO630 anionic surfactant, and then immediately removing the film from the solution. The resulting surfactant-coated microstructured film pieces were air-dried at ambient temperature and humidity. [Table 2]

[0194] Preparative Example 5. Procedure for Preparing Louvered Microstructured Films A diamond (220 micrometers deep) was used to cut a tool with multiple parallel linear grooves. The grooves were spaced at a pitch of 60 micrometers. Resin A was prepared by mixing the materials in Table 2 below. [Table 3]

[0195] A cast-and-cure microreplication process was performed using Resin A and the tooling described above. Line conditions were: resin temperature 150°F (65.6°C), die temperature 150°F (65.6°C), coater IR 120°F (48.9°C) edge / 130°F (54.4°C) center, tool temperature 100°F (37.8°C), and line speed 70 feet per minute (fpm) (0.36 meters per second (m / s)). Fusion D lamps (obtained from Fusion UV Systems, Gaithersburg, MD) with a peak wavelength of 385 nm were used for curing and were operated at 100% power. The resulting "louver" microstructured film had multiple protrusions (ribs) separated by channels, as shown in Figure 4A (detailed above). The microstructured film was a reverse of the tool's topography, with the protrusions in the microstructured film being negative replicas of the tool's grooves and the channels in the microstructured film being negative replicas of the uncut portions of the tool between the grooves. The microstructured film's protrusions (ribs) were evenly spaced with a height ("H") of 220 micrometers, a width ("W") of 30 micrometers, a pitch ("P") of 60 micrometers, and a wall angle θ of 91.5 degrees (resulting in the protrusions being slightly tapered (i.e., wider at the bottom and narrower at the top). The land layer ("L") of cured resin had a thickness of 8 micrometers. The base layer was a PET film (3M Company, St. Paul, MN) with a thickness of 74.4 micrometers. The side of the PET film in contact with the resin was primed with a thermosetting acrylic polymer (RHOPLEX 3208 polymer obtained from Dow Chemical, Midland, MI).

[0196] A surfactant coating was applied by cutting the microstructured film into 2.54 cm x 7.62 cm pieces, immersing each piece in a 0.1 weight percent aqueous solution of IPEGAL-CO630 anionic surfactant, and then immediately removing the film from the solution. The resulting surfactant-coated microstructured film pieces were air-dried at ambient temperature and humidity.

[0197] Preparative Example 6. Device Preparation Device 800 (shown in FIGS. 8A and 8B) was prepared by forming a laminate of three film sections. The cover sheet component (i.e., cover 820) of device 800 was prepared by laser cutting a 72 mm long by 20 mm wide section from a sheet of 3M Microfluidic Diagnostic Film 9962 (a polyester film (3.9 mil) with a hydrophilic coating on both sides, including the major surface 822 of cover 820 facing the microstructured substrate 830, obtained from 3M Company). A circular hole (5 mm diameter) was laser cut into the film so that the center of the hole was located 13.5 mm perpendicular to the narrow edge of the film and 10 mm perpendicular to the long edge of the film. The circular hole formed the first aperture 850 of device 800. The second film component of the device (i.e., adhesive layer 870) was prepared by laser cutting a 72 mm long by 20 mm wide section from a sheet of 3M 1522 Double-Sided Medical Tape (a clear, double-sided acrylic adhesive with a polyethylene backing, obtained from 3M Company). The total thickness of the double-sided tape, with the release liner removed, was measured to be 150 micrometers using a digital caliper. A rectangular opening 880 (60 mm long by 2.5 mm wide) was laser cut into the second film component, positioned so that the narrow edge of the opening was located 11 mm from the narrow edge of the film and the long edge of the opening was located 8.75 mm from the long edge of the film.

[0198] The third film component of the device (i.e., microstructured substrate 810) was a 72 mm long by 20 mm wide laser-cut section of the surfactant-coated microstructured film of Preparative Example 1. All laser cutting of the films was performed using a Muse Core CO2 Laser Cutter (Full Spectrum Laser, Las Vegas, Nevada).

[0199] Device 800 was assembled by removing all release liners from the films and then aligning the edges of the films to form a stack with the second film sandwiched between the cover sheet film and the microstructured film. The films were positioned so that the microstructured surface 830 of the third film faced the major surface 822 of the second film and cover. Adhesive lamination of the stack was completed by applying a 4-pound (1.8 kilogram) roller to the stack and moving the roller back and forth once. The second adhesive film formed a fluid seal (e.g., formed a sidewall) between the cover sheet and the top surface of the microstructured film around the edges of the rectangular opening and first aperture hole. In a final step, a razor blade was used to trim the stack at the edge distal to the first aperture (dashed line 862 in Figure 8B) so that the resulting device had overall dimensions of 60 mm long by 20 mm wide. This cut an opening in the second film, exposing the second aperture of the device as a 2.5 mm rectangular opening at the newly created edge of device 800.

[0200] Preparation Example 7. A device was prepared according to the same procedure as reported in Preparative Example 6, except that the second film component of the device was prepared using 3M 1513 Double-Sided Medical Tape (a clear, double-sided acrylic adhesive with a polyester backing, obtained from 3M Company). The total thickness of the double-sided tape, with the release liner removed, was measured to be 75 micrometers using a digital caliper.

[0201] Preparation Example 8. A device was prepared according to the same procedure as reported in Preparative Example 6, except that the third film component of the device was prepared using the surfactant-coated microstructured film of Preparative Example 2.

[0202] Preparation Example 9. A device was prepared according to the same procedure as reported in Preparative Example 6, except that the third film component of the device was prepared using the surfactant-coated microstructured film of Preparative Example 3.

[0203] Comparative example A. A device was prepared according to the same procedure reported in Preparative Example 6, except that the third film component of the device was prepared using the surfactant-coated microstructured film of Preparative Example 4.

[0204] Preparation Example 10. A device was prepared according to the same procedure reported in Preparative Example 6, except that the third film component of the device was prepared using the surfactant-coated microstructured film of Preparative Example 5.

[0205] Preparation Example 11. A device was prepared according to the same procedure as reported in Preparative Example 6, except that the third film component of the device was prepared using the surfactant-coated microstructured film of Preparative Example 5, and the second film component of the device was prepared using 3M 1513 Double-Sided Medical Tape. [Table 4]

[0206] Comparative example B. A device was prepared according to the same procedure as reported in Preparative Example 6, except that a non-microstructured polyethylene terephthalate (PET) film (MELINEX 454 film (3 mil), Dupont Teijin Films) was used as the third film component of the device to prepare the third film component of the device.

[0207] Comparative example C. A device was prepared according to the same procedure as reported in Preparative Example 6, except that the non-microstructured film of Comparative Example B (MELANEX 454 PET film) was used as the third film component to prepare the third film component of the device, and 3M 1513 Double-Sided Medical Tape was used to prepare the second film component of the device.

[0208] Reference Example 1. Method for separating red blood cells from blood Defibriminated sheep blood (obtained from Becton Dickinson, Franklin Lakes, NJ) was measured to have an undiluted hematocrit concentration of 40% using a Zip-IQ PCV Centrifuge (LW Scientific Incorporated, Lawrenceville, GA). In addition to the undiluted blood samples, diluted blood samples of 12%, 8%, and 4% hematocrit were prepared using 1× phosphate-buffered saline (PBS).

[0209] A module for delivering a blood sample to device 800 was prepared (shown in FIGS. 8C and 8D). The module components included a silicone gasket 844 (25 mm x 25 mm x 3.2 mm) prepared from SYLGARD 184 silicone elastomer (Dow Chemical, Midland, MI) with a 5 mm hole cut in the center of the gasket, and a sheet 842 (25 mm x 25 mm x 3.2 mm) of PLEXIGLAS polymethyl methacrylate (PMMA) film (Rohm GmbH, Darmstadt, Germany) with a 0.06 inch (1.52 mm) diameter hole punched in the center of the sheet. A section of ethyl vinyl acetate (EVA) plastic tubing 846 [0.02 inch (0.51 mm) inner diameter and 0.06 inch (1.52 mm) outer diameter, McMaster-Carr, Elmhurst, Ill.] was inserted into a hole in the PLEXIGLAS film sheet and secured in place with HARDMAN DOUBLE / BUBBLE epoxy (Royal Adhesives, Wilmington, Calif.) 847. The opposite end of the tubing was fitted with a 22.5-gauge needle attached to a 1 mL Luer-lock syringe.

[0210] A device 800 (selected from the devices of Preparative Examples 6-11 and Comparative Examples A-C) was placed on a horizontal surface. A blood sample (20 microliters to 50 microliters) was placed into the first aperture of the device, and the blood was wicked to the end of the device by capillary flow. Next, a silicone gasket 844 was placed on the cover sheet of the device and aligned so that the hole in the gasket was centered over the first aperture of the device. Next, a PLEXIGLAS sheet 842 (to which EVA plastic tubing 846 had been adhered as described above) was placed on the exposed surface of the gasket and aligned so that the hole in the sheet was centered over the hole in the gasket, forming the assembly shown in FIG. 8D. A syringe (not shown) attached to the other end of the EVA plastic tubing was placed into a syringe pump (Model NE-1600, New Era Pump Systems Inc., Farmingdale, NY) (not shown). The pump was operated with a flow rate set at 100 microliters / min. A blood sample exiting the device was collected through the second aperture 860. A 10-microliter sample of the collected blood was diluted with 90 microliters of 1x PBS. The diluted sample was pipetted into a C-CHIP Disposable Hemacytometer (Incyto, Republic of Korea) according to the manufacturer's instructions. For comparison, a sample of blood placed in the device was also pipetted into the C-CHIP Disposable Hemacytometer. The counting plate was analyzed using a Zeiss LSM 510 META module Axioplan 2 upright confocal microscope (Zeiss, Jena, Germany). Red blood cells were counted manually or using Image J image processing software (National Institutes of Health, Bethesda, MD). The percent red blood cell (RBC) reduction of a blood sample using the described method was calculated by comparing the RBC count of the blood sample taken from the device with the RBC count of the blood sample placed in the device according to Equation 1.Each device was tested in triplicate (n=3 devices) with a particular blood sample and the results in % RBC reduction are reported in Table 4 as the mean value.

[0211] Formula 1:

number

[0212] Preparation Example 12. CAD design files were used to fabricate a master for the device's microstructured substrate (Figure 2B) using the multiphoton exposure system described in U.S. Patent No. 8,605,256 (DeVoe et al.) and U.S. Patent No. 8,455,846 (Gates et al.). A negative contrast photoresist, as described in U.S. Patent No. 10,133,174 (Lee et al.), was photopatterned onto a silicon wafer substrate. Once scanning was complete, the substrate with the patterned structure was immersed in a developer solution of propylene glycol monomethyl ether acetate (obtained from Sigma-Aldrich) to remove unpolymerized photoresist. The master was then electroformed with nickel or nickel alloy to create the metal tool used for replication. The nickel-plated tool was used to create an impression molded specimen using polypropylene resin (C700-35 resin, Dow Chemical, Midland, MI). The platens of a Carver press (Carver, Wabash, Ind.) were heated to 170° C. and the tool and resin were pressed together at 1000 pound force (approximately 4450 Newtons) for 7 minutes, followed by cooling under pressure until the platen temperature reached 80° C. The pressure was released and the molded microstructured substrate was removed from the tool.

[0213] The molded microstructured substrate had top and bottom surfaces with overall dimensions of 25.4 mm (width), 76.2 mm (length), and 1 mm (depth). The substrate's microstructure was an array of linear prismatic microstructures recessed below the top surface of the substrate and positioned along the floor of a flow channel (3 mm wide, 60 mm long) with first and second open ends. The linear array of peak structures and adjacent valleys was oriented at a 0-degree angle with respect to the direction of liquid flow in the completed device. The characteristics of the linear prismatic microstructures are reported in Table 5. The prismatic features protruded above the floor of the flow channel. The surrounding walls of the flow channel extended toward the top surface 100 micrometers above the tips of the prismatic structures. The first open end of the flow channel was fluidically attached to a semicircular first cavity 100 micrometers deep from the top surface and with a volume of 2.88 microliters. The first cavity formed the first aperture of the device. The first aperture served as the liquid sample intake reservoir for the final device. The opposite, open end of the flow channel was fluidically attached to a rectangular second cavity (5 mm wide, 6 mm long, and 250 micrometers deep from the top surface). The second cavity formed the second aperture of the device. The second aperture served as the receiving reservoir for the liquid sample exiting the flow channel.

[0214] The cover component of the device was a section of 3M Microfluidic Diagnostic Film 9975R (obtained from 3M Company). The cover was positioned over the flow channel and liquid sample intake reservoir section and attached with an adhesive to the surface of the microstructured substrate surrounding the sample intake reservoir and flow channel section. Prior to application, a circular hole (5 mm diameter) was laser cut into the cover component and positioned so that the center of the hole was located over the center of the sample intake reservoir upon attachment of the cover to the microstructured substrate. The receiving reservoir was not covered. [Table 6]

[0215] Preparation Example 13. The device was prepared according to the same procedure as described in Preparative Example 12, except that the linear array of peak structures and adjacent valleys was oriented at a 45-degree angle relative to the direction of liquid flow in the completed device (the orientation shown in Figure 5D); the flow channel dimensions were 3 mm wide and 40 mm long; the first aperture cavity had a depth of 300 micrometers and a volume of 8.7 microliters, and the second aperture cavity had dimensions of 5 mm (width), 6 mm (length), and 300 micrometers (depth).

[0216] Preparation Example 14. The device was prepared according to the same procedure as described in Preparative Example 13, except that the linear array of peak structures and adjacent valleys was oriented at a 90-degree angle (i.e., perpendicular) to the direction of liquid flow in the completed device (the orientation shown in Figure 5C).

[0217] Preparation Example 15. The device was prepared according to the same procedure as described in Preparative Example 12, except that the array of linear prism microstructures was replaced with an array of fluidly connected wells having a circular shape connected to adjacent wells by vents (as shown in Figures 10A-10B). Each well had a diameter of 200 micrometers, a depth of 150 micrometers, and a draft angle of 5 degrees. The vents had a length of 29 micrometers, a width of 40 micrometers, a depth of 150 micrometers, and a draft angle of 5 degrees. Each well located in the center of the flow channel was connected to four other wells, each connection via a vent, with the spacing of the vents as shown in Figure 10B. The wall surrounding the periphery of the flow channel extended 100 micrometers above the top surface of the well. The flow channel dimensions were 3 mm wide and 40 mm long.

[0218] The first open end of the flow channel was fluidically attached to a semicircular first cavity having a depth of 100 micrometers from the top surface and a volume of 2.88 microliters. The first cavity formed the first aperture of the device. The opposite second open end of the flow channel was fluidically attached to a rectangular second cavity (5 mm wide, 6 mm long, and 250 micrometers deep from the top surface). The second cavity formed the second aperture of the device.

[0219] Reference Example 2. Method for separating red blood cells from blood Human blood was collected in BD VACUTAINER citrate tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) and used as citrated whole blood or as citrated whole blood diluted 1:1 with 1x phosphate-buffered saline (PBS).

[0220] Microstructured substrates were prepared as described in Preparative Examples 12-15, and a cover component modified with a blood drop tube was attached to each substrate. The modified cover component was a section of 3M Microfluidic Diagnostic Film 9975R (obtained from 3M Company), with a circular hole (5 mm diameter) laser-cut in the cover component. Plastic tubing (0.05 inch ID, 0.09 inch OD) was inserted into the hole and secured with epoxy adhesive (3M SCOTCH-WELD Epoxy Adhesive DP100 Plus Clear, obtained from 3M Company). Any tubing extending beyond the adhesive surface of the cover component was removed with a razor blade. The cover was positioned over the flow channel section and liquid sample capture reservoir section of the microstructured substrate and adhesively attached to the surface of the microstructured substrate surrounding the two sections. The cover component was positioned so that the center of the hole was located over the center of the sample capture reservoir when the cover was attached. The receiving reservoir was not covered. The tube extended approximately 3 cm in length from the outer surface of the cover.

[0221] Each resulting device was placed on a horizontal surface (with the underside of the device facing the horizontal surface). A blood sample (20 to 50 microliters) was added to the sample inlet reservoir through the tubing using a micropipette. The blood sample was either wicked to the end of the device by capillary flow or advanced using positive pressure from the micropipette. A sufficient amount of blood was introduced into the device to fill the open volume of the flow channel without allowing excess blood to pool in the intake reservoir. Any excess blood in the receiving reservoir was quickly removed from the reservoir using a micropipette or a KIMWIPE wiper (Kimberly-Clark Corporation, Irving, TX). After administration of the blood sample, each device was left on a horizontal surface for 5 minutes.

[0222] A 1 mL luer-lock syringe filled with mineral oil was attached to a section of plastic tubing, and the mineral oil was partially dispensed into the tubing, leaving a small air gap at the open end of the tubing (approximately 10 cm of tubing length). The syringe was placed on a syringe pump (model NE-1600, New Era Pump Systems Inc.). The open end of the syringe assembly's tubing was connected to the open end of the tubing extending from the device. The pump was operated at a flow rate set to 50 microliters / min to push the blood sample, along with any trapped air, out of the flow channel. The blood sample exiting the flow channel was collected in several 2- to 4-microliter aliquots. Each sample was collected as soon as the aliquot volume accumulated in the receiving reservoir.

[0223] For each collected sample, 1 / 10, 1 / 20, 1 / 100, and 1 / 200 diluted samples were prepared in 1x PBS. A 2-microliter aliquot of each diluted sample was loaded onto an Agilent Take3 microvolume plate (TAKE3-SN, Agilent Technologies, Santa Clara, CA) using a micropipette. The plate included at least one well as a 1x PBS blank, which was used for dilutions according to the manufacturer's instructions. Absorbance measurements were recorded at 406 nm, 414 nm, and 576 nm for each sample and the 1x PBS blank using an Agilent Synergy Neo2 plate reader (Agilent Technologies). Using the procedure in the "Method for Measuring Intact Red Blood Cell Content of Samples" section (described below), the percentage reduction of red blood cells from blood samples subjected to each device was calculated using the procedure described in this example.

[0224] Results using citrated human whole blood are shown in Table 6, and results using citrated human whole blood diluted 1:1 with 1x PBS are shown in Table 7. For each type of device, the results reported in Tables 5 and 6 are from three device technical replicates (n=3). The calculated percent red blood cell reduction for each replicate device was averaged from the total volume of aliquot samples taken from the device. [Table 7] [Table 8]

[0225] Method for determining the intact red blood cell content of a sample To simultaneously measure the intact and lysed red blood cell contents of samples, a dilution series calibration curve was constructed using known inputs of lysed and intact human red blood cells. Citrated human whole blood was used as the 100% intact red blood cell sample. To create the 0% intact red blood cell sample, an aliquot of whole blood was lysed by vortexing for 1 minute using a ZR BashingBead Lysing Tube (Product No. S6012-50, Zymo Research, Irvine, CA). The tube was centrifuged at 10,000 x g for 1 minute, and the supernatant, containing only the lysed cell contents, was transferred to a new 1.5 mL Eppendorf tube. Standard samples of intact to lysed red blood cells for the dilution series were prepared by mixing known intact and lysed cells in various ratios, ranging from 100% to 0% intact red blood cells. The intact red blood cell concentration of the standard samples was confirmed using a standard C-CHIP Disposable Hemacytometer according to the manufacturer's instructions.

[0226] Each standard sample was diluted with 1x PBS (so that the absorbance was within the dynamic range of the plate reader) and analyzed in technical triplicate using an Agilent Synergy Neo2 plate reader equipped with a Take 3 multivolume plate (sample volumes and blanks were selected according to the manufacturer's instructions). Absorbances at 406 nm, 414 nm, and 576 nm were measured for each standard sample.

[0227] Ratio A 414nm / A 576nm was plotted against known inputs of % intact red blood cells. 414nm A = absorbance of the sample at 414 nm, A 576nm = absorbance of the sample at 576 nm. For background subtraction, subtraction of a blank (1x PBS) was used. The data set was fitted to a logarithmic curve to generate Equation A. Equation A was used to calculate the percentage of intact red blood cells from the blood-containing suspension.

[0228] Formula A:

number

[0229] The absorbance signal at 406 nm for each sample was adjusted by the cell percentage ratio to calculate the signal from intact red blood cells using Equation B.

[0230] Formula B:

number

[0231] After the absorbance at 406 nm wavelength was adjusted for intact and lysed cells in Equation B, the absorbance signal of the blood sample placed in the device was compared to the absorbance signal of the corresponding blood aliquot taken from the device according to Equation C to calculate the percent red blood cell (RBC) reduction.

[0232] Formula C:

number

[0233] Example 1. Use of Nitrocellulose Membrane Collection Article The device of Preparative Example 10 was placed on a horizontal surface (with the underside of the device facing the horizontal surface). A sample of citrated human whole blood (20 microliters to 50 microliters) was placed into the first aperture of the device using a micropipette, and the blood was wicked to the end of the device by capillary flow. The device was left standing on the horizontal surface for 1 minute. Next, the edge of a strip (approximately 2.5 mm x 60 mm) of UNISTART CN nitrocellulose membrane (Sartorius Stedium Biotech, Goettingen, Germany) was placed in contact with the second aperture of the device. After contacting the nitrocellulose membrane with the second aperture, at least 10 percent of the blood in the device was visually observed to flow from the device to the nitrocellulose membrane by capillary action.

[0234] Example 2. Use of paper collection items The same procedure was followed as reported in Example 1, except that the nitrocellulose membrane collection article was replaced with a strip (approximately 2.5 mm x 60 mm) of Whatman 40 filter paper (product number 1440-110, GE Healthcare, Buckinghamshire, UK). After contacting the paper strip with the second aperture, at least 10 percent of the blood in the device was visually observed to flow from the device to the paper strip by capillary action.

[0235] Example 3. Use of Microstructured Film Collecting Articles The same procedure as reported in Example 1 was followed, except that the nitrocellulose membrane collection article was replaced with a strip (approximately 2.5 mm x 60 mm) of the microstructured film from Preparative Example 5. An edge of the microstructured film strip (approximately 2.5 mm x 60 mm) was placed in contact with the second aperture of the device. The microstructured film strip was positioned with the location of the microstructures on the microstructured film strip inverted relative to the location of the microstructures on the device, as shown in Figure 3B. After contacting the film strip with the second aperture, at least 10 percent of the blood in the device was visually observed to flow from the device to the microstructured film strip by capillary action.

[0236] Preparative Example 16. Microstructured Film with Upright Stems The polypropylene (PP) microstructured film shown in FIG. 13, having discrete stem structures with angled sidewalls, was prepared by a molding process according to Example 1 of U.S. Pat. No. 9,358,714 (Chandrasekaran), except that the beta-nucleating masterbatch was not included. The film was then corona treated using a BD-20AC Laboratory Corona Treater (Electro-Technic Products, Chicago, IL). Three-dimensional photomicrographs of the microstructured film were taken using a Keyence VK-X3100 3D Surface Profilometer (Keyence Corporation, Itasca, IL), and measurements were performed using the accompanying VK-X 3000 MultiFileAnalyzer software package. The microstructured film had a total thickness of approximately 345 micrometers and contained a staggered array of 2,000 stem features per square inch. The stem features had a generally flat surface at their apex (i.e., a truncated cone shape). The backing layer had a thickness of 83.5 micrometers. Tables 8 and 9 report the dimensions of stem height, stem diameter at base in the downweb direction, stem diameter at base in the crossweb direction, stem diameter at top in the downweb direction, stem diameter at top in the crossweb direction, center-to-center spacing (pitch) between stems in the downweb direction, and center-to-center spacing (pitch) between stems in the crossweb direction. [Table 9] [Table 10]

[0237] Preparative Example 17. Preparation of a device containing a microstructured film with upstanding stems The procedure described in Preparative Example 6 was modified with the following modifications: First, the microstructured film of Preparative Example 1 was replaced with the microstructured film of Preparative Example 16 as the third film component of the device. Second, epoxy was used to seal the two long edges of the device and the narrow edge adjacent to the first aperture. The narrow edge forming the second aperture was not sealed.

[0238] Preparative Example 18. Preparation of a device containing a microstructured film with an upstanding stem and a coagulant coating of a microstructured array A flocculant solution of polyethyleneimine (PEI) (branched, 70,000 Da molecular weight, 30% w / v aqueous solution, catalog number 00618, obtained from Polysciences, Inc., Warrington, PA) was further diluted in two steps: 1:10 (wt:vol) with deionized water, followed by 1:10 vol:vol deionized water. The diluted flocculant solution (30 microliters to 100 microliters) was pipetted into the rectangular opening 880 of the device of prepared Example 17. The applied PEI flocculant solution was allowed to air dry overnight at ambient conditions to provide a flocculant-coated microstructured array.

[0239] Preparative Example 19. Preparation of a device containing a microstructured film with an upstanding stem and a coagulant coating of a microstructured array An aqueous flocculant solution of guanylated polyethyleneimine (G-PEI) was prepared as described in Example 1 of U.S. Pat. No. 10,087,405 (Swanson et al.) without the addition of butanediol diglycidyl ether. The flocculant solution (30 microliters to 100 microliters) was pipetted into the rectangular opening 880 of the device of Prepared Example 17. The applied G-PEI flocculant solution was allowed to air dry overnight at ambient conditions to provide a flocculant-coated microstructured array.

[0240] Preparative Example 20: Preparation of a device with a microchannel fluid control film The procedure described in Preparative Example 6 was followed, with the modification that the microstructured film of Preparative Example 1 as the third film component of the device was replaced with a microchannel fluid control film made as described in the examples of U.S. Pat. No. 1,1392,899 (Halverson et al.) under "Preparation of a Microchannel Fluid Control Film."

[0241] Reference Example 3. Method for separating red blood cells from blood Human whole blood collected in EDTA tubes was obtained from the Oklahoma Blood Institute (Oklahoma City, OK). The initial hematocrit of the human blood samples was measured using a Zip-IQ PCV Centrifuge (LW Scientific Incorporated, Lawrenceville, GA).

[0242] This method used a module with a syringe pump for delivering a blood sample, as described in Reference Example 1. A device selected from Preparative Examples 17-19 was placed on a horizontal surface. A sample of human whole blood (50-100 microliters) was applied to the first aperture 850 of the device. The blood wicked to the end of the device by capillary flow, and the device was allowed to stand for 10 minutes to allow red blood cells to settle into the microstructure array section. The pump was then turned on with a flow rate set to 100 microliters / minute. The blood sample exiting the device was collected in a series of 5-microliter aliquots through the second aperture 860.

[0243] A standard calibration curve was generated from a dilution series of whole blood in water to correlate RBC count with absorbance at 406 nm. Each blood sample tested was diluted with water (to lyse cells and bring absorbance within the dynamic range of the plate reader) and analyzed in technical duplicate using an Agilent Synergy Neo2 plate reader equipped with a Take 3 multivolume plate (sample volumes and blanks were selected according to the manufacturer's instructions). All dilutions were prepared using deionized water with a resistivity greater than 18 megaohm-cm prepared from a SYNERGY UV Water Purification System (MilliporeSigma, Burlington, MA). The absorbance at 406 nm was measured for each sample. The percent red blood cell (RBC) reduction of a blood sample was calculated by comparing the absorbance of an aliquot of blood withdrawn from the device with the absorbance of the whole blood sample applied to the device according to Equation D.

[0244] Formula D:

number

[0245] In Equation D, "absorbance 406nm,output " = absorbance at 406 nm wavelength of the blood sample taken from the device. "Absorbance 406nm,input " = absorbance at 406 nm wavelength of the blood sample placed in the device. For each type of device, the results are reported in Table 10. A single device of each type was tested. The calculated percent reduction in red blood cells was averaged across all of the aliquot samples taken from the devices. Approximately 10 aliquot samples were taken from each device. [Table 11]

[0246] Reference Example 4. Method for separating red blood cells from blood The method of Reference Example 3 was followed using a single device (n=1) from Preparative Example 17 and a blood sample containing a PEI aggregating agent. For the blood sample, a flocculant solution of polyethyleneimine (PEI) (branched, 70,000 Da molecular weight, 30% w / v aqueous solution, catalog number 00618, obtained from Polysciences, Inc.) was prepared by performing a 1 / 10 dilution in PBS (e.g., 1 mL flocculant + 9 mL of 1x PBS). A 90 microliter aliquot of human whole blood was mixed with 10 microliters of the flocculant solution in a tube. The resulting blood sample was mixed using a pipette (3-5 times) and then incubated at room temperature for 10 minutes. The incubated blood sample was mixed using a pipette (3-5 times), and then approximately 100 microliters of the sample was placed in the first aperture of the device. A total of seven aliquots were taken from the device. The calculated percent RBC reduction was 64.4%. The calculated percent reduction in red blood cells was averaged across all aliquot samples taken from the device.

[0247] Reference Example 5. Method for separating red blood cells from blood containing a gelatin agglutinant The method of Reference Example 4 was followed using two devices from Preparation Example 20. One device (n=1) was used with a whole blood sample having a 40% hematocrit containing 1x PBS, and the second device (n=1) was used with a blood sample containing a gelatin agglutination solution, with all solutions heated to 37°C prior to the experiment. A 10% stock aqueous solution of bovine gelatin was prepared in 1x PBS and heated to 37°C. For Device 1, a 450 microliter aliquot of human whole blood was mixed with 50 microliters of 1x PBS, and approximately 100 microliters of blood was added to the first aperture of the device. For Device 2, a 450 microliter aliquot of human whole blood was mixed with 50 microliters of 10% gelatin, and approximately 100 microliters of blood was added to the first aperture of the device. The devices were incubated at 37°C for 15 minutes. A total of one aliquot was taken from each device. The calculated percent RBC reduction was 2.0% for the device containing 1x PBS. The calculated percent RBC reduction was 32.7% for the device containing 1% gelatin flocculating agent. The results are reported in Table 11. [Table 12]

[0248] Preparative Example 6. Preparation of Devices Containing Microstructured Films, Aggregation with Gelatin, and Adjusted Hematocrit The method of Reference Example 5 was followed using two devices of Preparative Example 20. One device (n=1) was used with a whole blood sample with hematocrit adjusted to 25% with donor plasma.

[0249] A total of one aliquot was taken from each device. The calculated percent RBC reduction was 62.3% for the device containing 1x PBS. The calculated percent RBC reduction was 79.8% for the device containing 1% gelatin flocculating agent. The results are reported in Table 12. [Table 13]

[0250] All of the above patents and patent applications are expressly incorporated herein by reference. The above-described embodiments are exemplary of the invention, and other configurations are possible. Accordingly, the present invention should not be deemed limited to the embodiments described in detail above and shown in the accompanying drawings, but instead should be deemed limited only by the fair scope of the following claims and their equivalents.

Claims

1. 1. A method for separating red blood cells from blood, comprising: a) 1) a microstructured substrate comprising a plurality of microstructures extending across a first surface of the microstructured substrate, at least a portion of an outer surface of the plurality of microstructures configured to allow capillary action; 2) a cover positioned a selected distance from the top of the first surface of the microstructured substrate; 3) at least one sidewall attaching the cover to the first surface of the microstructured substrate along a periphery of the first surface of the microstructured substrate; 4) a first aperture defined by at least one of the microstructured substrate or the cover; and 5) a second aperture defined by at least one of the microstructured substrate or the cover; and Equipped with obtaining a device wherein the first surface of the microstructured substrate, together with the at least one sidewall, defines a first open volume that is the sum of the open spaces located between the plurality of microstructures from the bottom to the top of each microstructure; and the cover, together with the top and at least one sidewall of the first surface of the microstructured substrate, defines a second open volume located adjacent to the first open volume, wherein the first open volume has a percentage of the 100% open volume that is greater than the volume percentage of red blood cells present in the blood, where the sum of the first open volume and the second open volume is 100% open volume; b) filling the device with a volume of blood through the first aperture by capillary action; c) waiting a sufficient time for at least a portion of the red blood cells to settle within the first open volume of the plurality of microstructures; d) fluidly coupling the device with a collection article at either the first aperture or the second aperture, thereby allowing at least 10% of an initial volume of blood from which at least a portion of the red blood cells have been drawn into the first open volume of the plurality of microstructures of the device to flow out of the device onto the collection article by capillary action; A method comprising:

2. 10. The method of claim 1, wherein at least 15%, at least 20%, or at least 30% of the blood exits the device upon contact with the collection article.

3. 3. The method of claim 1 or 2, wherein the time is sufficient for at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90% of the red blood cells to settle within the first open volume of the plurality of microstructures of the device.

4. The method of any one of claims 1 to 3, further comprising removing the blood from which at least a portion of the red blood cells have been drawn from the collection article via wicking.

5. The method of any one of claims 1 to 4, wherein the blood is undiluted.

6. The method of any one of claims 1 to 5, wherein the red blood cells sediment by gravity alone.

7. The method of any one of claims 1 to 6, wherein the volume of blood filled through the first aperture is up to 100 microliters of blood.

8. 8. The method of any one of claims 1-7, further comprising passing the blood through a filter before entering the device, passing the blood with at least a portion of the red blood cells drawn into the first open volume of the plurality of microstructures after exiting the device, or both.

9. The method of any one of claims 1 to 8, further comprising adding an agglutinating agent to the volume of blood before filling the device with the volume of blood.

10. 10. The method of claim 9, wherein the agglutinating agent is added and present in an amount of 0.01 micrograms to 5000 micrograms per mL of blood.

11. 1. A method for separating solid particles from a fluid, comprising: a) 1) a microstructured substrate comprising a plurality of microstructures extending across a first surface of the microstructured substrate, at least a portion of an outer surface of the plurality of microstructures configured to allow capillary action; 2) a cover positioned a selected distance from the top of the first surface of the microstructured substrate; 3) at least one sidewall attaching the cover to the first surface of the microstructured substrate along the periphery of the first surface of the microstructured substrate; 4) a first aperture defined by at least one of the microstructured substrate or the cover; and 5) a second aperture defined by at least one of the microstructured substrate or the cover; and Equipped with obtaining a device wherein the first surface of the microstructured substrate, together with the at least one sidewall, defines a first open volume that is the sum of the open spaces between the microstructures from the bottom to the top of each microstructure; and the cover, together with the top of the first surface of the microstructured substrate and the at least one sidewall, defines a second open volume adjacent to the first open volume, wherein the first open volume has a greater volume percentage of the 100% open volume than the volume percentage of particles present in the fluid, where the first open volume and the second open volume together represent 100% open volume; b) filling the device with a volume of the fluid through the first aperture by capillary action; c) waiting a sufficient time for at least a portion of the particles to settle within the first open volume of the plurality of microstructures; d) fluidly coupling the device with a collection article at either the first aperture or the second aperture, thereby causing at least 10% of the initial volume of fluid that has drawn at least a portion of the particles into the first open volume of the plurality of microstructures to flow out of the device and onto the collection article by capillary action; A method comprising:

12. A kit comprising a device and a collection item, wherein the device comprises: 1) a microstructured substrate comprising a plurality of microstructures extending across a first surface of the microstructured substrate, the microstructures covering at least 90% of the first surface of the microstructured substrate, and at least a portion of an outer surface of the plurality of microstructures configured to allow capillary action; 2) a cover positioned a selected distance from the top of the first surface of the microstructured substrate; 3) at least one sidewall attaching the cover to the first surface of the microstructured substrate along a periphery of the first surface of the microstructured substrate; 4) a first aperture defined by at least one of the microstructured substrate or the cover; and 5) a second aperture defined by at least one of the microstructured substrate or the cover; and Equipped with the first surface of the microstructured substrate together with the at least one sidewall defines a first open volume that is the sum of the open spaces located between the plurality of microstructures from the bottom to the top of each microstructure, and the cover together with the top of the first surface of the microstructured substrate and the at least one sidewall defines a second open volume located adjacent to the first open volume; the collection article having a surface that exhibits an advancing contact angle with water of less than 90 degrees; kit.

13. The collectible items are: a) a first polymer layer having a substantially planar first major surface and an opposite second major surface; b) a second polymer layer bonded to the first polymer layer, the second polymer layer having a first major surface and a second major surface, the first major surface of the second polymer layer conforming to the second major surface of the first polymer layer, the second major surface of the second polymer layer defining a cavity with at least one wall, the second polymer layer having a channel connecting the cavity to at least one edge of the second polymer layer or the first major surface of the first polymer layer, the surface of the cavity exhibiting an advancing contact angle with water of less than 90 degrees; The kit of claim 12, comprising:

14. 14. The kit of claim 12 or 13, wherein the collection article comprises a microstructured substrate comprising a plurality of microstructures extending across a first surface of the microstructured substrate, at least a portion of an outer surface of the plurality of microstructures having a surface that exhibits an advancing contact angle with water of less than 90 degrees.

15. 15. The kit of any one of claims 12-14, wherein the cavity of the collection device or at least a portion of the exterior surface of the plurality of microstructures of at least one of the device or collection article comprises a surfactant, a surface treatment, a hydrophilic polymer, a flocculating agent, or any combination thereof.

16. 16. The kit of claim 15, wherein the agglutinating agent is hydrophilic and non-hemolytic.

17. 17. The kit of claim 15 or 16, wherein the flocculating agent comprises a modified or unmodified amino polymer selected from the group consisting of polyethyleneimine, polylysine, polyaminoamide, polyallylamine, polyvinylamine, polydimethylamine-epichlorohydrin-ethylenediamine, polydiallyldimethylammonium chloride, cationic polyacrylamide (CPAM), polyaminosiloxane, and dendrimers formed from polyaminated amines (PAMAM) and polypropyleneimine.

18. 18. The kit of any one of claims 15 to 17, wherein the agglutinating agent comprises a modified or unmodified material selected from the group consisting of gelatin, collagen, fibrinogen, dextran, hydroxyethyl starch (HES), pentastarch, polyvinylpyrrolidone (PVP), and polyethylene glycol (PEG).

19. The kit of any one of claims 15 to 18, wherein the flocculating agent comprises a modified or unmodified polyethyleneimine polymer.

20. The kit of any one of claims 15 to 19, wherein the agglomerating agent comprises modified or unmodified gelatin.

21. 21. The kit of any one of claims 12-20, wherein the microstructure of at least one of the device or the collection article comprises a plurality of ribs alternating with channels extending across the first surface of the microstructured substrate, each of the ribs comprising a sidewall and a top surface, and each of the channels comprising a bottom surface.

22. 22. The kit of claim 21, wherein the top surface of each rib is the top of a cap disposed on the side wall, the cap having a width greater than the width between the opposing side walls.

23. 22. The kit of claim 21, wherein the collection article comprises a plurality of ribs alternating with channels, each channel comprising at least one secondary channel.

24. 21. The kit of any one of claims 12-20, wherein the microstructure of at least one of the device or the collection article comprises an array of peak structures and adjacent valleys, the valleys having a maximum width in the range of 10 microns to 250 microns, and the peak structures having an apex angle of greater than 5 degrees up to 90 degrees.

25. 25. The kit of claim 24, wherein the array of peak structures and adjacent valleys extending across the first surface of the microstructured substrate is disposed at an angle between 0 degrees and 90 degrees relative to a flow direction of the device.

26. 26. The kit of claim 24 or 25, wherein the array of peak structures and adjacent valleys of at least one of the device or collection article further comprises gaps between adjacent peak structures.

27. 27. The kit of any one of claims 12-26, wherein the microstructure of at least one of the device or the collection article comprises a two-dimensional (x-axis and y-axis) array of protrusions arranged across the first surface of the microstructured substrate, each of the protrusions comprising a base, an apex, and one or more sides connecting the apex to the base.

28. 21. The kit of any one of claims 12-20, wherein the microstructured substrate of at least one of the device or the collection article comprises a microstructured layer having first and second major surfaces, the microstructure comprising a plurality of cavities extending between the first and second major surfaces, each cavity comprising a first opening, a second opening, and at least one sidewall extending between the first opening and the second opening.

29. 21. The kit of any one of claims 12-20, wherein the microstructure of at least one of the device or the collection article comprises a facet and a sidewall that meets the facet at a ridge of the microstructure, the facet and the sidewall defining an oblique angle therebetween.

30. 21. The kit of any one of claims 12-20, wherein the microstructure of at least one of the device or collection article comprises an array of interconnected wells, at least 80% of the wells being fluidly connected to at least two adjacent wells, each connected via a vent.

31. The kit of any one of claims 12 to 20, wherein the microstructure comprises an array of upstanding stems extending across the first surface of the microstructured substrate.

32. 32. The kit of any one of claims 12 to 31, wherein the ratio of the first open volume to the second open volume is greater than 1:

1.

33. The kit of any one of claims 12 to 32, further comprising at least one filter.

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