Method and kit for removing particles from a fluid

Abstract

The present disclosure provides methods and kits for removing particles from a fluid. A method for removing red blood cells includes obtaining a device having a microstructured substrate including a microstructure extending across a first surface, where at least a portion of an outer surface of the microstructure is configured to allow capillary action. The apparatus further includes a cover disposed at 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 perimeter of the first surface of the microstructured substrate. The method further includes filling the device with a volume of blood through the first aperture via capillary action; waiting for a time sufficient to cause at least a portion of the erythrocytes to settle within the first open volume of the microstructures; and fluidly coupling the device with a collection article such that some of the initial volume of blood flows out of the device, where at least some of the erythrocytes from the initial volume of blood have retained within the first open volume of the microstructures. Also included is a kit comprising the device and the collection article.

Description

Background Art

[0001] The identification and quantification of biomarkers in blood typically require the removal of red blood cells prior to analysis. Red blood cells are present in whole blood at a concentration of 35% - 50%, reported as the hematocrit level. This high concentration can interfere with biomarker assays. The level of interference varies within the physiological hematocrit range, which results in variability in the reported concentration of the biomarker. Interference in optical detection assays (e.g., ELISA) can be caused by the light scattering effect and absorption of hemoglobin in the visible spectrum. Red blood cells can also interfere with electrochemical detection assay methods, such as those commonly used in glucose test strips. Chemical substances, such as oxygen present in red blood cells, can interfere with redox reactions.

[0002] In centralized hospitals or clinical laboratories, the separation of red blood cells is achieved by centrifugation. The separation process requires the collection of a large volume (e.g., milliliters) of blood by venipuncture in a test tube. During centrifugation, the red blood cells are aggregated at the bottom of the tube, leaving the remaining blood (e.g., cell-free plasma) accessible in the top layer for further analysis. In these centralized settings, biomarker detection is then typically performed on large, complex analyzers that are capable of automated liquid handling and can frequently verify assay performance through calibration.

[0003] Point-of-care blood analyzers used outside of central laboratories also require the removal of red blood cells prior to analysis. In situations where rapid results are needed, venipuncture to obtain a quantity of blood and bench-top centrifugation are often not available or are too time-consuming. Pricking a finger provides approximately 5 microliters of blood. Glucose test strips typically receive less than 1 microliter of blood and are subject to the interference mentioned above. Removing 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 that includes a microstructured substrate including 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 permit capillary action. The device further includes: a cover spaced apart from a top of the first surface of the microstructured substrate by a selected distance; and at least one sidewall attaching the cover to the first surface of the microstructured substrate along a perimeter of the first surface of the microstructured substrate. Further, the device includes a first orifice defined by at least one of the microstructured substrate or the cover and a second orifice 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 a sum of open spaces from a bottom to a top of each of the plurality of microstructures located between the plurality of microstructures, wherein 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, and taking a sum of the combined first open volume and the second open volume as 100% open volume, a percentage of the first open volume in the 100% open volume is greater than a volume percentage of red blood cells present in the blood. The method further includes filling the device with a volume of blood via capillary action through the first orifice and waiting for 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. Additionally, the method includes fluidly coupling the device to a collection article at the first orifice or the second orifice such that at least 10% of an initial volume of blood flows out of the device and via capillary action onto the collection article, wherein at least some of the red blood cells from the initial volume of blood have remained within the first open volume of the plurality of microstructures.

[0005] In a second aspect, a method for separating solid particles from a fluid is provided. The method includes obtaining a device that includes a microstructured substrate that includes 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 permit capillary action. The device further includes: a cover spaced apart from a top of the first surface of the microstructured substrate by a selected distance; and at least one sidewall attaching the cover to the first surface of the microstructured substrate along a perimeter of the first surface of the microstructured substrate. Further, the device includes a first orifice defined by at least one of the microstructured substrate or the cover and a second orifice 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 a sum of open spaces from a bottom to a top of each of the plurality of microstructures that are between the plurality of microstructures, wherein 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 positioned adjacent to the first open volume. A sum of the combined first open volume and the second open volume is taken as a 100% open volume, and a percentage of the first open volume in the 100% open volume is greater than a volume percentage of particles present in the fluid. The method further includes filling the device with a volume of the fluid through the first orifice by capillary action and waiting for a time sufficient for at least a portion of the particles to settle within the first open volume of the plurality of microstructures. Additionally, the method includes fluidly coupling the device to a collection article at the first orifice or the second orifice such that at least 10% of an initial volume of blood flows out of the device and flows by capillary action onto the collection article, wherein at least some of the red blood cells from the initial volume of blood have remained within the first open volume of the plurality of microstructures.

[0006] In a third aspect, a kit is provided. The kit includes a device and a collection article. The device includes a microstructured substrate that includes 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 an outer surface of the plurality of microstructures is configured to permit capillary action. The device further includes: a cover spaced apart from a top of the first surface of the microstructured substrate by a selected distance; and at least one sidewall attaching the cover to the first surface of the microstructured substrate along a perimeter of the first surface of the microstructured substrate. Further, the device includes a first orifice defined by at least one of the microstructured substrate or the cover and a second orifice 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 a sum of open spaces from a bottom to a top of each of the plurality of microstructures located between the plurality of microstructures, wherein 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 found that the devices, kits, and methods according to at least some embodiments of the present disclosure can provide for the removal of solid particles (e.g., red blood cells) from very small (e.g., microliter) volumes of fluid (e.g., blood).

[0008] The foregoing summary of the present disclosure is not intended to describe every disclosed embodiment or every implementation of the present disclosure. The following description more particularly exemplifies illustrative embodiments. Guidance is provided throughout the application by way of lists of examples, which may be used in various combinations. In each case, the recited lists are only used as representative groups and should not be construed as exclusive lists. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 is a general flowchart of an exemplary method.

[0010] Figure 2A is a perspective view of a general schematic of an exemplary device.

[0011] Figure 2B is a perspective view of a general schematic of a microstructured substrate used in an exemplary device.

[0012] Figure 3A is a scanning electron microscope (SEM) image of a cross-section of a portion of an exemplary device, which is labeled to illustrate a portion of an exemplary method.

[0013] Figure 3B is Figure 3ASEM images of a cross-section of this portion of an exemplary device and an exemplary collection article, which are marked to illustrate another portion of the exemplary method.

[0014] Figure 4A is a schematic cross-sectional view of a microstructured substrate having a plurality of ribs alternating with channels.

[0015] Figure 4B is a schematic perspective view of a microstructured substrate having a plurality of ribs alternating with channels, wherein the ribs include a top cover at their top surface.

[0016] Figure 5A is a schematic cross-sectional view of a microstructured substrate having an array of linear prisms.

[0017] Figure 5B is an SEM image of a cross-section of a portion of an exemplary device.

[0018] Figure 5C is a schematic perspective view of a portion of a microstructured substrate having an array of peak structures and adjacent valleys in a certain orientation.

[0019] Figure 5D is a schematic perspective view of a portion of a microstructured substrate having an array of peak structures and adjacent valleys in another orientation.

[0020] Figure 5E is a schematic cross-sectional view of a microstructured substrate having a faceted structure.

[0021] Figure 6A is a schematic cross-sectional view of a microstructured substrate having an array of two-dimensional protrusions.

[0022] Figure 6B is a top plan view of four representative engineered micropatterned regions for an array of two-dimensional protrusions.

[0023] Figure 7A is a schematic cross-sectional view of a microstructured substrate having a plurality of cavities extending between two main surfaces.

[0024] Figure 7B is a generalized schematic exploded view of a microstructured substrate having a plurality of cavities extending between two main surfaces.

[0025] Figure 8A is a generalized schematic exploded view of the device of Example 1.

[0026] Figure 8B is a generalized schematic top view of the device of Example 1.

[0027] Figure 8CIs a general schematic top view of two components of a pump that is attached and fixed to the device of Example 1.

[0028] Figure 8D Is a general schematic perspective view of a device of Example 1 that is adapted to be attached to a pump.

[0029] Figure 9 Is a general flow chart of another exemplary method.

[0030] Figure 10A Is a schematic perspective view of a part of a microstructured substrate having an array of fluidly connected wells.

[0031] Figure 10B Is a schematic top view of a part of a microstructured substrate having an array of circularly shaped fluidly connected wells.

[0032] Figure 11A Is a general schematic view of an exemplary collection article.

[0033] Figure 11B Is a general schematic view of another exemplary collection article.

[0034] Figure 12 Is a schematic cross-sectional view of yet another exemplary collection article.

[0035] Figure 13 Is a schematic cross-sectional view of a microstructured substrate having an array of generally vertical rods.

[0036] Although the above drawings illustrate several embodiments of the present disclosure, as noted in the description, other embodiments are also contemplated. The drawings are not necessarily to scale. In all cases, the present disclosure presents the invention in an exemplary, rather than a limiting, manner. Detailed Description

[0037] As used herein, the term "microreplication" refers to the production of a microstructured surface by a method in which individual feature fidelity is maintained for surface features during fabrication.

[0038] As used herein, the term "microstructure" encompasses both structures (i.e., features) that protrude above the main surface of a substrate and structures that are recessed below the main surface of the substrate. Combinations of protruding and recessed features may be considered. A microstructure also refers to the structure being a predefined molded structure (e.g., obtained by molding a thermoplastic resin against a mold surface that includes a negative image of the microstructure desired to be disposed on a first major side of the substrate), having dimensions in at least two orthogonal directions ranging from about 5 micrometers to about 3000 micrometers. One of these orthogonal directions may generally be perpendicular to the plane of the substrate (e.g., along the z-axis), and thus this dimension may include, for example, protrusion height or recess depth.

[0039] As used herein, the term "capillarity" refers to the flow of a fluid without the assistance of an external force (e.g., pressure, gravity, vacuum, etc.). Capillarity often occurs when an aqueous fluid contacts a hydrophilic surface. An aqueous fluid contains 50 volume % or more water.

[0040] As used herein, the term "hydrophilic" means that a surface is wetted by an aqueous solution and does not indicate whether the material absorbs the aqueous solution. "Wetting" means that when in contact with an aqueous fluid, the surface exhibits spontaneous wicking. "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.

[0041] As used herein, the term "hydrophobic" means that a surface lacks spontaneous wicking when in contact with an aqueous fluid. In some embodiments, a hydrophobic surface exhibits an advancing water contact angle of 70° or greater, preferably 90° or greater.

[0042] As used herein, "curing" means hardening or partially hardening a composition by any mechanism, such as by heat, light, radiation, electron beam, microwave, chemical reaction, or a combination thereof. As used herein, the term "curable" means that a material can be cured or solidified, for example, by heating to remove a solvent, heating to cause polymerization, chemical crosslinking, radiation-induced polymerization or crosslinking, etc. As used herein, "cured" means that a material or composition has been hardened or partially hardened (e.g., polymerized or crosslinked) by curing.

[0043] 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 the process of winding a material onto a support or unwinding it from a support and further processing it in some way. Examples of further processing include coating, cutting, die-cutting, and exposure to radiation, etc. Polymer films can be manufactured to various thicknesses, typically in the range of about 5 microns to 1000 microns.

[0044] As used herein, "solid" refers to the state of a substance that is not a liquid or a gas, and a solid has a stable three-dimensional shape.

[0045] As used herein, "fluid" refers to a composition that includes liquids (i.e., the state of a substance that is not a solid or a gas) and encompasses solutions, suspensions, and emulsions.

[0046] As used herein, the "outer surface" with respect to a microstructure refers to the outermost surface of the microstructure.

[0047] As used herein, "thermoplastic" refers to a polymer that flows when sufficiently heated above its glass transition temperature and becomes solid when cooled. In contrast, "thermosetting" refers to a polymer that permanently solidifies upon curing and does not flow upon subsequent heating. Thermosetting polymers are typically cross-linked polymers.

[0048] As used herein, the term "glass transition temperature" (T g ) of a polymer refers to the transition of the polymer from the glassy state to the rubbery state and can be measured using differential scanning calorimetry (DSC), such as at a heating rate of 10 °C per minute in a nitrogen gas stream. When referring to the T g of a monomer, it is the T g of the homopolymer of that monomer. The homopolymer must have a sufficiently high molecular weight such that the T g reaches a limiting value, as it is generally understood that the T g of a homopolymer will increase to a limiting value as the molecular weight increases. The homopolymer is also understood to be substantially free of moisture, residual monomers, solvents, and other contaminants that may affect the T g . Suitable DSC methods and analysis modes are described in Matsumoto, A. et al., Journal of Polymer Science, Part A, Polymer Chemistry (J. Polym. Sci. A., Polym. Chem.) 1993, 31, 2531-2539.

[0049] As used herein, "transparent" means that a material (e.g., a layer) has a transmittance of at least 50%, 70%, or optionally greater than 90% in the portion of the visible spectrum from at least 400 nanometers (nm) to 700 nm.

[0050] The terms "preferred" and "preferably" refer to embodiments of the present disclosure that can provide certain benefits in certain circumstances. However, in the same or other circumstances, other embodiments may also be preferred. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the present disclosure.

[0051] In this application, terms such as "a", "an", and "the" are not intended to refer only to a singular entity but include the general category for which a particular example can be used to illustrate. The terms "a", "an", and "the" are used interchangeably with the term "at least one". The phrases "at least one of... " and "comprising at least one of... " followed by a list refer to any one of the items in the list and any combination of two or more of the items in the list.

[0052] 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" means one or all of the listed elements or a combination of any two or more of the listed elements.

[0053] Also herein, it is assumed that all numbers are modified by the term "about" and preferably by the term "exactly". As used herein in connection with measured quantities, the term "about" means the variation in the measured quantity that would be expected by a person skilled in the art who has made the measurement and taken a level of care commensurate with the purpose of the measurement and the precision of the measuring equipment used.

[0054] As used herein as a modifier of a characteristic or property, unless otherwise specifically defined, the term "generally" means that the characteristic or property will be readily recognizable by a person of ordinary skill in the art but does not require absolute precision or a perfect match (e.g., within + / - 20% for a quantifiable characteristic). Unless otherwise specifically defined, the term "substantially" means highly approximate (e.g., within + / - 10% for a quantifiable characteristic), but again does not require absolute precision or a perfect match. Terms such as same, equal, consistent, constant, exact, etc. are understood to be within the normal tolerances or measurement errors applicable to a particular situation and do not require absolute precision or a perfect match.

[0055] In point-of-care biomarker analysis, there is a need to simply and effectively separate red blood cells from microliter volumes of blood without hemolysis, dilution, or significant loss of blood to dead space.

[0056] In a first aspect, the present disclosure provides a method for separating red blood cells from blood. The method includes: a) obtaining a device that includes: 1) a microstructured substrate that includes a plurality of microstructures extending across a first surface of the microstructured substrate, wherein at least a portion of an outer surface of the plurality of microstructures is configured to permit capillary action; 2) a cover that is disposed a selected distance above a top of the first surface of the microstructured substrate; 3) at least one sidewall that attaches the cover to the first surface of the microstructured substrate along a perimeter of the first surface of the microstructured substrate; 4) a first orifice defined by at least one of the microstructured substrate or the cover; and 5) a second orifice defined by at least one of the microstructured substrate or the cover; Wherein a first surface of the microstructured substrate and the at least one sidewall together define a first open volume, which is the sum of the open spaces from the bottom to the top of each micro-structure located between the plurality of micro-structures. Wherein the cover and the top of the first surface of the microstructured substrate and the at least one sidewall together define a second open volume located adjacent to the first open volume, and the sum of the combined first open volume and the second open volume is taken as 100% open volume, and the percentage of the first open volume in the 100% open volume is greater than the volume percentage of red blood cells present in the blood; b) filling the device with a volume of blood through the first orifice by capillary action; c) waiting for a time sufficient for at least a portion of the red blood cells to settle within the first open volume of the plurality of micro-structures; and d) fluidly coupling the device to a collection article at the first orifice or the second orifice such that at least 10% of an initial volume of the blood flows out of the device and onto the collection article by capillary action, wherein at least some of the red blood cells from the initial volume of the blood have remained within the first open volume of the plurality of micro-structures of the device.

[0057] Referring Figure 1 , the method includes obtaining a device 110 (wherein the device is as described above); filling the device 120 with a volume of blood through a first orifice by capillary action; waiting for a time 130 sufficient for at least a portion of the red blood cells to settle within the first open volume of the plurality of micro-structures; and fluidly coupling the device to a collection article at the first orifice or the second orifice such that at least 10% of an initial volume of the blood flows out of the device and onto the collection article by capillary action 140, wherein at least some of the red blood cells from the initial volume of the blood have remained within the first open volume of the plurality of micro-structures of the device. 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 micro-structures.

[0058] Referring to FIG. 2 and Figure 3A, an exemplary device 200 is shown. The device 200 includes a microstructured substrate 210, which includes a plurality of microstructures 230 extending across the first surface 202 of the microstructured substrate 210. At least a portion of the outer surface 232 of the plurality of microstructures 230 is configured to allow capillary action. The device 200 further includes a cover 220 spaced apart from the top 221 of the first surface of the microstructured substrate 210 by a selected distance D and at least one sidewall 240 attaching the cover 220 to the first surface 202 of the microstructured substrate 210 along the perimeter P of the first surface of the microstructured substrate 210. The perimeter P is jointly formed by each of the side edges Pa, Pb, Pc, Pd... Pn of the particular device. Figure 2A The illustrated device 200 has four side edges Pa, Pb, Pc, and Pd, so the perimeter P includes each of these four side edges. It is contemplated that devices having other shapes different from four side edges are possible.

[0059] Optionally, at least one sidewall 240 may include an adhesive layer (e.g., which attaches the cover to the substrate) disposed between the cover 220 and the microstructured substrate 210, such as a double-sided tape as used in Preparation 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 an adhesive film containing an adhesive polymer. Preferably, the adhesive contains an adhesive polymer and a crosslinking agent. The term "adhesive polymer" as used herein refers to a polymer that exhibits adhesiveness at ambient temperature (e.g., 20 °C - 25 °C). The adhesive polymer can be, for example, an acrylic polymer, a polyurethane, a polyolefin, or a polyester. In a selected embodiment, the adhesive layer includes a double-sided coated adhesive film. Some suitable commercially available double-sided coated adhesive films are available from the 3M Company (St. Paul, MN) under the trade names 3M Medical Silicone Tape 2477P and one of 3M Medical Tapes 1509, 1510, 1513, 1522, 9874, and 9877.

[0060] Further, the device includes a first orifice 250 defined by at least one of the microstructured substrate 210 or the cover 220 and a second orifice 260 defined by at least one of the microstructured substrate 210 or the cover 220. Figure 2A The illustrated device includes a first orifice 250 defined by the cover 220 and a second orifice 260 defined by both the microstructured substrate 210 and the cover 220.

[0061] In selected embodiments, the sidewall, the microstructured substrate, and the cover are hermetically sealed to each other at their points of contact (e.g., seams), which minimizes leakage of fluid sample from any seams between the three (e.g., fluid can enter and leave primarily or only through the first aperture and / or the second aperture). In some cases, the microstructured substrate itself includes a sidewall in its structure such that at least one sidewall is part of the microstructured substrate.

[0062] Suitable materials for use as the cover include, for example but not limited to, polyolefins (e.g., high density polyethylene (HDPE), medium density polyethylene (MDPE), or low density polyethylene (LDPE)), polyesters, polyamides, polyvinyl chloride, polyether esters, polyimides, polyester amides, polyacrylates, polyvinyl acetate, or hydrolytic derivatives of polyvinyl acetate. In certain embodiments, polyolefins are preferred because they have excellent physical properties, are easy to process, and are generally low cost. Additionally, polyolefins are generally tough, durable, and hold their shape well, and thus are easy to handle after article formation. In selected embodiments, the film layer comprises polyethylene terephthalate (PET). A suitable commercially available PET is a 5 mil (127 micron) thick PET sheet available from Tekra (New Berlin, WI) under the trade name "MELINEX 454". A suitable commercially available LDPE is available from Dow Chemical Company (Midland, MI) under the trade name "DOW 955I LDPE". Additionally, various additives may be included in the cover, such as surface energy modifiers (such as surfactants and hydrophilic polymers), plasticizers, antioxidants, pigments, mold release agents, antistatic agents, and the like.

[0063] 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 between the plurality of microstructures 230 from the bottom 236 to the top 221 of each microstructure 230. (For the sake of brevity, the arrow for 270 only points to the portion of the first open volume located between two adjacent microstructures 230). The cover 220, together with the top 221 of the first surface 202 of the microstructured substrate 210 and at least one sidewall 240, defines a second open volume 280 that is positioned adjacent to the first open volume 270, and the sum of the combined first open volume and the second open volume is taken as 100% open volume, and the percentage of the first open volume in the 100% open volume is greater than the volume percentage of red blood cells present in the blood. In order to adapt the device to blood containing a specific volume percentage of (e.g., solid) particles such as red blood cells, it can be determined that, considering the sum of the combined first open volume and the second open volume as 100% open volume, the percentage of the first open volume in the 100% open volume needs to be greater than the volume percentage of the particles present in the fluid. For example, if the volume percentage of the particles is 20% of the total volume of the fluid, the appropriate ratio of the first open volume to the second open volume will be greater than 1:4 (e.g., 1.1:4). If the volume percentage of the particles is 75% of the total volume of the fluid, the appropriate ratio of the first open volume to the second open volume will be greater than 3:1 (e.g., 3.1:1).

[0064] In some cases, the ratio of the first open volume 270 to the 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 at most 2.0:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, or at most 1.5:1. Brace 272 represents the height of the first open volume 270, and brace 282 represents the height of the second open volume 280, and the plus sign indicates that when the device 220 is oriented as shown in the z-axis and y-axis, in this device 220 the second volume 280 is directly stacked on top of the first open volume 270. Note that Figure 3A and Figure 3B the device 200 in is not drawn to scale for a ratio of the first open volume 270 to the second open volume 280 of 1:1 or greater, and is shown to assist in describing the devices and methods provided herein.

[0065] Optionally, it is also useful to select a specific relationship (e.g., ratio) between the average height of the plurality of microstructures and the average width of the spacing between each of the plurality of microstructures. Preferably, the first open volume is large enough so that if all the red blood cells settle, no red blood cells should protrude above the top of the microstructures.

[0066] As described above, at least a portion of the outer surface of the plurality of microstructures is configured to permit capillary action. Capillary action is well known in the art to refer to fluid flow without external force assistance, typically for aqueous fluids (having 50% or more by volume of water) in contact with a hydrophilic surface. Thus, once blood is introduced into the first orifice of the device, the blood is spontaneously transported along the outer surface of the microstructures, thereby diffusing within the region of the microstructures. Two general factors that affect the ability of the microstructures to spontaneously transport fluid are (i) the structure or topography of the surface (e.g., capillary action, shape of the cavities) and (ii) the nature of the surface (e.g., surface energy). To achieve a desired amount of fluid transport ability, the designer can adjust the structure or topography of the base layer and / or adjust the surface energy of the capillary microstructured surface. To achieve wicking, the surface of the capillary microstructures must be able to be "wetted" by the liquid to be transported (e.g., a liquid substance). Optionally, the sensitivity of the solid surface to be wetted by the liquid is characterized by the contact angle formed between the liquid deposited on a horizontally placed surface and stabilized thereon and the solid surface. This angle is sometimes referred to as the "static equilibrium contact angle" and is sometimes referred to herein simply as the "advancing contact angle". In some cases, if a material has an advancing contact angle of less than 90 degrees, the material is considered hydrophilic, and a hydrophilic surface exhibits an advancing (maximum) water contact angle of less than 90°, preferably 45° or less.

[0067] It is necessary to make the outer surfaces of a sufficient number of microstructures hydrophilic so that the microstructured substrate can perform capillary action, thereby transporting the fluid (e.g., blood) throughout the device once it has been introduced via the first orifice, so that solid particles can enter the open volume between the microstructures and thus settle out from the bulk fluid. In some cases, 50% or more of the surface area of the outer surface of the microstructures can perform capillary action, and 60%, 70%, 80%, 90% or 95% or more of the outer surface of the microstructures can perform capillary action. In a selected embodiment, at least a portion of the main surface 223 of the cover 220 facing the microstructured substrate 230 is hydrophilic to assist in capillary action of the fluid within the device 200. According to any of the devices described herein, the hydrophilicity of the outer surface of the microstructures and / or the main surface of the cover 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 outer surface comprising a surfactant, a surface treatment agent, a hydrophilic polymer, a flocculant, or any combination thereof. Suitable surfactants include, for example but not limited to, C8-C18 alkane sulfonates; C8-C18 secondary alkyl sulfonates; alkyl benzene sulfonates; C8-C18 alkyl sulfates; alkyl ether sulfates; sodium lauryl polyoxyethylene ether 4 sulfate; sodium lauryl polyoxyethylene ether 8 sulfate; dioctyl sulfosuccinate, sodium salt; lauroyl lactate; stearoyl lactate; or any combination thereof. One or more surfactants can be applied by conventional methods, such as by rubbing a surfactant coating onto the surface of the microstructures and drying the coating. Suitable surface treatments include hydrophilic coatings that comprise plasma-deposited silicon / oxygen materials and / or diamond-like glass (DLG) materials. For example, plasma deposition of each of the silicon / oxygen materials and DLG materials is described in PCT Publication No. WO 2007 / 075665 (Somasiri et al.). In addition, 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, for example but not limited to, polyesters, polyamides, polyurethanes, poly(vinyl alcohol), poly(alkylene glycols), poly(epoxides), poly(vinyl pyrrolidone), rubber elastomers, or any combination thereof.

[0068] The use of certain ionic polymers, particularly cationic polymers, for the flocculation of cells and / or cell debris and for the precipitation of proteins is known. When using a flocculant, a device having a microstructure that is larger than the microstructure when not using a flocculant (e.g., height and / or depth, spacing between adjacent microstructures, etc.) can be used. This is because when flocculated, the particles tend to agglomerate and have a larger size, such that a larger space may be required to hold the particles within the microstructure. Conversely, a device with a large microstructure may be less effective in retaining unflocculated particles (e.g., red blood cells) separated from a fluid (e.g., blood). Thus, the microstructured surface of the device can be selected in part by considering the expected size of the particles or flocculated particles.

[0069] The polymer used as a flocculant can be unmodified (e.g., polyethyleneimine) or modified (e.g., guanylated polyethyleneimine). In some embodiments, the suitable flocculant is hydrophilic and non-hemolytic (i.e., does not lyse red blood cells). Some suitable flocculants are described in detail in U.S. Patent Nos. 8,435,776 (Rasmussen et al.) and 10,005,814 (Rasmussen et al.), which are hereby incorporated by reference in their entireties. In certain cases, the flocculant comprises an unmodified or modified (e.g., functionalized) amino polymer. For example, suitable amino polymers can be selected from the group consisting of: polyethyleneimine, polylysine, polyaminoamide, polyallylamine, polyvinylamine, poly(dimethylamine-epichlorohydrin-ethylenediamine), poly(diallyldimethylammonium chloride), cationic polyacrylamide (CPAM), polyaminosiloxane, and dendrimers formed from polyamidoamine (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, poly(dimethylamine-epichlorohydrin-ethylenediamine), poly(diallyldimethylammonium chloride), CPAM, polyaminosiloxane, and dendrimers formed from PAMAM and polypropyleneimine. For example, the functionalization can include reacting the amino polymer with an alkylating, acylating, or amidinating reagent. In selected embodiments, the flocculant comprises a modified or unmodified polyethyleneimine polymer.

[0070] In some embodiments, the suitable flocculant 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). In selected embodiments, the flocculant comprises a modified or unmodified gelatin.

[0071] One or more flocculants can be applied by conventional methods, such as by applying a flocculant coating on the surface of the microstructure and drying the coating.

[0072] In some cases, the flocculant has a weight average molecular weight (Mw) of 5,000 grams per mole (g / mol) or greater, 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 greater as determined by gel permeation chromatography; and a weight average molecular weight of 500,000 g / mol or less. Sometimes, a high molecular weight (e.g., 50,000 g / mol or higher) can help flocculate particles (e.g., red blood cells).

[0073] Advantageously, after filling the device with a certain volume of fluid, at least some of the flocculant tends to dissolve, disperse, or dissolve and disperse into the fluid (e.g., blood). In some cases, the amount of flocculant employed is designed such that the concentration of the flocculant in a certain volume of fluid sample (e.g., blood) is 0.01 micrograms per milliliter (µg / mL) of fluid or greater, 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, 1000 µg / mL, or 1500 µg / mL or greater; and 5000 µg / mL or less, 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 or less. In other words, in some embodiments, the flocculant is present in a certain volume of fluid (e.g., blood) in an amount of 0.01 µg / mL to 5000 µg / mL. When in the form of a dry coating on the surface of the device, the amount of flocculant will vary based on the molecular weight (Mw) of the particular flocculant.

[0074] Figure 3A and Figure 3BIt also incorporates a cartoon depiction on the SEM image of the device to illustrate the concept of how the device 200 is typically used. For example, after the blood 295 has been filled into the device 200, using capillary action, the red blood cells 290 start to settle between the microstructures 230 into the first open volume 270. In Figure 3A , only three red blood cells 290 are depicted as being located in the first open volume 270. Referring to Figure 3B , the device 200 is depicted after waiting for a time sufficient for all the red blood cells 290 to settle within the first open volume 270 of the plurality of microstructures 230. Typically, red blood cells settle due to the action of gravity only. There is a volume of blood 295 in the second open volume 280 from which the red blood cells have been removed. In use, when the device is fluidly coupled to the collection article 100 at a first orifice (not shown) or at the second orifice 260, a quantity of an initial volume of blood flows out of the device 200 via capillary action and onto the collection article 100, wherein at least some of the red blood cells from the initial volume of blood have been retained within the first open volume 270 of the plurality of microstructures 230.

[0075] The collection article 100 is not particularly limited as long as the collection article is capable of capillary action. Various types of collection articles are described below. Figure 3B The collection article 100 depicted in

[0076] Optionally, the method further comprises passing a fluid (e.g., blood) through a filter before entering the device, after leaving the device, or both before entering the device and after leaving the device. For example, filtering the fluid can be used to remove at least one undesired component from the fluid. Suitable filters include, for example but not limited to, non-woven filters, textile filters, membrane filters, paper filters, and sponge filters. Exemplary suitable filters include, for example, fiberglass filters and asymmetric polysulfone / polyethersulfone filters (e.g., such as the VIVID plasma separation membrane available commercially from Pall Corporation (Port Washington, NY), or the Cobetter OneStep plasma separation membrane or Cobetter RB series, both available commercially from Cobetter Filtration Equipment Co., Ltd. (Hangzhou, China)).

[0077] In some cases, the method further comprises adding a flocculant to a volume of fluid (e.g., blood) before filling the device with the volume of fluid (e.g., blood). The flocculant can be as described in detail above, including the concentration present in the volume of fluid (e.g., an amount from 0.01 micrograms / mL to 5000 micrograms / mL of fluid).

[0078] Red blood cells typically account for 35%-50% of the total volume of whole blood. Having a ratio of the first open volume to the second open volume of 1:1 or greater provides sufficient space within the first open volume between the microstructures to retain up to all of the red blood cells of an undiluted whole blood sample, while leaving the second open volume free for blood containing fewer (to none) red blood cells. Thus, when the device is contacted with a collection article after sedimentation, the blood (in some cases plasma) from which the red blood cells have been removed preferentially exits the device because it is positioned closer to the orifice. In contrast, the sedimented red blood cells tend to remain between the microstructures within the first open volume and are less likely to be removed from the device upon contact with the collection article. In some cases, the ratio of the first open volume to the second open volume is selected to minimize the volume of blood that can sediment in the first open volume having red blood cells, so as to maximize the volume of blood (from which red blood cells have been removed) that can flow 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, it is generally not necessary to dilute whole blood to effectively separate red blood cells from the blood, such that the blood can be used in an undiluted form. However, in cases where the blood is diluted, a smaller ratio 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.

[0079] Advantageously, the devices according to at least some embodiments of the present disclosure effectively separate (at least a portion of) red blood cells from a small volume of blood while minimizing the residence time of the analyzable blood within the device. This is contrary to devices that employ a flowing stream or result in dead volume losses. Examples of red blood cell separation devices that require flowing blood include U.S. 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.), KR 2010 / 0048507 (Chun et al.), and TW I338134 (Chou et al.).

[0080] Preferably, when in contact with the collected article, at least 10%, 15%, 20%, 25%, 30%, 35%, or even at least 40% of the blood from which red blood cells have been removed exits the device. The greater this percentage, the more efficient the separation method using the device according to the present disclosure, and the smaller the volume of the analyzable sample lost in the device.

[0081] 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, the devices according to at least some embodiments of the present disclosure are suitable for removing solid particles (e.g., red blood cells) from a fluid (e.g., blood); 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 and second open volumes of the device can be between 5 microliters and 120 microliters.

[0082] In some cases, the first orifice is defined by a cover of the device, and a volume of fluid (e.g., blood) can be introduced into the device using the force of gravity and / or by wicking the fluid into the orifice. In other cases, the first orifice is defined by a microstructured substrate. For example, refer to Figure 2B, the first orifice 250 can be configured as a reservoir that is constructed to hold at least a certain minimum volume of fluid. The reservoir can be defined by a portion of the microstructured substrate 210 adjacent to an end 237 of the microstructure 230. Similarly, the second orifice 260 can be a reservoir that is constructed to hold the fluid when the fluid exits the microstructured surface 202 of the microstructured substrate 210. Generally, it is advantageous that the second orifice is positioned at a distance from the first orifice to easily allow the displacement gas (typically air) from the device to be discharged when the fluid is deposited into the device via the first orifice. Optionally, a positive pressure can be provided by a pump to deposit a certain volume of blood into the first orifice. Optionally, a certain volume of blood can be drawn into the device by a core through the first orifice. In some cases, it is preferred that the blood 295 is discharged from the second orifice 260. Louver structure

[0083] Referring again to Figure 3A and 3B , the microstructure 230 of the device of certain embodiments includes a "louver structure" that includes ribs separated by channels. This shape is also shown in Figure 4A , where the microstructure 230 includes a plurality of ribs 230 alternating with channels 201, the plurality of ribs extending across the first surface 202 of the microstructured substrate 210, and where each rib 230 includes sidewalls 233 and 234 and a top surface 221, and each channel 201 includes a bottom surface 205.

[0084] More specifically, the depicted microstructured substrate 210 includes a plurality of channels 201a - 201d on a base layer 213. As Figure 4A shown, a continuous platform layer "L" can be present between the bottom of the channel 205 and the top surface 202 of the base layer 213. Alternatively, the channels 201 can extend all the way through the microstructured substrate 210. In some cases (as Figure 4B shown), the bottom surface 205 of the trench can coincide with the top surface 202 of the base layer 213. In a typical embodiment, the base layer 213 is a preformed film including an organic polymeric material different from the ribs 230.

[0085] The height and width of the rib (e.g., protrusion) 230 are defined by adjacent channels (e.g., 201a and 201b). The rib 230 can be defined by a top surface 221, a bottom surface 231, and sidewalls 233 and 234 that couple the top surface 221 to the bottom surface 231. The sidewalls 233 and 234 can be parallel to each other. More typically, the sidewalls have a wall angle.

[0086] The rib 230 may be defined by a width "W". Generally, the rib 230 has a width parallel to the first surface of the microstructured substrate and a height orthogonal to the first surface of the microstructured substrate. Except for the platform region "L", the rib 230 typically has a height nominally the same as that of the channel 201. In a typical embodiment, the height "H" of the channel 201 and / or the rib 230 is at least 10 microns, 20 microns, 30 microns, 40 microns, 50 microns, 60 microns, 70 microns, 80 microns, 90 microns or 100 microns. In some embodiments, the height is not greater than 250 microns, 240 microns, 230 microns, 220 microns, 210 microns, 200 microns, 190 microns, 180 microns, 170 microns, 160 microns, 150 microns, 140 microns, 130 microns, 120 microns, 110 microns or 100 microns. In some embodiments, the height of the channel 201 and / or the rib 230 is in the range of 50 microns to 250 microns. The microstructured substrate typically includes a plurality of ribs 230 having nominally the same height and width. In some embodiments, the rib 230 has a height "H", a maximum width "W" at its widest part, 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 rib is at least 6, 7, 8, 9 or 10. In other embodiments, the aspect ratio of the rib is at least 15, 20, 25, 30, 35, 40, 45 or 50.

[0087] The channel 201 has a height "H" defined by the distance between the bottom surface 205 and the top surface 221, which is typically parallel to the top surface 202 of the base layer 213. The channel 201 has a maximum width "W" and is spaced apart at a pitch "P" along the microstructured surface 202. The width "W" of the channel at the base (i.e., adjacent to the bottom surface 205) is typically nominally the same as the width of the adjacent top surface 221 of the channel. However, when the width of the channel at the base is different from the width of the adjacent top surface, the width is defined by the maximum width. For an area of ​​interest, such as an area for calculating the first open volume, the maximum width of multiple channels can be averaged. The microstructured substrate may include multiple channels with nominally the same height and width. In a typical embodiment, the channel typically has a width of no more than 50 microns, 40 microns, 30 microns, 20 microns, 10 microns, 9 microns, 8 microns, 7 microns, 6 microns, 5 microns, 4 microns, 3 microns, 2 microns, or 1 micron. In some embodiments, the channel generally has a width of no greater than 900 microns, 800 microns, 700 microns, 600 microns, or 500 microns. In some embodiments, the channel has a width of at least 50 microns, 60 microns, 70 microns, 80 microns, 90 microns, or 100 microns.

[0088] In some embodiments, the wall angle θ is greater than 90 degrees, 91 degrees, 92 degrees, 93 degrees, 94 degrees, 95 degrees, 96 degrees, 97 degrees, 98 degrees, 99 degrees, or 100 degrees. In some embodiments, the wall angle is no greater than 110 degrees, 109 degrees, 108 degrees, 107 degrees, 106 degrees, 105 degrees, 104 degrees, 103 degrees, 102 degrees, 101 degrees, 100 degrees, 99 degrees, 98 degrees, 97 degrees, 96 degrees, or 95 degrees. In some embodiments, the wall angle is close to 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 comprising a first sidewall and a second sidewall, wherein the first sidewall has a wall angle at which a line parallel to the first surface of the microstructured substrate is 0 degrees to +10 degrees or 0 degrees to -10 degrees relative to the bottom surface of the microstructured substrate.

[0089] In some embodiments, ribs 230 have a pitch "P" of at least 10 microns. The pitch is as Figure 4AThe distance between the depicted starting points of the first rib and the second rib. The spacing can be at least 15 microns, 20 microns, 25 microns, 30 microns, 35 microns, 40 microns, 45 microns or 50 microns, 60 microns or 70 microns. This spacing is typically not greater than 1 mm. The spacing is typically not greater than 900 microns, 800 microns, 700 microns, 600 microns or 500 microns. In some embodiments, the spacing is typically not greater than 550 microns, 500 microns, 450 microns, 400 microns, 350 microns, 300 microns, 250 microns or 200 microns. In some embodiments, the spacing is not greater than 175 microns, 150 microns, 100 microns. In typical embodiments, the ribs are evenly spaced with a single spacing. Alternatively, the ribs can be spaced such that the spacing between adjacent ribs is not the same. In this subsequent embodiment, at least some and typically most (at least 50%, 60%, 70%, 80%, 90% or more of the total ribs) have the spacing just described. The spacing of the channels is in the same range as the ribs just described. Optionally, the channels have an average spacing of 10 microns to 200 microns. The spacing and height of the ribs are important for facilitating coating of the ribs with a coating. When the ribs are spaced too closely, it may be difficult to coat the sidewalls evenly. When the ribs are spaced too far apart, the coating may not effectively provide its intended function.

[0090] Figure 4B is depicted in Figure 4A a variant of the louver structure, where in some cases, the top surface 221 of each rib 230 is the top of a top cover 235 that is disposed on sidewalls 233, 234, and the top cover 235 has a width ("CW") that is greater than the width ("WW") between the opposing sidewalls 233 and 234. Without being bound by theory, it is believed that once the particles have settled in the device, the presence of the top cover (e.g., undercut feature) on the rib can help retain the particles in the channel.

[0091] The louver structure can be prepared by any suitable method. In one embodiment, the structure (e.g., Figure 4AThe microstructured substrate 210) shown can be prepared by a method comprising the following steps: (a) preparing a polymerizable composition; (b) depositing the polymerizable composition in an amount just sufficient to fill the cavities of a master mold onto a negative microstructured molding surface (e.g., a tool) of the master mold; (c) filling the cavities by moving beads of the polymerizable composition between a base layer (e.g., a preformed film) and the master mold, wherein at least one of the base layer and the master mold is flexible; and (d) curing the composition. The deposition temperature can range from ambient temperature to about 180°F (82°C). The master mold can be metallic, such as nickel, copper or brass plated with chromium or nickel, or can be a thermoplastic material that is stable under polymerization conditions and has a surface energy that allows complete removal of the polymeric material from the master mold. When the base layer is a preformed film, one or more surfaces of the film can optionally be primed or otherwise treated to promote adhesion to the organic material of the microstructures.

[0092] In one embodiment, the structure (e.g., Figure 4B the microstructured substrate 210) shown having a top cap on ribs can be prepared by known methods of making undercut features (e.g., partially disassembling a mold made of multiple parts after molding, thereby opening each cavity and allowing easy removal of the features; or by first molding straight ribs without undercuts and then capping these rods in a separate forming step after demolding). Alternatively, such microstructured substrates can be made according to the disclosure of PCT Publication No. WO2015 / 041844 (Rule et al.), wherein a polyolefin resin is deposited into a mold cavity to form a first layer that includes a plurality of undercut features on and extending from an integral backing, and the first layer is demolded from the mold cavity at a rate of at least 150 millimeters per minute (mm / min).

[0093] The polymerizable resin can comprise a combination of a first polymerizable component and a second polymerizable component selected from (meth)acrylate monomers, (meth)acrylate oligomers, and mixtures thereof. As used herein, a "monomer" or "oligomer" is any substance that can be converted into a polymer. Similarly, the term "(meth)acrylate" refers to acrylate and / or methacrylate compounds. In some cases, the polymerizable composition can comprise (meth)acrylated urethane oligomers, (meth)acrylated epoxy oligomers, (meth)acrylated polyester oligomers, (meth)acrylated phenolic oligomers, (meth)acrylated acrylic oligomers, and mixtures thereof.

[0094] The polymerizable resin can be a radiation-curable polymer resin, such as a UV-curable resin. In some cases, the polymerizable resin composition useful for the microstructured substrates of the present disclosure can include a polymerizable resin composition such as that described in U.S. Patent No. 8,012,567 (Gaides et al.).

[0095] The chemical composition and thickness of the base layer can depend on the end use of the microstructured substrate. In typical embodiments, 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 oriented films of polyethylene, polypropylene, and polycycloolefins), polyimide, and glass. Optionally, the base layer can contain mixtures or combinations of these materials. In some embodiments, the base layer can be multilayered or can contain dispersed components suspended or dispersed in a continuous phase.

[0096] Examples of base layer materials include polyethylene terephthalate (PET) and polycarbonate (PC). Examples of useful PET films include optical grade polyethylene terephthalate, available under the trade name “Melinex 618” from DuPont Films of Wilmington, Del. Examples of optical grade polycarbonate films include LEXAN polycarbonate film 8010, available from GE Polymershapes, Seattle, WA, and Panlite 1151, available from Teijin, Alpha, GA.

[0097] Alternatively, the microstructured substrate 210 can be prepared by melt extrusion, i.e., pouring a fluid resin composition onto a master negative microstructured molding surface (e.g., a tool) and allowing the composition to harden. In this embodiment, the ribs 230 are interconnected with the base layer 213 in a continuous layer. Each rib and the connections between them typically comprise the same thermoplastic material. The thickness of the platform layer (i.e., the thickness excluding the portions created by the replicated microstructures) is typically between 0.001 inches and 0.100 inches, and preferably between 0.003 inches and 0.010 inches. Suitable resin compositions for melt extrusion are transparent materials that are dimensionally stable, durable, weather resistant, and can be easily formed into the desired configuration. Examples of suitable materials include acrylic resins 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 trade name SURLYN by Dow Chemical (Midland, MI), E.I. Dupont de Nemours and Co., Inc.; (poly)ethylene-co-acrylic acid; polyesters; polyurethanes; and cellulose acetate butyrate.

[0098] In another embodiment, the master negative microstructured molding surface (e.g., a tool) can be used as an embossing tool, such as that described in U.S. Patent No. 4,601,861 (Pricone).

[0099] Further details regarding microstructured substrates having such louver structures and how to form such microstructured substrates are described in WO 2019 / 118685 (Schmidt et al.) and WO 2020 / 026139 (Schmidt et al.), each of which is incorporated herein by reference. Prismatic structure

[0100] Figure 5AAn alternative microstructured substrate 309 is shown that includes 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 planar surface 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 straight prisms. A straight prism means that the apex angle θ, 340, is typically about 90°. However, this angle can range from 5° to 90°, and can range from 20° to 80°. In selected embodiments, the microstructures include linear prisms having an apex angle less than or equal to 90 degrees. In some embodiments, the apex angle of the peak structure is typically twice the wall angle, especially when the facets of the peak structure are interconnected at the valleys between the peak structures. Thus, the apex angle is typically greater than 5 degrees, more typically at least 25 degrees, 30 degrees, 35 degrees, 40 degrees, 45 degrees, 50 degrees, 55 degrees, or 60 degrees. The apex angle of the peak structure is typically less than 90 degrees, more typically less than 85 degrees, 80 degrees, 75 degrees, 70 degrees, 65 degrees, 60 degrees, 55 degrees, 50 degrees, 45 degrees, 40 degrees, or 35 degrees. Optionally, there is a gap between adjacent peak structures 320, e.g., at the first planar surface 331 or parallel to the first planar surface.

[0101] These vertices can be sharp (as shown), rounded, or truncated. In some cases, it is advantageous to use sharp or rounded vertices because the likelihood of particles settling on top of these shapes is lower than that of truncated (e.g., flat) shapes. Preferably, the radius of the prism tip is less than the radius of the particles (e.g., red blood cells). The space between the peaks (e.g., prisms) can be characterized as the pitch ("P"). In this embodiment, the pitch is also equal to the maximum width of the valley. Thus, as previously described, the pitch is greater than 1 micron, 2 microns, 3 microns, 4 microns, 5 microns, 6 microns, 7 microns, 8 microns, 9 microns, or 10 microns, up to a range of 250 microns. The length ("L") of the prism microstructures is typically the largest dimension and can span the entire dimension of the microstructured surface, film, or article. The prism facets do not need to be the same, and the prisms can be inclined relative to each other. The facets of adjacent peak structures typically connect at the bottom of the valley, i.e., close to the planar base layer. The facets of the peak structures form a continuous surface in the same direction. For example, in Figure 5A , the facets 321 and 322 of the prism peak structures are continuous in the direction of the length (L) of the microstructure, or in other words in the y direction.

[0102] Optionally, a continuous platform layer 360 may be present between the bottom of the channel or valley and the top surface 331 of the base member 310 (e.g., a planar one). In some embodiments, such as when the microstructured surface is prepared by casting and curing a polymerizable resin composition, the thickness of the platform layer is typically at least 0.5 microns, 1 micron, 2 microns, 3 microns, 4 microns, or 5 microns, up to a maximum of 50 microns. In some embodiments, the thickness of the platform layer is not greater than 45 microns, 40 microns, 35 microns, 30 microns, 25 microns, 20 microns, 15 microns, or 10 microns.

[0103] Now referring to Figure 5B , there is provided a SEM image of a cross-section of a portion of an exemplary device of Preparation Example 12 having a prismatic microstructure. The first surface 302 of the microstructured substrate 325, together with at least one sidewall 340, defines a first open volume 370, which is the sum of the open spaces from the bottom 336 to the top 327 of each micro-structure 320 located between the plurality of micro-structures 320. (For the sake of brevity, the arrow for 370 only points to a portion of the first open volume located between two adjacent micro-structures 320). The cover 352, together with the top 327 of the first surface 302 of the microstructured substrate 325 and at least one sidewall 340, defines a second open volume 380 located adjacent to the first open volume 370, and taking the sum of the combined first open volume and the second open volume as 100% open volume, the percentage of the first open volume in the 100% open volume is greater than the volume percentage of red blood cells present in the blood.

[0104] In selected embodiments, the micro-structure includes an array of peak structures and adjacent valleys, where the valleys have a maximum width ranging from 10 microns to 250 microns, and the peak structures have an apex angle greater than 5 degrees and at most 90 degrees.

[0105] 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; they can be oriented at an angle between 0 degrees and 90 degrees relative to the flow direction of the device. More specifically, the peak structures and adjacent valleys can be oriented 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 Figure 5B the device 300, the peak structures 320 and valleys 301 are oriented at an angle of 0 degrees (e.g., in the same direction as the flow) relative to the flow direction of the device 300. In Figure 5COn the microstructured substrate 325, the peak structures 322 and valleys 301 are oriented at an angle of 90 degrees (e.g., perpendicular) relative to the flow direction (“F”) of the device that will include the microstructured substrate 325. At Figure 5D On the microstructured substrate 325, the peak structures 320 and valleys 301 are oriented at an angle of 45 degrees relative to the flow direction (“F”) of the device that will include the microstructured substrate 325. By selecting an orientation greater than 0 degrees (e.g., up to 90 degrees), the particles have a greater chance of contacting more than one microstructure in the direction of fluid inflow into the device and are potentially more likely to be trapped within the microstructure.

[0106] Further details regarding microstructured substrates having such arrays of peak structures and how to form such microstructured substrates are described in US 2021 / 0187819 (Connell et al.), which is incorporated herein by reference. Small face structure

[0107] Similar to the prism structure, Figure 5E An alternative microstructured substrate 700 having a faceted structure is shown. More specifically, Figure 5E A microstructured substrate 700 is shown that defines a bottom surface 705, a top surface 720, a first sidewall 732, and facets 733. In other words, the microstructure includes facets 733 and sidewalls 732 that meet the facets 733 at the ridges 720 of the microstructure. The facets 733 and sidewalls 732 typically define an angle of inclination therebetween.

[0108] In selected embodiments, the microstructure includes facets and sidewalls that meet the facets at the ridges of the microstructure, and wherein the facets and sidewalls define an angle of inclination therebetween.

[0109] Further details regarding microstructured substrates having such faceted structures and how to form such microstructured substrates are described in WO 2020 / 250180 (Kenney et al.), which is incorporated herein by reference. Protrusion array structure

[0110] Figure 6A Another alternative microstructured substrate 400 having an array structure of protrusions is shown. More specifically, Figure 6A Is a schematic side view of a microstructured substrate 400 having a two-dimensional (x-axis and y-axis) array of protrusions 410 disposed across a first surface 420. Each protrusion 410 includes a base 412, a top 414, and one or more sides 416, 418 that connect the top to the base. Optionally, each protrusion 410 is a spaced-apart post. For example, Figure 6Bis a top plan view of four representative engineered micropatterned regions of a raised two-dimensional array, including spaced-apart posts 410 present in all images except the bottom right image. Some microstructured surfaces may include protrusions having a range of aspect ratios, such as an array of protrusions having a constant height and variable width. In such cases, the surface is typically characterized by the maximum aspect ratio.

[0111] In selected embodiments, the microstructure includes a two-dimensional (x-axis and y-axis) array of protrusions disposed across a first surface of a microstructured substrate, wherein each protrusion includes a base, a top, and one or more sides connecting the top to the base.

[0112] Further details regarding microstructured substrates having such an array of protrusions and how to form such microstructured substrates are described in WO 2020 / 097319 (Wolk et al.), which is incorporated herein by reference. Cavity array structure

[0113] Figure 7A Shows another alternative microstructured substrate 500a having a cavity array structure. A "cavity array" is a cavity array having a discrete cavity density of at least about 100 / cm 2 , preferably at least about 10 / mm 2 . The cavities have a three-dimensional structure, the dimensions of which such as the diameter of the opening range between about 5 microns and 250 microns and the depth ranges between about 2 microns and 250 microns. The array can be any regular array, such as a closely packed array or a rectangular array, or the cavities can be randomly distributed. More specifically, Figure 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 includes a microstructured layer 510 having a first major surface 514 and a second major surface 516, wherein the microstructure includes a plurality of cavities 522 extending between the first major surface 514 and the second major surface 516. Each cavity includes 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 sidewalls 526 in the sidewall forms a sidewall angle θ with a line 515 perpendicular to the first major surface 514 of the microstructured layer 510. Each cavity 522 further includes a depth "D", which is the vertical distance between the first orifice 524 and the second orifice 528. Optionally, the microstructured substrate 500a further includes any one of an adhesive layer 540, a first substrate 530, or a second base layer 550.

[0114] Figure 7BIt 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 cavity 522 includes a cross-section parallel to the first major surface 514, and the cross-section of the cavity can be circular, elliptical, or polygonal. Optionally, the cross-section is dimensionally reduced in the direction from the first major surface 514 to the second major surface 516. This embodiment of the microstructured substrate 500b also includes a (e.g., flexible) substrate 530 coupled to the second major surface 516 of the microstructured layer 510.

[0115] In a selected embodiment, the microstructured substrate includes a microstructured layer having a first major surface and a second major surface, wherein the microstructure includes a plurality of cavities extending between the first major surface and the second major surface, and wherein each cavity includes a first opening, a second opening, and at least one sidewall extending between the first opening and the second opening.

[0116] Further details regarding microstructured substrates having such an array of cavities and how to form such microstructured substrates are described in U.S. Patent No. 9,329,311 (Halverson et al.), which is incorporated herein by reference. Connected well hole structure

[0117] Figures 10A to 10B Another alternative microstructured substrate 1025 is shown, wherein the microstructure includes an array of fluid-connected wells, wherein at least some of the wells are fluidly connected to at least two adjacent wells, and each well is connected via an exhaust port. For example, Figure 10A is a schematic perspective view of a portion of the microstructured substrate 1025 having an array of fluid-connected wells 1077 connected to each other via exhaust ports 1087.

[0118] Figure 10B is Figure 10A a schematic top view of a portion of the microstructured substrate 1025 having an array of fluid-connected wells 1077 having a circular shape connected to adjacent wells 1077 via exhaust ports 1087. In Figures 10A to 10BIn the specific structure shown, the well holes 1077 in the center of the microstructured substrate 1025 (e.g., having at least one other well hole between the well hole and the perimeter of the microstructured substrate) are each connected to four other well holes 1077, where each connection is via an exhaust port 1087. There are also well holes 1077 located adjacent to the perimeter 1095 that are connected via an exhaust port 1087 to only one or two adjacent well holes 1077. For example, referring to Figure 10A , well hole 1077c is located at the corner of the array and is attached via exhaust port 1087a to only one other well hole 1077d. Similarly, a well hole can be attached via an exhaust port to three other adjacent well holes, four, five, six, seven, eight, nine, ten, eleven, or twelve other adjacent well holes.

[0119] The shape of the well hole is not particularly limited and can include a curved shape, a polygonal shape, an irregular shape, or a combination thereof. In some embodiments, the well hole includes a circle, a triangle, a quadrilateral, an ellipse, or a combination thereof. In the case where the well hole includes a shape having corners, the exhaust port is optionally located at the corner (e.g., to reduce the likelihood of trapping air bubbles in the corner).

[0120] Typically, each well hole has an open volume large enough to hold at least one sedimented particle (e.g., red blood cell), such as 100 femtoliters or greater, 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, 700 nanoliters, 800 nanoliters, or 900 nanoliters or greater; 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. In other words, in some cases, each well hole has an open volume ranging from 100 femtoliters to 1 microliter or 500 femtoliters to 0.1 microliter.

[0121] Some typical dimensions of each well hole include the depth (i.e., the distance between the top surface 1027 of the microstructured substrate 2025 and the bottom surface of the well hole 1029, as Figure 10AAs shown), the depth is 50 microliters or greater, 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 greater; 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 also applies to the diameter of the well. The diameter is the longest line passing through the center point of the shape.

[0122] Preferably, at least some of the vent holes between the wells are located at the same depth as the bottom surface of the adjacent wells. This helps to encourage the bubbles to leave the wells rather than being trapped near the bottom of the wells. In some embodiments, the vent holes have a total depth equal to the total depth of the adjacent wells, but this is not required. When using a tool to form the microstructured substrate, having the vent holes at the same depth as the adjacent wells tends to be more practical than having the vent holes only connect the lower portions of two wells.

[0123] Referring again to Figure 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. One or more of the sidewalls 1087 have a height ("H") that extends 50 microns to 250 microns above the top surface of the plurality of microstructures.

[0124] A multi - photon exposure system as described in U.S. Patent No. 8,605,256 (DeVoe et al.) can be used to fabricate the connected well structure to produce a patterning tool. This structured polymer tool is then typically nickel - plated to produce a metallized tool. Next, using the nickel - plated tool, a press such as a Carver press (Carver, Wabash, Indiana) and a resin (e.g., polypropylene resin) are used to fabricate a mold sample. The platen is heated (e.g., to 170 °C), and then the tool and the resin are pressed together with a high force (e.g., 1000 pounds force) for several minutes (e.g., 5 minutes - 10 minutes). After cooling (e.g., until the platen temperature reaches 80 °C), the pressure is released and the molded sample can be removed. Using this method, a microstructured substrate in the form of a microstructure film can be provided.

[0125] Further details regarding the microstructured substrate having such connected wells and how to form such a microstructured substrate are described in co - owned application serial number 63 / 425,473, which is incorporated herein by reference. Rod net structure

[0126] Figure 13 Another alternative microstructured substrate 1320 having a rod network structure is shown. In such embodiments, the microstructures 1326 include an array of upright rods 1326 extending across the first surface 1324 of the microstructured substrate 1320. The microstructures 1326 are generally upright rods of various shapes. "Generally upright" means that the rods project away from the first surface 1324 (e.g., in a planar direction). The rods 1326 may project upwardly from the surface 1324 at a generally normal angle, or the rods 1326 may project at an angle away from the surface 1324. The rods may also be of irregular shape such that they may not project at any one uniform angle.

[0127] The microstructured substrate 1320 includes a backing layer 1321 having a first surface 1324 with an array of generally upright rods 1326. The rods 1326 may be arranged in a regular or irregular array. A variety of patterns of rods may be used, such as hexagonal, diagonal, sinusoidal, etc. The rods 1326 may be at least partially constructed of an elastomeric material. In some cases, the entire outer surface of the rods 1326 is an elastomeric material. In Figure 13 embodiments, the backing layer 1321 is formed integrally with the rods 1326. The combination of the backing layer 1321 and the rods 1326 is sometimes referred to as a rod network. Although the illustrated embodiments show the rods 1326 as generally cylindrical, the sides of the rods 1326 typically have a slight taper 1335 to facilitate removal from the mold. As shown, the taper 1335 is inward from the base 1302 to the tip 1304 of the rod 1326. It will be appreciated that the rods may be configured to have a taper outward from the base to the tip of the rod. A variety of non-cylindrical shapes may also be used, such as frustoconical or pyramidal, rectangular, hemispherical, square, hexagonal, octagonal, gumdrop-shaped, etc.

[0128] The backing layer 1321 from which the rods 1326 extend directly is typically about 0.05 millimeters to about 0.5 millimeters (0.002 inches to 0.02 inches) thick. Accordingly, an additional backing layer 1322 is optionally applied to the second surface 1325 to reinforce the backing layer 1321 and form a multi-layer base or backing structure. As used herein, the "backing" or "base" layer will be used to refer to the common backing or base structure. Such a structure may be single-layer or multi-layer (such as Figure 13 shown), having one or more layers that support the generally upright rods 1326, although typically at most one of these layers 1321 will be formed integrally with the rods 1326.

[0129] The rods typically have a height 1328 in the range of about 0.2 mm to about 3 mm, preferably about 0.2 mm to about 1.5 mm. The spacing or gap 1330 between adjacent rods 1326 is generally in the range of about 0.25 mm to about 2.5 mm, and more typically in the range of about 0.4 mm to about 1.0 mm. This spacing gap creates a certain percentage of free volume, which is the volume within the rod network that is not occupied by the rods. The percentage of free volume is typically 60% to 98% of the rod network, and more typically 85% to 95%. The rods 1326 have a maximum cross-sectional dimension 1329 of about 0.076 mm to about 0.76 mm. The rods 1326 are arranged on the backing at a density of at least 15.5 / square centimeter (100 / square inch), and more typically at least 50 / square centimeter. The rod density is generally at most about 1500 / square centimeter, and more typically at most about 500 / square centimeter. The rods have an aspect ratio of at least 1.25, preferably at least 1.5, and most preferably at least 2.0. The aspect ratio is the ratio of the rod height to the maximum cross-sectional dimension. For rods with a circular cross-section, the maximum cross-sectional dimension is the rod diameter.

[0130] Suitable elastomeric rod materials include elastomers such as anionic triblock copolymers, polyolefin-based thermoplastic elastomers, halogen-containing polyolefin-based thermoplastic elastomers, dynamic vulcanized elastomer-thermoplastic blend-based thermoplastic elastomers, thermoplastic polyether esters or polyester-based elastomers, polyamide or polyimide-based thermoplastic elastomers, ionomer thermoplastic elastomers, hydrogenated block copolymers in thermoplastic elastomer interpenetrating polymer networks, thermoplastic elastomers polymerized by carbocation, 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 rubbers such as polydimethylsiloxane, polyisoprene, polybutadiene, polyurethanes, 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 can be in linear, radial, or star configurations, and can 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 with elastomeric segments), which are available from KRATON Polymers Company of Houston, Texas under the name KRATON ™ and can be purchased.

[0131] Elastomeric resin materials, such as those described above, may also have any of a number of conventional additives added thereto, including, for example, plasticizers, tackifiers, fillers, antioxidants, UV absorbers, hindered amine light stabilizers (HALS), dyes or pigments, light blockers, and the like.

[0132] Suitable backing layer materials include thermoplastic polyurethanes, polyvinyl chlorides, polyamides, polyimides, polyolefins (such as polyethylene and polypropylene), polyesters (such as polyethylene terephthalate), polystyrenes, nylons, acetals, block polymers (such as polystyrene materials having elastomeric segments, available from KRATON Polymers Company of Houston, Texas under the name KRATON ™ and obtainable), polycarbonates, thermoplastic elastomers (such as polyolefin, polyester, or nylon types), and copolymers and blends thereof. In some cases, the entire web of rods is formed of 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, foaming agents, surfactants, pigments, dyes, coupling agents, plasticizers, suspending agents, hydrophilic / hydrophobic additives, adhesives, and the like.

[0133] Further details regarding microstructured substrates having such a web of rods and how to form such microstructured substrates are described in WO 2009 / 020811 (Tuman et al.), which is incorporated herein by reference. Collection article

[0134] As described above, the collection article is not particularly limited so long as the collection article is capable of capillary action. Thus, suitable collection articles include, for example, films, membranes, nonwoven fiber sheets, woven fiber sheets, foams, and the like. The collection article may include one or more cavities and / or microstructured surfaces for retaining a fluid (e.g., blood) in which at least some of the particles (e.g., red blood cells) from the fluid have been removed in the device. The materials described above in detail for the outer surface of the microstructure of the device that impart a configuration to allow capillary action are also applicable to the collection article. For example, the material forms at least a portion of a surface that exhibits an advancing contact angle with water of less than 90 degrees.

[0135] In some cases, the collection article includes: a) a first polymer layer having a first major surface that is substantially flat and an opposing second major surface; and b) A second polymer layer that is bonded to the first polymer layer, the second polymer layer having a first major surface and a second major surface, wherein the first major surface of the second polymer layer is conformal with the second major surface of the first polymer layer, wherein the second major surface of the second polymer layer defines a cavity including at least one wall, wherein the second polymer layer has a channel that connects the cavity to at least one edge of the second polymer layer or the first major surface of the first polymer layer, and wherein the surface of the cavity exhibits an advancing contact angle with water that is less than 90 degrees.

[0136] See Figures 11A to 11B , which provides a schematic overview of such a collection article 1100. A suitable collection article 1100 includes a first polymer layer 1110 having a substantially flat first major surface 1111 and an opposite second major surface 1113; and a second polymer layer 1120 that is bonded to the first polymer layer 1110, the second polymer layer 1120 having a first major surface 1121 and a second major surface 1123, wherein the first major surface 1121 of the second polymer layer 1120 is conformal with the second major surface 1113 of the first polymer layer 1110, wherein the second major surface 1123 of the second polymer layer 1120 defines a cavity 1150 including at least one wall 1154, wherein the second polymer layer 1120 has a channel 1160 (as Figure 11A shown) that connects the cavity 1150 to at least one edge 1125 of the second polymer layer 1120, or the second polymer layer 1120 has a channel 1160 (as Figure 11B shown) that connects the cavity 1150 to the first major surface 1111 of the first polymer layer 1110. The surface 1152 of the cavity 1150 exhibits an advancing contact angle with water that is less than 90 degrees.

[0137] In Figure 11A the embodiment shown, the channel 1160 also connects the cavity 1150 to a second edge of the second polymer layer 1120. In this embodiment, the channel 1160 is deep enough to extend through the second polymer layer and into the first polymer layer 1110, so the channel 1160 also connects the cavity 1150 to the second edge 1117 of the first polymer layer 1110. Also in Figure 1 the embodiment of A, optionally, the cavity 1150 has a first volume 1151, the channel 1160 of the second polymer layer 1120 has a second volume 1161, and the second volume 1161 is 0.1% to 10% of the first volume 1151. Figure 11A The first polymer layer of the embodiment of

[0138] In Figure 11B the illustrated embodiment, the channel 1160 has a generally cylindrical shape, which can be formed, for example, by drilling through the first polymer layer 1110 and the second polymer layer 1120 using a laser, respectively. Also in Figure 11B the embodiment, optionally, the cavity 1150 has a first volume 1151, the channel 1160 of the second polymer layer 1120 has a second volume 1161, and the second volume 1161 is 0.001% to 0.1% of the first volume 1151.

[0139] Further details regarding collection articles having such a structure and how to form collection articles having such a structure are described in PCT Publication No. WO 2020 / 261086 (Halverson et al.), which PCT publication is incorporated herein by reference.

[0140] Figure 12 is a schematic cross-sectional view of another exemplary collection article 1200. The collection article includes a fluid control membrane 1201. The fluid control membrane 1201 has a main channel 1230 and a secondary channel 1231 defined by a main ridge 1220 and a secondary ridge 1221, wherein the channels 1230, 1231 and the ridges 1220, 1221 extend along a channel axis that is angled with respect to the longitudinal axis (e.g., the x-axis) of the fluid control membrane 1201. Each main channel 1230 is defined by a set of main ridges 1220 (a first ridge and a second ridge) on either side of the main channel 1230. The main ridge 1220 has a height hp measured from the bottom surface 1230a of the channel 1230 to the top surface 1220a of the ridge 1220.

[0141] 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 be wicked into the collection article 1200 while still maintaining a robust structure. In some embodiments, the fluid control layer thickness tv is less than about 75 microns thick, or between about 20 microns and about 200 microns.

[0142] In some embodiments, microstructures are disposed within the main channel 1230. In some embodiments, the microstructures include secondary channels 1231 disposed between the first and second main ridges 1220 of the main channel 1230. Each secondary channel in the secondary channels 1231 is associated with at least one secondary ridge 1221. The secondary channels 1231 can be located between a set of secondary ridges 1221 or between the secondary ridges 1221 and the main ridges 1220. In other words, in some cases, the microstructures of the collection article include a plurality of ribs 1220 alternating with the channels 1230, and wherein each channel 1230 includes at least one secondary channel 1231.

[0143] Further details regarding collection articles having such fluid control structures and how to form collection articles having such structures are described in PCT Publication No. WO 2015 / 164632 (Halverson et al.), which is incorporated herein by reference.

[0144] In some embodiments, the collection article includes the same microstructures as the device. More specifically, the collection article can include Figures 4A to 7B or Figures 10A to 10B any of the microstructured substrates depicted in and described in detail above. In some cases, the microstructures of the device have a different size, a different shape, or both, from the microstructures of the collection article.

[0145] In a second aspect, the present disclosure provides a method of separating solid particles from a fluid. The method includes: a) obtaining a device that includes: 1) a microstructured substrate that includes a plurality of microstructures extending across a first surface of the microstructured substrate, wherein at least a portion of an outer surface of the plurality of microstructures is configured to permit capillary action; 2) a cover that is disposed a selected distance above a top of the first surface of the microstructured substrate; 3) at least one sidewall that attaches the cover to the first surface of the microstructured substrate along a perimeter of the first surface of the microstructured substrate; 4) a first orifice defined by at least one of the microstructured substrate or the cover; and 5) a second orifice defined by at least one of the microstructured substrate or the cover; wherein the first surface of the microstructured substrate and the at least one sidewall together define 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, wherein the cover and the top of the first surface of the microstructured substrate and the at least one sidewall together define a second open volume that is positioned adjacent to the first open volume, and taking the sum of the combined first open volume and second open volume as 100% open volume, the percentage of the first open volume in the 100% open volume being greater than the volume percentage of the particles present in the fluid. b) filling the device with a volume of fluid via capillary action through the first orifice; c) waiting for a time sufficient for at least a portion of the particles to settle within the first open volume of the plurality of microstructures; and d) Fluidly couple the device to a collection article at the first or second orifice such that at least 10% of an initial volume of fluid exits the device and flows by capillary action onto the collection article, wherein at least some of the particles from the initial volume of fluid have been retained within the first open volume of the plurality of microstructures.

[0146] Referring Figure 9 , the method includes obtaining device 910 (wherein the device is as directly described above); filling device 920 with a volume of fluid through a first orifice by capillary action; waiting for a time 930 sufficient for at least a portion of the particles to settle within the first open volume of the plurality of microstructures; and fluidly coupling the device to a collection article at the first orifice or at the second orifice such that at least 10% of an initial volume of fluid exits the device and flows by capillary action onto the collection article, wherein at least some of the particles from the initial volume of fluid have been retained within the first open volume of the plurality of microstructures 940. The characteristics, materials, structures, etc. of each of the device and the collection article for the method of the second aspect can be according to any of the embodiments of the device and the collection article as described in detail above with respect to the first aspect. Similarly, the method of the second aspect can be according to any of the embodiments of the method of the first aspect described in detail above.

[0147] In a third aspect, a kit is provided. The kit includes a device and a collection article, wherein the device includes: 1) A microstructured substrate including a plurality of microstructures extending across a first surface of the microstructured substrate, wherein the microstructures cover at least 90% of the first surface of the microstructured substrate, and wherein at least a portion of the outer surfaces of the plurality of microstructures are configured to permit capillary action; 2) A cover disposed at a selected distance above 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 perimeter of the first surface of the microstructured substrate; 4) A first orifice defined by at least one of the microstructured substrate or the cover; and 5) A second orifice defined by at least one of the microstructured substrate or the cover; Wherein the first surface of the microstructured substrate, together with the at least one sidewall, defines a first open volume, which is the sum of the open spaces from the bottom to the top of each micro-structure located between the plurality of micro-structures. 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. And Wherein the collection article has a surface that exhibits an advancing contact angle with water of less than 90 degrees.

[0148] The kit may include any number of each of the devices and collection articles. In some cases, the kit includes the same number of each of the devices and collection articles. The characteristics, materials, structures, etc. of each of the devices and collection articles for the kit may be according to any implementation of the devices and collection articles as described in detail above with respect to the first aspect. Exemplary embodiments

[0149] In a first embodiment, the present disclosure provides a method for separating red blood cells from blood. The method includes obtaining a device that includes a microstructured substrate including a plurality of microstructures extending across a first surface of the microstructured substrate, wherein at least a portion of an outer surface of the plurality of microstructures is configured to permit capillary action; a cover disposed to be spaced 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 perimeter of the first surface of the microstructured substrate; a first orifice defined by at least one of the microstructured substrate or the cover; and a second orifice 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 a sum of open spaces from a bottom to a top of each of the plurality of microstructures located between the plurality of microstructures, wherein 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, and a sum of the combined first open volume and the second open volume is taken as a 100% open volume, and a percentage of the first open volume in the 100% open volume is greater than a volume percentage of red blood cells present in the blood. The method further includes filling the device with a volume of blood via capillary action through the first orifice and waiting for 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. Additionally, the method includes fluidly coupling the device to a collection article at the first orifice or the second orifice such that at least 10% of an initial volume of the blood flows out of the device and via capillary action onto the collection article, wherein at least some of the red blood cells from the initial volume of the blood have remained within the first open volume of the plurality of microstructures.

[0150] In a second embodiment, the present disclosure provides the method according to the first embodiment, wherein the collection article has a surface that exhibits an advancing contact angle of less than 90 degrees with water.

[0151] 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 flat first major surface and an opposing second major surface; and 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, wherein the first major surface of the second polymer layer is conformal with the second major surface of the first polymer layer, wherein the second major surface of the second polymer layer defines a cavity including at least one wall, wherein the second polymer layer has 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 the surface of the cavity exhibits an advancing contact angle with water of less than 90 degrees.

[0152] 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 including a plurality of microstructures extending across a first surface of the microstructured substrate, wherein at least a portion of the outer surface of the plurality of microstructures has a surface that exhibits an advancing contact angle with water of less than 90 degrees, and wherein the collection article is oriented such that the plurality of microstructures are positioned closer to the plurality of microstructures of the device than to the second opposing surface of the microstructured substrate of the device.

[0153] In a fifth embodiment, the present disclosure provides a method according to any one of the first to fourth embodiments, wherein the at least one sidewall is part of the microstructured substrate of the device.

[0154] In a sixth embodiment, the present disclosure provides a method according to any one of the first to fifth embodiments, wherein upon contact with the collection article, at least 15%, at least 20% or at least 30% of the blood exits the device.

[0155] In a seventh embodiment, the present disclosure provides a method according to any one 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 sediment within the first open volume of the plurality of microstructures of the device.

[0156] In an eighth embodiment, the present disclosure provides a method according to any one of the first to seventh embodiments, wherein the microstructured substrate of at least one of the device or the collection article is a microstructured film.

[0157] In a ninth embodiment, the present disclosure provides a method according to any one of the first to eighth embodiments, wherein at least a portion of the outer surface of the cavity of the collection device or the plurality of microstructures of at least one of the device or the collection article comprises a surfactant, a surface treatment agent, a hydrophilic polymer, a flocculant, or any combination thereof.

[0158] In a tenth embodiment, the present disclosure provides a method according to any one of the first to ninth embodiments, wherein the flocculant is hydrophilic and non-hemolytic.

[0159] In an eleventh embodiment, the present disclosure provides a method according to the ninth or tenth embodiment, wherein the flocculant comprises a modified or unmodified amino polymer selected from the group consisting of polyethyleneimine, polylysine, polyaminoamide, polyallylamine, polyvinylamine, poly(dimethylamine-epichlorohydrin-ethylenediamine), poly(diallyldimethylammonium chloride), cationic polyacrylamide (CPAM), polyaminosiloxane, and dendrimers formed from polyamidoamine (PAMAM) and polypropyleneimine.

[0160] In a twelfth embodiment, the present disclosure provides a method according to any one of the ninth to eleventh embodiments, wherein the flocculant comprises a modified or unmodified polyethyleneimine polymer.

[0161] In a thirteenth embodiment, the present disclosure provides a method according to any one of the ninth to twelfth embodiments, wherein after filling the device with a certain volume of blood, at least a portion of the flocculant dissolves, disperses, or dissolves and disperses into the blood.

[0162] In a fourteenth embodiment, the present disclosure provides a method according to the thirteenth embodiment, wherein the flocculant is present in the volume of blood in an amount from 0.01 micrograms per milliliter of blood to 5000 micrograms per milliliter of blood.

[0163] In a fifteenth embodiment, the present disclosure provides a method according to any one of the first to fourteenth embodiments, the method further comprising removing the blood from the collection article via wicking, wherein at least some of the red blood cells from the blood have been retained.

[0164] In a sixteenth embodiment, the present invention provides a method according to any one of the first to fifteenth embodiments, wherein the microstructures of the device have a different size, a different shape, or both, from the microstructures of the collection article.

[0165] In a seventeenth embodiment, the present disclosure provides a method according to any one of the first to sixteenth embodiments, wherein the first orifice is defined by a covering.

[0166] In an eighteenth embodiment, the present disclosure provides a method according to any one of the first to seventeenth embodiments, wherein the second orifice is defined by the microstructured substrate or the covering of the device.

[0167] In a nineteenth embodiment, the present disclosure provides a method according to any one of the first to eighteenth embodiments, wherein the blood is undiluted.

[0168] In a twentieth embodiment, the present disclosure provides a method according to any one of the first to nineteenth embodiments, wherein red blood cells sediment solely due to gravity.

[0169] In a twenty-first embodiment, the present disclosure provides a method according to any one of the first to twentieth embodiments, wherein the device further includes an adhesive layer disposed between the covering and the microstructured substrate.

[0170] In a twenty-second embodiment, the present disclosure provides a method according to any one of the first to twenty-first embodiments, wherein the microstructure of at least one of the device or the collection article includes a plurality of ribs that alternate with channels extending across a first surface of the microstructured substrate, and wherein each rib of the ribs includes a sidewall and a top surface, and each channel of the channels includes a bottom surface.

[0171] In a twenty-third embodiment, the present disclosure provides a method according to any one of the first to twenty-second embodiments, wherein the top surface of each rib is the top of a cover disposed on the sidewall, and the width of the cover is greater than the width between the opposing sidewalls.

[0172] In a twenty-fourth embodiment, the present disclosure provides a method according to the twenty-second embodiment, wherein the microstructure of the collection article includes a plurality of ribs that alternate with channels, and wherein each channel includes at least one secondary channel.

[0173] In a twenty-fifth embodiment, the present disclosure provides a method according to any one of the first to twenty-first embodiments, wherein the microstructure of at least one of the device or the collection article includes an array of peak structures and adjacent valleys, wherein the valleys have a maximum width ranging from 10 micrometers to 250 micrometers, and the peak structures have an apex angle greater than 5 degrees and at most 90 degrees.

[0174] 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 oriented at an angle between 0 degrees and 90 degrees relative to the flow direction of the device.

[0175] In a twenty-seventh embodiment, the present disclosure provides a method according to the twenty-fifth or twenty-sixth embodiment, wherein the array of peak structures and adjacent valleys in at least one of the device or the collection article further includes a gap between adjacent peak structures.

[0176] In a twenty-eighth embodiment, the present disclosure provides a method according to any one of the first to twenty-first embodiments, wherein the microstructure in at least one of the device or the collection article includes a two-dimensional (x-axis and y-axis) array of protrusions disposed across the first surface of the microstructured substrate; wherein each protrusion includes a base, a top, and one or more sides connecting the top to the base.

[0177] In a twenty-ninth embodiment, the present disclosure provides a method according to any one of the first to twenty-first embodiments, wherein the microstructured substrate in at least one of the device or the collection article includes a microstructured layer having a first major surface and a second major surface, wherein the microstructure includes a plurality of cavities extending between the first major surface and the second major surface; wherein each cavity includes a first opening, a second opening, and at least one sidewall extending between the first opening and the second opening.

[0178] In a thirtieth embodiment, the present disclosure provides a method according to any one of the first to twenty-first embodiments, wherein the microstructure in at least one of the device or the collection article includes facets and sidewalls that meet at the ridges of the microstructure, and wherein the facets and the sidewalls define an inclination angle therebetween.

[0179] In a thirty-first embodiment, the present disclosure provides a method according to any one of the first to twenty-first embodiments, wherein the microstructure in at least one of the device or the collection article includes an array of fluidly connected wells, wherein at least some of the wells are fluidly connected to at least two adjacent wells, and each well is connected via an exhaust port.

[0180] In a thirty-second embodiment, the present disclosure provides a method according to any one of the first to twenty-first embodiments, wherein the microstructure includes an array of vertical rods extending across the first surface of the microstructured substrate.

[0181] In a thirty-third embodiment, the present disclosure provides a method according to any one of the first to thirty-first embodiments, wherein the volume of the blood filled through the first orifice is at most 100 microliters of blood.

[0182] In a thirty-fourth embodiment, the present disclosure provides a method according to any one 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.

[0183] In a thirty-fifth embodiment, the present disclosure provides a method according to any one of the first to thirty-fourth embodiments, the method further comprising: passing the blood through a filter before entering the device; passing the blood through a filter after leaving the device, wherein at least some of the red blood cells from the blood are retained within the first open volume of the plurality of microstructures; or both.

[0184] In a thirty-sixth embodiment, the present disclosure provides a method according to any one of the first to thirty-fifth embodiments, the method further comprising: adding a flocculant to the volume of blood before filling the device with the volume of blood.

[0185] In a thirty-seventh embodiment, the present disclosure provides a method according to the thirty-sixth embodiment, wherein the flocculant is added and present in an amount of 0.01 micrograms / mL of blood to 5000 micrograms / mL of blood.

[0186] In a thirty-eighth embodiment, the present disclosure provides a method for separating solid particles from a fluid. The method includes obtaining a device that includes a microstructured substrate that includes 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 permit capillary action. The device further includes: a cover spaced apart from a top of the first surface of the microstructured substrate by a selected distance; and at least one sidewall that attaches the cover to the first surface of the microstructured substrate along a perimeter of the first surface of the microstructured substrate. Further, the device includes a first orifice defined by at least one of the microstructured substrate or the cover and a second orifice 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 a sum of open spaces from a bottom to a top of each of the plurality of microstructures that are between the plurality of microstructures, wherein 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 that is positioned adjacent to the first open volume. A sum of the combined first open volume and the second open volume is taken as a 100% open volume, and a percentage of the first open volume in the 100% open volume is greater than a volume percentage of particles present in the fluid. The method further includes filling the device with a volume of the fluid through the first orifice via capillary action and waiting for a time sufficient for at least a portion of the particles to settle within the first open volume of the plurality of microstructures. Additionally, the method includes fluidly coupling the device to a collection article at the first orifice or the second orifice such that at least 10% of an initial volume of blood flows out of the device and flows via capillary action onto the collection article, wherein at least some of the red blood cells from the initial volume of blood have remained within the first open volume of the plurality of microstructures.

[0187] In a thirty-fourth embodiment, the present disclosure provides a kit. The kit includes a device and a collection article. The device includes a microstructured substrate that includes 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 an outer surface of the plurality of microstructures is configured to permit capillary action. The device further includes: a cover spaced apart from a top of the first surface of the microstructured substrate by a selected distance; and at least one sidewall attaching the cover to the first surface of the microstructured substrate along a perimeter of the first surface of the microstructured substrate. Further, the device includes a first orifice defined by at least one of the microstructured substrate or the cover and a second orifice defined by at least one of the microstructured substrate or the cover. The first surface of the microstructured substrate and the at least one sidewall together define a first open volume that is a sum of open spaces from a bottom to a top of each of the plurality of microstructures located between the plurality of microstructures, wherein the cover and the first surface of the microstructured substrate and the at least one sidewall together define 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.

[0188] In a fortieth embodiment, the present disclosure provides the kit according to the thirty-ninth embodiment, wherein the collection article includes a) a first polymer layer having a substantially flat first major surface and an opposite second major surface; and 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, wherein the first major surface of the second polymer layer is conformal with the second major surface of the first polymer layer, wherein the second major surface of the second polymer layer defines a cavity including at least one wall, wherein the second polymer layer has 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.

[0189] In a forty-first embodiment, the present disclosure provides the kit according to the thirty-ninth or fortieth embodiment, wherein the collection article includes a microstructured substrate that includes a plurality of microstructures extending across a first surface of the microstructured substrate, wherein 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.

[0190] In a forty-second embodiment, the present disclosure provides the kit according to the forty-first embodiment, wherein the microstructured substrate of at least one of the device or the collection article is a microstructured membrane.

[0191] In a forty-third embodiment, the present disclosure provides a kit according to any one of the thirty-ninth to forty-second embodiments, wherein at least a portion of the outer surface of the cavity of the collection device or the plurality of microstructures of at least one of the device or the collection article comprises a surfactant, a surface treatment agent, a hydrophilic polymer, a flocculant, or any combination thereof.

[0192] In a forty-fourth embodiment, the present disclosure provides a kit according to the forty-third embodiment, wherein the flocculant on at least a portion of the plurality of microstructures of the device is hydrophilic and non-hemolytic.

[0193] In a forty-fifth embodiment, the present disclosure provides a method according to the forty-third or forty-fourth embodiment, wherein the flocculant 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, poly(dimethylamine-epichlorohydrin-ethylenediamine), poly(diallyldimethylammonium chloride), cationic polyacrylamide (CPAM), polyaminosiloxane, and dendrimers formed from poly(amidoamine) (PAMAM) and polypropyleneimine.

[0194] In a forty-sixth embodiment, the present disclosure provides a method according to any one of the forty-third to forty-fifth embodiments, wherein the flocculant on at least a portion of the plurality of microstructures of the device comprises a modified or unmodified polyethyleneimine polymer.

[0195] In a forty-seventh embodiment, the present invention provides a kit according to any one of the forty-first to forty-sixth embodiments, wherein the microstructures of the device have a different size, a different shape, or both, from the microstructures of the collection article.

[0196] In a forty-eighth embodiment, the present disclosure provides a kit according to any one of the thirty-ninth to forty-seventh embodiments, wherein the at least one sidewall is part of the microstructured substrate of the device.

[0197] In a forty-ninth embodiment, the present disclosure provides a kit according to any one of the thirty-ninth to forty-eighth embodiments, wherein the first orifice is defined by a cover.

[0198] In a fiftieth embodiment, the present disclosure provides a kit according to any one of the thirty-ninth to forty-ninth embodiments, wherein the second orifice is defined by the microstructured substrate or the cover of the device.

[0199] In a fifty-first embodiment, the present disclosure provides a kit according to any one of the thirty-ninth to fiftieth embodiments, wherein the device further includes an adhesive layer disposed between the cover member and the microstructured substrate.

[0200] In a fifty-second embodiment, the present disclosure provides a kit according to any one of the thirty-ninth to fifty-first embodiments, wherein the microstructure of at least one of the device or the collection article includes a plurality of ribs that alternate with channels extending across a first surface of the microstructured substrate, and wherein each rib of the ribs includes a sidewall and a top surface, and each channel of the channels includes a bottom surface.

[0201] In a fifty-third embodiment, the present disclosure provides a kit according to the fifty-second embodiment, wherein the top surface of each rib is the top of a top cover disposed on the sidewall, and the width of the top cover is greater than the width between the opposing sidewalls.

[0202] In a fifty-fourth embodiment, the present disclosure provides a kit according to the fifty-second embodiment, wherein the microstructure of the collection article includes a plurality of ribs that alternate with channels, and each channel includes at least one secondary channel.

[0203] In a fifty-fifth embodiment, the present disclosure provides a kit according to any one of the thirty-ninth to fifty-first embodiments, wherein the microstructure of at least one of the device or the collection article includes an array of peak structures and adjacent valleys, wherein the valleys have a maximum width ranging from 10 micrometers to 250 micrometers, and the peak structures have an apex angle greater than 5 degrees and at most 90 degrees.

[0204] In a fifty-sixth embodiment, the present disclosure provides a kit according to the fifty-fifth embodiment, wherein the array of peak structures and adjacent valleys extending across the first surface of the microstructured substrate is oriented at an angle between 0 degrees and 90 degrees relative to the flow direction of the device.

[0205] In a fifty-seventh embodiment, the present disclosure provides a kit according to the fifty-fifth or fifty-sixth embodiment, wherein the array of peak structures and adjacent valleys of at least one of the device or the collection article further includes a gap between adjacent peak structures.

[0206] In a fifty-eighth embodiment, the present disclosure provides a kit according to any one of the thirty-ninth to fifty-first embodiments, wherein the microstructure of at least one of the device or the collection article includes a two-dimensional (x-axis and y-axis) array of protrusions disposed across a first surface of the microstructured substrate; wherein each protrusion includes a base, a top, and one or more sides connecting the top to the base.

[0207] In a fifty-ninth embodiment, the present disclosure provides a kit according to any one of the thirty-ninth to fifty-first embodiments, wherein the microstructured substrate of at least one of the device or the collection article includes a microstructured layer having a first major surface and a second major surface, wherein the microstructures include a plurality of cavities extending between the first major surface and the second major surface; wherein each cavity includes a first opening, a second opening, and at least one sidewall extending between the first opening and the second opening.

[0208] In a sixtieth embodiment, the present disclosure provides a kit according to any one of the thirty-ninth to fifty-first embodiments, wherein the microstructures of at least one of the device or the collection article include facets and sidewalls meeting the facets at the ridges of the microstructures, and wherein the facets and the sidewalls define an inclination angle therebetween.

[0209] In a sixty-first embodiment, the present disclosure provides a kit according to any one of the thirty-ninth to fifty-first embodiments, wherein the microstructures of at least one of the device or the collection article include an array of interconnected wells, wherein at least 80% of the wells are fluidly connected to at least two adjacent wells, and each well is connected via an exhaust port.

[0210] In a sixty-second embodiment, the present disclosure provides a method according to any one of the thirty-ninth to fifty-first embodiments, wherein the microstructures include an array of vertical rods extending across a first surface of the microstructured substrate.

[0211] In a sixty-third embodiment, the present disclosure provides a method according to any one of the thirty-ninth to sixty-second embodiments, wherein the ratio of the first open volume to the second open volume is greater than 1:1.

[0212] In a sixty-fourth embodiment, the present disclosure provides a kit according to any one of the thirty-ninth to sixty-third embodiments, the kit further including at least one filter.

[0213] In a sixty-fifth embodiment, the present disclosure provides a method according to any one of the forty-third to forty-sixth embodiments, wherein the flocculant on at least a portion of the plurality of microstructures of the device includes 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).

[0214] In a sixty-sixth embodiment, the present disclosure provides a method according to any one of the forty-third to forty-seventh embodiments or the sixty-fifth embodiment, wherein the flocculant on at least a portion of the plurality of microstructures of the device comprises modified or unmodified gelatin. Examples

[0215] The following examples further illustrate the objects and advantages of the present disclosure, but the specific materials and their amounts, as well as other conditions and details recited in these examples should not be construed as unduly limiting the present disclosure. Unless otherwise specified or apparent from the context, all parts, percentages, ratios, etc. in the examples and the remainder of the specification are by weight. The material table (below) lists the materials used in the examples and the sources of the materials. Preparation Examples 1 - Preparation Examples 4 . Procedure for preparing a prismatic-structured microstructured film

[0216] 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 mixed in a high-speed mixer, heated in an oven at about 70 °C for 24 hours, and then cooled to room temperature. A copper button was used as a template for preparing a linear prismatic film. Both the button and the compounded resin were heated in an oven at about 70 °C for 15 minutes. The warm resin was applied to the center of the warm button using a pipette. A MELINEX 618 PET support film (DuPont Teijin Films, Chester, VA) was placed on the applied resin, followed by a glass plate. The primed surface of the PET film was oriented to contact the resin. The glass plate was held in place by hand pressure until the resin completely covered the surface of the button. The glass plate was carefully removed. If any air bubbles were introduced, a rubber manual roller was used to remove the bubbles.

[0217] The sample was cured with UV light by passing it through a UV processor (model QC120233AN, having two Hg vapor lamps, from RPC Industries, Plainfield, IL) twice at a rate of 15.2 meters per minute in a nitrogen atmosphere. By gently pulling at a 90° angle, the sample with Figure 5AThe cured microstructured film of the array pattern shown is removed from the copper template. Optionally, an adhesive layer (8 mil thick, obtained as 3M 8188 optically clear adhesive from 3M Company) backed by a release liner is applied to the back surface (i.e., the non-microstructured surface) of the microstructured film using a manual roller. The characteristics of the prepared linear prism microstructured film are recorded in Table 1 as Preparation Example 1 - Example 4.

[0218] The microstructured film is cut into segments of 2.54 cm × 7.62 cm, and a surfactant coating is applied by immersing each segment into an aqueous solution of 0.1 weight percent IPEGAL - CO630 anionic surfactant and then immediately removing the film from the solution. The resulting surfactant-coated microstructured film segments are air-dried at ambient temperature and humidity. Preparation Example 5 . Procedure for preparing a louvered structure microstructured film

[0219] A tool with multiple parallel linear grooves (220 microns deep) is cut using diamond. The grooves are spaced 60 microns apart. Resin A is prepared by mixing the materials in Table 2 below.

[0220] A casting and curing microreplication process is carried out using Resin A and the above tool. The production line conditions are resin temperature 150°F (65.6°C), mold 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), line speed 70 feet per minute (fpm) (0.36 meters per second (m / s)). A Fusion D lamp with a peak wavelength of 385 nm (obtained from Fusion UV Systems, Gaithersburg, MD) is used for curing and is operated at 100% power. As Figure 4AAs shown (described in detail above), the resulting "louvered" microstructured film includes a plurality of protrusions (ribs) separated by channels. The microstructured film is a topographical inversion of the tool such that the protrusions of the microstructured film are negative replicas of the grooves of the tool and the channels of the microstructured film are negative replicas of the uncut portions between the grooves of the tool. The protrusions (ribs) of the microstructured film are uniformly spaced with a height ("H") of 220 microns, a width ("W") of 30 microns, a pitch ("P") of 60 microns, and a wall angle θ of 91.5 degrees, which results in the protrusions being slightly tapered (i.e., wider at the bottom surface and narrower at the top surface). The platform layer ("L") of the cured resin has a thickness of 8 microns. The base layer is a PET film (3M Company, St. Paul, MN) with a thickness of 74.4 microns. The side of the PET film that contacts the resin is primed with a thermosetting acrylic polymer (RHOPLEX 3208 polymer, obtained from Dow Chemical, Midland, MI).

[0221] The microstructured film is cut into segments of 2.54 cm × 7.62 cm, and a surfactant coating is applied by immersing each segment in an aqueous solution of 0.1 weight percent IPEGAL-CO630 anionic surfactant and then immediately removing the film from the solution. The resulting surfactant-coated microstructured film segments are air dried at ambient temperature and humidity. Preparation Example 6 . Device Preparation

[0222] Device 800 (such as Figure 8A and 8BAs shown), it is prepared by laminating three thin film sections. The cover sheet component of device 800 (i.e., cover 820) is prepared by laser cutting a 72 mm long by 20 mm wide section from a sheet of 3M microfluidic diagnostic membrane 9962 (a polyester membrane (3.9 mils) with hydrophilic coatings on both sides, including the main surface 822 of cover 820 facing the microstructured substrate 830, obtained from 3M Company). Circular holes (5 mm in diameter) are laser cut in the membrane such that the center of the hole is positioned 13.5 mm from the narrow edge of the membrane in the vertical direction and 10 mm from the long edge of the membrane in the vertical direction. The circular hole forms the first orifice 850 of device 800. The second membrane component of the device (i.e., adhesive layer 870) is prepared by laser cutting a 72 mm long × 20 mm wide section from a sheet of 3M 1522 double-sided medical tape (a transparent 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 is measured using a digital caliper to be 150 microns. A rectangular opening 880 (60 mm long × 2.5 mm wide) is laser cut into the second membrane component and oriented such that the narrow edge of the opening is located 11 mm from the narrow edge of the membrane and the long edge of the opening is located 8.75 mm from the long edge of the membrane.

[0223] The third membrane component of the device (i.e., microstructured substrate 810) is a 72 mm long × 20 mm wide laser cut section of the surfactant-coated microstructured membrane of Preparation Example 1. All laser cutting in the membrane is performed using a Muse Core CO2 laser cutter (Full Spectrum Laser, Las Vegas, Nevada).

[0224] Device 800 is constructed by removing any release liners from the membranes and then aligning the membrane edges into a stack, with the second membrane sandwiched between the cover sheet membrane and the microstructured membrane. The membranes are oriented such that the microstructured surface 830 of the third membrane faces the main surfaces 822 of the second membrane and the cover. The adhesive lamination of the stack is completed by applying a 4-pound (1.8 kg) roller to the stack and having the roller make one reciprocating motion. The second adhesive membrane forms a fluid seal around the edges of the rectangular opening and the first orifice hole (e.g., forming sidewalls) between the cover sheet and the top surface of the microstructured membrane. In the final step, the stack is trimmed ( Figure 8B along the dashed line 862) at an edge located away from the first orifice such that the resulting device has an overall size of 60 mm long by 20 mm wide. This cut through the opening in the second membrane exposes the second orifice of the device, a 2.5 mm rectangular opening in the newly created edge of device 800. Preparation Example 7 .

[0225] The device was prepared according to the same procedure reported in Preparation Example 6, except that the second membrane component of the device was prepared using 3M 1513 double-sided medical tape (a transparent double-sided acrylic adhesive with a polyester backing, obtained from 3M). The total thickness of the double-sided tape with the release liner removed was measured using a digital caliper to be 75 microns. Preparation Example 8 .

[0226] The device was prepared according to the same procedure reported in Preparation Example 6, except that the third membrane component of the device was prepared using the surfactant-coated microstructured membrane of Preparation Example 2. Preparation Example 9 .

[0227] The device was prepared according to the same procedure reported in Preparation Example 6, except that the third membrane component of the device was prepared using the surfactant-coated microstructured membrane of Preparation Example 3. Comparative Example A.

[0228] The device was prepared according to the same procedure reported in Preparation Example 6, except that the third membrane component of the device was prepared using the surfactant-coated microstructured membrane of Preparation Example 4. Preparation Example 10 .

[0229] The device was prepared according to the same procedure reported in Preparation Example 6, except that the third membrane component of the device was prepared using the surfactant-coated microstructured membrane of Preparation Example 5. Preparation Example 11 .

[0230] The device was prepared according to the same procedure reported in Preparation Example 6, except that the third membrane component of the device was prepared using the surfactant-coated microstructured membrane of Preparation Example 5, and the second membrane component of the device was prepared using 3M 1513 double-sided medical tape. Comparative Example B.

[0231] The device was prepared according to the same procedure reported in Preparation Example 6, except that a non-microstructured polyethylene terephthalate (PET) membrane (MELINEX 454 membrane (3 mils), Dupont Teijin Films) was used as the third membrane component of the device to prepare the third membrane component of the device. Comparative Example C.

[0232] The device was prepared according to the same procedure reported in Preparation Example 6, except that the non-microstructured film of Comparative Example B (MELINEX 454 film) was used as the third film component of the device 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. Reference Example 1 . Method for separating red blood cells from blood

[0233] The defibrinated 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 sample, diluted blood samples with hematocrits of 12%, 8%, and 4% were prepared using 1X phosphate-buffered saline (PBS).

[0234] Prepare a module for delivering a blood sample to device 800 (shown in Figure 8C and Figure 8D ). The module components include a silicone gasket 844 (25 mm × 25 mm × 3.2 mm) prepared from SYLGARD 184 silicone elastomer (Dow Chemical, Midland, MI), a 5 mm hole was cut in the center of the gasket, and a sheet 842 (25 mm × 25 mm × 3.2 mm) of PLEXIGLAS polymethyl methacrylate (PMMA) film (Röhm GmbH, Darmstadt, Germany), a 0.06-inch (1.52 mm) diameter hole was drilled in the center of the sheet. A section of ethylene vinyl acetate (EVA) plastic tube 846 (0.02-inch (0.51 mm) inner diameter and 0.06-inch (1.52 mm) outer diameter, McMaster-Carr, Elmhurst, IL) was inserted into the hole in the PLEXIGLAS film sheet and fixed in place with HARDMAN DOUBLE / BUBBLE epoxy resin (Royal Adhesives, Wilmington, CA) 847. The other end of the tube was fitted to a 22.5-gauge needle, which was attached to a 1 mL Luer-Lok syringe.

[0235] Place the device 800 (selected from the devices of Preparation Examples 6 to 11 and Comparative Examples A to C) on a horizontal surface. Add a blood sample (20 μL to 50 μL) to the first orifice of the device and allow the blood to wick through the capillary flow to the end of the device. Next, place the silicone washer 844 on the cover sheet of the device and align it so that the hole in the washer is centered above the first orifice of the device. Then place the PLEXIGLAS sheet 842 (with the EVA plastic tube 846 adhered thereto as described above) on the exposed surface of the gasket and align it so that the hole in the sheet is centered above the hole in the gasket, thereby forming Figure 8D the assembly. Place a syringe (not shown) attached to the other end of the EVA plastic tube in a syringe pump (model NE-1600, New Era Pump Systems Inc, Farmingdale, NY) (not shown). Start the pump at a flow rate set to 100 μL / min. Collect the blood sample flowing out of the device from the second orifice 860. Dilute 10 μL of the collected blood sample with 90 μL of 1X PBS. Pipette the diluted sample into a C-CHIP disposable hematology counter according to the manufacturer's instructions (Incyto, Republic of Korea). For comparison, pipette the blood sample added to the device into a C-CHIP disposable hematology counter as well. Analyze the counting slide using a Zeiss LSM510 Meta Module Axioplan 2 upright confocal microscope (Zeiss, Jena, Germany). Manually or using Image J image processing software (National Institutes of Health, Bethesda, MD) count the red blood cells. Calculate the percentage reduction in red blood cells (RBC) of the blood sample using the described method by comparing the RBC count of the blood sample collected from the device with the RBC count of the blood sample added to the device according to Equation 1. Each device was tested in triplicate (n = 3 devices) with the specified blood sample and the results expressed as % RBC reduction were reported as the average in Table 4. Equation 1: Preparation Example 12 .

[0236] 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.), use the CAD design file to fabricate the microstructured substrate for the device (Figure 2B A profiling mold as described in, for example, U.S. Patent No. US 10,133,174 (Lee et al.) is used. A negative contrast photoresist is photopatterned on a silicon wafer substrate. When the scanning is complete, the substrate with the patterned structure is immersed in a developing solution of propylene glycol monomethyl ether acetate (obtained from Sigma-Aldrich) to remove the unpolymerized photoresist. The profiling mold is then electroformed with nickel or a nickel alloy to fabricate a metal tool for replication. The nickel-plated tool is used to fabricate a molded sample with polypropylene resin (C700-35 resin, Dow Chemical, Midland, MI). The platen of a Carver press (Carver, Wabash, IN) is heated to 170 °C, and the tool and resin are pressed together at 1000 pounds force (about 4450 Newtons) for 7 minutes, followed by cooling under pressure until the temperature of the platen reaches 80 °C. The pressure is released and the molded microstructured substrate is removed from the tool.

[0237] The molded microstructured substrate has an upper surface and a lower surface, with a total size of 25.4 mm (width), 76.2 mm (length), and 1 mm (depth). The microstructure of the substrate is an array of linear prism microstructures located along the bottom surface of a flow channel (3 mm wide and 60 mm long), which is recessed below the upper surface of the substrate and has a first open end and a second open end. The linear array of peak structures and adjacent valleys is oriented at an angle of 0 degrees relative to the liquid flow direction of the finished device. The characteristics of the linear prism microstructures are reported in Table 5. The prism features protrude above the bottom surface of the flow channel. The walls surrounding the perimeter of the flow channel extend 100 micrometers above the tips of the prism structures to the upper surface. The first open end of the flow channel is fluidly attached to a semi-circular first cavity with a depth of 100 micrometers from the upper surface and a volume of 2.88 microliters. The first cavity forms the first orifice of the device. The first orifice serves as the liquid sample intake reservoir of the final device. The opposite second open end of the flow channel is fluidly attached to a rectangular second cavity (5 mm wide, 6 mm long, 250 micrometers deep from the upper surface). The second cavity forms the second orifice of the device. The second orifice serves as the receiving reservoir for the liquid sample leaving the flow channel.

[0238] The cover member of the device is part of a 3M microfluidic diagnostic membrane 9975R (obtained from 3M). The cover is placed over the flow channel and the liquid sample intake reservoir section and adhesively attached to the surface of the microstructured substrate surrounding the sample intake reservoir and the flow channel section. Before application, a circular hole (5 mm diameter) is laser cut into the cover member and oriented such that when the cover is attached to the microstructured substrate, the center of the hole is positioned above the center of the sample intake reservoir. The receiving reservoir is not covered. Preparation Example 13 .

[0239] The device was prepared according to the same procedure as described in Preparation Example 12, except that: the linear array of peak structures and adjacent valleys was oriented at an angle of 45 degrees with respect to the liquid flow direction of the finished device (oriented as shown in Figure 5D ); the flow channel had dimensions of 3 mm wide and 40 mm long; the first orifice body had a depth of 300 microns and a volume of 8.7 microliters; and the second orifice body had dimensions of 5 mm (width), 6 mm (length), and 300 microns (depth). Preparation Example 14 .

[0240] The device was prepared according to the same procedure as described in Preparation Example 13, except that the linear array of peak structures and adjacent valleys was oriented at an angle of 90 degrees (i.e., perpendicular) with respect to the liquid flow direction of the finished device (oriented as shown in Figure 5C ). Preparation Example 15 .

[0241] The device was prepared according to the same procedure as described in Preparation Example 12, except that the array of linear prismatic microstructures was replaced by an array of fluid-connected wells having a circular shape, and these wells were connected to adjacent wells through vent ports (as shown in Figures 10A to 10B ). Each well had a diameter of 200 microns, a depth of 150 microns, and a draft angle of 5 degrees. The vent ports had a length of 29 microns, a width of 40 microns, a depth of 150 microns, and a draft angle of 5 degrees. The wells located at the center of the flow channel were each connected to four other wells, and each well was connected via a vent port and the spacing of the vent ports was as shown in Figure 10B . The wall surrounding the perimeter of the flow channel extended 100 microns above the top surface of the wells. The flow channel had dimensions of 3 mm wide and 40 mm long.

[0242] The first open end of the flow channel was fluidly attached to a semi-circular first cavity having a depth of 100 microns from the upper surface and a volume of 2.88 microliters. The first cavity formed the first orifice of the device. The opposite second open end of the flow channel was fluidly attached to a rectangular second cavity (5 mm wide, 6 mm long, 250 microns deep from the upper surface). The second cavity formed the second orifice of the device. Reference Example 2 . Method for separating red blood cells from blood

[0243] Human blood was collected in BD VACUTAINER citrate tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) and used as citrated whole blood or citrated whole blood diluted 1:1 with 1X phosphate buffered saline (PBS).

[0244] The microstructured substrates described in Preparation Examples 12 to 15 were prepared, and a cover member modified with a tube for blood infusion was attached to each substrate. The modified cover member was a section of 3M Microfluidic Diagnostic Membrane 9975R (obtained from 3M Company), which had a circular hole (5 mm diameter) laser cut into the cover member. A plastic tube (0.05 inch ID, 0.09 inch OD) was inserted into the hole and fixed with an epoxy adhesive (3M SCOTCH-WELD Epoxy Adhesive DP100 Plus transparent, obtained from 3M Company). Any tube extending beyond the adhesive surface of the cover member was removed with a razor blade. The cover was placed over the flow channel and the liquid sample intake reservoir section of the microstructured substrate and adhesively attached to the surface of the microstructured substrate surrounding these two sections. The cover member was oriented such that when the cover was attached, the center of the hole was positioned above the center of the sample intake reservoir. The receiving reservoir was not covered. The tube extended approximately 3 cm in length from the outer surface of the cover.

[0245] Each resulting device was placed on a horizontal surface (oriented such that the lower surface of the device faced the horizontal surface). A blood sample (20 μL - 50 μL) was added to the sample inlet reservoir through the tube using a micropipette. The blood sample was allowed to wick by capillary flow to the end of the device or advanced using positive pressure from the micropipette. In the case where there was no excess blood in the intake reservoir, a sufficient volume of blood was added to the device to fill the open volume of the flow channel. 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 application of the blood sample, each device was left undisturbed on the horizontal surface for 5 minutes.

[0246] 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 to leave a small air gap (approx. 10 cm tube length) at the open end of the tubing. The syringe was placed in a syringe pump (Model NE-1600, New Era Pump Systems Inc). The open end of the tubing of the syringe assembly was connected to the open end of the tubing extending from the device. The pump was started at a flow rate set to 50 microliters per minute to force the blood sample out of the flow channel with a trapped air volume. The blood sample flowing out of the flow channel was collected as several 2 - 4 microliter aliquots. Each sample was collected once the aliquot volume had accumulated in the receiving reservoir.

[0247] For each collected sample, diluted samples of 1 / 10, 1 / 20, 1 / 100, and 1 / 200 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 1 well as a 1X PBS blank, which was used for dilution according to the manufacturer's instructions. For each sample and the 1X PBS blank, absorbance measurements at 406 nm, 414 nm, and 576 nm were recorded using an Agilent Synergy Neo2 plate reader (Agilent Technologies). The procedure in the section "Method for Determining the Total Red Blood Cell Content of a Sample" (described below) was used to calculate the percentage reduction of red blood cells from the blood samples submitted to each device using the procedure described in this example.

[0248] The results using citrated human whole blood are provided in Table 6, and the results using citrated human whole blood diluted 1:1 in 1X PBS are presented in Table 7. For each type of device, the results reported in Tables 5 and 6 are from technical replicates of three devices (n = 3). The calculated value of the percentage reduction of red blood cells for each replicate device is the average of the total volume of the aliquot samples collected from that device. Method for determining the intact red blood cell content of a sample

[0249] To simultaneously measure the intact red blood cell content and the lysed red blood cell content of a sample, a dilution series calibration curve is prepared using lysed and intact human red blood cells of known input. Citrated human whole blood is used as a sample of 100% intact red blood cells. To generate a sample of 0% intact red blood cells, an aliquot of whole blood is lysed by vortexing for 1 minute using a ZR BashingBead lysis tube (product number S6012-50, Zymo Research, Irvine, CA). The tube is centrifuged at 10,000 x g for 1 minute and the supernatant containing only the lysed cell contents is transferred to a new 1.5 mL Eppendorf tube. Standard samples of intact to lysed red blood cells for the dilution series are prepared by mixing different proportions of known intact and lysed cells (ranging from 100% to 0% intact red blood cells). The intact red blood cell concentration of the standard samples is confirmed using a C-CHIP disposable hematology counter according to the manufacturer's instructions.

[0250] Each standard sample is diluted in 1X PBS (such that the absorbance will be within the dynamic range of the plate reader) and analyzed in triplicate using an Agilent Synergy Neo2 plate reader with a Take 3 multi-volume plate (sample volume and blank are selected according to the manufacturer's instructions). The absorbance of each standard sample is measured at 406 nm, 414 nm, and 576 nm.

[0251] Ratio / is plotted against % intact red blood cells relative to the known input. = absorbance of the sample at 414 nm and = absorbance of the sample at 576 nm. Subtraction of the blank (1X PBS) is used for background subtraction. The data set is fit with a logarithmic curve to generate Equation A. Equation A is used to calculate the percentage of intact red blood cells from a suspension containing blood. Equation A : In Equation A, “% intact” = percentage of intact red blood cells in the suspension, “A 414nm ” = absorbance of the blood sample at 414 nm wavelength, “A PBS，414nm ” = absorbance of 1X PBS at 414 nm wavelength, “A 576nm ” = absorbance of the blood sample at 576 nm wavelength, and “A PBS，576nm ” = absorbance of 1X PBS at 576 nm wavelength.

[0252] The absorbance signal of each sample at 406 nm is adjusted by the ratio of the percentage of cells to calculate the signal from intact red blood cells using Equation B. Equation B : In Equation B, "Ai 406nm " = the absorbance at 406 nm wavelength of the blood sample attributable to intact red blood cells, "% intact" = the percentage of intact red blood cells in the suspension calculated by Equation A, "A 406nm " = the absorbance of the sample at 406 nm wavelength, and "A PBS，406nm " = the absorbance of 1X PBS at 406 nm wavelength.

[0253] After adjusting the absorbance at 406 nm wavelength for intact and lysed cells in Equation B, the absorbance signal of the blood sample added to the device is compared with the absorbance signal of the corresponding blood aliquot collected from the device to calculate the percentage reduction of red blood cells (RBC) according to Equation C. Equation C : In Equation C, "Ai 406nm，输入 " = the absorbance at 406 nm wavelength of the blood sample added to the device attributable to intact red blood cells calculated by Equation B, and "Ai 406nm，输出 " = the absorbance at 406 nm wavelength of the blood sample collected from the device attributable to intact red blood cells calculated by Equation B. Example 1 . Use of the nitrocellulose membrane collection article

[0254] Place the device of Preparation Example 10 on a horizontal surface (oriented such that the lower surface of the device faces the horizontal surface). Add a citrated human whole blood sample (20 μL to 50 μL) to the first orifice of the device using a micropipette and allow the blood to wick through the capillary flow to the end of the device. Maintain the device undisturbed on the horizontal surface for 1 minute. Then, place the edge of a UNISTART CN nitrocellulose membrane strip (about 2.5 mm by 60 mm from Sartorius Stedium Biotech, Goettingen, Germany) in contact with the second orifice of the device. After the nitrocellulose membrane contacts the second orifice, visually observe that at least 10% of the blood in the device wicks from the device to the nitrocellulose membrane by capillary action. Example 2 . Use of the paper collection article

[0255] Follow the same procedure reported in Example 1, except that Whatman 40 filter paper strips (product number 1440-110, GE Healthcare, Buckinghamshire, UK) (about 2.5 mm by 60 mm) were used instead of the nitrocellulose membrane to collect the product. After bringing the paper strip into contact with the second orifice, it was visually observed that at least 10% of the blood in the device flowed by capillary action from the device to the paper strip. Example 3 . Use of the microstructured membrane to collect the product

[0256] Follow the same procedure reported in Example 1, except that the microstructured membrane strip of Preparation Example 5 (about 2.5 mm by 60 mm) was used instead of the nitrocellulose membrane to collect the product. The edge of the microstructured membrane strip (about 2.5 mm by 60 mm) was placed in contact with the second orifice of the device. The microstructured membrane strip was oriented as Figure 3B shown, where the position of the microstructure of the microstructured membrane strip was inverted relative to the position of the microstructure of the device. After the membrane strip was brought into contact with the second orifice, it was visually observed that at least 10% of the blood in the device flowed by capillary action from the device to the microstructured membrane strip. Preparation Example 16 . Microstructured membrane with vertical rods

[0257] Prepare a discrete rod structure with angled sidewalls by the molding process of Example 1 of U.S. Patent No. 9,358,714 (Chandrasekaran) Figure 13A polypropylene (PP) microstructured membrane, except that it does not include a β-nucleating masterbatch, and then the membrane is corona-treated using a BD-20AC laboratory corona treater (Electro-Technic Products, Chicago, IL). 3D micrographs of the microstructured membrane are taken using a Keyence VK-X3100 3D surface profiler (Keyence Corporation, Itasca, IL), and measurements are made using the accompanying VK-X 3000 MultiFile Analyzer software package. The microstructured membrane has a total thickness of approximately 345 microns and an alternating array of 2000 rod features per square inch. The rod features have a generally flat surface at the vertices (i.e., a frustoconical shape). The thickness of the backing layer is 83.5 microns. In Tables 8 and 9, the following dimensions are reported: rod height, rod diameter at the base in the machine direction of the web, rod diameter at the base in the cross-machine direction of the web, rod diameter at the vertex in the machine direction of the web, rod diameter at the top in the cross-machine direction of the web, center-to-center distance (pitch) between rods in the machine direction of the web, and center-to-center distance (pitch) between rods in the cross-machine direction of the web. Preparation Example 17 . Preparation of an apparatus comprising a microstructured membrane having vertical rods

[0258] Follow the method described in Preparation Example 6, but with the following improvements. First, replace the microstructured membrane of Preparation Example 1 with the microstructured membrane of Preparation Example 16 as the third membrane component of the apparatus. Second, seal the two long edges and the narrow edge near the first orifice of the apparatus using epoxy resin. The narrow edge forming the second orifice is not sealed. Preparation Example 18 . Preparation of an apparatus comprising a microstructured membrane having vertical rods and a flocculant coating of a microstructured array

[0259] A flocculant solution of polyethyleneimine (PEI) (branched, MW 70,000 Da, 30% weight / volume aqueous solution, catalog number 00618, obtained from Polysciences, Inc., Warrington, PA) is diluted further in two steps with 1:10 (weight:volume) deionized water, and then with 1:10 volume:volume deionized water. The diluted flocculant solution (30 μL to 100 μL) is added to the rectangular opening 880 of the apparatus of Preparation Example 17 by pipette. The applied PEI flocculant solution is air-dried overnight under ambient conditions to provide a flocculant-coated microstructured array. Preparation Example 19 Preparation of a device comprising a microstructured film having vertical poles and a flocculant coating of a microstructured array

[0260] Prepare an aqueous solution of a flocculant of guanylated polyethyleneimine (G-PEI) as described in Example 1 of U.S. Patent No. 10,087,405 (Swanson et al.) without adding butanediol diglycidyl ether. Add the flocculant solution (30 μL to 100 μL) to the rectangular opening 880 of the device of Preparation Example 17 by pipette. Allow the applied G-PEI flocculant solution to air dry overnight under ambient conditions to provide a flocculant-coated microstructured array. Preparation Example 20: Preparation of a device with a microchannel fluid control membrane

[0261] Follow the procedure described in Preparation Example 6, with the modification that the microstructured film of Preparation Example 1, which is the third membrane component of the device, is replaced with a microchannel fluid control membrane prepared as described in "Preparation of a Microchannel Fluid Control Membrane" in the Examples of U.S. Patent No. 11,392,899 (Halverson et al.). Reference Example 3 Method for separating red blood cells from blood

[0262] Whole human blood collected in an EDTA tube was obtained from the Oklahoma Blood Institute, Oklahoma City, OK. The initial hematocrit of the human blood sample was measured using a Zip-IQ PCV centrifuge (LW Scientific Incorporated, Lawrenceville, GA).

[0263] In this method, a module having a syringe pump for delivering a blood sample as described in Reference Example 1 was used. Place the device selected from Preparation Example 17 to Preparation Example 19 on a horizontal surface. Add a whole human blood sample (50 μL to 100 μL) to the first orifice 850 of the device. Allow the blood to wick by capillary flow to the end of the device, and keep the device undisturbed for 10 minutes to allow the red blood cells to settle in the microstructured array section. Then, start the pump at a flow rate set to 100 μL / min. Collect the blood sample flowing out of the device from the second orifice 860 as a series of 5 μL aliquots.

[0264] A standard calibration curve was prepared from a dilution series of whole blood in water to correlate RBC count with absorbance at 406 nm. Each tested blood sample was diluted in water (to lyse the cells and keep the absorbance within the dynamic range of the plate reader) and analyzed in duplicate using an Agilent Synergy Neo2 plate reader with a Take 3 multi-volume plate (selecting sample volume and blank according to the manufacturer's instructions). All dilutions were prepared using deionized water with a resistivity greater than 18 megaohm-centimeters prepared by a SYNERGY ultraviolet water purification system (MilliporeSigma, Burlington, MA). The absorbance of each sample at 406 nm was measured. The percentage reduction of red blood cells (RBC) in the blood sample was calculated according to Equation D by comparing the absorbance of the blood aliquot collected from the device with the absorbance of the whole blood sample added to the device. Equation D : In Equation D, "Absorbance 406nm，输出 " = absorbance at 406 nm wavelength of the blood sample collected from the device. "Absorbance 406nm，输入 " = absorbance at 406 nm wavelength of the blood sample added to the device. For each type of device, the results are reported in Table 10. A single device of each type was tested. The calculated value of the percentage reduction of red blood cells was the average of all aliquot samples collected from the device. Approximately 10 aliquot samples were collected from each device. Reference Example 4 . Method for separating red blood cells from blood

[0265] Using the single device of Preparation Example 17 (n = 1) and a blood sample containing a PEI flocculant, the method of Reference Example 3 was followed. For the blood sample, a flocculant solution of polyethyleneimine (PEI) (branched, MW 70,000 Da, 30% weight / volume 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 1X PBS). An aliquot of 90 μL of whole human blood was mixed with 10 μL of the flocculant solution in a tube. The resulting blood sample was mixed using a pipette (3 to 5 times) and then incubated at room temperature for 10 minutes. The incubated blood sample was mixed using a pipette (3 to 5 times), and then approximately 100 μL of the sample was added to the first orifice of the device. A total of seven aliquots were collected from the device. The calculated percentage reduction in RBC was 64.4%. The calculated value of the percentage reduction in red blood cells is the average of all aliquot samples collected from the device. Reference Example 5. Method for Separating Red Blood Cells from Blood Using a Gelatin Flocculant

[0266] The method of Reference Example 4 was followed using two devices of Preparation Example 20. One device (n = 1) was used with a whole blood sample having a hematocrit of 40% and 1X PBS, and a second device (n = 1) was used with a blood sample having a gelatin flocculant solution, all solutions being heated to 37 °C prior to the experiment. An aqueous solution of 10% bovine gelatin stock solution was prepared in 1X PBS and heated to 37 °C. For Device 1, an aliquot of 450 μL of whole human blood was mixed with 50 μL of 1X PBS, and approximately 100 μL of the blood was added to the first orifice of the device. For Device 2, an aliquot of 450 μL of whole human blood was mixed with 50 μL of 10% gelatin, and approximately 100 μL of the blood was added to the first orifice of the device. The devices were incubated at 37 °C for 15 minutes. A total of one aliquot was collected from each device. For the device with 1X PBS, the calculated percentage reduction in RBC was 2.0%. For the device with 1% gelatin flocculant, the calculated percentage reduction in RBC was 32.7%. The results are reported in Table 11. Reference Example 6. Preparation of a Device Containing a Microstructured Membrane, Flocculation with Gelatin, and Adjusted Hematocrit

[0267] The method of Reference Example 5 was followed using two devices of Preparation Example 20. One device (n = 1) was used for a whole blood sample having its hematocrit adjusted to 25% with donor plasma.

[0268] Collect a total of one aliquot from each device. For the device with 1X PBS, the calculated percentage reduction in RBC was 62.3%. For the device with 1% gelatin flocculant, the calculated percentage reduction in RBC was 79.8%. The results are reported in Table 12.

[0269] All patents and patent applications mentioned above are hereby expressly incorporated by reference. The above embodiments are illustrative of the present invention and may be of other configurations. Therefore, the present invention should not be considered limited to the embodiments described in detail above and shown in the drawings, but only limited by the reasonable scope of the appended claims and their equivalents.

Claims

1. A method for separating red blood cells from blood, the method comprising: a) obtaining a device, the device comprising: 1) a microstructured substrate, the microstructured substrate comprising a plurality of microstructures extending across a first surface of the microstructured substrate, wherein at least a portion of an outer surface of the plurality of microstructures is configured to permit capillary action; 2) a cover, the cover being disposed at a selected distance above a top of the first surface of the microstructured substrate; 3) at least one sidewall, the at least one sidewall attaching the cover to the first surface of the microstructured substrate along a perimeter of the first surface of the microstructured substrate; 4) a first orifice, the first orifice being defined by at least one of the microstructured substrate or the cover; and 5) a second orifice, the second orifice being defined by at least one of the microstructured substrate or the cover; wherein the first surface of the microstructured substrate and the at least one sidewall together define a first open volume, the first open volume being a sum of open spaces from a bottom to a top of each of the plurality of microstructures that are located between the plurality of microstructures, wherein the cover, the top of the first surface of the microstructured substrate, and the at least one sidewall together define a second open volume that is positioned adjacent to the first open volume, and a sum of the combined first open volume and the second open volume is taken as a 100% open volume, and a percentage of the first open volume in the 100% open volume is greater than a volume percentage of red blood cells present in the blood; b) filling the device with a volume of blood via capillary action through the first orifice; c) waiting for 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; and d) fluidly coupling the device to a collection article at the first orifice or the second orifice such that at least 10% of an initial volume of blood flows out of the device and via capillary action onto the collection article, wherein at least some of the red blood cells from the initial volume of blood have been retained within the first open volume of the plurality of microstructures of the device.

2. The method according to claim 1, wherein when in contact with the collection article, causing at least 15%, at least 20%, or at least 30% of the blood to flow out of the device.

3. The method according to claim 1 or claim 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 according to any one of claims 1 to 3, the method further comprising removing the blood from the collection article via wicking, wherein at least some of the red blood cells from the blood have been retained.

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

6. The method according to any one of claims 1 to 5, wherein the red blood cells sediment only due to gravity.

7. The method according to any one of claims 1 to 6, wherein the volume of the blood filled through the first orifice is at most 100 microliters of blood.

8. The method according to any one of claims 1 to 7, the method further comprising: Pass the blood through a filter before it enters the device; Pass the blood through a filter after it exits the device, wherein at least some of the red blood cells from the blood are retained within the first open volume of the plurality of microstructures; or both.

9. The method according to any one of claims 1 to 8, the method further comprising: Add a flocculant to the volume of blood before filling the device with the volume of blood.

10. The method according to claim 9, wherein the flocculant is added and present in an amount of 0.01 micrograms / mL of blood to 5000 micrograms / mL of blood.

11. A method for separating solid particles from a fluid, the method comprising: a) Obtaining a device, the device comprising: 1) A microstructured substrate, the microstructured substrate comprising a plurality of microstructures extending across a first surface of the microstructured substrate, wherein at least a portion of the outer surface of the plurality of microstructures is configured to permit capillary action; 2) A cover, the cover being disposed at a selected distance above the top of the first surface of the microstructured substrate; 3) At least one sidewall, the at least one sidewall attaching the cover to the first surface of the microstructured substrate along a perimeter of the first surface of the microstructured substrate; 4) A first orifice, the first orifice being defined by at least one of the microstructured substrate or the cover; and 5) A second orifice, the second orifice being defined by at least one of the microstructured substrate or the cover; wherein the first surface of the microstructured substrate and the at least one sidewall together define a first open volume, the first open volume being the sum of the open spaces from the bottom to the top of each of the plurality of microstructures located between the plurality of microstructures, wherein the cover and the top of the first surface of the microstructured substrate and the at least one sidewall together define a second open volume located adjacent to the first open volume, and taking the sum of the combined first open volume and the second open volume as the 100% open volume, the percentage of the first open volume in the 100% open volume being greater than the volume percentage of the particles present in the fluid; b) Filling the device with a volume of the fluid through the first orifice by capillary action; c) Waiting for a time sufficient for at least a portion of the particles to sediment within the first open volume of the plurality of microstructures; and d) Fluidly coupling the device to a collection article at the first orifice or the second orifice such that at least 10% of an initial volume of the fluid flows out of the device and onto the collection article by capillary action, wherein at least some of the particles from the initial volume of the fluid have been retained within the first open volume of the plurality of microstructures.

12. A kit, the kit comprising a device and a collection article, wherein the device comprises: 1) A microstructured substrate, the microstructured substrate comprising a plurality of microstructures extending across a first surface of the microstructured substrate, wherein the microstructures cover at least 90% of the first surface of the microstructured substrate, and wherein at least a portion of the outer surface of the plurality of microstructures is configured to permit capillary action; 2) A cover, the cover being disposed at a selected distance from the top of the first surface of the microstructured substrate; 3) At least one sidewall, the at least one sidewall attaching the cover to the first surface of the microstructured substrate along a perimeter of the first surface of the microstructured substrate; 4) A first orifice, the first orifice being defined by at least one of the microstructured substrate or the cover; And 5) A second orifice, the second orifice being defined by at least one of the microstructured substrate or the cover; wherein the first surface of the microstructured substrate and the at least one sidewall together define a first open volume, the first open volume being the sum of the open spaces from the bottom to the top of each microstructure located between the plurality of microstructures, and wherein the cover, the top of the first surface of the microstructured substrate, and the at least one sidewall together define a second open volume located adjacent to the first open volume; and wherein the collection article has a surface with an advancing contact angle with water of less than 90 degrees.

13. The kit according to claim 12, wherein the collection article comprises: a) A first polymer layer having a substantially flat first major surface and an opposite second major surface; And 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, wherein the first major surface of the second polymer layer is conformal with the second major surface of the first polymer layer, wherein the second major surface of the second polymer layer defines a cavity comprising at least one wall, wherein the second polymer layer has 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 the surface of the cavity has an advancing contact angle with water of less than 90 degrees.

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

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

16. The kit according to claim 15, wherein the flocculant is hydrophilic and non-hemolytic.

17. The kit according to claim 15 or claim 16, wherein the flocculant comprises a modified or unmodified amino polymer selected from the group consisting of: polyethyleneimine, polylysine, polyaminoamide, polyallylamine, polyvinylamine, poly(dimethylamine-epichlorohydrin-ethylenediamine), polydiallyldimethylammonium chloride, cationic polyacrylamide (CPAM), polyaminosiloxane, and dendrimers formed from polyamidoamine (PAMAM) and polypropyleneimine.

18. The kit according to any one of claims 15 to 17, wherein the flocculant 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 according to any one of claims 15 to 18, wherein the flocculant comprises a modified or unmodified polyethyleneimine polymer.

20. The kit according to any one of claims 15 to 19, wherein the flocculant comprises a modified or unmodified gelatin.

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

22. The kit according to claim 21, wherein the top surface of each rib is the top of a top cover disposed on the sidewall, and the width of the top cover is greater than the width between the opposing sidewalls.

23. The kit according to claim 21, wherein the collection article includes a plurality of ribs alternating with channels, and wherein each channel includes at least one secondary channel.

24. The kit according to any one of claims 12 to 20, wherein the microstructure of at least one of the device or the collection article includes an array of peak structures and adjacent valleys, wherein the valleys have a maximum width ranging from 10 microns to 250 microns, and the peak structures have an apex angle greater than 5 degrees and at most 90 degrees.

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

26. The kit according to claim 24 or claim 25, wherein the array of peak structures and adjacent valleys of at least one of the device or the collection article further includes a gap between adjacent peak structures.

27. The kit according to any one of claims 12 to 26, wherein the microstructure of at least one of the device or the collection article includes a two-dimensional (x-axis and y-axis) array of protrusions disposed across a first surface of the microstructured substrate; wherein each protrusion of the plurality of protrusions includes a base, a top, and one or more sides connecting the top to the base.

28. The kit according to any one of claims 12 to 20, wherein the microstructured substrate of at least one of the device or the collection article comprises a microstructured layer having a first major surface and a second major surface, wherein the microstructures comprise a plurality of cavities extending between the first major surface and the second major surface; wherein each cavity comprises a first opening, a second opening, and at least one sidewall extending between the first opening and the second opening.

29. The kit according to any one of claims 12 to 20, wherein the microstructures of at least one of the device or the collection article comprise facets and sidewalls meeting the facets at the ridges of the microstructures, and wherein the facets and the sidewalls define an inclination angle therebetween.

30. The kit according to any one of claims 12 to 20, wherein the microstructures of at least one of the device or the collection article comprise an array of interconnected wells, wherein at least 80% of the wells are fluidly connected to at least two adjacent wells, and each well is connected via an exhaust port.

31. The kit according to any one of claims 12 to 20, wherein the microstructures comprise an array of upright posts extending across a first surface of the microstructured substrate.

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

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