Method and device for removing particles from fluid

Abstract

The present disclosure provides methods and apparatus for removing particles from a fluid. In one embodiment, 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 also 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 settle at least a portion of the red blood cells within the first open volume of the microstructure, and applying pressure to the device such that some of the initial volume of blood flows out of the device, at least some of the erythrocytes in the initial volume of blood have been retained within the first open volume of the microstructure.

Description

Background Art

[0001] The identification and quantification of biomarkers in blood typically requires 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 determinations. The level of interference varies within the physiological hematocrit range, which results in variability in the reported concentrations of biomarkers. Interference in optical detection assays (e.g., ELISA) can be caused by the light scattering effects and absorption of hemoglobin in the visible spectrum. Red blood cells can also interfere with electrochemical detection assays, such as those commonly used in glucose test strips. Chemicals, such as oxygen present in red blood cells, can interfere with redox reactions.

[0002] In centralized hospitals or clinical laboratories, separation of red blood cells is achieved by centrifugation. The separation process requires the collection of large volumes (e.g., milliliters) of blood in test tubes via veins. During centrifugation, red blood cells are gathered 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 environments, 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 required, intravenous collection of a volume of blood and benchtop centrifugation are often unavailable or too time-consuming. A finger prick provides a volume of blood of about 5 microliters. Glucose test strips typically receive less than 1 microliter of blood and are subject to the interferences 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 including a microstructured substrate, the microstructured substrate including a plurality of microstructures extending across a first surface of the microstructured substrate. At least a portion of the outer surface of the plurality of microstructures is configured to allow capillary action. The device also includes a cover disposed at a selected distance from the top of the first surface of the microstructured substrate and at least one sidewall that attaches the cover to the first surface of the microstructured substrate along the periphery of the first surface of the microstructured substrate. In addition, the device includes a first hole defined by at least one of the microstructured substrate or the cover and a second hole defined by at least one of the microstructured substrate or the cover. The first surface of the microstructured substrate together with at least one sidewall defines a first open volume, which is the sum of the open spaces between the plurality of microstructures from the bottom to the top of each microstructure, wherein the cover together with the top of the first surface of the microstructured substrate and at least one sidewall defines a second open volume positioned 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 100% open volume is greater than the volume percentage of red blood cells present in the blood. The method also includes filling the device with a certain volume of blood through the first hole via capillary action, and waiting a time sufficient for at least a portion of the red blood cells to settle in the first open volume of the plurality of microstructures. In addition, the method includes applying pressure to the device so that at least 10% of the initial volume of blood flows out of the device through the first hole or the second hole, and at least some of the red blood cells in the initial volume of blood have been retained in the first open volume of the plurality of microstructures.

[0005] In a second aspect, a device is provided. The device includes a microstructured substrate including a plurality of microstructures extending across a first surface of the microstructured substrate. The microstructures cover at least 90% of the first surface of the microstructured substrate, and at least a portion of the outer surface of the plurality of microstructures is configured to allow capillary action. The device also includes a cover disposed at a selected distance from the top of the first surface of the microstructured substrate and at least one sidewall that attaches the cover to the first surface of the microstructured substrate along the periphery of the first surface of the microstructured substrate. In addition, the device includes a first hole defined by at least one of the microstructured substrate or the cover and a second hole defined by at least one of the microstructured substrate or the cover. The first surface of the microstructured substrate defines a first open volume together with at least one sidewall, 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, wherein the cover defines a second open volume positioned adjacent to the first open volume together with the top of the first surface of the microstructured substrate and at least one sidewall.

[0006] In a third aspect, a method for separating solid particles from a fluid is provided. The method includes obtaining a device including a microstructured substrate, the microstructured substrate including a plurality of microstructures extending across a first surface of the microstructured substrate. At least a portion of the outer surface of the plurality of microstructures is configured to allow capillary action. The device also includes a cover disposed at a selected distance from the top of the first surface of the microstructured substrate and at least one sidewall that attaches the cover to the first surface of the microstructured substrate along the periphery of the first surface of the microstructured substrate. In addition, the device includes a first hole defined by at least one of the microstructured substrate or the cover and a second hole defined by at least one of the microstructured substrate or the cover. The first surface of the microstructured substrate defines a first open volume together with at least one sidewall, 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, wherein the cover defines a second open volume positioned adjacent to the first open volume together with the top of the first surface of the microstructured substrate and at least one sidewall. 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 100% open volume is greater than the volume percentage of particles present in the fluid. The method also includes filling the device with a certain volume of fluid through the first hole via capillary action, and waiting a time sufficient for at least a portion of the particles to settle within the first open volume of the plurality of microstructures. In addition, the method includes applying pressure to the device so that at least 10% of the fluid flows out of the device through the first hole or the second hole, and at least some of the particles in the fluid have been retained in the first open volume of the plurality of microstructures.

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

[0008] The above summary of the present disclosure is not intended to describe each disclosed embodiment or each specific implementation of the present disclosure. The following description more specifically illustrates exemplary embodiments. In several places throughout the application, guidance is provided by example lists, which can be used in various combinations. In each case, the narrated list is used only as a representative group and should not be interpreted as an exclusive list.

[0009] BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 is a generalized flow chart of an exemplary method.

[0011] Figure 2A is a perspective view of a generalized schematic diagram of an exemplary apparatus.

[0012] Figure 2Bis a perspective view of a generalized schematic diagram of a microstructured substrate used in an exemplary device.

[0013] Figure 3A is a scanning electron microscope (SEM) image of a cross-section of a portion of an exemplary apparatus, which is labeled to illustrate a portion of an exemplary method.

[0014] Figure 3B yes Figure 3A SEM image of a cross section of a portion of an exemplary device, which is labeled to illustrate another portion of an exemplary method.

[0015] Figure 4A is a schematic cross-sectional view of a microstructured substrate having a plurality of ribs alternating with channels.

[0016] Figure 4B is a schematic perspective view of a microstructured substrate having a plurality of ribs alternating with channels, wherein the ribs include caps at their top surfaces.

[0017] Figure 5A is a schematic cross-sectional view of a microstructured substrate having an array of linear prisms.

[0018] Figure 5B is a SEM image of a cross-section of a portion of an exemplary device of Example 8.

[0019] 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.

[0020] 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.

[0021] Figure 5E is a schematic cross-sectional view of a microstructured substrate having a faceted structure.

[0022] Fig. 6A is a schematic cross-sectional view of a microstructured substrate having a two-dimensional array of protrusions.

[0023] Figure 6B are top plan views of four representative engineered micropatterned areas for two-dimensional protrusion arrays.

[0024] Fig. 7A is a schematic cross-sectional view of a microstructured substrate having a plurality of cavities extending between two major surfaces.

[0025] Figure 7Bis a generalized schematic exploded view of a microstructured substrate having a plurality of cavities extending between two major surfaces.

[0026] Fig. 8A It is an exploded schematic diagram of the device of Example 1.

[0027] Figure 8B is a generalized schematic top view of the device of Example 1.

[0028] Figure 8C is a generalized schematic top view of two components for attaching a pump fixed to the device of Example 1.

[0029] Fig.8D is a generalized schematic perspective view of the device of Example 1 suitable for attachment to a pump.

[0030] Fig. 9 is a generalized flow chart of another exemplary method.

[0031] Fig. 10A is a schematic perspective view of a portion of a microstructured substrate having an array of fluidically connected wells.

[0032] Fig. 10B is a schematic top view of a portion of a microstructured substrate having an array of circular, fluidically connected wells.

[0033] Fig.11 is a schematic cross-sectional view of a microstructured substrate having an array of generally upstanding rods.

[0034] Although the above drawings illustrate several embodiments of the present disclosure, other embodiments are also contemplated as indicated in the description. The drawings are not necessarily drawn to scale. In all cases, the present disclosure presents the invention by way of example and not limitation. DETAILED DESCRIPTION

[0035] As used herein, the term "microreplication" refers to the production of a microstructured surface by a process in which the structured surface features maintain the fidelity of individual features during fabrication.

[0036] As used herein, the term "microstructure" encompasses both structures (i.e., features) that protrude above a major surface of a substrate and structures that are recessed below a major surface of a substrate. Combinations of protruding features and recessed features are contemplated. A microstructure also refers to a structure that is a predetermined molded structure (e.g., obtained by molding a polymerized 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 microns to about 3000 microns. One of these orthogonal directions may generally be perpendicular to the plane of the substrate (e.g., along the z-axis), so the dimension may include, for example, a protrusion height or a recess depth.

[0037] As used herein, the term "capillary action" refers to the flow of a fluid without the assistance of an external force (eg, pressure, gravity, vacuum, etc.). Capillary action often occurs when an aqueous fluid contacts a hydrophilic surface. The aqueous fluid contains 50% or more water by volume.

[0038] As used herein, the term "hydrophilic" refers to a surface that is wetted by an aqueous solution and does not indicate whether the material absorbs the aqueous solution. "Wetting" means that the surface exhibits spontaneous wicking when in contact with an aqueous fluid. "Spontaneous" means occurring without external forces. In some embodiments, a hydrophilic surface exhibits an advancing (maximum) water contact angle of less than 90°, preferably 45° or less.

[0039] As used herein, the term "hydrophobic" refers to a surface that 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.

[0040] As used herein, "curing" refers to 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 "hardenable" refers to a material that can be cured or solidified, such as by heating to remove solvents, heating to cause polymerization, chemical crosslinking, radiation-induced polymerization or crosslinking, etc. As used herein, "cured" refers to a material or composition that has been hardened or partially hardened by curing (e.g., polymerization or crosslinking).

[0041] As used herein, a polymer "film" is a polymer material in the form of a generally flat sheet that is sufficiently flexible and strong to be processed in a roll-to-roll manner. Roll-to-roll refers to the process of winding a material onto or unwinding it from a support and further processing it in some manner. Examples of further processing include coating, cutting, punching, and exposure to radiation, among others. Polymer films can be manufactured in a variety of thicknesses, typically ranging from about 5 microns to 1000 microns.

[0042] As used herein, "solid" refers to a state of matter that is not liquid or gas, and a solid has a stable three-dimensional shape.

[0043] As used herein, "fluid" refers to compositions that include liquids (ie, a state of matter that is not solid or gas) and encompass solutions, suspensions, and emulsions.

[0044] As used herein, "external surface" with respect to a microstructure refers to the outermost surface of the microstructure.

[0045] As used herein, "thermoplastic" refers to a polymer that flows when heated sufficiently above its glass transition temperature and becomes solid when cooled. In contrast, "thermoset" refers to a polymer that is permanently solidified after curing and does not flow upon subsequent heating. Thermosetting polymers are typically cross-linked polymers.

[0046] As used herein, the term "glass transition temperature" (T g ) refers to the transition of a polymer from a glassy state to a 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 stream. When referring to the T of a monomer, g When it is the T of the homopolymer of this monomer g The homopolymer must have a high enough molecular weight so that T g reaches a limiting value, because it is generally understood that the T g The molecular weight will increase to a limit. Homopolymers are also understood to be essentially free of moisture, residual monomers, solvents and other g Suitable DSC methods and analysis modes are described in Matsumoto, A. et al., J. Polym. Sci. A., Polym. Chem. 1993, 31, 2531-2539.

[0047] As used herein, "transparent" refers to a material (eg, layer) having at least 50% transmittance, 70% transmittance, or optionally greater than 90% transmittance in at least the 400 nanometers (nm) to 700 nm portion of the visible spectrum.

[0048] The words "preferred" and "preferably" refer to embodiments of the present disclosure that may provide certain benefits in certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. In addition, the recitation of one or more preferred embodiments does not mean that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the present disclosure.

[0049] In this application, terms such as "a", "an", and "the" are not intended to refer to only a singular entity, but include general categories of which specific examples may be used for illustration. The terms "a", "an", and "the" are used interchangeably with the term "at least one". The phrases "at least one of" and "including at least one of" followed by a list refer to any one of the items in the list and any combination of two or more items in the list.

[0050] As used herein, the term "or" is generally used in its ordinary sense, including "and / or", unless the content 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.

[0051] Also herein, it is assumed that all numbers are modified by the term "about," and preferably by the term "exactly." As used herein in conjunction with measured amounts, the term "about" refers to the variation in the amount of the measurement that would be expected by a skilled artisan making the measurement and making the measurement with a level of care commensurate with the purpose of the measurement and the precision of the measuring device used.

[0052] As used herein as a modifier of a property or attribute, unless otherwise specifically defined, the term "substantially" means that the property or attribute will be readily recognized by a person of ordinary skill but does not require absolute precision or a perfect match (e.g., within + / -20% for quantifiable properties). Unless otherwise specifically defined, the term "substantially" means a high degree of similarity (e.g., within + / -10% for quantifiable properties), but again does not require absolute precision or a perfect match. Terms such as identical, equal, consistent, constant, exact, etc. are understood to be within the usual tolerance or measurement error applicable to the particular situation, without requiring absolute precision or a perfect match.

[0053] In point-of-care biomarker analysis, there is a need for simple and efficient separation of red blood cells from microliter amounts of blood without hemolysis, dilution, or significant loss of blood to dead space.

[0054] In a first aspect, the present disclosure provides a method for separating red blood cells from blood. The method comprises:

[0055] a) obtaining a device, the device comprising:

[0056] 1) a 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 allow capillary action;

[0057] 2) a cover disposed a selected distance from the top of the first surface of the microstructured substrate;

[0058] 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;

[0059] 4) a first aperture defined by at least one of the microstructured substrate or the cover; and

[0060] 5) a second aperture defined by at least one of the microstructured substrate or the cover;

[0061] wherein the first surface of the microstructured substrate together with the at least one sidewall defines a first open volume, the first open volume being the sum of the open spaces between the plurality of microstructures from the bottom to the top of each microstructure, 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, and taking the sum of the combined first and second open volumes as 100% open volume, the percentage of the first open volume in 100% open volume is greater than the volume percentage of red blood cells present in blood;

[0062] b) filling the device with a volume of blood via capillary action through the first aperture;

[0063] c) waiting a time sufficient to allow at least a portion of the red blood cells to settle within the first open volume of the plurality of microstructures; and

[0064] d) applying pressure to the device so that at least 10% of an initial volume of blood, at least some red blood cells of which have been retained within the first open volume of the plurality of microstructures, flows out of the device through the first or second apertures.

[0065] In a second aspect, the present disclosure provides a device. The device comprises:

[0066] 1) a 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 an outer surface of the plurality of microstructures is configured to allow capillary action;

[0067] 2) a cover disposed a selected distance from the top of the first surface of the microstructured substrate;

[0068] 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;

[0069] 4) a first aperture defined by at least one of the microstructured substrate or the cover; and

[0070] 5) a second aperture defined by at least one of the microstructured substrate or the cover;

[0071] wherein the first surface of the microstructured substrate together with at least one sidewall defines 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, wherein the cover together with the top of the first surface of the microstructured substrate and at least one sidewall defines a second open volume positioned adjacent to the first open volume.

[0072] The following disclosure relates to both the first aspect and the second aspect.

[0073] Reference Figure 1 , the method includes obtaining a device 110 (where the device is as described above); filling the device with a volume of blood through a first aperture via capillary action 120; waiting a time sufficient to allow at least a portion of the red blood cells to settle within a first open volume of the plurality of microstructures 130; and applying pressure to the device so that at least 10% of an initial volume of blood flows out of the device through the first aperture or the second aperture, at least some of the red blood cells in the initial volume of blood having been retained within the first open volume of the plurality of microstructures 140. In some cases, the waiting time is sufficient to allow at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even at least 95% of the red blood cells to settle within the first open volume of the plurality of microstructures.

[0074] Refer to Figure 2 and Figure 3A , an exemplary device 200 is shown. The device 200 includes a microstructured substrate 210 including a plurality of microstructures 230 extending across a first surface 202 of the microstructured substrate 210. At least a portion of an outer surface 232 of the plurality of microstructures 230 is configured to allow capillary action. The device 200 also includes a cover 220 disposed a selected distance D from a top 221 of the first surface of the microstructured substrate 210 and at least one sidewall 240 attaching the cover 220 to the first surface 202 of the microstructured substrate 210 along a perimeter P of the first surface of the microstructured substrate 210. The perimeter P is collectively comprised of each of the side edges Pa, Pb, Pc, Pd, ... Pn of this particular device. Figure 2A The device 200 shown has four sides Pa, Pb, Pc and Pd, so the perimeter P includes each of these four sides. Devices with other shapes than four sides are envisioned.

[0075] Optionally, at least one sidewall 240 may include an adhesive layer disposed between the cover 220 and the microstructured substrate 210 (e.g., which attaches the cover to the substrate), such as the double-sided tape used in Example 1 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 comprises an adhesive polymer and a crosslinking agent. The term "adhesive polymer" as used herein refers to a polymer that exhibits adhesion at ambient temperature (e.g., 20°C-25°C). The adhesive polymer may 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-coated adhesive films are available from 3M Company (St. Paul, Minnesota) under the trade names 3M Medical Silicone Tape 2477P and one of 3M Medical Tapes 1509, 1510, 1513, 1522, 9874, and 9877.

[0076] Additionally, the device includes a first aperture 250 defined by at least one of the microstructured substrate 210 or the cover 220 and a second aperture 260 defined by at least one of the microstructured substrate 210 or the cover 220 . Figure 2A The device shown includes a first aperture 250 defined by the cover 220 and a second aperture 260 defined by both the microstructured substrate 210 and the cover 220 .

[0077] In selected embodiments, the sidewalls, microstructured substrate, and cover are hermetically sealed to one another at their contact points (e.g., seams), which minimizes leakage of the fluid sample from any seams between the three (e.g., the fluid may enter and exit primarily or only through the first aperture and / or the second aperture). In some cases, the microstructured substrate itself includes the sidewalls in its structure, such that at least one sidewall is part of the microstructured substrate.

[0078] Suitable materials for use as covers include, for example, but are not limited to, polyolefins (e.g., high density polyethylene (HDPE), medium density polyethylene (MDPE), or low density polyethylene (LDPE)), polyesters, polyamides, polyvinyl chloride, polyetheresters, polyimides, polyesteramides, polyacrylates, polyvinyl acetate, or hydrolyzed derivatives of polyvinyl acetate. In certain embodiments, polyolefins are preferred because they have excellent physical properties, are easy to process, and are generally low in cost. In addition, polyolefins are generally tough, durable, and retain their shape well, and are therefore easy to handle after the article is formed. In selected embodiments, the film layer includes polyester polyethylene terephthalate (PET). A suitable commercially available PET is a 5 mil (127 micron) thick PET sheet purchased from Tekra (New Berlin, Wisconsin) under the trade name "MELINEX 454". A suitable commercially available LDPE is purchased from Dow Chemical Company (Midland, Michigan) under the trade name "DOW 955I LDPE". Furthermore, various additives may be contained in the cover, for example, a surface energy modifier (such as a surfactant and a hydrophilic polymer), a plasticizer, an antioxidant, a pigment, a release agent, an antistatic agent, and the like.

[0079] The first surface 202 of the microstructured substrate 210, together with the 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 simplicity, the arrow for 270 only points to a portion of the first open volume between two adjacent microstructures 230). The cover 220, together with the top 221 of the first surface 202 of the microstructured substrate 210 and the at least one sidewall 240, defines a second open volume 280 positioned adjacent to the first open volume 270, and the sum of the combined first and second open volumes is taken as 100% open volume, the percentage of the first open volume in 100% open volume being greater than the volume percentage of red blood cells present in the blood. In order to adapt the device to blood containing a certain volume percentage of (e.g., solid) particles (such as red blood cells), it can be determined that the percentage of the first open volume in 100% open volume needs to be greater than the volume percentage of the particles present in the fluid, considering the sum of the combined first open volume and the second open volume to be 100% open volume. For example, if the volume percentage of particles is 20% of the total volume of the fluid, then a suitable ratio of the first open volume to the second open volume would be greater than 1:4 (e.g., 1.1:4). If the volume percentage of particles is 75% of the total volume of the fluid, then a suitable ratio of the first open volume to the second open volume would be greater than 3:1 (e.g., 3.1:1).

[0080] 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 up to 2.0:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, or up to 1.5:1. Brackets 272 represent the height of the first open volume 270, and brackets 282 represent the height of the second open volume 280, with the plus sign indicating that the second volume 280 is stacked directly on top of the first open volume 270 in the device 220 when the device 220 is oriented as shown by the z-axis and y-axis. Note that Figure 3A and Figure 3B The device 200 in FIG. 2 is not drawn to scale for a ratio of first open volume 270 to second open volume 280 of 1:1 or greater, and is shown to aid in describing the devices and methods provided herein.

[0081] 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 red blood cells settle, no red blood cells should protrude above the top of the microstructures.

[0082] As described above, at least a portion of the outer surface of the plurality of microstructures is configured to allow capillary action. Capillary action is well known in the art to refer to fluid flow without the assistance of external forces, typically for aqueous fluids (having 50% or more volume of water) in contact with hydrophilic surfaces. Therefore, once blood is introduced into the first hole of the device, the blood is spontaneously transported along the outer surface of the microstructure, thereby diffusing in the area of ​​the microstructure. Two general factors that affect the ability of the microstructure to spontaneously transport fluids are (i) the structure or morphology of the surface (e.g., capillary action, the shape of the cavity) and (ii) the properties of the surface (e.g., surface energy). In order to achieve the desired amount of fluid transport capacity, the designer can adjust the structure or morphology of the substrate and / or adjust the surface energy of the capillary microstructured surface. In order to achieve wicking, the surface of the capillary microstructure must be able to be "wetted" by the liquid to be transported (e.g., liquid substance). Optionally, the sensitivity of the solid surface to being wetted by the liquid is characterized by the contact angle formed by the liquid after being deposited on a horizontally placed surface and stabilized thereon with 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, a material is considered hydrophilic if it has an advancing contact angle of less than 90 degrees, while hydrophilic surfaces exhibit advancing (maximum) water contact angles of less than 90°, preferably 45° or less.

[0083] Sufficient microstructures need to be hydrophilic on the outer surface to enable the microstructured substrate to perform capillary action, thereby transporting the fluid (e.g., blood) throughout the device once the fluid has been introduced by the first hole, so that solid particles can enter the open volume between the microstructures, thereby being able to settle out of the bulk fluid. In some cases, 50% or more of the area of ​​the outer surface of the microstructure is capable of capillary action, and 60%, 70%, 80%, 90%, or 95% or more of the outer surface of the microstructure is capable of capillary action. In selected embodiments, at least a portion of the major surface 223 of the cover 220 facing the microstructured substrate 230 is hydrophilic to facilitate the capillary action of the fluid within the device 200. According to any device described herein, the hydrophilicity of the outer surface of the microstructure and / or the major surface of the cover can be achieved by one or more of material selection, additives contained in the material, or surface treatment. In some embodiments, the microstructure has 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 are not limited to, C8-C18 alkane sulfonates; C8-C18 secondary alkyl sulfonates; alkylbenzene sulfonates; C8-C18 alkyl sulfates; alkyl ether sulfates; sodium laureth 4 sulfate; sodium laureth 8 sulfate; dioctyl sulfosuccinate, sodium salt; lauroyl lactylate; stearoyl lactylate; or any combination thereof. One or more surfactants may be applied by conventional methods, such as by rubbing a coating of surfactant on the surface of the microstructure and allowing the coating to dry. Suitable surface treatments include hydrophilic coatings comprising plasma deposited silicon / oxygen materials and / or diamond-like glass (DLG) materials. For example, plasma deposition of each of 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. Pat. 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 are not limited to, polyesters, polyamides, polyurethanes, poly(vinyl alcohol), poly(alkylene glycol), poly(alkylene oxide), poly(vinyl pyrrolidone), rubber elastomers, or any combination thereof.

[0084] The use of certain ionic polymers, particularly cationic polymers, for flocculation of cells and / or cell fragments and for the precipitation of proteins is known. When using a flocculant, a device having a microstructure larger than that when a flocculant is not used (e.g., height and / or depth, spacing between adjacent microstructures, etc.) can be used. This is because particles tend to clump and have a larger size when flocculating, so that a larger space may be required to keep particles in the microstructure. On the contrary, 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). Therefore, the microstructured surface of the device can be partially selected by considering the expected size of the particles or flocculated particles.

[0085] The polymer used as a flocculant can be unmodified (e.g., polyethyleneimine) or modified (e.g., guanylated polyethyleneimine). In some embodiments, a suitable flocculant is hydrophilic and non-hemolytic (i.e., does not dissolve red blood cells). Some suitable flocculants are described in detail in U.S. Patent Nos. 8435776 (Rasmussen et al.) and 10005814 (Rasmussen et al.), the entire contents of which are incorporated herein by reference. In some cases, the flocculant comprises an unmodified or modified (e.g., functionalized) amino polymer. For example, a suitable amino polymer can be selected from polyethyleneimine, polylysine, polyaminoamide, polyallylamine, polyethyleneamine, polydimethylamine-epichlorohydrin-ethylenediamine, polydiallyldimethylamine chloride, cationic polyacrylamide (CPAM), polyaminosiloxane, and a dendrimer formed by polyaminoamine (PAMAM) and polypropylamine. Suitable modified amino polymers can be prepared by functionalizing one or more amino polymers selected from polyethyleneimine, polylysine, polyaminoamide, polyallylamine, polyethyleneamine, polydimethylamine-epichlorohydrin-ethylenediamine, polydiallyldimethylammonium chloride, CPAM, polyaminosiloxanes, and dendrimers formed from PAMAM and polypropyleneimine. For example, functionalization can include reacting the amino polymer with an alkylating, acylation, or amidinating agent. In selected embodiments, the flocculant comprises a modified or unmodified polyethyleneimine polymer.

[0086] The one or more flocculants can be applied by conventional methods, such as by applying a coating of the flocculant on the surface of the microstructures and allowing the coating to dry.

[0087] In some cases, the flocculant has a weight average molecular weight (Mw) of 5,000 grams per mole (g / mol) or more, 10,000 g / mol, 20,000 g / mol, 30,000 g / mol, 40,000 g / mol, 50,000 g / mol, 60,000 g / mol, 70,000 g / mol, 80,000 g / mol, 90,000 g / mol, 100,000 g / mol, 110,000 g / mol, 120,000 g / mol, 130,000 g / mol, 140,000 g / mol, or 150,000 g / mol or more; and a weight average molecular weight of 500,000 g / mol or less. Sometimes, a high molecular weight (eg, 50,000 g / mol or higher) can help flocculate particles (eg, red blood cells).

[0088] Advantageously, after filling the device with a 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 so that the concentration of the flocculant in a volume of a 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 , 250μg / mL, 500μg / mL, 750μg / mL, 1000μg / mL or 1500μg / mL or more; and 5000μg / mL or less, 4000μg / mL, 3000μg / mL, 2000μg / mL, 1000μg / mL, 500μg / mL, 200μg / mL, 100μg / mL, 50μg / mL, 10μg / mL or 2μg / mL or less. Stated otherwise, in some embodiments, the flocculant is present in a certain volume of fluid (e.g., blood) in an amount of 0.01μg / mL to 5000μg / mL. When on the surface of the device in the form of a dry coating, the amount of the flocculant will vary based on the molecular weight (Mw) of a specific flocculant.

[0089] Figure 3A and Figure 3B A cartoon depiction on top of the SEM image of the device is also incorporated to illustrate the concept of how the device 200 is typically used. For example, after blood 295 has filled the device 200, using capillary action, red blood cells 290 begin to settle between the microstructures 230 into the first open volume 270. Figure 3A, only three red blood cells 290 are depicted as being located in the first open volume 270. Figure 3B , depicts the device 200 after waiting a time sufficient for all of the red blood cells 290 to settle within the first open volume 270 of the plurality of microstructures 230. Typically, the red blood cells settle simply due to gravity. In the second open volume 280 there is a volume of blood 295 from which the red blood cells have been removed. In use, when pressure is applied to the device, an initial volume of blood is caused to flow out of the device through the first aperture 250 or the second aperture 260, at least some of the red blood cells in the initial volume of blood having been retained within the first open volume 270 of the plurality of microstructures 230. In some cases, it is preferred that the blood 295 be discharged from the second aperture 260.

[0090] Optionally, the method also includes making fluid (for example, blood) before entering the device, after leaving the device or before entering the device and after leaving the device all pass through a filter. For example, filtered fluid can be used for removing at least one undesirable component from fluid. Suitable filter includes, for example, but not limited to nonwoven filter, textile filter, membrane filter, paper filter and sponge filter. Exemplary suitable filter includes, for example, glass fiber filter and asymmetric polysulfone / polyethersulfone filter (for example, VIVID plasma separation membrane commercially available from Pall Corporation (Port Washington, NY), or Cobetter OneStep plasma separation membrane or Cobetter RB series, both of which can be commercially available from CobetterFiltration Equipment Co., Ltd. (Hangzhou, China)).

[0091] In some cases, the method also includes 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 a concentration present in the volume of fluid (e.g., an amount of 0.01 micrograms / mL to 5000 micrograms / mL of fluid).

[0092] Red blood cells tend to account for 35%-50% of the total volume of whole blood. Making the ratio of the first open volume to the second open volume 1:1 or greater provides enough space in the first open volume between the microstructures for retaining up to all red blood cells of the undiluted whole blood sample, while making the second open volume free for blood containing fewer (to no) red blood cells. Therefore, when pressure is applied to the device after sedimentation, blood (plasma in some cases) that has been removed from red blood cells preferentially leaves the device because it is positioned closer to the hole. In contrast, the settled red blood cells tend to remain between the microstructures in the first open volume, and less can be removed from the device by applying pressure. 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 be settled in the first open volume with red blood cells, so as to maximize the volume of blood (red blood cells have been removed therefrom) that can be flowed out of the device for analysis. By selecting a ratio of the first open volume to the second open volume of 1:1 or greater, it is usually not necessary to dilute the whole blood to effectively separate red blood cells from the blood, so that the blood can be used in an undiluted form. However, in the case of diluted blood, smaller ratios may 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.

[0093] Advantageously, the device according to at least some embodiments of the present disclosure effectively separates (at least a portion of) red blood cells from a small volume of blood while minimizing the retention of analyzable blood within the device. This is in contrast to devices that employ a flowing stream or result in a loss of dead volume. 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.).

[0094] Preferably, at least 10%, 15%, 20%, 25%, 30%, 35% or even at least 40% of the blood from which red blood cells have been removed flows out of the device when pressure is applied. The greater this percentage, the more efficient the separation method using the device according to the present disclosure and the less analyzable sample volume is lost in the device.

[0095] 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) when the sample volume is 120 microliters or less, 110 microliters, 100 microliters, 90 microliters, 80 microliters, 70 microliters, 60 microliters, 50 microliters, 40 microliters, 30 microliters, 20 microliters, 10 microliters, or even 5 microliters or less; and 1 microliter or more, 2 microliters, 3 microliters, 4 microliters, 5 microliters, 6 microliters, 7 microliters, 8 microliters, 9 microliters, 10 microliters, 12 microliters, 15 microliters, 25 microliters, 35 microliters, or 45 microliters or more. For example, the sum of the first open volume and the second open volume of the device can be between 5 microliters and 120 microliters.

[0096] In some cases, the first hole is defined by the cover of the device, and a volume of fluid (e.g., blood) can be introduced into the device using gravity and / or wicking of the fluid into the hole. In other cases, the first hole is defined by the microstructured substrate. For example, referring to Figure 2B , the first hole 250 can be provided as a reservoir configured to hold at least a certain minimum volume of fluid. The reservoir can be defined by a portion of one end 237 of the microstructured substrate 210 adjacent to the microstructure 230. Similarly, the second hole 260 can be a reservoir configured to hold the fluid when the fluid leaves the microstructured surface 202 of the microstructured substrate 210. Generally, it is advantageous that the second hole is positioned a distance away from the first hole 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 hole. Optionally, a positive pressure can be provided by a pump to deposit a certain volume of blood in the first hole, and then after at least some red blood cells have settled in the first open volume, the pump can be engaged again to push the blood out of the second open volume from the second hole. Optionally, a certain volume of blood can be wicked into the device through the first hole, and then the positive pressure provided by the user's finger or the negative pressure provided by the vacuum source can be used to push the blood out of the first hole or the second hole from the second open volume. Thus, the pressure pushing the blood (from which some red blood cells have been removed) can be either positive or negative.

[0097] Louver structure

[0098] Reference again 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 , wherein 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 wherein each rib 230 includes sidewalls 233 and 234 and a top surface 221, and each channel 201 includes a bottom surface 205.

[0099] More specifically, the depicted microstructured substrate 210 includes a plurality of channels 201a-201d on a base layer 213. Figure 4A As shown, a continuous platform layer "L" can exist between the bottom of the channel 205 and the top surface 202 of the base layer 213. Alternatively, the channel 201 can extend all the way through the microstructured substrate 210. In some cases (such as Figure 4B ), the bottom surface 205 of the groove 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.

[0100] The height and width of the rib (e.g., protrusion) 230 are defined by adjacent channels (e.g., 201a and 201b). The rib 230 may be defined by a top surface 221, a bottom surface 231, and side walls 233 and 234 that join the top surface 221 to the bottom surface 231. The side walls 233 and 234 may be parallel to each other. More typically, the side walls have a wall angle.

[0101] The ribs 230 can be defined by a width "W". Typically, the ribs 230 have a width parallel to the first surface of the microstructured substrate and a height orthogonal to the first surface of the microstructured substrate. The ribs 230 typically have the same nominal height as the channels 201, except for the land areas "L". In typical embodiments, the height "H" of the channels 201 and / or ribs 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 no greater than 500 microns, 475 microns, 450 microns, 425 microns, 400 microns, 375 microns, 350 microns, 325 microns, 300 microns, 275 microns, 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 channel 201 and / or rib 230 is in the range of 50 microns to 500 microns. The microstructured substrate typically includes a plurality of ribs 230 having nominally the same height and width. In some embodiments, rib 230 has a height "H", a maximum width "W" at its widest portion, and an aspect ratio H / W of at least 1.5. In some embodiments, H / W is at least 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.5, 4.0, 4.5, or 5.0. In other embodiments, the aspect ratio of the ribs is at least 6, 7, 8, 9, or 10. In other embodiments, the aspect ratio of the ribs is at least 15, 20, 25, 30, 35, 40, 45, or 50.

[0102] 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.

[0103] 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.

[0104] In some embodiments, ribs 230 have a pitch "P" of at least 10 microns. The pitch is as Figure 4AThe distance between the starting point of the first rib depicted and the starting point of the second rib. The spacing may 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. The spacing is usually 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 a typical embodiment, the ribs are evenly spaced apart with a single spacing. Alternatively, the ribs may be spaced apart so 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 the ribs with the coating. When the ribs are too closely spaced, it may be difficult to evenly coat the sidewalls. When the ribs are too far apart, the coating may not be able to effectively provide its intended function.

[0105] Figure 4B Depicted in Figure 4A A variation of a louver structure of the present invention is provided, wherein in some cases, the top surface 221 of each rib 230 is the top of a cap 235 disposed on the sidewalls 233, 234, and the cap 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 the presence of the cap (e.g., undercut feature) on the rib can help retain the particle in the channel once the particle has settled in the device.

[0106] 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 including the following steps: (a) preparing a polymerizable composition; (b) depositing the polymerizable composition onto a negative microstructure molding surface (e.g., a tool) of the master in an amount just sufficient to fill the cavities of the master; (c) filling the cavities by moving beads of the polymerizable composition between a base layer (e.g., a preformed film) and the master, wherein at least one of the base layer and the master is flexible; and (d) curing the composition. The deposition temperature can range from ambient temperature to about 180°F (82°C). The master can be metallic, such as nickel, chrome-plated or nickel-plated copper or brass, or can be a thermoplastic material that is stable under polymerization conditions and has a surface energy that allows the polymerized material to be completely removed from the master. 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 microstructure.

[0107] In one embodiment, a structure (e.g., Figure 4B The microstructured substrate 210 with caps on the ribs shown in Figure 2 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 the rods in a separate forming step after demolding). Alternatively, such a microstructured substrate 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, the first layer including multiple 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).

[0108] The polymerizable resin may include 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, "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 may include (meth)acrylated urethane oligomers, (meth)acrylated epoxy oligomers, (meth)acrylated polyester oligomers, (meth)acrylated phenolic oligomers, (meth)acrylated acrylic oligomers, and mixtures thereof.

[0109] The polymerizable resin can be a radiation curable polymer resin, such as a UV curable resin.In some cases, polymerizable resin compositions useful in the microstructured substrates of the present disclosure can include polymerizable resin compositions such as those described in US Pat. No. 8,012,567 (Gaides et al.).

[0110] The chemical composition and thickness of the base layer may depend on the end use of the microstructured substrate. In a typical embodiment, the thickness of the base layer may be at least about 0.025 millimeters (mm) and may be about 0.05 mm to about 0.25 mm. Available 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 naphthalene dicarboxylic acid, materials based on polyolefins (such as cast or oriented films of polyethylene, polypropylene and polycycloolefins), polyimides and glass. Optionally, the base layer may include a mixture or combination of these materials. In some embodiments, the base layer may be multilayered or may contain a dispersed component suspended or dispersed in a continuous phase.

[0111] 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, Wilmington, Delaware. Examples of optical grade polycarbonate films include LEXAN polycarbonate film 8010 available from GE Polymershapes, Seattle, Washington, and Panlite 1151 available from Teijin Kasei, Alpharetta, Georgia.

[0112] Alternatively, microstructured substrate 210 can be prepared by melt extrusion, that is, fluid resin composition is poured onto negative microstructured molding surface (such as tool) of master and the composition is allowed to harden. In this embodiment, rib 230 is interconnected with base layer 213 in a continuous layer. Each rib and the connection therebetween generally include the same thermoplastic material. The thickness of the platform layer (that is, excluding the thickness of the part produced by the replicated microstructure) is typically between 0.001 inch and 0.100 inch, and preferably between 0.003 inch and 0.010 inch. Suitable resin composition for melt extrusion is a transparent material, which is dimensionally stable, durable, weather-resistant and can be easily shaped into a desired structure. 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) EI Dupont de Nemours and Co., Inc.; (poly)ethylene-co-acrylic acid; polyesters; polyurethanes; and cellulose acetate butyrate.

[0113] In another embodiment, a master negative microstructured molding surface (eg, a tool) can be used as an embossing tool, such as described in US Pat. No. 4,601,861 (Pricone).

[0114] Further details regarding microstructured substrates having such louvered 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.

[0115] Prismatic structure

[0116] 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 to the first surface. In some embodiments, the prisms are right prisms. A right prism means that the top angle θ, 340, is typically about 90°. However, the angle can be in the range of 5° to 90°, and can be in the range of 20° to 80°. In selected embodiments, the microstructure includes linear prisms having a top angle less than or equal to 90 degrees. In some embodiments, the top angle of the peak structure is typically twice the wall angle, particularly 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, 30, 35, 40, 45, 50, 55, or 60 degrees. The apex angle of the peak structure is typically less than 90 degrees, more typically less than 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, or 35 degrees. Optionally, there is a gap between adjacent peak structures 320, for example at or parallel to the first planar surface 331.

[0117] 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 the tops of these shapes is lower than that of truncated (e.g., flat) shapes. Preferably, the radius of the prism top is less than the radius of the particle (e.g., red blood cell). The space between the peaks (e.g., prisms) can be characterized as a spacing ("P"). In this embodiment, the spacing is also equal to the maximum width of the valley. Therefore, as previously described, the spacing is greater than 1 micron, 2 microns, 3 microns, 4 microns, 5 microns, 6 microns, 7 microns, 8 microns, 9 microns, or 10 microns, ranging up to 500 microns. The length ("L") of the prismatic microstructure is typically the largest dimension and can span the entire size of the microstructured surface, film, or article. The prism facets do not need to be identical, and the prisms can be tilted relative to each other. The facets of adjacent peak structures are typically connected 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 In FIG. 5 , facets 321 and 322 of the prismatic peak structures are continuous in the direction of the length (L) of the microstructure, or in other words, in the y-direction.

[0118] Optionally, a continuous platform layer 360 may be present between the bottom of the channel or valley and the top surface 331 of the (e.g., planar) base member 310. 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, ranging up to 50 microns. In some embodiments, the thickness of the platform layer is no greater than 45 microns, 40 microns, 35 microns, 30 microns, 25 microns, 20 microns, 15 microns, or 10 microns.

[0119] Reference now Figure 5B , provides an SEM image of a cross section of a portion of an exemplary device of Example 8 having prismatic microstructures. 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 microstructure 320 located between the plurality of microstructures 320. (For simplicity, the arrow for 370 only points to a portion of the first open volume located between two adjacent microstructures 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 the sum of the combined first and second open volumes is taken as 100% open volume, and the percentage of the first open volume in 100% open volume is greater than the volume percentage of red blood cells present in the blood.

[0120] In selected embodiments, the microstructures include an array of peak structures and adjacent valleys, wherein the valleys have a maximum width ranging from 10 microns to 500 microns, and the peak structures have a top angle greater than 5 degrees and up to 90 degrees.

[0121] 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 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 or more, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 degrees or more; and 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, or 15 degrees or less. For example, in Figure 5B In the device 300 of FIG. 3 , the peak structures 320 and the valleys 301 are oriented at an angle of 0 degrees relative to the flow direction of the device 300 (eg, in the same direction as the flow). 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 including the microstructured substrate 325. Figure 5D On a microstructured substrate 325 of FIG. 3 , the peak structures 320 and valleys 301 are oriented at an angle of 45 degrees relative to the flow direction (“F”) of a device that will include the microstructured substrate 325. By selecting an orientation greater than 0 degrees (e.g., up to 90 degrees), particles have a greater chance of contacting more than one microstructure in the direction of fluid flow into the device and are potentially more likely to be trapped within a microstructure.

[0122] Further details regarding microstructured substrates having such arrays of peak structures and how to form such microstructured substrates are described in US2021 / 0187819 (Connell et al.), which is incorporated herein by reference.

[0123] Small face structure

[0124] Similar to the prismatic structure, Figure 5E An alternative microstructured substrate 700 having a faceted structure is shown. More specifically, Figure 5E A microstructured substrate 700 is shown defining a bottom surface 705, a top surface 720, a first sidewall 732, and a facet 733. Stated another way, the microstructure includes a facet 733 and a sidewall 732 that meets the facet 733 at a ridge 720 of the microstructure. The facet 733 and the sidewall 732 typically define an oblique angle therebetween.

[0125] In selected embodiments, the microstructure includes a facet and a sidewall that meets the facet at a ridge of the microstructure, and wherein the facet and the sidewall define a slope angle therebetween.

[0126] 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.

[0127] Protrusion array structure

[0128] Fig. 6A Another alternative microstructured substrate 400 having a protrusion array structure is shown. More specifically, Fig. 6A 4 is a schematic side view of a microstructured substrate 400 having a two-dimensional (x-axis and y-axis) array of protrusions 410 arranged across a first surface 420. Each protrusion 410 includes a base 412, a top 414, and one or more sides 416, 418 connecting the top to the base. Optionally, each protrusion 410 is a spaced-apart column. For example, Figure 6B4 are top plan views of four representative engineered micropatterned regions of a two-dimensional array of protrusions, including spaced-apart pillars 410 present in all images except the lower right image. Some microstructured surfaces may include protrusions having a range of aspect ratio values, such as an array of protrusions having a constant height and a variable width. In this case, the surface is typically characterized by the largest aspect ratio value.

[0129] In selected embodiments, the microstructures include 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.

[0130] Further details regarding microstructured substrates having such protrusion arrays and how to form such microstructured substrates are described in WO 2020 / 097319 (Wolk et al.), which is incorporated herein by reference.

[0131] Cavity array structure

[0132] Fig. 7A Another alternative microstructured substrate 500a having a cavity array structure is shown. A "cavity array" is a microstructured substrate having at least about 100 / cm 2 , preferably at least about 10 / mm 2 The cavity array has a density of discrete cavities. The cavity has a three-dimensional structure with dimensions such as an opening diameter in the range of about 5 microns to 250 microns and a depth in the range of about 2 microns to 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, Fig. 7A 5 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 forms a sidewall angle θ with a line 515 perpendicular to the first major surface 514 of the microstructured layer 510. Each cavity 522 also includes a depth "D", which is the vertical distance between the first hole 524 and the second hole 528. Optionally, the microstructured substrate 500a further includes any one of the adhesive layer 540, the first substrate 530, or the second substrate layer 550.

[0133] Figure 7B5 is a 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 a 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. The cross-section optionally decreases in size 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.

[0134] In selected embodiments, the microstructured substrate comprises a microstructured layer having a first major surface and a second major surface, wherein the microstructure comprises 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.

[0135] Further details regarding microstructured substrates having such arrays of cavities and how to form such microstructured substrates are described in US Pat. No. 9,329,311 (Halverson et al.), which is incorporated herein by reference.

[0136] Connected well structure

[0137] Fig. 10A -B shows yet another alternative microstructured substrate 1025, wherein the microstructures include an array of fluidically connected wells, wherein at least some of the wells are fluidically connected to at least two adjacent wells, each connection being via a vent. For example, Fig. 10A is a schematic perspective view of a portion of a microstructured substrate 1025 having an array of fluidically connected wells 1077 connected to each other by vents 1087.

[0138] Fig. 10B for Fig. 10A Schematic top view of a portion of a microstructured substrate 1025 having an array of fluidically connected wells 1077 having a circular shape connected to adjacent wells 1077 through vents 1087. Fig. 10AIn the particular configuration shown in FIG-B, a well 1077 in the center of the microstructured substrate 1025 (e.g., having at least one other well between the well and the perimeter of the microstructured substrate) is each connected to four other wells 1077, each connection being via a vent 1087. There are also wells 1077 located adjacent to the perimeter 1095 that are connected to only one or two adjacent wells 1077 via vents 1087. For example, referring to FIG. Fig. 10A , well 1077c is located at a corner of the array and is attached to only one other well 1077d via exhaust port 1087a. Similarly, the well may be attached to three other adjacent wells via exhaust ports, to four, five, six, seven, eight, nine, ten, eleven or twelve other adjacent wells via exhaust ports.

[0139] The shape of the well is not particularly limited and may include a curved shape, a polygonal shape, an irregular shape, or a combination thereof. In some embodiments, the well includes a circle, a triangle, a quadrilateral, an ellipse, or a combination thereof. In the case where the well includes a shape with corners, the vent is optionally located at the corner (e.g., to reduce the possibility of trapping bubbles in the corner).

[0140] Typically, each well has an open volume large enough to accommodate at least one sedimenting particle (e.g., red blood cells), such as 100 femtoliters or more, 250 femtoliters, 500 femtoliters, 750 femtoliters, 1 picoliter, 100 picoliters, 250 picoliters, 500 picoliters, 750 picoliters, 1 nanoliter, 100 nanoliters, 200 nanoliters, 300 nanoliters, 400 nanoliters, 500 nanoliters , 600 nanoliters, 700 nanoliters, 800 nanoliters, or 900 nanoliters or more; and 1 microliter or less, 900 nanoliters, 800 nanoliters, 700 nanoliters, 600 nanoliters, 500 nanoliters, 400 nanoliters, 300 nanoliters, 200 nanoliters, 100 nanoliters, 1 nanoliter, 750 picoliters, 500 picoliters, 250 picoliters, 1 picoliter, 750 femtoliters, or 500 femtoliters or less. Stated in other ways, in some cases, each well has an open volume ranging from 100 femtoliters to 1 microliter or 500 femtoliters to 0.1 microliters.

[0141] Some typical dimensions for each well include a depth (i.e., the distance between the top surface 1027 of the microstructured substrate 2025 and the bottom surface of the well 1029, e.g., 50 μl or more, 75 μl, 100 μl, 125 μl, 150 μl, 175 μl, 200 μl, 225 μl, 250 μl, 275 μl, 300 μl, 325 μl, or 350 μl or more) of 50 μl or more. Fig. 10Aand 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 through the center point of the shape.

[0142] Preferably, at least some of the vents between the wells are located at the same depth as the bottom surface of the adjacent wells. This helps to encourage bubbles to leave the wells rather than being trapped near the bottom of the wells. In some embodiments, the vents have a total depth equal to the total depth of the adjacent wells, but this is not required. When using a tool to form a microstructured substrate, it is often more practical to have the vents have the same depth as the adjacent wells than to have the vents only connect the lower portions of the two wells.

[0143] Reference again Fig. 10A In some embodiments, the microstructured substrate 1025 further comprises at least one sidewall 1097 disposed along a perimeter 1095 of the first surface 1027 of the microstructured substrate 1025. The one or more sidewalls 1087 have a height ("H") extending from 50 microns to 250 microns beyond the top surfaces of the plurality of microstructures.

[0144] The well structure of the connection can be made using a multiphoton exposure system as described in U.S. Patent No. 8,605,256 (DeVoe et al.), to produce a patterned tool. Such a 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 (Indiana Wabash Carver (Carver)) and a resin (e.g., polypropylene resin) are used to make a pressed sample. The press plate is heated (e.g., heated to 170°C), and then the tool and resin are pressed together for several minutes (e.g., 5 minutes-10 minutes) with a high force (e.g., 1000 pounds of force). After cooling (e.g., until the press plate 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.

[0145] Further details regarding microstructured substrates having such connected wells and how to form such microstructured substrates are described in commonly owned application serial number 63 / 425,473 (docket number PA100766US01).

[0146] Pole network structure

[0147] Fig.11Another alternative microstructured substrate 1120 having a rod network structure is shown. In such an embodiment, the microstructure 1126 includes an array of upright rods 1126 extending across the first surface 1124 of the microstructured substrate 1120. The microstructures 1126 are generally upright rods of various shapes. "Generally upright" means that the rods protrude away from the first surface 1124 (e.g., in a planar direction). The rods 1126 can protrude upward from the surface 1124 at a generally right angle, or the rods 1126 can protrude at an angle away from the surface 1124. The rods can also be irregularly shaped so that they may not protrude at any one uniform angle.

[0148] The microstructured substrate 1120 includes a backing layer 1121 having a first surface 1124 having a series of generally upright rods 1126. The rods 1126 can be arranged in a regular or irregular array. Various patterns of rods can be used, such as hexagonal, diagonal, sinusoidal, etc. The rods 1126 can be at least partially composed of an elastic material. In some cases, the entire outer surface of the rods 1126 is an elastomeric material. Fig.11 In the embodiment of the present invention, backing layer 1121 is formed integrally with rod 1126. The combination of backing layer 1121 and rod 1126 is sometimes referred to as a rod net. Although the illustrated embodiment shows that rod 1126 is roughly cylindrical, the side of rod 1126 typically has a slight taper 1135 to facilitate removal from the mold. As shown, taper 1135 is inward from the base 1102 of rod 1126 to the top 1104. It can be clearly envisioned that the rod can be configured to have a taper from the base of the rod to the top outward. A variety of non-cylindrical shapes can also be used, such as truncated cones or pyramids, rectangles, hemispheres, squares, hexagons, octagons, glue drop shapes, etc.

[0149] The backing layer 1121 from which the rods 1126 extend directly is typically about 0.05 mm to about 0.5 mm (0.002 inches to 0.02 inches) thick. Therefore, an additional backing layer 1122 is optionally applied to the second surface 1125 to reinforce the backing layer 1121 and form a multi-layer base or backing construction. As used herein, a "backing" or "base" layer will be used to refer to a common backing or base construction. This construction can be a single layer or multiple layers (such as Figure 1 ), having one or more layers supporting a generally upright rod 1126, although typically at most one of these layers 1121 will be integrally formed with the rod 1126.

[0150] The rods typically have a height 1128 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 1130 between adjacent rods 1126 is roughly in the range of about 0.25 mm to about 2.5 mm, more typically in the range of about 0.4 mm to about 1.0 mm. The spacing gap creates a certain percentage of free volume, which is the volume in the rod network that is not occupied by the rods. The percentage of free volume is typically 60% to 98% of the rod network, more typically 85% to 95%. The rods 1126 have a maximum cross-sectional dimension 1129 of about 0.076 mm to about 0.76 mm. The rods 1126 are arranged on the backing at a density of at least 15.5 per square centimeter (100 per square inch), more typically at least 50 per square centimeter. The rod density is roughly at most about 1500 per square centimeter, more typically at most about 500 per square centimeter. The rod has an aspect ratio of at least 1.25, preferably at least 1.5, and most preferably at least 2.0. The aspect ratio refers to the ratio of the rod height to the largest cross-sectional dimension. For a rod with a circular cross-section, the largest cross-sectional dimension is the rod diameter.

[0151] Suitable elastomeric rod materials include elastomers such as anionic triblock copolymers, polyolefin-based thermoplastic elastomers, thermoplastic elastomers based on halogen-containing polyolefins, thermoplastic elastomers based on dynamically vulcanized elastomer-thermoplastic blends, thermoplastic polyetheresters or polyester-based elastomers, thermoplastic elastomers based on polyamides or polyimides, ionomer thermoplastic elastomers, hydrogenated block copolymers in thermoplastic elastomer interpenetrating polymer networks, thermoplastic elastomers polymerized by carbocations, 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, polyurethane, ethylene / propylene / diene terpolymer elastomers, chloroprene rubber, styrene-butadiene copolymers (random or block), styrene-isoprene copolymers (random or block), acrylonitrile-butadiene copolymers, mixtures thereof, and copolymers thereof. The block copolymers may be linear, radial or star-shaped configurations and may be diblock (AB) or triblock (ABA) copolymers or mixtures thereof. Blends of these elastomers with each other or with modified non-elastomers are also contemplated. Commercially available elastomers include block polymers (e.g., polystyrene materials with elastomeric segments), available from KRATON Polymers Company of Houston, Texas, under the name KRATON TM Purchased.

[0152] 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, opacifiers, and the like.

[0153] Suitable backing layer materials include thermoplastic polyurethanes, polyvinyl chlorides, polyamides, polyimides, polyolefins (e.g., polyethylene and polypropylene), polyesters (e.g., polyethylene terephthalate), polystyrenes, nylons, acetals, block polymers (e.g., polystyrene materials with elastomeric segments available from Kraton Polymers Company of Houston, Texas under the name KRATON TM The thermoplastic material may be a thermoplastic material such as ...

[0154] Further details on microstructured substrates having such a rod network array and how to form such a microstructured substrate are described in WO 2009 / 020811 (Tuman et al.), which is incorporated herein by reference.

[0155] In a third aspect, the present disclosure provides a method of separating solid particles from a fluid.

[0156] The method includes:

[0157] a) obtaining a device, the device comprising:

[0158] 1) a 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 allow capillary action;

[0159] 2) a cover disposed a selected distance from the top of the first surface of the microstructured substrate;

[0160] 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;

[0161] 4) a first aperture defined by at least one of the microstructured substrate or the cover; and

[0162] 5) a second aperture defined by at least one of the microstructured substrate or the cover;

[0163] wherein the first surface of the microstructured substrate together with at least one sidewall defines 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, wherein the cover together with the top of the first surface of the microstructured substrate and at least one sidewall defines a second open volume positioned adjacent to the first open volume, and the sum of the combined first and second open volumes is taken as 100% open volume, the percentage of the first open volume in 100% open volume being greater than the volume percentage of particles present in the fluid.

[0164] b) filling the device with a volume of fluid through the first aperture via capillary action;

[0165] c) waiting a time sufficient to allow at least a portion of the particles to settle within the first open volume of the plurality of microstructures; and

[0166] d) applying pressure to the device such that at least 10% of the fluid, at least some of the particles in the fluid having been retained within the first open volume of the plurality of microstructures, flows out of the device through the first aperture or the second aperture.

[0167] Reference Fig. 9 , the method includes obtaining a device 910 (where the device is as described directly above); filling the device with a volume of fluid via a first hole via capillary action 920; waiting a time sufficient for at least a portion of the particles to settle within a first open volume of a plurality of microstructures 930; and applying pressure to the device so that at least 10% of the initial volume of fluid flows out of the device through the first hole or the second hole, at least some of the particles in the initial volume of fluid having been retained within the first open volume of the plurality of microstructures 940. The characteristics, materials, structure, etc. of the device used in the method of the third aspect can be according to any embodiment of the device as described in detail above with respect to the first and second aspects. Similarly, the method of the third aspect can be according to any embodiment of the method of the first aspect described in detail above.

[0168] Exemplary embodiments

[0169] In a first embodiment, the present disclosure provides a method for separating red blood cells from blood. The method includes obtaining a device, the device including a microstructured substrate, the 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 allow capillary action; a cover, the cover being disposed a selected distance from a top of the first surface of the microstructured substrate; 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; a first hole, the first hole being defined by at least one of the microstructured substrate or the cover; a second hole, the second hole being defined by the microstructured substrate; At least one of a substrate or a cover is defined; the first surface of the microstructured substrate and at least one sidewall together define a first open volume, the first open volume is the sum of the open spaces from the bottom to the top of each microstructure located between the plurality of microstructures, wherein the cover together with the top of the first surface of the microstructured substrate and at least one sidewall defines a second open volume positioned 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 100% open volume is greater than the volume percentage of red blood cells present in the blood. The method also includes filling the device with a certain volume of blood through a first hole via capillary action, and waiting for a time sufficient for at least a portion of the red blood cells to settle in the first open volume of the plurality of microstructures. In addition, the method includes applying pressure to the device so that at least 10% of the initial volume of blood flows out of the device through the first hole or the second hole, and at least some of the red blood cells in the initial volume of blood have been retained in the first open volume of the plurality of microstructures.

[0170] In a second embodiment, the present disclosure provides a method according to the first embodiment, wherein at least one sidewall is part of a microstructured substrate.

[0171] In a third embodiment, the present disclosure provides a method according to the first or second embodiment, wherein at least 15%, at least 20%, or at least 30% of the blood flows out of the device when pressure is applied.

[0172] In a fourth embodiment, the present disclosure provides a method according to any one of the first to third embodiments, wherein the time is sufficient to allow 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.

[0173] In a fifth embodiment, the present disclosure provides a method according to any one of the first to fourth embodiments, wherein the microstructured substrate is a microstructured film.

[0174] In a sixth embodiment, the present disclosure provides a method according to any one of the first to fifth embodiments, wherein at least a portion of the outer surfaces of the plurality of microstructures comprises a surfactant, a surface treatment agent, a hydrophilic polymer, a flocculant, or any combination thereof.

[0175] In a seventh embodiment, the present disclosure provides a method according to any one of the first to sixth embodiments, wherein the flocculant is hydrophilic and non-hemolytic.

[0176] In an eighth embodiment, the present disclosure provides a method according to the sixth embodiment or the seventh embodiment, wherein the flocculant comprises a modified or unmodified amino polymer selected from the group consisting of polyethyleneimine, polylysine, polyaminoamide, polyallylamine, polyethyleneamine, polydimethylamine-epichlorohydrin-ethylenediamine, polydiallyldimethylammonium chloride, cationic polyacrylamide (CPAM), polyaminosiloxane, and a dendrimer formed from polyaminoamine (PAMAM) and polypropylamine.

[0177] In a ninth embodiment, the present disclosure provides a method according to any one of the sixth to eighth embodiments, wherein the flocculant comprises a modified or unmodified polyethyleneimine polymer.

[0178] In a tenth embodiment, the present disclosure provides a method according to any one of the sixth to ninth embodiments, wherein after filling the device with a volume of blood, at least a portion of the flocculant dissolves, disperses, or both dissolves and disperses in the blood.

[0179] In an eleventh embodiment, the present disclosure provides a method according to the tenth embodiment, wherein the flocculant is present in the volume of blood in an amount of 0.01 micrograms to 5000 micrograms per milliliter of blood.

[0180] In a twelfth embodiment, the present disclosure provides a method according to any one of the first to eleventh embodiments, wherein the pressure is a positive pressure.

[0181] In a thirteenth embodiment, the present disclosure provides a method according to any one of the first to twelfth embodiments, wherein the pressure is a negative pressure.

[0182] In a fourteenth embodiment, the present disclosure provides a method according to any one of the first to thirteenth embodiments, wherein the first hole is defined by a cover.

[0183] In a fifteenth embodiment, the present disclosure provides a method according to any one of the first to fourteenth embodiments, wherein the second aperture is defined by a microstructured substrate or cover.

[0184] In a sixteenth embodiment, the present disclosure provides a method according to any one of the first to fifteenth embodiments, wherein the blood is undiluted.

[0185] In a seventeenth embodiment, the present disclosure provides a method according to any one of the first to sixteenth embodiments, wherein the red blood cells settle due to gravity alone.

[0186] In an eighteenth embodiment, the present disclosure provides a method according to any one of the first to seventeenth embodiments, wherein the device further comprises an adhesive layer disposed between the cover and the microstructured substrate.

[0187] In a nineteenth embodiment, the present disclosure provides a method according to any one of the first to eighteenth embodiments, wherein the microstructure comprises a plurality of ribs alternating with channels extending across the first surface of the microstructured substrate, and wherein each of the ribs comprises a sidewall and a top surface, and each channel comprises a bottom surface.

[0188] In a twentieth embodiment, the present disclosure provides a method according to the nineteenth embodiment, wherein a top surface of each rib is a top of a cap disposed on the side wall, and a width of the cap is greater than a width between opposing side walls.

[0189] In a twenty-first embodiment, the present disclosure provides a method according to any one of the first to eighteenth embodiments, wherein the microstructure comprises an array of peak structures and adjacent valleys, wherein the valleys have a maximum width ranging from 10 microns to 500 microns, and the peak structures have a top angle greater than 5 degrees and a maximum of 90 degrees.

[0190] In a twenty-second embodiment, the present disclosure provides a method according to the twenty-first 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 and 90 degrees relative to the flow direction of the device.

[0191] In a twenty-third embodiment, the present disclosure provides a method according to the twenty-first embodiment or the twenty-second embodiment, further comprising gaps between adjacent peak structures.

[0192] In a twenty-fourth embodiment, the present disclosure provides a method according to any one of the first to eighteenth embodiments, wherein the microstructure comprises a two-dimensional (x-axis and y-axis) array of protrusions arranged across the first surface of the microstructured substrate; wherein each protrusion comprises a base, a top, and one or more sides connecting the top to the base.

[0193] In a twenty-fifth embodiment, the present disclosure provides a method according to any one of the first to eighteenth embodiments, wherein the microstructured substrate comprises a microstructured layer having a first major surface and a second major surface, wherein the microstructure comprises 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.

[0194] In a twenty-sixth embodiment, the present disclosure provides a method according to any one of the first to thirteenth embodiments, wherein the microstructure includes a facet and a sidewall that meets the facet at a ridge of the microstructure, and wherein the facet and the sidewall define an inclination angle therebetween.

[0195] In a twenty-seventh embodiment, the present disclosure provides a method according to any one of the first to eighteenth embodiments, wherein the microstructure comprises an array of fluidly connected wells, wherein at least some of the wells are fluidly connected to at least two adjacent wells, each connection being via a vent.

[0196] In a twenty-eighth embodiment, the present disclosure provides a method according to any one of the first to eighteenth embodiments, wherein the microstructures comprise an array of upstanding stems extending across the first surface of the microstructured substrate.

[0197] In a twenty-ninth embodiment, the present disclosure provides a method according to any one of the first to twenty-eighth embodiments, wherein the volume of blood filled through the first hole is at most 100 microliters of blood.

[0198] In a thirtieth embodiment, the present disclosure provides a method according to any one of the first to twenty-ninth embodiments, wherein the ratio of the first open volume to the second open volume is greater than 1:1.

[0199] In a thirty-first embodiment, the present disclosure provides a method according to any one of the first to thirtieth embodiments, further comprising passing the blood through a filter before entering the device, after leaving the device, or both before entering the device and after leaving the device, at least some red blood cells in the blood being retained in a first open volume of the plurality of microstructures.

[0200] In a thirty-second embodiment, the present disclosure provides a method according to any one of the first to thirty-first embodiments, further comprising adding a flocculant to the volume of blood before filling the device with the volume of blood.

[0201] In a thirty-third embodiment, the present disclosure provides a method according to the thirty-second embodiment, wherein the flocculant is added and present in an amount of 0.01 micrograms / mL to 5000 micrograms / mL of blood.

[0202] In a thirty-fourth embodiment, the present disclosure provides a device. The device includes a microstructured substrate including a plurality of microstructures extending across a first surface of the microstructured substrate. The microstructures cover at least 90% of the first surface of the microstructured substrate, and at least a portion of the outer surface of the plurality of microstructures is configured to allow capillary action. The device also includes a cover disposed at a selected distance from the top of the first surface of the microstructured substrate and at least one sidewall that attaches the cover to the first surface of the microstructured substrate along the periphery of the first surface of the microstructured substrate. In addition, the device includes a first hole defined by at least one of the microstructured substrate or the cover and a second hole defined by at least one of the microstructured substrate or the cover. The first surface of the microstructured substrate defines a first open volume together with at least one sidewall, 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, wherein the cover defines a second open volume positioned adjacent to the first open volume together with the top of the first surface of the microstructured substrate and at least one sidewall.

[0203] In the thirty-fifth embodiment, the present disclosure provides a method for separating solid particles from a fluid. The method includes obtaining a device including a microstructured substrate, the microstructured substrate including a plurality of microstructures extending across a first surface of the microstructured substrate. At least a portion of the outer surface of the plurality of microstructures is configured to allow capillary action. The device also includes a cover disposed at a selected distance from the top of the first surface of the microstructured substrate and at least one side wall that attaches the cover to the first surface of the microstructured substrate along the periphery of the first surface of the microstructured substrate. In addition, the device includes a first hole defined by at least one of the microstructured substrate or the cover and a second hole defined by at least one of the microstructured substrate or the cover. The first surface of the microstructured substrate defines a first open volume together with at least one side wall, 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, wherein the cover defines a second open volume positioned adjacent to the first open volume together with the top of the first surface of the microstructured substrate and at least one side wall. 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 100% open volume is greater than the volume percentage of particles present in the fluid. The method also includes filling the device with a certain volume of fluid through the first hole via capillary action, and waiting a time sufficient for at least a portion of the particles to settle within the first open volume of the plurality of microstructures. In addition, the method includes applying pressure to the device so that at least 10% of the fluid flows out of the device through the first hole or the second hole, and at least some of the particles in the fluid have been retained in the first open volume of the plurality of microstructures.

[0204] Example

[0205] The following examples further illustrate the objects and advantages of the present disclosure, but the specific materials and amounts thereof listed in these examples, as well as other conditions and details, should not be construed as unduly limiting the present disclosure. Unless otherwise indicated or apparent from the context, all parts, percentages, ratios, etc. in the examples and the remainder of the specification are by weight. The Materials Table (below) lists the materials used in the examples and the sources of the materials.

[0206] Material

[0207]

[0208] Preparation Example 1-Example 4. Procedure for Preparing Prismatic Microstructured Films

[0209] 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 prism 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, Virginia) 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 bubbles were introduced, a rubber hand roller was used to remove the bubbles.

[0210] The samples were cured with UV light by passing them through a UV processor (Model QC120233AN with two Hg vapor lamps, obtained from RPC Industries, Plainfield, IL) twice at a rate of 15.2 m / min under a nitrogen atmosphere. Figure 5A The cured microstructured film of the array pattern shown was removed from the copper template. A release liner-backed adhesive layer (8 mil thick, obtained from 3M Company as 3M 8188 optically clear adhesive) was optionally applied to the back surface (i.e., non-microstructured surface) of the microstructured film using a hand roller. The characteristics of the prepared linear prism microstructured films are recorded in Table 1 as Preparation Examples 1-4.

[0211] The microstructured film was cut into 2.54 cm x 7.62 cm sections and a surfactant coating was applied by dipping each section into a 0.1 weight percent aqueous solution of IPEGAL-CO630 anionic surfactant and then immediately removing the film from the solution. The resulting surfactant coated microstructured film sections were air dried at ambient temperature and humidity.

[0212] Table 1 .

[0213] Microstructured films Peak height (μm) Pitch (micrometer) Vertex angle (degrees) Vertex Shape Preparation Example 1 50 27 30 sharp Preparation Example 2 50 58 60 sharp Preparation Example 3 50 100 90 sharp Preparation Example 4 50 173 120 sharp

[0214] Preparation Example 5. Procedure for Preparing Louvered Microstructured Films

[0215] A tool with multiple parallel linear grooves was cut using diamond (220 micrometers deep). The grooves were spaced at 60 micrometers apart. Resin A was prepared by mixing the materials in Table 2 below.

[0216] Table 2. Composition of Resin A used to prepare the microstructured film of Preparative Example 5

[0217]

[0218] The casting and curing microreplication process was performed using Resin A and the above tooling. Line conditions were 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)). Fusion D lamps with a peak wavelength of 385 nm (obtained from Fusion UV Systems, Gaithersburg, Maryland) were used for curing and operated at 100% power. Figure 4A As shown in Figure 1 (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 a negative replication of the grooves of the tool, and the channels of the microstructured film are a negative replication of the uncut portions between the grooves of the tool. The protrusions (ribs) of the microstructured film are evenly 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 causes the protrusions to be 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, Minnesota) having a thickness of 74.4 microns. The resin-contacting side of the PET film was primed with a thermosetting acrylic polymer (RHOPLEX 3208 polymer, available from Dow Chemical, Midland, Michigan).

[0219] The microstructured film was cut into 2.54 cm x 7.62 cm sections and a surfactant coating was applied by dipping each section into a 0.1 weight percent aqueous solution of IPEGAL-CO630 anionic surfactant and then immediately removing the film from the solution. The resulting surfactant coated microstructured film sections were air dried at ambient temperature and humidity.

[0220] Example 1. Device preparation

[0221] Device 800 (such as Fig. 8A and 8B830, obtained from 3M Company). The total thickness of the double-sided tape with the release liner removed was measured using a digital caliper to be 150 microns. A rectangular opening 880 (60 mm long x 2.5 mm wide) was laser cut into the second film component and oriented so that the narrow edge of the opening was located 11 mm from the narrow edge of the film and the long edge of the opening was located 8.75 mm from the long edge of the film.

[0222] The third film component of the apparatus (i.e., microstructured substrate 810) was a 72 mm long x 20 mm wide laser cut section of the surfactant coated microstructured film of Preparative Example 1. All laser cutting in the film was performed using a Muse Core CO2 laser cutter (Full Spectrum Laser, Las Vegas, Nevada).

[0223] The device 800 is constructed by removing any release liner from the films and then aligning the edges of the films into a stack with the second film sandwiched between the cover sheet film and the microstructured film. The films are oriented so that the microstructured surface 830 of the third film faces the second film and the major surface 822 of the cover. Adhesive lamination of the stack is accomplished by applying a 4 lb (1.8 kg) roller to the stack and performing a reciprocating motion of the roller. The second adhesive film forms a fluid seal between the cover sheet and the top surface of the microstructured film around the edges of the rectangular opening and the first hole (e.g., forming the sidewalls). In a final step, the stack is trimmed at the edges away from the first hole using a razor blade ( Figure 8B The dotted line 862 of the second film is shown in Figure 864, so that the resulting device has an overall size of 60 mm long by 20 mm wide. This cut through the opening in the second film exposes the second hole of the device, which is a 2.5 mm rectangular opening in the newly created edge of the device 800.

[0224] Example 2 .

[0225] The device was prepared according to the same procedure as reported in Example 1, except that 3M 1513 double-sided medical tape (transparent double-sided acrylic adhesive with polyester backing, obtained from 3M Company) was used to prepare the second film part of the device. The total thickness of the double-sided tape with the release liner removed was measured using a digital caliper and was 75 microns.

[0226] Example 3 .

[0227] The device was prepared according to the same procedure as reported in Example 1, except that the surfactant-coated microstructured film of Preparation Example 2 was used to prepare the third film part of the device.

[0228] Example 4 .

[0229] The device was prepared according to the same procedure as reported in Example 1, except that the surfactant-coated microstructured film of Preparation Example 3 was used to prepare the third film part of the device.

[0230] Comparative Example A .

[0231] The device was prepared according to the same procedure as reported in Example 1, except that the surfactant-coated microstructured film of Preparation Example 4 was used to prepare the third film part of the device.

[0232] Example 5 .

[0233] The device was prepared according to the same procedure as reported in Example 1, except that the surfactant-coated microstructured film of Preparation Example 5 was used to prepare the third film part of the device.

[0234] Example 6 .

[0235] The device was prepared according to the same procedure as reported in Example 1, except that the third film part of the device was prepared using the surfactant-coated microstructured film of Preparation Example 5, and the second film part of the device was prepared using 3M 1513 double-sided medical tape.

[0236] Table 3. Summary of device features and dimensions

[0237]

[0238] Comparative Example B .

[0239] The device was prepared according to the same procedure as reported in Example 1, except that a non-microstructured polyethylene terephthalate (PET) film (MELINEX 454 film (3 mil), Dupont Teijin Films) was used as the third film part of the device.

[0240] Comparative Example C .

[0241] The device was prepared according to the same procedure as reported in Example 1, except that the non-microstructured film of Comparative Example B (MELINEX 454 film) was used as the third film part to prepare the third film part of the device, and 3M 1513 double-sided medical tape was used to prepare the second film part of the device.

[0242] Example 7. Method for separating red blood cells from blood

[0243] Defibrinated sheep blood (obtained from Becton Dickinson, Franklin Lakes, NJ) had an undiluted hematocrit concentration of 40% measured using a Zip-IQ PCV centrifuge (LW Scientific Incorporated, Lawrenceville, Ga.) In addition to the undiluted blood samples, diluted blood samples of 12%, 8%, and 4% hematocrit were prepared using 1X phosphate buffered saline (PBS).

[0244] Prepare a module for delivering a blood sample to the device 800 (in Figure 8C and Fig.8D The module components included a silicone gasket 844 (25 mm x 25 mm x 3.2 mm) made from SYLGARD 184 silicone elastomer (Dow Chemical, Midland, Michigan) with a 5 mm hole cut in the center of the gasket, and a PLEXIGLAS polymethyl methacrylate (PMMA) film (Rohm GmbH, Darmstadt, Germany). GmbH)) was cut into a sheet 842 (25 mm x 25 mm x 3.2 mm) with a 0.06 inch (1.52 mm) diameter hole drilled in the center of the sheet. A length of ethyl vinyl acetate (EVA) plastic tubing 846 (0.02 inch (0.51 mm) inner diameter and 0.06 inch (1.52 mm) outer diameter, McMaster-Carr, Elmhurst, IL) was inserted into the hole in the PLEXIGLAS membrane sheet and secured in place with HARDMAN DOUBLE / BUBBLE epoxy (Royal Adhesives, Wilmington, CA) 847. The other end of the tubing was fitted with a 22.5 gauge needle attached to a 1 mL Luer-Lok syringe.

[0245] The device 800 (selected from the devices of Examples 1-6 and Comparative Examples A-C) is placed on a horizontal surface. A blood sample (20 microliters to 50 microliters) is added to the first hole of the device and the blood is allowed to wick to the end of the device by capillary flow. Next, a silicone gasket 844 is placed on the cover piece of the device and aligned so that the hole in the gasket is centered over the first hole of the device. A PLEXIGLAS sheet 842 (with an EVA plastic tube 846 adhered thereto as described above) is then placed on the exposed surface of the gasket and aligned so that the hole in the sheet is centered over the hole in the gasket, thereby forming a Fig.8DComponents. The syringe (not shown) attached to the other end of the EVA plastic tube is placed in a syringe pump (model NE-1600, New Era Pump Systems Inc, Farmingdale, New York) (not shown). The pump is started with a flow rate set to 100 microliters / minute. Blood samples flowing out of the device from the second hole 860 are collected. 10 microliters of collected blood samples are diluted with 90 microliters of 1X PBS. The diluted samples are pipetted into a C-CHIP disposable blood cell counter according to the manufacturer's instructions (Incyto, Republic of Korea). For comparison, the blood samples added to the device are also pipetted into a C-CHIP disposable blood cell counter. Counting slides are analyzed using a Zeiss LSM 510 element module Axioplan 2 upright confocal microscope (Zeiss, Jena, Germany). Red blood cells are counted manually or using Image J image processing software (National Institutes of Health, Bethesda, Maryland). The percent RBC reduction of a blood sample using the described method was calculated by comparing the RBC count of the blood sample collected from the device to 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 designated blood sample and the results expressed as % RBC reduction are reported as the mean in Table 4.

[0246] Equation 1:

[0247]

[0248] Table 4 .

[0249]

[0250] Example 8 .

[0251] The CAD design files were used to fabricate the device using the multiphoton exposure system described in U.S. Pat. No. 8,605,256 (DeVoe et al.) and U.S. Pat. No. 8,455,846 (Gates et al.). Figure 2BA microstructured substrate of the invention is a mold. A negative contrast photoresist such as described in US Pat. No. 10,133,174 (Lee et al.) is photopatterned on a silicon wafer substrate. When scanning is completed, 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 mold is then electroformed with nickel or nickel alloy to make a metal tool for replication. Nickel-plated tools are used to make a pressed sample with polypropylene resin (C700-35 resin, Dow Chemical, Midland, Michigan). The press plate of the Carver press (Carver, Indiana) is heated to 170°C, and the tool and resin are pressed together for 7 minutes under 1000 pounds of force (about 4450 Newtons), followed by cooling under pressure until the temperature of the press plate reaches 80°C. The pressure is released and the molded microstructured substrate is removed from the tool.

[0252] 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 prismatic microstructures positioned along the bottom surface of the flow channel (3 mm wide, 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 prismatic microstructures are reported in Table 5. The prismatic features protrude above the bottom surface of the flow channel. The wall surrounding the periphery of the flow channel extends 100 microns to the upper surface above the tip of the prismatic structure. The first open end of the flow channel is fluidically attached to a semicircular first cavity having a depth of 100 microns from the upper surface and a volume of 2.88 microliters. The first cavity forms the first hole of the device. The first hole is used as a liquid sample intake storage portion 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 microns deep from the upper surface). The second cavity forms a second aperture of the device. The second aperture serves as a receiving reservoir for the liquid sample exiting the flow channel.

[0253] The cover component of the device was a portion of 3M Microfluidic Diagnostic Film 9975R (obtained from 3M Company). The cover was placed over the flow channel and liquid sample intake reservoir section and adhesively attached to the surface of the microstructured substrate surrounding the sample intake reservoir and flow channel section. Prior to application, a circular hole (5 mm diameter) was laser cut into the cover component and oriented so that when the cover was attached to the microstructured substrate, the center of the hole was positioned over the center of the sample intake reservoir. The receiving reservoir was not covered.

[0254] Table 5 .

[0255] Microstructured surface Peak height (μm) Pitch (micrometer) Vertex angle (degrees) Vertex Shape Example 8 200 107.7 30 sharp

[0256] Example 9 .

[0257] The device was prepared according to the same procedure as described in Example 8, except that the linear array of peak structures and adjacent valleys were oriented at an angle of 45 degrees relative to the direction of liquid flow in the finished device (oriented at Figure 5D ); the dimensions of the flow channel are 3 mm wide and 40 mm long; the first hole cavity has a depth of 300 microns and a volume of 8.7 microliters; and the second hole cavity has dimensions of 5 mm (width), 6 mm (length), 300 microns (depth).

[0258] Example 10 .

[0259] The device was prepared according to the same procedure as described in Example 9, except that the linear array of peak structures and adjacent valleys were oriented at an angle of 90 degrees (perpendicular) relative to the direction of liquid flow in the finished device (oriented at Figure 5C ).

[0260] Embodiment 11 .

[0261] The device was prepared according to the same procedure as described in Example 8, except that the linear prismatic microstructure array was replaced by an array of circularly shaped fluidically connected wells connected to adjacent wells (e.g. Figures 10A-10B ). Each well has a diameter of 200 microns, a depth of 150 microns, and a draft angle of 5 degrees. The vents have 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 in the center of the flow channel are each connected to four other wells, each connection being via a vent and the vents are spaced as shown. Fig. 10B The wall surrounding the perimeter of the flow channel extends 100 microns above the top surface of the well. The dimensions of the flow channel are 3 mm wide and 40 mm long.

[0262] The first open end of the flow channel is fluidly attached to a semicircular first cavity having a depth of 100 microns from the upper surface and a volume of 2.88 microliters. The first cavity forms a first hole of the 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 microns deep from the upper surface). The second cavity forms a second hole of the device.

[0263] Example 12. Method for separating red blood cells from blood

[0264] Human blood was collected in BD VACUTAINER citrate tubes (Becton, Dickinson, Franklin Lakes, NJ) and used as citrated whole blood or diluted 1:1 with IX phosphate buffered saline (PBS).

[0265] The microstructured substrate described in Example 8-Example 11 is prepared, and the cover part modified with the tube for blood dripping is attached to each substrate. The modified cover part is a part of 3M microfluidic diagnostic film 9975R (obtained from 3M Company), which has a circular hole (5mm diameter) laser cut in the cover part. A plastic tube (0.05 inch ID, 0.09 inch OD) is inserted into the hole and fixed with an epoxy adhesive (3M SCOTCH-WELD epoxy adhesive DP100 Plus transparent, obtained from 3M Company). Remove any tube extending beyond the adhesive surface of the cover part with a razor blade. The cover is placed above the flow channel and liquid sample intake storage section of the microstructured substrate, and is adhesively attached to the surface of the microstructured substrate surrounding the two sections. The cover part is oriented so that when the cover is attached, the sample intake storage section located in the center of the hole is above the center. The receiving storage section is not covered. The tube extends about 3cm long from the outer surface of the cover.

[0266] Each obtained device is placed on a horizontal surface (oriented so that the lower surface of the device faces the horizontal surface). A blood sample (20 microliters-50 microliters) is added to the sample inlet storage portion through a tube using a micropipette. Allow the blood sample to wick to the end of the device by capillary flow or advance using the positive pressure from the micropipette. In the case of no excessive blood in the intake storage portion, a sufficient volume of blood is added to the device to fill the open volume of the flow channel. Any excess blood in the receiving storage portion is quickly removed from the storage portion using a micropipette or a KIMWIPE wiper (Kimberly-Clark Corporation, Irving, Texas). After applying the blood sample, each device is kept undisturbed for 5 minutes on a horizontal surface.

[0267] The 1mL Luer lock syringe filled with mineral oil will be attached to a section of plastic tubing, and mineral oil will be partially distributed in the tube, to leave a small air gap (about 10cm tube length) at the open end of the tube. The syringe is placed in a syringe pump (model NE-1600, New Era Pump Systems Inc). The open end of the tube of the syringe assembly is connected to the open end of the tube extending from the device. Start the pump with a flow rate set to 50 microliters / minute to force the blood sample to flow out from the flow channel with trapped air volume. The blood sample flowing out of the flow channel is collected as several 2 microliters-4 microliters of aliquots. Once the aliquot volume is accumulated in the receiving storage portion, each sample is collected.

[0268] For each sample collected, 1 / 10, 1 / 20, 1 / 100 and 1 / 200 dilution samples were prepared in 1X PBS. 2 microliters of aliquots of each diluted sample were loaded onto Agilent Take3 microvolume plates (TAKE3-SN, Agilent Technologies (Agilent Technologies), Santa Clara, California) with micropipette. The plate includes at least 1 well as 1X PBS blank, which is used for dilution according to manufacturer's instructions. For each sample and 1X PBS blank, the absorbance measurements at 406nm, 414nm and 576nm were recorded using Agilent Synergy Neo2 plate reader (Agilent Technologies (Agilent Technologies)). The program in chapters and sections "the method for measuring the complete red blood cell content of a sample" (described below) is used to calculate the percentage reduction of the red blood cells from the blood sample submitted to each device using the program described in the present embodiment.

[0269] The results using citrated whole human blood are provided in Table 6, and the results using citrated whole human blood diluted 1:1 in 1X PBS are presented in Table 7. The device of Comparative Example B (i.e., a device without a microstructured surface) was also tested. The results reported in Tables 6 and 7 are from technical replicates of three devices (n=3) for each type of device. The calculated value for the percent reduction in red blood cells for each replicate device is the average of the total volume of the aliquot sample collected from that device.

[0270] Table 6 .

[0271]

[0272] Table 7 .

[0273] Devices fabricated with microstructured substrates were derived from The percentage of RBC reduction Standard Deviation Example 8 42.1％ 0.3％ Example 9 8.4％ 16.5％ Example 10 10.6％ 23.9％ Embodiment 11 35.0％ 9.7％ Comparative Example B 4.7％ 10.9％

[0274] Method for determining the intact red blood cell content of a sample

[0275] In order to measure the complete red blood cell content and the lysed red blood cell content of the sample simultaneously, the calibration curve of the dilution series is prepared with the lysed and complete human red blood cells of known input. The human whole blood of citric acidification is used as the sample of 100% complete red blood cells. In order to produce 0% complete red blood cell sample, the whole blood of aliquots is dissolved by vortexing for 1 minute using ZR BashingBead dissolving tube (product number S6012-50, Enzyme Research Institute of Irvine, California (Zymo Research)). The tube is centrifuged for 1 minute at 10,000xg, and the supernatant containing only the lysed cell content is transferred to a new 1.5mL Eppendorf tube. By mixing the known complete and lysed cells (ranging from 100% to 0% complete red blood cells) of different ratios, the standard sample of complete to lysed red blood cells for dilution series is prepared. According to the manufacturer's instructions, the complete red blood cell concentration of the standard sample is confirmed using C-CHIP disposable blood cell counter.

[0276] Each standard sample was diluted in 1X PBS (so that the absorbance would be within the dynamic range of the plate reader) and analyzed in triplicate using an Agilent Synergy Neo2 plate reader with a Take 3 multivolume plate (sample volumes and blanks were selected according to the manufacturer's instructions). The absorbance of each standard sample was determined at 406 nm, 414 nm, and 576 nm.

[0277] Scale A 414nm / A 576nm Plotted relative to known input of % intact red blood cells. 414nm = absorbance of the sample at 414 nm and A 576nm = Absorbance of sample at 576 nm Subtraction of blank (1X PBS) was used for background subtraction. A logarithmic curve was fit to the data set to generate equation A. Equation A was used to calculate the percentage of intact red blood cells from a suspension containing blood.

[0278] Equation A:

[0279]

[0280] In equation A, "% intact" = the percentage of intact red blood cells in the suspension, "A 414nm = Absorbance of blood sample at 414 nm wavelength, “A PBS,414nm = Absorbance of 1X PBS at 414 nm, "A 576nm = absorbance of the blood sample at 576 nm, and PBS,576nm = absorbance of 1X PBS at 576 nm.

[0281] The absorbance signal at 406 nm for each sample was adjusted by the ratio of cell percentage to calculate the signal from intact erythrocytes using Equation B.

[0282] Equation B:

[0283]

[0284] In equation B, “Ai 406nm = absorbance at 406 nm of a blood sample attributable to intact red blood cells, "% intact" = percentage of intact red blood cells in the suspension calculated by equation A, "A 406nm = absorbance of the sample at 406 nm, and PBS,406nm = absorbance of 1X PBS at 406 nm.

[0285] 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 to the absorbance signal of the corresponding blood aliquot collected from the device to calculate the percent red blood cell (RBC) reduction according to Equation C.

[0286] Equation C:

[0287]

[0288] In equation C, “Ai 406nm，输入 = the absorbance at 406 nm of the blood sample added to the device attributable to intact red blood cells calculated by Equation B, and "Ai 406nm，输出 = absorbance at 406 nm of the blood sample collected from the device attributable to intact red blood cells calculated by Equation B.

[0289] Preparation Example 6. Microstructured Film with Upstanding Stems

[0290] A discrete rod structure with sloping side walls was prepared by a molding process according to Example 1 of U.S. Pat. No. 9,358,714 (Chandrasekaran) Fig.11A polypropylene (PP) microstructured film of the invention was prepared except that the beta nucleation master was not included and the film was then corona treated using a BD-20AC laboratory corona treater (Electro-Technic Products, Chicago, Illinois). A 3-dimensional micrograph of the microstructured film was taken using a Keyence VK-X3100 3D surface profiler (Keyence Corporation, Itasca, Illinois) and measured using the attached VK-X 3000 MultiFileAnalyzer software package. The microstructured film had a total thickness of about 345 microns and a staggered array of 2000 rod features per square inch. The rod features had a generally flat surface (i.e., a truncated cone shape) at the apex. The thickness of the backing layer was 83.5 microns. In Tables 8 and 9, the following dimensions are reported: rod height, rod diameter at the base in the longitudinal direction of the web, rod diameter at the base in the transverse direction of the web, rod diameter at the apex in the longitudinal direction of the web, rod diameter at the top in the transverse direction of the web, center-to-center distance (pitch) between rods in the longitudinal direction of the web, and center-to-center distance (pitch) between rods in the transverse direction of the web.

[0291] Table 8. Rod feature dimensions of the microstructured film of Preparation Example 6

[0292]

[0293] Table 9. Rod feature dimensions of the microstructured film of Preparation Example 6

[0294]

[0295] Example 13. Preparation of a device comprising a microstructured film having upstanding stems

[0296] The following modifications were made according to the method described in Example 1. First, the microstructured film of Preparative Example 6 was used instead of the microstructured film of Preparative Example 1 as the third film component of the device. Second, epoxy resin was used to seal the two long edges of the device and the narrow edge near the first hole. The narrow edge forming the second hole was not sealed.

[0297] Example 14. Device comprising a microstructured film having upstanding stems and a flocculant coating of a microstructured array preparation

[0298] A flocculant solution of polyethyleneimine (PEI) (branched, MW 70,000Da, 30% weight / volume aqueous solution, catalog number 00618, obtained from Polysciences, Inc., Warrington, Pennsylvania) was further diluted with 1:10 (weight:volume) deionized water, followed by 1:10 volume:volume deionized water in two steps. The diluted flocculant solution (30 microliters-100 microliters) was added to the rectangular opening 880 of the device of Example 13 with a pipette. The applied PEI flocculant solution was air dried overnight under ambient conditions to provide a flocculant-coated microstructured array.

[0299] Example 15. Device comprising a microstructured film having upstanding stems and a flocculant coating of a microstructured array preparation

[0300] An aqueous flocculant solution of guanylated polyethyleneimine (G-PEI) was prepared as described in Example 1 of U.S. Pat. No. 10,087,405 (Swanson et al.) without the addition of butanediol diglycidyl ether. The flocculant solution (30 μL-100 μL) was added to the rectangular opening 880 of the device of Example 13 using a pipette. The applied G-PEI flocculant solution was air dried overnight under ambient conditions to provide a flocculant-coated microstructured array.

[0301] Example 16. Method for separating red blood cells from blood

[0302] Whole human blood collected in EDTA tubes was obtained from the Oklahoma Blood Institute, Oklahoma City, Okla. Initial hematocrit of human blood samples was measured using a Zip-IQ PCV centrifuge (LW Scientific Incorporated, Lawrenceville, GA).

[0303] In this method, a module with a syringe pump for delivering a blood sample described in Example 7 is used. The device selected from Example 13-Example 15 is placed on a horizontal surface. A whole human blood sample (50 microliters-100 microliters) is added to the first hole 850 of the device. Allow the blood to wick to the end of the device by capillary flow, and keep the device undisturbed for 10 minutes to allow red blood cells to settle in the microstructured array section. Then, start the pump with a flow rate set to 100 microliters / minute. Collect the blood sample that flows out of the device from the second hole 860 as a series of 5 microliter aliquots.

[0304] Prepare standard calibration curve from the dilution series of whole blood in water to associate RBC count with the absorbance at 406nm.Dilute each blood sample tested in water (to dissolve cells and make absorbance within the dynamic range of plate reader) and use Agilent Synergy Neo2 plate reader with Take 3 multi-volume plates to carry out technical analysis in duplicate (select sample volume and blank according to manufacturer's instructions).Deionized water with a resistivity greater than 18 megohm-cm prepared by SYNERGY ultraviolet water purification system (MilliporeSigma, Burlington, Massachusetts) is used to prepare all dilutions.Measure the absorbance of each sample at 406nm.According to equation D, the red blood cell (RBC) reduction percentage of blood sample is calculated by comparing the absorbance of the blood aliquot collected from the device with the absorbance of the whole blood sample added to the device.

[0305] Equation D:

[0306]

[0307] In equation D, "absorbance 406nm，输出 = Absorbance at 406 nm of the blood sample collected from the device. 406nm，输入 = Absorbance at 406 nm of the blood sample added to the device. The results are reported in Table 10 for each type of device. A single device of each type was tested. The calculated value for percent red blood cell reduction was the average of all aliquot samples collected from the device. Approximately 10 aliquot samples were collected from each device.

[0308] Table 10 .

[0309] Device The percentage of RBC reduction Example 13 20.9％ Embodiment 14 84.4％ Embodiment 15 79.6％

[0310] Example 17. Method for separating red blood cells from blood

[0311] The method of Example 16 was performed using a single device (n=1) of Example 13 and a blood sample containing a PEI flocculant. For the blood sample, a flocculant solution of polyethyleneimine (PEI) (branched, MW 70,000Da, 30% weight / volume aqueous solution, catalog number 00618, obtained from Polysciences, Inc.) was prepared by diluting 1 / 10 in PBS (e.g., 1 mL flocculant + 9 mL 1X PBS). An aliquot of 90 microliters of whole human blood was mixed with 10 microliters of flocculant solution in a tube. The resulting blood sample was mixed using a pipette (3-5 times) and then incubated at room temperature for 10 minutes. The incubated blood sample was mixed with a pipette (3-5 times), and then approximately 100 microliters of the sample was added to the first well of the device. A total of seven aliquots were collected from the device. The calculated RBC reduction percentage was 64.4%. The calculated value of the red blood cell reduction percentage is the average of all aliquot samples collected from the device.

[0312] All patents and patent applications mentioned above are expressly incorporated herein by reference. The above embodiments are illustrative of the present invention, and other configurations are also possible. Therefore, the present invention should not be considered to be limited to the embodiments described in detail above and shown in the accompanying drawings, but only limited by the reasonable scope of the attached 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 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 allow capillary action; 2) a cover disposed a selected distance from the top of the first surface of the microstructured substrate; 3) at least one sidewall attaching the cover to the first surface of the microstructured substrate along a perimeter of the first surface of the microstructured substrate; 4) a first aperture defined by at least one of the microstructured substrate or the cover; and 5) a second aperture defined by at least one of the microstructured substrate or the cover; wherein the first surface of the microstructured substrate together with the at least one sidewall defines 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, 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 the sum of the combined first and second open volumes is taken as 100% open volume, the percentage of the first open volume in the 100% open volume being greater than the 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 aperture; c) waiting a time sufficient to allow at least a portion of the red blood cells to settle within the first open volumes of the plurality of microstructures; and d) applying pressure to the device so that at least 10% of an initial volume of blood, at least some red blood cells of which have been retained within the first open volume of the plurality of microstructures, flows out of the device through the first or second apertures.

2. The method according to claim 1, wherein: The at least one sidewall is part of the microstructured substrate.

3. The method according to claim 1 or claim 2, wherein: At least 15%, at least 20%, or at least 30% of the blood is caused to flow out of the device upon application of pressure.

4. The method according to any one of claims 1 to 3, wherein: The time is sufficient to allow 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.

5. The method according to any one of claims 1 to 4, wherein: The microstructured substrate is a microstructured film.

6. The method according to any one of claims 1 to 5, wherein: At least a portion of the outer surfaces of the plurality of microstructures comprises a surfactant, a surface treatment agent, a hydrophilic polymer, a flocculant, or any combination thereof.

7. The method according to claim 6, wherein: The flocculant is hydrophilic and non-hemolytic.

8. The method according to claim 6 or claim 7, wherein: The flocculant comprises a modified or unmodified amino polymer, wherein the amino polymer is selected from polyethyleneimine, polylysine, polyaminoamide, polyallylamine, polyvinylamine, polydimethylamine-epichlorohydrin-ethylenediamine, polydiallyldimethylammonium chloride, cationic polyacrylamide (CPAM), polyaminosiloxane, and a dendrimer formed by polyaminoamine (PAMAM) and polypropylamine.

9. The method according to any one of claims 6 to 8, wherein: The flocculant comprises a modified or unmodified polyethyleneimine polymer.

10. The method according to any one of claims 6 to 9, wherein: After filling the device with the volume of blood, at least a portion of the flocculant dissolves, disperses, or both dissolves and disperses in the blood.

11. The method according to claim 10, wherein: The flocculant is present in the volume of blood in an amount of 0.01 micrograms to 5000 micrograms per milliliter of blood.

12. The method according to any one of claims 1 to 11, wherein: The pressure is a positive pressure.

13. The method according to any one of claims 1 to 11, wherein: The pressure is negative pressure.

14. The method according to any one of claims 1 to 13, wherein: The first aperture is defined by the cover.

15. The method according to any one of claims 1 to 14, wherein: The second aperture is defined by the microstructured substrate and the cover.

16. The method according to any one of claims 1 to 15, wherein: The blood was undiluted.

17. The method according to any one of claims 1 to 16, wherein: The red blood cells settle simply due to gravity.

18. The method according to any one of claims 1 to 17, wherein: The device also includes an adhesive layer disposed between the cover and the microstructured substrate.

19. The method according to any one of claims 1 to 18, wherein: The microstructures include a plurality of ribs alternating with channels extending across the first surface of the microstructured substrate, and wherein each of the ribs includes a sidewall and a top surface, and each of the channels includes a bottom surface.

20. The method according to claim 19, wherein: The top surface of each rib is the top of a top cover provided on the side wall, and the width of the top cover is greater than the width between the opposing side walls.

21. The method according to any one of claims 1 to 18, wherein: The microstructure includes an array of peak structures and adjacent valleys, wherein the valleys have a maximum width ranging from 10 microns to 500 microns, and the peak structures have a top angle greater than 5 degrees and a maximum of 90 degrees.

22. The method according to claim 21, 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 and 90 degrees relative to a flow direction of the device.

23. The method of claim 11 or claim 22, wherein: The array of peak structures and adjacent valleys also includes gaps between adjacent peak structures.

24. The method according to any one of claims 1 to 18, wherein: The microstructure comprises a two-dimensional (x-axis and y-axis) array of protrusions arranged across the first surface of the microstructured substrate; wherein each of the protrusions comprises a base, a top, and one or more sides connecting the top to the base.

25. The method according to any one of claims 1 to 18, wherein: The microstructured substrate comprises a microstructured layer having a first major surface and a second major surface, wherein the microstructure comprises a plurality of cavities extending between the first major surface and the second major surface; Each cavity includes a first opening, a second opening, and at least one side wall extending between the first opening and the second opening.

26. The method according to any one of claims 1 to 18, wherein: The microstructure includes a facet and a sidewall that meets the facet at a ridge of the microstructure, and wherein the facet and the sidewall define a tilt angle therebetween.

27. The method according to any one of claims 1 to 18, wherein: The microstructure includes an array of fluidly connected wells, wherein at least some of the wells are fluidly connected to at least two adjacent wells, each connection being via a vent.

28. The method according to any one of claims 1 to 18, wherein: The microstructures include an array of upstanding stems extending across the first surface of the microstructured substrate.

29. The method according to any one of claims 1 to 28, wherein: The volume of the blood filled through the first hole is at most 100 microliters of blood.

30. The method according to any one of claims 1 to 29, wherein: A ratio of the first open volume to the second open volume is greater than 1:

1.

31. The method of any one of claims 1 to 30, further comprising passing the blood through a filter before entering the device, after leaving the device, or both before entering the device and after leaving the device, wherein at least some red blood cells in the blood are retained within the first open volume of the plurality of microstructures.

32. The method of any one of claims 1 to 31, further comprising adding a flocculant to the volume of blood prior to filling the device with the volume of blood.

33. The method of claim 32, wherein: The flocculant is added and present in an amount of 0.01 micrograms / mL to 5000 micrograms / mL of blood.

34. An apparatus, comprising: 1) a 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 an outer surface of the plurality of microstructures is configured to allow capillary action; 2) a cover disposed a selected distance from the top of the first surface of the microstructured substrate; 3) at least one sidewall attaching the cover to the first surface of the microstructured substrate along a perimeter of the first surface of the microstructured substrate; 4) a first aperture defined by at least one of the microstructured substrate or the cover; and 5) a second aperture defined by at least one of the microstructured substrate or the cover; wherein the first surface of the microstructured substrate together with the at least one sidewall defines 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, 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.

35. A method for separating solid particles from a fluid, the method comprising: a) obtaining a device, the device comprising: 1) a 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 allow capillary action; 2) a cover disposed a selected distance from the top of the first surface of the microstructured substrate; 3) at least one sidewall attaching the cover to the first surface of the microstructured substrate along a perimeter of the first surface of the microstructured substrate; 4) a first aperture defined by at least one of the microstructured substrate or the cover; and 5) a second aperture defined by at least one of the microstructured substrate or the cover; wherein the first surface of the microstructured substrate together with the at least one sidewall defines a first open volume, the first open volume being the sum of the open spaces between the plurality of microstructures from the bottom to the top of each microstructure, 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, and the sum of the combined first and second open volumes is taken as 100% open volume, the percentage of the first open volume in the 100% open volume being greater than the volume percentage of particles present in the fluid; b) filling the device with a volume of fluid through the first aperture via capillary action; c) waiting a time sufficient to allow at least a portion of the particles to settle within the first open volumes of the plurality of microstructures; and d) applying pressure to the device so that at least 10% of the fluid, at least some of the particles in the fluid having been retained within the first open volumes of the plurality of microstructures, flows out of the device through the first or second apertures.

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