Method and device for removing particles from a fluid

Abstract

The present disclosure provides a method and a device for removing particles from a fluid. The method for removing red blood cells includes obtaining a device having a microstructured substrate including a microstructure extending across a first surface, at least a portion of an outer surface of the microstructure being configured to enable capillary action. The device also includes a cover disposed at a selected distance away from a top of the first surface of the microstructured substrate and at least one sidewall portion for 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 a first aperture by capillary action, waiting for a time sufficient for at least a portion of the red blood cells to settle within a first open volume of the microstructure, and applying pressure to the device, thereby causing a portion of an initial volume of the blood in which at least a portion of the red blood cells are retained within the first open volume of the microstructure to flow out of the device.

Description

Technical Field

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

Background Art

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

[0003] Point-of-care blood analyzers used outside of central laboratories also require the removal of red blood cells prior to analysis. In situations where rapid results are needed, access to venous blood volume and benchtop centrifugation are often not available or are too time-consuming. With finger prick, a blood volume of approximately 5 microliters is collected. Glucose test strips typically accept a blood volume of less than 1 microliter and are subject to the above-mentioned interference. 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 having a plurality of microstructures extending across a first surface of the microstructured substrate. At least a portion of the outer surface of the plurality of microstructures is configured to enable capillary action. The device also includes a cover disposed at a selected distance from the top of the first surface of the microstructured substrate and at least one sidewall portion for attaching the cover to the first surface of the microstructured substrate along the perimeter of the first surface of the microstructured substrate. Further, the device includes a first aperture defined by at least one of the microstructured substrate or the cover and a second aperture defined by at least one of the microstructured substrate or the cover. The first surface of the microstructured substrate, together with at least one sidewall portion, defines a first open volume that is the sum of the open spaces located between the plurality of microstructures from the bottom to the top of each microstructure. The cover, together with the top of the first surface of the microstructured substrate and at least one sidewall portion, defines a second open volume located adjacent to the first open volume. If the sum of the first open volume and the second open volume is defined as 100% open volume, the first open volume has a percentage greater than the volume percentage of red blood cells present in the blood out of the 100% open volume. The method further includes filling the device with a volume of blood through the first aperture by capillary action and waiting for a time sufficient for at least a portion of the red blood cells to settle within the first open volume of the plurality of microstructures. Additionally, the method includes applying pressure to the device, thereby causing at least 10% of the initial volume of the blood in which at least a portion of the red blood cells are retained within the first open volume of the plurality of microstructures to flow out of the device through either the first aperture or the second aperture.

[0005] In a second aspect, a device is provided. The device includes a microstructured substrate having a plurality of microstructures extending across a first surface of the microstructured substrate. The microstructures cover at least 90% of the first surface of the microstructured substrate, and at least a portion of the outer surfaces of the plurality of microstructures is configured to enable 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 portion for attaching the cover to the first surface of the microstructured substrate along the perimeter of the first surface of the microstructured substrate. Further, the device includes a first aperture defined by at least one of the microstructured substrate or the cover, and a second aperture defined by at least one of the microstructured substrate or the cover. The first surface of the microstructured substrate, together with at least one sidewall portion, defines a first open volume that is the sum of the open spaces located between the plurality of microstructures from the bottom to the top of each microstructure, and the cover, together with the top of the first surface of the microstructured substrate and at least one sidewall portion, defines a second open volume located adjacent to the first open volume.

[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 having a plurality of microstructures extending across a first surface of the microstructured substrate. At least a portion of the outer surfaces of the plurality of microstructures is configured to enable 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 portion for attaching the cover to the first surface of the microstructured substrate along the periphery of the first surface of the microstructured substrate. Further, the device includes a first aperture defined by at least one of the microstructured substrate or the cover and a second aperture defined by at least one of the microstructured substrate or the cover. The first surface of the microstructured substrate, together with at least one sidewall portion, defines a first open volume that is the sum of the open spaces located between the plurality of microstructures from the bottom to the top of each microstructure. The cover, together with the top of the first surface of the microstructured substrate and at least one sidewall portion, defines a second open volume located adjacent to the first open volume. When the sum of the first open volume and the second open volume is defined as 100% open volume, the first open volume has a percentage greater than the volume percentage of the particles present in the fluid out of the 100% open volume. The method further includes filling the device with a volume of fluid through the first aperture by capillary action and waiting for a time sufficient for at least a portion of the particles to settle within the first open volume of the plurality of microstructures. Additionally, the method includes applying pressure to the device, thereby causing at least 10% of the fluid in which at least a portion of the particles is retained within the first open volume of the plurality of microstructures to flow out of the device through either the first aperture or the second aperture.

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

[0008] The above summary of the disclosure is not intended to describe every embodiment or all implementations disclosed in the disclosure. The following description illustrates exemplary embodiments in more detail. Throughout several places in this application, guidance is provided through lists of examples, and these examples can be used in various combinations. In each instance, the listed lists function only as representative groups and should not be construed as exclusive lists.

Brief Description of the Drawings

[0009]

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[0010] The figures identified above depict some embodiments of the present disclosure, but as noted in the description, other embodiments are also contemplated. The drawings are not necessarily drawn to scale. In all cases, the present disclosure presents the invention by way of illustration and not limitation.

DETAILED DESCRIPTION OF THE INVENTION

[0011] As used herein, the term "microreplication" means the generation of a microstructured surface by a process in which the structured surface features maintain individual feature fidelity during manufacture.

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

[0013] As used herein, the term "capillary action" refers to the flow of a fluid without the assistance of an external force (e.g., pressure, gravity, vacuum, etc.). Often, capillary action occurs with respect to an aqueous fluid in contact with a hydrophilic surface. The aqueous fluid contains 50 volume % or more water.

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

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

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

[0017] As used herein, a polymeric "film" is a polymeric material in the form of a generally flat sheet having sufficient flexibility and strength to be processed in a roll-to-roll manner. Roll-to-roll means a process in which the material is wound onto or unwound from a support and further processed in some way. Examples of further processes include coating, slitting, blanking, and exposure to radiation. Polymeric films can generally be manufactured with various thicknesses in the range of about 5 micrometers to 1000 micrometers.

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

[0019] As used herein, "fluid" refers to a composition that includes a liquid (i.e., a state of matter that is neither a solid nor a gas), and encompasses solutions, suspensions, and emulsions.

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

[0021] As used herein, "thermoplastic" refers to a polymer that flows when heated to a temperature well above its glass transition point and becomes solid when cooled. In contrast, "thermosetting" refers to a polymer that cures permanently upon curing and does not flow upon subsequent heating. Thermosetting polymers are typically crosslinked polymers.

[0022] As used herein, the term "glass transition temperature" (T g ) of a polymer refers to the transition of the polymer from a glassy state to a rubbery state and can be measured using differential scanning calorimetry (DSC), for example, at a heating rate of 10 °C per minute in a nitrogen stream. When the T g of a monomer is mentioned, it is the T g of the homopolymer of that monomer. The homopolymer must have a sufficiently high molecular weight such that the T g reaches a limiting value, because it is generally recognized that the T g of the homopolymer increases to the limiting value with increasing molecular weight. It is also understood that the homopolymer is substantially free of water, residual monomer, solvent, and other contaminants that can affect the T g . Suitable DSC methods and analysis modes are as described in Matsumoto, A. et al., J. Polym. Sci. A., Polym. Chem. 1993, 31, 2531 - 2539.

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

[0024] The terms "preferred" and "preferably" refer to embodiments of the present disclosure that may provide certain benefits under certain circumstances. However, in the same or other circumstances, other embodiments may also be preferred. Further, the recitation of one or more preferred embodiments does not mean that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the present disclosure.

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

[0026] As used herein, the term "or" is generally used in its ordinary sense including "and / or" unless the content clearly indicates otherwise. The term "and / or" means one or all of the recited elements, or any combination of two or more of the recited elements.

[0027] Also, in this specification, all numbers are assumed to be modified by the term "about", preferably by the term "exactly". When used herein in connection with a measured quantity, the term "about" refers to the variation in the measured quantity that would be expected by a person of ordinary skill in the art who takes due care in making the measurement and in view of the purpose of the measurement and the accuracy of the measuring device used.

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

[0029] In point-of-care biomarker analysis, it is necessary to simply and efficiently separate red blood cells from a microliter volume of blood without hemolysis, dilution, or significant loss of blood to dead space.

[0030] In a first aspect, the present disclosure provides a method for separating red blood cells from blood. The method comprises a) 1) a microstructured substrate comprising a plurality of microstructures extending across a first surface of the microstructured substrate, wherein at least a portion of the outer surface of the plurality of microstructures is configured to enable capillary action; 2) a cover disposed at a selected distance from the top of the first surface of the microstructured substrate; 3) at least one sidewall portion for attaching the cover to the first surface of the microstructured substrate along the 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; 5) a second aperture defined by at least one of the microstructured substrate or the cover. When the first surface of the microstructured substrate, together with the at least one side wall portion, defines a first open volume that is the sum of the open spaces located between a plurality of microstructures from the bottom to the top of each microstructure, and the cover, together with the top of the first surface of the microstructured substrate and the at least one side wall portion, defines a second open volume located adjacent to the first open volume, and when the sum of the first open volume and the second open volume is defined as 100% open volume, the first open volume has a percentage greater than the volume percentage of red blood cells present in the blood among the 100% open volume, obtaining a device, b) filling the device with a volume of blood through the first aperture by capillary action; c) waiting for a time sufficient for at least a part of the red blood cells to settle within the first open volume of the plurality of microstructures; d) applying pressure to the device, whereby at least 10% of the initial volume of blood in which at least a part of the red blood cells is retained within the first open volume of the plurality of microstructures flows out of the device through either the first aperture or the second aperture.

[0031] In a second aspect, the present disclosure provides a device. The device 1) a microstructured substrate comprising a plurality of microstructures extending across a first surface of the microstructured substrate, the microstructures covering at least 90% of the first surface of the microstructured substrate, and at least a part of the outer surfaces of the plurality of microstructures being configured to enable capillary action; 2) a cover disposed at a distance selected from the top of the first surface of the microstructured substrate; 3) at least one side wall portion for attaching the cover to the first surface of the microstructured substrate along the periphery of the first surface of the microstructured substrate; 4) a first aperture defined by at least one of the microstructured substrate or the cover; 5) a second aperture defined by at least one of the microstructured substrate or the cover; The first surface of the microstructured substrate, together with the at least one side wall portion, defines a first open volume that is the sum of the open spaces located between a plurality of microstructures from the bottom to the top of each microstructure. The cover, together with the top of the first surface of the microstructured substrate and the at least one side wall portion, defines a second open volume located adjacent to the first open volume.

[0032] The following disclosure relates to both the first and second aspects.

[0033] Referring to FIG. 1, the method includes obtaining a device (the device is as described above), 110; filling the device with a volume of blood through a first aperture by capillary action, 120; waiting for a time sufficient for at least a portion of the red blood cells to settle within the first open volume of the plurality of microstructures, 130; applying pressure to the device, thereby causing at least 10% of the initial volume of blood in which at least a portion of the red blood cells are retained within the first open volume of the plurality of microstructures to flow out of the device through either the first aperture or the second aperture, 140. Optionally, the waiting time is a time sufficient for at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even at least 95% of the red blood cells to settle within the first open volume of the plurality of microstructures.

[0034] Referring to FIGS. 2 and 3A, an exemplary device 200 is shown. The device 200 includes a microstructured substrate 210 having a plurality of microstructures 230 extending across a first surface 202 of the microstructured substrate 210. At least a portion of the outer surface 232 of the plurality of microstructures 230 is configured to enable capillary action. The device 200 also includes a cover 220 disposed at a selected distance D from the top of the first surface 221 of the microstructured substrate 210, and at least one sidewall portion 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 formed from each side Pa, Pb, Pc, Pd,... Pn of a particular device. The device 200 shown in FIG. 2A has four sides Pa, Pb, Pc, and Pd, and thus the perimeter P includes each of these four sides. Other shapes of devices having a number of sides other than four are contemplated.

[0035] Optionally, at least one sidewall portion 240 may include an adhesive layer (e.g., an adhesive for attaching the cover to the substrate) disposed between the cover 220 and the microstructured substrate 210, such as the double-sided tape used in Example 1 below. This can 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 made by coating a film of an adhesive containing an adhesive polymer. Preferably, the adhesive includes an adhesive polymer and a crosslinking agent. As used herein, the term "adhesive polymer" refers to a polymer that exhibits adhesiveness at ambient temperature (e.g., 20 - 25 °C). The adhesive polymer can be, for example, an acrylic polymer, a polyurethane, a polyolefin, or a polyester. In a selected embodiment, the adhesive layer includes a double-sided coated adhesive film. Some suitable commercially available double-sided coated adhesive films are sold by 3M Company (St. Paul, MN) under the trade names 3M Medical Silicone Tape 2477P, and 3M Medical Tape 1509, 1510, 1513, 1522, 9874, and 9877.

[0036] Furthermore, the device includes a first aperture 250 defined by at least one of the microstructured substrate 210 or the cover 220, and a second aperture 260 defined by at least one of the microstructured substrate 210 or the cover 220. The device shown in FIG. 2A includes a first aperture 250 defined by the cover 220 and a second aperture 260 defined by both the microstructured substrate 210 and the cover 220.

[0037] In the selected embodiments, the sidewall portion, the microstructured substrate, and the cover are hermetically sealed to each other at their points of contact (e.g., seams), which minimizes leakage of fluid samples from any seams between the three (e.g., fluid can enter and exit primarily through or only through the first aperture and / or the second aperture). Optionally, the microstructured substrate itself includes a sidewall portion within its structure such that at least one sidewall portion is part of the microstructured substrate.

[0038] Suitable materials for use as the cover include, for example, polyolefins (e.g., high density polyethylene (HDPE), medium density polyethylene (MDPE), or low density polyethylene (LDPE)), polyesters, polyamides, poly(vinyl chloride), polyether esters, polyimides, polyester amides, polyacrylates, polyvinyl acetates, or hydrolytic derivatives of polyvinyl acetate, but are not limited thereto. In certain embodiments, polyolefins are preferred because of their excellent physical properties, ease of processing, and typically low cost. Also, polyolefins are generally tough, durable, and retain their shape well, making them easy to handle after article formation. In the selected embodiments, the film layer includes polyethylene terephthalate (PET) of polyester. One suitable commercially available PET is a 5 mil (127 micrometer) thick PET sheet sold under the trade name "MELINEX 454" by Tekra (New Berlin, WI). A suitable commercially available LDPE is commercially available under the trade name "DOW 955I LDPE" from The Dow Chemical Company (Midland, MI). Further, various additives such as surface energy modifiers (e.g., surfactants and hydrophilic polymers), plasticizers, antioxidants, pigments, release agents, antistatic agents, etc. may be included in the cover layer.

[0039] The first surface 202 of the microstructured substrate 210, together with at least one sidewall portion 240, defines a first open volume 270 that is the sum of the open spaces located between the plurality of microstructures 230 from the bottom 236 to the top 221 of each microstructure 230. (For simplicity, the arrow for 270 only indicates a part 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 at least one sidewall portion 240, defines a second open volume 280 that is adjacent to the first open volume 270. When the total of the first open volume and the second open volume is defined as 100% open volume, the first open volume has a percentage greater than the volume percentage of red blood cells present in the blood out of that 100% open volume. To adjust the device for blood containing particles such as a specific volume percentage of red blood cells (e.g., solids), considering that the total of the first open volume and the second open volume is 100% open volume, it can be concluded that the first open volume needs to have a percentage greater than the volume percentage of the particles present in the fluid out of that 100% open volume. For example, when the volume percentage of the particles is 20% of the total volume of the fluid, a suitable ratio of the first open volume to the second open volume is greater than 1:4 (e.g., 1.1:4). When the volume percentage of the particles is 75% of the total volume of the fluid, a suitable ratio of the first open volume to the second open volume is greater than 3:1 (e.g., 3.1:1).

[0040] In some cases, the ratio of the first open volume 270 to the second open volume 280 is 1:1 or more, 1.1:1, 1.2:1, 1.3:1, or 1.4:1 or more, and at most 2.0:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, or 1.5:1. The square brackets 272 indicate the height of the first open volume 270, the square brackets 282 indicate the height of the second open volume 280, and in this device 220, when the device 220 is oriented as shown by the z-axis and the y-axis, it shows that the second volume 280 is directly stacked on top of the first open volume 270. Note that the devices 200 of FIGS. 3A and 3B are not to scale for the case where the ratio of the first open volume 270 to the second open volume 280 is 1:1 or more and are shown to assist in the description of the devices and methods provided herein.

[0041] Optionally, it may also be useful to select a specific relationship (e.g., ratio) between the average height of a plurality of microstructures and the average width of the pitch between each of the plurality of microstructures. Preferably, the first open volume is large enough such that when all red blood cells have sedimented, there are no red blood cells protruding above the top of the microstructures.

[0042] As described above, at least a part of the outer surface of the plurality of microstructures is configured to enable capillary action. Capillary action is well known in the art to refer to the flow of a fluid without the aid of an external force for an aqueous fluid (having 50% by volume or more of water) typically in contact with a hydrophilic surface. Thus, when blood is introduced into the first aperture of the device, the blood is spontaneously transported along the outer surface of the microstructures and thereby spreads within the region of the microstructures. Two general factors that affect the ability of the microstructures to spontaneously transport fluid are: (i) the structure or topography of the surface (e.g., capillary-like, cavity shape), and (ii) the nature of the surface (e.g., surface energy). To achieve a desired amount of fluid transport ability, the designer may adjust the structure or topography of the substrate layer and / or the surface energy of the capillary microstructured surface. To achieve wicking, the surface of the capillary microstructures must be capable of being "wetted" by the liquid being transported (e.g., a substance in a liquid state). Optionally, the ease of wetting of a solid surface by a liquid is characterized by the contact angle formed between the solid surface and the liquid after the liquid has been deposited on and stabilized on a horizontally disposed surface. This angle may also be referred to as the "static equilibrium contact angle" or simply the "advancing contact angle" herein. In some cases, a material is considered hydrophilic when it has an advancing contact angle of less than 90 degrees, while a hydrophilic surface exhibits an advancing (maximum) water contact angle of less than 90°, preferably 45° or less.

[0043] When fluid is introduced through the first aperture, a sufficient portion of the outer surface of the microstructured substrate needs to be hydrophilic so that the microstructured substrate can cause capillary action to transport the fluid (e.g., blood) throughout the device, whereby solid particles can access the open volume between the microstructures and settle out of most of the fluid. In some cases, 50% or more of the outer surface area of the microstructures can cause capillary action, and 60%, 70%, 80%, 90%, or 95% or more of the outer surface of the microstructures can cause capillary action. In a selected embodiment, at least a portion of the main surface 223 of the cover 220 facing the microstructured substrate 230 is hydrophilic to assist in the capillary action of the fluid inside the device 200. The hydrophilicity of the outer surface of the microstructures and / or the main surface of the cover can be achieved by one or more of material selection, additives included in the material, or surface treatment according to any of the devices described herein. In some embodiments, the microstructures have an outer surface that includes a surfactant, a surface treatment agent, a hydrophilic polymer, a flocculant, or any combination thereof. Suitable surfactants include, for example, C8-C18 alkane sulfonates, C8-C18 secondary alkane sulfonates; alkyl benzene sulfonates; C8-C18 alkyl sulfates; alkyl ether sulfates; sodium lauryl sulfate; sodium lauryl octyl sulfate; dioctyl sulfosuccinate, sodium salt; lauroyl lactylate; stearoyl lactylate; or any combination thereof, but are not limited thereto. One or more surfactants can be applied by conventional methods, for example, by spreading a coating of the surfactant on the surface of the microstructures and drying the coating. Suitable surface treatments include hydrophilic coatings including plasma deposited silicon / oxygen materials and / or diamond-like glass (DLG) materials. Each plasma deposition of the silicon / oxygen material and the DLG material is described, for example, in PCT International Publication No. WO 2007 / 075665 (Somasiri et al.).Furthermore, examples of suitable DLG materials are disclosed in U.S. Patent Nos. 6,696,157 (David et al.), 6,881,538 (Haddad et al.), and 8,664,323 (Iyer et al.). Suitable hydrophilic polymers include, for example, but are not limited to, polyesters, polyamides, polyurethanes, poly(vinyl alcohol), poly(alkylene glycols), poly(alkylene oxides), poly(vinyl pyrrolidone), rubber elastomers, or any combination thereof.

[0044] For the aggregation of cells and / or cell fragments and for the precipitation of proteins, it is known to use certain ionic polymers, particularly cationic polymers. When an aggregating agent is used, a device having a larger microstructure (e.g., height and / or depth, pitch between adjacent microstructures, etc.) than when no aggregating agent is used can be used. This is because the particles tend to form agglomerates and have a larger size when aggregated, so a larger space may be required to hold the particles within the microstructure. In contrast, a device having a large microstructure may not be as effective in holding particles (e.g., red blood cells) separated from a non-aggregated fluid (e.g., blood). Thus, the microstructured surface of the device may be partially selected considering the expected size of the particles or aggregated particles.

[0045] The polymers used as flocculants can be unmodified (e.g., polyethyleneimine) or modified (e.g., guanylated polyethyleneimine). In some embodiments, suitable flocculants are hydrophilic and non-hemolytic (i.e., do not lyse red blood cells). Some suitable flocculants are described in detail in U.S. Patent Nos. 8,435,776 (Rasmussen et al.) and 10,005,814 (Rasmussen et al.), the entireties of which are incorporated herein by reference. In certain cases, the flocculant comprises an unmodified or modified (e.g., functionalized) amino polymer. Suitable amino polymers can be selected from the group consisting of, for example, polyethyleneimine, polylysine, polyaminoamide, polyallylamine, polyvinylamine, polydimethylamine-epichlorohydrin-ethylenediamine, polydiallyldimethylammonium chloride, cationic polyacrylamide (CPAM), polyaminosiloxane, and dendrimers formed from polyamidoamine (PAMAM) and polypropyleneimine. Suitable modified amino polymers can be prepared by functionalizing one or more amino polymers selected from the group consisting of polyethyleneimine, polylysine, polyaminoamide, polyallylamine, polyvinylamine, polydimethylamine-epichlorohydrin-ethylenediamine, polydiallyldimethylammonium chloride, CPAM, polyaminosiloxane, and dendrimers formed from PAMAM and polypropyleneimine. For example, the functionalization can include reacting the amino polymer with an alkylating agent, an acylating agent, or a guanylating agent. In selected embodiments, the flocculant comprises a modified or unmodified polyethyleneimine polymer.

[0046] One or more flocculants can be applied by conventional methods, for example, by applying a coating of the flocculant onto the surface of the microstructure and drying the coating.

[0047] In some cases, when measured by gel permeation chromatography, the aggregating agent has a weight average molecular weight (Mw) of 5,000 grams per mole (g / mol) or more, 10,000 g / mol, 20,000 g / mol, 30,000 g / mol, 40,000 g / mol, 50,000 g / mol, 60,000 g / mol, 70,000 g / mol, 80,000 g / mol, 90,000 g / mol, 100,000 g / mol, 110,000 g / mol, 120,000 g / mol, 130,000 g / mol, 140,000 g / mol, or 150,000 g / mol or more, and 500,000 g / mol or less per mole. Sometimes, a high molecular weight, for example, 50,000 g / mol or more, may help aggregate particles (e.g., red blood cells).

[0048] Advantageously, at least a portion of the aggregating agent tends to dissolve, disperse, or a combination thereof in a fluid (e.g., blood) after the device is filled with a certain volume of the fluid. In some cases, the amount of the aggregating agent used is designed to result in an aggregating agent concentration in a certain volume of a fluid sample (e.g., blood) of 0.01 microgram per milliliter (μg / mL) or more, 0.1 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, 25 μg / mL, 50 μg / mL, 75 μg / mL, 100 μg / mL, 150 μg / mL, 250 μg / mL, 500 μg / mL, 750 μg / mL, 1000 μg / mL, or 1500 μg / mL or more, and 5000 μg / mL or less, 4000 μg / mL, 3000 μg / mL, 2000 μg / mL, 1000 μg / mL, 500 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL, 10 μg / mL, or 2 μg / mL or less. In other words, in some embodiments, the aggregating agent is present in a certain volume of a fluid (e.g., blood) in an amount of 0.01 μg / mL to 5000 μg / mL. The amount of the aggregating agent in the form of a dry coating on the surface of the device varies based on the molecular weight (Mw) of the particular aggregating agent.

[0049] Also shown in FIGS. 3A and 3B are illustrative depictions on SEM images of the device to show concepts regarding how the device 200 is typically used. For example, utilizing capillary action, after the blood 295 fills the device 200, the red blood cells 290 begin to sediment into the first open volume 270 among the microstructures 230. In FIG. 3A, only three red blood cells 290 are depicted as being located within the first open volume 270. Referring to FIG. 3B, the device 200 is depicted after sufficient time has elapsed for all of the red blood cells 290 to sediment within the first open volume 270 of the plurality of microstructures 230. In many cases, the red blood cells sediment due to gravity alone. A volume of blood 295 from which the red blood cells have been removed is present in the second open volume 280. In use, when pressure is applied to the device, at least some amount of the initial volume of blood in which at least a portion of the red blood cells are retained within the first open volume 270 of the plurality of microstructures 230 flows out of the device through either the first aperture 250 or the second aperture 260. In some cases, it is preferred that the blood 295 exits through the second aperture 260.

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

[0051] In some cases, the method further comprises adding a flocculant to a volume of fluid (e.g., blood) before filling the device with the volume of fluid (e.g., blood). The flocculant can be as described in detail above, including the concentration present in a volume of fluid (e.g., an amount of 0.01 - 5000 micrograms per mL of fluid).

[0052] Red blood cells tend to constitute 35-50% of the total volume of whole blood. When the ratio of the first release volume to the second release volume is 1:1 or more, sufficient space is provided between the microstructures within the first release volume to hold all of the red blood cells of the undiluted whole blood sample at maximum, while the second release volume remains available for blood that contains few (or none at all) red blood cells. Thus, when pressure is applied to the device after sedimentation, the blood (or in some cases plasma) from which the red blood cells have been removed is located closer to the aperture and thus exits the device preferentially. In contrast, the sedimented red blood cells tend to be retained between the microstructures within the first release volume and tend to be less likely to be removed from the device by the application of pressure. In some cases, the ratio of the first release volume to the second release volume is selected to minimize the volume of blood that might sediment into the first release volume along with the red blood cells, in order to maximize the volume of blood (from which the red blood cells have been removed) that can flow out of the device for analysis. By selecting a ratio of the first release volume to the second release volume of 1:1 or more, it is typically not necessary to dilute the whole blood for effective separation of red blood cells from the blood, and thus the blood can be used in its undiluted form. However, in the case of diluted blood, it is possible to use a smaller ratio, such as 0.9:1 or less, 0.8:1, 0.7:1, 0.6:1, or even 0.5:1 or less.

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

[0054] 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 larger the percentage, the higher the efficiency of the separation method using the device according to the present disclosure, and the smaller the analyzable sample volume lost by the device.

[0055] The device according to at least certain embodiments of the present disclosure is suitable for use in 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 and second open volumes of the device can be from 5 microliters to 120 microliters.

[0056] In some cases, the first aperture is defined by a cover of the device and can introduce a volume of fluid (e.g., blood) into the device using gravity and / or wicking into the aperture. In other cases, the first aperture is defined by a microstructured substrate. For example, referring to FIG. 2B, the first aperture 250 can be provided as a reservoir configured to hold at least a specific minimum volume of fluid. The reservoir can be defined by a portion of the microstructured substrate 210 adjacent to one end 237 of the microstructure 230. Similarly, the second aperture 260 can be a reservoir configured to hold fluid as the fluid exits the microstructured surface 202 of the microstructured substrate 210. In many cases, it is advantageous for the second aperture to be located at a distance from the first aperture so that replacement gas (typically air) from the device can be easily expelled when the fluid is deposited on the device through the first aperture. Optionally, a positive pressure can be provided by a pump to deposit a volume of blood into the first aperture, and then, after at least a portion of the red blood cells have sedimented into the first open volume, the pump can be operated again to push the blood out of the second open volume and out of the second aperture. Optionally, a volume of blood can be wicked through the first aperture into the device and then the blood can be pushed out of the second open volume and out of either the first aperture or the second aperture using either a positive pressure provided by the user's finger or a negative pressure provided by a vacuum source. Thus, the pressure resulting in blood (with some red blood cells removed) can be either positive or negative pressure.

[0057] Louver structure Referring again to FIGS. 3A and 3B, the microstructure 230 of the device of a particular embodiment includes a "louver structure" with ribs separated by channels. This shape is also shown in FIG. 4A, where the microstructure 230 comprises a plurality of ribs 230 alternating with channels 201 extending across the first surface 202 of the microstructured substrate 210. Each of the ribs 230 comprises side walls 233 and 234 and a top surface 221, and each of the channels 201 comprises a bottom surface 205.

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

[0059] The height and width of the rib (e.g., protrusion) 230 are defined by the adjacent channels (e.g., 201a and 201b). The rib 230 can be defined by a top surface 221, a bottom surface 231, and side walls 233 and 234 joining 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.

[0060] The rib 230 can be defined by a width "W". In many cases, the rib 230 has a width parallel to the first surface of the microstructured substrate and a height perpendicular to the first surface of the microstructured substrate. Except for the land region "L", the rib 230 typically has a height nominally the same as that of the channel 201. In an exemplary embodiment, the height "H" of the channel 201 and / or the rib 230 is at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 micrometers. In some embodiments, the height is 500, 475, 450, 425, 400, 375, 350, 325, 300, 275, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, or 100 micrometers or less. In some embodiments, the height of the channel 201 and / or the rib 230 ranges from 50 to 500 micrometers. The microstructured substrate typically comprises a plurality of ribs 230 having nominally the same height and width. In some embodiments, the rib 230 has a height "H", a maximum width "W" at its widest part, and an aspect ratio H / W of at least 1.5. In some embodiments, H / W is at least 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.5, 4.0, 4.5 or 5.0. In other embodiments, the aspect ratio of the rib is at least 6, 7, 8, 9, or 10. In other embodiments, the aspect ratio of the rib is at least 15, 20, 25, 30, 35, 40, 45, or 50.

[0061] Channel 201 has a height "H" defined by the distance between a bottom surface 205 and a top surface 221, and such top and bottom surfaces are typically parallel to the top surface 202 of the base layer 213. Channel 201 has a maximum width "W" and is spaced apart by 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 channel adjacent to the top surface 221. However, if the width of the channel at the base is different from the width adjacent to the top surface, the width is defined by the maximum width. The maximum widths of a plurality of channels can be averaged in a target area such as an area for calculating a first open volume. The microstructured substrate can comprise a plurality of channels having nominally the same height and width. In typical embodiments, the channels generally have a width of 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 micrometer or less. In some embodiments, the channels generally have a width of 900, 800, 700, 600, or 500 micrometers or less. In some embodiments, the channels have a width of at least 50, 60, 70, 80, 90, or 100 micrometers.

[0062] In some embodiments, the wall angle θ is greater than 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 degrees. In some embodiments, the wall angle is 110, 109, 108, 107, 106, 105, 104, 103, 102, 101, 100, 99, 98, 97, 96, or 95 degrees or less. In some embodiments, the wall angle approaches 90 degrees. When the wall angle is 90 degrees, the angle between 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 side wall can be described as including a first and a second side wall, and the first side wall has a wall angle with respect to a line parallel to the first surface of the microstructured substrate of 0 degrees to +10 degrees or 0 degrees to -10 degrees with respect to the bottom surface of the microstructured substrate.

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

[0064] A variation of the louver structure of Figure 4A is depicted in Figure 4B, where in some cases, the top surface 221 of each rib 230 is the 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 sidewalls 233 and 234 on both sides. Without wishing to be bound by theory, the presence of caps on the ribs (e.g., undercut features) is thought to assist in retaining particles in the channels when the particles settle onto the device.

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

[0066] In one embodiment, a structured substrate 210, such as that shown in FIG. 4B having caps on ribs, can be prepared by known methods for creating undercut features (e.g., by partially disassembling a mold made of multiple parts after molding to open each of the cavities and make the features easily removable, or by first molding straight ribs without undercuts and then forming caps on their stems in a separate shaping step after demolding). Alternatively, such a structured substrate may be prepared according to the disclosure of PCT International Publication No. WO 2015 / 041844 (Rule et al.), in which case a polyolefin resin is deposited in a mold cavity to form a first layer including a plurality of undercut features on and extending from an integral backing, and the first layer is demolded from the mold cavity at a rate of at least 150 millimeters per minute (mm / min).

[0067] The curable resin can include a combination of first and second curable components 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. The term "(meth)acrylate" refers to both acrylate and methacrylate compounds. Optionally, the curable composition can include (meth)acrylated urethane oligomers, (meth)acrylated epoxy oligomers, (meth)acrylated polyester oligomers, (meth)acrylated phenol oligomers, (meth)acrylated acrylic oligomers, and mixtures thereof.

[0068] The curable resin can be a radiation curable polymer resin such as a UV curable resin. Optionally, examples of curable resin compositions useful for the microstructured substrate of the present disclosure can include curable resin compositions as described in U.S. Patent No. 8,012,567 (Gaides et al.).

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

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

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

[0072] In yet another embodiment, the master negative microstructured forming surface (e.g., tool) can be used as an embossing tool as described in U.S. Patent No. 4,601,861 (Pricone).

[0073] Further details regarding such microstructured substrates having such louver structures and methods of forming them are described in International Publication No. WO 2019 / 118685 (Schmidt et al.) and International Publication No. WO 2020 / 026139 (Schmidt et al.), each of which is incorporated herein by reference.

[0074] Prism structure FIG. 5A shows an alternative microstructured substrate 309 comprising a linear array of regular prisms 320. Each prism has a first facet 321 and a second facet 322. The prisms are typically formed on a base member 310 (e.g., a preformed polymer film) having a first plane 331 in which the prisms are formed and a second surface 332 that is substantially flat or planar and on the opposite side of the first surface. In some embodiments, the prisms are right-angled columns. A right-angled column means that the apex angle θ, 340 is typically about 90°. However, this angle may range from 5° to 90°, or may range from 20° to 80°. In a selected embodiment, the microstructure comprises linear prisms having an apex angle of 90 degrees or less. In some embodiments, particularly when the facets of the peak structure are interconnected by valleys between the peak structures, the apex angle of the peak structure is typically twice the wall angle. 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, for example, in the first plane 331 or parallel thereto, between adjacent peak structures 320.

[0075] These apexes may be pointed (as shown), rounded, or have a shape with a truncated tip. In some cases, a pointed or rounded apex shape may have a lower potential for particles to settle on the shape compared to a (e.g., flat) shape with a truncated apex, so it is advantageous to use a pointed or rounded apex. Preferably, the radius of the prism tip is smaller than the radius of the particles (e.g., red blood cells). The distance between (e.g., prism) peaks can be characterized as a pitch ("P"). In this embodiment, the pitch is also equal to the maximum width of the valley. Thus, the pitch is greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 microns as described above and ranges up to 500 microns. The length of the prism microstructure ("L") is typically the maximum dimension and can span the entire dimension of the microstructured surface, film, or article. The prism facets need not be the same and the prisms may be inclined 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 FIG. 5A, the facets 321 and 322 of the prism peak structure are continuous in the direction of the length of the microstructure (L), i.e., the y-direction.

[0076] Optionally, the continuous land layer 360 can be present between the bottom of the channel or valley and the top surface 331 of the (e.g., flat) base member 310. In some embodiments, for example, when the microstructured surface is prepared from the casting and curing of a polymerizable resin composition, the thickness of the land layer typically ranges from at least 0.5, 1, 2, 3, 4, or 5 micrometers up to 50 micrometers. In some embodiments, the thickness of the land layer is 45, 40, 35, 30, 25, 20, 15, or 10 microns or less.

[0077] Referring to FIG. 5B, a SEM image of a cross-section is provided of a portion of an exemplary device of Example 8 having a prism microstructure. The first surface 302 of the microstructured substrate 325 defines a first open volume 370, which is the sum of the open spaces located between a plurality of microstructures 320 from the bottom 336 to the top 327 of each microstructure 320, together with at least one side wall portion 340. (For simplicity, the arrows in 370 only indicate a part of the first open volume between two adjacent microstructures 320). The cover 352, together with the top 327 and at least one side wall portion 340 of the first surface 302 of the microstructured substrate 325, defines a second open volume 380 adjacent to the first open volume 370. When the total of the first open volume and the second open volume is defined as 100% open volume, the first open volume has a percentage greater than the volume percentage of red blood cells present in the blood out of the 100% open volume.

[0078] In a selected embodiment, the microstructure comprises an array of peak structures and adjacent valleys, the valleys having a maximum width in the range of 10 microns to 500 microns, and the peak structures having an apex angle of greater than 5 degrees up to a maximum of 90 degrees.

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

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

[0081] Facet structure Similar to the prism structure, FIG. 5E shows an alternative microstructured substrate 700 having a faceted structure. More specifically, FIG. 5E shows a microstructured substrate 700 that defines a bottom surface 705, a top surface 720, a first side wall 732, and a facet 733. In other words, the microstructure comprises a facet 733 and a side wall 732 that intersects the facet at the ridge 720 of the microstructure. The facet 733 and the side wall 732 typically define an oblique angle therebetween.

[0082] In a selected embodiment, the microstructure comprises a facet and a side wall that intersects the facet at the ridge of the microstructure, and the facet and the side wall define an oblique angle therebetween.

[0083] Further details regarding microstructured substrates having such a faceted structure and methods of forming them are described in International Publication No. WO 2020 / 250180 (Kenney et al.), which is incorporated herein by reference.

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

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

[0086] Further details regarding microstructured substrates having such an array of protrusions and methods of forming them are described in International Publication No. 2020 / 097319 (Wolk et al.), which is incorporated herein by reference.

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

[0088] FIG. 7B 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 opposite second major surface 516. The first major surface 514 includes an array of discrete cavities 522. In a particular embodiment, each of the cavities 522 includes a cross-section parallel to the first major surface 514 that can be circular, elliptical, or polygonal. The cross-section decreases in size in the direction from the first major surface 514 to the second major surface 516, as necessary. This embodiment of the microstructured substrate 500b further includes a substrate 530 (e.g., flexible) coupled to the second major surface 516 of the microstructured layer 510.

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

[0090] Further details regarding microstructured substrates having such cavity arrays and methods of forming them are described in U.S. Patent No. 9,329,311 (Halverson et al.), which is incorporated herein by reference.

[0091] Connected well structure FIGS. 10A - 10B show yet another alternative microstructured substrate 1025, the microstructure comprising an array of fluid-connected wells, at least some of the wells being fluid-connected to at least two adjacent wells, each being connected via a vent. For example, FIG. 10A is a schematic perspective view of a portion of a microstructured substrate 1025 having an array of fluid-connected wells 1077 connected to each other by vents 1087.

[0092] Figure 10B is a schematic top view of a portion of the microstructured substrate 1025 of FIG. 10A having an array of fluid-connected wells 1077 having a circular shape connected by vents 1087 to adjacent wells 1077. In the particular structure shown in FIGS. 10A-10B, the central well 1077 of the microstructured substrate 1025 (e.g., having at least one other well located between it 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, which are connected via vents 1087 to one or two adjacent wells 1077. For example, referring to FIG. 10A, well 1077c is at the corner of the array and is attached via vent 1087a to only one other well 1077d. Similarly, wells can be attached via vents to three other adjacent wells, or to four, five, six, seven, eight, nine, ten, eleven, or twelve other adjacent wells via vents.

[0093] The shape of the wells is not particularly limited and can include curved shapes, polygonal shapes, irregular shapes, or combinations thereof. In some embodiments, the wells are circular, triangular, quadrilateral, elliptical, or combinations thereof. If the wells include shapes having corners, the vents are located at the corners as necessary (e.g., to reduce the possibility of trapping air bubbles at the corners).

[0094] Typically, each well has an open volume large enough to hold at least one sedimented particle (e.g., red blood cell), 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. In other words, in some cases, each well has an open volume in the range of 100 femtoliters to 1 microliter or 500 femtoliters to 0.1 microliter.

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

[0096] Preferably, at least some of the vents between the wells are located at the same depth as the bottom surfaces of the adjacent wells. This helps to encourage the bubbles to exit the wells without being trapped near the bottom of the wells. In some embodiments, the vents have an overall depth equal to the overall depth of the adjacent wells, but this is not essential. When the microstructured substrate is formed using a tool, creating vents having the same depth as the adjacent wells tends to be more practical than creating vents that connect only the lower portions of two wells.

[0097] Referring again to FIG. 10A, in some embodiments, the microstructured substrate 1025 further includes at least one sidewall portion 1097 disposed along the periphery 1095 around the first surface 1027 of the microstructured substrate 1025. The one or more sidewall portions 1087 have a height ("H") that extends more than 50 - 250 micrometers beyond the top surfaces of the plurality of microstructures.

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

[0099] Further details regarding such microstructured substrates having such connected wells and methods of forming them are described in co-owned patent application Ser. No. 63 / 425,473 (Docket No. PA100766US01).

[0100] Stem web structure FIG. 11 shows a further alternative microstructured substrate 1120 having a stem web structure. In such an embodiment, the microstructure 1126 comprises an array of upright stems 1126 extending across a first surface 1124 of the microstructured substrate 1120. The microstructure 1126 is a plurality of substantially upright stems of various shapes. "Substantially upright" means that the stem projects from the first surface 1124 (e.g., in a planar direction). The stem 1126 may project upwardly from the surface 1124 at a substantially normal angle, or the stem 1126 may project at an angle away from the surface 1124. The stems may also be irregular in shape such that they do not project at any one uniform angle.

[0101] The microstructured substrate 1120 includes a backing layer 1121 having a first surface 1124 that includes an array of generally upright stems 1126. The stems 1126 may be arranged in a regular or irregular array. Stems of various patterns such as hexagonal, diagonal, sine wave, etc. may be used. The stems 1126 may be at least partially composed of an elastomeric material. In some cases, the entire outer surface of the stems 1126 is an elastomeric material. In the embodiment of FIG. 11, the backing layer 1121 is formed integrally with the stems 1126. The combination of the backing layer 1121 and the stems 1126 is sometimes referred to as a stem web. The illustrated embodiment shows the stems 1126 as generally cylindrical, but the sides of the stems 1126 typically have a slight taper 1135 to facilitate removal from the mold. As shown, the taper 1135 is inwardly directed from the base 1102 to the tip 1104 of the stem 1126. It is expressly contemplated that the stem may be configured to have an outward taper from the base to the tip of the stem. Various non-cylindrical shapes such as frustum of a cone or pyramid, rectangle, hemisphere, square, hexagon, octagon, gumdrop, etc. may also be utilized.

[0102] The backing layer 1121 on which the stems 1126 directly extend is typically about 0.05 millimeters to about 0.5 millimeters (0.002 inches to 0.02 inches) thick. Thus, an additional backing layer(s) 1122 may be applied to the second surface 1125 as needed to reinforce the backing layer 1121 and form a multi-layer base or backing structure. As used herein, the "backing" or "base" layer is used to refer to the collective backing or base structure. Such a structure may be a single layer or multiple layers (shown in FIG. 1) having one or more layers that support the generally upright stems 1126, but typically at most one of these layers 1121 is formed integrally with the stems 1126.

[0103] The stem typically has 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 separation or gap 1130 between adjacent stems 1126 is generally 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. This separation gap creates a percentage of free volume, which is the volume within the stem web not occupied by the stems. The percentage of free volume is typically 60 - 98% of the stem web, more typically 85 - 95%. The stem 1126 has a maximum cross-sectional dimension 1129 in the range of about 0.076 mm to about 0.76 mm. The stems 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 stem density is generally at most about 1500 per square centimeter, more typically at most about 500 per square centimeter. The stem has an aspect ratio of at least 1.25, preferably at least 1.5, most preferably at least 2.0. The aspect ratio refers to the ratio of the height of the stem to its maximum cross-sectional dimension. For a stem with a circular cross-section, the maximum cross-sectional dimension is the diameter of the stem.

[0104] Suitable elastomeric stem materials include elastomer classes such as anionic triblock copolymers, polyolefin-based thermoplastic elastomers, halogen-containing polyolefin-based thermoplastic elastomers, dynamically vulcanized elastomer-thermoplastic blend-based thermoplastic elastomers, thermoplastic polyether esters or polyester-based elastomers, polyamide or polyimide-based thermoplastic elastomers, ionomer thermoplastic elastomers, hydrogenated block copolymers in thermoplastic elastomer interpenetrating polymer networks, thermoplastic elastomers by carbocationic polymerization, polymer blends containing styrene / hydrogenated butadiene block copolymers, and polyacrylate-based thermoplastic elastomers. Some specific examples of elastomers are natural rubber, butyl rubber, EPDM rubber, silicone rubber 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 in a linear, radial or star configuration and may be diblock (AB) or triblock (ABA) copolymers or mixtures thereof. Blends of these elastomers with each other or with modified non-elastomers are also contemplated. Commercially available elastomers include block polymers (e.g., polystyrene materials containing elastomeric segments) available under the name KRATON™ from KRATON Polymers Company in Houston, Texas.

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

[0106] 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 containing elastomeric segments available under the name KRATON™ from KRATON Polymers Company, Houston, Texas), polycarbonates, thermoplastic elastomers (e.g., polyolefin, polyester, or nylon types), and copolymers and blends thereof. In some cases, the entire stem web is formed from one or more thermoplastic materials, such as those listed above. The thermoplastic material may also contain additives including, but not limited to, fillers, fibers, antistatic agents, lubricants, wetting agents, foaming agents, surfactants, pigments, dyes, coupling agents, plasticizers, suspending agents, hydrophilic / hydrophobic additives, adhesives, and the like.

[0107] Further details regarding such microstructured substrates having a stem web and methods of forming them are described in International Publication No. WO 2009 / 020811 (Tuman et al.), which is incorporated herein by reference.

[0108] In a third aspect, the present disclosure provides a method of separating solid particles from a fluid. The method comprises a) 1) a microstructured substrate comprising a plurality of microstructures extending across a first surface of the microstructured substrate, wherein at least a portion of the outer surface of the plurality of microstructures is configured to enable capillary action; 2) a cover disposed at a selected distance from the top of the first surface of the microstructured substrate; 3) at least one sidewall portion for attaching the cover to the first surface of the microstructured substrate along the 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; 5) a second aperture defined by at least one of the microstructured substrate or the cover, and the first surface of the microstructured substrate, together with the at least one side wall portion, defines a first open volume that is the sum of the open spaces located between a plurality of microstructures from the bottom to the top of each microstructure; the cover, together with the top of the first surface of the microstructured substrate and the at least one side wall portion, defines a second open volume located adjacent to the first open volume; when the sum of the first open volume and the second open volume is defined as 100% open volume, the first open volume has a percentage greater than the volume percentage of the particles present in the fluid among the 100% open volume, to obtain a device; b) filling the device with a volume of fluid through the first aperture by capillary action; c) waiting for a time sufficient for at least a part of the particles to settle within the first open volume of the plurality of microstructures; d) applying pressure to the device, thereby causing at least 10% of the fluid in which at least a part of the particles is held within the first open volume of the plurality of microstructures to flow out of the device through either the first aperture or the second aperture.

[0109] Referring to FIG. 9, the method includes obtaining a device (the device being as described above), 910; filling the device with a volume of fluid through a first aperture by capillary action, 920; waiting for a time sufficient for at least a portion of the particles to settle within a first open volume of the plurality of microstructures, 930; applying pressure to the device, thereby causing at least 10% of an initial volume of the fluid in which at least a portion of the particles are retained within the first open volume of the plurality of microstructures to flow out of the device through either the first aperture or the second aperture, 940. The characteristics, materials, structures, etc. of the device for the method of the third aspect can be according to any embodiment of the device described in detail above with respect to the first and second aspects. Similarly, the method of the third aspect can be according to any of the embodiments of the method of the first aspect described in detail above.

[0110] Exemplary embodiments In a first embodiment, the present disclosure provides a method for separating red blood cells from blood. The method includes obtaining a device including a microstructured substrate having a plurality of microstructures extending across a first surface of the microstructured substrate, at least a portion of an outer surface of the plurality of microstructures being configured to enable capillary action; a cover disposed at a selected distance away from a top of the first surface of the microstructured substrate; at least one sidewall portion attaching the cover to the first surface of the microstructured substrate along a perimeter of the first surface of the microstructured substrate; a first aperture defined by at least one of the microstructured substrate or the cover; and a second aperture defined by at least one of the microstructured substrate or the cover. The first surface of the microstructured substrate, together with at least one sidewall portion, defines a first open volume that is a sum of open spaces located between the plurality of microstructures from a bottom to a top of each microstructure. The cover, together with the top of the first surface of the microstructured substrate and at least one sidewall portion, defines a second open volume located adjacent to the first open volume. When a sum of the first open volume and the second open volume is defined as a 100% open volume, the first open volume has a percentage greater than a volume percentage of red blood cells present in blood out of the 100% open volume. The method further includes filling the device with a volume of blood through the first aperture by capillary action and waiting for a time sufficient for at least a portion of the red blood cells to settle within the first open volume of the plurality of microstructures. Additionally, the method includes applying pressure to the device, thereby causing at least 10% of an initial volume of the blood in which at least a portion of the red blood cells are retained within the first open volume of the plurality of microstructures to flow out of the device through either the first aperture or the second aperture.

[0111] In a second embodiment, the present disclosure provides a method according to the first embodiment, wherein at least one sidewall portion is part of the microstructured substrate.

[0112] 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 upon application of pressure.

[0113] In a fourth embodiment, the present disclosure provides a method according to any of the first to third embodiments, wherein the time is sufficient for at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90% of the red blood cells to settle within the first open volume of the plurality of microstructures.

[0114] In a fifth embodiment, the present disclosure provides a method according to any of the first to fourth embodiments, wherein the microstructured substrate is a microstructured film.

[0115] In a sixth embodiment, the present disclosure provides a method according to any of the first to fifth embodiments, wherein at least a portion of the outer surface of the plurality of microstructures comprises a surfactant, a surface treatment agent, a hydrophilic polymer, a flocculant, or any combination thereof.

[0116] In a seventh embodiment, the present disclosure provides a method according to any of the first to sixth embodiments, wherein the flocculant is hydrophilic and non-hemolytic.

[0117] In an eighth embodiment, the present disclosure provides a method according to the sixth or seventh embodiment, wherein the flocculant comprises a modified or unmodified amino polymer selected from the group consisting of polyethyleneimine, polylysine, polyaminoamide, polyallylamine, polyvinylamine, polydimethylamine-epichlorohydrin-ethylenediamine, polydiallyldimethylammonium chloride, cationic polyacrylamide (CPAM), polyaminosiloxane, and dendrimers formed from polyamine dendrimers (PAMAM) and polypropyleneimine.

[0118] In a ninth embodiment, the present disclosure provides a method according to any of the sixth to eighth embodiments, wherein the flocculant comprises a modified or unmodified polyethyleneimine polymer.

[0119] In the tenth embodiment, the present disclosure provides a method according to any one of the sixth to ninth embodiments, and at least a part of the flocculant is dissolved, dispersed, or a combination thereof in the blood after filling the device with a certain volume of blood.

[0120] In the eleventh embodiment, the present disclosure provides a method according to the tenth embodiment, and the flocculant is present in a volume of blood in an amount of 0.01 to 5000 micrograms per milliliter of blood.

[0121] In the twelfth embodiment, the present disclosure provides a method according to any one of the first to eleventh embodiments, and the pressure is a positive pressure.

[0122] In the thirteenth embodiment, the present disclosure provides a method according to any one of the first to twelfth embodiments, and the pressure is a negative pressure.

[0123] In the fourteenth embodiment, the present disclosure provides a method according to any one of the first to thirteenth embodiments, and the first aperture is defined by a cover.

[0124] In the fifteenth embodiment, the present disclosure provides a method according to any one of the first to fourteenth embodiments, and the second aperture is defined by a microstructured substrate or a cover.

[0125] In the sixteenth embodiment, the present disclosure provides a method according to any one of the first to fifteenth embodiments, and the blood is not diluted.

[0126] In the seventeenth embodiment, the present disclosure provides a method according to any one of the first to sixteenth embodiments, and the red blood cells settle by gravity only.

[0127] In the eighteenth embodiment, the present disclosure provides a method according to any one of the first to seventeenth embodiments, and the device further comprises an adhesive layer disposed between the cover and the microstructured substrate.

[0128] In the 19th embodiment, the present disclosure provides a method according to any one of the 1st to 18th embodiments, the microstructure comprising a plurality of ribs alternately arranged with channels extending across a first surface of a microstructured substrate, each of the ribs comprising a side wall and a top surface, and each of the channels comprising a bottom surface.

[0129] In the 20th embodiment, the present disclosure provides a method according to the 19th embodiment, the top surface of each rib being the top of a cap disposed on the side wall, the cap having a width greater than the width between the side walls on both sides.

[0130] In the 21st embodiment, the present disclosure provides a method according to any one of the 1st to 18th embodiments, the microstructure comprising an array of peak structures and adjacent valleys, the valleys having a maximum width in the range of 10 micrometers to 500 micrometers, and the peak structures having a vertex angle of greater than 5 degrees up to a maximum of 90 degrees.

[0131] In the 22nd embodiment, the present disclosure provides a method according to the 21st embodiment, the array of peak structures and adjacent valleys extending across a first surface of the microstructured substrate being oriented at an angle of 0 to 90 degrees with respect to the flow direction of the device.

[0132] In the 23rd embodiment, the present disclosure provides a method according to the 21st or 22nd embodiment, further comprising a gap between adjacent peak structures.

[0133] In the 24th embodiment, the present disclosure provides a method according to any one of the 1st to 18th embodiments, the microstructure comprising a two-dimensional (x-axis and y-axis) array of protrusions arranged across a first surface of a microstructured substrate, each of the protrusions comprising a base, a top, and one or more sides connecting the top to the base.

[0134] In the 25th embodiment, the present disclosure provides a method according to any one of the 1st to 18th embodiments. The microstructured substrate includes a microstructured layer having first and second main surfaces. The microstructure includes a plurality of cavities extending between the first and second main surfaces. Each cavity includes a first opening, a second opening, and at least one side wall extending between the first opening and the second opening.

[0135] In the 26th embodiment, the present disclosure provides a method according to any one of the 1st to 13th embodiments. The microstructure includes a facet and a side wall intersecting the facet at a ridge of the microstructure. The facet and the side wall define an oblique angle therebetween.

[0136] In the 27th embodiment, the present disclosure provides a method according to any one of the 1st to 18th embodiments. The microstructure includes an array of fluid-connected wells. At least some of the wells are fluid-connected to at least two adjacent wells, and each is connected via a vent.

[0137] In the 28th embodiment, the present disclosure provides a method according to any one of the 1st to 18th embodiments. The microstructure includes an array of upright stems extending across a first surface of the microstructured substrate.

[0138] In the 29th embodiment, the present disclosure provides a method according to any one of the 1st to 28th embodiments. The volume of blood filled through the first aperture is up to 100 microliters of blood.

[0139] In the 30th embodiment, the present disclosure provides a method according to any one of the 1st to 29th embodiments. The ratio of the first open volume to the second open volume is greater than 1:1.

[0140] In the 31st embodiment, the present disclosure provides a method according to any one of the 1st to 30th embodiments, further including passing blood through a filter before entering the device, passing blood in which at least a part of red blood cells is retained in a first open volume of a plurality of microstructures after exiting the device, or both.

[0141] In the 32nd embodiment, the present disclosure provides a method according to any one of the 1st to 31st embodiments, further including adding a flocculant to a certain volume of blood before filling the device with the certain volume of blood.

[0142] In the 33rd embodiment, the present disclosure provides a method according to the 32nd embodiment, wherein the flocculant is added and present in an amount of 0.01 to 5000 micrograms per milliliter of blood.

[0143] In the 34th embodiment, the present disclosure provides a device. The device includes a microstructured substrate having a plurality of microstructures extending across a first surface of the microstructured substrate. The microstructures cover at least 90% of the first surface of the microstructured substrate, and at least a part of the outer surfaces of the plurality of microstructures is configured to enable 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 portion for attaching the cover to the first surface of the microstructured substrate along the periphery of the first surface of the microstructured substrate. Further, the device includes a first aperture defined by at least one of the microstructured substrate or the cover, and a second aperture defined by at least one of the microstructured substrate or the cover. The first surface of the microstructured substrate, together with at least one sidewall portion, defines a first open volume that is the sum of the open spaces located between the plurality of microstructures from the bottom to the top of each microstructure, and the cover, together with the top of the first surface of the microstructured substrate and at least one sidewall portion, defines a second open volume located adjacent to the first open volume.

[0144] In the 35th embodiment, the present disclosure provides a method for separating solid particles from a fluid. The method includes obtaining a device including a microstructured substrate having a plurality of microstructures extending across a first surface of the microstructured substrate. At least a part of the outer surface of the plurality of microstructures is configured to enable capillary action. The device also includes a cover disposed at a selected distance away from the top of the first surface of the microstructured substrate, and at least one sidewall portion for attaching the cover to the first surface of the microstructured substrate along the periphery of the first surface of the microstructured substrate. Further, the device includes a first aperture defined by at least one of the microstructured substrate or the cover, and a second aperture defined by at least one of the microstructured substrate or the cover. The first surface of the microstructured substrate, together with at least one sidewall portion, defines a first open volume that is the sum of the open spaces located between the plurality of microstructures from the bottom to the top of each microstructure. The cover, together with the top of the first surface of the microstructured substrate and at least one sidewall portion, defines a second open volume located adjacent to the first open volume. When the sum of the first open volume and the second open volume is defined as 100% open volume, the first open volume has a percentage larger than the volume percentage of the particles present in the fluid among the 100% open volume. The method further includes filling the device with a volume of fluid through the first aperture by capillary action, and waiting for a time sufficient for at least a part 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, thereby causing at least 10% of the fluid in which at least a part of the particles is retained within the first open volume of the plurality of microstructures to flow out of the device through either the first aperture or the second aperture.

Example

[0145] The objects and advantages of the present disclosure are further illustrated by the following examples, but the specific materials and their amounts listed in these examples, as well as other conditions and details, should not be construed as unduly limiting the present disclosure. Unless otherwise specified, all parts, percentages, ratios, etc. in the examples and the rest of the specification are based on weight. The material table (below) lists the materials used in the examples and their suppliers.

[0146] Material [Table 1]

[0147] Preparation Examples 1 to 4. Procedure for preparing a prism structure microstructured film A UV curable resin was prepared from PHOTOMER 6210 aliphatic urethane diacrylate oligomer (75 parts), SR238 1,6 - hexanediol diacrylate (25 parts), and LUCIRIN TPO photoinitiator (0.5%). The components were blended with 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 the linear prism film. Both the button and the compounded resin were heated in an oven at about 70 °C for 15 minutes. The heated resin was applied to the center of the heated button using a transfer pipette. A section of MELINEX 618 PET support film (DuPont Teijin Films (Chester, VA)) was placed on top of the applied resin, and then a glass plate was placed. The primed surface of the PET film was oriented to contact the resin. The glass plate was held in place with hand pressure until the resin completely covered the surface of the button. The glass plate was carefully removed. If there were air bubbles, a rubber hand roller was used to remove the air bubbles.

[0148] The sample was cured with UV light by passing it twice through a UV processor (model QC 120233AN equipped with two Hg lamps obtained from RPC Industries (Plainfield, IL)) at a rate of 15.2 meters per minute under a nitrogen atmosphere. The cured microstructured film having the array pattern shown in Figure 5A was removed from the copper template by gently peeling it off at an angle of 90°. An adhesive layer lined with a release liner (8 mil thick, obtained as 3M 8188 Optically Clear Adhesive from 3M Corporation) was applied as needed to the back surface (i.e., the non-microstructured surface) of the microstructured film using a hand roller. The characteristics of the prepared linear prism microstructured film are reported in Table 1 as Preparation Examples 1 to 4.

[0149] The microstructured film was cut into 2.54 cm × 7.62 cm sections, and each section was immersed in an aqueous solution of 0.1 weight percent of the IPEGAL-CO630 anionic surfactant, and then the film was immediately taken out of the solution to apply a surfactant coating. The obtained sections of the microstructured film coated with the surfactant were air-dried at ambient temperature and ambient humidity.

[0150] [Table 2]

[0151] Preparation Example 5. Procedure for preparing a louver structure microstructured film Using a diamond (depth 220 micrometers), a tool having a plurality of parallel straight grooves was cut. The grooves were spaced at a pitch of 60 micrometers. Resin A was prepared by mixing the materials in Table 2 below.

[0152] [Table 3]

[0153] The cast-and-cure microreplication process was performed using Resin A and the above tools. The line conditions were a resin temperature of 150°F (65.6°C), a die temperature of 150°F (65.6°C), a coater IR of 120°F (48.9°C) at the edges / 130°F (54.4°C) at the center, a tool temperature of 100°F (37.8°C), and a line speed of 70 feet per minute (fpm) (0.36 meters per second (m / s)). A Fusion D lamp (obtained from Fusion UV Systems (Gaithersburg, MD)) having a peak wavelength of 385 nm was used for curing and operated at 100% output. The resulting "louver" microstructured film had multiple protrusions (ribs) separated by channels, as shown in Figure 4A (detailed above). The microstructured film was a topography inversion of the tool, where the protrusions of the microstructured film were a negative replica of the grooves of the tool and the channels of the microstructured film were a negative replica of the uncut portions of the tool between the grooves. The protrusions (ribs) of the microstructured film were equally spaced with a height ("H") of 220 micrometers, a width ("W") of 30 micrometers, a pitch ("P") of 60 micrometers, and a wall angle θ of 91.5 degrees [as a result, the protrusions were slightly tapered (i.e., wider at the bottom and narrower at the top)]. The land layer ("L") of the cured resin had a thickness of 8 micrometers. The base layer was a PET film (3M Company (St. Paul, MN)) with a thickness of 74.4 micrometers. The side of the PET film in contact with the resin was primed with a thermosetting acrylic polymer (RHOPLEX 3208 polymer obtained from Dow Chemical (Midland, MI)).

[0154] The microstructured film was cut into 2.54 cm × 7.62 cm sections, and each section was immersed in a 0.1 weight percent aqueous solution of the IPEGAL-CO630 anionic surfactant and then immediately removed from the solution to apply the surfactant coating. The resulting sections of the microstructured film coated with the surfactant were air dried at ambient temperature and ambient humidity.

[0155] Example 1. Preparation of the Device Device 800 (shown in FIGS. 8A and 8B) was prepared by forming a laminate of three film sections. The cover sheet component of device 800 (i.e., cover 820) was prepared by laser cutting a section 72 mm in length × 20 mm in width from a sheet of 3M Microfluidic Diagnostic Film 9962 (a polyester film (3.9 mils) with hydrophilic coatings on both sides, including the main surface 822 of cover 820 facing the microstructured substrate 830, obtained from 3M Company). A circular hole (5 mm in diameter) was laser cut in the film such that the center of the hole was located 13.5 mm in a direction perpendicular to the narrow edge of the film and 10 mm in a direction perpendicular to the long edge of the film. The circular hole formed the first aperture 850 of device 800. The second film component of the device (i.e., adhesive layer 870) was prepared by laser cutting a section 72 mm in length × 20 mm in width from a sheet of 3M 1522 Double-Sided Medical Tape (a transparent double-sided acrylic adhesive with a polyethylene backing, obtained from 3M Company). The total thickness of the double-sided tape with the release liner removed was measured to be 150 micrometers using a digital caliper. A rectangular opening 880 (60 mm in length × 2.5 mm in width) was laser cut in the second film component and oriented such 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.

[0156] The third film component of the device (i.e., microstructured substrate 810) was a laser cut section 72 mm in length × 20 mm in width of the microstructured film coated with the surfactant of Preparation Example 1. All laser cutting of the films was performed using a Muse Core CO2 Laser Cutter (Full Spectrum Laser (Las Vegas, Nevada)).

[0157] Device 800 was assembled by removing all release liners from the film and then aligning the edges of the film to form a stack having a second film sandwiched between a cover sheet film and a microstructured film. The film was oriented such that the microstructured surface 830 of the third film faced the main surfaces 822 of the second film and the cover. The adhesive lamination of the stack was completed by applying a 4-pound (1.8-kilogram) roller to the stack and making one reciprocating motion of the roller. The second adhesive film formed a fluid seal (e.g., formed sidewall portions) between the cover sheet and the top surface of the microstructured film around the edges of the rectangular opening and the first aperture hole. In the final step, the stack was trimmed at the edge located distally from the first aperture using a razor blade (dashed line 862 in FIG. 8B) so that the resulting device had an overall dimension of 60 mm in length and 20 mm in width. This cut the opening of the second film, exposing the second aperture of the device as a 2.5-mm rectangular opening at the newly created edge of device 800.

[0158] Example 2 A device was prepared following the same procedure reported in Example 1, except that the second film component of the device was prepared using 3M 1513 Double-Sided Medical Tape (a transparent double-sided acrylic adhesive having a polyester backing, obtained from 3M Company). The total thickness of the double-sided tape with the release liner removed was measured to be 75 micrometers using a digital caliper.

[0159] Example 3 A device was prepared following the same procedure reported in Example 1, except that the third film component of the device was prepared using a microstructured film coated with the surfactant of Preparation Example 2.

[0160] Example 4 The device was prepared according to the same procedure reported in Example 1, except that the third film component of the device was prepared using a microstructured film coated with the surfactant of Preparation Example 3.

[0161] Comparative Example A The device was prepared according to the same procedure reported in Example 1, except that the third film component of the device was prepared using a microstructured film coated with the surfactant of Preparation Example 4.

[0162] Example 5 The device was prepared according to the same procedure reported in Example 1, except that the third film component of the device was prepared using a microstructured film coated with the surfactant of Preparation Example 5.

[0163] Example 6 The device was prepared according to the same procedure reported in Example 1, except that the third film component of the device was prepared using a microstructured film coated with the surfactant of Preparation Example 5, and the second film component of the device was prepared using 3M 1513 Double-Sided Medical Tape.

[0164]

Table 4

[0165] Comparative Example B The device was prepared according to the same procedure reported in Example 1, except that the third film component of the device was prepared using a non-microstructured polyethylene terephthalate (PET) film (MELINEX 454 film (3 mil), Dupont Teijin Films) as the third film component of the device.

[0166] Comparative Example C The third film component of the device was prepared using the non-microstructured film of Comparative Example B (MELANEX 454 PET film) as the third film component, and the second film component of the device was prepared using 3M 1513 Double-Sided Medical Tape. The device was prepared according to the same procedure as reported in Example 1, except for the above.

[0167] Example 7. Method for Separating Red Blood Cells from Blood Defibrinated sheep blood (obtained from Becton Dickinson (Franklin Lakes, NJ)) was measured to have an undiluted hematocrit concentration of 40% using a Zip-IQ PCV Centrifuge (LW Scientific Incorporated (Lawrenceville, GA)). In addition to the undiluted blood sample, diluted blood samples with hematocrits of 12%, 8%, and 4% were prepared using 1X phosphate-buffered saline (PBS).

[0168] A module for delivering a blood sample to device 800 was prepared (shown in FIGS. 8C and 8D). The module components included a silicone gasket 844 (25 mm × 25 mm × 3.2 mm) prepared from SYLGARD 184 silicone elastomer (Dow Chemical (Midland, MI)), with a 5 mm hole cut in the center of the gasket, and a sheet 842 (25 mm × 25 mm × 3.2 mm) of PLEXIGLAS polymethyl methacrylate (PMMA) film (Roehm GmbH (Darmstadt, Germany)), with a 0.06 inch (1.52 mm) diameter hole drilled in the center of the sheet. A portion of an ethyl vinyl acetate (EVA) plastic tube 846 [inner diameter 0.02 inch (0.51 mm) and outer diameter 0.06 inch (1.52 mm), McMaster-Carr (Elmhurst, IL)] was inserted into the hole in the PLEXIGLAS film sheet and secured in place with HARDMAN DOUBLE / BUBBLE epoxy (Royal Adhesives (Wilmington, CA)) 847. The opposite end of the tube was adapted to a 22.5 gauge needle attached to a 1 mL Luer lock syringe.

[0169] Device 800 (selected from the devices of Examples 1 to 6 and Comparative Examples A to C) was placed on a horizontal plane. A blood sample (20 to 50 microliters) was placed into the first aperture of the device, and the blood was wicked to the end of the device by capillary flow. Next, a silicone gasket 844 was placed on the cover sheet of the device and aligned so that the hole in the gasket was centered over the first aperture of the device. Next, a PLEXIGLAS sheet 842 (to which the EVA plastic tube 846 was adhered as described above) was placed on the exposed surface of the gasket and aligned so that the hole in the sheet was centered over the hole in the gasket, forming the assembly of FIG. 8D. A syringe (not shown) attached to the other end of the EVA plastic tube was set in a syringe pump (model NE-1600, New Era Pump Systems Inc, (Farmingdale, NY)) (not shown). The pump was operated with the flow rate set at 100 microliters / minute. A blood sample flowing out of the device was collected from the second aperture 860. A 10 microliter sample of the collected blood was diluted using 90 microliters of 1X PBS. The diluted sample was pipetted into a C-CHIP Disposable Hemacytometer according to the instructions of the manufacturer (Incyto (Republic of Korea)). For comparison, a sample of the blood placed in the device was also pipetted into the C-CHIP Disposable Hemacytometer. The counting plate was analyzed using a Zeiss LSM 510 META module Axioplan 2 upright confocal microscope (Zeiss (Jena, Germany)). Red blood cells were counted manually or by using Image J image processing software (National Institutes of Health (Bethesda, MD)). The percent decrease in red blood cells (RBC) of the blood sample using the described method was calculated by comparing the number of RBCs in the blood sample collected from the device with the number of RBCs in the blood sample placed in the device according to Equation 1. Each device was tested in triplicate (n = 3 devices) using a specific blood sample, and the results in percent RBC decrease were reported in Table 4 as the average value.

[0170]

Number

[0171]

Table 5

[0172] Example 8 A CAD design file was used to fabricate a prototype for the microstructured substrate of Figure 2B of the device using the multiphoton exposure system described in U.S. Patent No. 8,605,256 (DeVoe et al.) and U.S. Patent No. 8,455,846 (Gates et al.). A negative contrast photoresist as described in U.S. Patent No. 10,133,174 (Lee et al.) was photopatterned onto a silicon wafer substrate. Once the scanning was complete, the substrate with the patterned structure was immersed in a developing solution of propylene glycol monomethyl ether acetate (obtained from Sigma-Aldrich) to remove the unpolymerized photoresist. The prototype was then electroformed with nickel or a nickel alloy to create a metal tool for replication. An impression molding sample was made using polypropylene resin (C700-35 resin, Dow Chemical (Midland, MI)) with a nickel plating tool. The platen of a Carver press (Carver (Wabash, IN)) was heated to 170 °C, and the tool and resin were pressed together at 1000 pounds force (about 4450 Newtons) for 7 minutes, followed by cooling under pressure until the platen temperature reached 80 °C. The pressure was released, and the molded microstructured substrate was removed from the tool.

[0173] The formed microstructured substrate had upper and lower surfaces with overall dimensions of 25.4 mm (width), 76.2 mm (length), and 1 mm (depth). The microstructure of the substrate was an array of linear prism microstructures located along the floor of a flow channel (3 mm wide, 60 mm long) that indented below the upper surface of the substrate and had first and second open ends. The linear array of peak structures and adjacent valleys was oriented at an angle of 0 degrees with respect to the liquid flow direction of the completed device. The characteristics of the linear prism microstructures are reported in Table 5. The prism features protruded upward from the floor of the flow channel. The walls surrounding the flow channel extended from 100 micrometers above the tip of the prism structure to the upper surface. The first open end of the flow channel was fluidly attached to a semi-circular first cavity having a depth of 100 micrometers from the upper surface and a volume of 2.88 microliters. The first cavity formed the first aperture of the device. The first aperture functioned as a reservoir for taking in the liquid sample of the final device. The second open end on the opposite side of the flow channel was fluidly attached to a rectangular second cavity (5 mm wide, 6 mm long, 250 micrometers deep from the top surface). The second cavity formed the second aperture of the device. The second aperture functioned as a receiving reservoir for the liquid sample exiting the flow channel.

[0174] The cover component of the device was a section of 3M Microfluidic Diagnostic Film 9975R (obtained from 3M Company). The cover was positioned over the flow channel and liquid sample intake reservoir section and attached with an adhesive to the surface of the microstructured substrate surrounding the sample intake reservoir and flow channel section. Prior to application, a circular hole (5 mm in diameter) was laser cut into the cover component and oriented so that the center of the hole was positioned over the center of the sample intake reservoir when the cover was attached to the microstructured substrate. The receiving reservoir was not covered.

[0175]

Table 6

[0176] Example 9 The device was prepared according to the same procedure as described in Example 8, except that a linear array of peak structures and adjacent valleys was oriented at an angle of 45 degrees with respect to the liquid flow direction of the completed device (the orientation shown in FIG. 5D); the dimensions of the flow channel were 3 mm in width and 40 mm in length; the first aperture cavity had a depth of 300 micrometers and a volume of 8.7 microliters, and the second aperture cavity had dimensions of 5 mm (width), 6 mm (length), and 300 micrometers (depth).

[0177] Example 10 The device was prepared according to the same procedure as described in Example 9, except that a linear array of peak structures and adjacent valleys was oriented at an angle of 90 degrees (i.e., a right angle) with respect to the liquid flow direction of the completed device (the orientation shown in FIG. 5C).

[0178] Example 11 The device was prepared according to the same procedure as described in Example 8, except that the linear array of prism microstructures was replaced by an array of fluid-connected wells having a circular shape connected to adjacent wells by vents (shown in FIGS. 10A - 10B). Each well had a diameter of 200 micrometers, a depth of 150 micrometers, and a taper of 5 degrees. The vents had a length of 29 micrometers, a width of 40 micrometers, a depth of 150 micrometers, and a taper of 5 degrees. The wells located at the center of the flow channel were each connected to four other wells, each connection being via a vent, and the vent spacing was as shown in FIG. 10B. The wall surrounding the flow channel extended 100 micrometers above the top surface of the wells. The dimensions of the flow channel were 3 mm in width and 40 mm in length.

[0179] The first open end of the flow channel was fluidly attached to a semi-circular first cavity having a depth of 100 micrometers from the upper surface and a volume of 2.88 microliters. The first cavity formed the first aperture of the device. The second open end on the opposite side of the flow channel was fluidly attached to a rectangular second cavity (width 5 mm, length 6 mm, depth 250 micrometers from the top surface). The second cavity formed the second aperture of the device.

[0180] Example 12 Method for Separating Red Blood Cells from Blood Human blood was collected into BD VACUTAINER citrate tubes (Becton, Dickinson and Company (Franklin Lakes, NJ)) and used as citrate whole blood or citrate whole blood diluted 1:1 with 1X phosphate buffered saline (PBS).

[0181] The microstructured substrates described in Examples 8 - 11 were prepared, and a modified cover component with a tube for blood dripping was attached to each substrate. The modified cover component was a section of 3M Microfluidic Diagnostic Film 9975R (obtained from 3M Company), with a circular hole (diameter 5 mm) laser cut in the cover component. A plastic tube (0.05 inch ID, 0.09 inch OD) was inserted into the hole and fixed with an epoxy adhesive (3M SCOTCH-WELD Epoxy Adhesive DP100 Plus Clear, obtained from 3M Company). Any tube extending beyond the adhesive surface of the cover component was removed with a pair of scissors. The cover was positioned over the flow channel section and the liquid sample intake reservoir section of the microstructured substrate and attached with an adhesive to the surface of the microstructured substrate surrounding the two sections. The cover component was oriented such that the center of the hole was positioned above the center of the sample intake reservoir when the cover was attached. The receiving reservoir was not covered. The tube extended approximately 3 cm from the outer surface of the cover.

[0182] Each of the obtained devices was placed on a horizontal plane (oriented such that the lower surface of the device faced the horizontal plane). Using a micropipette, a blood sample (20 - 50 microliters) was added through a tube to the sample inlet reservoir. The blood sample was wicked to the end of the device by capillary flow or advanced using positive pressure from the micropipette. A sufficient amount of blood was introduced into the device to fill the open volume of the flow channel without accumulating excess blood in the capture reservoir. Excess blood in the receiving reservoir was quickly removed from the reservoir using a micropipette or a KIMWIPE wiper (Kimberly-Clark Corporation (Irving, TX)). After administration of the blood sample, each device was left stationary on a horizontal surface for 5 minutes.

[0183] A 1 mL Luer-lock syringe filled with mineral oil was attached to a portion of a plastic tube, and the mineral oil was partially dispensed into the tube, leaving a small air gap (about 10 cm in length of the tube) at the open end of the tube. The syringe was set on a syringe pump (Model NE-1600, New Era Pump Systems Inc.). The open end of the tube of the syringe assembly was connected to the open end of the tube extending from the device. The pump was operated at a flow rate set at 50 microliters / minute to push the blood sample out of the flow channel together with the volume of air trapped. The blood sample flowing out of the flow channel was collected as several 2 - 4 microliter aliquots. Each sample was collected as soon as the aliquot volume accumulated in the receiving reservoir.

[0184] For each sample taken, diluted samples of 1 / 10, 1 / 20, 1 / 100, and 1 / 200 in 1X PBS were prepared. 2 microliter aliquots of each diluted sample were loaded onto an Agilent Take3 microvolume plate (Take3-SN, Agilent Technologies (Santa Clara, CA)) using a micropipette. The plate included at least 1 well as a 1X PBS blank used for dilution according to the manufacturer's instructions. For each sample and 1X PBS blank, absorbance measurements were recorded at 406 nm, 414 nm, and 576 nm using an Agilent Synergy Neo2 plate reader (Agilent Technologies). Using the procedure described in the section "Method for Measuring the Intact Red Blood Cell Content of a Sample" (described below), the percentage decrease in red blood cells from the blood samples subjected to each device was calculated using the procedure described in this example.

[0185] The results using citrated human whole blood are shown in Table 6, and the results using citrated human whole blood diluted 1:1 with 1X PBS are shown in Table 7. The device of Comparative Example B (i.e., the device without a microstructured surface) was also tested. For each type of device, the results reported in Tables 6 and 7 are from technical replicates of 3 devices (n = 3). The percentage decrease in red blood cells calculated for each replicate device was averaged over the total volume of the aliquot samples taken from the device.

[0186]

Table 7

[0187]

Table 8

[0188] Method for Measuring the Intact Red Blood Cell Content of a Sample To simultaneously measure the content of intact red blood cells and lysed red blood cells in a sample, a calibration curve for the dilution series was created using known inputs of lysed human red blood cells and intact human red blood cells. Citrate-treated human whole blood was used as a 100% intact red blood cell sample. To create a 0% intact red blood cell sample, an aliquot of whole blood was lysed by vortexing for 1 minute using a ZR BashingBead lysis tube (product number S6012-50, Zymo Research (Irvine, CA)). The tube was centrifuged at 10,000 x g for 1 minute, and the supernatant containing only the lysed cell contents was transferred to a new 1.5 mL Eppendorf tube. Standard samples of intact red blood cells versus lysed red blood cells for the dilution series were prepared by mixing known intact cells and lysed cells at various ratios in the range of 100% to 0% intact red blood cells. The intact red blood cell concentration of the standard samples was confirmed using a standard C-CHIP Disposable Hemacytometer according to the manufacturer's instructions.

[0189] Each standard sample was diluted with 1X PBS (so that the absorbance was within the dynamic range of the plate reader), and analyzed in triplicate technically using an Agilent Synergy Neo2 plate reader equipped with a Take 3 multi-volume plate (sample volume and blank were selected according to the manufacturer's instructions). For each standard sample, the absorbance was measured at 406 nm, 414 nm, and 576 nm).

[0190] Ratio A 414nm / A 576nm was plotted against the known input of % intact red blood cells. A 414nm is the absorbance of the sample at 414 nm, and A 576nm is the absorbance of the sample at 576 nm. For background subtraction, subtraction of the blank (1X PBS) was used. The dataset was fitted to a logarithmic curve to create Equation A. Equation A was used to calculate the percentage of intact red blood cells from a suspension containing blood.

[0191]

Number

[0192] In Formula A, “% Intact” = the percentage of red blood cells in the suspension that were intact, “A 414nm ” = the absorbance of the blood sample at a wavelength of 414 nm, “A PBS,414nm ” = the absorbance of 1X PBS at a wavelength of 414 nm, “A 576nm ” = the absorbance of the blood sample at a wavelength of 576 nm, and “A PBS,576nm ” = the absorbance of 1X PBS at a wavelength of 576 nm.

[0193] The absorbance signal at 406 nm for each sample was adjusted by the ratio of cell percentages, and the signal from intact red blood cells was calculated using Formula B.

[0194]

Number

[0195] In Formula B, “Ai 406nm ” = the absorbance of the blood sample at a wavelength of 406 nm attributable to intact red blood cells, “% Intact” = the percentage of red blood cells in the suspension that are intact calculated by Formula A, “A 406nm ” = the absorbance of the sample at a wavelength of 406 nm, and “A PBS,406nm ” = the absorbance of 1X PBS at a wavelength of 406 nm.

[0196] After adjusting the absorbance at 406 nm for intact cells and lysed cells in Formula B, the absorbance signal of the blood sample placed in the device was compared with the absorbance signal of the corresponding blood aliquot taken from the device in accordance with Formula C to calculate the percent decrease in red blood cells (RBC).

[0197]

Number

[0198] In Formula C, "Ai" 406nm,input " = the absorbance at a wavelength of 406 nm of the blood sample placed in the device due to intact red blood cells, calculated by Formula B, and "Ai" 406nm,output " = the absorbance at a wavelength of 406 nm of the blood sample taken from the device due to intact red blood cells, calculated by Formula B.

[0199] Preparation Example 6. Microstructured Film with Upright Stems The polypropylene (PP) microstructured film of FIG. 11 having a discrete stem structure with angled sidewalls was prepared by the molding process according to Example 1 of U.S. Patent No. 9,358,714 (Chandrasekaran) (however, no beta nucleating masterbatch was included), and then the film was corona treated using a BD-20AC Laboratory Corona Treater (Electro-Technic Products (Chicago, IL)). A three-dimensional micrograph of the microstructured film was taken using a Keyence VK-X3100 3D Surface Profilometer (Keyence Corporation (Itasca, IL)), and measurements were made using the attached VK-X 3000 MultiFileAnalyzer software package. The microstructured film had a total thickness of about 345 micrometers and had a staggered array of 2000 stem features per square inch. The stem features had a substantially flat surface at the top portion (i.e., frustum of a cone shape). The thickness of the backing layer was 83.5 micrometers. Tables 8 and 9 report the dimensions of the stem height, the diameter of the stem at the base in the down-web direction, the diameter of the stem at the base in the cross-web direction, the diameter of the stem at the top portion in the down-web direction, the diameter of the stem at the top portion in the cross-web direction, the center-to-center distance (pitch) between the stems in the down-web direction, and the center-to-center distance (pitch) between the stems in the cross-web direction.

[0200] [Table 9]

[0201]

Table 10

[0202] Example 13. Preparation of a device containing a microstructured film having an upright stem The following changes were made to the procedure described in Example 1. First, the microstructured film of Preparation Example 1 was replaced with the microstructured film of Preparation Example 6 as the third film component of the device. Second, epoxy was used to seal the two long edges of the device and the narrow edge adjacent to the first aperture. The narrow edge where the second aperture was formed was not sealed.

[0203] Example 14. Preparation of a device containing a microstructured film having an upright stem and a flocculant coating of a microstructured array A flocculant solution of polyethyleneimine (PEI) (branched, molecular weight 70,000 Da, 30 wt / vol% aqueous solution, catalog number 00618, obtained from Polysciences, Inc. (Warrington, PA)) was further diluted in two steps with 1:10 (weight: volume) deionized water, followed by 1:10 volume: volume deionized water. The diluted flocculant solution (30 - 100 microliters) was pipetted into the rectangular opening 880 of the device of Example 13. The applied PEI flocculant solution was air-dried overnight under ambient conditions to provide a microstructured array coated with the flocculant.

[0204] Example 15. Preparation of a device containing a microstructured film having an upright stem and a flocculant coating of a microstructured array An aqueous solution of a guanylated polyethyleneimine (G-PEI) flocculant was prepared as described in Example 1 of U.S. Patent No. 10,087,405 (Swanson et al.), except that butanediol diglycidyl ether was not added. A flocculant solution (30 - 100 microliters) was pipetted into the rectangular opening 880 of the device of Example 13. The applied G-PEI flocculant solution was air-dried overnight under ambient conditions to provide a microstructured array coated with the flocculant.

[0205] Example 16. Method for separating red blood cells from blood Human whole blood collected in an EDTA tube was obtained from the Oklahoma Blood Institute (Oklahoma City, OK). The initial hematocrit of the human blood sample was measured using a Zip-IQ PCV Centrifuge (LW Scientific Incorporated, Lawrenceville, GA).

[0206] In this method, a module having a syringe pump for delivering the blood sample described in Example 7 was used. A device selected from Examples 13 - 15 was placed on a horizontal surface. A sample of human whole blood (50 - 100 microliters) was placed into the first aperture 850 of the device. The blood was wicked to the end of the device by capillary flow, and the device was left standing for 10 minutes to allow the red blood cells to sediment into the microstructured array section. Next, the pump was operated with the flow rate set to 100 microliters / min. The blood sample flowing out of the device from the second aperture 860 was collected as a series of 5-microliter aliquots.

[0207] A standard calibration curve was created from a dilution series of whole blood in water to correlate the RBC count with the absorbance at 406 nm. Each blood sample to be tested was diluted with water (to lyse the cells and bring the absorbance within the dynamic range of the plate reader), and analyzed in duplicate technically using an Agilent Synergy Neo2 plate reader equipped with a Take 3 multi-volume plate (sample volume and blank were selected according to the manufacturer's instructions). All dilutions were prepared using deionized water with a resistivity greater than 18 megaohm-cm prepared from a SYNERGY UV Water Purification System (MilliporeSigma (Burlington, MA)). The absorbance at 406 nm was measured for each sample. The percent reduction of red blood cells (RBC) in the blood sample was calculated by comparing the absorbance of an aliquot of blood taken from the device with the absorbance of the whole blood sample placed in the device according to Equation D.

[0208] [Number]

[0209] In Equation D, "Absorbance 406nm,output " = the absorbance at 406 nm wavelength of the blood sample taken from the device. "Absorbance 406nm,input " = the absorbance at 406 nm wavelength of the blood sample placed in the device. For each type of device, the results are reported in Table 10. A single device of each type was tested. The calculated percent reduction of red blood cells was averaged over all the aliquot samples taken from the device. Approximately 10 aliquot samples were taken from each device.

[0210] [Table 11]

[0211] Example 17. Method for Separating Red Blood Cells from Blood Using the single device (n = 1) of Example 13 and a blood sample containing a PEI aggregating agent, the method of Example 16 was followed. For the blood sample, an aggregating agent solution of polyethyleneimine (PEI) (branched, MW of 70,000 Da, 30% weight / volume aqueous solution, catalog number 00618, obtained from Polysciences, Inc.) was prepared by diluting it 1 / 10 in PBS (e.g., 1 mL aggregating agent + 9 mL of 1X PBS). A 90 microliter aliquot of human whole blood was mixed with 10 microliters of the aggregating agent 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 placed into the first aperture of the device. A total of seven aliquots were taken from the device. The calculated percent RBC reduction was 64.4%. The calculated percent reduction of red blood cells was averaged over all of the aliquot samples taken from the device.

[0212] All of the above patents and patent applications are hereby expressly incorporated by reference herein. The above-described embodiments are illustrative of the present invention, and other configurations are 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 rather should be considered to be limited only by the fair scope of the following claims and their equivalents.

Claims

1. A method for separating red blood cells from blood, A microstructured substrate comprising a plurality of microstructures extending across a first surface thereof, wherein at least a portion of the outer surface of the plurality of microstructures is configured to enable capillary action, A cover is positioned at a distance selected from the top of the first surface of the microstructured substrate, At least one side wall portion is provided along the periphery of the first surface of the microstructured substrate for attaching the cover to the first surface of the microstructured substrate, A first aperture defined by at least one of the microstructured substrate or the cover, A second aperture defined by at least one of the microstructured substrate or the cover, The first surface of the microstructured substrate, together with the at least one side wall portion, defines a first open volume which is the sum of the open spaces located between the plurality of microstructures from the bottom to the top of each microstructure; the cover, together with the top of the first surface of the microstructured substrate and the at least one side wall portion, defines a second open volume located adjacent to the first open volume; and if the sum of the first open volume and the second open volume is considered to be 100% open volume, then the first open volume has a percentage greater than the volume percentage of red blood cells present in the blood, thereby obtaining a device. The device is filled with a certain volume of blood through the first aperture by capillary action, A sufficient amount of time is allowed for at least a portion of the red blood cells to settle into the first open volume of the plurality of microstructures. A method comprising applying pressure to the device, thereby causing at least 10% of the initial volume of blood in which at least a portion of the red blood cells are held within the first open volume of the plurality of microstructures to flow out of the device through either the first aperture or the second aperture.

2. The method according to claim 1, wherein the at least one side wall portion is part of the microstructured substrate.

3. The method according to claim 1, wherein at least 15% of the blood flows out of the device when the pressure is applied.

4. The method according to claim 1, wherein the time is sufficient for at least 20% of the red blood cells to settle into the first open volume of the plurality of microstructures.

5. The method according to claim 1, wherein the microstructured substrate is a microstructured film.

6. The method according to claim 1, wherein at least a portion of the outer surface of the plurality of microstructures contains a substance selected from the group consisting of surfactants, surface treatment agents, hydrophilic polymers, flocculants, and any combination thereof.

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

8. The method according to claim 6, wherein the flocculant comprises a modified or unmodified amino polymer selected from the group consisting of polyethyleneimine, polylysine, polyaminoamide, polyallylamine, polyvinylamine, polydimethylamine-epichlorohydrin-ethylenediamine, polydiallyldimethylammonium chloride, cationic polyacrylamide (CPAM), polyaminosiloxane, and dendrimers formed from polyaminediamine (PAMAM) and polypropyleneimine.

9. The method according to claim 6, wherein the flocculant comprises a modified or unmodified polyethyleneimine polymer.

10. The method according to claim 6, wherein at least a portion of the coagulant is dissolved in the blood after the device has been filled with the volume of blood.

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

12. The method according to claim 1, wherein the pressure is positive pressure.

13. The method according to claim 1, wherein the pressure is negative pressure.

14. The method according to claim 1, wherein the first aperture is defined by the cover.

15. The method according to claim 1, wherein the second aperture is defined by the microstructured substrate and the cover.

16. The method according to claim 1, wherein the blood is not diluted.

17. The method according to claim 1, wherein the red blood cells settle solely by gravity.

18. The method according to claim 1, wherein the device further comprises an adhesive layer disposed between the cover and the microstructured substrate.

19. The method according to claim 1, wherein the microstructure comprises a plurality of ribs arranged alternately with channels extending across the first surface of the microstructured substrate, each of the ribs having a side wall and a top surface, and each of the channels having a bottom surface.

20. The method according to claim 19, wherein the top surface of each rib is the top of a cap positioned on a side wall, and the cap has a width greater than the width between the side walls on both sides.

21. The method according to claim 1, wherein the microstructure comprises an array of peak structures and adjacent valleys, the valleys having a maximum width in the range of 10 microns to 500 microns, and the peak structures having an apex angle of more than 5 degrees to 90 degrees.

22. The method according to claim 21, wherein the array of peak structures extending across the first surface of the microstructured substrate and adjacent valleys is oriented at an angle of 0 to 90 degrees with respect to the flow direction of the device.

23. The method according to claim 11, wherein the array of the peak structure and adjacent valleys further comprises gaps between adjacent peak structures.

24. The method according to claim 1, wherein the microstructure comprises a two-dimensional (x-axis and y-axis) array of protrusions arranged across the first surface of the microstructured substrate, and 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 claim 1, wherein the microstructured substrate comprises a microstructured layer having first and second main surfaces, the microstructure comprises a plurality of cavities extending between the first and second main surfaces, each cavity comprising 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 claim 1, wherein the microstructure comprises facets and side walls that intersect the facets at the ridges of the microstructure, and the facets and the side walls define an oblique angle between them.

27. The method according to claim 1, wherein the microstructure comprises an array of fluid-connected wells, at least some of the wells being fluid-connected to at least two adjacent wells, each connected via a vent.

28. The method according to claim 1, wherein the microstructure comprises an array of upright stems extending across the first surface of the microstructured substrate.

29. The method according to claim 1, wherein the volume of blood filled through the first aperture is a maximum of 100 microliters of blood.

30. The method according to claim 1, wherein the ratio of the first open volume to the second open volume is greater than 1:

1.

31. The method according to claim 1, further comprising: passing the blood through a filter before it enters the device; passing the blood, in which at least a portion of the red blood cells are retained within the first open volume of the plurality of microstructures, through the device after it has left the device; or both.

32. The method according to claim 1, further comprising adding a coagulant to the volume of blood before filling the device with the volume of blood.

33. The method according to claim 32, wherein the coagulant is added and present in an amount of 0.01 to 5000 micrograms per 1 mL of blood.

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