Microstructured substrate containing connected wells - Patent Application 20070122997

Abstract

The present disclosure provides a microstructured substrate. The microstructured substrate includes microstructures extending across a surface of the microstructured substrate. The microstructures comprise an array of interconnected wells, at least some of which are fluidly connected to at least two adjacent wells, each connection via a vent. Each well has an open volume ranging from 100 femtoliters to 1 microliter. At least a portion of the exterior surfaces of the plurality of microstructures are configured to allow capillary action.

Description

[Technical Field]

[0001] Many devices (e.g., diagnostic devices, fluid transport films, etc.) utilize capillary action to move aqueous fluids. Further development of substrates that utilize capillary motion is desirable. Summary of the Invention

[0002] In a first aspect, a microstructured substrate is provided. The microstructured substrate comprises a plurality of microstructures extending across a first surface of the microstructured substrate. The microstructures comprise an array of interconnected wells, at least some of the wells being fluidly connected to at least two adjacent wells, each connection being via a vent. Each well has an open volume ranging from 100 femtoliters to 1 microliter. At least a portion of the exterior surface of the plurality of microstructures is configured to allow capillary action.

[0003] It has been discovered that devices and methods according to at least certain embodiments of the present disclosure can provide for the removal of solid particles (e.g., red blood cells) from very small (e.g., microliter) volumes of fluid (e.g., blood).

[0004] The above summary of the present disclosure is not intended to describe each embodiment or every implementation disclosed in the present disclosure. The following description more particularly illustrates exemplary embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list. [Brief explanation of the drawings]

[0005] [Figure 1A] 1 is a schematic perspective view of a portion of an exemplary microstructured substrate having an array of fluidically connected wells. [Figure 1B]FIG. 1 is a schematic top view of a portion of a microstructured substrate having an array of fluidically connected wells having circular shapes. [Figure 2] 1 is a schematic cross-sectional view of a portion of an exemplary microstructured substrate having an array of fluidically connected wells. [Figure 3A] FIG. 1 is a schematic top view of a portion of a microstructured substrate having an array of fluidly connected wells with circular shapes and triangular vents. [Figure 3B] 3B is a schematic perspective view of a portion of the microstructured substrate of FIG. 3A. [Figure 4A] FIG. 1 is a schematic top view of a portion of a microstructured substrate having an array of fluidly connected wells with circular shapes and rounded bottoms. [Figure 4B] 4B is a schematic perspective view of a portion of the microstructured substrate of FIG. 4A. [Figure 5A] FIG. 1 is a schematic top view of a portion of a microstructured substrate having an array of fluidically connected wells having triangular shapes. [Figure 5B] FIG. 5B is an enlarged view of a portion of FIG. 5A. [Figure 5C] 5B is a schematic perspective view of a portion of the microstructured substrate of FIG. 5A. [Figure 6A] FIG. 1 is a schematic top view of a portion of a microstructured substrate having an array of fluidically connected wells having square shapes. [Figure 6B] 6B is a schematic perspective view of a portion of the microstructured substrate of FIG. 6A. [Figure 7A] FIG. 1 is a schematic top view of a portion of a microstructured substrate having an array of fluidically connected wells having hexagonal shapes. [Figure 7B] 7B is a schematic side view of a portion of the microstructured substrate of FIG. 7A. [Figure 8A] 1 is a generalized exploded schematic diagram of a device in which an exemplary microstructured substrate may be used. [Figure 8B] FIG. 8B is a generalized schematic top view of the device of FIG. 8A. [Figure 8C]FIG. 8C is a generalized schematic top view of two components used to attach a fixed pump to the device of FIGS. 8A-8B. [Figure 8D] FIG. 8C is a generalized schematic perspective view of the device of FIGS. 8A-8B adapted to be attached to a pump. [Figure 9] FIG. 1 is a perspective view of a generalized schematic of a microstructured substrate for use in a device. DETAILED DESCRIPTION OF THE INVENTION

[0006] The above-identified figures set forth several embodiments of the present disclosure; however, as noted in the description, other embodiments are also contemplated. The figures are not necessarily drawn to scale. In all cases, this disclosure presents the invention by way of representation and not limitation.

[0007] As used herein, the term "microreplication" refers to the production of microstructured surfaces by a process in which the structured surface features retain individual feature fidelity during fabrication.

[0008] As used herein, the term "microstructure" encompasses both structures (i.e., features) that protrude above a major surface of a substrate and structures that are recessed below the major surface of the substrate. Combinations of protruding and recessed features are contemplated. Microstructure further means that the structure is a predetermined molded structure (e.g., as obtained by molding a polymeric thermoplastic against a tool surface that has a negative of the microstructure desired to be provided on the first major surface of the substrate) having dimensions in at least two orthogonal directions ranging from about 5 micrometers to about 3000 micrometers. One of these orthogonal directions can 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.

[0009] As used herein, the term "capillary action" refers to the flow of a fluid without the assistance of an external force (e.g., pressure, gravity, vacuum, etc.). Capillary action often occurs for aqueous fluids in contact with hydrophilic surfaces. Aqueous fluids contain 50% or more water by volume.

[0010] As used herein, the term "hydrophilic" refers to a surface that is wetted by aqueous solutions, and does not describe whether the material absorbs the aqueous solution. "Wetting" means that the surface exhibits spontaneous wicking when contacted with aqueous fluids. "Spontaneous" means that it occurs without an external force. In some embodiments, a hydrophilic surface exhibits an advancing (maximum) water contact angle of less than 90°, preferably 45° or less.

[0011] As used herein, the term "hydrophobic" refers to a surface that lacks spontaneous wicking when contacted with aqueous fluids. In some embodiments, a hydrophobic surface exhibits an advancing water contact angle of 70° or greater, preferably 90° or greater.

[0012] As used herein, "curing" means solidifying or partially solidifying a composition by any mechanism, e.g., heat, light, radiation, electron beam, microwave, chemical reaction, or a combination thereof. As used herein, the term "solidifiable" refers to a material that can be hardened or solidified by, for example, heating to remove solvent, heating to cause polymerization, chemical crosslinking, radiation-induced polymerization or crosslinking, etc. As used herein, "cured" refers to a material or composition that has been hardened or partially solidified (e.g., polymerized or crosslinked) by curing.

[0013] As used herein, a polymer "film" is a polymer material in the form of a generally flat sheet that is flexible and strong enough to be processed in a roll-to-roll manner. Roll-to-roll refers to a process in which the material is wound onto or unwound from a support and further processed in some way. Examples of further processing include coating, slitting, blanking, and exposure to radiation. Polymer films can be produced in a variety of thicknesses, generally ranging from about 5 micrometers to 1000 micrometers.

[0014] As used herein, "solid" refers to a state of matter that is neither a liquid nor a gas, and a solid has a stable three-dimensional form.

[0015] As used herein, "fluid" refers to a composition that contains a liquid (i.e., a state of matter that is neither solid nor gas), and includes solutions, suspensions, and emulsions.

[0016] As used herein, "outer surface" with respect to a microstructure refers to the outermost surface of the microstructure.

[0017] As used herein, "thermoplastic" refers to a polymer that flows when heated sufficiently above its glass transition point and becomes solid when cooled. In contrast, "thermoset" refers to a polymer that becomes permanently hardened upon curing and does not flow upon subsequent heating. Thermoset polymers are typically crosslinked polymers.

[0018] As used herein, the "glass transition temperature" (T g The term T refers to the transition of a polymer from a glassy to a rubbery state and can be measured using Differential Scanning Calorimetry (DSC), for example, at a heating rate of 10°C per minute in a nitrogen stream. g When a reference is made to a T, it refers to the T of the homopolymer of that monomer. g The homopolymer is T gThe molecular weight must be high enough so that the T g It is generally recognized that the T increases with increasing molecular weight up to a critical value. Homopolymers also contain moisture, residual monomers, solvents, and g It is also understood that the DSC is substantially free of other contaminants that may affect the performance of the polymer. A suitable DSC method and analysis mode is as described in Matsumoto, A. et al., J. Polym. Sci. A., Polym. Chem. 1993, 31, 2531-2539.

[0019] As used herein, "transparent" refers to a material (e.g., a layer) that has at least 50% transmittance, 70% transmittance, or optionally greater than 90% transmittance over at least the 400 nanometer (nm) to 700 nm portion of the visible light spectrum.

[0020] The words "preferred" and "preferably" refer to embodiments of the present disclosure that may offer certain benefits, under particular circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, or is intended to exclude other embodiments from the scope of the present disclosure.

[0021] In this application, terms such as "a," "an," and "the" are not intended to refer only to a singular entity, but include a general class 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 "including at least one of," followed by a list, refer to any one of the items in the list, and any combination of two or more items in the list.

[0022] As used herein, the term "or" is generally used in its ordinary sense, including "and / or," unless the context clearly dictates otherwise. The term "and / or" refers to one or all of the listed elements or a combination of any two or more of the listed elements.

[0023] Also, all numbers herein are intended to be modified by the term "about," and preferably by the term "exactly." When used herein in connection with a measured quantity, the term "about" refers to the variation in the measured quantity that would be expected by one of ordinary skill in the art making the measurement and exercising a level of care commensurate with the purpose of the measurement and the precision of the measuring device used.

[0024] The term "generally," when used herein as a modifier to a characteristic or attribute, unless specifically defined otherwise, means that the characteristic or attribute is readily recognizable by one of ordinary skill in the art, but does not require absolute precision or perfect agreement (e.g., within + / - 20% for quantifiable characteristics). The term "substantially," unless specifically defined otherwise, means a high degree of approximation (e.g., within + / - 10% for quantifiable characteristics), but again does not require absolute precision or perfect agreement. Terms such as same, equal, uniform, constant, exactly, etc., are understood to be within normal tolerances or measurement errors applicable to the particular circumstances, rather than requiring absolute precision or perfect agreement.

[0025] As described above, at least a portion of the outer surface of the plurality of microstructures is configured to allow capillary action. Capillary action is known in the art to refer to the flow of fluid without the assistance of an external force, typically for aqueous fluids (having 50% or more water by volume) in contact with a hydrophilic surface. Thus, when a fluid is introduced into a microstructured substrate, the fluid spontaneously transports along the outer surface of the microstructure, thereby spreading within the area of ​​the microstructure. Two general factors affect the ability of a microstructure to spontaneously transport fluid are (i) the structure or topography of the surface (e.g., capillary, cavity shape) and (ii) the properties of the surface (e.g., surface energy). To achieve a desired amount of fluid transport capability, designers may adjust the structure or topography of the substrate layer and / or the surface energy of the capillary microstructured surface. To achieve wicking, the surface of the capillary microstructure must be "wettable" by the liquid (e.g., a substance in a liquid state) to be transported. Optionally, the wettability of a solid surface by a liquid is characterized by the contact angle that a liquid makes with the solid surface after being deposited on a horizontally positioned surface and allowed to settle thereon. This angle is sometimes referred to as the "static equilibrium contact angle," or simply the "advancing contact angle" herein. In some cases, a material is considered hydrophilic if it has an advancing contact angle of less than 90 degrees, while a hydrophilic surface exhibits an advancing (maximum) water contact angle of less than 90 degrees, preferably 45 degrees or less.

[0026] A sufficient portion of the exterior surface of the microstructures must be hydrophilic so that when fluid is introduced through the first aperture, the microstructured substrate can undergo capillary action to transport the fluid (e.g., blood) throughout the device, allowing solid particles to access the open volume between the microstructures and settle out of the bulk of the fluid. For example, with reference to FIG. 9 , the first aperture 950 can be provided as a reservoir configured to hold at least a certain minimum volume of fluid. The reservoir can be defined by a portion of the microstructured substrate 910 adjacent one end 937 of the microstructure 930. Similarly, the second aperture 960 can be a reservoir configured to hold fluid as it exits the microstructured surface 902 of the microstructured substrate 910. Note that the microstructured surface 902 does not depict interconnected wells, but rather a general representation of linear microstructures.

[0027] In some cases, 50% or more of the area of ​​the exterior surface of the microstructure can undergo capillary action, and 60%, 70%, 80%, 90%, or 95% or more of the exterior surface of the microstructure can undergo capillary action. The hydrophilicity of the exterior surface of the microstructure, according to any device described herein, can be achieved by one or more of material selection, additives included in the material, or surface treatment. In some embodiments, the microstructure has an exterior surface comprising a surfactant, a surface treatment, a hydrophilic polymer, or any combination thereof. Suitable surfactants include, but are not limited to, for example, C8-C18 alkane sulfonate, C8-C18 secondary alkane sulfonate, alkyl benzene sulfonate, C8-C18 alkyl sulfate, alkyl ether sulfate, sodium laureth 4 sulfate, sodium laureth 8 sulfate, dioctyl sulfosuccinate, sodium salt, lauroyl acylate, stearoyl lactylate, or any combination thereof. The one or more surfactants can be applied by conventional methods, for example, by spreading a coating of the surfactant on the surface of the microstructure and allowing the coating to dry. Suitable surface treatments include hydrophilic coatings comprising plasma-deposited silicon / oxygen materials and / or diamond-like glass (DLG) materials. Plasma deposition of silicon / oxygen materials and DLG materials is described, for example, in PCT Publication WO 2007 / 075665 (Somasiri et al.). Further, examples of suitable DLG materials are disclosed in U.S. Patent Nos. 6,696,157 (David et al.), 6,881,538 (Haddad et al.), and 8,664,323 (Iyer et al.). Suitable hydrophilic polymers include, but are not limited to, polyesters, polyamides, polyurethanes, poly(vinyl alcohols), poly(alkylene glycols), poly(alkylene oxides), poly(vinylpyrrolidones), rubber elastomers, or any combination thereof.

[0028] In a first aspect, the present disclosure provides a microstructured substrate comprising a plurality of microstructures extending across a first surface of the microstructured substrate, the microstructures comprising an array of interconnected wells, at least some of the wells fluidly connected to at least two adjacent wells, each connection being via a vent, each well having an open volume in the range of 100 femtoliters to 1 microliter, and at least a portion of an outer surface of the plurality of microstructures configured to allow capillary action.

[0029] Various devices, fluid transport films, and the like utilize capillary action to move aqueous fluids, as described, for example, in PCT Publication Nos. WO 2000 / 042958 (Johnston et al.), WO 2012 / 1589990 (Ludowise et al.), WO 2015 / 164632 (Halverson et al.), WO 2015 / 164468 (Meuler et al.), and WO 2021 / 124165 (Swanson et al.), which are incorporated herein by reference.

[0030] Microstructured substrates according to at least certain embodiments of the present disclosure can be used for aqueous fluid transport by capillary action. Another exemplary use of articles that provide capillary flow is for the separation of particles from fluids, as described in commonly owned applications 63 / 425468 (Docket No. PA100124US02) and 63 / 425483 (Docket No. PA100776US01).

[0031] In addition to particles in general, red blood cells may be removed from blood. In centralized hospital or clinical laboratories, red blood cell separation is performed via centrifugation. The separation process requires a large volume (e.g., milliliters) of blood drawn from a vein into a test tube. During centrifugation, the red blood cells are packed at the bottom of the tube, while the remaining blood (e.g., cell-free plasma) remains accessible in the upper layer for further analysis. In these centralized settings, biomarker detection is then typically performed on large, complex analyzers capable of automated liquid handling and accessible for frequent verification of assay performance via calibration. Point-of-care hematology analyzers used outside of central laboratories also require red blood cell removal before analysis. Access to intravenous blood volumes and benchtop centrifugation are often unavailable or too time-consuming in situations where rapid results are required. A fingerstick draws approximately 5 microliters of blood. Glucose test strips typically accept blood volumes less than 1 microliter and are subject to the interferences described above. Removing red blood cells from these small volumes remains a challenge.

[0032] Point-of-care biomarker analysis requires simple and efficient separation of red blood cells from microliter volumes of blood without hemolysis, dilution, or significant loss of blood to dead space. One suitable use of microstructured substrates according to the present disclosure is to provide wells into which small blood samples can be spread and into which red blood cells can settle and separate from the blood.

[0033] Without wishing to be bound by theory, it is believed that microstructured substrates according to at least certain embodiments of the present disclosure minimize the occurrence of trapped air bubbles as small amounts of fluid move through the microstructures of the substrate by capillary action.

[0034] Referring to FIG. 1A, a schematic perspective view of a portion of an exemplary microstructured substrate 1025 is provided, in which a plurality of microstructures 1060 comprises an array of fluidly connected wells 1077, at least some of which are fluidly connected to at least two adjacent wells 1077, each connected via a vent 1087. FIG. 1B is a schematic top view of a portion of the microstructured substrate 1025 of FIG. 1A having an array of fluidly connected wells 1077 having circular shapes connected to adjacent wells 1077 by vents 1087. Sidewalls 1079 of the wells 1077 are also shown in FIG. 1B. The plurality of microstructures 1060 includes the walls, top surface, bottom surface, etc. of the microstructured substrate 1025, defining the voids of the wells 1077 and vents 1087.

[0035] With respect to two wells, "adjacent" means a (e.g., first) well that is next to another (e.g., second) well, without any other (e.g., third) well located between the two (e.g., first and second) wells. As shown in FIG. 1B, the two adjacent wells are fluidly connected to each other by a vent 1087 that has a width ("W") that is smaller than the diameter ("D") of the well 1077. The diameter D is the longest line that passes through the center point ("C") of the well 1077. Typically, the vent 1087 also has a length ("L") that is shorter than the diameter D of the well 1077.

[0036] In some embodiments, at least some of the vents have a width of 1 micrometer or more, 2 micrometers, 3 micrometers, 4 micrometers, 5 micrometers, 6 micrometers, 7 micrometers, 8 micrometers, 9 micrometers, 10 micrometers, 11 micrometers, 12 micrometers, 13 micrometers, 14 micrometers, or 15 micrometers or more and 40 micrometers or less, 38 micrometers, 36 micrometers, 34 micrometers, 32 micrometers, 30 micrometers, 28 micrometers, 26 micrometers, 24 micrometers, 20 micrometers, 18 micrometers, 16 micrometers, 14 micrometers, 12 micrometers, or 10 micrometers or less, for example, in the range of 1 micrometer to 40 micrometers.

[0037] 1A-1B, each central well 1077 of the microstructured substrate 1025 is connected to four other wells 1077, each connection via a vent 1087 (e.g., the central well has at least one other well located between it and the periphery of the microstructured substrate). There are also wells 1077 located adjacent to the periphery 1095, which are connected to only one or two adjacent wells 1077 via vents 1087. For example, referring to FIG. 1A, well 1077c is at a corner of the array and is attached to only one other well 1077d via vent 1087a. Similarly, a well could be attached to three other adjacent wells via vents, and to four, five, six, seven, eight, nine, ten, eleven, or twelve other adjacent wells via vents.

[0038] 2, a schematic cross-sectional view of a portion of an exemplary microstructured substrate 1025 is provided. The microstructured substrate 1025 has an array of fluidly connected wells 1077, although the fluid connections are not shown in this view. In some cases, a land thickness (“L”) of substrate material exists between the bottom surface 1029 of the wells 1077 and the bottom surface 1023 of the microstructured substrate 1025. The land thickness L can provide the microstructured substrate 1025 with dimensional stability useful for robust handling of the microstructured substrate 1025, and is typically between 2 millimeters (mm) and 250 mm, e.g., between 10 mm and 50 mm.

[0039] In this particular embodiment, one or more wells 1077 have at least one sidewall 1079 that has an angled slope relative to at least one of the top surface 1027 of the well 1077 or the bottom surface 1023 of the microstructured substrate 1025. An angled slope means that the sidewall 1079 is not perpendicular (e.g., within ±10 degrees of perpendicular) to at least one of the top surface 1027 of the well 1077 or the bottom surface 1023 of the microstructured substrate 1025. If a microstructured substrate 1025 having wells 1077 with angled sidewalls 1079 such that the diameter of the well is larger at the top surface 1027 than at the bottom 1029 of the well is made by certain methods (e.g., such as by molding a polymer thermoplastic against a tool surface with a negative of the microstructure desired to be provided on the first major surface of the substrate), it may be easier to remove the tool surface from the formed wells.

[0040] The shape of the wells is not particularly limited and can include curved, polygonal, irregular, or a combination thereof. In some embodiments, the wells are circular, triangular, quadrilateral, elliptical, or a combination thereof. When the wells have cornered shapes, the vents are optionally located at the corners (e.g., to reduce the likelihood of trapping air bubbles at the corners). The shape of the vents can also vary. Some exemplary suitable shapes of the microstructured substrate are described in detail below with respect to Figures 3A-7B.

[0041] Typically, each well has an open volume large enough to hold at least one sedimenting particle (e.g., red blood cells), for example, 100 femtoliters or more, 250 femtoliters, 500 femtoliters, 750 femtoliters, 1 picoliter, 100 picoliters, 250 picoliters, 500 picoliters, 750 picoliters, 1 nanoliter, 100 nanoliters, 200 nanoliters, 300 nanoliters, 400 nanoliters, 500 nanoliters, 600 nanoliters, The wells have an open volume of 700 nanoliters, 800 nanoliters, or 900 nanoliters or more, and 1 microliter or less, 900 nanoliters, 800 nanoliters, 700 nanoliters, 600 nanoliters, 500 nanoliters, 400 nanoliters, 300 nanoliters, 200 nanoliters, 100 nanoliters, 1 nanoliter, 750 picoliters, 500 picoliters, 250 picoliters, 1 picoliter, 750 femtoliters, or 500 femtoliters or less. Stated another way, in some cases, each well has an open volume in the range of 100 femtoliters to 1 microliter, or 500 femtoliters to 0.1 microliters.

[0042] Some typical dimensions for each well include a depth (i.e., the distance between the top surface 1027 of the microstructured substrate 2025 and the bottom surface 1029 of the well, as shown in FIGS. 1A and 2) of 50 microliters or more, 75 microliters, 100 microliters, 125 microliters, 150 microliters, 175 microliters, 200 microliters, 225 microliters, 250 microliters, 275 microliters, 300 microliters, 325 microliters, or 350 microliters or more, and 500 microliters or less, 475 microliters, 450 microliters, 425 microliters, 400 microliters, 375 microliters, 350 microliters, 325 microliters, 300 microliters, 275 microliters, 250 microliters, 225 microliters, 200 microliters, 175 microliters, or 150 microliters or less. This same range of distances is also applicable to the diameter of the well. As stated above, the diameter is the longest line passing through the center point of the shape.

[0043] Preferably, at least some of the vents between wells are located at the same depth as the bottom of the adjacent wells. This helps encourage air bubbles to exit the wells without becoming trapped near the bottom of the wells. In some embodiments, the vents have a total depth equal to the total depth of the adjacent wells, although this is not required. When the microstructured substrate is formed using a tool, creating vents that have the same depth as the adjacent wells tends to be more practical than creating vents that connect only the bottoms of two wells. In contrast, when the microstructured substrate is made by laminating two or more layers together, it may be practical to include vents in only a single layer, such as the layer that includes the bottom of the well.

[0044] 1A and 2, in some embodiments, the microstructured substrate 1025 further includes at least one sidewall 1097 disposed along the perimeter 1095 of the first surface 1027 of the microstructured substrate 1025. The one or more sidewalls 1097 have a height (“H”) that extends between 50 micrometers and 250 micrometers beyond the top surface 1027 of the plurality of microstructures 1060. In selected embodiments, the first surface 1027 of the microstructured substrate 1025, together with at least one sidewall 1097, defines a first open volume (“V1”) that is the sum of the open spaces located within the well 1077 and the vent 1087, and the top surface 1099 of the at least one sidewall 1095, together with the top surface 1027 of the plurality of microstructures 1060, defines a second open volume (“V2”) adjacent to the first open volume V1, and the first open volume V1 is larger than the second open volume V2.

[0045] Referring to FIG. 3A, a schematic top view of a portion of a microstructured substrate 3025 is provided having an array of fluidly connected wells 3077 having circular shapes and a triangular vent 3087 connecting three wells 3077 together. For example, as shown in the figure, vent 3087a connects the three wells together, thereby allowing fluid flow between each of wells 3077x, 3077y, and 3077z. FIG. 3B provides a perspective view of the portion of the microstructured substrate 3025 of FIG. 3A to provide another view of the microstructures 3060. One way to provide microstructures 3060, such as 3060a, that are located at the center (e.g., not adjacent to the periphery) of the microstructured substrate 3025 is to form a series of spaced (e.g., symmetrical) curved trilobe segments.

[0046] Referring to Figure 4A, a schematic top view of a portion of a microstructured substrate 4025 is provided having an array of fluidly connected wells 4077 having circular shapes and rounded bottoms 4029 connected by vents 4087. Figure 4B provides a schematic perspective view of a portion of microstructured substrate 4025 of Figure 4A to provide another view of microstructures 4060.

[0047] Referring to Figure 5A, a schematic top view of a portion of a microstructured substrate 5025 is provided having an array of fluidically connected wells 5087 having triangular shapes connected by vents 5087. Figure 5B is an enlarged view of a portion of Figure 5A, clearly showing that each vent 5087 connects six wells 5077. Thus, when fluid exits well 5077a in a particular flow direction ("FD"), the fluid can enter up to five other adjacent wells 5077. Furthermore, in this embodiment, each well 5077 can be fluidly connected to up to 12 adjacent wells.

[0048] Figure 5C provides a schematic perspective view of a portion of the microstructured substrate 5025 of Figure 5A to provide another view of the microstructures 5060. One way to provide microstructures 5060, such as 5060a, that are located in the center (e.g., not adjacent to the periphery) of the microstructured substrate 5025 is to form a series of spaced apart planar panels having beveled edges 5062 (as shown in Figures 5B and 5C, respectively).

[0049] Referring to FIG. 6A, a schematic top view of a portion of a microstructured substrate 6025 is provided having an array of fluidically connected wells 6077 having square shapes connected by vents 6087 at the corners of the squares. Further, in this embodiment, each well 6077 may be fluidly connected to up to six adjacent wells. FIG. 6B provides a schematic perspective view of a portion of the microstructured substrate 6025 of FIG. 6A to provide another view of the microstructures 6060. Similar to the triangular-shaped wells of FIGS. 5A-5B, one way to provide microstructures 6060, such as 6060a, located in the center (e.g., not adjacent to the periphery) of the microstructured substrate 6025 is to form a series of spaced-apart planar panels with beveled edges 6062.

[0050] Referring to FIG. 7A, a schematic top view of a portion of a microstructured substrate 7025 is provided having an array of fluidly connected wells 7077 having hexagonal shapes connected by vents 7087 at the corners of the hexagons. Further, in this embodiment, each well 7077 can be fluidly connected to up to six adjacent wells. FIG. 7B provides a schematic perspective view of a portion of the microstructured substrate 7025 of FIG. 7A to provide another view of the microstructures 7060. Similar to the circular-shaped wells of FIGS. 3A-3B, one way to provide microstructures 7060, such as 7060a, located in the center (e.g., not adjacent to the periphery) of the microstructured substrate 7025 is to form a series of spaced-apart, flat panel (e.g., symmetrical) trilobe segments having beveled edges 7062.

[0051] Variations on the specific structures described herein are expressly contemplated.

[0052] Devices including microstructured substrates according to at least certain embodiments of the present disclosure are suitable for use in removing solid particles (e.g., red blood cells) from fluids (e.g., blood) when the sample volume is 120 microliters or less, 110 microliters, 100 microliters, 90 microliters, 80 microliters, 70 microliters, 60 microliters, 50 microliters, 40 microliters, 30 microliters, 20 microliters, 10 microliters, or even 5 microliters or less, and 1 microliter or more, 2 microliters, 3 microliters, 4 microliters, 5 microliters, 6 microliters, 7 microliters, 8 microliters, 9 microliters, 10 microliters, 12 microliters, 15 microliters, 25 microliters, 35 microliters, or 45 microliters or more. For example, the sum of the first open volume and the second open volume can be between 5 microliters and 120 microliters.

[0053] The interconnected well structure can be fabricated using a multiphoton exposure system, such as that described in U.S. Pat. No. 8,605,256 (DeVoe et al.), to create a patterned tool. Such a structured polymer tool is then typically plated with nickel to create a metallized tool. The nickel-plated tool is then used to create an impression-molded specimen using a press, such as a Carver Press (Carver, Wabash, IN), and resin (e.g., polypropylene resin). The press platens are heated (e.g., to 170°C), and the tool and resin are then pressed together with high force (e.g., 1000 pound force) for several minutes (e.g., 5-10 minutes). After cooling (e.g., until the platen temperature reaches 80°C), the pressure is released and the molded specimen can be removed. Using such a method, microstructured substrates can be optionally provided in the form of microstructured films.

[0054] In one embodiment, a microstructured substrate can be prepared by a method comprising: (a) preparing a polymerizable composition; (b) depositing the polymerizable composition onto a negative microstructured molding surface (e.g., a tool) of a master in an amount just sufficient to fill the cavities of the master; (c) filling the cavities by transferring a bead of the polymerizable composition between a base layer (e.g., a preformed film), at least one of which is flexible, and the master; and (d) curing the composition. Deposition temperatures can range from ambient to about 180°F (82°C). The master can be a metal, such as nickel, chromium, or nickel-plated copper or brass, or a thermoplastic material with a surface energy that is stable under polymerization conditions and allows clean removal of the polymerized material from the master. If the base layer is a preformed film, one or more of the film's surfaces can optionally be primed or otherwise treated to promote adhesion with the microstructured organic material.

[0055] The polymerizable resin can include a combination of first and second polymerizable components selected from (meth)acrylate monomers, (meth)acrylate oligomers, and mixtures thereof. As used herein, "monomer" or "oligomer" refers to any substance that can be converted into a polymer. The term "(meth)acrylate" refers to both acrylate and methacrylate compounds. In some cases, the polymerizable composition can include (meth)acrylated urethane oligomers, (meth)acrylated epoxy oligomers, (meth)acrylated polyester oligomers, (meth)acrylated phenolic oligomers, (meth)acrylated acrylic oligomers, and mixtures thereof.

[0056] The polymerizable resin may be a radiation-curable polymer resin, such as a UV-curable resin. In some cases, polymerizable resin compositions useful in the microstructured substrates of the present disclosure may include those described in U.S. Pat. No. 8,012,567 (Gaides et al.).

[0057] Alternatively, microstructured substrates can be prepared by melt extrusion, i.e., casting a fluid resin composition onto a master negative microstructured molding surface (e.g., a tool) and allowing the composition to solidify. Resin compositions suitable for melt extrusion are dimensionally stable, durable, weather-resistant, and transparent materials that can be easily formed into the desired configuration. Examples of suitable materials include acrylics, such as Plexiglas brand resins manufactured by Rohm and Haas Company (Philadelphia, PA), polycarbonates, reactive materials, such as thermosetting acrylates and epoxy acrylates, polyethylene-based ionomers, such as those sold under the trade name SURLYN by Dow Chemical (Midland, MI) EI Dupont de Nemours and Co., Inc., (poly)ethylene-co-acrylic acid, polyesters, polyurethanes, and cellulose acetate butyrate.

[0058] In yet another embodiment, the master negative microstructured molding surface (eg, tool) can be used as an embossing tool as described in US Pat. No. 4,601,861 (Pricone).

[0059] In some cases, the microstructured substrates disclosed herein may be useful for inclusion in devices and / or methods for separating particles from a fluid, such as those described in commonly owned applications 63 / 425468 (Docket No. PA100124US02) and 63 / 425483 (Docket No. PA100776US01), which are incorporated herein by reference in their entireties. For example, referring to FIGS. 8A-8D, FIG. 8A is a generalized, exploded, schematic diagram of one device in which an exemplary microstructured substrate may be used. Note that the microstructured surface 810 does not show interconnected wells, but rather a general representation of linear microstructures 830. FIG. 8B is a generalized, schematic, top view of the device of FIG. 8A. FIG. 8C is a generalized, schematic, top view of two components used to attach a pump secured to the device of FIGS. 8A-8B. FIG. 8D is a generalized, schematic, perspective view of the device of FIGS. 8A-8B adapted to be attached to a pump. 8A-8D are described in further detail in Example 1 of commonly owned application Ser. No. 63 / 425468 (Docket No. PA100124US02).

[0060] Illustrative Embodiments In a first embodiment, the present disclosure provides a microstructured substrate. The microstructured substrate comprises a plurality of microstructures extending across a first surface of the microstructured substrate. The microstructures comprise an array of interconnected wells, at least some of the wells being fluidly connected to at least two adjacent wells, each connection being via a vent. Each well has an open volume ranging from 100 femtoliters to 1 microliter. At least a portion of the exterior surface of the plurality of microstructures is configured to allow capillary action.

[0061] In a second embodiment, the present disclosure provides a microstructured substrate according to the first embodiment, wherein at least some of the wells are fluidly connected to three adjacent wells or four adjacent wells, each connection being via a vent.

[0062] In a third embodiment, the present disclosure provides a microstructured substrate according to the first or second embodiment, wherein the wells have a depth in the range of 50 micrometers to 500 micrometers.

[0063] In a fourth embodiment, the present disclosure provides a microstructured substrate according to any of the first to third embodiments, wherein the wells have a diameter in the range of 50 micrometers to 500 micrometers.

[0064] In a fifth embodiment, the present disclosure provides a microstructured substrate according to any of the first to fourth embodiments, wherein at least some of the vents have a width in the range of 1 micrometer to 40 micrometers.

[0065] In a sixth embodiment, the present disclosure provides a microstructured substrate according to any of the first to fifth embodiments, wherein at least some of the vents between the wells are located at the same depth as the bottoms of adjacent wells.

[0066] In a seventh embodiment, the present disclosure provides a microstructured substrate according to any of the first to sixth embodiments, wherein at least some of the vents between the wells have a total depth equal to the total depth of the adjacent wells.

[0067] In an eighth embodiment, the present disclosure provides a microstructured substrate according to any of the first to seventh embodiments, wherein the wells comprise a curved shape, a polygonal shape, an irregular shape, or a combination thereof.

[0068] In a ninth embodiment, the present disclosure provides a microstructured substrate according to any of the first to eighth embodiments, wherein the wells comprise a circular shape, a triangular shape, a square shape, an oval shape, or a combination thereof.

[0069] In a tenth embodiment, the present disclosure provides a microstructured substrate according to any of the first to ninth embodiments, wherein the wells have a circular shape.

[0070] In an eleventh embodiment, the present disclosure provides a microstructured substrate according to any of the first to ninth embodiments, wherein the well has a shape that includes a corner, and the vent is located at the corner.

[0071] In a twelfth embodiment, the present disclosure provides a microstructured substrate according to any of the first to eleventh embodiments, wherein each well has an open volume in the range of 500 femtoliters to 0.1 microliters.

[0072] In a thirteenth embodiment, the present disclosure provides a microstructured substrate according to any of the first to twelfth embodiments, wherein at least a portion of an outer surface of the plurality of microstructures comprises a surfactant, a surface treatment, a hydrophilic polymer, or any combination thereof.

[0073] In a fourteenth embodiment, the present disclosure provides a microstructured substrate according to any of the first to thirteenth embodiments, wherein the microstructured substrate is a microstructured film.

[0074] In a fifteenth embodiment, the present disclosure provides a microstructured substrate according to any of the first to fourteenth embodiments, further comprising at least one sidewall disposed along the periphery of the first surface of the microstructured substrate.

[0075] In a sixteenth embodiment, the present disclosure provides a microstructured substrate according to any of the first to fifteenth embodiments, wherein at least one sidewall has a height that extends 50 micrometers to 250 micrometers beyond the top surface of the plurality of microstructures.

[0076] In a seventeenth embodiment, the present disclosure provides a microstructured substrate according to the sixteenth embodiment, wherein a first surface of the microstructured substrate, together with at least one sidewall, defines a first open volume that is the sum of the open spaces located within the well and the vent, and an upper surface of the at least one sidewall, together with an upper surface of the plurality of microstructures, defines a second open volume adjacent to the first open volume, and the first open volume is larger than the second open volume. [Example]

[0077] Objects and advantages of the present disclosure are further illustrated by the following examples, although the particular materials and amounts thereof recited in these examples, as well as other conditions and details, should not be construed to unduly limit the present disclosure. Unless otherwise specified or otherwise apparent from the context, all parts, percentages, ratios, etc. in the examples and the remainder of the specification are by weight.

[0078] Example 1. Microstructured Substrate CAD design files were used to fabricate masters of microstructured substrates using multiphoton exposure systems as described in U.S. Patent No. 8,605,256 (DeVoe et al.) and U.S. Patent No. 8,455,846 (Gates et al.). A negative contrast photoresist, as described in U.S. Patent No. 10,133,174 (Lee et al.), was photopatterned onto a silicon wafer substrate. Once scanning was complete, the substrate with the patterned structures was immersed in a developer solution of propylene glycol monomethyl ether acetate (obtained from Sigma-Aldrich) to remove unpolymerized photoresist. The master was then electroformed with nickel or nickel alloy to create the metal tool used for replication. The nickel-plated tool was used to create impression molded specimens using polypropylene resin (C700-35 resin, Dow Chemical, Midland, MI). The platens of a Carver press (Carver, Wabash, IN) were heated to 170° C. and the tool and resin were pressed together at 1000 pound force (approximately 4450 Newtons) for 7 minutes, followed by cooling under pressure until the platen temperature reached 80° C. The pressure was released and the molded microstructured substrate was removed from the tool.

[0079] The molded microstructured substrate had upper and lower surfaces with overall dimensions of 25.4 mm (width), 76.2 mm (length), and 1 mm (depth). The substrate's microstructure was an array of fluidically connected wells having a circular shape connected to adjacent wells by vents (shown in Figures 1A and 1B) positioned within a flow channel (3 mm wide, 40 mm long) recessed below the upper surface of the substrate and having first and second open ends. Each well had a diameter of 200 micrometers, a depth of 150 micrometers, and a draft angle of 5 degrees. The vents had a length of 29 micrometers, a width of 40 micrometers, a depth of 150 micrometers, and a draft angle of 5 degrees. Each well located in the center of the flow channel was connected to four other wells, each connection via a vent, with the spacing of the vents as shown in Figure 1B. The walls surrounding the periphery of the flow channel extended 100 micrometers above the top surface of the well.

[0080] The first open end of the flow channel was fluidically attached to a semicircular first cavity having a depth of 100 micrometers from the top surface and a volume of 2.88 microliters. The first cavity formed the first aperture of the device. The first aperture served as a liquid sample intake reservoir in the final device. The opposite second open end of the flow channel was fluidically attached to a rectangular second cavity (5 mm wide, 6 mm long, and 250 micrometers deep from the top surface). The second cavity formed the second aperture of the device. The second aperture served as a receiving reservoir for the liquid sample exiting the flow channel.

[0081] Comparative example A A device without a microstructured surface within the flow channel was prepared as Comparative Example A for testing using the method of Example 2. Each device was prepared by forming a laminate of three film sections. The cover sheet component of the device was prepared by laser cutting a 72 mm long x 20 mm wide section from a sheet of 3M Microfluidic Diagnostic Film 9962. A circular hole (5 mm diameter) was laser cut into the film so that the center of the hole was located 13.5 mm perpendicular to the narrow edge and 10 mm perpendicular to the long edge of the film. The first circular hole formed the first aperture of the device. The second film component of the device was prepared by laser cutting a 72 mm long x 20 mm wide section from a sheet of 3M 1522 medical double-sided tape (a clear, double-sided acrylic adhesive with a polyethylene backing, obtained from 3M Company). The total thickness of the double-sided tape, with the release liner removed, was measured to be 150 micrometers using a digital caliper. A rectangular opening (60 mm long x 2.5 mm wide) was laser cut into the second film component and oriented so that the narrow edge of the opening was located 11 mm from the narrow edge of the film and the long edge of the opening was located 8.75 mm from the long edge of the film.

[0082] The third film component of the device was a 72 mm long x 20 mm wide laser-cut section of non-microstructured polyethylene terephthalate (PET) film (MELINEX 454 film (3 mil), Dupont Teijin Films). All laser cutting of the films was performed using a Muse Core CO2 laser cutter (Full Spectrum Laser, Las Vegas, Nevada).

[0083] The device was assembled by removing all release liners from the films and then aligning the edges of the films to form a stack with the second film sandwiched between the cover sheet film and the microstructured film. Adhesive lamination of the stack was completed by applying a 4-pound (1.8 kilogram) roller to the stack and rolling it back and forth once. The second adhesive film formed a fluid seal (e.g., formed a sidewall) between the cover sheet and the top surface of the microstructured film around the edges of the rectangular opening and the first aperture hole. In a final step, a razor blade was used to trim the stack at the edge distal to the first aperture, so that the resulting device had overall dimensions of 60 mm long by 20 mm wide. This cut an opening in the second film, exposing the second aperture of the device as a 2.5 mm rectangular opening at the newly created edge of the device.

[0084] Example 2. 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 citrated whole blood or as citrated whole blood diluted 1:1 with 1X phosphate-buffered saline (PBS) (GIBCO 1X phosphate-buffered saline, pH 7.4, obtained from Thermo Fisher Scientific, Waltham, MA).

[0085] Microstructured substrates were prepared as described in Example 1, and a cover component modified with a blood drop tube was attached to each substrate. The modified cover component was a section of 3M Microfluidic Diagnostic Film 9975R (available from 3M Company), with a circular hole (5 mm diameter) laser-cut in the cover component. Plastic tubing (0.05 inch ID, 0.09 inch OD) was inserted into the hole and secured with epoxy adhesive (3M SCOTCH-WELD Epoxy Adhesive DP100 Plus Clear, available from 3M Company). Any tubing extending beyond the adhesive surface of the cover component was removed with a razor blade. The cover was positioned over the flow channel section and liquid sample capture reservoir section of the microstructured substrate and adhesively attached to the surface of the microstructured substrate surrounding the two sections. The cover component was oriented so that the center of the hole was located over the center of the sample capture reservoir when the cover was attached. The receiving reservoir was not covered. The tube extended approximately 3 cm in length from the outer surface of the cover.

[0086] Each resulting device was placed on a horizontal surface (oriented with the underside of the device facing the horizontal surface). A blood sample (20–50 μL) was added to the sample inlet reservoir through the tubing using a micropipette. The blood sample was either wicked to the end of the device by capillary flow or advanced using positive pressure from the micropipette. A sufficient volume of blood was added to the device to fill the open volume of the flow channel without allowing excess blood to pool in the intake 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 on a horizontal surface for 5 minutes.

[0087] A 1 mL luer-lock syringe filled with mineral oil was attached to a section of plastic tubing, and the mineral oil was partially dispensed into the tubing, leaving a small air gap at the open end of the tubing (approximately 10 cm of tubing length). The syringe was placed in a syringe pump (model NE-1600, New Era Pump Systems Inc.). The open end of the syringe assembly's tubing was connected to the open end of the tubing extending from the device. The pump was operated with a flow rate set at 50 microliters / min to push the blood sample, along with any trapped air, out of the flow channel. The blood sample exiting the flow channel was collected in several 2- to 4-microliter aliquots. Each sample was collected as soon as the aliquot volume accumulated in the receiving reservoir.

[0088] For each collected sample, 1 / 10, 1 / 20, 1 / 100, and 1 / 200 diluted samples were prepared in 1X PBS. A 2-microliter aliquot of each diluted sample was loaded onto an Agilent Take3 microvolume plate (TAKE3-SN, Agilent Technologies, Santa Clara, CA) using a micropipette. The plate included at least one well as a 1X PBS blank, which was used for dilutions according to the manufacturer's instructions. Absorbance measurements were recorded at 406 nm, 414 nm, and 576 nm for each sample and the 1X PBS blank using an Agilent Synergy Neo2 plate reader (Agilent Technologies). Using the procedure in the "Method for Measuring Intact Red Blood Cell Content of Samples" section (described below), the percentage reduction of red blood cells from blood samples subjected to each device was calculated using the procedure described in this example. The same procedure was performed using the device of Comparative Example A by delivering blood to the first opening of the device through an adhesively attached tube.

[0089] Results using citrated human whole blood are shown in Table 1, and results using citrated human whole blood diluted 1:1 with 1X PBS are shown in Table 2. The results reported in Tables 1 and 2 are from three device technical replicates (n=3). The calculated percent red blood cell reduction for each replicate device was averaged from the total volume of aliquot samples collected from the device.

[0090] [Table 1]

[0091] [Table 2]

[0092] Method for determining the intact red blood cell content of a sample To simultaneously measure the intact and lysed red blood cell contents of samples, a dilution series calibration curve was generated using known inputs of lysed and intact human red blood cells. Citrated human whole blood was used as the 100% intact red blood cell sample. To create the 0% intact red blood cell sample, an aliquot of whole blood was lysed by vortexing for 1 minute using a ZR BashingBead Lysing Tube (Product No. S6012-50, Zymo Research, Irvine, CA). The tube was centrifuged at 10,000 x g for 1 minute, and the supernatant, containing only the lysed cell contents, was transferred to a new 1.5 mL Eppendorf tube. Standard samples of intact versus lysed red blood cells for the dilution series were prepared by mixing known intact and lysed cell ratios ranging from 100% to 0% intact red blood cells. The intact red blood cell concentration of the standard samples was confirmed using a C-CHIP disposable hemocytometer according to the manufacturer's instructions.

[0093] Each standard sample was diluted with 1X PBS (so that the absorbance was within the dynamic range of the plate reader) and analyzed in technical triplicate using an Agilent Synergy Neo2 plate reader equipped with a Take 3 multivolume plate (sample volumes and blanks were selected according to the manufacturer's instructions). Absorbances at 406 nm, 414 nm, and 576 nm were measured for each standard sample.

[0094] Ratio A 414nm / A 576nm was plotted against known inputs of % intact red blood cells. 414nm A = absorbance of the sample at 414 nm, A 576nm = absorbance of the sample at 576 nm. Subtraction of a blank (1X PBS) was used for background subtraction. The data set was fitted to a logarithmic curve to generate Equation A. Equation A was used to calculate the percentage of intact red blood cells from the blood-containing suspension.

[0095]

number

[0096] In Equation A, "% Intact" = the percentage of red blood cells in the suspension that were intact, "A 414nm ” = absorbance of the blood sample at 414 nm wavelength, “A PBS,414nm " = absorbance at 414 nm wavelength of 1X PBS, "A 576nm ” = absorbance of the blood sample at 576 nm wavelength, and “A PBS,576nm " = absorbance of 1X PBS at 576 nm wavelength.

[0097] The absorbance signal at 406 nm for each sample was adjusted by the cell percentage ratio to calculate the signal from intact red blood cells using Equation B.

[0098]

number

[0099] In formula B, "Ai 406nm " = absorbance at 406 nm wavelength of the blood sample due to intact red blood cells, "% Intact" = percentage of red blood cells in the suspension that are intact calculated by formula A, "A 406nm ” = absorbance of the sample at 406 nm wavelength, and “A PBS,406nm " = absorbance at 406 nm wavelength of 1X PBS.

[0100] After the absorbance at 406 nm wavelength was adjusted for intact and lysed cells in Equation B, the absorbance signal of the blood sample applied to the device was compared to the absorbance signal of the corresponding blood aliquot collected from the device according to Equation C to calculate the percent red blood cell (RBC) reduction.

[0101]

number

[0102] In formula C, "Ai 406nm,input " = absorbance at 406 nm wavelength of the blood sample added to the device due to intact red blood cells, calculated by Equation B, and "Ai 406nm,output ” = absorbance at 406 nm wavelength of the blood sample drawn from the device due to intact red blood cells, calculated by Equation B.

[0103] All of the above-referenced patents and patent applications are expressly incorporated herein by reference. The above-described embodiments are illustrative of the invention, and other configurations are possible. Accordingly, the invention should not be deemed limited to the embodiments described in detail above and illustrated in the accompanying drawings, but instead should be deemed limited only by the fair scope of the following claims and their equivalents.

Claims

1. 1. A microstructured substrate comprising a plurality of microstructures extending across a first surface of the microstructured substrate, the microstructures comprising an array of interconnected wells, at least some of the wells fluidly connected to at least two adjacent wells, each connection being via a vent, each well having an open volume in the range of 100 femtoliters to 1 microliter, and at least a portion of an outer surface of the plurality of microstructures configured to allow capillary action.

2. 10. The microstructured substrate of claim 1, wherein at least some of the wells are fluidly connected to three adjacent wells or four adjacent wells, each connection being via a vent.

3. The microstructured substrate of claim 1 or 2, wherein the wells have a depth in the range of 50 micrometers to 500 micrometers.

4. The microstructured substrate of any one of claims 1 to 3, wherein the wells have a diameter in the range of 50 micrometers to 500 micrometers.

5. The microstructured substrate of any one of claims 1 to 4, wherein at least some of the vents have a width in the range of 1 micrometer to 40 micrometers.

6. The microstructured substrate of any one of claims 1 to 5, wherein at least some of the vents between wells are located at the same depth as the bottoms of the adjacent wells.

7. The microstructured substrate of any one of claims 1 to 6, wherein at least some of the vents between wells have a total depth equal to the total depth of the adjacent wells.

8. The microstructured substrate of any one of claims 1 to 7, wherein the wells comprise a curved shape, a polygonal shape, an irregular shape, or a combination thereof.

9. The microstructured substrate of any one of claims 1 to 8, wherein the wells comprise a circular shape, a triangular shape, a quadrilateral shape, an oval shape, or a combination thereof.

10. The microstructured substrate of any one of claims 1 to 9, wherein the wells have a circular shape.

11. The microstructured substrate of any one of claims 1 to 9, wherein the well comprises a shape that includes a corner, and the vent is located at the corner.

12. The microstructured substrate of any one of claims 1 to 11, wherein each well has an open volume in the range of 500 femtoliters to 0.1 microliters.

13. The microstructured substrate of any one of claims 1 to 12, wherein at least a portion of the outer surface of the plurality of microstructures comprises a surfactant, a surface treatment, a hydrophilic polymer, or any combination thereof.

14. The microstructured substrate of any one of claims 1 to 13, wherein the microstructured substrate is a microstructured film.

15. The microstructured substrate of any one of claims 1 to 14, further comprising at least one sidewall disposed along a periphery of the first surface of the microstructured substrate.

16. 16. The microstructured substrate of claim 15, wherein the at least one sidewall has a height that extends between 50 micrometers and 250 micrometers beyond the top surface of the plurality of microstructures.

17. 17. The microstructured substrate of claim 16, wherein the first surface of the microstructured substrate, together with the at least one sidewall, defines a first open volume that is the sum of the open spaces located within the well and the vent, and a top surface of the at least one sidewall, together with the top surfaces of the plurality of microstructures, defines a second open volume adjacent to the first open volume, and the first open volume is larger than the second open volume.

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