Devices, kits and methods for sample processing

Abstract

Described herein is a device including a substrate; and a first patterned template layer on the substrate. The first patterned template layer has a plurality of apertures, and a fluid introduced into an aperture of the plurality of apertures settles on an upper surface of the substrate and forms a fluid droplet. Also described is a method of using the device to process a fluid sample, as well as a kit for assembling the device.

Description

[0001] DEVICES, KITS AND METHODS FOR SAMPLE PROCESSING

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] The present application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 607,224, filed December 07, 2023, which is incorporated herein by reference in its entirety.

[0004] BACKGROUND

[0005] In certain scientific applications, it is sometimes desirable to subdivide and physically deposit fluid samples into an array of small volume samples. Doing so allows for high throughput assaying of these fluid samples in, for example diagnostic / prognostic tests, biological analyses, biochemical analyses, and / or chemical analyses. Another benefit of simultaneously analyzing samples is the reduction of technical replicate issues (e.g., batch effects) by performing the same assay on the same substrate in parallel.

[0006] Currently, there are only limited solutions to provide such arrays of small volume fluid samples, and the existing solutions are often incompatible with emerging techniques such as fluidics, robotics, and / or automation workflows.

[0007] There is thus a need for novel devices and methods for providing the patterned and / or small volume sample arrays that are compatible with the newer high-throughput analysis automation technology. The present invention addresses this need.

[0008] SUMMARY

[0009] In some aspects, the present invention is directed to the following non-limiting embodiments:

[0010] Device

[0011] In some aspects, the present invention is directed to a device.

[0012] In some embodiments, the device comprises a substrate; and a first patterned template layer on the substrate.

[0013] In some embodiments, the first patterned template layer has a plurality of apertures.

[0014] In some embodiments, a fluid introduced into an aperture of the plurality of apertures settles on an upper surface of the substrate and forms a fluid droplet. In some embodiments, the upper surface of the substrate is coated with a hydrophobic coating, a hydrophilic coating, or a charged coating.

[0015] In some embodiments, the upper surface of the substrate is coated with a poly-L- lysine coating.

[0016] In some embodiments, a coating applied on the upper surface of the substrate has a pattern.

[0017] In some embodiments, the pattern of the coating corresponds to a pattern of the plurality of apertures in first patterned template layer.

[0018] In some embodiments, the pattern of the coating creates a channel for a plurality of fluid droplets on an upper surface of the substrate to flow between spots on the substrate.

[0019] In some embodiments, the device further comprises a wick between the substrate and the first patterned template layer.

[0020] In some embodiments, the wick has a plurality of openings corresponding to the plurality of apertures in the first patterned template layer.

[0021] In some embodiments, the wick absorbs a liquid in the fluid droplet and leaves a substance of interest in the fluid droplet on the upper surface of the substrate.

[0022] In some embodiments, the device comprises a filter layer on and above the first patterned template layer.

[0023] In some embodiments, the filter layer removes a debris in the fluid, but does not remove a liquid and a substance of interest in the fluid.

[0024] In some embodiments, the filter is capable of holding droplets of fluid on an upper surface thereof by surface tension prior to an application of a centrifugal force.

[0025] In some embodiments, the device comprises a second patterned template layer above the first patterned template layer.

[0026] In some embodiments, the second patterned template layer has a window region, the window region configured to provide access to or visual inspection of some or all of the apertures in the first patterned template layer.

[0027] In some embodiments, the second patterned template layer has a plurality of apertures corresponding to the plurality of apertures in the first patterned template layer, and the plurality of apertures in the second patterned template layer providing access to or visual inspection of some or all of the apertures in the first patterned template layer.

[0028] In some embodiments, the device further comprises a lid above the first patterned template layer, the lid sealing the plurality of apertures of the first patterned template layer. In some embodiments, the substrate or the first patterned template layer comprises an alignment feature for aligning elements of the device.

[0029] In some embodiments, the device further comprises a second patterned template layer, the second patterned template layer comprising an alignment feature for aligning elements of the device.

[0030] In some embodiments, the device further comprises a lid, the lid comprising an alignment feature for aligning elements of the device.

[0031] Method of processing fluid sample

[0032] In some aspects, the present invention is directed to a method of processing a fluid sample. In some embodiments, the method uses the device herein.

[0033] In some embodiments, the method comprises: forming a droplet of the fluid sample on the upper surface of the substrate by introducing the fluid sample into an aperture of the plurality of the apertures in the first patterned template layer.

[0034] In some embodiments, the fluid sample is introduced directly into the aperture of the patterned template layer.

[0035] In some embodiments, the device further comprises a filter layer above the first patterned template layer and covering the aperture of the patterned template layer, and the fluid sample is introduced into the aperture of the patterned template layer by passing through the filter.

[0036] In some embodiments, forming the droplet of the fluid sample on the upper surface of the substrate comprises subjecting the device holding the fluid sample to a centrifugal force.

[0037] In some embodiments, the method further comprises removing a liquid from the droplet of the fluid sample and leaving a substance of interest on a spot of the substrate as defined by the droplet location.

[0038] In some embodiments, the liquid is removed from the droplet of the fluid sample by evaporation, such as by subjecting the substrate to an air flow or an elevated temperature. In some embodiments, before the evaporation step, some liquid of the droplet is removed by the wick when the droplet passes through the wick layer.

[0039] In some embodiments, the device further comprises a wick between the substrate and the first patterned template layer, the wick has an opening corresponding to the aperture in the first patterned template, and the wick absorbs the liquid from the droplet of the fluid sample. Kit

[0040] In some aspects, the present invention is directed to a kit. In some embodiments, the kit is for assembling a device. In some embodiments, the device is the same as or similar to those described herein.

[0041] In some embodiments, the kit comprises: a substrate; a first patterned template layer, wherein the first patterned template layer has a plurality of apertures; and an instruction manual.

[0042] In some embodiments, the instruction manual instructs that: the first patterned template layer is to be placed on top of the substrate; and a fluid is to be introduced into the plurality of apertures, settles on an upper surface of the substrate, and forms a fluid droplet.

[0043] In some embodiments, the upper surface of the substrate is coated with a hydrophobic coating, a hydrophilic coating, or a charged coating.

[0044] In some embodiments, the upper surface of the substrate is coated with a poly-L- lysine coating.

[0045] In some embodiments, a coating applied on the upper surface of the substrate has a pattern.

[0046] In some embodiments, the pattern of the coating applied on the upper surface of the substrate corresponds to a pattern of the plurality of apertures in first patterned template layer.

[0047] In some embodiments, the pattern of the coating applied on the upper surface of the substrate creates a channel for a plurality of fluid droplets on the upper surface of the substrate to flow between spots on the substrate.

[0048] In some embodiments, the kit further comprises a wick having a plurality of openings corresponding to the plurality of apertures in the first patterned template layer.

[0049] In some embodiments, the instruction manual instructs that the wick is to be placed between the substrate and the first patterned template layer, and the wick and the first patterned template layer are to be properly aligned such that the plurality of openings of in wick and the plurality of the apertures in the first patterned template layer are aligned.

[0050] In some embodiments, the wick is made from an absorbent material that absorbs a liquid in the fluid droplets and leaves a substance of interest in the fluid droplets.

[0051] In some embodiments, further comprises a filter layer, wherein the instruction manual instructs that the filter layer is to be placed on and above the first patterned template layer.

[0052] In some embodiments, the filter layer removes a debris in the fluid, but does not remove a liquid and a substance of interest in the fluid. In some embodiments, the filter is capable of holding droplets of fluid on an upper surface thereof by surface tension when not subjected to a downward centrifugal force.

[0053] In some embodiments, the kit further comprises a second patterned template layer.

[0054] In some embodiments, the instruction manual instructs that the second patterned template layer is to be placed above the first patterned template layer.

[0055] In some embodiments, the second patterned template layer has a window region, the window region providing access to or visual inspection of some or all of the apertures in the first patterned template layer.

[0056] In some embodiments, the second patterned template layer has a plurality of apertures corresponding to the plurality of apertures in the first patterned template layer, the plurality of apertures in the second patterned template layer providing access to or visual inspection of some or all of the apertures in the first patterned template layer.

[0057] In some embodiments, the kit further comprises a lid, wherein the instruction manual instructs that the lid is to be placed above the first patterned template layer and seals the plurality of apertures of the first patterned template layer.

[0058] In some embodiments, the substrate or the first patterned template layer comprises an alignment feature for aligning elements of the device.

[0059] In some embodiments, the device further comprises a second patterned template layer, the second patterned template layer comprising an alignment feature for aligning elements of the device.

[0060] In some embodiments, the device further comprises a lid, the lid comprising an alignment feature for aligning elements of the device.

[0061] In some embodiments, the instruction manual instructs that the elements of the device are to be aligned using the aligning feature as a guide.

[0062] BRIEF DESCRIPTION OF THE DRAWINGS

[0063] The following detailed description of exemplary embodiments will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating, non-limiting embodiments are shown in the drawings. It should be understood, however, that the instant specification is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0064] Figs. 1A-1B illustrate some elements of the device herein, in accordance with some embodiments. Figs. 2A-2B, 3 A-3B, 4A-4B, 5A-5B, and 6-9 illustrate the first patterned template layer of the device herein, in accordance with some embodiments.

[0065] Figs. 10A-10B and 11 illustrate the second patterned template layer of the device herein, in accordance with some embodiments.

[0066] Fig. 12 illustrates the second patterned template layer of the device herein, in accordance with some embodiments.

[0067] Figs. 13 and 14A-14B are cross-sectional illustrations of the first patterned template layer having funnel-shaped apertures, in accordance with some embodiments.

[0068] Fig. 15 shows two examples of the first patterned template layers, which were printed with poly-lactic acid, in accordance with some embodiments. The thicknesses of the two first patterned template layers are both 3 mm. The nominal diameters of the apertures on the two examples are 3mm and 6mm, respectively. The alignment marks are nominally 3.75 mm thick.

[0069] Fig. 16 shows two examples of the second patterned template layers, which were printed using poly-lactic acid, in accordance with some embodiments. The thicknesses of the two second patterned template layers are both 3 mm.

[0070] Figs. 17A-17C show certain aspects of an example of a sample process using an exemplary device in accordance with some embodiments disclosed herein. Fig. 17A: the first patterned template layer of the device, holding fluid droplets by surface tension. Fig. 17B: the substrate of the device, after the fluid droplets were transferred thereon. Fig. 17C: the substrate of the device, after the fluid droplets thereon have been dried.

[0071] Figs. 18A-18F show certain aspects of an example of a sample process using an exemplary device in accordance with some embodiments disclosed herein. Fig. 18 A: the exemplary device before assembly, which includes a substrate, a wick, a first patterned template layer having 10 apertures, a filter, a second patterned template layer having a window region which allows the apertures in the first patterned template layer to be accessed, and a lid. Fig. 18B: the exemplary device, after assembly (without the lid). Fig. 18C: fluid droplets loaded on the filter of the device. Fig. 18D: the exemplary device, with fluid droplets loaded and completed with the lid. Fig. 18E: the exemplary device, secured with a clip. Fig. 18F: the exemplary device, loaded with fluid droplets, completed, and placed in a centrifuge.

[0072] Fig. 19 illustrate a non-limiting example of a lid, in accordance with some embodiments. DETAILED DESCRIPTION

[0073] The following disclosure provides many different embodiments, or examples, for implementing different features of the provided subject matter. Specific examples of components and arrangements are described below to simplify the present disclosure. These are, of course, merely examples and are not intended to be limiting. For example, the formation of a first feature over or on a second feature in the description that follows may include embodiments in which the first and second features are formed in direct contact, and may also include embodiments in which additional features may be formed between the first and second features, such that the first and second features may not be in direct contact. In addition, the present disclosure may repeat reference numerals and / or letters in the various examples. This repetition is for the purpose of simplicity and clarity and does not in itself dictate a relationship between the various embodiments and / or configurations discussed.

[0074] In a variety of scientific applications, it is desirable to consider ways to standardize analyses, reduce batch effects, and scale analytical output. One way to achieve this would be to produce a designed and / or spatially patterned array of fluids into stereotypical “spots” (i.e., biological fluids, chemical fluids, and the like) onto a variety of substrates. Doing this successfully would allow for high throughput assaying of these fluids for the purposes of screening (i.e., diagnostic / prognostic tests) as well as performing biological, biochemical, and chemical analyses in a highly parallel fashion. The ability to affix multiple samples on a single substrate, for example, using a variety of glass microscope slides as the substrate, could greatly reduce the cost of performing experiments (e.g., through reduction of the volume of reagents required for carrying out multiple single runs) as well as provides significant time savings. Another benefit of simultaneously analyzing samples would be the reduction of technical replicate issues (i.e., batch effects) by performing the same assay on the same substrate in parallel.

[0075] Existing devices for preparing arrays of small volume fluid samples have certain shortcomings. For example, some existing devices require significant amount of manual labor to produce the array. Some have sample bleeding issues. Some produce arrays of spots with an inconsistent fluid distribution. Some leave residue on the slide / substrate around areas where the fluid spots are produced, which impedes reagent flow. Some devices include slides patterned with hydrophobic and hydrophilic regions, which affect flow of fluids. These shortcomings limit the utility of these devices in newer technologies, such as those utilizing fluidics, robotics, or other automation workflows. To address these challenges, the present disclosure describes an adaptable device, as well as a method using the same, which can be customized to arrange fluid spots / droplets in arrays of any pattern, on any sized substrate. The device and method herein allow the accommodation of layering such that processing steps (e.g., micro-, ultra-, nanofiltration; heated or cooled wells, etc.) can be added or subtracted, as needed.

[0076] The device herein (a) allows good adherence between the fluid sample and the substrate on which the array is formed, (b) allows for high throughput assays that can be customized in size, number, and dimension (e.g, fiducial framing), (c) provides improved consistency in fluid distribution within a spot and between spots (patterning), (d) leaves the substrate free from debris or other contaminants, (e) offers flexibility for use in a wide variety of instrumentation systems, (f) allows for increased flexibility for the materials to be used (e.g., allows materials under test or observation to remain in suspension as opposed to directly being deposited from vessel to substrate without observation or other processing), and (g) provides a flexible framework for assembling layers of materials to customize and stage assays in order to allow for “lab-in-suspension” approach before depositing processed fluids to be deposited on a substrate.

[0077] The device and method herein allow the formation of arrays of fluid droplets / spots using common laboratory systems such as pipettes and centrifuges, and is compatible with automation such as robotics, liquid or bioprinting, or other lab-in-suspension assays.

[0078] Definitions

[0079] As used herein, each of the following terms has the meaning associated with it in this section. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Generally, the nomenclature used herein are those well-known and commonly employed in the art. It should be understood that the order of steps or order for performing certain actions is immaterial, so long as the present teachings remain operable. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference.

[0080] In the application, where an element or component is said to be included in and / or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components and can be selected from a group consisting of two or more of the recited elements or components.

[0081] In the methods described herein, the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0082] In this document, the terms "a," "an," or "the" are used to include one or more than one unless the context clearly dictates otherwise. The term "or" is used to refer to a nonexclusive "or" unless otherwise indicated. The statement "at least one of A and B" or "at least one of A or B" has the same meaning as "A, B, or A and B."

[0083] "About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, in certain embodiments ±5%, in certain embodiments ±1%, in certain embodiments ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0084] As used herein, the term “top,” “bottom,” “upper,” “lower,” “above,” “below,” “upward,” “downward,” etc., when referring to positions of elements, surfaces of elements herein or directions, do not indicate the absolute positions / directions in the real world. Rather, these terms should be interpreted based on the drawings herein. For example, the an “upper surface” of an element is the surface which is higher than the “lower surface” of the same element in the direction of the vertical axis (z-axis) as shown in the figures. For another example, the term “downward” means a direction opposite to the direction of the z- axis.

[0085] Device

[0086] In some aspects, the present invention is directed to a device.

[0087] In some embodiments, the device arranges a plurality of fluid droplets according to a predetermined pattern, such as on a substrate of the device.

[0088] In some embodiments, some or all of the fluid droplets of the plurality of fluid droplets includes a substance to be collected and a liquid. In some embodiments, the device collects the substance from the plurality of fluid droplets and / or according to the predetermined pattern. For example, the substance may be collected from the plurality of fluid droplets in the predetermined pattern on the substrate thereof. In some embodiments, the device is configured to be centrifuged. In some embodiments, during the centrifugation, a centrifugal force is applied to the fluid droplets in a direction pointing roughly downwardly of the device.

[0089] Referring to Fig. 1 A, in some embodiments, the device 100 includes a substrate 110, and a first patterned template layer 130 on and above the substrate 110.

[0090] In some embodiments, the substrate 110 holds the plurality of fluid droplets, and / or the substance collected from the fluid droplets, and the first patterned template layer 130 arranges the fluid droplets (and thereby the collected substance) according to the predetermined pattern.

[0091] In some embodiments, the substrate 110 is configured to hold the substance, such as a liquid, a cell, or the like, that is being collected from the plurality of fluid droplets.

[0092] In some embodiments, the substrate 110 is a slide, such as a microscope slide. In some embodiments, the slide is charged and / or coated. In some embodiments, the slide is a Fisherbrand™ Superfrost™ Plus Microscope Slide (catalog no. 12-550-15) (which has a width of 25 mm and a length of 75 mm in accordance with some embodiments).

[0093] In some embodiments, the substrate 110 is coated. In some embodiments, the coating on the substrate 110 is hydrophilic, hydrophobic, and / or charged. In some embodiments, the coating on the substrate 110 functionalizes the coated area. In some embodiments, the entirety of the upper surface of the substrate 110 is coated. In some embodiments, only a portion of the upper surface of the substrate 110 is coated. In some embodiments, the substrate 110 is coated with more than one type of material having different properties (e.g., two or more of hydrophilic, hydrophobic, positively charged, or negatively charged). In some embodiments, the coating has a pattern. For example, in some embodiments, the substrate 110 is coated with a first material that retains the fluid droplets in areas in which the fluid droplets stay, and with a second material that expels the fluid in areas in which the fluid droplets are not supposed to be.

[0094] In some embodiments, the substrate 110 is coated with a poly-l-lysine. In some embodiments, the substrate 110 is coated with a poly-l-lysine solution (e.g., a 0.1 % (w / v) poly-l-lysine aqueous solution, such as that commercially available from Sigma Aldrich with product number P8920-100mL). In some embodiments, the charging and / or coating promotes adhesion between the plurality of fluid droplets and the substrate 100, thereby retaining the deposited droplets in specific areas of the substrate 110.

[0095] In some embodiments, the coating on the substrate 110 has a pattern. In some embodiments, the patterned coating allows the substrate 110 to retain the deposited fluid droplets at specific spatial locations. In some embodiments, the patterned coating creates channels for fluid to flow between spots.

[0096] In some embodiments, the first patterned template layer 130 is on and in direct contact with the substrate 110. In some embodiments, the device 110 further comprises a wick 120 (described elsewhere herein) sandwiched between and in direct contact with the substrate 110 and the first patterned template layer 130.

[0097] In some embodiments, the first patterned template layer 130 has a plurality of apertures 132. In some embodiments, the plurality of apertures 132 are arranged according to the predetermined pattern such that the plurality of fluid droplets can be arranged on the substrate 110 with the same pattern.

[0098] In some embodiments, the apertures 132 extends through the thickness of the first patterned template layer 130. In some embodiments, the apertures 132 define regions through which the fluid can be introduced onto the substrate 110, thereby allowing the patterned array of fluid droplets to be formed. In some embodiments, a volume of the apertures 132 is calculated based on the thickness thereof in the z-direction and the lateral dimensions in the xy plane.

[0099] In some embodiments, a diameter (or an equivalent diameter in the case that the aperture 132 is not round along the xy plane) of the aperture 132 in the xy plane is about 1 mm, such as about 1.5 mm, about 2 mm, about 2.5 mm, about 3 mm, about 5 mm, about 8 mm, about 10 mm, about 15 mm, about 20 mm, or any ranges therebetween.

[0100] In some embodiments, the aperture 132 is sized to provide a droplet volume between about 0.5 pl and about 100 pl, such as between about 5 pl and about 50 pl, between about 8 pl and about 25 pl, or between 10 pl and 20 pl. In some embodiments, the aperture 132 is sized to provide a droplet volume of about 0.5 pl, about 1 pl, about 2 pl, about 5 pl, about 8 pl, about 10 pl, about 15 pl, about 20 pl, about 25 pl, about 50 pl, about 80 pl, about 100 pl, or any ranges therebetween.

[0101] In some embodiments, all or some of the apertures 132 are within a window area 134, which remains accessible when a second patterned template layer 150 (described elsewhere herein) is attached.

[0102] In some embodiments, the first patterned template layer 130 provides a template for holding the plurality of fluid droplets in place, such as before, during and after a centrifugation. In some embodiments, the first patterned template layer 130 provides an opening through which fluid flows, such as when the fluid is being collected or removed from the device via, e.g., a filter. Additional descriptions of the first patterned template layer 130 are provided elsewhere herein.

[0103] In some embodiments, the device 100 further comprises the wick 120 between the substrate 110 and the first patterned template layer 130. In some embodiments, the wick 120 is in direct contact with the substrate 110, the first patterned template layer 130, or both.

[0104] In some embodiments, the wick 120 has a plurality of openings 122. In some embodiments, the openings 122 in the wick 120 allows the fluid droplets in the first patterned template layer 120 to access the substrate 110. The sizes, shapes and / or patterns openings 122 can be used / formed according to the design of the first patterned template layer 130.

[0105] In some embodiments, the locations of the openings 122 in the wick 120 correspond to the locations of the apertures 132 in the first patterned template layer 130. In some embodiments, each corresponding opening 122 and aperture 132 are concentric with each other.

[0106] In some embodiments, the wick openings 122 are of about same size as the apertures 132 on first patterned template 130. In some embodiments, the holes 122 are slightly larger than the apertures 132, such as about 1% larger, about 2% larger, about 5% larger, about 10% larger, about 20% larger, or about 50% larger in diameter. In some embodiments, the slightly larger holes 122 provide the space for capillary action, such as capillary action to remove the liquid from the fluid droplet.

[0107] In some embodiments, the wick 120 absorbs the liquid in the fluid droplets, thereby separating the liquid from the substance of interest on the substrate 110. In some embodiments, the wick 120 soaks up fluids between samples (fluid droplets) so that samples do not cross-contaminate. In some examples, the inclusion of the wick 120 allows the elimination of the coating / modification on the top surface of the substrate 110. In some embodiments, the wick 120 works in conjunction with the first template layer 130 to prevent the dispersion or spreading of fluids on the substrate 110.

[0108] The choice of material for the wick 120 is not limited. Non-limiting examples of materials for the wick 120 includes filter paper or blotting paper, sponge, cloth, and the like. In some embodiments, the wick 120 is made from Whatman filter paper Grade 1 (with 0.30 mm thickness) or VWR® Blotting Paper, Grade 703 (catalog number 28298-028). Other materials, including but not limited to sponge, cloth, or the like, with different thicknesses can be used.

[0109] In some embodiments, without the application of the centrifugal force, surface tension alone is sufficient to retain the fluid droplets in the apertures 132. In some embodiments, the device 100 further comprises a filter layer 140 on and above the first template layer 130. In some embodiments, the filter layer 140 is in direct contact with the first template layer 130.

[0110] In some embodiments, the filter 140 holds the fluid droplets on an upper surface thereof when a centrifugal force is not applied. In some embodiments, the filter 140 sorts the substance in the fluid droplets according to a size of a mesh of the filter. In some embodiments, the filter 140 filters the fluid, removing large debris from the fluid and allowing liquid and smaller particles to pass through, land on the substrate 110, and form fluid droplets inside the apertures 132 or on substrate 110.

[0111] The material of the filter 140 is not limited. Non-limiting examples of materials of the filter 140 include nylon, polyester, polypropylene, poly ether ether ketone (PEEK), stainless steel, combinations thereof, or the like. In some embodiments, the material of the filter 140 is hydrophobic. In some embodiments, the material of the filter 140 is hydrophilic. In some embodiments, the material of the filter 140 is charged.

[0112] The thickness and mesh size of the filter 140 is not limited and can be chosen according to, e.g., the nature of the sample, the material and / or coatings of the substrate 110, the size and / or shapes of the apertures 132, the thickness of the second patterned template layer 150 (described elsewhere herein), and the like. In some embodiments, the thickness and / or mesh size of the filter 140 are chosen such that the fluid sample is retained on the upper surface of the filter 140 prior to the application of centrifugal force, and is allowed to pass through the mesh of the filter 140 to reach the lower side of the filter 140, such as to reach the apertures 132 and / or the upper surface of the substrate 110 during the application of the centrifugal force.

[0113] In some embodiments, a thickness of the filter 140 ranges from about 2 pm to 2000 pm, such as from about 5 pm to 1000 pm, from about 10 pm to 500 pm, from about 20 pm to 200 pm, or from about 50 pm to 100 pm. In some embodiments, the thickness of the filter 140 is about 2 pm, about 5 pm, about 10 pm, about 20 pm, about 50 pm, about 100 pm, about 200 pm, about 500 pm, about 1000 pm, about 2000 pm, or any ranges therebetween.

[0114] In some embodiments, a mesh size of the filter 140 ranges from about 2 pm to 2000 pm, such as from about 5 pm to 1000 pm, from about 10 pm to 500 pm, from about 20 pm to 200 pm, or from about 50 pm to 100 pm. In some embodiments, the mesh size of the filter 140 is about 2 pm, about 5 pm, about 10 pm, about 20 pm, about 50 pm, about 100 pm, about 200 pm, about 500 pm, about 1000 pm, about 2000 pm, or any ranges therebetween. In some embodiments, the filter 140 is a nylon mesh filter having a thickness of about 70 gm and a mesh size of about 70 gm (e.g., Spectrum Laboratiories product no. 145801).

[0115] In some embodiments, the filter 140 absorbs the liquid in the fluid sample. In some embodiments, the filter 140 does not absorb the liquid, or does not substantially absorb the liquid.

[0116] In some embodiments, the filter 140 is constructed from a material having resistance against corrosives, alkalis, organics, and / or acids. In some embodiments, the filter 140 has a pH resistance between pH values of about 2 and 14 or between about 3 and 10. In some embodiments, the filter 140 is considered to have pH resistance at a given pH value if the filter 140 is able perform its intended function (e.g., retaining fluid at either side thereof, filtering fluid, or the like) for 1 hour or more.

[0117] In some embodiments, the filter 140 has a thermal stability at about 130 °C or higher, such as about 150 °C or higher, about 160 °C or higher, about 180 °C or higher, about 200 °C or higher, about 230 °C or higher, or about 250 °C or higher, for 1 hour or more.

[0118] In some embodiments, the filter 140 is sterilizable by irradiation sterilization or autoclaving, without causing substantial damage to the its intended function.

[0119] In some embodiments, the device 100 further comprises a second patterned template layer 150 on and above the first patterned template 130. In some embodiments, the second patterned template layer 150 is in direct contact with the first patterned template 130. In some embodiments, the second patterned template layer 150 is above and in direct contact with the filter 140.

[0120] In some embodiments, the second patterned template layer 150 has a window area 154. In some embodiments, the window area provides and / or allows the visual inspection of and / or access to some or all of the apertures 132 in the first patterned template 130.

[0121] In some embodiments, the second patterned template layer 150 provides a thickness to the stack of layers in device 100 without touching or interfering with the fluid samples. In some embodiments, a thickness of the second patterned template layer 150 is equal to or larger than a height of the fluid droplets formed on either the optional filter 140 or a height of a portion of the fluid droplets in the apertures 132 that is above the upper surface of the first patterned template layer 130, such that the stacking of any subsequent layer, if any, does not interact with the fluid droplets.

[0122] In some embodiments, the second patterned template layer 150 stabilizes the stack so that the sections fit together without bulges or gaps. In some embodiments, the second pattern template layer 150 facilitates the introduction of the fluid into the lower (underlying) layers, such as the substrate 110, the first patterned template layer 130, the wick 120, and the filter 140.

[0123] In some embodiments, the second patterned template layer 150 comprises features on a lower surface thereof that can retain the optional filter 140 layer in place.

[0124] Additional descriptions of the second patterned template layer 150 are provided elsewhere herein.

[0125] In some embodiments, the device further comprises a lid 160 on and above the first patterned template layer 130. In some embodiments, the lid 160 is in direct contact with the second patterned template layer 150 (if present), in direct contact with the filter 140 (if present and the second patterned template layer 150 is not present), or in direct contact with the first pattern template layer 130 (if both the filter 140 and the second patterned template layer 150 are absent).

[0126] In some embodiments, the lid 160 tops off the stack of layers of the device 100, and prevents fluid loss. In some embodiments, the lid 160 provides a surface for screws, clips, and / or clamps to attach to the device 100 without disrupting the sample collection area as defined by the substrate 110, the aperture 132, the optional openings 122, and the optional window area 154. In some embodiments, the lid 160 provides a mechanical structure that allow the device 100 to be placed into a centrifuge.

[0127] The material for making the lid 160 is not limited. In some embodiments, the lid 160 is a glass lid, a metal lid, a plastic lid, a polymer lid, or the like. In some embodiments, the lid 160 is a glass microscope slide with the same size as the microscope slide used for substrate 110. In some embodiments, lid 160 is formed using the same materials as the patterned template layers 130 and / or 150.

[0128] In some embodiments, the device 100 further comprises a stretch film (such as a polyolefin-based film like parafilm), a screw, a clip, and / or a clamp for securing the layers of the device 100 together and keeps the proper alignment of the layers.

[0129] In some embodiments, all the layers of the device 100 are mechanically clipped together using a stretch film, a screw, a clip (e.g., a glass clip), a clamp, and the like. One example of the glass clip is those made by the Guangzhou Open Find Electronic Commerce Co, LTD, with the catalog number A170826WQ001. This clip is a zinc alloy rectangle adjustable clip clamp, with an internal bracket holder to support a device between 6- 10mm thickness.

[0130] In some embodiments, elements for securing and aligning the layers of the device 100 are built into the first patterned template 130, the second patterned template 150, and / or the lid 160. In some embodiments, elements for securing and aligning the layers of the device 100 are built into the lid 160.

[0131] In some embodiments, the lid 160 comprises features that allow lid 160 to mechanically combine with the substrate 110, and thereby securing all the layers sandwiched between the lid 160 and the substrate 110, without the need of a screw / clip / clamp. In some embodiments, the lid 160 is mechanically combinable with the first patterned template layer 130, the second patterned template layer 150, the wick 120 and / or the filter 140 (provided that these layers are part of the device 100).

[0132] Referring to Fig. 19, a non-limiting example of the lid 2200 is shown. In some embodiments, the lid 2200 comprises a base 2202, sidewalls 2204, and retaining features 2206. In some embodiments, the lid 2202 slides over the previously-combined structure (e.g., the substrate, the patterned template layer(s), the wick, and the filter), and secures all the layers in a properly aligned state.

[0133] Referring back to Fig. 1 A, in some embodiments, the device 100 comprises the substrate 110, the wick 120 (optional), the first patterned template layer 130, the filter 140 (optional), the second patterned template layer 150 (optional), and the lid 160 (optional) from bottom to top in this order, and each of the layers is in direct contact with the adjacent layer(s). For example, for a device 100 that comprises substrate 110, the first patterned template layer 130, and the lid 160 but does not comprise the wick 120 or the filter 140, the substrate 110 at the bottom is in direct contact with the first patterned template layer 130 in the middle, and the first patterned template layer 130 in the middle is also in direct contact with the lid 160 on the top.

[0134] Referring to Fig. IB, in some embodiments, the device 100’ further comprises a funnel 170’ inserted in to the aperture 132’ of the first patterned template layer 130’. In some embodiments, the funnel 170’ extends beyond an upper surface (in relative to the direction of the z-axis) of the first patterned template layer 130’ or a lower surface (in relative to the direction of the z-axis) of the first patterned template layer 130’

[0135] In some embodiments, the funnel 170’ is fabricated as a part of the first patterned template layer 130’. In some embodiments, the funnel 170’ is a separate element from the first patterned template layer 130’ and aligns with the apertures 132’.

[0136] In some embodiments, the maximum volume the funnel 170’ is able to hold is about 10 pl, about 20 pl, about 50 pl, about 100 pl, about 200 pl, about 500 pl, or about 1000 pl. In some embodiments, the inclusion of the funnel 170’ allows the first patterned template layer 130’ to hold larger volume of fluid than in the case where the funnel 170’ is absence. First patterned template layer

[0137] Referring to Figs. 2A and 2B, in some embodiments, the first patterned template layer 200 has an upper surface 202, and a lower surface 204. A thickness of the first patterned template layer 200 in the z direction is Z200. The first patterned template layer 200 has a first dimension X202 and a second dimension y202. first patterned template layer 200 has a plurality of apertures 206 that extend through the thickness of the substrate. The apertures 206 have a dimension X206 and y206 in the plane of the upper and lower surfaces.

[0138] In some embodiments, the plurality of apertures 206 is in a window region 208, which has dimensions dimension X208 and y208. The first patterned template layer 200, the window region 208, and the plurality of apertures 206 can have any dimension and / or shape. The location as well as the dimensions in the x-y plane of the window region 208 is configured depending on the imaging tool that is to be used.

[0139] Figs. 3 A and 3B illustrate another configuration of the first patterned template layer 300, which is similar to that of the first patterned template layer 200, but with the addition of alignment features 310a, 310b, 310c and 3 lOd to assist in the alignment of the template with layers above or below the first patterned template layer 300 (e.g., substrate, wick, filter, lid, etc.).

[0140] As shown in Figs. 3 A and 3B, the alignment features 310a and 310c are along one of the longitudinal edges of the template 300, and the alignment features 310b and 3 lOd are along the two lateral edges. The alignment features 310a-3 lOd have a thickness t2io greater than the thickness tioo of the first patterned template layer 300, to facilitate the alignment of the first patterned template layer 300 with other layers of the device herein.

[0141] One of ordinary skill in the art, reading in light of the instant specification, would understand that the shape and placement of the alignment features are not limited and there are many ways to configure the alignment features to facilitate the alignment of the layers.

[0142] For example, Figs. 4A and 4B illustrate another configuration of the first patterned template layer 400, with a set of alignment features 401a, 401b and 401c.

[0143] Figs. 5A and 5B illustrate yet another configuration of the first patterned template layer 500 having yet another set of alignment features 510a and 510b.

[0144] The first patterned template layer can be made of any suitable material. In some embodiments, the first patterned template layer is made from glass, stainless steel, polytetrafluoroethylene (Teflon), nylon, a plastic or a polymeric material, a poly-lactic acid (PLA), a PEEK, or combinations thereof. In some embodiments, the first patterned template layer is formed using an additive manufacturing (such as a 3D printing), which is able to create the apertures in the manufacturing process. In some embodiments, the apertures are made by machining an unpatterned sheet of material. In some embodiments, the first patterned template layer is formed using injection molding.

[0145] In some embodiments, a combination of the material type and the thickness of the material provides sufficient rigidity to the first patterned template layer, such as allowing the upper and lower surfaces thereof to remain flat during the production and / or use.

[0146] The first patterned template layer may be of any shape, size, and / or dimensions. In some embodiments, the first patterned template layer is sized to match the other layers of the device, such as the substrate, the lid, etc, in the xy plane. In some embodiments, the first patterned template layer has dimensions in the xy plane that match a rectangular microscope slide, which can be used as the substrate and / or the lid. For example, if a Fisherbrand™ Superfrost™ Plus Microscope Slide (catalog no. 12-550-15) with a width of 25 mm and a length of 75 mm is used as the substrate and / or lid, the width and length of the first patterned template layer can be 25 mm and 75 mm, respectively.

[0147] Referring to Figs. 6 and 7, in some embodiments, the first patterned template layer is square or circular in the xy plane.

[0148] In some embodiment, the window region, which is defined by the instrumentation with which the substrate-to-be-pattemed will be used, takes any shape that fits within the area of the substrate surface. As shown in Figs. 2A, 2B, 3 A, 3B, 4A, 4B, 5A, 5B, 6, 7, and 8, the window region (indicated by a dashed line) can be rectangular, square, or circular, though other shapes may be used according to the instrumentation used to analyze the droplets on the slide.

[0149] It is worth noting that the apertures of the first patterned template layer do not have to be round in the xy plane, and that the window region does not have to be rectangular; rather, they can take any shape. Referring to Fig. 8, the first patterned template layer 800 has square apertures 806 that fit within a circular window 808.

[0150] It is worth noting that the alignment features can extend beyond one or both of the surfaces of the first patterned template. In Figs. 2A, 2B, 3 A, 3B, 4A, 4B, 5A, and 5B, the alignment features extend above one of the two surfaces of the first patterned template only. As shown in Fig. 9, in the first patterned template layer 900, the alignment features 910 extends beyond both the lower surface 904 and the upper surface 902 (t9io > t9oo). In some embodiments, the first patterned template layer has features on the upper surface thereof that allows the retention of the optional filter layer in place. In some embodiments, these features could include internal clamps within the layer, interlocking pieces between layers, protrusions, designs that support a taut filter layer, or the like.

[0151] In some embodiments, the xy plane cross-sections of an aperture in the first patterned template layer do not have the same size and / or shape along the vertical direction (the z-axis) (i.e., having an “irregular shape”). In some embodiments, the first patterned template layer, in the regions near the apertures, comprises guide structures that extend beyond one or both of the surfaces of the first patterned template layer.

[0152] In some embodiments, the irregular shape of the aperture and / or the guide structures provides better confinement of fluid droplets as they are formed on the substrate below. In some embodiments, the irregular shape of the aperture and / or the guide structures provides better confinement of the fluid in the aperture that extends beyond a level of the upper surface of the first patterned template layer and prevents leakage and / or spreading of fluid across separating regions between the apertures.

[0153] Referring to Fig. 13, a cross-section of an embodiment of the first patterned template layer 1300 is shown. In the first patterned template layer 1300, a plurality of apertures 1306 are arranged inside a window region 1308, which has a dimension of XBOS in the x-direction. Each of the apertures 1306 has a first dimension xi306a at an upper surface 1302 of the first patterned template layer 1300, and a second dimension xi306b at the lower surface 1304.

[0154] In some embodiments, the first dimension xi306a is larger than the second dimension xi306b. In some embodiments, the apertures 1306 have a funnel shape, such as a circular conical funnel shape. In some embodiments, the funnel shape allows the fluid to be held at a top side of the aperture 1306 by surface tension before application of the centrifugal force, while also allowing the fluid to move to the bottom side of the aperture 1306 when the centrifugal force is applied. In some embodiments, the funnel design allows for more fluid to be deposited within the layer. Any shape of the funnel in both the xy plane and in the z direction may be chosen to control the fluid delivery. In some embodiments, the cross-section of the aperture at the xy plane may have a circular shape, a square shape, a rectangular shape, an elliptical shape or any other shape. In some embodiments, in the z-direction, the lateral or longitudinal dimension changes linearly (e.g., a conical funnel shape in the xz or yz plane) or non-linearly (e.g., an hourglass shape in the xz or yz plane). In some embodiments, the apertures 1306 have regions where the lateral / longitudinal dimension is varying in the z- direction, and regions where the lateral / longitudinal dimension is fixed in the z-direction. Referring to Fig. 14A, in some embodiments, the first patterned template layer 1400 comprises, near the apertures 1406 (such as funnel shaped aperture 1406), retaining protrusions 1412 above an upper surface 1402. In some embodiments, the retaining protrusions 1414 confine a deposited fluid in apertures 1406 above a level of the lower surface 1404, such that the fluid does not spread between adjacent apertures 1406.

[0155] Referring to Fig. 14B, in some embodiments, the first patterned template layer 1400’ comprises, near the apertures 1406’ (such as funnel shaped aperture 1406’), protrusions 1414’ below surface 1404’. In some embodiments, the protrusions 1414’ confine deposited fluid on the underlying substrate directly under aperture 1406, preventing spreading.

[0156] Referring to Fig. 15, two non-limiting examples of the first patterned template layers 1600 and 1650 are shown. The exemplary first patterned template layers are 3D printed using PLA. Each first patterned template layer is approximately 3mm thick. The apertures in 1600 are approximately 3 mm in diameter, and the apertures in 1650 are approximately 6 mm in diameter. Alignment marks are nominally 3.75 mm thick. The apertures fit within the window region of the second template layer (1700, shown in Fig. 16). Openings in an underlying (optional) wick layer are concentric with the apertures in the first patterned template layers. The openings are the same size as, or larger than the aperture sizes in the first patterned template layer. Where the aperture dimension varies in the z-direction, the opening is the same as or larger than the dimension of the aperture where it is in contact with the wick layer.

[0157] Second patterned template layer

[0158] Referring to Figs. 10A and 10B, in some embodiments, the second patterned template layer 1000 is sized identically with the first patterned template layer, so that the two patterned temple layers can be aligned.

[0159] In some embodiments, the two patterned template layers are aligned using alignment marks.

[0160] In some embodiments, the two patterned template layers are aligned through alignment features incorporated into at least one of the first and second patterned template layers to facilitate their alignment and integration. In some embodiments, the alignment features include a clip or a “runner” that allows one layer to slide over or under the other.

[0161] In some embodiments, the second patterned template layer 1000 has a window region 1008, which is sized the same as or larger than the window region of the first patterned template layer (such as the window region 134 of the first patterned template layer 130 as shown in Fig. 1 A). In some embodiments, the window region 1008 provides and / or allows access to some or all of the apertures of the first patterned template layer.

[0162] Referring to Fig. 11, an example of the second template layer 1000 comprising additional alignment features is shown. The location of the alignment features on the second patterned template layer 1000 is not limited and is the same as or similar to those as described for the first patterned template layer in some embodiments.

[0163] Referring to Fig. 12, an example of the second patterned template layer 1200 that is compatible with the first template layer 600 of Fig. 6 is shown. As opposed to an open window, the second patterned template layer 1200 of Fig. 12 is patterned with an array of holes 1206 that fit within window area 1208. The size (e.g., diameter or equivalent diameter) of the apertures 1206, dl206, is chosen to be equal to or greater than the size (diameter or equivalent diameter) of the apertures 606 (d606) in the first patterned template layer 600 shown in Fig. 6. In some embodiments, the apertures 1206 of Fig. 12 and the apertures 606 of Fig. 6 are concentric when the two patterned template layers are properly aligned, and the size of the apertures 1206 in the second patterned template layer 1200 is equal to or larger than the size of the apertures 606 in the first patterned template layer 600.

[0164] The shapes and profiles of the apertures in the second patterned template layer are not limited, as long as they are compatible with the apertures in the first patterned template layer.

[0165] The thickness of the second patterned template layer is not limited, as long as such a thickness is sufficient to provide rigidity. One of ordinary skill in the art would be able to determine the thickness based on the material used for constructing the second patterned template layer, as well as the shape, size, number and arrangement of cut-out sections, such as the window region or the aperture in the second patterned template layer.

[0166] In some embodiments, the manufacture of the second patterned template layer is similar to that of the first patterned template layer, which is described elsewhere herein.

[0167] Referring to Fig. 16, two non-limiting examples the second patterned template layers 1700 and 1750 are shown. The two second patterned template layers 1700 and 1750 3D are printed using PLA. Each second patterned template layer is approximately 3mm in thickness. The window size in the second patterned template layer 1700 is approximately 18 mm by 34.9 mm, which corresponds to an optical measurement area of a particular analysis tool (this analysis tool is not considered part of the device herein), and the window size in the second patterned template layer 1750 is approximately 21 mm by 37 mm.

[0168] Method In some aspects, the present invention is directed to a method of processing a sample with the device herein.

[0169] In some embodiments, the method comprises loading a fluid sample onto the device.

[0170] In some embodiments, the device does not comprise the filter, and the fluid is loaded directly into the apertures of the first patterned template layer.

[0171] In some embodiments, the device comprises the filter, and the fluid is loaded on an upper side of the filter opposite to the first patterned template layer. In some embodiments, the fluid passes through the filter and enters the aperture in the first patterned template layer without the application of a centrifugal force. In some embodiments, surface tension holds the fluid droplets on the filter and a centrifugal force is applied before the fluid droplets passes through the filter and enter the aperture in the first patterned template layer.

[0172] In some embodiments, the fluid droplet on the filter or in the apertures of the first patterned template layer are settled on an upper surface of the substrate, via gravity, charge- charge / hydrophilic / hydrophobic interaction between the fluid droplet and the substrate, and / or the application of a centrifugal force.

[0173] In some embodiments, the method further comprises drying the fluid droplets transferred to the substrate. The method of drying the fluid droplets is not limited. In some embodiments, the drying includes evaporation, such as, but not limited to, subjecting the substrate to a flow of air or a temperature at least 20 degrees Celsius above room temperature. In some embodiments, the wick between the substrate and the first patterned template layer removes all or a part of the liquid in the fluid droplet.

[0174] Figs. 17A-17C illustrate a non-limiting example of the method herein.

[0175] Referring to Fig. 17A, fluid droplets were applied on a non-limiting first patterned template layer having a 5 x 8 array of apertures by pipetting, and were held in the apertures by the surface tension alone.

[0176] Referring to Fig. 17B, the fluid droplets were transferred onto the surface of the substrate (a microscope slide having a frosted region) by stacking the first patterned template layer of Fig. 17 on top of the substrate, and applying a centrifugal force downwardly. As shown in Fig. 17B, the fluid droplets were confined to the substrate in regions under the apertures, with no observable spreading or cross-contamination.

[0177] Referring to Fig. 17C, the array of fluid droplets shown in Fig. 17B were dried by placing the substrate on a slide warmer of about 65 °C for about one hour. After drying, an array of solid substance spots was formed on the substrate, which can be subjected to further analysis. For example, in the case that the fluid droplets are saliva samples from subjects, the solid substance spots can be stained with antibodies or dyes for specific types of immune cells, enabling the identification and counting of the immune cells present in the saliva samples.

[0178] Figs. 18A-18F illustrate another non-limiting example of the method herein.

[0179] Referring to Fig. 18 A, a non-limiting device according to some embodiments herein comprises a substrate, a wick, a first patterned template layer with apertures, a filter, a second patterned template layer with a window region, and a lid.

[0180] Referring to Fig. 18B, the layers of the non-limiting device, apart from the lid, were assembled using parafilm as a fastening mean.

[0181] Referring to Fig. 18C, droplets of fluid samples were introduced onto the filter at locations corresponding to the apertures in the first patterned template layer with a pipette, which accessed the filter through the window region of the second patterned template. Due to surface tension, the fluid droplets are held on the filter.

[0182] Referring to Fig. 18D, the lid was placed on top of the assembly of Fig. 18C.

[0183] Referring to Fig. 18E, a clip with screws was used to secure the layers together.

[0184] Referring to Fig. 18F, the assembly of Fig. 18E was place in a centrifuge, which can apply a centrifugal force to the assembly and settle the fluid droplets on the filter on the bottom substrate layer.

[0185] Enumerated Embodiments:

[0186] In some aspects, the present invention is directed to the following non-limiting embodiments:

[0187] Embodiment 1 : A device, comprising: a substrate; and a first patterned template layer on the substrate, wherein the first patterned template layer has a plurality of apertures, wherein a fluid introduced into an aperture of the plurality of apertures settles on an upper surface of the substrate and forms a fluid droplet.

[0188] Embodiment 2: The device of Embodiment 1, wherein at least one of the following applies:

[0189] (a) the upper surface of the substrate is coated with a hydrophobic coating, a hydrophilic coating, or a charged coating;

[0190] (b) the upper surface of the substrate is coated with a poly-L-lysine coating;

[0191] (b) a coating applied on the upper surface of the substrate has a pattern. Embodiment 3: The device of any one of Embodiments 1-2, wherein the coating applied on the upper surface of the substrate has the pattern, and wherein at least one of the following applies:

[0192] (a) the pattern of the coating corresponds to a pattern of the plurality of apertures in first patterned template layer;

[0193] (b) the pattern of the coating creates a channel for a plurality of fluid droplets on an upper surface of the substrate to flow between spots on the substrate.

[0194] Embodiment 4: The device of any one of Embodiments 1-3, further comprises a wick between the substrate and the first patterned template layer, wherein the wick has a plurality of openings corresponding to the plurality of apertures in the first patterned template layer.

[0195] Embodiment 5: The device of Embodiment 4, wherein the wick absorbs a liquid in the fluid droplet and leaves a substance of interest in the fluid droplet on the upper surface of the substrate.

[0196] Embodiment 6: The device of any one of Embodiments 1-5, further comprises a filter layer on and above the first patterned template layer.

[0197] Embodiment 7: The device of Embodiment 6, wherein at least one of the following applies:

[0198] (a) the filter layer removes a debris in the fluid, but does not remove a liquid and a substance of interest in the fluid;

[0199] (b) the filter is capable of holding droplets of fluid on an upper surface thereof by surface tension prior to an application of a centrifugal force.

[0200] Embodiment 8: The device of Embodiment 1, further comprises a second patterned template layer above the first patterned template layer, wherein at least one of the following applies:

[0201] (a) the second patterned template layer has a window region, and the window region allows an access to or a visual inspection of some or all of the apertures in the first patterned template layer,

[0202] (b) the second patterned template layer has a plurality of apertures corresponding to the plurality of apertures in the first patterned template layer, and the plurality of apertures in the second patterned template layer allow an access to or a visual inspection of some or all of the apertures in the first patterned template layer.

[0203] Embodiment 9: The device of any one of Embodiments 1-8, further comprises a lid above the first patterned template layer and seals the plurality of apertures of the first patterned template layer. Embodiment 10: The device of any one of Embodiments 1-9, wherein at least one of the following applies:

[0204] (a) the substrate or the first patterned template layer comprises an alignment feature for aligning elements of the device;

[0205] (b) the device further comprises a second patterned template layer, and the second patterned template layer comprises an alignment feature for aligning elements of the device;

[0206] (c) the device further comprises a lid, and the lid comprises an alignment feature for aligning elements of the device.

[0207] Embodiment 11 : A method of processing a fluid sample with the device of any one Embodiments 1-10, the method comprising: forming a droplet of the fluid sample on the upper surface of the substrate by introducing the fluid sample into an aperture of the plurality of the apertures in the first patterned template layer.

[0208] Embodiment 12: The method of Embodiment 11, wherein at least one of the following applies:

[0209] (a) the fluid sample is introduced directly into the aperture of the patterned template layer;

[0210] (b) the device further comprises a filter layer above the first patterned template layer covering the aperture of the patterned template layer, and the fluid sample is introduced into the aperture of the patterned template layer by passing the filter.

[0211] Embodiment 13: The method of any one of Embodiment 11-12, wherein forming the droplet of the fluid sample on the upper surface of the substrate comprises subjecting the device holding the fluid sample to a centrifugal force.

[0212] Embodiment 14: The method of any one of Embodiments 11-13, further comprises removing a liquid from the droplet of the fluid sample and leaving a substance of interest on a spot of the droplet.

[0213] Embodiment 15: The method of Embodiment 14, wherein at least one of the following applies:

[0214] (a) the liquid is removed from the droplet of the fluid sample by subjecting the substrate to an air flow or an elevated temperature;

[0215] (b) the device further comprises a wick between the substrate and the first patterned template layer, the wick has an opening corresponding to the aperture in the first patterned template, and the wick absorbs the liquid from the droplet of the fluid sample.

[0216] Embodiment 16: A kit for assembling a device, comprising: a substrate; a first patterned template layer, wherein the first patterned template layer has a plurality of apertures; and an instruction manual instructs that: the first patterned template layer is to be placed on top of the substrate; and a fluid is to be introduced into the plurality of apertures, settles on an upper surface of the substrate, and forms a fluid droplet.

[0217] Embodiment 17: The kit of Embodiment 16, wherein at least one of the following applies:

[0218] (a) the upper surface of the substrate is coated with a hydrophobic coating, a hydrophilic coating, or a charged coating;

[0219] (b) the upper surface of the substrate is coated with a poly-L-lysine coating;

[0220] (c) a coating applied on the upper surface of the substrate has a pattern.

[0221] Embodiment 18: The kit of Embodiment 17, wherein the coating applied on the upper surface of the substrate has the pattern, and wherein at least one of the following applies:

[0222] (a) the pattern of the coating corresponds to a pattern of the plurality of apertures in first patterned template layer;

[0223] (b) the pattern of the coating creates a channel for a plurality of fluid droplets on the upper surface of the substrate to flow between spots on the substrate.

[0224] Embodiment 19: The kit of any one of Embodiments 16-19, further comprises a wick having a plurality of openings corresponding to the plurality of apertures in the first patterned template layer, wherein the instruction manual instructs that the wick is to be placed between the substrate and the first patterned template layer, and the wick and the first patterned template layer are to be properly aligned such that the plurality of openings of in wick and the plurality of the apertures in the first patterned template layer are aligned.

[0225] Embodiment 20: The kit of Embodiment 19, wherein the wick is made from an absorbent material that absorbs a liquid in the fluid droplets and leaves a substance of interest in the fluid droplets.

[0226] Embodiment 21 : The kit of any one of Embodiments 16-20, further comprises a filter layer, wherein the instruction manual instructs that the filter layer is to be placed on and above the first patterned template layer.

[0227] Embodiment 22: The kit of Embodiment 21, wherein at least one of the following applies: (a) the filter layer removes a debris in the fluid, but does not remove a liquid and a substance of interest in the fluid;

[0228] (b) the filter is capable of holding droplets of fluid on an upper surface thereof by surface tension when not subjected to a downward centrifugal force.

[0229] Embodiment 23: The kit of any one of Embodiments 16-22, further comprises a second patterned template layer, wherein the instruction manual instructs that the second patterned template layer is to be placed above the first patterned template layer, and wherein at least one of the following applies:

[0230] (a) the second patterned template layer has a window region, and the window region allows an access to or a visual inspection of some or all of the apertures in the first patterned template layer,

[0231] (b) the second patterned template layer has a plurality of apertures corresponding to the plurality of apertures in the first patterned template layer, and the plurality of apertures in the second patterned template layer allow an access to or a visual inspection of some or all of the apertures in the first patterned template layer.

[0232] Embodiment 24: The kit of any one of Embodiments 16-23, further comprises a lid, wherein the instruction manual instructs that the lid is to be placed above the first patterned template layer and seals the plurality of apertures of the first patterned template layer.

[0233] Embodiment 25: The kit of any one of Embodiments 16-24, wherein at least one of the following applies:

[0234] (a) the substrate or the first patterned template layer comprises an alignment feature for aligning elements of the device;

[0235] (b) the device further comprises a second patterned template layer, and the second patterned template layer comprises an alignment feature for aligning elements of the device;

[0236] (c) the device further comprises a lid, and the lid comprises an alignment feature for aligning elements of the device, and. wherein the instruction manual instructs that the elements of the device are to be aligned using the aligning elements as guide.

[0237] Identifiers Used in Drawings

[0238] Device: 100, 100’

[0239] Substrate: 110

[0240] Wick: 120

[0241] Opening(s) in wick: 122 First patterned template layer: 130, 130’, 200, 300, 400, 500, 550, 600, 700, 800, 900, 1200, 1300, 1400, 1400’, 1600, 1650

[0242] Upper surface of first patterned template layer: 202, 302, 402, 602, 702, 802, 902, 1202, 1302, 1402, 1402’

[0243] Lower surface of first patterned template layer: 204, 304, 404, 904, 1304, 1404, 1404’

[0244] Apertures in first patterned template layer: 132, 132’, 206, 306, 406, 606, 706, 806, 906, 1206, 1306, 1406, 1406’

[0245] Apertures in first patterned template layer at upper surface: 1306a, 1406a, 1406a’

[0246] Apertures in first patterned template layer at lower surface: 1306b, 1406b, 1406b’

[0247] Retaining protrusions above upper surface: 1412

[0248] Retaining protrusions below lower surface: 1414’

[0249] Window area in first patterned template layer: 134, 208, 308, 408, 608, 708, 808, 1208, 1308, 1408, 1408’

[0250] Alignment feature(s) in first patterned template layer: 310a, 310b, 310c, 3 lOd, 410a, 410b, 410c, 510a, 510b, 560a, 560b, 910

[0251] Filter: 140

[0252] Second patterned template layer: 150, 1000, 1100, 1700, 1750

[0253] Upper surface of second patterned template layer: 1002, 1102

[0254] Lower surface of second patterned template layer: 1004, 1101

[0255] Window area in second patterned template layer: 154, 1008, 1108

[0256] Alignment feature(s) in second patterned template layer: 1110

[0257] Lid: 160, 2200

[0258] Base of lid: 2202

[0259] Sidewall(s) of lid: 2204

[0260] Retaining feature(s) of lid: 2206

[0261] Funnel 170’ x???: Dimension of ??? in the x axis (e.g., X206 is the dimension of 206 in the x axis) y???: Dimension of ??? in the y axis (e.g., y206 is the dimension of 206 in the y axis) z???: Dimension of ??? in the z axis (e.g., Z200 is the dimension of 200 in the z axis) d???: Dimension of ??? in the xy plane (e.g., diameter or equivalent diameter) (e.g., deoe is the dimension of 606 in the xy plane)

[0262] The foregoing outlines features of several embodiments so that those skilled in the art may better understand the aspects of the present disclosure. Those skilled in the art should appreciate that they may readily use the present disclosure as a basis for designing or modifying other processes and structures for carrying out the same purposes and / or achieving the same advantages of the embodiments introduced herein. Those skilled in the art should also realize that such equivalent constructions do not depart from the spirit and scope of the present disclosure, and that they may make various changes, substitutions, and alterations herein without departing from the spirit and scope of the present disclosure.

Claims

CLAIMSWhat is claimed is:

1. A device, comprising: a substrate; and a first patterned template layer on the substrate, the first patterned template layer having a plurality of apertures, wherein a fluid introduced into an aperture of the plurality of apertures settles on an upper surface of the substrate and forms a fluid droplet.

2. The device of claim 1, further comprising a coating applied on the upper surface of the substrate, wherein at least one of the following applies:(a) the coating is a hydrophobic coating, a hydrophilic coating, a charged coating, or a combination thereof;(b) the coating includes a poly-L-lysine coating;(c) the coating includes a patterned coating.

3. The device of claim 2, wherein the coating includes the patterned coating, and wherein at least one of the following applies:(a) the pattern of the coating corresponds to a pattern of the plurality of apertures in first patterned template layer;(b) the pattern of the coating creates a channel for a plurality of fluid droplets on an upper surface of the substrate to flow between spots on the substrate.

4. The device of claim 1, further comprising a wick between the substrate and the first patterned template layer, wherein the wick has a plurality of openings corresponding to the plurality of apertures in the first patterned template layer.

5. The device of claim 4, wherein the wick absorbs a liquid in the fluid droplet and leaves a substance of interest in the fluid droplet on the upper surface of the substrate.

6. The device of claim 1, further comprises a filter layer on and above the first patterned template layer.

7. The device of claim 6, wherein at least one of the following applies:(a) the filter layer removes a debris in the fluid, but does not remove a liquid and a substance of interest in the fluid;(b) the filter is capable of holding droplets of fluid on an upper surface thereof by surface tension prior to an application of a centrifugal force.

8. The device of claim 1, further comprises a second patterned template layer above the first patterned template layer, wherein at least one of the following applies:(a) the second patterned template layer has a window region, the window region providing access to or visual inspection of some or all of the apertures in the first patterned template layer,(b) the second patterned template layer has a plurality of apertures corresponding to the plurality of apertures in the first patterned template layer, the plurality of apertures in the second patterned template layer providing access to or visual inspection of some or all of the apertures in the first patterned template layer.

9. The device of claim 1, further comprises a lid above the first patterned template layer, the lid sealing the plurality of apertures of the first patterned template layer.

10. The device of claim 1, wherein at least one of the following applies:(a) the substrate or the first patterned template layer comprises an alignment feature for aligning elements of the device;(b) the device further comprises a second patterned template layer, and the second patterned template layer comprises an alignment feature for aligning elements of the device;(c) the device further comprises a lid, and the lid comprises an alignment feature for aligning elements of the device.

11. A method of processing a fluid sample with the device of claim 1, the method comprising: forming a droplet of the fluid sample on the upper surface of the substrate by introducing the fluid sample into an aperture of the plurality of the apertures in the first patterned template layer.

12. The method of claim 11, wherein at least one of the following applies:(a) the fluid sample is introduced directly into the aperture of the patterned template layer;(b) the device further comprises a filter layer above the first patterned template layer covering the aperture of the patterned template layer, and the fluid sample is introduced into the aperture of the patterned template layer by passing through the filter.

13. The method of claim 11, wherein forming the droplet of the fluid sample on the upper surface of the substrate comprises subjecting the device holding the fluid sample to a centrifugal force.

14. The method of claim 11, further comprising removing a liquid from the droplet of the fluid sample and leaving a substance of interest on a spot of the substrate.

15. The method of claim 14, wherein at least one of the following applies:(a) the liquid is removed from the droplet of the fluid sample by subjecting the substrate to an air flow or an elevated temperature;(b) the device further comprises a wick between the substrate and the first patterned template layer, the wick has an opening corresponding to the aperture in the first patterned template, and the wick absorbs the liquid from the droplet of the fluid sample.

16. A kit for assembling a device, comprising: a substrate; a first patterned template layer having a plurality of apertures; and an instruction manual instructing that: the first patterned template layer is to be placed on top of the substrate; and a fluid is to be introduced into the plurality of apertures, settles on an upper surface of the substrate, and forms a fluid droplet.

17. The kit of claim 16, further comprising a coating applied on the upper surface of the substrate, wherein at least one of the following applies:(a) the coating includes a hydrophobic coating, a hydrophilic coating, a charged coating, or a combination thereof;(b) the coating includes a poly-L-lysine coating;(c) the coating includes a patterned coating.

18. The kit of claim 17, wherein the coating includes the patterned coating, and wherein at least one of the following applies:(a) the pattern of the coating corresponds to a pattern of the plurality of apertures in first patterned template layer;(b) the pattern of the coating creates a channel for a plurality of fluid droplets on the upper surface of the substrate to flow between spots on the substrate.

19. The kit of claim 16, further comprises a wick having a plurality of openings corresponding to the plurality of apertures in the first patterned template layer, wherein the instruction manual instructs that the wick is to be placed between the substrate and the first patterned template layer, and the wick and the first patterned template layer are to be properly aligned such that the plurality of openings in the wick and the plurality of the apertures in the first patterned template layer are aligned.

20. The kit of claim 19, wherein the wick is made from an absorbent material that absorbs a liquid in the fluid droplets and allows a substance of interest in the fluid droplet to pass.

21. The kit of claim 16, further comprising a filter layer, wherein the instruction manual instructs that the filter layer is to be placed on and above the first patterned template layer.

22. The kit of claim 21, wherein at least one of the following applies:(a) the filter layer removes a debris in the fluid, but does not remove a liquid and a substance of interest in the fluid;(b) the filter is capable of holding droplets of fluid on an upper surface thereof by surface tension when not subjected to a downward centrifugal force.

23. The kit of claim 16, further comprising a second patterned template layer, wherein the instruction manual instructs that the second patterned template layer is to be placed above the first patterned template layer, and wherein at least one of the following applies:(a) the second patterned template layer has a window region, the window region providing access to or visual inspection of some or all of the apertures in the first patterned template layer,(b) the second patterned template layer has a plurality of apertures corresponding to the plurality of apertures in the first patterned template layer, the plurality of apertures in the second patterned template layer providing access to or visual inspection of some or all of the apertures in the first patterned template layer.

24. The kit of claim 16, further comprising a lid, wherein the instruction manual instructs that the lid is to be placed above the first patterned template layer, sealing the plurality of apertures of the first patterned template layer.

25. The kit of claim 16, wherein at least one of the following applies:(a) the substrate or the first patterned template layer comprises an alignment feature for aligning elements of the device;(b) the device further comprises a second patterned template layer, and the second patterned template layer comprises an alignment feature for aligning elements of the device;(c) the device further comprises a lid, and the lid comprises an alignment feature for aligning elements of the device; wherein the instruction manual instructs that the elements of the device are to be aligned using the aligning feature as guide.

Images