Two-dimensional matrix droplet array

Abstract

The present disclosure provides a device that may be used for a range of different assays such as immunoassays, nucleic acid analysis, metabolite analysis, clinical chemistry, and complete blood cell count. The device optionally contains a sample analysis region to analyze the samples processed in the device. The device comprises a top substrate bound to a bottom substrate wherein the top substrate bound to the bottom substrate forms two or more primary zones separated by one or more secondary zones, and wherein the top substrate has an opening in one or more of the primary zones.

Description

TWO-DIMENSIONAL MATRIX DROPLET ARRAYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefit to the filing date of U.S. Provisional Patent Application Serial No. 63 / 601,393, filed on November 21, 2023, and PCT Patent Application Serial No. PCT / US2024 / 036808, filed on July 3, 2024, the disclosure of which application are herein incorporated by reference in their entirety.INTRODUCTION

[0002] Analyte analysis is usually performed by carrying out sample preparation step that is either performed manually or using complicated robotics. After sample preparation, the assaying of an analyte in the prepared sample further involves use of expensive and complicated systems for transporting the prepared sample to a machine that then performing analysis of an analyte in the prepared sample.

[0003] Devices that can be used to prepare a sample for multiple types of assays and assay the prepared sample are highly desirable in the field of analyte analysis. Such devices would offer a low cost option and would considerably increase the ease of performing analyte analysis, especially in clinical applications, such as point-of-care applications.

[0004] As such, there is an interest in devices for sample preparation because they allow for reduced sample volumes and reagent volumes, potential for higher sensitivity, and faster time to result.SUMMARY

[0005] The present disclosure is defined by the appendant claims.

[0006] The present disclosure provides a device that may be used for a range of different assays such as immunoassays, nucleic acid analysis, metabolite analysis, clinical chemistry, and complete blood cell count. The device optionally contains a sample analysis region to analyze the samples processed in the device.

[0007] In an aspect, the disclosure provides a device that comprises a top substrate bound to a bottom substrate wherein the top substrate bound to the bottom substrate forms two or more primary zones separated by one or more secondary zones, and wherein the top substrate has an opening in one or more of the primary zones.

[0008] In another aspect, the disclosure provides a sample processing device comprising a top substrate, a bottom substrate attached to the top substrate so as to form an interior volume, whereinthe top substrate and / or bottom substrate substantially define a first plane, a sample processing region within the interior volume, wherein the sample processing region comprises two or more primary zones and one or more secondary zones, wherein each primary zone is separated from an adjacent primary zone in a direction substantially along the first plane by a secondary zone of the one or more secondary zones, wherein the top substrate comprises an opening into a respective primary zone of the two or more primary zones.

[0009] In other aspects, the disclosure provides methods of using the discussed devices and sample processing devices.

[0010] Also provided is an optional reagent delivery device that may be used with a version of the device. A pressure sample mixing device is also provided.

[0011] As will be discussed further below, the sample processing device of the disclosure may comprise an internal volume formed by the top substrate and bottom substrate when attached to one another. This internal volume may itself be split into, or comprise, different regions which are connected to one another. For instance, the device may comprise a sample processing region within, or forming part of, the internal volume. This sample processing region may be used to perform one or more processing steps on a sample attached to a microparticle as the microparticle is moved through the sample processing region.

[0012] The sample processing region may be connected to a sample analysis region, which sample analysis region can be used to analyze the processed sample. This analysis may be performed using external apparatus to, e.g. digitally image, the sample analysis region. In this context, a connection between regions is intended to mean that a microparticle can be moved between said regions, e.g. a path or channel exists for the microparticle to move from one region to another. The sample processing region may additionally or alternatively be connected to a sample mixing region. For instance, in use, a microparticle may be able to travel from a sample mixing region, into a sample processing region, and then into a sample analysis region.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 illustrates a top view of the top of the sample processing device according to one embodiment.

[0014] FIG. 2 illustrates a top view of the top of the sample processing device according to another embodiment.

[0015] FIG. 3 illustrates a top view of the top of the sample processing device according to another embodiment.

[0016] FIG. 4 illustrates a top view of the top of the sample processing device according to another embodiment.

[0017] FIG. 5 illustrates a top view of the top of the sample processing device according to another embodiment.

[0018] FIG. 6 illustrates a top view of the top of the sample processing device according to another embodiment.

[0019] FIG. 7 illustrates an isometric view of the top of the sample processing device according to one embodiment.

[0020] FIG. 8 illustrates an isometric view of the top of the sample processing device according to another embodiment.

[0021] FIG. 9 illustrates an enlargement of the sample analysis region according to one embodiment.

[0022] FIG. 10 illustrates an exemplary method of mixing the sample using the device according to one embodiment.

[0023] FIG. 11 illustrates an isometric view of the top of the sample processing device according to another embodiment.

[0024] FIG. 12 illustrates a top view of the top of the sample processing device according to another embodiment.

[0025] FIG. 13 illustrates a top view of the top of the sample processing device according to another embodiment.

[0026] FIG. 14 illustrates a bottom view of the top of the sample processing device, i.e., the top substrate, according to another embodiment.

[0027] FIG. 15 illustrates a bottom view of the top of the sample processing device, i.e., the top substrate, according to another embodiment.

[0028] FIG. 16 illustrates a cross-section two exemplary primary zones of the device according to an embodiment.

[0029] FIG. 17 illustrates an exemplary sample processing path according to an embodiment.

[0030] FIG. 18 illustrates an exemplary sample processing path according to an embodiment.

[0031] FIGs. 19A-19E illustrate an exemplary sample processing path according to an embodiment.

[0032] FIG. 20 illustrates an isometric view of the reagent delivery device according to an embodiment.

[0033] FIG. 21 illustrates a deconstructed view of the reagent delivery device according to an embodiment.

[0034] FIG. 22 illustrates a deconstructed view of a component of the reagent delivery device.

[0035] FIG. 23 illustrates a cross-section of the internal components of the reagent delivery device in the non-activated position according to an embodiment.

[0036] FIG. 24 illustrates a cross section of the internal components of the reagent delivery device in the activated position according to an embodiment.

[0037] FIG. 25 illustrates a cross-section of the internal components of a component of the reagent delivery device according to an embodiment.

[0038] FIGs. 26A-26D illustrate a cross-section of the primary zone with sample present.

[0039] FIGs. 27A-27B illustrate exemplary pad designs.

[0040] FIGs. 28A-28K illustrate exemplary fluid retention features.

[0041] FIG. 29 illustrates an exemplary sample mixing device.

[0042] FIGs. 30A-30C illustrates exemplary sample mixing devices.

[0043] FIG. 31 illustrates exemplary secondary features for fixed primary zones.

[0044] FIGs. 32A-32B. A. illustrates an exemplary method of mixing the sample using the device according to one embodiment. B. Illustrates a bottom view of the top of the sample processing device, i.e., the top substrate, according to another embodiment.

[0045] FIGs. 33A-33O. A-G. illustrates exemplary sample analysis regions. H-N. illustrates exemplary substrate retention features. O. illustrates an exemplary barrier feature.

[0046] FIG. 34 illustrates an exemplary sample analysis region of the embodiment depicted in FIG. 8 and FIG. 9.

[0047] FIGs. 35A-35H illustrate moving microparticles or microparticles and assisting particles across an array of wells and loading microparticles into wells of the array using a magnet in accordance with an aspect of the disclosed subject matter.

[0048] FIG. 36 is a diagram illustrating the magnet force upon a plurality of microparticles or microparticles and assisting particles.

[0049] FIGs. 37A-37C are diagrams of the cross-section of exemplary embodiments of the device.

[0050] FIGs. 38A-38B illustrates an exemplary embodiment of the device.

[0051] FIG. 39 illustrates an exemplary sample mixing region.

[0052] FIG. 40A-40B illustrates an exemplary embodiment of the device containing a waste disposal region..

[0053] FIGs. 41A-41B illustrates an exemplary pre-treatment region.

[0054] FIG. 42 illustrates an exemplary method of mixing the sample using the device according to one embodiment.

[0055] FIG. 43 illustrates an exemplary substrate stopper feature.

[0056] FIGs. 44A-44D illustrates exemplary features of the sample mixing region.

[0057] FIG. 45 illustrates an exemplary sample analysis region.

[0058] FIG. 46 illustrates an exemplary sample processing path according to an embodiment.

[0059] FIG. 47 illustrates an exemplary embodiment of the device containing surface finishes.

[0060] FIG. 48 illustrates an exemplary voice coil mixing method.

[0061] FIG. 49 illustrates exemplary particle patterns produced by voice coil mixing.

[0062] FIG. 50 illustrates exemplary particle mixing and movement produced by voice coil mixing.

[0063] FIG. 51 illustrates exemplary particle mixing produced by voice coil mixing at varying frequencies.

[0064] FIG. 52 illustrates the use of beads to capture target nucleic acids in a sample in a microwell prior to recombinase polymerase amplification (RPA) as described in Example 1. Specifically, FIG. 52A shows that when beads, such as polyethyleneimine (PEI)-coated beads, were used in microwells containing a target nucleic acid (a positive sample), a time-dependent fluorescence intensity increase in some microwells was observed, while almost no fluorescent microwells were observed for samples that did not contain any target nucleic acid (a negative sample). FIG. 52B shows a fluorescent well count of 5,000 cp for microwells containing target DNA (a positive sample) was sufficiently distinguished from microwells that did not contain any target DNA (a negative sample; 0 cp in assay).

[0065] FIG. 53 illustrates that the use of beads to capture target nucleic acids in a sample in a micro well prior to amplification, such as RPA, was successful to shorten the amplification time for such target nucleic acids when compared to amplification using general PCR as described in Example 1.

[0066] FIG. 54 illustrates an exemplary embodiment of the device.DETAILED DESCRIPTION

[0067] A device is disclosed. The device optionally contains a sample analysis region to analyze the samples processed in the device. Also provided herein are exemplary methods for using the device. Also provided is an optional reagent delivery device that may be used in conjunction with the device. Also provided are sample mixing devices and methods.

[0068] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to a particular embodiment described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0069] It must be noted that as used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, refer to “a primary zone” includes plurality of such primary zones and reference to “the well” includes reference to one or more wells and equivalents thereof known to those skilled in the art, and so forth.

[0070] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The present disclosure is controlling to the extent there is a contradiction between the present disclosure and a publication incorporated by reference.DETAILED DESCRIPTION

[0071] Embodiments of the present disclosure relate to methods, and devices for analysis of analyte(s) in a sample. The sample may be a range of different samples including, without limitation, a biological sample, an environmental sample, a food sample, a water sample, etc. In some embodiments, the biological sample is a liquid sample or a liquid extract of a solid sample. Non-limiting examples of biological samples include bodily fluid, blood, veinous blood, capillary blood, serum, plasma, urine, saliva, sweat, sputum, semen, mucus, lacrimal fluid, tears, dermal fluid, lymph fluid, amniotic fluid, interstitial fluid, intestinal fluid, gastrointestinal fluid, lung lavage, spinal fluid, cerebrospinal fluid, feces, nasal mucus, virginal discharge, tissue, organ, or like. In some embodiments, tissues may include, but are not limited to, skeletal muscle tissue, liver tissue, lung tissue, kidney tissue, myocardial tissue, brain tissue, bone marrow, cervix tissue, skin, etc. In certain cases, the source of the sample may be an organ or tissue, such as a biopsy sample, which may be solubilized by tissue disintegration / cell lysis.Definitions

[0072] Before the embodiments of the present disclosure are described, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0073] The modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context. When used in the context of a range, the modifier “about” should also be considered as disclosing the range defined by the absolute values of thetwo endpoints. For example, the range of from about “2 to about 10” also discloses the range “from 2 to 10.” The term “about” may refer to plus or minus 10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9-1.1.

[0074] It should be noted that many of the terms used herein are relative terms. For example, the terms “upper” and “lower” are relative to each other in location, i.e., an upper component is located at a higher elevation than a lower component in a given orientation, but these terms can change if the component is flipped. The terms “inlet” and “outlet” are relative to a fluid flowing through them with respect to a given structure, e.g., a fluid flows through the inlet into the structure and flows through the outlet out of the structure.

[0075] The terms “horizontal” and “vertical” are used to indicate direction relative to an absolute reference, i.e., ground level. However, these terms should not be construed to require structures to be absolutely parallel or absolutely perpendicular to each other. For example, a first vertical structure and a second vertical structure are not necessarily parallel to each other. The terms “top” and “bottom” are used to refer to surfaces where the top is always higher than the bottom relative to an absolute reference, i.e., the surface of the earth. The terms “upwards” and “downwards” are also relative to an absolute reference; upwards is always against the gravity of the earth while downwards is always towards the gravity of the earth.

[0076] “Comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0077] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0078] ‘ ‘Microbead” and “microparticle” are used herein interchangeably and refer to a substantially spherical solid support. The microbead or microparticle is a substantially spherical solid support that is influenced by a magnetic field such that the magnetic field can attract or repulse the microparticle or magnetic particle. A microbead or microparticle may occupy or settle in an array of wells, such as, for example, in an array of wells in a detection module. The microparticle and microbead may contain at least one specific binding member that binds to an analyte of interest and at least one detectable label. Alternatively, the microparticle and microbeadmay contain a first specific binding member that binds to the analyte and a second specific binding member that also binds to the analyte and contains at least one detectable label.

[0079] "Non-functional bead," "helper bead," and "assisting particle" are used interchangeably and refers to a substantially spherical assisting solid support, that is larger in diameter than a microparticle, which is configured to be chemically inert with respect to other components of an assay. As used herein, an assisting particle refers to a spherical particle which generally does not chemically interact with other particles (including a microparticle, conjugate, and / or reagent), but which is magnetic or paramagnetic. In certain exemplary embodiments, assisting particle may be coated so as to chemically interact with interf erents, that is, any materials which would interfere with assay or analysis of an analyte of interest within the targeted sample. In such embodiments, the assisting particles can also improve binding efficiency of the microparticles including, for the purpose of illustration and not limitation, by binding with interferents. Additionally and alternatively, the shape of a solid support can be roughly spherical, though not limited to such shapes.

[0080] The term “adjacent” is used with respect to the zones and regions of the device and sample processing device. The term "adjacent" may mean that the adjacent entities neighbor one another, even if separated by another entity. For instance, if two primary zones are adjacent one another they may be neighboring one another, even if they are separated by, e.g. an intervening secondary zone. Similarly, two regions of the device may be described as adjacent if they neighbor one another even if they are separated by, e.g., a channel or opening. If the device is planar, e.g. has a width and length significantly greater than its thickness (such that the width and length define a first plane), adjacent may be taken to mean that the zones or regions in question are located next to each other in the width and / or length direction (e.g. along a plane of the device), e.g. they may be side by side in the width and / or length direction.

[0081] The term “height” is used in respect of the zones and regions of the device. The “height” may be measured as, e.g., the shortest distance between the interior surface of the top substrate and the interior surface of the bottom substrate at a given location. For instance, if the height of a given primary zone is to be measured, this may be measured as the shortest distance between the interior surface of the top substrate at that primary zone and the interior surface of the bottom substrate at that primary zone. If the device is planar, e.g. has a width and length significantly greater than its thickness (such that the width and length define a first plane), the height may be measured in a direction substantially parallel to the thickness (e.g. perpendicular to the first plane).

[0082] The assisting solid supports can be larger in diameter than the other support mediums within the storage region and configured so as to not chemically interact with any othercomponents within the mixing region. Specifically, the diameter of the assisting solid supports (e.g. helper beads) can be at least 1 %, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least about 11 %, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 50%, at least 75%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, at least 600%, at least 650%, at least 700%, at least 750%, at least 800%, at least 850%, at least 900%, at least 950%, at least 1000% greater or larger than the diameter of other support mediums (e.g., microparticles).

[0083] The assisting particles can be configured such that they do not chemically bond or pair with other components of the targeted solution, such as the microparticles, target conjugates, and / or the target analyte. In certain exemplary embodiments, both microparticles and assisting particles can be magnetic, paramagnetic, or superparamagnetic particles (or any combination therein). In such exemplary embodiments, both microparticles and assisting particles, under the influence of a magnetic field or force, can form into chains of connected particles which facilitates mixing within the targeted solution.

[0084] As shown in FIG. 36, such a configuration can be achieved by the inclusion of a plurality of assisting particles within the sample. These assisting particles are larger than the microparticles. In certain embodiments, the assisting particles can have a diameter of between about 5 pm and about 15 pm, and in certain exemplary embodiments, about 8 pm and about 12 pm, preferably about 10 pm. In certain exemplary embodiments, the assisting particles do not affect immunoreactions or other interactions of the microparticles with an analyte of interest, antigen, antibody, or other particle. The assisting particles can also be magnetic or paramagnetic, and thus contribute to the strength of the effective magnetic force which acts to move the sample (which contains both the assisting particles and microparticles). In certain exemplary embodiments, the assisting particles can include a negative surface charge, for example and not limitation, greater than or equal to -30 mV. The assisting particles can also be sized so as not to interfere with the assay of the targeted microparticles. This combination of active microparticles and inactive assisting particles can achieve the advantages of both 1) strong magnetic force coupling with the sample to enable movement through an assay surface and 2) high detection sensitivity resulting from a reduced (overall) amount of microparticles in the sample.

[0085] For purpose of example and not limitation, the microparticles and assisting particles can include a ratio of microparticles to assisting particles of between about 1:1 and 100:1. In some embodiments, the microparticles and assisting particles can include a ratio of microparticles toassisting particles of between about 1:1 and 50:1. In some embodiments, the microparticles and assisting particles can include a ratio of microparticles to assisting particles of between about 1:1 and 25:1. In some embodiments, the microparticles and assisting particles can include a ratio of microparticles to assisting particles of between about 1:1 and about 20:1. In some embodiments, the microparticles and assisting particles can include a ratio of microparticles to assisting particles of between about 1:1 and about 15:1. In some embodiments, the microparticles and assisting particles can include a ratio of microparticles to assisting particles of between about 1 : 1 and about 10:1. In some embodiments, the microparticles and assisting particles can include a ratio of microparticles to assisting particles of between about 1:1 and about 5:1. In some embodiments, the microparticles and assisting particles can include a ratio of microparticles to assisting particles of between about 5:1 and about 25:1 In some embodiments, the microparticles and assisting particles can include a ratio of microparticles to assisting particles of between about 10:1 and about 20:1. In some embodiments, the microparticles and assisting particles can include a ratio of microparticles to assisting particles of about 20:1. In some embodiments, the microparticles and assisting particles can include a ratio of microparticles to assisting particles of about 10: 1. In some embodiments, the microparticles and assisting particles can include a ratio of microparticles to assisting particles of about 9:1. In some embodiments, the microparticles and assisting particles can include a ratio of microparticles to assisting particles of about 8:1. In some embodiments, the microparticles and assisting particles can include a ratio of microparticles to assisting particles of about 7:1. In some embodiments, the microparticles and assisting particles can include a ratio of microparticles to assisting particles of about 6:1. In some embodiments, the microparticles and assisting particles can include a ratio of microparticles to assisting particles of about 5:1. In some embodiments, the microparticles and assisting particles can include a ratio of microparticles to assisting particles of about 4:1. In some embodiments, the microparticles and assisting particles can include a ratio of microparticles to assisting particles of about 3:1. In some embodiments, the microparticles and assisting particles can include a ratio of microparticles to assisting particles of about 2:1. In some embodiments, the microparticles and assisting particles can include a ratio of microparticles to assisting particles of about 1:1.

[0086] “Component,” “components,” or “at least one component,” refer generally to a capture antibody, a detection reagent or conjugate, a calibrator, a control, a sensitivity panel, a container, a buffer, a diluent, a salt, an enzyme, a co-factor for an enzyme, a detection reagent, a pretreatment reagent / solution, a substrate (e.g., as a solution), a stop solution, and the like that can be included in a kit for assay of a test sample, such as a patient urine, serum, whole blood, tissue aspirate, or plasma sample, in accordance with the methods described herein and other methodsknown in the art. Some components can be in solution or lyophilized for reconstitution for use in an assay.

[0087] ‘ ‘Label” or “detectable label” as used interchangeably herein refers to a moiety attached to a specific binding member or analyte to render the reaction between the specific binding member and the analyte detectable, and the specific binding member or analyte so labeled is referred to as “detectably labeled.” A label can produce a signal that is detectable by visual or instrumental means. Various labels include: (i) a tag attached to a specific binding member or analyte by a cleavable linker; or (ii) signal-producing substance, such as chromagens, fluorescent compounds, enzymes, chemiluminescent compounds, radioactive compounds, and the like. Representative examples of labels include moieties that produce light, e.g., acridinium compounds, and moieties that produce fluorescence, e.g., fluorescein. Other labels are described herein. In this regard, the moiety, itself, may not be detectable but may become detectable upon reaction with yet another moiety. Use of the term “detectably labeled” is intended to encompass such labeling.

[0088] “Specific binding partner” or “specific binding member” as used interchangeably herein refer to one of two different molecules that specifically recognizes the other molecule compared to substantially less recognition of other molecules. The one of two different molecules has an area on the surface or in a cavity, which specifically binds to and is thereby defined as complementary with a particular spatial and polar organization of the other molecule. The molecules may be members of a specific binding pair. For example, a specific binding member may include, but not limited to, a protein, such as a receptor, an enzyme, an antibody and an aptamer, a peptide, a nucleotide, oligonucleotide, a nucleic acid, a polynucleotide and combinations thereof.

[0089] “Specific binding” or “specifically binding” as used herein may refer to the interaction of an antibody, a protein, or a peptide with a second chemical species, wherein the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.

[0090] The terms “polynucleotide” and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi- stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer including purine and pyrimidine bases or othernatural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The terms “polynucleotide” and “nucleic acid” should be understood to include, as applicable to the embodiment being described, single- stranded (such as sense or antisense) and double-stranded polynucleotides.

[0091] By "hybridizable" or “complementary” or “substantially complementary" it is meant that a nucleic acid (e.g. RNA, DNA) contains a sequence of nucleotides that enables it to non- covalently bind, i.e. form Watson-Crick base pairs and / or G / U base pairs, “anneal”, or “hybridize,” to another nucleic acid in a sequence- specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength. Standard Watson-Crick base-pairing includes: adenine / adenosine) (A) pairing with thymidine / thymidine (T), A pairing with uracil / uridine (U), and guanine / guanosine) (G) pairing with cytosine / cytidine (C). Inosine (I) bases pair with cytosine / cytidine. In addition, for hybridization between two RNA molecules (e.g., dsRNA), and for hybridization of a DNA molecule with an RNA molecule (e.g., when a DNA target nucleic acid base pairs with a guide RNA, etc.): G can also base pair with U. For example, G / U basepairing is partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anti-codon base-pairing with codons in mRNA. Thus, in the context of this disclosure, a G (e.g., of a protein-binding segment (e.g., dsRNA duplex) of a guide RNA molecule; of a target nucleic acid (e.g., target DNA or RNA) base pairing with a sensor RNA) is considered complementary to both a U and to C. For example, when a G / U base-pair can be made at a given nucleotide position of a protein-binding segment (e.g., dsRNA duplex) of a sensor RNA molecule, the position is not considered to be non-complementary, but is instead considered to be complementary.

[0092] Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible. The conditions appropriate for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, variables well known in the art. The greater the degree of complementarity between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences. Typically, the length for a hybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more).

[0093] It is understood that the sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, a polynucleotide mayhybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure, a ‘bulge’, and the like). A polynucleotide can include 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence complementarity to a target region within the target nucleic acid sequence to which it will hybridize. For example, an antisense nucleic acid in which 18 of 20 nucleotides of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. The remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides. Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined using any convenient method. Example methods include BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), e.g., using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).

[0094] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

[0095] "Analyte", " target analyte", "analyte of interest" as used interchangeably herein, refers to a substance, material or chemical constituent the presence, absence and / or amount of which is being analyzed in a biological sample obtained from a subject. In some aspects, the analyte is a biomolecule. Non-limiting examples of biomolecules include macromolecules such as, proteins, lipids, and carbohydrates. In certain instances, the analyte may be hormones, antibodies, growth factors, cytokines, enzymes, receptors (e.g., neural, hormonal, nutrient, and cell surface receptors) or their ligands, cancer markers (e.g., PSA, TNF-alpha), markers of myocardial infarction (e.g., troponin, creatine kinase, Creatinine kinase-cardiac muscle biomarker (CK-MB), B-type natriuretic peptide (also known as brain natriuretic peptide; BNP), N-terminal prohormone of brain natriuretic peptide (NT-proBNP) and the like), toxins, drugs (e.g., drugs of addiction), metabolic agents (e.g., including vitamins), and the like. Non-limiting examples of protein analytes include peptides, polypeptides, protein fragments, protein complexes, fusion proteins, recombinant proteins, phosphoproteins, glycoproteins, lipoproteins, or the like.

[0096] “Antibody” and “antibodies” as used herein refers to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies such as, but not limited to, a bird (for example, a duck or a goose), a shark, a whale, and a mammal, including a non-primate (for example, a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, etc.) or a nonhuman primate (for example, a monkey, a chimpanzee, etc.), recombinant antibodies, chimeric antibodies, single-chain Fvs (“scFv”), single chain antibodies, single domain antibodies, Fab fragments, F(ab') fragments, F(ab')2 fragments, disulfide-linked Fvs (“sdFv”), and anti-idiotypic (“anti-Id”) antibodies, dual-domain antibodies, dual variable domain (DVD) or triple variable domain (TVD) antibodies (dual-variable domain immunoglobulins and methods for making them are described in Wu, C., et al., Nature Biotechnology, 25(11): 1290-1297 (2007) and PCT International Application WO 2001 / 058956, the contents of each of which are herein incorporated by reference), and functionally active epitope-binding fragments of any of the above. Antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, namely, molecules that contain an analyte-binding site. Immunoglobulin molecules can be of any type (for example, IgG, IgE, IgM, IgD, IgA, and IgY), class (for example, IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass. For simplicity sake, an antibody against an analyte is frequently referred to herein as being either an “anti-analyte antibody” or merely an “analyte antibody”.

[0097] “Antibody fragment” as used herein refers to a portion of an intact antibody comprising the antigen-binding site or variable region. The portion does not include the constant heavy chain domains (i.e., CH2, CH3, or CH4, depending on the antibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv) molecules, single-chain polypeptides containing only one light chain variable domain, single-chain polypeptides containing the three CDRs of the light-chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and singlechain polypeptides containing the three CDRs of the heavy chain variable region.

[0098] “Epitope,” or “epitopes,” or “epitopes of interest” refer to a site(s) on any molecule that is recognized and can bind to a complementary site(s) on its specific binding partner. The molecule and specific binding partner are part of a specific binding pair. For example, an epitope can be on a polypeptide, a protein, a hapten, a carbohydrate antigen (such as, but not limited to, glycolipids, glycoproteins or lipopolysaccharides), or a polysaccharide. Its specific binding partner can be, but is not limited to, an antibody.

[0099] Capillary pressure barriers are meniscus pinning structures that create capillary stop structures and achieve meniscus alignment. Meniscus pinning occurs when energy has to be applied in order to advance the meniscus over its pinning position. Typically, a sharp ridge is used inside a channel or chamber to create a stable meniscus alignment feature that forces the meniscus to deform such that advancement of the meniscus becomes energetically disadvantageous. The meniscus then tends to align along the resulting capillary pressure barrier unless additional energy, in the form of e.g. an increase in fluid pressure, is applied.

[0100] A phaseguide is defined as a capillary pressure barrier that spans the complete length of an advancing phase front, such that the advancing front aligns itself along the phaseguide before crossing it. Phaseguide’s primarily function is defining a moveable meniscus and controlling the flow of the moveable meniscus. The location, shape, advancement, or some other physical characteristic can be influenced by the combined effects of the design of the stable capillary pressure barrier and energy (typically fluid pressure) applied to a fluid that exists on one or other of the sides of the meniscus. The primary zones and secondary zones of the present disclosure differ from phaseguides in that primary and secondary zones do not define moveable menisci or control the flow of advancing phase fronts. The primary and secondary zones pin droplets in stationary zones and do not advance, flow, or move from the zones once deposited.

[0101] ‘ ‘Contact angle” as used herein refers to the angle where a liquid surface meets a solid surface. The contact angle describes the shape of a liquid drop resting on the surface of the solid substrate, and is the angle of contact of the liquid on the surface of the solid substrate, measured within the liquid at the contact line where liquid, solid, and gas meet. A surface with a contact angle of <90° is considered to be hydrophilic and a contact angle of >90° is considered hydrophobic. As the contact angle of a surface increases, the surface is considered more hydrophobic. Unless stated otherwise, when the term contact angle is used it refers to contact angle between HPLC water and a specific surface at room temperature.

[0102] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety to disclose and describe the methods and / or materials in connection with which the publications are cited. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.1. OVERVIEW

[0103] Provided herein is a device, also known as a sample processing device, that may be used for sample processing and analyte detection. To provide structure to the description of the device, first, the overall device design will be disclosed. Second, the uses of the device will be disclosed. Third, the benefits of the device will be disclosed. Following an overview of the basic design of the device, a detailed disclosure of the device’s design will be disclosed which includes the arrangement of the sample processing region and the arrangement of the optional sample analysis region. Following the detailed disclosure of the device’s design, an exemplary optional reagent delivery device will be disclosed. In some embodiments, bulk reagent delivery may be used in place of the optional reagent delivery device. Following the disclosure of the exemplary optional reagent delivery device, exemplary sample mixing devices and methods will be disclosed.

[0104] I. Device design

[0105] The device comprises a top substrate bound to a bottom substrate where the top substrate bound to the bottom substrate forms two or more primary zones separated by two or more secondary zones. Each primary zone contains an opening in the top substrate. In some embodiments, each secondary zone contains an opening in the top substrate. In some embodiments, each secondary zone does not contain an opening in the top substrate. The sample processing region is formed between the top and the bottom substrate. The sample processing region is unbounded throughout the region (i.e., the interior of the device containing the primary and secondary zones). In some embodiments, the top substrate and bottom substrate form a first distance between the top substrate and bottom substrate within the primary zones and a second distance between the top substrate and the bottom substrate within the secondary zones. In some embodiments, the first distance is less than the second distance.

[0106] The first distance may be less than the second distance because the pad of the primary zone protrudes into the sample processing region in at least some primary zones. This means that the distance from the pad to the interior surface of the bottom substrate is less than the distance from the interior surface of the top substrate to the interior surface of the bottom substrate in a secondary zone.

[0107] The top substrate may be substantially planar and the top substrate may have a width, length, and thickness, wherein the width and length are significantly greater than the thickness. The bottom substrate may be substantially planar and the bottom substrate may have a width, length, and thickness, wherein the width and length are significantly greater than the thickness. The width and length of the top substrate may be substantially identical to the width and length of the bottom substrate or the width and / or length may differ.

[0108] The device may be described as a “two-dimensional matrix droplet array” in that the device comprises a plurality of primary zones and secondary zones arranged substantially along two dimensions of a plane. At least some of the primary zones and secondary zones are configured to receive droplets of a fluid.

[0109] In some embodiments, the device comprises a first substrate and a second substrate positioned on the first substrate. The second substrate comprises a sidewall about at least a portion of a periphery of the second substrate where the first substrate, sidewall, and second substrate define a central chamber therebetween. The second substrate comprises a surface facing the central chamber comprising a plurality of recessed elements, and a plurality of protruding elements wherein primary zones are defined between a surface of the plurality of protruding elements facing the first substrate and a surface of the first substrate facing the second substrate and wherein secondary zones are defined between a surface of the recessed element facing the first substrate and the surface of the first substrate facing the second substrate. The second substrate has an opening in one or more of the primary zones.

[0110] In some embodiments, the device comprises a first substrate and a second substrate positioned on the first substrate. The first substrate comprises a sidewall about at least a portion of a periphery of the first substrate where the first substrate, sidewall, and second substrate define a central chamber therebetween. The second substrate comprises a surface facing the central chamber comprising a plurality of recessed elements, and a plurality of protruding elements wherein primary zones are defined between a surface of the plurality of protruding elements facing the first substrate and a surface of the first substrate facing the second substrate and wherein secondary zones are defined between a surface of the recessed element facing the first substrate and the surface of the first substrate facing the second substrate. The second substrate has an opening in one or more of the primary zones.

[0111] In some embodiments, the device comprises a first substrate, a spacer layer positioned on a surface of the first substrate wherein the spacer layer is disposed about at least a portion of a periphery of the first substrate, and a second substrate positioned on the first substrate. The first substrate comprises a sidewall about at least a portion of a periphery of the second substrate where the first substrate, sidewall, and second substrate define a central chamber therebetween. The second substrate comprises a surface facing the central chamber comprising a plurality of recessed elements, and a plurality of protruding elements wherein primary zones are defined between a surface of the plurality of protruding elements facing the first substrate and a surface of the first substrate facing the second substrate and wherein secondary zones are defined between a surface of the recessed element facing the first substrate and the surface of the first substrate facing thesecond substrate. The second substrate has an opening in one or more of the primary zones. In some embodiments, the spacer layer is selected from the group consisting of: an adhesive layer, a shim layer, and a raised feature layer. In some embodiments, the spacer layer is an adhesive layer. In some embodiments, the spacer layer is a shim layer. In some embodiments, the spacer layer is a first adhesive layer, a shim layer, and a second adhesive layer. In some embodiments, the spacer layer is a first adhesive layer, a raised feature layer, and a second adhesive layer. In some embodiments, the adhesive is a ultraviolet (UV) bonding adhesive.

[0112] The term “top substrate” may be used interchangeably with the term “second substrate”. The term “bottom substrate” may be used interchangeably with the term “first substrate”. The term “pad” may be used interchangeably with the term “protruding element”.

[0113] The device comprises a sample processing region in addition to an optional sample analysis region. The sample processing region is configured to allow the processing of a sample in a modular manner. By a “modular manner” it is meant that depending on the assay, the primary and / or secondary zones may be filled with reagents (e.g., wash buffer, lysis buffer, conjugation reagents, detection reagents, amplification reagent, etc.) particular for a given assay and the device is compatible with different types of assays (e.g., immunoassays, nucleic acid analysis, metabolite analysis, clinical chemistry, etc.) and different ways of performing the assays (e.g., modulating the number of wash steps, conjugation steps, etc.). The sample or components of the sample are moved from one primary zone to another primary zone using microparticles. In general, a sample is added to a primary zone in the sample processing region. Microparticles are added at the same time, before, or after the sample is added to the same primary and / or secondary zone that the sample is added to or will be added to. In some embodiments, the microparticles are already present in the primary and / or secondary zone to which the sample is added to. In some embodiments, the microparticles are microparticles and assisting particles. The sample or component of the sample is then bound to the microparticles and the microparticles or microparticles and assisting particles are moved using a magnetic field to a different primary zone. After sample processing is complete, the microparticles or microparticles and assisting particles are moved to a sample analysis region on the device or off the device.

[0114] FIG. 1 discloses an illustration of an embodiment of the device. In this embodiment, the device 100 comprises a sample processing region 110. The sample processing region 110 is unbounded. By unbounded it is meant that there is no physical barrier separating the primary zones 101 and the secondary zones 107. While there is only a single element label for the primary zone 101, each square (101) is to represent an individual primary zone. While there is only a single element label for the secondary zone 107, each space between the squares is to represent anindividual secondary zone. The sample processing region comprises a primary zone 101 that has an opening 103 in the primary zone. While there is only a single element label for the opening in the primary zone 103, each opening (103) in the square (101) is meant to represent an individual opening in the primary zone. The primary zone is adjacent to the secondary zone 107. The secondary zone may comprise an opening 104. While there is only a single element label for the opening in the secondary zone 107, each opening (104) in between the squares represents an individual opening in the secondary zone. The sample processing region 110 may be connected to a tertiary zone (i.e., the sample detection zone) 112 by a transition zone 113. The device may further comprise a quaternary zone 114 (i.e., a hydrophilic liquid well) that is connected to the tertiary zone. The top and the bottom substrate are bound by adhesive or clips 111. In some embodiments, the top substrate and bottom are bound by an adhesive layer between the top and bottom substrate. In some embodiments, the adhesive is a ultraviolet (UV) bonding adhesive. In some embodiments, the top and bottom substrate are bound using laser welding.

[0115] An alternative embodiment of FIG. 1 is disclosed below. In this embodiment, the device100 comprises a sample processing region 110. The device 100 comprises a second substrate positioned on a first substrate. The second substrate comprises a surface facing a central chamber comprising a plurality of recessed elements, and a plurality of protruding elements. The primary zones 101 are defined between a surface of the plurality of protruding elements facing the first substrate and a surface of the first substrate facing the second substrate. The secondary zones 107 are defined between a surface of the plurality of recessed elements facing the first substrate and the surface of the first substrate facing the second substrate. The sample processing region 110 is unbounded. By unbounded it is meant that there is no physical barrier separating the primary zones101 and the secondary zones 107. While there is only a single element label for the primary zone 101, each square (101) is to represent an individual primary zone. In some embodiments, the primary zones are discrete. By “discrete” it is meant that each primary zone is physically separated from each other primary zone. While there is only a single element label for the secondary zone 107, each space between the squares is to represent an individual secondary zone. In some embodiments, the secondary zones are connected. By “connected” it is meant that secondary zones are physically associated with each other. The sample processing region comprises a primary zone 101 that has an opening 103 in the primary zone. While there is only a single element label for the opening in the primary zone 103, each opening (103) in the square (101) is meant to represent an individual opening in the primary zone. The primary zone is adjacent to the secondary zone 107. The secondary zone may comprise an opening 104. While there is only a single element label for the opening in the secondary zone 107, each opening (104) in between the squares represents anindividual opening in the secondary zone. The sample processing region 110 may be positioned adjacent to a sample analysis region comprising a tertiary zone (i.e., the sample detection zone) 112, a quaternary zone 114 (i.e., a hydrophilic liquid well), and a quinary zone 115 (i.e., a hydrophobic liquid well) wherein the tertiary zone 112, the quaternary zone 114, and the quinary zone 115 are connected. The sample analysis region is defined by the surface of the second substrate facing the first substrate and the surface of the first substrate facing the second substrate. The quinary zone 115 is located at a first end of the sample analysis region. The quinary zone comprises an opening spanning the second substrate. The quaternary zone 114 is located at a second end of the sample analysis region. The quaternary zone comprises a cylindrical opening spanning the second substrate. The tertiary zone 112 is located at a midpoint between the first end (e.g., the quinary zone 115) and the second end (e.g., the quaternary zone 115) of the of the sample analysis region. The sample analysis region may be connected to the sample processing region 110 by a transition zone 113. The second substrate positioned over the first substrate are bound together by adhesive or clips 111. In some embodiments, the second substrate and the first substrate are bound by an adhesive layer between the second substrate and the first substrate. In some embodiments, the adhesive is a ultraviolet (UV) bonding adhesive. In some embodiments, the second substrate and the first substrate are bound using laser welding.

[0116] FIG. 26B discloses a schematic representation of a cross-section of the sample processing region in an embodiment. In FIG. 26B, the sample processing region 2605 contains a primary zone 2608 and two secondary zones 2607. The sample processing region 2605 is formed from the top substrate 2609 bound to the bottom substrate 2610. The primary zone 2608 contains an opening 2603. The secondary zone contains an opening 2604. The sample or reagent 2602 may be added through the opening 2603 of the primary zone 2608 wherein the sample or reagent 2602 is held in place through capillary forces generated by the opening 2603 and surface tension facilitated by the edges of a pad 2601 in the primary zone 2608. Generally, the secondary zones comprise air and serve as hydrophobic zones that assist in the holding of the sample or reagents in the primary zones. In some embodiments, the secondary zones also contain reagents or samples. In some embodiments, the secondary zones do not contain the sample or reagent. In some embodiments, a portion of the secondary zones contain the sample or reagent and a portion of the secondary zones do not contain the sample or reagents.

[0117] An alternative embodiment of FIG. 26B is described below. FIG. 26B discloses a schematic representation of a cross-section of the sample processing region in an embodiment. In FIG. 26B, the sample processing region 2605 contains a primary zone 2608 and two secondary zones 2607. The sample processing region 2605 is defined by a second substrate 2609 positionedon a first substrate 2610. Either the first substrate 2610 or the second substrate 2609 comprises a sidewall about at least a portion of a periphery of the first substrate 2610. The first substrate 2610, the sidewall, and the second substrate 2609 define a central chamber therebetween (i.e., the space occupied by 2607 and 2608). The second substrate comprises a surface (i.e., the interior portion of 2609) facing the central chamber (i.e., the space occupied by 2607 and 2608) comprising a protruding element 2601 and recessed elements (i.e., the upper portion of 2607). While only a single protruding element is shown it is to be understood that the device comprises a plurality of protruding elements. While only two recessed elements are shown it is to be understood that the device comprises a plurality of recessed elements. The primary zone 2608 is defined between the surface of the protruding element (i.e., the interior portion of 2601) facing the first substrate 2610 and a surface of the first substrate facing the second substrate (i.e., the interior portion of 2610). The secondary zones 2607 are defined between a surface of the recessed elements (i.e., the upper portion of 2607) and the surface of the first substrate facing the second substrate (i.e., the interior portion of 2610). The second substrate 2609 comprises an opening 2603 in the primary zone. The second substrate 2609 comprises an opening 2604 in the secondary zones. The sample or reagent 2602 may be added through the opening 2603 of the primary zone 2608 wherein the sample or reagent 2602 is held in place through capillary forces generated by the opening 2603 and surface tension facilitated by the protruding element 2601 in the primary zone 2608. Generally, the secondary zones comprise air and serve as hydrophobic zones that assist in the holding of the sample or reagents in the primary zones. In some embodiments, the secondary zones also contain reagents or samples. In some embodiments, the secondary zones do not contain the sample or reagent. In some embodiments, a portion of the secondary zones contain the sample or reagent and a portion of the secondary zones do not contain the sample or reagents.

[0118] FIG. 37A discloses a schematic representation of a cross-section of the sample processing region of the device in an embodiment where the second substrate comprises a sidewall. In FIG. 37A, the device 3700 comprises a first substrate 3702 and second substrate 3701 positioned on the first substrate 3702. The second substrate 3701 comprises a sidewall 3703 about at least a portion of a periphery of the first substrate. The first substrate 3702, the sidewall 3703, and the second substrate 3701 define a central chamber 3704 therebetween. The second substrate comprises a surface facing the central chamber 3704 comprising a plurality of protruding elements (3705a, 3705b, and 3705c) and a plurality of recessed elements (3706a, 3706b, and 3706c). The border of the sidewall 3703 of the second substrate and the first substrate 3702 is defined by 3707 (black line). The primary zones (3710a, 3710b, and 3710c; bounded by thick black lines) are defined between the surface of the plurality of protruding elements (3705a, 3705b, and 3705c)facing the first substrate and the surface of the first substrate facing the second substrate (interior surface of 3702). The secondary zones (3711a, 3711b, 3711c, and 371 Id; bounded by thick black lines) are defined between the surface of the plurality of recessed elements (3706a, 3706b, and 3706c) facing the first substrate and the surface of the first substrate facing the second substrate (interior surface of 3702). The second substrate 3701 has an opening 3708 in the primary zones. The second substrate 3701 has an opening 3709 in one or more of the secondary zones.

[0119] FIG. 37B discloses a schematic representation of a cross-section of the sample processing region of the device in an embodiment where the first substrate comprises a sidewall. In FIG. 37B, the device 3720 comprises a first substrate 3722 and second substrate 3721 positioned on the first substrate 3722. The first substrate 3722 comprises a sidewall 3723 about at least a portion of a periphery of the first substrate. The first substrate 3722, the sidewall 3723, and the second substrate 3721 define a central chamber 3724 therebetween. The second substrate comprises a surface facing the central chamber comprising a plurality of protruding elements (3725a, 3725b, and 3725c) and a plurality of recessed elements (3726a, 3726b, and 3726c). The border of the sidewall 3723 of the first substrate and the second substrate 3722 is defined by 3727 (black line). The primary zones (3730a, 3730b, and 3730c; bounded by thick black lines) are defined between the surface of the plurality of protruding elements (3725a, 3725b, and 3725c) facing the first substrate and the surface of the first substrate facing the second substrate (interior surface of 3722). The secondary zones (3731a, 3731b, 3731c, and 373 Id; bounded by thick black lines) are defined between the surface of the plurality of recessed elements (3726a, 3726b, and 3726c) facing the first substrate and the surface of the first substrate facing the second substrate (interior surface of 3722). The second substrate 3721 has an opening 3728 in the primary zones. The second substrate 3721 has an opening 3729 in one or more of the secondary zones.

[0120] FIG. 37C discloses a schematic representation of a cross-section of the sample processing region of the device in an embodiment where the device comprises a spacer layer. In FIG. 37C, the device 3740 comprises a first substrate 3742 and a spacer layer 3743 positioned on the first substrate 3742 where the spacer layer is disposed about at least a portion of a periphery of the first substrate 3742. In some embodiments, the at least a portion of the periphery comprises at least two, at least three, or at least four peripheral sides of the second substrate. In some embodiments, the spacer layer is selected from the group consisting of: an adhesive layer, a shim layer, and a raised feature layer. In some embodiments, the spacer layer is an adhesive layer. In some embodiments, the spacer layer is a shim layer. In some embodiments, the spacer layer is a first adhesive layer, a shim layer, and a second adhesive layer. A second substrate 3741 positioned on the spacer layer 3743. In some embodiments, the adhesive is a ultraviolet (UV) bondingadhesive. The first substrate 3742, the spacer layer 3743, and the second substrate 3741 define a central chamber 3744 therebetween. The second substrate comprises a surface facing the central chamber comprising a plurality of protruding elements (3745a, 3745b, and 3745c) and a plurality of recessed elements (3746a, 3746b, and 3746c). The border of the spacer layer 3743, the first substrate 3742, and the second substrate 3741 is defined by 3747 (black lines). The primary zones (3750a, 3750b, and 3750c; bounded by thick black lines) are defined between the surface of the plurality of protruding elements (3745a, 3745b, and 3745c) facing the first substrate and the surface of the first substrate facing the second substrate (interior surface of 3742). The secondary zones (3751a, 3751b, 3751c, and 3751d; bounded by thick black lines) are defined between the surface of the plurality of recessed elements (3746a, 3746b, and 3746c) facing the first substrate and the surface of the first substrate facing the second substrate (interior surface of 3742). The second substrate 3741 has an opening 3748 in the primary zones. The second substrate 3741 has an opening 3749 in one or more of the secondary zones.

[0121] The schematics disclosed in FIG. 37A, FIG. 37B, and FIG. 37C may be applied to any of the devices disclosed herein. The schematics disclosed in FIG. 37A, FIG. 37B, and FIG. 37C may be applied to the devices disclosed in FIG. 1-15, FIG. 17-19, and FIG. 32.

[0122] As discussed above, the device may use capillary forces and surface tension to retain fluid within the various zones of the device. This effect occurs because of the small size of the device and, e.g., the small dimensions associated with the primary and secondary zones. In particular, the behavior of a contained fluid is dominated by capillary forces and surface tension rather than gravity. This means, for instance, that an opening may provide capillary force to retain fluid within a given zone and that the edges of the pad may provide surface tension to retain fluid within a primary zone.

[0123] The top and bottom substrates of the present disclosure may be made of a range of different materials such that the materials facilitate the methods and designs disclosed herein. The material may be rigid or flexible. The rigidity and flexibility may be controlled both by the material used and the thickness of the material in the top and bottom substrate. In some embodiments, the top and bottom substrate are made of the same material. In some embodiments, the top and bottom substrates are made from different materials. In some embodiments, the entire top substrate is made of the same material. In some embodiments, the top substrate is made from a combination of materials where a portion of the top substrate is made from one material and a different portion of the top substrate is made from a different material. In some embodiments, the bottom substrate is made from a combination of materials where a portion of the bottom substrate is made from one material and a different portion of the bottom substrate is made from a differentmaterial. For instance, materials that find use in the present disclosure include, without limitation, glass, silicon, ceramic, metal, polymethyl methacrylate (PMMA), polystyrene (PS), polycarbonate (PC), cyclic olefin copolymer (COC), cyclic olefin polymer (COP), polypropylene (PP), polyurethane (PU), polytetrafluoroethylene (PTFE), polyvinylchloride (PVC), polydimethylsiloxane (PDMS), acrylonitrile butadiene styrene (ABS), poly(lactic acid) (PLA), thermoplastic PU, clear resin, polyethylene glycol diacrylate (PEGDA), thin film, etc. In some embodiments, the top substrate (or second substrate) is the same hydrophobicity as the bottom substrate (or first substrate). In some embodiments, the top substrate (second substrate) is a different hydrophobicity than the bottom substrate (or first substrate).

[0124] In some embodiments, all or a portion of the top substrate is made of glass. In some embodiments, all or a portion of the top substrate is made of silicon. In some embodiments, all or a portion of the top substrate is made of ceramic. In some embodiments, all or a portion of the top substrate is made of metal. In some embodiments, all or a portion of the top substrate is made of polymethyl methacrylate (PMMA). In some embodiments, all or a portion of the top substrate is made of polystyrene (PS). In some embodiments, all or a portion of the top substrate is made of polycarbonate (PC). In some embodiments, all or a portion of the top substrate is made of cyclic olefin copolymer (COC). In some embodiments, all or a portion of the top substrate is made of cyclic olefin polymer (COP). In some embodiments, all or a portion of the top substrate is made of polypropylene (PP). In some embodiments, all or a portion of the top substrate is made of polyurethane (PU). In some embodiments, all or a portion of the top substrate is made of polytetrafluoroethylene (PTFE). In some embodiments, all or a portion of the top substrate is made of polyvinylchloride (PVC). In some embodiments, all or a portion of the top substrate is made of polydimethylsiloxane (PDMS). In some embodiments, all or a portion of the top substrate is made of acrylonitrile butadiene styrene (ABS). In some embodiments, all or a portion of the top substrate is made of poly(lactic acid) (PLA). In some embodiments, all or a portion of the top substrate is made of thermoplastic PU. In some embodiments, all or a portion of the top substrate is made of clear resin. In some embodiments, all or a portion of the top substrate is made of polyethylene glycol diacrylate (PEGDA).

[0125] In some embodiments, all or a portion of the bottom substrate is made of glass. In some embodiments, all or a portion of the bottom substrate is made of silicon. In some embodiments, all or a portion of the bottom substrate is made of ceramic. In some embodiments, all or a portion of the bottom substrate is made of metal. In some embodiments, all or a portion of the bottom substrate is made of polymethyl methacrylate (PMMA). In some embodiments, all or a portion of the bottom substrate is made of polystyrene (PS). In some embodiments, all or a portion of thebottom substrate is made of polycarbonate (PC). In some embodiments, all or a portion of the bottom substrate is made of cyclic olefin copolymer (COC). In some embodiments, all or a portion of the bottom substrate is made of cyclic olefin polymer (COP). In some embodiments, all or a portion of the bottom substrate is made of polypropylene (PP). In some embodiments, all or a portion of the bottom substrate is made of polyurethane (PU). In some embodiments, all or a portion of the bottom substrate is made of polytetrafluoroethylene (PTFE). In some embodiments, all or a portion of the bottom substrate is made of polyvinylchloride (PVC). In some embodiments, all or a portion of the bottom substrate is made of polydimethylsiloxane (PDMS). In some embodiments, all or a portion of the bottom substrate is made of acrylonitrile butadiene styrene (ABS). In some embodiments, all or a portion of the bottom substrate is made of poly(lactic acid) (PLA). In some embodiments, all or a portion of the bottom substrate is made of thermoplastic PU. In some embodiments, all or a portion of the bottom substrate is made of clear resin. In some embodiments, all or a portion of the bottom substrate is made of polyethylene glycol diacrylate (PEGDA). In some embodiments, all or a portion of the bottom substrate is made of a thin film. Thin films provide the advantage of enhanced mixing of fluids in the device due to the deformability of the thin film.

[0126] 1(a). Sample processing region

[0127] The sample processing region of the devices disclosed herein have an arrangement of primary zones and secondary zones. In some embodiments, the primary zones are discrete. By “discrete” it is meant that each primary zone is physically separated from each other primary zone. In some embodiments, the secondary zones are connected. By “connected” it is meant that secondary zones are physically associated with each other. There is a range in the number of primary and secondary zones that is dependent on the particular embodiment being described. The arrangement of the primary and secondary zones is also variable and dependent on the particular embodiment discussed.

[0128] The device comprises a range in the number of primary and secondary zones. For example, the device may comprise 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, or 24 or more primary zones. The device may comprise 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, 36 ormore, or 37 or more secondary zones. Each adjacent primary zone is separated by 1 secondary zone such that if there were 3 primary zones in a straight line there would be a 1stsecondary zone between the 1stand 2ndprimary zone and 2ndsecondary zone between the 2ndand 3rdprimary zone.

[0129] The primary and secondary zones may be arranged in a number of different patterns. For instance, the pattern may be a grid, lines, or a non-grid pattern. When the pattern is a grid, the grid may be 2x4, 2x5, 2x6, 2x7, 2x8, 2x9, 2x10, 3x4, 3x5, 3x6, 3x7, 3x8, 3x9, 3x10, 4x2, 4x3, 4x4, 4x5, 4x6, 4x8, 4x9, or 4x10 where the first number indicates the number of columns, and the second number indicates the number of rows. When the pattern is a line, the line may be 1x3, 1x4, 1x5, 1x6, 1x7, 1x8, 1x9, or 1x10. When the pattern is a non-grid, the non-grid may be in the form of a honeycomb pattern such as depicted in FIG. 11. When the pattern is a non-grid, the non-grid may be in the form of an irregular pattern such as depicted in FIG. 27A.

[0130] The pads or protruding elements in the primary zones may be separated by a particular distance such that fluids that may be contained within one primary zone does not bridge to another primary zone. The distance between primary zones may be a range of values. For instance, the distance between primary zones may be about 1mm to about 2mm. In some embodiments, the distance between primary zones may be greater than 2mm. The range of distance or specific distance may be any intervening distance. For instance, the distance between primary zones may be at least about 1.00mm, at least about 1.01mm, at least about 1.02mm, at least about 1.03mm, at least about 1.04mm, at least about 1.05mm, at least about 1.06mm, at least about 1.07mm, at least about 1.08mm, at least about 1.09mm, at least about 1.10mm, at least about 1.11mm, at least about 1.12mm, at least about 1.13mm, at least about 1.14mm, at least about 1.15mm, at least about1.16mm, at least about 1.17mm, at least about 1.18mm, at least about 1.19mm, at least about1.20mm, at least about 1.21mm, at least about 1.22mm, at least about 1.23mm, at least about1.24mm, at least about 1.25mm, at least about 1.26mm, at least about 1.27mm, at least about1.28mm, at least about 1.29mm, at least about 1.30mm, at least about 1.31mm, at least about1.32mm, at least about 1.33mm, at least about 1.34mm, at least about 1.35mm, at least about1.36mm, at least about 1.37mm, at least about 1.38mm, at least about 1.39mm, at least about1.40mm, at least about 1.41mm, at least about 1.42mm, at least about 1.43mm, at least about1.44mm, at least about 1.45mm, at least about 1.46mm, at least about 1.47mm, at least about1.48mm, at least about 1.49mm, at least about 1.50mm, at least about 1.51mm, at least about1.52mm, at least about 1.53mm, at least about 1.54mm, at least about 1.55mm, at least about1.56mm, at least about 1.57mm, at least about 1.58mm, at least about 1.59mm, at least about1.60mm, at least about 1.61mm, at least about 1.62mm, at least about 1.63mm, at least about1.64mm, at least about 1.65mm, at least about 1.66mm, at least about 1.67mm, at least about1.68mm, at least about 1.69mm, at least about 1.70mm, at least about 1.71mm, at least about 1.72mm, at least about 1.73mm, at least about 1.74mm, at least about 1.75mm, at least about 1.76mm, at least about 1.77mm, at least about 1.78mm, at least about 1.79mm, at least about 1.80mm, at least about 1.81mm, at least about 1.82mm, at least about 1.83mm, at least about 1.84mm, at least about 1.85mm, at least about 1.86mm, at least about 1.87mm, at least about 1.88mm, at least about 1.89mm, at least about 1.90mm, at least about 1.91mm, at least about 1.92mm, at least about 1.93mm, at least about 1.94mm, at least about 1.95mm, at least about1.96mm, at least about .97mm, at least about 1.98mm, at least about 1.99mm, or at least about 2mm.

[0131] The sample may be added to a fixed primary zone or a variable primary zone. By “fixed” primary zone it is meant that the primary zone for sample addition is fixed in place, i.e., a specific primary zone. In some embodiments, the fixed primary zone has one or more openings. For instance, there may be one or more, two or more, three or more, four or more, or five or more openings. By “variable” primary zone it is meant that the sample may be added to any of the primary zones present in the device. The terms “fixed primary zone” and “variable primary zone” are solely used for the purposes of referring to primary zones to which the sample is added and not primary zones to which the sample is not directly added.

[0132] In some embodiments, the sample processing region comprises a fixed primary zone. When the sample processing region comprises a fixed primary zone, the fixed primary zone may be larger than the primary zones. In some embodiments, the fixed primary zone is the same size as the primary zones. In some embodiments, the fixed primary zone is separate from the primary zones. In some embodiments, the fixed primary zone is a sample mixing region. In some embodiments, the fixed primary zone has one or more openings. For instance, there may be one or more, two or more, three or more, four or more, or five or more openings. In some embodiments, the sample processing region comprises two or more fixed primary zones. The fixed primary zones may have one or more secondary features that assist in the mixing of fluids contained therein. There may be a range in the number of secondary features in the fixed primary zone. For instance, there may be one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, or twenty or more. In some embodiments, the secondary features are according to FIG. 31. The secondary features may have any shape that assists in mixing that may occur in the fixed primary zone. Non-limiting examples of the shape include, without limitation, arectangle, a circle, a triangle, a pentagon, a hexagon, a heptagon, an octagon, a decagon, a dodecagon, an amoeboid, a non-regular shape, etc.

[0133] In some embodiments, the fixed primary zone is formed from a laterally extended protruding element in the second substrate facing the first substrate wherein the laterally extended protruding element has a surface facing the first substrate that has a greater surface area than the surface of the plurality of protruding elements facing the first substrate. The laterally extended protruding element may be referred to as a tertiary protruding element. When the sample processing region comprises a fixed primary zone, the fixed primary zone may be larger than the primary zones. In some embodiments, the fixed primary zone has a surface facing the first substrate that has a greater surface area than the surface of the plurality of protruding elements facing the first substrate. In some embodiments, the fixed primary zone is separate from the primary zones. In some embodiments, the fixed primary zone is a sample mixing region. In some embodiments, the fixed primary zone is a cantilever wherein only a portion of the fixed primary zone is attached to the second substrate. In some embodiments, the fixed primary zone has one or more openings. For instance, there may be one or more, two or more, three or more, four or more, or five or more openings. In some embodiments, the sample processing region comprises two or more fixed primary zones. The fixed primary zones may have one or more secondary features that assist in the mixing of fluids contained therein. There may be a range in the number of secondary features in the fixed primary zone. For instance, there may be one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, or twenty or more. In some embodiments, the secondary features are according to FIG. 31. The secondary features may have any shape that assists in mixing that may occur in the fixed primary zone. Non-limiting examples of the shape include, without limitation, a rectangle, a circle, a triangle, a pentagon, a hexagon, a heptagon, an octagon, a decagon, a dodecagon, an amoeboid, a non-regular shape, etc.

[0134] In some embodiments, the sample processing region comprises a variable primary zone. The variable primary zone may be any of the primary zones present in the sample processing region. The sample may be added to the variable primary zone before, after, or at the same time as any other reagent added to the device.

[0135] 1(b). Sample analysis region

[0136] The optional sample analysis region of the devices disclosed herein comprises one or more sample detection zones. In some embodiments, the sample analysis region is present on the device. In some embodiments, the device does not contain a sample analysis region. When thedevice does not contain a sample analysis region, the analysis of the sample or analytes contained therein are analyzed off the device. There is both a range in the types of the sample detection zones and the number of sample detection zones present. The types of sample detection zones include, without limitation, wells or micro wells, one or more chambers, nanopores, etc. In some embodiments, the sample analysis region comprises hydrophobic liquid and hydrophilic liquid wells.

[0137] In some embodiments, the sample analysis region has a first end laterally separated from a second end and is defined by the surface of the second substrate facing the first substrate and the surface of the first substrate facing the second substrate. In some embodiments, the surface of the second substrate facing the first substrate in the sample analysis region comprises an enlarged protruding element where a surface of the enlarged protruding element facing the first substrate has a greater surface area than a surface of the plurality of protruding elements facing the first substrate, and the enlarged protruding element extends from the first end to the second end. The enlarged protruding element may be referred to as a secondary protruding element.

[0138] In some embodiments, the sample analysis region comprises a sample detection zone comprising wells. The wells are generally designed to contain one or more microparticles. In some embodiments, the wells are only able to contain a single microparticle. In some embodiments, the wells are able to contain two microparticles. In some embodiments, the wells are able to contain three or more microparticles. There may be a range of wells present in the sample detection zone. For instance, the sample detection zone may contain about 100 or more, about 200 or more, about 500 or more, about 1000 or more, about 2000 or more, about 3000 or more, about 4000 or more 5000 or more, about 6000 or more, about 7000 or more, about 8000 or more, about 9000 or more, about 10000 or more, about 20000 or more, about 30000 or more, about 40000 or more 50000 or more, about 60000 or more, about 70000 or more, about 80000 or more, about 90000 or more, about 100000 or more, about 200000 or more, about 300000 or more, about 400000 or more, about 500000 or more, about 600000 or more, about 700000 or more, about 800000 or more, about 900000 or more, about or 1000000 or more wells or microwells. In some embodiments, the sample detection zone comprises 100000 or more wells or microwells. In some embodiments, the sample detection zone comprises 300000 or more wells or microwells. In some embodiments, the portion of the sample detection region containing wells is optically transparent.

[0139] In some embodiments, the sample analysis region comprises a sample detection zone comprising a chamber. In some embodiments, the chamber is a reaction vessel. In some embodiments, the chamber is an imaging chamber. In some embodiments, the chamber is an imaging chamber and a reaction vessel. The chamber may be a range of different sizes and shapessuch that the chamber is suitable for the detection of the sample or the analytes contained therein. The chamber may accommodate a reaction mixture having a volume of from 1 microliter to 1 milliliter. For example, the chamber may be sized to contain a volume of from about 1 to about 10 pF, about 10 to about 50 pL, about 50 to about 100 pL, about 100 to about 200 pL, about 200 to about 300 pL, about 300 to about 400 pL, about 400 to about 500 pL, about 500 to about 600 pL, about 600 to about 700 pL, about 700 to about 800 pL, about 800 to about 900 pL, about or from about 900 to about 1000 pF. When the chamber is an imaging chamber, one or more of the sides of the chamber may be optically transparent. For instance, the top of the chamber, the bottom of the chamber, the sides of sides of the chamber, or any combination thereof may be optically transparent.

[0140] In some embodiments, the sample analysis region comprises a sample detection zone comprising nanopores. When the sample detection zone comprises nanopores, the sample comprise nucleic acids. In some embodiments, the nanopores are designed to only allow the translocation of a single nucleic acid at a time. The nucleic acid may be single- or double- stranded. When the nucleic acid is single-stranded, the nanopore has a diameter such that only a single single-stranded nucleic can be translocated. When the nucleic acid is double stranded, the nanopore has a diameter such that only a single double- stranded nucleic can be translocated. The nanopores may be any type of nanopore including, without limitation, biological nanopores, solid state nanopores, etc.

[0141] The sample detection zone of the present disclosure may be a single sample detection zone, multiple sample detection zones of the same type, or multiple sample detection zones of two or more different types.

[0142] When the sample detection zone is a single sample detection zone, the sample detection zone may be any of those described above or throughout the present disclosure. In some embodiments, the sample detection zone comprises wells. In some embodiments, the sample detection zone comprises microwells. In some embodiments, the sample detection zone comprises nanopores or nanochannels. In some embodiments, the sample detection zone comprises a chamber.

[0143] When the sample detection zone comprises wells or microwells, the wells or microwells may be specific to electrochemical detection, imaging analysis, or absorbance-based measurements. In some embodiments, the wells or micro wells are specific to electrochemical detection. In some embodiments, the wells or microwells are specific to image analysis. In some embodiments, the wells or microwells are specific to absorbance-based measurements. In some embodiments, the wells or microwells are specific to electrochemical detection and image analysis.In some embodiments, the wells or micro wells are specific to electrochemical detection and absorbance based measurements. In some embodiments, the wells or microwells are specific to image analysis and absorbance-based measurements. In some embodiments, the wells or microwells are specific to electrochemical detection, image analysis, and absorbance-based measurements.

[0144] When the sample detection zone comprises wells or microwells, the wells or microwells may be contained in a chamber or in an open region. In some embodiments, the wells or micro wells are contained in a chamber. In some embodiments, the wells or micro wells are in an open region.

[0145] When the sample detection zone comprises a chamber, imaging chamber, or reaction vessel, the chamber, imaging chamber, or reaction vessel may be specific to electrochemical detection, imaging analysis, or absorbance-based measurements. In some embodiments, the chamber or reaction vessel is specific to electrochemical detection. In some embodiments, the chamber, imaging chamber, or reaction vessel is specific to image analysis. In some embodiments, the chamber, imaging chamber, or reaction vessel is specific to absorbance-based measurements. In some embodiments, the chamber or reaction vessel is specific to electrochemical detection and image analysis. In some embodiments, the chamber, imaging chamber, or reaction vessel is specific to electrochemical detection and absorbance-based measurements. In some embodiments, the chamber, imaging chamber, or reaction vessel is specific to image analysis and absorbancebased measurements. In some embodiments, the chamber, imaging chamber, or reaction vessel is specific to electrochemical detection, image analysis, and absorbance-based measurements.

[0146] When the sample detection zone comprises nanopores or nanochannels, the nanopores or nanochannels may be contained in a chamber or in an open region. In some embodiments, the nanopores or nanochannels are contained in a chamber. In some embodiments, the nanopores or nanochannels are in an open region.

[0147] When the sample detection zone contains multiple sample detection zones it may be referred to as a combined sample detection zone. When the sample detection zone contains multiple sample detection zones, the sample detection zone may contain multiple sample detection zones of the same type or multiple sample detection zones of different types.

[0148] In some embodiments, the sample detection zone contains multiple sample detection zones of the same type, such as any of those described above. There is a range of different numbers of sample detections zones of the same type that may be in the combined sample detection zone. For example, the combined sample detection zone may contain 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more sample detection zones. Thesample detection zones in the combined sample detection zone may be in an open region or in discrete chambers that are linked in series, parallel, or in a grid-like fashion. In some embodiments, the sample detection zones in the combined sample detection zone are in an open region. In some embodiments, the sample detection zones in the combined sample detection zones are in discrete chambers that are linked in series. In some embodiments, the sample detection zones in the combined sample detection zones are in discrete chambers that are linked in parallel. In some embodiments, the sample detection zones in the combined sample detection zones are in discrete chambers that are linked in a grid-like fashion.

[0149] In some embodiments, the sample detection zone contains multiple sample detection zones of the same type, such as any of those described above. There is a range of different numbers of sample detections zones of the same type that may be in the combined sample detection zone. For example, the combined sample detection zone may contain 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more sample detection zones. The sample detection zones in the combined sample detection zone may be in an open region or in discrete chambers that are linked in series, parallel or in a grid-like fashion. In some embodiments, the sample detection zones in the combined sample detection zone are in an open region. In some embodiments, the sample detection zones in the combined sample detection zones are in discrete chambers that are linked in series. In some embodiments, the sample detection zones in the combined sample detection zones are in discrete chambers that are linked in parallel. In some embodiments, the sample detection zones in the combined sample detection zones are in discrete chambers that are linked in a grid-like fashion.

[0150] In some embodiments, the sample detection zone contains multiple sample detection zones of different types, such as any of those described above. There is a range of different numbers of sample detections zones of the different types that may be in the combined sample detection zone. For example, the combined sample detection zone may contain 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more sample detection zones. The combined sample detection zone may contain multiple sample detection zones of the same and different types. For instance, the combined sample detection zone may contain 2 sample detection zones comprising wells or microwells and two sample detection zones comprising chambers or reaction vessels. In some embodiments, the combined sample detection zone comprises microwells or wells, and nanopores or nanochannels. In some embodiments, the combined sample detection zone comprises microwells or wells, and chambers, imaging chambers, or reaction vessels. In some embodiments, the combined sample detection zone comprises nanopores or nanochannels, and chambers or reaction vessels. The sample detection zones in thecombined sample detection zone may be in an open region or in discrete chambers that are linked in series, parallel, or in a grid-like fashion. In some embodiments, the sample detection zones in the combined sample detection zone are in an open region. In some embodiments, the sample detection zones in the combined sample detection zones are in discrete chambers that are linked in series. In some embodiments, the sample detection zones in the combined sample detection zones are in discrete chambers that are linked in parallel. In some embodiments, the sample detection zones in the combined sample detection zones are in discrete chambers that are linked in a grid-like fashion.

[0151] In some embodiments, the sample analysis region comprises hydrophobic liquid and hydrophilic liquid wells. When the sample analysis region comprises hydrophobic liquid and hydrophilic liquid wells, the sample detection zone comprises wells or microwells. The hydrophilic liquid well holds and / or delivers hydrophilic liquid to the wells or micro wells. The hydrophobic liquid well holds and / or delivers hydrophobic liquid to the wells or micro wells. The hydrophobic liquid and / or hydrophilic liquid may be pre-filled in, or added to, the device. When the hydrophobic liquid and / or hydrophilic liquid are added to the device, the addition may be performed manually or robotically. In general, hydrophilic liquid is added before the hydrophobic liquid is added. The substrate generally interacts with a component in the wells when microparticles and an analyte that is present such that the interaction produces a detectable signal. In some embodiments, the hydrophilic liquid is a substrate solution. The substrate solution may be any substrate solution that reacts with a specific binding member (e.g., the second specific binding member or the detectably labeled second specific binding member) to produce a detectable signal. In some embodiments, the hydrophobic liquid is oil. The oil may be any oil deemed useful. In certain cases, the hydrophobic liquid is selected based on its low affinity for water to decrease mixing of the hydrophobic liquid with the substrate solution. In certain cases, the hydrophobic liquid is an oil. In certain cases, the hydrophobic liquid is 3M FC-40 oil, a hydrocarbon oil, a vegetable oil, or silicone liquids (e.g., a silicone oil). In certain cases, the oil is a fluorocarbon oil. In certain cases, the oil is Novec 7500, FC-40, or Galden HT200. The device, sample analysis region, and methods of using the same may be according to the devices, sample analysis regions, and methods of use as disclosed in U.S. Provisional Patent Application Serial No. 63 / 601,654, and International Patent Application Attorney Docket No. ADDV-145WO Titled “Sample Analysis Device and Methods” filed on November 8, 2024, and U.S. Provisional Patent Application Attorney Docket No. ADDV-155PRV Titled “Automated Assay Processing Unit” filed on November 8, 2024, each of which are specifically incorporated by reference herein.

[0152] II. Uses of the devices

[0153] The devices disclosed herein may be used for a number of different assay types and the assays may be performed in different ways. The different types of assays and the different ways that the assays are performed are based on the primary and secondary zones are configured (e.g., the reagents contained in each zone). The assays are conducted using a sample path where the microparticles are moved from one primary zone to another primary zone using a magnetic field. In some embodiments, the microparticles are microparticles and assisting particles. The different types of assays include, without limitation, immunoassays, nucleic acid analysis, metabolite analysis, clinical chemistry, complete blood count (CBC), etc.

[0154] In some embodiments, the device may be used to perform immunoassays. Any immunoassay may be utilized. The immunoassay may be an enzyme-linked immunoassay (ELISA), a competitive inhibition assay, such as forward or reverse competitive inhibition assays, or a competitive binding assay, for example. In some embodiments, a detectable label (e.g., such as one or more fluorescent labels one or more tags attached by a cleavable linker which can be cleaved chemically or by photocleavage) is attached to the capture antibody and / or the detection antibody.

[0155] In some embodiments, the device may be used to perform nucleic acid analysis. The device may employ various forms of nucleic acid analysis to detect analytes of interest, e.g., a nucleic acid, a non-nucleic acid containing a nucleic acid tag, or a nucleic acid produced from the analyte, including, without limitation, PCR, isothermal amplification, etc.

[0156] In some embodiments, the device may be used to perform metabolite analysis. In some cases, the clinical chemistry panels include metabolic panels. The metabolite analysis helps evaluate, for example, the body's electrolyte balance and / or the status of several major body organs. Examples of metabolite analyses that may be employed by the device include, but are not limited to, basic metabolic panel (BMP), comprehensive metabolic panel (CMP), electrolyte panel, lipid panel, liver panel, renal panel, and thyroid function panel. The basic metabolic panel (BMP) includes 8 tests, all of which are found in the CMP. The BMP provides information about the current health of kidneys and respiratory system as well as electrolyte and acid / base balance and level of blood glucose. The CMP measurement is used for liver and kidney health, level of blood glucose, acid / base balance in blood, fluid and electrolyte balance, and important blood proteins. In some cases, the CMP measures glucose, calcium, total amount of albumin and globulins, bilirubin, BUN (blood urea nitrogen), creatinine, albumin, sodium, potassium, bicarbonate, chloride, alkaline phosphatase (ALP), alanine transaminase (ALT), and aspartate aminotransferase (AST). The electrolyte panel is used to detect a problem with the body’s fluid and electrolyte balance. For example, the electrolyte panel measures the blood levels of carbondioxide, chloride, potassium, and sodium. The lipid panel is used to assess a subject’s risk of developing cardiovascular disease. For example, the lipid panel measures the amount of cholesterol and other fats in blood, such as total cholesterol, LDL (low-density lipoprotein), HDL (high-density lipoprotein), and triglycerides. The liver panel (hepatic function panel) is used to screen for, detect, evaluate, and monitor acute and chronic liver inflammation (hepatitis), liver disease and / or damage. The liver panel measures different enzymes, proteins, and other substances made by liver. For example, the liver panel includes albumin, total protein, ALP, ALT, AST, gamma-glutamyl transferase (GGT), bilirubin, Lactate dehydrogenase (LD), Prothrombin time (PT). The renal panel (kidney function panel) includes tests such as albumin, creatinine, BUN, eGFR to evaluate kidney function. The thyroid Function Panel is used to evaluate thyroid gland function and to help diagnose thyroid disorders. The thyroid function panel measure thyroid hormone such as thyroxine (T4), triiodothyronine (T3), and thyroid stimulating hormone (TSH). In some cases, a high TSH level indicates that the thyroid gland is not making enough thyroid hormone (primary hypothyroidism). The opposite situation, in which the TSH level is low, usually indicates that the thyroid is producing too much thyroid hormone (hyperthyroidism). In other cases, the finding of an elevated TSH and low free T4 (FT4) or free T4 index (FTI) indicates primary hypothyroidism due to disease in the thyroid gland. A low TSH and low FT4 or FTI indicate hypothyroidism due to a problem involving the pituitary gland. A low TSH with an elevated FT4 or FTI is found in individuals who have hyperthyroidism. These clinical chemistry panels are well known in the art and are further described in the assay portion of the present disclosure.

[0157] In some embodiments, the device may be used to perform clinical chemistry. In certain cases, clinical chemistry may involve detection of electrochemical species or chromogenic reaction product generated by action of an enzyme on a substrate. For example, the substrate may be an analyte present in a sample and the enzyme may be specific for the analyte and may catalytically react with the analyte to generate an electrochemical species or a colored reaction product. In other cases, clinical chemistry may involve capturing the analyte using a first binding member to generate a first complex comprising the analyte and the first binding member; contacting the complex with a second binding member, that binds to the analyte, to generate a second complex comprising the analyte, the first binding member, and the second binding member. The second binding member is conjugated to an enzyme that generates an electrochemical species or chromogenic reaction product upon exposure to a suitable substrate.

[0158] In some embodiments, the device may be used to perform complete blood counts of blood cells or blood cell types. Blood cells and blood cell types that may be detecting by thedevices disclosed herein include, without limitation, red blood cells, hemoglobin, white blood cells (including neutrophils, lymphocytes, monocytes, eosinophils, and basophils), platelets, reticulocytes, and nucleated red blood cells. Various measurements of different blood components may be performed, including, but not limited to, cell count, cell size, cell complexity, granularity, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration. In some embodiments, the above disclosed measurements may be performed using stain independent methods in the absence of histological staining.

[0159] In order to perform different types of assays or perform the assays in different ways, the primary zones may be filled with reagents. The reagents may be added to the primary zones may be added manually by the user, robotically by a system employing the device, or through the use of a reagent delivery device. In some embodiments, reagents are added to the secondary zones in addition to the primary zones.

[0160] FIG. 26A-26D illustrates a cross-section of the sample processing region where the sample and / or reagents are added. FIG. 26A and FIG. 26B illustrates a simplified depiction of a sample processing region where the sample processing region contains one primary zone and two secondary zones and the primary zone is filled with a sample or reagent. The primary zone 2608 contains an opening 2603. The secondary zone 2607 contains an opening 2604. The sample or reagent 2602 may be added through the opening 2603 of the primary zone 2608 wherein the sample or reagent 2602 is held in place through capillary forces generated by the opening 2603 and surface tension facilitated by the edges of a square pad 2601 in the primary zone 2608.

[0161] FIG. 26C illustrates a simplified depiction of a sample processing region where the sample processing region contains three primary zones and four secondary zones and two of the primary zones are filled with sample or reagent.

[0162] FIG. 26D illustrates a simplified depiction of a sample processing region where the sample processing region contains three primary zones and four secondary zones and two of the primary zones and one of the secondary zones are filled with sample or reagent. In this embodiment, the sample or reagent is added through the opening in two of the primary zones and one of the secondary zones. The addition of the sample or reagent in three adjacent zones (i.e., two primary zones and one secondary) causes the zones to merge and create a singular zone of increased volume.

[0163] The primary zones of the present disclosure are capable of holding a range of different volumes while retaining the volume within the primary zone without spilling into the secondary zone or beyond. For instance, the primary zone may hold about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about49, about 50, or more than about 50 pL of fluid. In some embodiments, the primary zone is capable of holding between 10-24 pL of fluid.

[0164] When the primary and the adjacent secondary zone are filled with the sample or fluid, the primary and the adjacent secondary zone are capable of holding a range of different volumes while retaining the volume within the primary and secondary zone without spilling into another zone. For instance, the primary and secondary zone collectively may hold about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100 or more than about 100 pF of fluid. In some embodiments, the primary and secondary zone is capable of holding between 20-50 pL of fluid.

[0165] When two primary and one secondary zones are filled with sample or fluid, the two primary and one secondary zones are capable of holding a range of different volumes while retaining the volume within the primary and secondary zone without spilling into another zone. For instance, the two primary and one secondary zones collectively may hold about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113,about 114, about 115, about 116, about 117, about 118, about 119, about 120, about 121, about 122, about 123, about 124, about 125, about 126, about 127, about 128, about 129, about 130, about 131, about 132, about 133, about 134, about 135, about 136, about 137, about 138, about 139, about 140, about 141, about 142, about 143, about 144, about 145, about 146, about 147, about 148, about 149, about 150, or more than aboutl50 pF of fluid. In some embodiments, the primary and secondary zone is capable of holding between 30-150 pF of fluid.

[0166] The assays used with the device are conducted using a sample path where the microparticles or microparticles and assisting particles are moved from one primary zone to another primary zone using a magnetic field. Exemplary sample paths are disclosed in FIG. 17- 19 using different embodiments of the device.

[0167] In FIG. 17, an exemplary sample path is shown. The sample and the microparticles are present in a fixed primary zone 1701 (e.g., a sample mixing zone). In some embodiments, microparticles are microparticles and assisting particles. The microparticles or microparticles and assisting particles are moved (black arrow) from the fixed primary zone to a primary zone 1702 containing a reagent (e.g., wash buffer). The microparticles or microparticles and assisting particles are then moved through two additional primary zones containing reagent 1703 and 1704 to a fourth primary zone containing reagent (e.g., conjugate) 1705. The microparticles or microparticles and assisting particles are eventually moved from the fourth primary zone 1705 to the sample analysis region 1706.

[0168] In FIG. 18, discloses short, medium, and long sample paths using the same exemplary device. In the short sample path, the sample and the microparticles or microparticles and assisting particles are present in a variable primary zone (S) and the microparticles or microparticles and assisting particles are then moved through a primary zone 1801 containing reagent (e.g., wash buffer) to a primary zone contain reagent (C; e.g., conjugate). The microparticles or microparticles and assisting particles are then moved from the primary zone containing reagent (C) through another primary zone and finally end in the sample analysis region 1802. In the medium path, the sample and the microparticles or microparticles and assisting particles are present in a variable primary zone (S) and the microparticles or microparticles and assisting particles are moved through four primary zones containing reagent (e.g., wash buffer) to a primary zone containing reagent (C; e.g., conjugate). The microparticles or microparticles and assisting particles are then moved through an additional four primary zones containing reagent (e.g., wash buffer) and finally end in the sample analysis region. In the long sample path, the sample and the microparticles are present in a variable primary zone (S) and the microparticles are moved through seven primary zones containing reagent (e.g., wash buffer) to a primary zone containing reagent (C; e.g.,conjugate). The microparticles or microparticles and assisting particles are then moved through an additional seven primary zones containing reagent (e.g., wash buffer) and finally ending in the sample analysis region.

[0169] FIG. 19, discloses exemplary paths through an embodiment of the device. In FIG. 19A, the sample and the microparticles or microparticles and assisting particles are present in a fixed primary zone 1901 (e.g., a mixing zone) and moved through two primary and one secondary zones merged with reagent (e.g., merged zone; spotted) 1902 to a primary zone containing a reagent 1903 (dark stripped; e.g., conjugate). The microparticles or microparticles and assisting particles are then moved through another two primary and one secondary zones merged (dotted) with reagent to finally end in the sample analysis region 1904. In FIG. 19B, the sample and the microparticles or microparticles and assisting particles are present in a fixed primary zone and moved through four primary zones containing reagent (dotted, e.g., wash buffer) to a primary zone containing regent (dark stripped; e.g., conjugate). The microparticles or microparticles and assisting particles are then moved through four primary zones with reagent (spotted) to finally end in the sample analysis region. In FIG. 19C, the sample and the microparticles or microparticles and assisting particles are present in a fixed primary zone and moved through two merged zones containing reagent (dotted, e.g., wash buffer) to a primary zone containing regent (dark stripped; e.g., conjugate). The microparticles or microparticles and assisting particles are then moved through two merged zones with reagent (spotted) to finally end in the sample analysis region. In FIG. 19D, the sample and the microparticles or microparticles and assisting particles are present in a fixed primary zone and moved through two primary zones containing reagent (dotted, e.g., wash buffer) to a primary zone containing regent (dark stripped; e.g., conjugate). The microparticles or microparticles and assisting particles are then moved through two primary zones with reagent (spotted) to finally end in the sample analysis region. In FIG. 19E, the sample and the microparticles or microparticles and assisting particles are present in a fixed primary zone and moved through three primary zones containing reagent (dotted, e.g., wash buffer) to a primary zone containing regent (dark stripped; e.g., conjugate). The microparticles or microparticles and assisting particles are then moved through three primary zones with reagent (spotted) to finally end in the sample analysis region.

[0170] FIG. 46, discloses exemplary paths through an embodiment of the device. In FIG. 46A, the sample and the microparticles or microparticles and assisting particles are present in a fixed primary zone 4601 (e.g., a mixing zone) and are moved through three primary zones containing reagents (e.g., wash buffer) to a primary zone containing a reagent 4602 (dark stripped; e.g., conjugate). The microparticles or microparticles and assisting particles are then moved throughthree primary zones with reagents (e.g., wash buffer) to the sample analysis region. The microparticles or microparticles and assisting particles are then moved from quaternary zone (e.g., hydrophilic well or reservoir) through the tertiary zone (e.g. sample detection zone) and the quinary zone (e.g., hydrophobic well or reservoir) 4605 and away from the sample analysis region.

[0171] For purpose of illustration and example and not limitation, reference is made to the exemplary sample detection region 2750 depicted in FIG. 35 A. In an embodiment, the sample detection region 2750 comprises wells 2753 defined therein. The wells 2753 have a well size. As described above and throughout, microparticles or microparticles and assisting particles can be moved across the array of wells 2753. In some embodiments, the microparticles include (i) a plurality of microparticles, each microparticle having a microparticle diameter smaller than the well size, and (ii) a plurality of assisting particles, each assisting particle having an assisting particle diameter larger than the well size. Specifically, the diameter of the assisting particle (e.g. helper beads) can be at least about 1 %, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11 %, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, or at least 35% greater or larger than the diameter of the well size. Moving the microparticles or microparticles and assisting particles across the array of wells 2753 further includes seeding a plurality of the microparticles or microparticles and assisting particles into wells of the array.

[0172] As embodied herein, moving the microparticles or microparticles and assisting particles across the array of wells 2753 includes moving a magnet 2759 along the array of wells. The magnet can be moved along the array of wells in any suitable configuration. For example and not limitation, the magnet can be located above the sample analysis region 2750. Additionally or alternatively, the magnet can be located adjacent to the sample analysis region. Additionally or alternatively, and as embodied herein, the magnet can be located below the sample analysis region 2750.

[0173] The position of the magnet relative to the sample analysis region 2750 and the array of wells 2753 can be selected based on the desired strength of the magnetic field to be applied to the microparticles or microparticles and assisting particles to move the microparticles or microparticles and assisting particles across the array of wells 2753. For purpose of example, and as described further herein, the distance between the magnet and the microparticles or microparticles and assisting particles can be selected based on the desired strength of the magnetic field to be applied to the microparticles or microparticles and assisting particles. For purpose of example and as embodied herein, the magnet can be moved along the array of wells at a magnetdistance defined between a bottom surface 2754 of the array of wells and the magnet 2759. For example and not limitation, the magnet distance can be between about Omm and about 10mm. Additionally or alternatively, and as embodied herein, the magnet can be moved along the array of wells 2753 with the magnet 2759 in contact with a bottom surface 2754 of the array of wells.

[0174] As further embodied herein, the shape and orientation of the magnet can also be selected to achieve the desired magnetic field. For example and not limitation, the magnet 2759 can be angled relative to the bottom surface 2754 of the array of wells as the magnet is moved along the bottom surface of the array of wells. For illustration and example and not limitation, reference is made to the exemplary sample analysis region 2750 depicted in FIG. 35B. A magnet axis 2763 can be defined between a first magnet end 2761 and a second magnet end 2762, and as the magnet 2759 is moved along the bottom surface 2754 of the array of wells. For purpose of example and not limitation, the magnet axis 2763 can be positioned at an angle 2760 of between about 0 degrees and about 80 degrees relative to the bottom surface of the array of wells. For example and not limitation, the magnet axis 2763 can be positioned at an angle 2760 of between about 10 degrees and about 30 degrees. For example and not limitation, and as embodied herein, the magnet axis 2763 can be positioned at an angle 2760 of about 20 degrees.

[0175] Additionally or alternatively, the magnetic element can be moved along the array of wells to achieve desired movement and seeding of microparticles or microparticles and assisting particles in the array of wells. For purpose of example and as embodied herein, the magnet 2759 can be moved in a direction 2755 parallel to the bottom surface 2754 of the array of wells. Additionally or alternatively, the magnetic element can be moved along the array of wells at any suitable speed to achieve desired movement and seeding of microparticles in the array of wells. For example, the speed of the magnet can be selected to achieve desired process times and microparticle loss during movement of the microparticles or microparticles and assisting particles across the array. For purpose of example and not limitation, magnet 2759 can be moved in a direction 2755 parallel to a bottom surface 2754 of the array of wells 2753 at a speed of between about 0.3 mm / s and about 10 mm / s. Additionally or alternatively, magnet 2759 can be moved in a direction 2755 parallel to a bottom surface 2754 of the array of wells 2753 at a speed of between about 0.3 mm / s and about 6 mm / s. Additionally or alternatively, magnet 2759 can be moved in a direction 2755 parallel to a bottom surface 2754 of the array of wells 2753 at a speed of between about 2 mm / s and about 6 mm / s. Additionally or alternatively, magnet 2759 can be moved in a direction 2755 parallel to a bottom surface 2754 of the array of wells 2753 at a speed of between about 4 mm / s and about 6 mm / s. For purpose of example and as embodied herein, the magnet 2759 can move in direction 2755 at an angle 2760 as described above, and the direction 2760 canbe selected to align with the direction of the acute angle defined between the magnet axis 2763 and the bottom surface 2754 of the array of wells. Although reference is made to the magnet 2759 moving along the array of wells, relative motion between the magnet 2759 and the array of wells can additionally or alternatively be achieved by moving the array of wells relative to the magnet, as described above.

[0176] Additionally or alternatively, the type and shape of the magnet can be selected to provide the desired magnetic field. The magnet 2759 can be a permanent magnet or an electromagnet. Any suitable magnet shape can be selected. For example and not limitation, the magnet can define a corner. Additionally or alternatively, and as embodied herein, the corner 2764 of the magnet 2759 can be in contact with the bottom surface 2754 of the array of wells as the magnet moves along the array of wells. For purpose of example and not limitation, the magnet can have a cylindrical, triangular, square, spherical, or other suitable shape. Additionally or alternatively, and as embodied herein, the magnet can have a rectangular shape.

[0177] As described above, the shape, orientation, and position of the magnetic element can be selected to provide a desired magnetic field. With reference to FIG. 35C-FIG. 35H, properties of the magnetic field in an exemplary detection region 2750 having magnet 2759, including a plurality of magnetic field lines which emit from the magnet, are depicted for purpose of illustration and explanation and not limitation. In exemplary sample detection region 2750, magnet 2759 was positioned with corner 2764 of the magnet 2759 in contact with the bottom surface 2754 of the array of wells, such that the magnetic field lines of the magnet 2759 extent through the bottom surface 2754 of the array of wells, with the magnetic field lines concentrated proximate to the comer 2764 of the magnet 2759. Additionally, exemplary magnet was positioned with magnet axis 2763 at an angle 2760 of about 20 degrees relative to the bottom surface 2754. Additionally or alternatively, exemplary magnet can be positioned with magnet axis 2763 at an angle 2760 of between about 0 degrees and 80 degrees. Additionally or alternatively, exemplary magnet can be positioned with magnet axis 2763 at an angle 2760 of between about 10 degrees and 30 degrees. With reference to FIG. 35B, an array axis "X" can be defined along top surface 2751 of the array of wells, and a second axis "Y" can be defined perpendicular to the array axis. For illustration and as embodied herein, a zero point on the array axis "X" is defined at the location on the array axis "X" where the comer 2764 of magnet 2759 contacts the bottom surface 2754 of the array of wells.

[0178] Additionally, and a zero point on the second axis "Y" is defined at the top surface 2751 of the array of wells.

[0179] With reference to FIG. 35D, magnetic force measured in pN in the array axis "X" and the second axis "Y" is shown as a function of position along the array axis "X" for the exemplary sample analysis region 2750 and magnet 2759 configuration described above. As shown, a negative magnetic force is generated in the second axis Y where the comer 2764 of magnet 2759 contacts the bottom surface 2754 of the array of wells. As embodied herein, the negative magnetic force acts on the microparticles or microparticles and assisting particles to pull the microparticles down into wells of the array of wells to load or seed the microparticles into the wells.

[0180] With reference to FIGs. 35E and 35F, magnetic force measured in pN is shown separately for the second axis "Y" and the array axis "X" as a function of position along the array axis "X" for the exemplary detection region 2750 and magnet 2759 configuration described above. As embodied herein, the magnetic force in the second axis "Y" can define a generally parabolic shape when plotted and can include a sharp negative peak in magnetic field strength where the comer 2764 of magnet 2759 contacts the bottom surface 2754 of the array of wells. Additionally, as embodied herein the magnitude of the magnetic force in the second axis "Y" can be greater than 2000 pN. As described above, the parabolic magnetic field and strong negative magnetic field can act on the microparticles to pull the microparticles into wells of the array to seed the microparticles within wells of the array. Additionally and as embodied herein, the magnetic field in the second axis "Y" can remain negative across the array axis "X" as shown. For example and as embodied herein, the magnetic field in the second axis

[0181] "Y" can remain negative over at least 6mm as measured in the array axis "X" and as depicted in FIG. 35E. The negative magnetic field in the second axis "Y" across a wide portion of the array of wells can retain microparticles that have been seeded in wells of the array within the wells.

[0182] With reference to FIG. 35G, magnetic field strength measured in H (AIM) is shown as a function of position along the array axis "X" for the exemplary detection region 2750 and magnet 2759 configuration described above. As embodied herein, the magnet 2759 can provide a strong magnetic field with a generally parabolic shape. As embodied herein, the magnetic field strength can be about 400,000 H (AIM) where the corner 2764 of magnet 2759 contacts the bottom surface 2754 of the array of wells. The magnetic field strength can seed and retain microparticles in wells of the array of wells as described herein.

[0183] With reference to FIG. 35H, magnetic force measured in pN in the array axis "X" (Fx) and in the second axis "Y" (Fy) is shown as a function of position along the array axis "X" and along the second axis "Y" for the exemplary detection region 2750 and magnet 2759 configuration described above.

[0184] With continued reference to FIGs.35B-35E, the magnetic field achieved can provide benefits for seeding and sealing microparticles into wells. For example and not limitation, the magnitude of the y-component of magnetic force relative to the x- component is high at the location where the comer 2764 of magnet 2759 contacts the bottom surface 2754 of the array of wells. The relatively high magnitude of the y-component can help to ensure microparticles are loaded (i.e., seeded) into wells and remain in wells during the sealing process. Additionally, the magnitude of the magnetic field is high, and the distribution of the magnetic field has a high peak. Having a distribution of magnetic field with a high peak can facilitate aggregation of microparticles or microparticles and assisting particles as the microparticles or microparticles and assisting particles are moved across the array of wells. Increased aggregation of microparticles or microparticles and assisting particles during movement across the array of wells can result in fewer excess microparticles left on the top of the array. Additionally, the magnitude of the x-component of magnetic force can be large enough to continuously pull excess microparticles along the surface of the array of wells. The increased magnitude of the magnetic force in the array axis "X" can help to further remove excess microparticles left on the top of array.

[0185] Assays that may be performed using the exemplary sample paths discussed above are described further in section for methods of detecting a target analyte in a sample.

[0186] II. Benefits of the devices

[0187] The devices of the present disclosure provide certain benefits over devices known in the art. The benefits of the devices of the present disclosure include high levels of modularity allowing for flexibility in the workflow used to process a sample by allowing different regions to be filled by different reagents depending on the assay, the ability to use small volumes allowing for scalability and faster reaction times, and provide the ability to analyze multiple sample types simultaneously. In terms of the modularity, the multiple exemplary embodiments of the devices disclosed herein provide high levels of configurability through the usage of multiple primary zones for sample or reagent addition, multiple secondary zones for hydrophobic separation (e.g., through the presence of air) or for merging to create large sample or reagent zones, many different types of sample analysis regions configured to analyze proteins or antigens (erg, immunoassays and clinical chemistry), nucleic acids (e.g., nucleic acid analysis), metabolites (e.g., clinical chemistry), or cells or cells types (e.g., immunoassays, clinical chemistry, nucleic acids, or CBC). The primary and secondary zones can be configured by the user to fit any type of assay or the particular steps of a given assay. The ability to perform these assays using small volumes allows for reduced costs of reagents and reduced consumption of sample which allows for the conservation of difficult to obtain samples. The modularity of the device also allows assays to be customized to the user’spreference and also allows multiple assays (e.g., immunoassays and nucleic acid analysis, or any combination of the assays disclosed above) to be performed simultaneously or sequentially on the same device for the same sample or multiple samples contained on a single device.2. DEVICE DESIGN

[0188] The devices of the present disclosure have a range of different layouts with varying numbers of primary and secondary zones, different arrangements of primary and secondary zones, the presence or absence of sample mixing zones, and the presence or absence of fixed primary zones. Exemplary embodiments of the devices are disclosed in FIG. 1-9, 11-19, 32, and 38-41.

[0189] FIG. 1 discloses an illustration of an embodiment of the device. In this embodiment, the device 100 contains a 3x8 grid of primary zones 101 and secondary zones 107. The device comprises a sample processing region 110. The sample processing region 110 is unbounded. By unbounded it is meant that there is no physical barrier separating the primary zones 101 and the secondary zones 107. The sample processing region comprises a primary zone 101 that has an opening 103 in the primary zone. The primary zone is adjacent to the secondary zone 107. The secondary zone may comprise an opening 104. The sample processing region 110 may be connected to a tertiary zone (i.e., the sample detection zone) 112 by a transition zone 113. The device may further comprise a quaternary zone 114 (i.e., a hydrophilic liquid well) that is connected to the tertiary zone. The device may further comprise a quinary zone 115 (i.e., a hydrophobic liquid well) that is connected to the tertiary zone. The top and the bottom substrate are bound by adhesive or clips 111. In some embodiments, the top substrate and bottom are bound by an adhesive layer between the top and bottom substrate. In some embodiments, the adhesive is a ultraviolet (UV) bonding adhesive. In some embodiments, the top and bottom substrate are bound using laser welding.

[0190] An alternative embodiment to FIG. 1 is disclosed below. In this embodiment, the device 100 contains a 3x8 grid of primary zones 101 and secondary zones 107. The device 100 comprises a second substrate positioned on a first substrate. The second substrate comprises a surface facing a central chamber comprising a plurality of recessed elements, and a plurality of protruding elements. The primary zones 101 are defined between a surface of the plurality of protruding elements facing the first substrate and a surface of the first substrate facing the second substrate. The secondary zones are defined between a surface of the plurality of recessed elements facing the first substrate and the surface of the first substrate facing the second substrate. The device comprises a sample processing region 110. The sample processing region 110 is unbounded. By unbounded it is meant that there is no physical barrier separating the primary zones 101 and the secondary zones 107. While there is only a single element label for the primary zone 101, eachsquare (101) is to represent an individual primary zone. In some embodiments, the primary zones are discrete. While there is only a single element label for the secondary zone 107, each space between the squares is to represent an individual secondary zone. In some embodiments, the secondary zones are connected. The sample processing region comprises a primary zone 101 that has an opening 103 in the primary zone. While there is only a single element label for the opening in the primary zone 103, each opening (103) in the square (101) is meant to represent an individual opening in the primary zone. The primary zone is adjacent to the secondary zone 107. The secondary zone may comprise an opening 104. While there is only a single element label for the opening in the secondary zone 107, each opening (104) in between the squares represents an individual opening in the secondary zone. The sample processing region 110 may be positioned adjacent to a sample analysis region comprising a tertiary zone (i.e., the sample detection zone) 112, a quaternary zone 114 (i.e., a hydrophilic liquid well), and a quinary zone 115 (i.e., a hydrophobic liquid well) wherein the tertiary zone 112, the quaternary zone 114, and the quinary zone 115 are connected. The sample analysis region is defined by the surface of the second substrate facing the first substrate and the surface of the first substrate facing the second substrate. The quinary zone 115 is located at a first end of the sample analysis region. The quinary zone comprises an opening spanning the second substrate The quaternary zone 114 is located at a second end of the sample analysis region. The quaternary zone comprises a cylindrical opening spanning the second substrate. The tertiary zone 112 is located at a midpoint between the first end (e.g., the quinary zone 115) and the second end (e.g., the quaternary zone 115) of the sample analysis region. The sample analysis region may be connected to the sample processing region 110 by a transition zone 113. The second substrate positioned over the first substrate are bound together by adhesive or clips 111. In some embodiments, the second substrate and the first substrate are bound by an adhesive layer between the second substrate and the first substrate. In some embodiments, the adhesive is an ultraviolet (UV) bonding adhesive. In some embodiments, the second substrate and the first substrate are bound using laser welding. The alternative embodiment disclosed for FIG.l may be applied to FIG. 2-6 such that the descriptions above describe the elements of the devices disclosed in FIG. 2-6.

[0191] FIG. 2 discloses an illustration of an embodiment of the device. In this embodiment, the device 200 contains a 2x4 grid of primary zones 201 and secondary zones 207. The device comprises a sample processing region 210. The sample processing region 210 is unbounded. By unbounded it is meant that there is no physical barrier separating the primary zones 201 and the secondary zones 207. The sample processing region comprises a primary zone 201 that has an opening 203 in the primary zone. The primary zone is adjacent to the secondary zone 207. Thesecondary zone may comprise an opening 204. The sample processing region 210 may be connected to a tertiary zone (i.e., the sample detection zone) 212 by a transition zone 213. The device may further comprise a quaternary zone 214 (i.e., a hydrophilic liquid well) that is connected to the tertiary zone. The device may further comprise a quinary zone 215 (i.e., a hydrophobic liquid well) that is connected to the tertiary zone. The top and the bottom substrate are bound by adhesive or clips 211. In some embodiments, the top substrate and bottom are bound by an adhesive layer between the top and bottom substrate. In some embodiments, the adhesive is an ultraviolet (UV) bonding adhesive. In some embodiments, the top and bottom substrate are bound using laser welding.

[0192] FIG. 3 discloses an illustration of an embodiment of the device. In this embodiment, the device 300 contains a 3x6 grid of primary zones 301 and secondary zones 307. The device comprises a sample processing region 310. The sample processing region 310 is unbounded. By unbounded it is meant that there is no physical barrier separating the primary zones 301 and the secondary zones 307. The sample processing region comprises a primary zone 301 that has an opening 303 in the primary zone. The primary zone is adjacent to the secondary zone 307. The secondary zone may comprise an opening 304. The sample processing region 310 may be connected to a tertiary zone (i.e., the sample detection zone) 312 by a transition zone 313. The device may further comprise a quaternary zone 314 (i.e., a hydrophilic liquid well) that is connected to the tertiary zone. The device may further comprise a quinary zone 315 (i.e., a hydrophobic liquid well) that is connected to the tertiary zone. The top and the bottom substrate are bound by adhesive or clips 311. In some embodiments, the top substrate and bottom are bound by an adhesive layer between the top and bottom substrate. In some embodiments, the top and bottom substrate are bound using laser welding.

[0193] FIG. 3 discloses an illustration of an embodiment of the device. In this embodiment, the device 300 contains a 3x6 grid of primary zones 301 and secondary zones 307. The device comprises a sample processing region 310. The sample processing region 310 is unbounded. By unbounded it is meant that there is no physical barrier separating the primary zones 301 and the secondary zones 307. The sample processing region comprises a primary zone 301 that has an opening 303 in the primary zone. The primary zone is adjacent to the secondary zone 307. The secondary zone may comprise an opening 304. The sample processing region 310 may be connected to a tertiary zone (i.e., the sample detection zone) 312 by a transition zone 313. The device may further comprise a quaternary zone 314 (i.e., a hydrophilic liquid well) that is connected to the tertiary zone. The device may further comprise a quinary zone 315 (i.e., a hydrophobic liquid well) that is connected to the tertiary zone. The top and the bottom substrateare bound by adhesive or clips 311. In some embodiments, the top substrate and bottom are bound by an adhesive layer between the top and bottom substrate. In some embodiments, the adhesive is an ultraviolet (UV) bonding adhesive. In some embodiments, the top and bottom substrate are bound using laser welding.

[0194] FIG. 4 discloses an illustration of an embodiment of the device. In this embodiment, the device 400 contains a 3x4 grid of primary zones 401 and secondary zones 407. The device comprises a sample processing region 410. The sample processing region 410 is unbounded. By unbounded it is meant that there is no physical barrier separating the primary zones 401 and the secondary zones 407. The sample processing region comprises a primary zone 401 that has an opening 403 in the primary zone. The primary zone is adjacent to the secondary zone 407. The secondary zone may comprise an opening 404. The sample processing region 410 may be connected to a tertiary zone (i.e., the sample detection zone) 412 by a transition zone 413. The device may further comprise a quaternary zone 414 (i.e., a hydrophilic liquid well) that is connected to the tertiary zone. The device may further comprise a quinary zone 415 (i.e., a hydrophobic liquid well) that is connected to the tertiary zone. The top and the bottom substrate are bound by adhesive or clips 411. In some embodiments, the top substrate and bottom are bound by an adhesive layer between the top and bottom substrate. In some embodiments, the top and bottom substrate are bound using laser welding.

[0195] FIG. 5 discloses an illustration of an embodiment of the device. In this embodiment, the device 500 contains a 2x8 grid of primary zones 501 and secondary zones 507. The device comprises a sample processing region 510. The sample processing region 510 is unbounded. By unbounded it is meant that there is no physical barrier separating the primary zones 501 and the secondary zones 507. The sample processing region comprises a primary zone 501 that has an opening 503 in the primary zone. The primary zone is adjacent to the secondary zone 507. The secondary zone may comprise an opening 504. The sample processing region 510 may be connected to a tertiary zone (i.e., the sample detection zone) 512 by a transition zone 513. The device may further comprise a quaternary zone 514 (i.e., a hydrophilic liquid well) that is connected to the tertiary zone. The device may further comprise a quinary zone 515 (i.e., a hydrophobic liquid well) that is connected to the tertiary zone. The top and the bottom substrate are bound by adhesive or clips 511. In some embodiments, the top substrate and bottom are bound by an adhesive layer between the top and bottom substrate. In some embodiments, the top and bottom substrate are bound using laser welding.

[0196] FIG. 6 discloses an illustration of an embodiment of the device. In this embodiment, the device 600 contains a 2x6 grid of primary zones 601 and secondary zones 607. The devicecomprises a sample processing region 610. The sample processing region 610 is unbounded. By unbounded it is meant that there is no physical barrier separating the primary zones 601 and the secondary zones 607. The sample processing region comprises a primary zone 601 that has an opening 603 in the primary zone. The primary zone is adjacent to the secondary zone 607. The secondary zone may comprise an opening 604. The sample processing region 610 may be connected to a tertiary zone (i.e., the sample detection zone) 612 by a transition zone 613. The device may further comprise a quaternary zone 614 (i.e., a hydrophilic liquid well) that is connected to the tertiary zone. The device may further comprise a quinary zone 615 (i.e., a hydrophobic liquid well) that is connected to the tertiary zone. The top and the bottom substrate are bound by adhesive or clips 611. In some embodiments, the top substrate and bottom are bound by an adhesive layer between the top and bottom substrate. In some embodiments, the adhesive is an ultraviolet (UV) bonding adhesive. In some embodiments, the top and bottom substrate are bound using laser welding.

[0197] FIG. 7 discloses an illustration of an isometric view of an embodiment of the device. The dotted lines represent interior portions of the device whereas solid lines represent exterior portions of the device. In this embodiment, the device 700 contains a 3x4 grid of primary zones 703 and secondary zones 705 in addition to a fixed primary zone 702. In some instances, but not all instances, the fixed primary zone 702 is also a sample mixing zone. The fixed primary zone is physically connected to a hooked portion 708. The boundaries of the top outside portion of the fixed primary zone is not attached to the rest of the device except for the portion furthest from the primary and secondary zones. When a vertical force is applied to the hooked portion 708, the top substrate (i.e., the roof) compresses and decompresses fluid (e.g., sample) that may be in the fixed primary zone 702 thereby mixing the fluid. The device comprises a sample processing region 701. The sample processing region 701 is unbounded. By unbounded it is meant that there is no physical barrier separating the primary zones 703 and the secondary zones 705. The sample processing region comprises a primary zone 703 that has an opening 704 in the primary zone. The primary zone is adjacent to a secondary zone such as 705. The secondary zone may comprise an opening 706. The sample processing region 701 be connected to a tertiary zone (i.e. the sample detection zone) 710 by a transition zone 707. The device may further comprise a quaternary zone 711 (i.e., a hydrophilic liquid well) that is connected to the tertiary zone. The device may further comprise a quinary zone 709 (i.e., a hydrophobic liquid well) that is connected to the tertiary zone.

[0198] An alternative embodiment to FIG. 7 is disclosed below. The dotted lines represent interior portions of the device whereas solid lines represent exterior portions of the device. In this embodiment, the device 700 contains a 3x4 grid of primary zones 703 and secondary zones 705in addition to a fixed primary zone 702. The device 700 comprises a second substrate positioned on a first substrate. The second substrate comprises a surface facing a central chamber comprising a plurality of recessed elements, and a plurality of protruding elements. The primary zones 701 are defined between a surface of the plurality of protruding elements facing the first substrate and a surface of the first substrate facing the second substrate. The secondary zones are defined between a surface of the plurality of recessed elements facing the first substrate and the surface of the first substrate facing the second substrate. In some instances, but not all instances, the fixed primary zone 702 is also a sample mixing zone. The fixed primary zone 702 is formed from a laterally extended protruding element in the second substrate facing the first substrate wherein the laterally extended protruding element has a surface facing the first substrate that has a greater surface area than the surface of the plurality of protruding elements facing the first substrate. The fixed primary zone 707 comprises hooked portion 708 joined to a surface of the extended protruding element opposite the surface of the second substrate facing the first substrate. The boundaries of the top outside portion of the fixed primary zone are not attached to the rest of the device except for the portion furthest from the primary and secondary zones. When a vertical force is applied to the hooked portion 708, the second substrate (i.e., the roof) compresses and decompresses fluid (e.g., sample) that may be in the fixed primary zone 702 thereby mixing the fluid. The device comprises a sample processing region 701. The sample processing region 701 is unbounded. By unbounded it is meant that there is no physical barrier separating the primary zones 703 and the secondary zones 705. The sample processing region comprises a primary zone 703 that has an opening 704 in the primary zone. The primary zone is adjacent to a secondary zone such as 705. The secondary zone may comprise an opening 706. The sample processing region 701 may be positioned adjacent to a sample analysis region comprising a tertiary zone (i.e., the sample detection zone) 710, a quaternary zone 711 (i.e., a hydrophilic liquid well), and a quinary zone 709 (i.e., a hydrophobic liquid well) wherein the tertiary zone 707, the quaternary zone 711, and the quinary zone 709 are connected. The sample analysis region is defined by the surface of the second substrate facing the first substrate and the surface of the first substrate facing the second substrate. The quinary zone 709 is located at a first end of the sample analysis region. The quinary zone comprises an opening spanning the second substrate. The quaternary zone 711 is located at a second end of the sample analysis region. The quaternary zone comprises a cylindrical opening spanning the second substrate. The tertiary zone 710 is located at a midpoint between the first end (e.g., the quinary zone 709) and the second end (e.g., the quaternary zone 711) of the of the sample analysis region. The sample analysis region may be connected to the sample processing region701 by a transition zone 707. The alternative embodiment disclosed for FIG.7 may be applied to FIG. 15 such that the descriptions above describe the elements of the devices disclosed in FIG. 15.

[0199] FIG. 8 discloses an illustration of an isometric view of an embodiment of the device. The dotted lines represent interior portions of the device whereas solid lines represent exterior portions of the device. In this embodiment, the device 800 contains a 2x4 grid of primary zones 803 and secondary zones 806 with in addition to one additional primary zone and a fixed primary zone 802. In some instances, but not all instances, the fixed primary zone 802 is also a sample mixing zone. The fixed primary zone is physically connected to a hooked portion 807. The boundaries of the top outside portion of the fixed primary zone is not attached to the rest of the device except for the portion furthest from the primary and secondary zones. When a vertical force is applied to the hooked portion 807, the top substrate (i.e., the roof) compresses and decompresses fluid (e.g., sample) that may be in the fixed primary zone 802 thereby mixing the fluid. The device comprises a sample processing region 801. The sample processing region 801 is unbounded. By unbounded it is meant that there is no physical barrier separating the primary zones 803 and the secondary zones 806. The sample processing region comprises a primary zone 803 that has an opening 804 in the primary zone. The primary zone is adjacent to a secondary zone such as 806. The secondary zone may comprise an opening 805.

[0200] An alternative embodiment to FIG. 8 is disclosed below. The dotted lines represent interior portions of the device whereas solid lines represent exterior portions of the device. In this embodiment, the device 800 contains a 2x4 grid of primary zones 803 and secondary zones 806 with in addition to one additional primary zone and a fixed primary zone 802. The device 800 comprises a second substrate positioned on a first substrate. The second substrate comprises a surface facing a central chamber comprising a plurality of recessed elements, and a plurality of protruding elements. The primary zones are defined between a surface of the plurality of protruding elements facing the first substrate and a surface of the first substrate facing the second substrate. The secondary zones are defined between a surface of the plurality of recessed elements facing the first substrate and the surface of the first substrate facing the second substrate. In some instances, but not all instances, the fixed primary zone 802 is also a sample mixing zone. The fixed primary zone 802 is formed from a laterally extended protruding element in the second substrate facing the first substrate wherein the laterally extended protruding element has a surface facing the first substrate that has a greater surface area than the surface of the plurality of protruding elements facing the first substrate. The fixed primary zone 802 comprises hooked portion 807 joined to a surface of an extended protruding element opposite the surface of the second substrate facing the first substrate. The boundaries of the top outside portion of the fixed primary zone isnot attached to the rest of the device except for the portion furthest from the primary and secondary zones. When a vertical force is applied to the hooked portion 807, the second substrate (i.e., the roof) compresses and decompresses fluid (e.g., sample) that may be in the fixed primary zone 802 thereby mixing the fluid. The device comprises a sample processing region 801. The sample processing region 801 is unbounded. By unbounded it is meant that there is no physical barrier separating the primary zones 803 and the secondary zones 806. The sample processing region comprises a primary zone 803 that has an opening 804 in the primary zone. The primary zone is adjacent to a secondary zone such as 806. The secondary zone may comprise an opening 805. The alternative embodiment disclosed for FIG.8 may be applied to FIG. 14 such that the descriptions above describe the elements of the devices disclosed in FIG. 14.

[0201] FIG. 9 discloses an illustration of an exemplary sample analysis region of the embodiment depicted in FIG. 8. The sample processing region 800 may be connected to a sample analysis region 900 comprising a tertiary zone (i.e., the sample detection zone) 902. The device may further comprise a quaternary zone 903 (i.e., a hydrophilic liquid well) that is connected to the tertiary zone. The quaternary zone 903 (i.e., a hydrophilic liquid well) may be surrounded by substrate retention features 904. The device may further comprise a quinary zone 901 (i.e., a hydrophobic liquid well) that is connected to the tertiary zone. The sample analysis region may be a pad such as those in the primary zones. In some embodiments, when the sample analysis region is a pad, the pad has the same thickness as the pads in the primary zones. In some embodiments, when the sample analysis region is a pad, the pad has a different thickness as the pads in the primary zones. A substrate retention feature may be configured to assist in the removal of the hydrophilic liquids disclosed herein.

[0202] An alternative embodiment to FIG. 9 is disclosed below. The sample processing region 800 may be connected to a sample analysis region 900 comprising a tertiary zone (i.e., the sample detection zone) 902. The sample analysis region comprises an enlarged protruding element where a surface of the enlarged protruding element facing the first substrate has a greater surface area than a surface of the plurality of protruding elements facing the first substrate, and the enlarged protruding element extends from the first end (e.g., a quinary zone 901) to the second end (e.g., a quaternary zone 903). The device may further comprise a quaternary zone 903 (i.e., a hydrophilic liquid well) that is connected to the tertiary zone. The quaternary zone 903 (i.e., a hydrophilic liquid well) may be surrounded by substrate retention features 904. The quaternary zone comprises a cylindrical opening spanning the second substrate. The device may further comprise a quinary zone 901 (i.e., a hydrophobic liquid well) that is connected to the tertiary zone. The quinary zone comprises an opening spanning the second substrate. The sample analysis region 900 is adjacentto the sample processing region 800. The sample analysis region is not physically separated from the sample processing region.

[0203] FIG. 38A and 38B discloses an illustration of an embodiment of the device. The embodiment of FIG. 38A shows the surface of the second substrate facing the central chamber or first substrate. The embodiment of FIG. 38B shows the surface of the second substrate facing away from the central chamber. The dotted lines represent interior portions of the device whereas solid lines represent exterior portions of the device. In this embodiment, the device 3800 contains a 2x6 grid of primary zones 3803 and secondary zones 3806 and a fixed primary zone 3802. The device 3800 comprises a second substrate positioned on a first substrate. The second substrate comprises a surface facing a central chamber comprising a plurality of recessed elements, and a plurality of protruding elements. The primary zones are defined between a surface of the plurality of protruding elements facing the first substrate and a surface of the first substrate facing the second substrate. The secondary zones are defined between a surface of the plurality of recessed elements facing the first substrate and the surface of the first substrate facing the second substrate. In some instances, but not all instances, the fixed primary zone 3802 is also a sample mixing zone. The fixed primary zone 3802 is formed from a laterally extended protruding element in the second substrate facing the first substrate wherein the laterally extended protruding element has a surface facing the first substrate that has a greater surface area than the surface of the plurality of protruding elements facing the first substrate. The laterally extended protruding element may be referred to as a tertiary protruding element. The fixed primary zone 3802 comprises a chamfered end 3812 laterally separated from a connected end 3813. By “chamfered end” it is meant that the edge of the chamfered end has a symmetrical slope on both the surface of the second substrate facing the central chamber and the surface of the second substrate facing away from the central chamber. In some embodiments, the chamfered end is crescent shaped. The boundaries of the top outside portion of the fixed primary zone is not attached to the rest of the device except for the connected end 3813. When a vertical force is applied to the chamfered end 3812, the second substrate (i.e., the roof) compresses and decompresses fluid (e.g., sample) that may be in the fixed primary zone 3802 thereby mixing the fluid. The device comprises a sample processing region 3801. The sample processing region 3801 is unbounded. By unbounded it is meant that there is no physical barrier separating the primary zones 3803 and the secondary zones 3806. The sample processing region comprises a primary zone 3803 that has an opening 3804 in the primary zone. The primary zone is adjacent to a secondary zone such as 3806. The secondary zone may comprise an opening 3805. The device 3800 comprises a sample analysis region 3807. In some embodiments, the device 3800 comprises a waste disposal region 3808. The waste disposal region3808 is at an end of the sample processing region 3801 that is furthest from the sample analysis region 3807. The waste disposal region 3808 comprises an opening in the second substrate and has a wedged portion on a side of the opening closest to an edge of the device. In some embodiments, the device 3800 comprises a clearance port 3809. The clearance port 3809 is an opening in the second substrate medially separated from the waste disposal region 3808.

[0204] FIG. 34 discloses an illustration of an exemplary sample analysis region of the embodiment depicted in FIG. 8 and FIG. 9. The thin dotted lines represent interior portions of the device whereas solid lines represent exterior portions of the device. The sample analysis region 3400 (bounded by thick dashed lines) is formed by the top substrate 3401 bound to the bottom substrate 3402. A pad 3406 defines the sample analysis region. The sample analysis region 3400 comprises a tertiary zone 3403 (i.e., sample detection zone). The device may further comprise a quaternary zone 3405 (i.e., a hydrophilic liquid well) that is connected to the tertiary zone. By connected is meant that the zones are adjacent to each other. The quaternary zone 3405 (i.e., a hydrophilic liquid well) may be surrounded by substrate retention regions(not shown; as depicted in FIG. 9 904). The device may further comprise a quinary zone 3404 (i.e., a hydrophobic liquid well) that is connected to the tertiary zone.

[0205] FIG. 45 discloses an illustration of an exemplary sample analysis region of the embodiment depicted in FIG. 38. The sample processing region may be connected to a sample analysis region 4500 comprising a tertiary zone (i.e., the sample detection zone) 4501. The sample analysis region comprises an enlarged protruding element 4502 where a surface of the enlarged protruding element facing the first substrate has a greater surface area than a surface of the plurality of protruding elements facing the first substrate, and the enlarged protruding element extends from the first end (e.g., a quinary zone 4504) to the second end (e.g., a quaternary zone 4503). The quaternary zone 4503 (i.e., a hydrophilic liquid well) may be surrounded by substrate retention features 4505. The quaternary zone comprises a cylindrical opening spanning the second substrate. The quinary zone 4504 comprises an opening spanning the second substrate. The enlarged protruding element 4502 of the sample analysis region 4500 contains a pinning wall 4506 about the perimeter of the enlarged protruding element 4502 excluding the second end containing the quinary zone 4504. The pining wall 4406 is a raised edge where the raised edge protrudes from the enlarged protruding element 4502. The pinning wall assists in retaining the hydrophilic liquid in the sample analysis region when the hydrophilic liquid is deposited or removed from the sample analysis region. The sample analysis region 4500 is adjacent to the sample processing region. The sample analysis region is not physically separated from the sample processing region. The sample analysis region comprises an anti-sink feature 4507 where the anti-sink feature is a slopeddepression in the surface of the second substrate facing away from the first substrate. The antisink feature provides the benefit of creating uniform second substrate thickness in the sample analysis region and providing increased optical clarity relative to non-sloped depressions. The uniform second substrate thickness increases reproducibility of the device during the manufacturing process. The pinning wall in FIG. 45 may be applied to any other embodiment of the device, e.g., the embodiments disclosed in FIG. 1-9, 11-19, 32, and 38-41. The sample analysis region depicted in FIG. 45 may be applied to any other embodiment of the device, e.g., the embodiments disclosed in FIG. 1-9, 11-19, 32, and 38-41.

[0206] FIG. 33A-G discloses illustrations of exemplary sample analysis regions. FIG. 33 A discloses an illustration of the sample analysis regions depicted in FIG. 8 and 9. The dotted lines represent interior portions of the device whereas solid lines represent exterior portions of the device. The sample analysis region 3300 comprises a tertiary zone 3301 (e.g., a sample detection zone) that is the width of the sample analysis region 3300. The sample analysis region 3300 comprises a quaternary zone 3304 (i.e., hydrophilic liquid well) that has substrate retention features 3305a-d which are quarter circle shaped and protrude into the quaternary zone 3304. The sample analysis region comprises a quinary zone 3306 (e.g., a hydrophobic liquid well). The left end 3302 has an oval shape that has a smaller area than the right end 3303 which has a larger area and is a circular shape.

[0207] FIG. 33B discloses an illustration of an exemplary sample analysis region. The sample analysis region 3307 comprises a tertiary zone 3308 (e.g., a sample detection zone) that is the width of the sample analysis region 3307. The sample analysis region 3307 comprises a quaternary zone 3311 (i.e., hydrophilic liquid well) that has substrate retention features 3312a-d which are quarter circle shaped and protrude into the quaternary zone 3311. The sample analysis region comprises a quinary zone 3313 (e.g., a hydrophobic liquid well). The left end 3309 has an oval shape and the right end 3310 has an oval shape that is the same size as the left end.

[0208] FIG. 33C discloses an illustration of an exemplary sample analysis region. The sample analysis region 3314 comprises a tertiary zone 3315 (e.g., a sample detection zone) where the width of the sample analysis region is larger than the tertiary zone. The sample analysis region 3314 comprises a quaternary zone 3318 (i.e., hydrophilic liquid well) that has substrate retention features 3319a-d which are quarter circle shaped and protrude into the quaternary zone 3318. The sample analysis region comprises a quinary zone 3320 (e.g., a hydrophobic liquid well). The left end 3316 has an oval shape that has a smaller area than the right end 3317 which has a larger area and is a circular shape.

[0209] FIG. 33D discloses an illustration of an exemplary sample analysis region. The sample analysis region 3321 comprises a tertiary zone 3322 (e.g., a sample detection zone) that is the width of the sample analysis region 3321. The sample analysis region 3321 comprises a quaternary zone 3325 (i.e., hydrophilic liquid well) that has substrate retention features 3326a-d which are quarter circle shaped and protrude into the quaternary zone 325. The sample analysis region comprises a quinary zone 3327 (e.g., a hydrophobic liquid well). The left end 3323 has an oval shape that has a smaller area than the right end 3324 which has a larger area and is a circular shape.

[0210] FIG. 33E discloses an illustration of an exemplary sample analysis region. The sample analysis region 3328 comprises a tertiary zone 3329 (e.g., a sample detection zone) wherein the width of the sample analysis region 3321 is smaller on the edge of the tertiary zone 3329 closest to the left end 3330 than on the edge of the tertiary zone 3329 closest to the left end 3331. The sample analysis region 3328 comprises a quaternary zone 3332 (i.e., hydrophilic liquid well) that has substrate retention features 3333a-d which are quarter circle shaped and protrude into the quaternary zone 3332. The sample analysis region comprises a quinary zone 3334 (e.g., a hydrophobic liquid well). The left end 3330 has an oval shape that has a smaller area than the right end 3331 which has a larger area and is an oval shape.

[0211] FIG. 33F discloses an illustration of an exemplary sample analysis region. The sample analysis region 3335 comprises a tertiary zone 3336 (e.g., a sample detection zone) that is the width of the sample analysis region 3335. The sample analysis region 3335 comprises a quaternary zone 3339 (i.e., hydrophilic liquid well) that has substrate retention features 3340a-d which are quarter circle shaped and protrude into the quaternary zone 3339. The sample analysis region comprises a quinary zone 3341 (e.g., a hydrophobic liquid well). The left end 3337 has an oval shape that has a smaller area than the right end 3338 which has a larger area and is a circular shape with a step reduction in the width prior to the tertiary zone 3336. By “step reduction in width” it is meant that there is a symmetrical reduction in the width that is immediate and not gradual.

[0212] FIG. 33G discloses an illustration of an exemplary sample analysis region. The sample analysis region 3342 comprises a tertiary zone 3343 (e.g., a sample detection zone) where the width of the sample analysis region is larger than the tertiary zone. The sample analysis region 3342 comprises a quaternary zone 3346 (i.e., hydrophilic liquid well) that has substrate retention features 3347a-d which are quarter circle shaped and protrude into the quaternary zone 3346. The sample analysis region comprises a quinary zone 3348 (e.g., a hydrophobic liquid well). The left end 3344 has an oval shape that has a smaller area than the right end 3345 which has a larger area and is a circular shape. The left end has a step reduction in the width prior to the tertiary zone 3343.

[0213] FIG. 33H-N discloses illustrations of exemplary substrate retention features. In FIG. 33H, the sample analysis region 3349 comprises a quaternary zone 3353 (i.e., hydrophilic liquid well) that has substrate retention features 3351 and 3352. The substrate retention features 3351 and 3352 may protrude from the bottom substrate to the top substrate or from the top substrate to the bottom substrate. The substrate retention feature 3351 is centrally located on the right end 3354 and is quarter circle shaped. The substrate retention feature 3352 is located on the right end 3354 of the sample analysis region 3349 and is horseshoe shaped with tapered edges. The substrate retention features of FIG. 33H may be applied to any sample analysis region disclosed herein.

[0214] In FIG. 331, the sample analysis region 3356 comprises a quaternary zone 3359 (i.e., hydrophilic liquid well) that has substrate retention features 3357 and 3358. The substrate retention features 3357 and 3358 may protrude from the bottom substrate to the top substrate or from the top substrate to the bottom substrate. The substrate retention feature 3357 is centrally located on the right end 3360 and is horseshoe shaped without tapered edges. The substrate retention feature 3358 is located on the right end 3360 of the sample analysis region 3356 and is horseshoe shaped without tapered edges. The substrate retention features 3357 and 3358 are concentric such that the substrate retention feature 3357 is nested in the substrate retention feature 3358. The substrate retention features of FIG. 331 may be applied to any sample analysis region disclosed herein.

[0215] In FIG. 33 J, the sample analysis region 3361 comprises a quaternary zone 3362 (i.e., hydrophilic liquid well) that has substrate retention features 3364a-d. The substrate retention features 3364a-d are grooves in either the top substrate or bottom substrate of the sample analysis region 3361. The substrate retention features 3364a-d are located on the right end 3363 of the sample analysis region 3361 and is horseshoe shaped. The substrate retention features 3364a-d are concentric such that the substrate retention feature 3364a is nested in the substrate retention feature 3364b, substrate retention feature 3364b is nested in the substrate retention feature 3364c, and substrate retention feature 3364c is nested in the substrate retention feature 3364d. The substrate retention features of FIG. 331 may be applied to any sample analysis region disclosed herein.

[0216] In FIG. 33K, the sample analysis region 3365 comprises a quaternary zone 3367 (i.e., hydrophilic liquid well) that has substrate retention features 3368a-d and 3369. The substrate retention features 3368a-d are quarter circle shaped and protrude into the quaternary zone 3367 and are located on the right end 3366. The substrate retention feature 3369 is located on the right end 3366 of the sample analysis region 3365 and is horseshoe shaped. The substrate retention feature 3369 may protrude from the bottom substrate to the top substrate or from the top substrateto the bottom substrate. The substrate retention features of FIG. 33K may be applied to any sample analysis region disclosed herein.

[0217] In FIG. 33L, the sample analysis region 3370 comprises a quaternary zone 3372 (i.e., hydrophilic liquid well) that has substrate retention features 3373a-d and 3374a-b. The substrate retention features 3373a-d are quarter circle shaped and protrude into the quaternary zone 3372 and are located on the right end 3371. The substrate retention feature 3374a-b is located prior to the right end 3371 of the sample analysis region 3370 and are rectangular shaped. The substrate retention features 3374a-b may protrude from the bottom substrate to the top substrate or from the top substrate to the bottom substrate. The substrate retention features of FIG. 33L may be applied to any sample analysis region disclosed herein.

[0218] In FIG. 33M, the sample analysis region 3375 comprises a quaternary zone 3377 (i.e., hydrophilic liquid well) that has substrate retention features 3378a-d and 3379a-b. The substrate retention features 3378a-d are quarter circle shaped and protrude into the quaternary zone 3377 and are located on the right end 3376. The substrate retention feature 3379a-b is located prior to the right end 3376 of the sample analysis region 3375 and are circular shaped. The substrate retention features 3379a-b may protrude from the bottom substrate to the top substrate or from the top substrate to the bottom substrate. The substrate retention features of FIG. 33M may be applied to any sample analysis region disclosed herein.

[0219] In FIG. 33N, the sample analysis region 3380 comprises a quaternary zone 3382 (i.e., hydrophilic liquid well) that has substrate retention features 3383a-d and 3384. The substrate retention features 3383a-d are quarter circle shaped and protrude into the quaternary zone 3382 and are located on the right end 3381. The substrate retention feature 3384 is located on the right end 3381 of the sample analysis region 3380 and is horseshoe shaped. The substrate retention feature 3384 may protrude from the bottom substrate to the top substrate or from the top substrate to the bottom substrate. The substrate retention features of FIG. 33K may be applied to any sample analysis region disclosed herein.

[0220] FIG. 330 discloses an illustration of an exemplary barrier feature. The sample analysis region 3385 comprises a quinary zone 3386 (e.g., a hydrophobic liquid well) on the left end 3387. The sample analysis zone also comprises a barrier feature 3388. The barrier feature 3388 is half circle shaped. The barrier feature 3388 may protrude from the bottom substrate to the top substrate or from the top substrate to the bottom substrate. The barrier feature of FIG. 330 may be applied to any sample analysis region disclosed herein. A barrier feature may be configured to assist with the sealing of wells using the hydrophobic liquids disclosed herein.

[0221] FIG. 43 discloses an exemplary substrate stopper feature. In some embodiments, the sample analysis region 4301 (dashed box) comprises a substrate stopper feature 4302. The sample analysis region comprises an enlarged protruding element 4304 where a surface of the enlarged protruding element facing the first substrate has a greater surface area than a surface of the plurality of protruding elements facing the first substrate, and the enlarged protruding element extends from the first end (e.g., a quinary zone 4303) to the second end (e.g., a quaternary zone 4305). The substrate stopper feature comprises a ridge that creates a difference in height between the quinary zone 4303 (i.e., a hydrophobic liquid well) and the quaternary zone 4305 (i.e., hydrophilic liquid well). When the hydrophilic liquid is present in the sample analysis region, the substrate stopper assists in retaining the hydrophilic liquid outside of the quinary zone. The substrate stopper feature may be presented in any embodiment of the sample analysis region comprising an enlarged protruding element.

[0222] FIG. 11 discloses an illustration of an isometric view of an embodiment of the device. The dotted lines represent interior portions of the device whereas solid lines represent exterior portions of the device. In this embodiment, the device 1100 contains a non-grid pattern (i.e., a honeycomb pattern) of primary zones 1103 and secondary zones 1106 in addition to a fixed primary zone 1108. In some instances, but not all instances, the fixed primary zone 1108 is also a sample mixing zone. The device comprises a reagent / sample injection port 1109. The device comprises a sample processing region 1105. The sample processing region 1105 is unbounded. By unbounded it is meant that there is no physical barrier separating the primary zones 1103 and the secondary zones 1106. The sample processing region comprises a primary zone 1103 that has an opening 1104 in the primary zone. The primary zone is adjacent to a secondary zone such as 1106. The sample processing region 1105 may be connected to a tertiary zone (i.e., the sample detection zone) 1102. The device may further comprise a quaternary zone 1107 (i.e., a hydrophilic liquid well) that is connected to the tertiary zone. The device may further comprise a quinary zone 1101 (i.e., an hydrophobic liquid well) that is connected to the tertiary zone.

[0223] FIG. 12 depicts an illustration of the top view of the embodiment of the device depicted in FIG. 11. 1201 refers to the reagent / sample injection port. 1202 refers to the fixed primary zone. 1206 refers to a primary zone. 1207 refers to a secondary zone. 1205 refers to a sample analysis region. 1203 refers to the hydrophilic liquid well. 1204 refers to the hydrophobic liquid well.

[0224] FIG. 13 depicts an embodiment of the device of FIG.12 with reduced size and primary zones. 1301 refers to the reagent / sample injection port. 1302 refers to the fixed primary zone. 1306 refers to a primary zone. 1307 refers to a secondary zone. 1305 refers to a sample analysis region. 1303 refers to the hydrophilic liquid well. 1304 refers to the hydrophobic liquid well.

[0225] FIG. 14 depicts an illustration of the bottom view of the top substrate of the device according to FIG. 8. 1401 refers to the sample processing region. 1402 refers to the fixed primary zone. 1403 refers to a primary zone. 1404 refers to the opening of a primary zone. 1407 refers to a secondary zone. 1406 refers to an opening in a secondary zone.

[0226] FIG. 15 depicts an illustration of the bottom view of the top substrate of the device according to FIG. 7. 1501 refers to the sample processing region. 1502 refers to the fixed primary zone. 1503 refers to a primary zone. 1504 refers to the opening of a primary zone. 1507 refers to a secondary zone. 1506 refers to an opening in a secondary zone. 1408 refers to the sample detection region

[0227] FIG. 32B depicts an illustration of the bottom view of the top substrate of the device according to FIG. 32A. 3210 refers to the sample processing region. 3211 refers to the fixed primary zone. 3212 refers to a primary zone. 3213 refers to the opening of a primary zone. 3214 refers to a secondary zone. 3215 refers to an opening in a secondary zone.

[0228] FIG. 39 discloses an illustration of an embodiment of a sample mixing zone of the device depicted in FIG. 38. The embodiment of FIG. 39 shows the surface of the second substrate facing the central chamber or first substrate. In this embodiment, the device 3900 comprises a sample mixing zone comprising a fixed primary zone 3901 (red box). The fixed primary zone 3901 is formed from a laterally extended protruding element 3902 in the second substrate facing the first substrate wherein the laterally extended protruding element has a surface facing the first substrate that has a greater surface area than the surface of the plurality of protruding elements facing the first substrate. The fixed primary zone 3901 comprises an opening 3903 where the sample may be deposited through. The fixed primary zone 3901 comprises a chamfered end 3904 laterally separated from a connected end 3905. By “chamfered end” it is meant that the edge of the chamfered end 3904 has a symmetrical slope on both the surface of the second substrate facing the central chamber and the surface of the second substrate facing away from the central chamber. The chamfered end 3904 may be joined with a vibration source that vibrates vertically such that the second substrate (i.e., the roof) compresses and decompresses fluid (e.g., sample) that may be in the fixed primary zone 3902 thereby mixing the fluid. The boundaries of the top outside portion of the fixed primary zone is not attached to the rest of the device except for the connected end 3905. In some embodiments, the laterally extended protruding element comprises a raised beveled perimeter 3907. By “raised beveled perimeter” it is meant that the perimeter of the laterally extended protruding element has a raised beveled edge. The raised beveled edge assists in retaining the sample in the sample mixing zone during the mixing process. In some embodiments, the laterally extended protruding element comprises a plurality of secondary features 3906a,3906b, 3906c. The plurality of secondary features 3906a, 3906b, 3906c are raised pillars that assist in mixing the sample that may be present in the sample mixing region. The sample mixing zone of FIG. 39 may be applied to any other embodiment of the device, e.g., the embodiments disclosed in FIG. 1-9, 11-19, 32, and 38-41.

[0229] FIG. 40A and FIG. 40B disclose an illustration of exemplary waste disposal region of the device depicted in FIG. 38. The embodiment of FIG. 40A and FIG. 40B shows the waste disposal region on the surface of the second substrate facing away from the central chamber. The waste disposal region 4001 is at an end of the sample processing region 4005 that is furthest from the sample analysis region 4006. The waste disposal region 4001 comprises an opening in the second substrate. The opening of the waste disposal region 4001 has a wedged portion 4002 extended into the central chamber. The wedge portion 4002 allows for the wicking of waste 4003 from a probe that may dispense waste into the waste port. The wedge portion 4002 of the waste disposal region 4001 directs the waste 4003 into the central chamber. The waste disposal region provides the benefit of being able to dispose of waste directly into the device without the need of external disposal. The waste disposal region of FIG. 40A and FIG. 40B may be applied to any other embodiment of the device, e.g., the embodiments disclosed in FIG. 1-9, 11-19, 32, and 38- 41.

[0230] FIG. 41A and FIG. 41B disclose an illustration of an exemplary of a pre-treatment region of the device. The embodiment of FIG. 41A shows the surface of the second substrate facing away from the central chamber. The embodiment of FIG. 41B shows the surface of the second substrate facing the central chamber or first substrate. In some embodiments, the device 4101 comprises a pre-treatment region 4102. The pre-treatment region is formed between the surface of the second substrate facing the first substrate and the surface of the first substrate facing the second substrate. In some embodiments, the pre-treatment region is a square. The pretreatment region comprises a plurality of elongated openings 4104a, 4104b, 4104c, and 4104d about the perimeter of the pre-treatment region 4102. The elongated openings 4104a, 4104b, 4104c, and 4104d about the perimeter of the pre-treatment region 4102 are separated from each other by a plurality connected regions 4107a, 4107b, 4107c, and 4107d. The plurality of the connected regions may be located at each comer of the pre-treatment region 4102. The elongated openings serve as a hydrophobic barrier between the pre-treatment region 4102 and the sample processing region 4103, and weakens the mechanical connection between pre-treatment region to the second substrate to enhance the mechanical vibration of pre-treatment region during mixing.. The pre-treatment region 4102 comprises a raised edge 4106 about the perimeter of the pretreatment region 4102 enclosed by the plurality of elongated openings 4104a, 4104b, 4104c, and4104d. The raised edge assists in retaining fluid deposited into the pre-treatment region. The pretreatment region 4102 comprises a plurality of openings 4105a, 4105b, and 4105c. The plurality of openings creates air-liquid interfaces that assist in mixing any fluid deposited within the pretreatment region. In some embodiments, the pre-treatment region is at an end of the sample processing region 4103 that is furthest from the sample analysis region. In some embodiments, when the device comprises a pre-treatment region, the device does not comprise a sample mixing region. In some embodiments, when the device comprises a pre-treatment region, fluids on the device are mixed through vibrating the device as described below. The pre-treatment region allows for pre-processing of the sample prior to introduction into the sample processing region. For instance, the pre-treatment region may be used for the dilution of the sample, lysis of the sample, modification of the sample or target analyte to allow for capture of the target analyte, etc. In some embodiments, the pre-treatment region contains one or more pre-treatment reagents, such as one or more detergents, surfactants, reducing agents, or any combinations thereof. In yet further embodiments, the sample is contacted with a pre-treatment reagent prior to introduction of the sample into the sample processing region.

[0231] FIG. 54 discloses an illustration of an exemplary embodiment of the device containing a pre-treatment region. In this embodiment, the device 5400 contains a 2x3 grid of primary zones 5403 and secondary zones 5406 and a fixed primary zone 5402. The device 5400 comprises a second substrate positioned on a first substrate. The second substrate comprises a surface facing a central chamber comprising a plurality of recessed elements, and a plurality of protruding elements. The primary zones are defined between a surface of the plurality of protruding elements facing the first substrate and a surface of the first substrate facing the second substrate. The secondary zones are defined between a surface of the plurality of recessed elements facing the first substrate and the surface of the first substrate facing the second substrate. In some instances, but not all instances, the fixed primary zone 5402 is also a sample mixing zone. The fixed primary zone 5402 is formed from a laterally extended protruding element in the second substrate facing the first substrate wherein the laterally extended protruding element has a surface facing the first substrate that has a greater surface area than the surface of the plurality of protruding elements facing the first substrate. The laterally extended protruding element may be referred to as a tertiary protruding element. The fixed primary zone 5402 comprises a chamfered end 5412 laterally separated from a connected end 5413. By “chamfered end” it is meant that the edge of the chamfered end has a symmetrical slope on both the surface of the second substrate facing the central chamber and the surface of the second substrate facing away from the central chamber. In some embodiments, the chamfered end is crescent shaped. The boundaries of the top outsideportion of the fixed primary zone is not attached to the rest of the device except for the connected end 5413. When a vertical force is applied to the chamfered end 5412, the second substrate (i.e., the roof) compresses and decompresses fluid (e.g., sample) that may be in the fixed primary zone 5402 thereby mixing the fluid. The device comprises a sample processing region 5401. The sample processing region 5401 is unbounded. By unbounded it is meant that there is no physical barrier separating the primary zones 5403 and the secondary zones 5406. The sample processing region comprises a primary zone 5403 that has an opening 5404 in the primary zone. The primary zone is adjacent to a secondary zone such as 5406. The secondary zone may comprise an opening 5405. The device 5400 comprises a sample analysis region 5407. In some embodiments, the device 5400 comprises a waste disposal region 5408. The waste disposal region 5408 is at an end of the sample processing region 5401 that is furthest from the sample analysis region 5407. The waste disposal region 5408 comprises an opening in the second substrate and has a wedged portion on a side of the opening closest to an edge of the device.

[0232] In some embodiments, the device 5400 comprises a pre-treatment region 5416. The pretreatment region is formed between the surface of the second substrate facing the first substrate and the surface of the first substrate facing the second substrate. In some embodiments, the pretreatment region is a square. The pre-treatment region 5416 comprises a raised edge 5414 about the perimeter of the pre-treatment region 5416. The raised edge assists in retaining fluid deposited into the pre-treatment region. The pre-treatment region 5416 comprises a plurality of openings 5415a, 5415b, and 5415c. The plurality of openings creates air-liquid interfaces that assist in mixing any fluid deposited within the pre-treatment region. In some embodiments, the pretreatment region is at an end of the sample processing region 5401 that is furthest from the sample analysis region. In some embodiments, when the device comprises a pre-treatment region, fluids on the device are mixed through vibrating the device as described below. The pre-treatment region allows for pre-processing of the sample prior to introduction into the sample processing region. For instance, the pre-treatment region may be used for the dilution of the sample, lysis of the sample, modification of the sample or target analyte to allow for capture of the target analyte, etc. In some embodiments, the pre-treatment region contains one or more pre-treatment reagents, such as one or more detergents, surfactants, reducing agents, or any combinations thereof. In yet further embodiments, the sample is contacted with a pre-treatment reagent prior to introduction of the sample into the sample processing region.

[0233] I. Arrangement of the sample processing region

[0234] The sample processing regions of the present disclosure have a range of different arrangements such as those described above. The sample processing regions disclosed herein alsohave a range in the number of primary zones present. For example, the device may comprise 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, or 24 or more primary zones.

[0235] The primary zones of the present disclosure have pads that are formed from the top substrate. The pads found in the primary zones may have a wide range of shapes including, without limitation, a rectangle, a circle, a triangle, a pentagon, a hexagon, a heptagon, an octagon, a decagon, a dodecagon, an amoeboid, a non-regular shape, or a circular shape with points. In some embodiments, the pad is a rectangle or square such as those depicted in FIG. 1-7. In some embodiments, the pad is a circle such as those depicted in FIG.8 and 14. In some embodiments, the pad is a hexagon such as those depicted in FIG. 11-13. In some embodiments, the pad is an amoeboid such as those depicted in FIG. 27 A. In some embodiments, the pad is a non-regular shape. In some embodiments, the pad is a circular shape with points such as those depicted in FIG. 27A. The pads in the primary zone have edges. In some embodiments, the edge is sharp. In some embodiments, the edge is curved such that the edge has a rounded shape. The pads of the primary zone may have a particular profile. In some embodiments, the pad is flat. In some embodiments, the pad has protrusions on the edges that are closer to the bottom substrate than the center of the pad such as depicted FIG. 16.

[0236] One or more surfaces of the pads may be designed in such a way that they retain fluid. The term “surface of the pad” refers to any surface of the pad that is facing the interior of the device such as the side of the pad that is facing another pad, the side of the pad that is facing the edge of the interior of the device, or the side of the pad that is facing the bottom substrate in the interior of the device. Surface of the pad may also refer to the surface of the plurality of protruding elements facing the first substrate. Alternative terms for the surfaces of the pad are “leading face” and “side surfaces”, wherein “leading face” refers to the surface of the pad that is facing the bottom substrate and “side surfaces” refer to the surfaces other than the “leading face”; when using this terminology and the device is planar, the “leading face” may be substantially parallel to the first plane and the “side surfaces” may be substantially perpendicular to the first plane. For instance, the one or more surfaces of the pads may contain serrations or grooves that occupy all or a portion of a given pad. The one or more surfaces of the pads may be two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or all of the surfaces of pad. The serrations or grooves may be in a range of different patterns including, without limitation, waves, straight lines that are perpendicular or parallel to the edge of the pad, diagonal to the edge of the pad, cross or hatch pattern, any combination thereof, etc. In someembodiments, the serrations or grooves are in a wave pattern such as depicted in FIG. 28G. In some embodiments, the serrations or grooves are in straight lines that are perpendicular or parallel to the edge of the pad such as depicted in FIG. 28A-B. In some embodiments, the serrations or grooves are in straight lines that are diagonal to the edge of the pad such as depicted in FIG. 28C- D. In some embodiments, the serrations or grooves are in a cross or hatch pattern such that two or more serrations or grooves cross one another such as depicted in FIG. 28E-F. In some embodiments, the serrations or grooves are uniformly spaced. In some embodiments, the serrations or grooves are not uniformly spaced. In some embodiments, all of the pads in the sample processing region contain serration or grooves. In some embodiments, only a portion of the pads in the sample processing region contain grooves or serrations.

[0237] One or more of the surfaces of the device may have a surface finish. A surface finish provides the advantage of allowing for a consistent surface roughness on surfaces that have the finish and provide consistent fluid behavior across multiple surfaces and devices sharing the same surface finish. By “surface finish” it is meant that the surface of the pad may have a certain smoothness or polish. Surfaces finished may be described as a Society of the Plastics Industry (SPI) finish or a Verein Deutscher Ingenieure (VDI) finish. VDI finish may also be referred to VDI 3400. The surface of the pad may have any finish deemed useful. For instance, the surface of the pad may have a VDI 3400 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or a VDI 3400 45. The surface of the pad may have an SPI Al, A2, A3, Bl, B2, B3, Cl, C2, C3, DI, D2, or a SPI D3. The surface finish may be a range of surface finishes. For instance, the surface finish may be from SPI Al to SPI to A3, from SPI Bl to SPI to B3, from SPI Cl to SPI C3, or from SPI DI to D3. The surface finish may be from VDI 0 to VDI 5, VDI 6 to VDI 10, VDI 11 to VDI 15, VDI 16 to VDI 20, VDI 21 to VDI 25, VDI 26 to VDI 30, VDI 31 to VDI 35, VDI 36 to VDI 40, or from VDI 41 to VDI 45.

[0238] The surface finish may be defined by an average surface roughness measurement (RA) in microns (pm). For instance, an SPI Al may have an RA of about 0.012 to about 0.025 m, an SPI A2 may have an RA of about 0.025 to about 0.05 pm, an SPI A3 may have an RA of about 0.05 to about 0.10 pm, an SPI B 1 may have an RA of about 0.05 to about 0.1 pm, an SPI B2 may have an RA of about 0.1 to about 0.15 pm, an SPI B3 may have an RA of about 0.28 to about 0.32 pm, an SPI Cl may have an RA of about 0.35 to about 0.40 pm, an SPI C2 may have an RA of about 0.45 to about 0.55 pm, an SPI C3 may have an RA of about 0.63 to about 0.70 pm, an SPI DI may have an RA of about 0.80 to about 1.00 pm, an SPI D2 may have an RA of about 1.00 to about 2.80 pm, and an SPI D3 may have an RA of about 3.20 to about 18.00 pm. A VDI 3400 0may have a RA of about 0.100 m, a VDI 3400 1 may have a RA of about 0.112 pm, a VDI 3400 2 may have a RA of about 0.126 pm, a VDI 3400 3 may have a RA of about 0.140 pm, a VDI 3400 4 may have a RA of about 0.160 pm, a VDI 3400 5 may have a RA of about 0.180 pm, a VDI 34006 may have a RA of about 0.200 pm, a VDI 34007 may have a RA of about 0.220 pm, a VDI 3400 8 may have a RA of about 0.250 pm, a VDI 3400 9 may have a RA of about 0.290 pm, a VDI 3400 10 may have a RA of about 0.320 pm, a VDI 3400 11 may have a RA of about 0.350 pm, a VDI 3400 12 may have a RA of about 0.400 pm, a VDI 3400 13 may have a RA of about 0.450 pm, a VDI 3400 14 may have a RA of about 0.500 pm, a VDI 3400 15 may have a RA of about 0.560 pm, a VDI 3400 16 may have a RA of about 0.630 pm, a VDI 3400 17 may have a RA of about 0.700 pm, a VDI 3400 18 may have a RA of about 0.800 pm, a VDI 3400 19 may have a RA of about 0.900 pm, a VDI 3400 20 may have a RA of about 1.000 pm, a VDI 340021 may have a RA of about 1.120 pm, a VDI 340022 may have a RA of about 1.260 pm, a VDI 3400 23 may have a RA of about 1.400 pm, a VDI 3400 24 may have a RA of about 1.600 pm, a VDI 3400 25 may have a RA of about 1.800 pm, a VDI 3400 26 may have a RA of about 2.000 pm, a VDI 3400 27 may have a RA of about 2.200 pm, a VDI 3400 28 may have a RA of about 2.500 pm, a VDI 3400 29 may have a RA of about 2.800 pm, a VDI 3400 30 may have a RA of about 3.200 pm, a VDI 3400 31 may have a RA of about 3.500 pm, a VDI 3400 32 may have a RA of about 4.000 pm, a VDI 3400 33 may have a RA of about 4.500 pm, a VDI 3400 34 may have a RA of about 5.000 pm, a VDI 3400 36 may have a RA of about 6.300 pm, a VDI 3400 37 may have a RA of about 7.000 pm, a VDI 3400 38 may have a RA of about 8.000 pm, a VDI 3400 39 may have a RA of about 9.000 pm, a VDI 340040 may have a RA of about 10.000 pm, a VDI 340041 may have a RA of about 11.200 pm, a VDI 340042 may have a RA of about 12.600 pm, a VDI 3400 43 may have a RA of about 14.000 pm, a VDI 3400 44 may have a RA of about 16.000 pm, and a VDI 3400 45 may have a RA of about 18.000 pm.

[0239] In some embodiments, the surface of the pad has a surface finish. In some embodiments, the surface of the plurality of protruding elements facing the first substrate has a surface finish. In some embodiments, the surface of the enlarged protruding element of the sample analysis region facing the first substrate has a surface finish. In some embodiments, the surface of the laterally extended protruding element in the second substrate facing the first substrate in the sample mixing region has a surface. In some embodiments, the surface of the plurality of protruding elements facing the first substrate has a surface finish and the surface of the enlarged protruding element of the sample analysis region facing the first substrate has a surface finish. In some embodiments, the surface of the plurality of protruding elements facing the first substrate has a surface finish,the surface of the enlarged protruding element of the sample analysis region facing the first substrate has a surface finish, and the surface of the laterally extended protruding element in the second substrate facing the first substrate in the sample mixing region has a surface finish.

[0240] In some embodiments, the surface finish of the surface of the enlarged protruding element of the sample analysis region has a higher surface finish than the surface finish of the surface of the plurality of protruding elements facing the first substrate. By “higher surface finish” it is meant that the average surface roughness (RA) is lower. For example, a surface finish of 0.012 m is higher than a surface finish of 0.05 pm and a surface finish of SPI Al is higher than a surface finish of SPI A3. A higher surface finish of the enlarged protruding element of the sample analysis region provides the added benefit of increased optical clarity which assists in the detection of an analyte in the sample detection zone. In some embodiments, the surface finish of the enlarged protruding element of the sample analysis region has a surface finish from about SPI Al to about SPI A3. In some embodiments, the surface of the plurality of protruding elements facing the first substrate has a surface finish from about SPI Bl to about SPI C3. In some embodiments, the surface of the laterally extended protruding element facing the first substrate has a surface finish from about SPI Bl to about SPI C3. In some embodiments, the surface finish of the enlarged protruding element of the sample analysis region has a surface finish from about 0.012 pm to about 0.10 pm. In some embodiments, the surface of the plurality of protruding elements facing the first substrate has a surface finish from about 0.15 pm to about 0.70 pm. In some embodiments, the surface of the laterally extended protruding element facing the first substrate has a surface finish from about 0.15 pm to about 0.70 pm.

[0241] FIG. 47 depicts an illustration of an embodiment of the device 4700 having a surface finish on one or more surfaces. In this embodiment, the surface of the plurality of protruding elements facing the first substrate 4701a, 4701b, and 4701c has a surface finish, the surface of the enlarged protruding element 4702 of the sample analysis region facing the first substrate has a surface finish, and the surface of the laterally extended protruding element 4703 in the second substrate facing the first substrate in the sample mixing region has a surface finish. In some embodiments, the enlarged protruding element 4702 of the sample analysis region facing the first substrate has a higher surface finish than the surface of the plurality of protruding elements facing the first substrate 4701a, 4701b, and 4701c. In some embodiments, the enlarged protruding element 4702 of the sample analysis region facing the first substrate has a higher surface finish than the surface of the laterally extended protruding 4703 element in the second substrate facing the first substrate in the sample mixing region. In some embodiments, the enlarged protruding element 4702 of the sample analysis region has substrate retention features 4704. In someembodiments, the enlarged protruding element 4702 of the sample analysis region has a pinning wall 4705 about the perimeter of the enlarged protruding element 4702 excluding the second end containing the quinary zone (not shown). The pining wall 4705 is a raised edge where the raised edge protrudes from the enlarged protruding element 4702. In some embodiments, the substrate retention features 4704 have a surface finish. In some embodiments, the pinning wall has a surface finish.

[0242] FIG. 281 depicts an illustration of a pad in a primary zone with serrations or grooves and a pad in a primary zone without serrations or grooves. The top substrate 2801 and the bottom substrate 2802 define the interior of the device. The pad on the left has surfaces 2803a and 2803b that do not have serrations or grooves. The pad has three additional surfaces not depicted, one surface opposite surface 2803a, one opposite surface 2803b, and one surface facing the bottom substrate 2802. The pad on the right has serrations or grooves that are diagonal to the edge of the pad on surface 2804a and 2804b. The pad may have additional surfaces with serrations or grooves such as the surface of the pad facing the bottom surface 2802 such as depicted in FIG. 28J, the surface of the pad opposite 2804a, and the surface of the pad opposite 2804b.

[0243] In some embodiments, the one or more surfaces of the pad contain dimples or divots such as depicted in FIG. 28H. In some embodiments, the dimples or divots are concave. In some embodiments, the dimples or divots are convex (e.g., protrusions). In some embodiments, a portion of the dimples or divots are convex and portion of the dimples or divots are concave. In some embodiments, the dimples or divots are circular in shape. In some embodiments, the dimples or divots are triangular in shape. In some embodiments, the dimples or divots are rectangular in shape. In some embodiments, the dimples or divots are pentagonal in shape. In some embodiments, the dimples or divots are cylindrical in shape. In some embodiments, the dimples or divots are hexagonal in shape. In some embodiments, the dimples or divots are heptagonal in shape. In some embodiments, the dimples or divots are octagonal in shape. In some embodiments, the dimples or divots are decagonal in shape. In some embodiments, the dimples or divots are dodecagonal in shape. In some embodiments, the dimples or divots are amoeboid in shape. In some embodiments, the dimples or divots are non-regular in shape. In some embodiments, the dimples or divots are uniformly spaced. In some embodiments, the dimples or divots are not uniformly spaced. In some embodiments, the serrations or grooves are not uniformly spaced. In some embodiments, all of the pads in the sample processing region contain dimples or divots. In some embodiments, only a portion of the pads in the sample processing region contain dimples or divots. In some embodiments, a portion of the pads in the sample processing region contain dimple or divots anda portion of the pads contain serrations or grooves. In some embodiments, all of or a portion of the pads in the sample processing region contain serrations or grooves and dimples or divots.

[0244] FIG. 16 depicts an illustration of a cross-section of the primary zones and pads of an embodiment of the device. A primary zone 1607 (between the dotted lines) is formed by the top substrate 1601 and the bottom substrate 1602. The primary zone contains a pad 1604 or 1605. In some embodiments, the pad is flat (1604). In some embodiments, the pad has a protrusion 1605 around the entire edge such that the protrusion 1605 is closer to the bottom substrate 1602 than the center of the pad 1606.

[0245] The pads of the present disclosure have a range of thickness. For instance, the pad may about 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, or about 1.0 mm. In some embodiments, the pad has a thickness between 0.3 mm and 0.8 mm.

[0246] The pads in the primary zone and the bottom substrate in the primary zone may have a particular patterning. The patterning may be designed to retain fluid in the primary zone. In some embodiments, the pad and the bottom substrate have hydrophilic patterning. In some embodiments, the pad and the bottom substrate in the primary zone do not have any patterning.

[0247] The sample processing regions disclosed herein also have a range in the number of secondary zones present. The device may comprise 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, 36 or more, or 37 or more secondary zones.

[0248] The secondary zones of the present disclosure are formed by the top and bottom substrates of the device. The secondary zones are present between one or more primary zones. In some embodiments, the secondary zone is between two primary zones. The secondary zones have a distance from the top substrate to the bottom substrate that is greater than the distance from the top substrate to the bottom substrate in the primary zones. The difference in the height between the primary zone and secondary zone creates surface tension that is able to hold a fluid in the primary zone without spilling into the secondary zone. The difference in the height between the primary zone and the secondary zone is depicted in FIG. 16 and FIG. 26. In some embodiments, the top substrate and the bottom substrate of the secondary zone have hydrophobic patterning. In some embodiments, the top substrate and the bottom substrate of the secondary zones have no patterning. In some embodiments, the top substrate and the bottom substrate of the secondary zones have uniform hydrophobicity.

[0249] The secondary zones may be designed in such a way that they retain fluid. For instance, the roof of the secondary zone (i.e., the bottom side of the top substrate facing the interior of the device) may contain serrations or grooves that occupy all or a portion of a given roof of a secondary zone. In some embodiments, the serrations or grooves are in a wave pattern such as depicted in FIG. 28G and FIG. 28K. In some embodiments, the serrations or grooves are in straight lines that are perpendicular or parallel to the edge of the pad adjacent to the secondary zone such as depicted in FIG. 28A-B. In some embodiments, the serrations or grooves are in straight lines that are diagonal to the edge of the pad adjacent to the secondary zone such as depicted in FIG. 28C-D. In some embodiments, the serrations or grooves are in a cross or hatch pattern such that two or more serrations or grooves cross one another such as depicted in FIG. 28E-F. In some embodiments, the serrations or grooves are uniformly spaced. In some embodiments, the serrations or grooves are not uniformly spaced. In some embodiments, all of the roofs of the secondary zones in the sample processing region contain serration or grooves. In some embodiments, only a portion of the roofs of the secondary zones in the sample processing region contain grooves or serrations.

[0250] In some embodiments, the roof of the secondary zone contains dimples or divots such as depicted in FIG. 28H. In some embodiments, the dimples or divots are concave. In some embodiments, the dimples or divots are convex (e.g., protrusions). In some embodiments, a portion of the dimples or divots are convex and portion of the dimples or divots are concave. In some embodiments, the dimples or divots are circular in shape. In some embodiments, the dimples or divots are triangular in shape. In some embodiments, the dimples or divots are rectangular in shape. In some embodiments, the dimples or divots are pentagonal in shape. In some embodiments, the dimples or divots are cylindrical in shape. In some embodiments, the dimples or divots are hexagonal in shape. In some embodiments, the dimples or divots are heptagonal in shape. In some embodiments, the dimples or divots are octagonal in shape. In some embodiments, the dimples or divots are decagonal in shape. In some embodiments, the dimples or divots are dodecagonal in shape. In some embodiments, the dimples or divots are amoeboid in shape. In some embodiments, the dimples or divots are non-regular in shape. In some embodiments, the dimples or divots are uniformly spaced. In some embodiments, the dimples or divots are not uniformly spaced. In some embodiments, all of the roofs of the secondary zones in the sample processing region contain dimples or divots. In some embodiments, only a portion of the roofs of the secondary zones in the sample processing region contain dimples or divots. In some embodiments, a portion of the roofs of the secondary zones in the sample processing region contain dimple or divots and a portion of the roofs of the secondary zones contain serrations or grooves. In some embodiments, all of or aportion of the roofs in the secondary zones in the sample processing region contain serrations or grooves and dimples or divots.

[0251] In some embodiments, the top substrate has uniform hydrophobicity such that the entire surface of the top substrate is hydrophobic, hydrophilic, or is not hydrophobic or hydrophilic. In some embodiments, the bottom substrate has uniform hydrophobicity such that the entire surface of the bottom substrate is hydrophobic, hydrophilic, or is not hydrophobic or hydrophilic. In some embodiments, the top substrate (e.g., second substrate) is hydrophobic. In some embodiments, the top substrate (e.g., second substrate) is hydrophilic. In some embodiments, the bottom substrate (e.g., first substrate) is hydrophobic. In some embodiments, the bottom substrate (e.g., first substrate) is hydrophilic. In some embodiments, the top substrate (e.g., second substrate) is hydrophobic and the bottom substrate (e.g., first substrate) is hydrophobic. In some embodiments, the top substrate (e.g., second substrate) is hydrophilic and the bottom substrate (e.g., first substrate) is hydrophobic. In some embodiments, the top substrate (e.g., second substrate) is hydrophobic and the bottom substrate (e.g., first substrate) is hydrophilic. In some embodiments, the top substrate (e.g., second substrate) is hydrophilic and the bottom substrate (e.g., first substrate) is hydrophilic. In embodiments where the top substrate (e.g., second substrate) and the bottom substrate (e.g., first substrate) are both hydrophobic, the top substrate (e.g., second substrate) may have a higher hydrophobicity than the bottom substrate (e.g., first substrate). In embodiments where the top substrate (e.g., second substrate) and the bottom substrate (e.g., first substrate) are both hydrophobic, the bottom substrate (e.g., first substrate) may have a higher hydrophobicity than the top substrate (e.g., second substrate). In embodiments where the top substrate (e.g., second substrate) and the bottom substrate (e.g., first substrate) are both hydrophilic, the top substrate (e.g., second substrate) may have a higher hydrophilicity than the bottom substrate (e.g., first substrate). In embodiments where the top substrate (e.g., second substrate) and the bottom substrate (e.g., first substrate) are both hydrophilic, the bottom substrate (e.g., first substrate) may have a higher hydrophilicity than the top substrate (e.g., second substrate).

[0252] In some embodiments, there is a difference in the hydrophobicity or the hydrophilicity between the top substrate and the bottom substrate. A difference in the hydrophobicity or the hydrophilicity of the top substrate (e.g., second substrate) or the bottom substrate (e.g., first substrate) enhances the retention of the fluids in the primary zones when compared to top substrates (e.g., second substrates) and the bottom substrates (e.g., first substrates) that do not have differences in the hydrophobicity or hydrophilicity. In some embodiments, there is a difference in the hydrophobicity or the hydrophilicity between the second substrate and the first substrate. Thedifference in the hydrophobicity or hydrophilicity may be a difference in the contact angle between the top substrate (e.g., second substrate) and the bottom substrate (e.g., first substrate). There may be a range in the difference of the contact angle between the top substrate (e.g., second substrate) and the bottom substrate (e.g., first substrate). For instance, there may be about a 5%, about a 10%, about a 15%, about a 20%, about a 25%, about a 30%, about a 35%, about a 40%, about a 45%, about a 50%, about a 55%, about a 60%, about a 65%, about a 70%, or about a 75% difference between the contact angles of top substrate (e.g., second substrate) and the bottom substrate (e.g., first substrate) or bottom substrate (e.g., second substrate) and the top substrate (e.g., first substrate).

[0253] In some embodiments, the contact angle of the first substrate or bottom substrate is about 5% to about 10% different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 10% to about 15% different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 15% to about 20% different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 20% to about 25% different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 25% to about 30% different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 30% to about 35% different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 35% to about 40% different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 40% to about 45% different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 45% to about 50% different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 50% to about 55% different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 55% to about 60% different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 60% to about 65% different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 65% to about70% different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 70% to about 75% different from the contact angle of the second substrate or top substrate.

[0254] In some embodiments, the contact angle of the first substrate or bottom substrate is about 10% to about 20% different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 20% to about 30% different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 30% to about 40% different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 40% to about 50% different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 50% to about 60% different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 60% to about 70% different from the contact angle of the second substrate or top substrate.

[0255] In some embodiments, the contact angle of the first substrate or bottom substrate is about 10% to about 70% different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 10% to about 60% different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 10% to about 50% different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 10% to about 40% different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 10% to about 30% different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 10% to about 20% different from the contact angle of the second substrate or top substrate.

[0256] In some embodiments, the contact angle of the first substrate or bottom substrate is about 20% to about 70% different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 20% to about 60% different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 20% to about 50% different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 20% to about40% different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 20% to about 30% different from the contact angle of the second substrate or top substrate.

[0257] There may be a range in the difference of the contact angle between the top substrate (e.g., second substrate) and the bottom substrate (e.g., first substrate). For instance, there may be about a 1°, about a 2°, about a 3°, about a 4°, about a 5°, about a 6°, about a 7°, about a 8°, about a 9°, about a 10°, about a 11°, about a 12°, about a 13°, about a 14°, about a 15°, about a 16°, about a 17°, about a 18°, about a 19°, about a 20°, about a 21°, about a 22°, about a 23°, about a 24°, about a 25°, about a 26°, about a 27°, about a 28°, about a 29°, about a 30°, about a 31°, about a 32°, about a 33°, about a 34°, about a 35°, about a 36°, about a 37°, about a 38°, about a 39°, about a 40°, about a 41°, about a 42°, about a 43°, about a 44°, about a 45°, about a 46°, about a 47°, about a 48°, about a 49°, about a 50°, about a 51°, about a 52°, about a 53°, about a 54°, about a 55°, about a 56°, about a 57°, about a 58°, about a 59°, about a 60°, about a 61°, about a 62°, about a 63°, about a 64°, about a 65°, about a 66°, about a 67°, about a 68°, about a 69°, about a 70°, about a 71°, about a 72°, about a 73°, about a 74°, about a 75°, or greater than a 75° difference between the contact angles of top substrate (or second substrate) and the bottom substrate (first substrate) or bottom substrate (or first substrate) and the top substrate (second substrate).

[0258] In some embodiments, the contact angle of the first substrate or bottom substrate is about 5° to about 10° different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 10° to about 15° different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 15° to about 20° different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 20° to about 25° different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 25° to about 30° different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 30° to about 35° different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 35° to about 40° different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 40° to about 45° different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 45° toabout 50° different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 50° to about 55° different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 55° to about 60° different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 60° to about 65° different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 65° to about 70° different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 70° to about 75° different from the contact angle of the second substrate or top substrate.

[0259] In some embodiments, the contact angle of the first substrate or bottom substrate is about 10° to about 20° different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 20° to about 30° different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 30° to about 40° different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 40° to about 50° different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 50° to about 60° different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 60° to about 70° different from the contact angle of the second substrate or top substrate.

[0260] In some embodiments, the contact angle of the first substrate or bottom substrate is about 5° to about 70° different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 5° to about 60° different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 5° to about 50° different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 5° to about 40° different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 5° to about 30° different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottomsubstrate is about 5° to about 20° different from the contact angle of the second substrate or top substrate.

[0261] In some embodiments, the contact angle of the first substrate or bottom substrate is about 10° to about 70° different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 10° to about 60° different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 10° to about 50° different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 10° to about 40° different from the contact angle of the second substrate or top substrate. In some embodiments, the contact angle of the first substrate or bottom substrate is about 10° to about 30° different from the contact angle of the second substrate or top substrate.

[0262] In some embodiments, the contact angle of the first substrate is greater than a specific contact angle. For instance, the contact angle of the first substrate may be greater than about 60°, about 65°, about 70°, about 75°, about 80°, about 85°, about 90°, about 95°, or greater than about 100°. In some embodiments, the contact angle of the first substrate is greater than about 60-100°. In some embodiments, the contact angle of the first substrate is greater than about 60-80°. In some embodiments, the contact angle of the first substrate is greater than about 65-75°. In some embodiments, the contact angle of the second substrate is less than a specific contact angle. For instance, the contact angle of the second substrate may be less than about 100°, about 95°, about 90°, about 85°, about 80°, about 75°, about 70°, about 65°, about 60°, or less than about 55°. In some embodiments, the contact angle of the second substrate is less than about 60-100°. In some embodiments, the contact angle of the second substrate is less than about 60-80°. In some embodiments, the contact angle of the second substrate is less than about 65-75°.

[0263] The primary zones of the present disclosure have an opening in the top substrate such that air or fluid may be added to the primary zone through the opening, either passively or actively. The opening may also produce capillary forces when a fluid is present in the primary zone such that the capillary forces hold the fluid in the primary zone without it spilling into the secondary zone. The opening in the primary zones has a range of different diameters. The diameter of opening may be about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1. 1.2, about 1.3, about 1.4, about 1.5, or greater than about 1.5 mm.

[0264] In some embodiments, the secondary zones have an opening in the top substrate. In some embodiments, the secondary zones do not have an opening in the top substrate. When the secondary zone has an opening in the top substrate, the opening is such that air or fluid may beadded to the secondary zone, either passively or actively. The opening in the secondary zones has a range of different diameters. The diameter of opening may be about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1. 1.2, about 1.3, about 1.4, about 1.5, or greater than about 1.5 mm.

[0265] In some embodiments, the sample processing region contains a sample mixing zone. In some embodiments, the sample mixing zone and the fixed primary zone are the same. In some embodiments, the sample processing region comprises two or more sample mixing zones. In some embodiments, the sample processing region comprises a fixed primary zone that is a sample mixing zone and one or more additional sample mixing zones. In some embodiments, the sample processing region comprises a fixed primary zone that is not a sample mixing zone and a sample mixing zone. FIG. 7, 8, and 10 disclose exemplary sample mixing zones.

[0266] FIG. 10 discloses an illustration of exemplary sample mixing zone. In this embodiment, the sample mixing zone comprises a fixed primary zone 1006 containing a sample 1005. The sample is added through the opening 1004 in the fixed primary zone. The fixed primary zone 1006 contains a hooked portion 1002. The hooked portion is capable of joining with a vibration source 1001. The vibration source provides vertical motion 1003 that compresses and decompresses the sample 1005. The compression and decompression of the sample 1005 results in the mixing of the sample. The fixed primary zone 1006 is a cantilever in that only the portion opposite the opening 1004 is attached to the top substrate. All other regions around the fixed primary zone are detached from the top substrate.

[0267] FIG. 32A discloses an alternative embodiment to FIG. 10 where the sample mixing zone does not contain a hooked portion. In this embodiment, the sample mixing zone comprises a fixed primary zone 3200. The sample is added through the opening 3201 in the fixed primary zone In this embodiment, the fixed primary zone contains two openings. In some embodiments, the fixed primary zone one or more openings. In some embodiments, the fixed primary zone contains three or more openings. The fixed primary zone 3200 does not contain a hooked portion. A vibration source comes in contact with the end 3202 of the fixed primary zone. The vibration source provides vertical motion that compresses and decompresses the sample. The compression and decompression of the sample results in the mixing of the sample. The fixed primary zone 3200 is a cantilever in that only the portion opposite the end 3202 is attached to the top substrate. All other regions around the fixed primary zone are detached from the top substrate.

[0268] FIG. 42 discloses an illustration of exemplary sample mixing zone. The sample mixing zone is the sample mixing according to FIG. 39. In this embodiment, the sample mixing zone comprises a fixed primary zone 4200 containing a laterally extended protruding element 4205 inthe second substrate facing the first substrate wherein the laterally extended protruding element has a surface facing the first substrate that has a greater surface area than the surface of the plurality of protruding elements facing the first substrate. The fixed primary zone 4200 does not comprise a hooked portion. The fixed primary zone 4200 comprises a chamfered end 4201 laterally separated from a connected end 4202. The sample is added through the opening 4206 in the fixed primary zone. The chamfered end 4201 is capable of joining with a vibration source comprising a spool 4203. The vibration source provides vertical motion 4204 on the spool 4203 joined to the chamfered end 4201 that compresses and decompresses the sample. The compression and decompression of the sample results in the mixing of the sample. The fixed primary zone 4200 is a cantilever in that only the portion opposite the opening 4202 is attached to the second substrate. All other regions around the fixed primary zone are detached from the second substrate.

[0269] FIG. 44A discloses an illustration of an exemplary sample mixing zone. The sample mixing zone is illustrated from the perspective directed to the surface of the second substrate facing the first substrate. In this embodiment, the sample mixing zone comprises a fixed primary zone 4400 containing a laterally extended protruding element 4401 in the second substrate facing the first substrate wherein the laterally extended protruding element has a surface facing the first substrate that has a greater surface area than the surface of the plurality of protruding elements facing the first substrate. The sample mixing zone comprises a fixed primary zone 4400. The fixed primary zone 4400 comprises a hooked portion (not shown). The fixed primary zone 4400 comprises an opening 4402 at an end laterally separated from a connected end 4403. The sample is added through the opening 4402 in the fixed primary zone. The fixed primary zone 4400 contains a pinning wall 4404 about the perimeter of the fixed primary zone 4400. The pining wall 4404 is a raised edge where the raised edge protrudes from the laterally extended protruding element about the perimeter of the laterally extended protruding element excluding the connected end 4403. The pinning wall assists in retaining the sample when the sample is mixed. The fixed primary zone 4400 is a cantilever in that only the connected end 4403 is attached to the second substrate. All other regions around the fixed primary zone are detached from the second substrate.

[0270] FIG. 44B discloses an illustration of an exemplary sample mixing zone. The sample mixing zone is illustrated from the perspective directed to the surface of the second substrate facing the first substrate. In this embodiment, the sample mixing zone comprises a fixed primary zone 4410 containing a laterally extended protruding element 4411 in the second substrate facing the first substrate wherein the laterally extended protruding element has a surface facing the first substrate that has a greater surface area than the surface of the plurality of protruding elements facing the first substrate. The sample mixing zone comprises a fixed primary zone 4410. The fixedprimary zone 4410 comprises a hooked portion (not shown). The fixed primary zone 4410 comprises an opening 4412 at an end laterally separated from a connected end 4413. The sample is added through the opening 4412 in the fixed primary zone. The fixed primary zone 4400 contains a plurality of pillars 4414a, 4414b, and 4414c spaced apart about the surface of the laterally extended protruding element facing the first substrate. The plurality of pillars 4414a, 4414b, and 4414c are raised cylinders that protrude from the laterally extended protruding element. The plurality of pillars 4414a, 4414b, and 4414c assists in mixing the sample when the fixed primary zone is compressed and decompress and facilitates the retention of liquids during mixing. The fixed primary zone 4410 is a cantilever in that only the connected end 4413 is attached to the second substrate. All other regions around the fixed primary zone are detached from the second substrate.

[0271] FIG. 44C discloses an illustration of an exemplary sample mixing zone. The sample mixing zone is illustrated from the perspective directed to the surface of the second substrate facing the first substrate. In this embodiment, the sample mixing zone comprises a fixed primary zone 4420 containing a laterally extended protruding element 4421 in the second substrate facing the first substrate wherein the laterally extended protruding element has a surface facing the first substrate that has a greater surface area than the surface of the plurality of protruding elements facing the first substrate. The sample mixing zone comprises a fixed primary zone 4420. The fixed primary zone 4420 comprises a hooked portion (not shown). The fixed primary zone 4420 comprises an opening 4422 at an end laterally separated from a connected end 4413. The sample is added through the opening 4422 in the fixed primary zone. The fixed primary zone 4420 contains a plurality of ridges 4424a, 4424b, and 4424c spaced apart about the surface of the laterally extended protruding element facing the first substrate. The plurality of ridge 4424a, 4424b, and 4424c are raised edges where the raised edges protrude from the laterally extended protruding element. The plurality of ridge 4424a, 4424b, and 4424c assists in mixing the sample when the fixed primary zone is compressed and decompress and facilitates the retention of liquids during mixing. The fixed primary zone 4420 is a cantilever in that only the connected end 4423 is attached to the second substrate. All other regions around the fixed primary zone are detached from the second substrate.

[0272] FIG. 44D discloses an illustration of an exemplary sample mixing zone. The sample mixing zone is illustrated from the perspective directed to the surface of the second substrate facing the first substrate. In this embodiment, the sample mixing zone comprises a fixed primary zone 4430 containing a laterally extended protruding element 4431 in the second substrate facing the first substrate wherein the laterally extended protruding element has a surface facing the firstsubstrate that has a greater surface area than the surface of the plurality of protruding elements facing the first substrate. The sample mixing zone comprises a fixed primary zone 4430. The fixed primary zone 4430 comprises a hooked portion (not shown). The fixed primary zone 4430 comprises an opening 4432 at an end laterally separated from a connected end 4433. The sample is added through the opening 4432 in the fixed primary zone. The fixed primary zone 4430 contains a plurality of pillars 4434a, 4434b, and 4434c spaced apart about the surface of the laterally extended protruding element facing the first substrate. The plurality of pillars 4434a, 4434b, and 4434c are raised cylinders that protrude from the laterally extended protruding element. The fixed primary zone 4430 contains a laterally extended protruding element 4431 containing a recessed beveled edge 4435 about the entire periphery of the laterally extended protruding element 4431. The plurality of pillars 4434a, 4434b, and 4434c assists in mixing the sample when the fixed primary zone is compressed and decompressed and facilitates the retention of liquids during mixing. The fixed primary zone 4430 is a cantilever in that only the connected end 4433 is attached to the second substrate. All other regions around the fixed primary zone are detached from the second substrate.

[0273] II. Arrangement of the sample analysis region

[0274] The optional sample analysis region of the devices of the present disclosure contains a number of different regions including, without limitation, a transition zone, a tertiary zone (e.g., a sample detection zone), a quaternary zone (e.g., hydrophilic liquid well or reservoir), a quinary zone (e.g., a hydrophobic liquid well or reservoir), or any combination thereof.

[0275] In some embodiments, the sample analysis region comprises a transition zone. The transition zone facilities the transfer of the microparticles comprising the sample or analytes contained in the sample from the sample processing region to the sample analysis region. In some embodiments, the transition zone is air-filled. The transition zone may be any length that sufficiently separates the sample processing region from the sample analysis region such that little or no fluid is transferred from the sample processing region to the sample analysis region.

[0276] In some embodiments, the sample analysis region comprises a tertiary zone. In some embodiments, the tertiary zone is a sample detection zone. The types of sample detection zones include, without limitation, wells or microwells, chambers, nanopores, etc. In some embodiments, the sample analysis region comprises hydrophobic liquid and hydrophilic liquid wells.

[0277] In some embodiments, the sample analysis region comprises a sample detection zone comprising wells. The wells may have sub-femtoliter volume, femtoliter volume, sub-nanoliter volume, nanoliter volume, sub-microliter volume, or microliter volume. For example, wells may be femoliter wells, nanoliter wells, or microliter wells. In certain embodiments, the wells in anarray may all have substantially the same volume. The array of wells may have a volume up to 100 microliter, e.g., about 0.1 femtoliter, about 1 femtoliter, about 10 femtoliter, about 25 femtoliter, about 50 femtoliter, about 100 femtoliter, about 0.1 pL, about 1 pL, about 10 pL, about 25 pL, about 50 pL, about 100 pL, about 0.1 nL, about 1 nL, about 10 nL, about 25 nL, about 50 nL, about 100 nL, about 0.1 microliter, about 1 microliter, about 10 microliter, about 25 microliter, about 50 microliter, about or 100 microliter.

[0278] In some examples, the wells are an array of wells that include a plurality of individual wells. The array of wells may include a plurality of wells that may range from 109to 10 in number per 1mm2. In certain cases, an array of about 100,000 to 500,000 wells (e.g., femtoliter wells) covering an area approximately 12 mm2may be fabricated. Each well may measure about 4.2 m wide X 3.2 pm deep (volume approximately 50 femtoliters) and may be capable of holding a single microparticle (about 3 pm diameter). At this density, the femtoliter wells are spaced at a distance of approx. 7.4 pm from each other. In some examples, the well array may be fabricated to have individual wells with a diameter of 10 nm to 10,000 nm.

[0279] The placement of single microparticles bound to the analyte molecules in the wells allows for either a digital readout or analog readout. For example, for a low number of positive wells (<~70% positive) Poisson statistics can be used to quantitate the analyte concentration in a digital format; for high numbers of positive wells (>~70%) the relative intensities of signalbearing wells are compared to the signal intensity generated from a single microparticle bound to the analyte molecule, respectively, and used to generate an analog signal. A digital signal may be used for lower analyte concentrations, whereas an analog signal may be used for higher analyte concentrations. A combination of digital and analog quantitation may be used, which may expand the linear dynamic range. In some embodiments, the signal intensity of a well increases overtime indicating the presence of more than analyte in the well. In some embodiments, the signal intensity in a well is stagnant and of higher intensity of wells containing a single analyte indicating the presence of more than analyte in the well As used herein, a “positive well” refers to a well that has a signal related to presence of a microparticle bound to the analyte molecule, which signal is above a threshold value. As used herein, a “negative well” refers to a well that may not have a signal related to presence of a microparticle bound to the analyte molecule. In certain embodiments, the signal from a negative well may be at a background level, i.e., below a threshold value.

[0280] The wells may be any of a variety of shapes, such as, cylindrical with a flat bottom surface, cylindrical with a rounded bottom surface, cubical, cuboidal, frustoconical, inverted frustoconical, pentagonal, triangular, pyramidal, or conical. In certain cases, the wells may includea sidewall that may be oriented to facilitate the receiving and retaining of a microparticle present liquid droplets that have been moved over the well array. In certain cases, the wells may include a sidewall that may be oriented to facilitate the receiving and retaining of a microparticle that is not present in a liquid droplet that have been moved over the well array. In some examples, the wells may include a first sidewall and a second sidewall, where the first sidewall may be opposite the second side wall. In some examples, the first sidewall is oriented at an obtuse angle with reference to the bottom of the wells and the second sidewall is oriented at an acute angle with reference to the bottom of the wells. In some embodiments, the movement of the droplets is in a direction parallel to the bottom of the wells and from the first sidewall to the second sidewall. In some embodiments, the movement of the microparticle is in a direction parallel to the bottom of the wells and from the first sidewall to the second sidewall.

[0281] In some embodiments, the sample analysis region comprises a sample detection zone comprising a chamber. The chamber may be a range of different sizes and shapes such that the chamber is suitable for the detection of the analyte. The chamber may accommodate a reaction mixture having a volume of from 5 microliters to 1 milliliter. For example, the reaction chamber may be sized to contain a reaction mixture having a volume of from 5 microliters to 500 microliters. According to certain embodiments, the reaction chamber is sized to contain a reaction mixture having a volume of from 5 microliters to 100 microliters. In some embodiments, the fluid capacity of the chamber is about 1 mL or less, about 750 pL or less, about 500 pL or less, about 400 pL or less, about 300 pL or less, about 250 pL or less, about 200 pL or less, about 150 pL or less, about 100 pL or less, about 50 pL or less, or about 25 pL or less.

[0282] The chamber may be a range of different shapes in order to retain the analyte within or without a reaction mixture. In some embodiments, the shape of the chamber is a cube. In some embodiments, the shape of the chamber is a cylinder. In some embodiments, the shape of the chamber is hexagonal. In some embodiments, the shape of the chamber is a sphere. In some embodiments, the shape of the chamber is octagonal. In some embodiments, the shape of the chamber is conical. In some embodiments, the shape of the chamber is cuboidal.

[0283] The bottom, sides, or top of the chamber may be optical transparent such that the analyte may be detected through optical means. In some embodiments, all sides of the chamber are optically transparent such that the analyte may be detected through optical means. In some embodiments, only the top and the bottom of the chamber are optically transparent such that the analyte may be detected through optical means. In some embodiments, the top of the chamber is reflective and the bottom of the chamber is transparent. In some embodiments, the bottom of the chamber is transparent and the bottom of the chamber is reflective.

[0284] In some embodiments, the chamber is a reaction vessel. The term “reaction vessel” as used herein generally refers to a container within which an amplification reaction, an immunoassay, clinical chemistry, or complete blood component analysis is performed. The reaction vessel may be obtained from commercial sources, e.g., as off-the-shelf components such as a microamp tube or microamp tubes joined together in a 96 well format, or may be custom manufactured. Reaction vessels useful in nucleic acid amplification reactions will generally be capable of rapidly transferring heat across the vessel, e.g., through the use of highly conductive materials (e.g., thermally conductive plastics) or physical modifications of the vessel (e.g., thin walls). Common reaction vessels include but are not limited to e.g., tubes, vials, multi-well plates, and the like. Reaction vessels may be constructed of a variety of materials including but not limited to e.g., polymeric materials.

[0285] The top opening of the reaction vessel may be any convenient shape. In certain aspects, the top opening of the reaction chamber of the reaction vessel is circular. The shape of the reaction chamber may vary. According to certain embodiments, the reaction chamber has a conical shape. The bottom of the reaction vessel may be flat. In other aspects, the reaction vessel has a round bottom.

[0286] In certain aspects, the wall of the reaction chamber is straight, whereby “straight” is meant the wall does not include a “step” (or “ridge”). In other aspects, the wall of the reaction chamber includes one or more (e.g., 2 or more, 3 or more, 4 or more, etc.) steps. The one or more steps may be complementary to the shape of the a cap for the reaction vessel. For example, according to certain embodiments, the reaction vessel includes a step that forms an upper region and a lower region of the reaction vessel, where the shape of the upper region is complementary to the shape of the reaction vessel cap.

[0287] The volume of the reaction vessel may vary. In certain aspects, the reaction chamber is sized to contain a reaction mixture having a volume of from 1 microliter to 500 microliters. For example, the reaction chamber may be sized to contain a reaction mixture having a volume of from 1 microliters to 10 microliters, 10 microliters to 50 microliters, 50 microliters to 100 microliters, 100 microliters to 200 microliters, 200 microliters to 300 microliters, 300 microliters to 400 microliters, 400 microliters to 500 microliters, etc. According to certain embodiments, the reaction chamber is sized to contain a reaction mixture having a volume of from 1 microliters to 200 microliters.

[0288] In certain aspects, the fluid capacity of the reaction vessel is 500 mL or less, 450 pL or less, 400 pL or less, 350 pL or less, 300 pL or less, 250 pL or less, 200 pL or less, 150 pL or less,100 pL or less, 50 pL or less, 25 pL or less, 20 pL or less, 15 pL or less, 10 pL or less, or 5 pL or less.

[0289] The external surface of the reaction vessel may include a variety of shapes and features. In certain aspects, the bottom surface of the reaction vessel is round. In other aspects, the bottom surface of the reaction vessel is flat.

[0290] The bottom, sides, or top of the reaction vessel may be optical transparent such that the analyte may be detected through optical means. In some embodiments, all sides of the reaction vessel are optically transparent such that the analyte may be detected through optical means. In some embodiments, only the top and the bottom of the reaction vessel are optically transparent such that the analyte may be detected through optical means. In some embodiments, only the top of the reaction vessel is optically transparent such that the analyte may be detected through optical means. In some embodiments, only the bottom of the reaction vessel is optically transparent such that the analyte may be detected through optical means.

[0291] In certain instances, the reaction vessels may be those described in US Patent No. 10,648,018 which is specifically incorporated by reference herein.

[0292] In some embodiments, the sample analysis region contains nanopores. When the sample detection zone comprises nanopores, the analyte or analyte molecule (e.g., a nucleic acid, a non- nucleic acid containing a nucleic acid tag, or a nucleic acid produced from the analyte) is detected by translocating the nucleic acid through or across a nanopore . In some embodiments, detecting the analyte may be carried out by translocating the nucleic acid through or across at least one or more nanopores . In some embodiments, at least two or more nanopores are presented side by side or in series. In some embodiments, the nanopore is dimensioned for translocation of not more than one nucleic acid at a time. Thus, the dimensions of the nanopore in some embodiments will typically depend on the dimensions of the nucleic acid to be examined. A nucleic acid with a double-stranded region can require a nanopore dimension greater than those sufficient for translocation of a nucleic acid which is entirely single- stranded. In addition, a microparticle- associated nucleic acid such as a microparticle tag can require larger nanopores than oligomer tags. Typically, a nanopore of about 1 nm diameter can permit passage of a single- stranded polymer, while nanopore dimensions of 2 nm diameter or larger will permit passage of a double-stranded nucleic acid. In some embodiments, the nanopore is selective for a single-stranded tag (e.g., from about 1 nm to less than 2 nm diameter) while in other embodiments, the nanopore is of a sufficient diameter to permit passage of double- stranded polynucleotides (e.g., 2 nm or larger). The chosen nanopore size provides an optimal signal-to-noise ratio for the analyte of interest.

[0293] In some embodiments, the nanopore may be between about 0.1 nm and about 1000 nm in diameter, between about 50 nm and about 1000 nm, between about 100 nm and 1000 nm, between about 0.1 nm and about 700 nm, between about 50 nm and about 700 nm, between about 100 nm and 700 nm, between about 0.1 nm and about 500 nm, between about 50 nm and about 500 nm, or between about 100 nm and 500 nm. For example, the nanopore may be about 0.1 nm, about 0.2 nm, about 0.3 nm, about 0.4 nm, about 0.5 nm, about 0.6 nm, about 0.7 nm, about 0.8 nm, about 0.9 nm, about 1.0 nm, about 1.5 nm, about 2.0 nm, about 2.5 nm, about 3.0 nm, about 3.5 nm, about 4.0 nm, about 4.5 nm, about 5.0 nm, about 7.5 nm, about 10 nm, about 15 nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 3500 nm, about 400 nm, about 450 nm, about 500 nm, about 550 nm, about 600 nm, about 650 nm, about 700 nm, about 750 nm, about 800 nm, about 850 nm, about 900 nm, about 950 nm, or about 1000 nm in diameter.

[0294] Various types of nanopores may be used for analyzing the nucleic acid present in a sample. These include, among others, biological nanopores that employ a biological nanopore embedded in a membrane. Another type of nanopore layer is a solid state nanopore in which the nanopore is made whole or in part from a fabricated or sculpted solid-state component, such as silicon. In some embodiments, the nanopore is a solid-state nanopore produced using controlled dielectric breakdown. In some embodiments, the nanopore is a solid-state nanopore produced by a method other than controlled dielectric breakdown.

[0295] In certain embodiments, the length of a nanopore may be up to about 200 nm, e.g., from about O.lnm to about 30 nm, from about 10 to about 80 nm, from about 1 to about 50 nm, from about 0.1 nm to about 0.5 nm, from about 0.3 nm to about 1 nm, from about 1 nm to about 2 nm, from about 0.3 nm to about 10 nm, or from about 10 to about 30 nm. The number of nanopores in a nanopore layer may be about 1, about 2, about 3, about 4, about 5, about 10, about 30, about 100, about 300, about 1000, about 3000, about 10000, about 30000, about 100000, about 300000 or more. The distance between nanopores in a layer between center to center may be about 100 nm to about 300 nm, about 300 nm to about 500 nm, about 500 nm to about 1000 nm, for example, 100 nm, 150 nm, 200 nm, or 300 nm.

[0296] In certain embodiments, multiple nanopore layers, each containing one or more nanopores, can be arranged in series with each other, for detecting and / or counting the tag (e.g., polymer, aptamer, microparticle). In this case, detecting and / or counting the nucleic acid may be carried out by translocating the nucleic acid through or across each nanopore layer. As such,counting the number of nucleic acids translocating through or across a nanopore in a layer / sheet / membrane refers to counting multiple nucleic acids translocating through or across one or more nanopores in one or more layer / sheet / membrane. In certain embodiments, when more than one nanopore layers are present (e.g., one, two, three, four, five, six, or other number of nanopore layers as technically feasible), optionally they are present in series wherein at least one nanopore in one layer is separate from or stacked onto (e.g., above or on top of) another nanopore in another layer, etc.). Where the nanopore layers are in series, at least two electrodes can be used to create an electric field to drive tags through the pores and, optionally, additional electrodes positioned between the nanopore layers can further provide driving current.

[0297] Biological Nanopores

[0298] For detecting and, optionally, counting the nucleic acid, any biological nanopore with channel dimensions that permit translocation of the nucleic can be used. Two broad categories of biological nanopores are suitable for the methods disclosed herein. Non- voltage gated nanopores allow passage of molecules through the nanopore without requiring a change in the membrane potential to activate or open the channel. On the other hand, voltage gated nanopores require a particular range of membrane potential to activate nanopore opening. Most studies with biological nanopores have used a-hemolysin, a mushroom- shaped homo-oligomeric heptameric channel of about 10 nm in length found in Staphylococcus aureus. Each subunit contributes two beta strands to form a 14 strand anti-parallel beta barrel. The nanopore formed by the beta barrel structure has an entrance with a diameter of approximately 2. 6 nm that contains a ring of lysine residues and opens into an internal cavity with a diameter of about 3 .6 nm. The stem of the hemolysin nanopore, which penetrates the lipid bilayer, has an average inside diameter of about 2.0 nm with a 1.5 nm constriction between the vestibule and the stem. The dimensions of the stem are sufficient for passage of single- stranded nucleic acids but not double- stranded nucleic acids. Thus, a-hemolysin nanopores may be used as a nanopore selective for single- stranded polynucleotides and other polymers of similar dimensions.

[0299] In other embodiments, the biological nanopore is of a sufficient dimension for passage of polymers larger than a single-stranded nucleic acid. An exemplary nanopore is mitochondrial porin protein, a voltage-dependent anion channel (VDAC) localized in the mitochondrial outer membrane. Porin protein is available in purified form and, when reconstituted into artificial lipid bilayers, generates functional channels capable of permitting passage of double-stranded nucleic acids (Szabo et al., 1998, F ASEB J. 12:495-502). Structural studies suggest that porin also has a beta-barrel type structure with 13 or 16 strands (Rauch et al., 1994, Biochem Biophys Res Comm 200:908-915). Porin displays a larger conductance compared conductance of nanopores formedby a-hemolysin, maltoporin (LamB), and gramicidin. The larger conductance properties of porin support studies showing that the porin channel is sufficiently dimensioned for passage of doublestranded nucleic acids. Nanopore diameter of the porin molecule is estimated at 4 nm. The diameter of an uncoiled double-stranded nucleic acid is estimated to be about 2 nm.

[0300] Another biological nanopore that may be suitable for scanning double stranded polynucleotides are channels found in B. subtilis (Szabo et al., 1997, J. Biol. Chem. 272:25275- 25282). Plasma membrane vesicles made from B. subtilis and incorporated into artificial membranes allow passage of double- stranded DNA across the membrane. Conductance of the nanopore formed by B. subtilis membrane preparations is similar to those of mitochondrial porin. Although there is incomplete characterization (e.g., purified form) of these nanopore, it is not necessary to have purified forms for the purposes herein. Diluting plasma membrane preparations, either by solubilizing in appropriate detergents or incorporating into artificial lipid membranes of sufficient surface area, can isolate single nanopores in a detection apparatus. Limiting the duration of contact of the membrane preparations (or protein preparations) with the artificial membranes by appropriately timed washing provides another method for incorporating single nanopores into the artificial lipid bilayers. Conductance properties may be used to characterize the nanopores incorporated into the bilayer.

[0301] In certain cases, the nanopores may be hybrid nanopores, where a biological nanopore is introduced in a solid state nanopore, e.g., a nanopore fabricated in a non-biological material. For example, a-haemolysin nanopore may be inserted into a solid state nanopore. In certain cases, the nanopores may be a hybrid nanopore described in Hall et al., Nature Nanotechnology, 28 November 2010, vol. 5, pg. 874-877.

[0302] Solid State Nanopores

[0303] In other embodiments, analysis of the nucleic acid is carried out by translocating the tag through or across a nanopore fabricated from non-biological materials. Nanopores can be made from a variety of solid-state materials using a number of different techniques, including, among others, chemical deposition, electrochemical deposition, electroplating, electron beam sculpting, ion beam sculpting, nanolithography, chemical etching, laser ablation, focused ion beam, atomic layer deposition, and other methods well known in the art (see, e.g., Li et al., 2001, Nature 412:166-169; and WO 2004 / 085609).

[0304] In particular embodiments, the nanopores may be the nanopores described in WO13167952A1 or WO 13167955 AL As described in WO13167952A1 or WO 13167955 Al, nanopores having an accurate and uniform nanopore size may be formed by precisely enlarging a nanopore formed in a membrane. The method may involve enlarging a nanopore by applying ahigh electric potential across the nanopore; measuring current flowing through the nanopore; determining size of the nanopore based in part on the measured current; and removing the electric potential applied to the nanopore when the size of the nanopore corresponds to a desired size. In certain cases, the applied electric potential may have a pulsed waveform oscillating between a high value and a low value, the current flowing through the nanopore may be measured while the electric potential is being applied to the nanopore at a low value.

[0305] Solid state materials include, by way of example and not limitation, any known semiconductor materials, insulating materials, and metals coated with insulating material. Thus, at least part of the nanopore(s) may comprise without limitation silicon, silica, silicene, silicon oxide, graphene, silicon nitride, germanium, gallium arsenide, or metals, metal oxides, and metal colloids coated with insulating material.

[0306] To make a nanopore of nanometer dimensions, various feedback procedures can be employed in the fabrication process. In embodiments where ions pass through a hole, detecting ion flow through the solid state material provides a way of measuring pore size generated during fabrication (see, e.g., U.S. Published Application No. 2005 / 0126905). In other embodiments, where the electrodes define the size of the pore, electron tunneling current between the electrodes gives information on the gap between the electrodes. Increases in tunneling current indicate a decrease in the gap space between the electrodes. Other feedback techniques will be apparent to the skilled artisan.

[0307] In some embodiments, the nanopore is fabricated using ion beam sculpting, as described in Li et al., 2003, Nature Materials 2:611-615. In some embodiments, the nanopore is fabricated using high current, as described in WO13167952A1 or WO13167955A1. In other embodiments, the nanopores may be made by a combination of electron beam lithography and high energy electron beam sculpting (see, e.g., Storm et al., 2003, Nature Materials 2:537-540). A similar approach for generating a suitable nanopore by ion beam sputtering technique is described in Heng et al., 2004, Biophy J 87:2905-2911. The nanopores are formed using lithography with a focused high energy electron beam on metal oxide semiconductor (CMOS) combined with general techniques for producing ultrathin films. In other embodiments, the nanopore is constructed as provided in U.S. Pat. Nos. 6,627,067; 6,464,842; 6,783,643; and U.S. Publication No. 2005 / 0006224 by sculpting of silicon nitride.

[0308] In some embodiments, the nanopores can be constructed as a gold or silver nanopore. These nanopores are formed using a template of porous material, such as polycarbonate filters prepared using a track etch method, and depositing gold or other suitable metal on the surface of the porous material. Track etched polycarbonate membranes are typically formed by exposing asolid membrane material to high energy nuclear particles, which creates tracks in the membrane material. Chemical etching is then employed to convert the etched tracks to nanopores. The formed nanopores have a diameter of about 10 nm and larger. Adjusting the intensity of the nuclear particles controls the density of nanopores formed in the membrane. Nanopores are formed on the etched membrane by depositing a metal, typically gold or silver, into the track etched nanopores via an electroless plating method (Menon et al., 1995, Anal Chem 67:1920-1928). This metal deposition method uses a catalyst deposited on the surface of the nanopore material, which is then immersed into a solution containing Au(I) and a reducing agent. The reduction of Au(I) to metallic Au occurs on surfaces containing the catalyst. Amount of gold deposited is dependent on the incubation time such that increasing the incubation time decreases the inside diameter of the pores in the filter material. Thus, the nanopore size may be controlled by adjusting the amount of metal deposited on the pore. The resulting nanopore dimension is measured using various techniques, for instance, gas transport properties using simple diffusion or by measuring ion flow through the pores using patch clamp type systems. The support material is either left intact, or removed to leave gold nanopores. Electroless plating technique is capable of forming nanopore sizes from less than about 1 nm to about 5 nm in diameter, or larger as required. Gold nanopores having nanopore diameter of about 0.6 nm appears to distinguish between Ru(bpy)2+2 and methyl viologen, demonstrating selectivity of the gold nanopores (Jirage et al., 1997, Science 278:655-658). Modification of a gold nanopore surface is readily accomplished by attaching thiol containing compounds to the gold surface or by derivatizing the gold surface with other functional groups. This features permits attachment of nanopore modifying compounds as well as sensing labels, as discussed herein. Devices, such as the cis / trans apparatuses used for biological nanopores described herein, can be used with the gold nanopores to analyze single coded molecules.

[0309] Where the mode of detecting the analyte (e.g., a nucleic acid, a non-nucleic acid containing a nucleic acid tag, or a nucleic acid produced from the analyte) involves current flow through the analyte (e.g., electron tunneling current), the solid state membrane may be metalized by various techniques. The conductive layer may be deposited on both sides of the membrane to generate electrodes suitable for interrogating the tag along the length of the chain, for example, longitudinal electron tunneling current. In other embodiments, the conductive layer may be deposited on one surface of the membrane to form electrodes suitable for interrogating the analyte across the nanopore, for example, transverse tunneling current. Various methods for depositing conductive materials are known, including, sputter deposition (i.e., physical vapor deposition), non-electrolytic deposition (e.g., colloidal suspensions), and electrolytic deposition. Other metal deposition techniques are filament evaporation, metal layer evaporation, electron-beamevaporation, flash evaporation, and induction evaporation, and will be apparent to the skilled artisan.

[0310] In some embodiments, the detection electrodes are formed by sputter deposition, where an ion beam bombards a block of metal and vaporizes metal atoms, which are then deposited on a wafer material in the form of a thin film. Depending on the lithography method used, the metal films are then etched by means of reactive ion etching or polished using chemical-mechanical polishing. Metal films may be deposited on preformed nanopores or deposited prior to fabrication of the pore.

[0311] In some embodiments, the detection electrodes are fabricated by electrodeposition (see, e.g., Xiang et al., 2005, Angew. Chem. Int. Ed. 44:1265-1268; Li et al., Applied Physics Lett. 77(24):3995-3997; and U.S. Publication Application No. 2003 / 0141189). This fabrication process is suitable for generating a nanopore and corresponding detection electrodes positioned on one face of the solid state film, such as for detecting transverse electron tunneling. Initially, a conventional lithographic process is used to form a pair of facing electrodes on a silicon dioxide layer, which is supported on a silicon wafer. An electrolyte solution covers the electrodes, and metal ions are deposited on one of the electrodes by passing current through the electrode pair. Deposition of metal on the electrodes over time decreases the gap distance between the electrodes, creating not only detection electrodes but a nanometer dimensioned gap for translocation of coded molecules. The gap distance between the electrodes may be controlled by a number of feedback processes.

[0312] Where the detection is based on imaging of charge induced field effects, a semiconductor can be fabricated as described in U.S. Pat. No. 6,413,792 and U.S. published application No. 2003 / 0211502. The methods of fabricating these nanopore devices can use techniques similar to those employed to fabricate other solid state nanopores.

[0313] Detection of the analyte, such as a polynucleotide, is carried out as further described below. Lor analysis of the analyte, the nanopore may be configured in various formats. In some embodiments, the device comprises a membrane, either biological or solid state, containing the nanopore held between two reservoirs, also referred to as cis and trans chambers (see, e.g., U.S. Patent No. 6,627,067). A conduit for electron migration between the two chambers allows electrical contact of the two chambers, and a voltage bias between the two chambers drives translocation of the tag through the nanopores. A variation of this configuration is used in analysis of current flow through nanopores, as described in U.S. Patent Nos.: 6,015,714 and 6,428,959; and Kasianowiscz et al., 1996, Proc Natl Acad Sci USA 93:13770-13773, the disclosures of which are incorporated herein by reference.

[0314] Variations of above the device are disclosed in U.S. application publication no. 2003 / 0141189. A pair of nanoelectrodes, fabricated by electrodeposition, is positioned on a substrate surface. The electrodes face each other and have a gap distance sufficient for passage of a single nucleic acid. An insulating material protects the nanoelectrodes, exposing only the tips of the nanoelectrodes for the detection of the nucleic acid. The insulating material and nanoelectrodes separate a chamber serving as a sample reservoir and a chamber to which the polymer is delivered by translocation. Cathode and anode electrodes provide an electrophoresis electric field for driving the tag from the sample chamber to the delivery chamber.

[0315] The current bias used to drive the analyte through the nanopore can be generated by applying an electric field directed through the nanopore. In some embodiments, the electric field is a constant voltage or constant current bias. In other embodiments, the movement of the tag is controlled through a pulsed operation of the electrophoresis electric field parameters (see, e.g., U.S. Patent Application No. 2003 / 141189 and U.S. Pat. No. 6,627,067). Pulses of current may provide a method of precisely translocating one or only a few bases of an oligonucleotide tag for a defined time period through the pore and to briefly hold the tag within the pore, and thereby provide greater resolution of the electrical properties of the tag.

[0316] The nanopore devices may further comprise an electric or electromagnetic field for restricting the orientation of the analyte as it passes through the nanopore. This holding field can be used to decrease the movement of the analyte within the nanopore. In some embodiments, an electric field that is orthogonal to the direction of translocation is provided to restrict the movement of the tag molecule within the nanopore. This is illustrated in U.S. Application Publication No. 2003 / 0141189 through the use of two parallel conductive plates above and beneath the sample plate. These electrodes generate an electric field orthogonal to the direction of translocation of an analyte molecule, and thus holding the tag molecule to one of the sample plates. A negatively charged backbone of a DNA, or nucleic acid modified to have negative charges on one strand, will be oriented onto the anodic plate, thereby limiting the motion of the tag molecule.

[0317] In still other embodiments, controlling the position of the analyte is carried out by the method described in U.S. Application Publication No. 2004 / 0149580, which employs an electromagnetic field created in the nanopore via a series of electrodes positions near or on the nanopore. In these embodiments, one set of electrodes applies a direct current voltage and radio frequency potential while a second set of electrodes applies an opposite direct current voltage and a radio frequency potential that is phase shifted by 180 degrees with respect to the radio frequency potential generated by the first set of electrodes. This radio frequency quadrupole holds a charged particle (e.g., nucleic acid) in the center of the field (i.e., center of the pore).

[0318] In exemplary embodiments, the nanopore membrane may be a multilayer stack of conducting layers and dielectric layers, where an embedded conducting layer or conducting layer gates provides well-controlled and measurable electric field in and around the nanopore through which the tag translocates. In an aspect, the conducting layer may be graphene. Examples of stacked nanopore membranes are found in US20080187915 and US20140174927, for example.

[0319] It is understood that the nanopore may be located in a membrane, layer or other substrate, which terms have been used interchangeably to describe a two-dimensional substrate comprising a nanopore.

[0320] In certain embodiments, the nanopore may be formed as part of the assay process for detecting and / or determining concentration of an analyte using the nanopore. Specifically, a device for detecting and / or determining concentration of an analyte using a nanopore may initially be provided without a nanopore formed in a membrane or layer. The device may include a membrane separating two chambers on the opposite sides of the membrane (a cis and a trans chamber). The cis and the trans chambers may include a salt solution and may be connected to a source of electricity. When a nanopore is to be created in the membrane, a voltage is applied to the salt solution in the cis and trans chamber and conductance through the membrane measured. Prior to the creation of a nanopore, there is no or minimal current measured across the membrane. Following creation of a nanopore, the current measured across the membrane increases. The voltage may be applied for an amount of time sufficient to create a nanopore of the desired diameter. Following the creation of a nanopore, an analyte or tag may be translocated through the nanopore and the translocation event detected. In certain embodiments, the same salt solution may be used for nanopore creation as well as for detection of translocation of an analyte or tag through the nanopore. Any suitable salt solution may be utilized for nanopore creation and / or translocation of an analyte or tag through the nanopore. Any salt solution that does not damage the counting label can be used. Exemplary salt solutions include lithium chloride, potassium chloride, sodium chloride, calcium chloride, magnesium chloride and the like. The concentration of the salt solution may be selected based on the desired conductivity of the salt solution. In certain embodiments, the salt solution may have a concentration ranging from about ImM to about 10 M, e.g., about 10 mM-10 M, about 30 mM-10 M, about 100 mM-10 M, about 1 M-10 M, about 10 mM-5 M, about 10 mM-3 M, about 10 mM-1 M, about 30 mM-5 M, about 30 mM-3 M, about 30 mM-1 M, about 100 mM-5 M, about 100 mM-3 M, about 100 mM-1 M, about 500 mM-5 M, about 500 mM-3 M, about or 500 mM-1 M, about such as, about 10 mM, about 30 mM, about 100 mM, about 500 mM, about 1 M, about 3 M, about 5 M, or about 10 M.

[0321] In some embodiments, the nanopore may become blocked, and the blocked nanopore is cleared by modulating the pattern of voltage applied by the electrodes across the nanopore layer or membrane. In some cases, a blocked nanopore is cleared by reversing polarity of the voltage across the nanopore layer or membrane. In some cases, a blocked nanopore is cleared by increasing the magnitude of the voltage applied across the nanopore layer or membrane. The increase in voltage may be transitory increase, lasting 10 seconds (s) or less, e.g., 8 s or less, 6 s or less, 5 s or less, 4 s or less, 3 s or less, 2 s or less, 1 s or less, 0.5 s or less, 0.4 s or less, 0.3 s or less, 0.2 s or less, including 0.1 s or less.

[0322] In some embodiments, the sample analysis region comprises a quintenary zone. In some embodiments, the quintenary zone is a hydrophilic liquid well. In some embodiments, the quintenary zone is a hydrophilic liquid reservoir. When the quintenary zone is a hydrophilic liquid well, the hydrophilic liquid is directly added to the well after the microparticles or microparticles and assisting particles are added to the sample detection zone. In some embodiments, when the sample detection zone comprises wells or microwells, the hydrophilic liquid is added after the microparticles are seeded into the wells or micro wells. When the quintenary zone is a hydrophilic liquid reservoir, the hydrophilic liquid may be pulled from a reservoir though a pump, gravity, suction, etc. In some embodiments, the hydrophilic liquid is a substrate solution. The substrate solution may be any substrate solution that reacts with a specific binding member (e.g., the second specific binding member or the detectably labeled second specific binding member) to produce a detectable signal.

[0323] In some embodiments, the quaternary zone comprises one or more substrate retention features. The substrate retention features of the present disclosure may be a range of different features and may be any combination of substrate features described herein. In some embodiments, the substrate retention feature is a first, a second, a third and a fourth substrate retention feature wherein each substrate retention feature is quarter circle shaped and protrudes into the quaternary zone such as depicted in FIG. 33G. In some embodiments, the substrate retention feature is centrally located on the right end of the sample analysis region, is quarter circle shaped, and protrudes from the top substrate or bottom substrate as depicted in FIG. 33H. In some embodiments, the substrate retention feature is located on the right end of the sample analysis region, is horseshoe shaped with tapered edges, and protrudes from the top substrate or bottom substrate as depicted in FIG. 33H. In some embodiments, the substrate retention feature is a first substrate retention feature located on the right end of the sample analysis region, is horseshoe shaped without tapered edges, and is concentric with a second substrate retention feature that ishorseshoe shaped without tapered edges wherein the first and the second substrate retention protrude from the top or bottom substrates such as depicted in FIG. 331.

[0324] In some embodiments, the substrate retention feature is a first, a second, a third and a fourth substrate retention feature wherein the first through fourth substrate retention features are grooves on the top or bottom substrates, are located on the right end of the sample analysis region, and are concentric with each other such as depicted in FIG. 33J. In some embodiments, the substrate retention feature is located on the right end of the sample analysis region, is horseshoe shaped without tapered ends, and protrudes from the top or bottom substrate as depicted in FIG. 33K. In some embodiments, the substrate retention feature is a first and a second substrate retention feature wherein the first and second substrate retention features are located prior to the right end of the sample analysis region, are rectangular shaped, the first substrate retention is opposite the second substrate retention feature, and the first and second substrate retention feature protrude from the top or bottom substrate such as depicted in FIG. 33L. In some embodiments, the substrate retention feature is a first and a second substrate retention feature wherein the first and second substrate retention features are located prior to the right end of the sample analysis region, are circular shaped, the first substrate retention is opposite the second substrate retention feature, and the first and second substrate retention feature protrude from the top or bottom substrate such as depicted in FIG. 33M.

[0325] In some embodiments, the sample analysis region comprises a quinary zone. In some embodiments, the quinary zone is a hydrophobic liquid well. In some embodiments, the quinary zone is a hydrophobic liquid reservoir. When the quinary zone is a hydrophobic liquid well, the hydrophobic liquid is directly added to the well after the microparticles or microparticles and assisting particles are added to the sample detection zone and following the addition of the hydrophilic liquid. In some embodiments, when the sample detection zone comprises wells or micro wells, the hydrophobic liquid is added after the microparticles or microparticles and assisting particles are seeded into the wells or microwells following hydrophilic liquid addition to seal the wells or microwells for detection. When the quinary zone is a hydrophobic liquid reservoir, the hydrophobic liquid may be pulled from a reservoir though a pump, gravity, suction, etc. In some embodiments, the hydrophobic liquid is oil. The oil may be any oil deemed useful. In certain cases, the hydrophobic liquid is selected based on its low affinity for water to decrease mixing of the hydrophobic liquid with the substrate solution. In certain cases, the hydrophobic liquid is an oil. In certain cases, the hydrophobic liquid is 3M FC-40 oil, a hydrocarbon oil, a vegetable oil, or silicone liquids (e.g., a silicone oil). In certain cases, the oil is a fluorocarbon oil. In certain cases, the oil is Novec 7500, FC-40, or Galden HT200.

[0326] In some embodiments, the quinary zone comprises a barrier feature. In some embodiments, the barrier feature is on the left end of the sample analysis region, is half circle shaped, and protrudes from the top or the bottom substrate.3. REAGENT DELIVERY CARTRIDGE

[0327] The present disclosure also provides an optional regent delivery device. The reagent delivery device contains a sample collection portion, a reagent cartridge, a frame, a seal and an integrated sample processing device.

[0328] In some embodiments, one or more reagents are added to the device using the reagent delivery device. In some embodiments, one or more reagents are added to the device by individually adding the one or more reagents to the device. In some embodiments, one or more reagents are added to the device using bulk reagent delivery. "Bulk reagent delivery” as used herein refers to the addition of reagents to the device using a storage container containing a reagent at a volume that exceeds the volume of the reagent necessary to perform any of the assays disclosed herein once. For instance, the storage container contains a sufficient volume of the reagent such that two or more of any of the assays disclosed may be performed without refiling the storage container. The storage container may sufficient volume of the reagent such that 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 100 or more, 200 or more, 300 or more, 400 or more, 500 or more, 1000 or more, 2000 or more, 3000 or more, 4000 or more, 5000 or more, 6000 or more, 7000 or more, 8000 or more, 9000 or more, or 10000 or more to of any of the assays disclosed may be performed without refiling the storage container.

[0329] FIG. 20 depicts an illustration of a partially assembled reagent delivery device. The reagent delivery contains a sample collection portion 2002. The sample collection portion contains an opening 2001 that is connected to a capillary portion that collects the sample. The sample collection portion is connected to the frame 2003. The frame holds the reagent cartridge 2005. In the reagent cartridge is an independent plunger 2004. The reagent delivery device connects to exemplary sample processing devices 2006 or 2007. The reagent delivery device may connect to any of the devices disclosed herein.

[0330] FIG. 21 depicts an illustration of a deconstructed view of the reagent delivery device. The reagent delivery device contains a sample collection portion 2102. The sample collection portion is connected a hollow capillary portion 2103. The capillary portion may be contacted to a sample to collect sample in the hollow capillary portion 2103. The reagent cartridge contains a plunger portion 2104, an independent plunger 2105 and a reagent containing portion 2106. The reagent cartridge connects to the interior of the frame 2107. The sample collection portion 2102connects to exterior of the frame 2107. A seal 2108 is connected to a sample processing device 2109. The seal 2108 and sample processing portion are connected to the bottom interior of the frame 2107.

[0331] FIG. 22 discloses an exemplary embodiment of an upside down view of the reagent cartridge. The reagent cartridge contains a seal 2201 that is connected to the reagent containing portion 2203. The seal 2201 prevents the reagents from leaving the reagent containing portion 2203. The plunger portion contains one or more plungers that are physically connected such that depression of the plunger portion depresses the one or more plungers. In some embodiments, the plunger portion contains both independent and connected plungers. The reagent cartridge contains an independent plunger that fits into openings 2205 and 2202. In some embodiments, the independent plunger dispenses a hydrophobic liquid from the reagent containing portion 2203.

[0332] FIG. 23 depicts an illustration of a cross-section of the reagent delivery device in a nonactivated state. The reagent delivery device has a sample collection portion 2202 physically connected to the frame. The sample collection portion contains an opening 2201 that is connected to the capillary portion. When the capillary portion contains a sample and pressure is applied to the device, the sample is delivered to a variable or fixed primary zone of the sample processing device. The reagent containing portion contains reagents 2208 that are dispensed into primary, secondary, quaternary and / or quinary zones following depression of the plunger portion. The seal 2309 of the reagent cartridge becomes pierced by film piercing features 2205 and allow reagents to be delivered to the sample processing device.

[0333] FIG. 24 depicts an illustration of a cross-section of the reagent delivery device in an activated state. In the activated state, the plunger portion 2403 is depressed and the film piercing features 2407 thereby releasing reagents into the sample processing device 2406.

[0334] FIG. 25 depicts an alternative design of the reagent cartridge. In place of the film piercing features disclosed in FIG. 23 and 24, there are film piercing features connected directed to the plunger portion and are present in the fluid chamber 2507. Upon depression, the film piercing features 2504 pierce the seal 2505 and allow reagents to be dispensed in the sample processing device.4. METHODS AND DEVICES FOR MIXING ON OR OFF THE DEVICE

[0335] The present disclosure provides methods and devices for mixing the fluids in the device. The fluids for use in the device may be mixed in the device itself or prior to addition to the device.

[0336] In some embodiments, the fluids in the device are mixed by vibrating the entire device. In these embodiments, the vibration source contacts the device and vibrates the entire device. In some embodiments, the vibration is in a vertical motion. In some embodiments, the vibration is ina horizontal motion. The vibration source may be a range of different sources including, without limitation, a voice coil, a vibration motor, a piezo actuator, a motor, a gas pump, etc.

[0337] In some embodiments, the fluids on the device are mixed by vibrating the entire device. In some embodiments, the entire device is vibrated using a voice coil. Vibrating the entire device with a voice coil provides particular advantages including without limitation, the ability to mixing any location on the device, the ability to create standing waves in stationary fluids which produces less fluidic disruption compared to other mixing methods, allows for mixing in low-volumes on the device, the ability to independently control of frequency and amplitude for precise tuning of mixing. In embodiments where fluids in the device are mixed by vibrating the entire device, the device lacks specified sample mixing regions.

[0338] FIG. 48 discloses an illustration of an exemplary voice coil mixing method and device. In this embodiment, the device 4800 is contacted with a voice coil 4801. The fluids 4802 on the device 4800 are vibrated vertical 4803. The vertical vibration 4803 is at a frequency that is substantially resonant with fluids on the device thereby producing standing waves (e.g., faraday waves). Standing waves produce efficient homogenization of the fluids with minimal splashing. The voice coil allows for independent tuning of the frequency of vibration to better resonate with the fluids 4802 to produce standing waves while also allowing controlling of the amplitude for focused mixing. Continuous variation of waveform frequencies and amplitude results in shifting patterns (e.g., such as the patterns disclosed in FIG. 49) that can be optimized for specific mixing applications.

[0339] FIG. 50 discloses an illustration of the mixing process when using standing waves. In this embodiment, in the absence of vibration, microparticles 5000 aggregate in one or more locations. In the presence of vibrations at a resonant frequency, standing waves 5001 are produced. The standing waves have nodes 5002a, 5002b, and 5002c and anti-nodes 5003a, 5003b, and 5003c. Microparticles 5004a, 5004b, and 5004c align along nodes 5002a, 5002b, and 5002c of the standing wave 5001 thereby mixing the fluids and the microparticles in the fluids.

[0340] FIG. 51 discloses microparticle mixing using standing waves in an embodiment of the device. In this embodiment, when the device is not vibrated microparticles are stationary in the fluid 5100. When the device is vibrated at a frequency that is not a resonance frequency 5101, microparticles mix, however, there is still microparticle aggregation without complete mixing. When the device is vibrated at a frequency that is a resonance frequency 5102, microparticles completely mix with maximal dispersion in the fluid.

[0341] In some embodiments, the fluids are mixed by vibrating only the top substrate. The vibration is achieved by contacting the top substrate with the vibration device. In someembodiments the entire top substrate is vibrated. In some embodiments, the entire top substrate is vibrated vertical. In some embodiments, a portion of the top substrate is vibrated. In some embodiments, the portion of the top substrate is a primary zone. In some embodiments, the primary zone is in a sample mixing zone.

[0342] FIG. 10 discloses an illustration of an exemplary sample mixing zone and method of mixing. In this embodiment, the sample mixing zone comprises a fixed primary zone 1006 containing a sample 1005. The sample is added through the opening 1004 in the fixed primary zone. In some embodiments, the fixed primary zone may contain two or more openings. In some embodiments, the fixed primary zone may contain three or more openings. The fixed primary zone 1006 contains a hooked portion 1002. The hooked portion is capable of joining with a vibration source 1001. The vibration source provides vertical motion 1003 that compresses and decompresses the sample 1005. The compression and decompression of the sample 1005 results in the mixing of the sample. The fixed primary zone 1006 is a cantilever in that only the portion opposite the opening 1004 is attached to the top substrate. All other regions around the fixed primary zone are detached from the top substrate.

[0343] FIG. 32A discloses an alternative embodiment to FIG. 10 where the sample mixing zone does not contain a hooked portion. In this embodiment, the sample mixing zone comprises a fixed primary zone 3200. The sample is added through the opening 3201 in the fixed primary zone In this embodiment, the fixed primary zone contains two openings. In some embodiments, the fixed primary zone one or more openings. In some embodiments, the fixed primary zone contains three or more openings. The fixed primary zone 3200 does not contain a hooked portion. A vibration source comes in contact with the end 3202 of the fixed primary zone. The vibration source provides vertical motion that compresses and decompresses the sample. The compression and decompression of the sample results in the mixing of the sample. The fixed primary zone 3200 is a cantilever in that only the portion opposite the end 3202 is attached to the top substrate. All other regions around the fixed primary zone are detached from the top substrate.

[0344] In some embodiments, the fluids in the device are mixed by vibrating only the bottom substrate. The vibration is achieved by contacting the bottom substrate with the vibration device. In some embodiments, the bottom substrate is vibrated. In some embodiments, the entire bottom substrate is vibrated vertically. In some embodiments, a portion of the bottom substrate is vibrated. In some embodiments, the portion of the bottom substrate is a primary zone. In some embodiments, the primary zone is in a sample mixing zone.

[0345] FIG. 29 discloses an illustration of an exemplary pressure sample mixing device and method. In this embodiment, the sample mixing device 2901 contacts a fixed or variable primaryzone at contact point 2906a and 2906b. In some embodiments, the sample mixing device 2901 contacts a fixed or variable primary zone at contact point 2906a and 2906b with a suction cup. In some embodiments, the sample mixing device 2901 contacts a fixed or variable primary zone at contact point 2906a and 2906b with a gasket. In some embodiments, the sample mixing device 2901 contacts a fixed or variable primary zone at contact point 2906a and 2906b in the absence of a suction cup or gasket. The sample mixing device 2901 contains a chamber 2902 (i.e. a bottom portion) which comprises air and a top portion 2908 separated by a diaphragm. The chamber has an open bottom. Above the chamber 2902 that contains air is a diaphragm 2903. The diaphragm may be moved or oscillated by a deflection amount 2905a and 2905b that compresses the air in the chamber 2902. The compression in the air results in movement in the fluid 2907 in the primary zone thereby mixing the fluid. The sample mixing device 2901 comprises one or more openings 2904 that allow pressure relief in the sample mixing device 2901 when the diaphragm 2903 is moving or oscillating.

[0346] The diaphragm of the sample mixing device may be made of a range of different materials including, without limitation, piezoelectric material, metal, a compliant material that deforms under heat or pressure, etc. The diaphragm of the sample mixing device may be moved or oscillated in a number of different ways. In some embodiments, the diaphragm is made of a piezoelectric material. In some embodiments, the piezoelectric material is moved or...

Claims

CLAIMS1. A device, comprising: a first substrate, and a second substrate positioned on the first substrate, wherein the second substrate comprises a sidewall about at least a portion of a periphery of the second substrate, wherein the first substrate, sidewall, and second substrate define a central chamber therebetween, wherein the second substrate comprises a surface facing the central chamber comprising a plurality of recessed elements, and a plurality of protruding elements; wherein primary zones are defined between a surface of the plurality of protruding elements facing the first substrate and a surface of the first substrate facing the second substrate; wherein secondary zones are defined between a surface of the plurality of recessed elements facing the first substrate and the surface of the first substrate facing the second substrate, wherein the second substrate has an opening in one or more of the primary zones, and wherein the first substrate and the second substrate have a difference in hydrophobicity or hydrophilicity.

2. The device of claim 1, wherein the at least a portion of the periphery comprises at least two, at least three, or at least four peripheral sides of the second substrate3. The device of claims 1 or 2, wherein the difference in hydrophobicity or hydrophilicity is a difference in the contact angle of the first substrate and the contact angle in the second substrate.

4. The device of claim 3, wherein the difference in the contact angle of the first substate and the contact angle of the second substrate is about a 10% to about a 60% difference in the contact angle.

5. The device of claim 4, wherein the difference in the contact angle of the first substate and the contact angle of the second substrate is about a 20% to about a 40% difference in the contact angle.

6. The device of claim 3, wherein the difference in the contact angle of the first substate and the contact angle of the second substrate is about a 5° to about a 60° difference in the contact angle.

7. The device of claim 6, wherein the difference in the contact angle of the first substate and the contact angle of the second substrate is about a 5° to about a 40° difference in the contact angle.

8. The device of any of claims 3, or 6-7, wherein the difference in the contact angle of the first substate and the contact angle of the second substrate is about a 5° to about a 30° difference in the contact angle.

9. The device of any of claims 1-8, wherein the first substrate is hydrophobic and the second substrate is hydrophilic.

10. The device of any of claims 1-8, wherein the first substrate is hydrophilic and the second substrate is hydrophobic.

11. The device of any of claims 1-8, wherein the first substrate is hydrophobic and the second substrate is hydrophobic.

12. The device of any of claims 1-8, wherein the first substrate is hydrophilic and the second substrate is hydrophilic.

13. The device of any of claims 1-12, wherein primary zones are discrete.

14. The device of any of claims 1-13, wherein secondary zones are connected.

15. The device of any of claim 1-14, wherein the second substrate has an opening in one or more of the secondary zones.

16. The device of any of claims 1-15, wherein the second substrate has an opening in each of the plurality of primary zones.

17. The device of any of claims 1-16, wherein the surface of the plurality of the protruding elements facing the first substrate and the surface of the first substrate facing the second substrate in the primary zones are separated by a first distance and the surface of the recessed element facing the first substrate and the surface of the first substrate facing the second substrate in the secondary zones are separated by a second distance.

18. The device of claim 17, wherein the first distance is less than the second distance.

19. The device of any of claims 1-18, wherein the protruding elements are a shape selected from the group consisting of a rectangle, a circle, a triangle, a pentagon, a hexagon, a heptagon, an octagon, a decagon, a dodecagon, an amoeboid, and a non-regular shape.

20. The device of any of claims 1-19, wherein the device comprises three or more primary zones separated by two or more secondary zones.

21. The device of any of claims 1-20, wherein the device comprises six or more primary zones separated by seven or more secondary zones.

22. The device of any of claims 1-21, wherein the device comprises nine or more primary zones separated by twelve or more secondary zones.

23. The device of any of claims 1-22, wherein the device comprises twelve or more primary zones separated by seventeen or more secondary zones.

24. The device of any of claims 1-23, wherein the device comprises fifteen or more primary zones separated by twenty-two or more secondary zones.

25. The device of any of claims 1-24, wherein the device comprises eighteen or more primary zones separated by twenty-seven or more secondary zones.

26. The device of any of claims 1-25, wherein the device comprises twenty-one or more primary zones separated by thirty-two or more secondary zones.

27. The device of any of claims 1-26, wherein the device comprises twenty-four or more primary zones separated by thirty- seven or more secondary zones.

28. The device of any of claims 1-27, further comprising a sample analysis region configured to analyze a sample.

29. The device of claim 28, wherein said sample analysis region is positioned adjacent to the central chamber on the first substrate.

30. The device of claims 28 or 29, wherein the sample analysis region is physically separated from the central chamber.

31. The device of claims 28 or 29, wherein the sample analysis region is not physically separated from the central chamber.

32. The device of claim 30, further comprising a transition zone wherein the transition zone fluidically connects the central chamber to the sample analysis region.

33. The device of any of claims 28-32, wherein the sample analysis region has a first end laterally separated from a second end and is defined by the surface of the second substrate facing the first substrate and the surface of the first substrate facing the second substrate.

34. The device of claim 33, wherein the surface of the second substrate facing the first substrate in the sample analysis region comprises an enlarged protruding element wherein: a surface of the enlarged protruding element facing the first substrate has a greater surface area than a surface of the plurality of protruding elements facing the first substrate, and the enlarged protruding element extends from the first end to the second end.

35. The device of claim 33 or 34, wherein the sample analysis region comprises a tertiary zone located at a midpoint between the first end and the second end of the of the sample analysis region wherein the tertiary zone is defined by: a) the surface of the second substrate facing the first substrate and the surface of the first substrate facing the second substrate, orb) the surface of the enlarged protruding element facing the first substrate and the surface of the first substrate facing the second substrate.

36. The device of claim 35, wherein the tertiary zone comprises at least one of selected from the group consisting of: one or more wells in the surface of the first substrate facing the second substrate, one or more nanopores, and one or more chambers.

37. The device of claim 36, wherein the one or more wells is an array of wells.

38. The device of any of claims 28-37, wherein the sample analysis region comprises two or more tertiary zones.

39. The device of claim 38, wherein the two or more tertiary zones are of the same type.

40. The device of claim 38, wherein the two or more tertiary zones are of different types.

41. The device of any of claims 1-40, wherein the plurality of primary zones are unbounded around the perimeter of each primary zone.

42. The device of any of claims 28-41, wherein the sample analysis region comprises a quaternary zone wherein the quaternary zone comprises a cylindrical opening spanning the second substrate and the quaternary zone is located at the second end of the sample analysis region.

43. The device of any of claims 28-42, wherein the sample analysis region comprises a quinary zone wherein the quinary zone is an opening spanning the second substrate and the quinary zone is located at the first end of the sample analysis region.

44. The device of claim 42, wherein the quaternary zone is a hydrophilic liquid well or reservoir.

45. The device of claim 43, wherein the quinary zone is a hydrophobic liquid well or reservoir.

46. The device of any of claims 42-45, wherein the quaternary zone comprises one or more substrate retention features.

47. The device of claim 46, wherein the one or more substrate retention features are selected from the group consisting of: quarter circle protrusions protruding from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate, horse shaped protrusions with tapered edges protruding from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate, horse shaped protrusions without tapered edges protruding from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate, horse shaped grooves without tapered edges protruding from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate, rectangular protrusions protruding from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate, and circular protrusions protruding from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate.

48. The device of any of claims 43-47, wherein the sample analysis region comprises a substrate stopper feature comprising a ridge on the surface of the enlarged protruding element facing the first substrate that creates a difference in the height between the quinary zone and the quaternary zone.

49. The device of any of claims 34-48, wherein the enlarged protruding element comprises a pinning wall comprising a raised edge about the perimeter of the enlarged protruding element excluding the end comprising the quinary zone50. The device of any of claims 43-49, wherein the quinary zone comprise a barrier feature wherein the barrier feature is a half-circle that protrudes from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate.

51. The device of any of claims 1-50, wherein one or more of the primary zones comprises a fluid.

52. The device of claim 51, wherein the primary zone retains the fluid through surface tension and capillary forces.

53. The device of any of claims 1-52, wherein one or more secondary zones contains a fluid.

54. The device of any of claims 1-53, wherein the device comprises a pre-treatment region configured to pre-treat a sample prior to processing of the sample.

55. The device of claim 54, wherein the pre-treatment region comprises a plurality of elongated openings about a perimeter of the pre-treatment region wherein the elongated openings are separated from each other by a plurality of connected regions wherein the pretreatment region is defined by the surface of the second substrate facing the first substrate and the surface of the first substrate facing the second substrate enclosed by the plurality of elongated openings.

56. The device of claim 55, wherein the pre-treatment region comprises a plurality of openings in the second substrate.

57. The device of claims 55 or 56, wherein the pre-treatment region comprises a plurality of openings in the second substrate.

58. The device of any of claims 55-57, wherein the pre-treatment region comprises a raised edge about the perimeter of the pre-treatment region enclosed by the plurality of elongated openings.

59. The device of any of claims 54-58, wherein the pre-treatment region is at an end of the sample processing region that is furthest from the sample analysis region.

60. The device of any of claims 1-59, wherein the device comprises one or more fixed primary zones wherein the one or more fixed primary zones are formed from a laterally extended protruding element in the second substrate facing the first substrate wherein the laterally extended protruding element has a surface facing the first substrate that has a greater surface area than the surface of the plurality of protruding elements facing the first substrate.

61. The device of any of claims 1-60, wherein the plurality of protruding elements have a surface finish.

62. The device of any of claims 1-61, wherein the laterally extended protruding element has a surface finish.

63. The device of any of claims 1-62, wherein the enlarged protruding element has a surface finish.

64. The device of claim 63, wherein the surface finish of the enlarged protruding element is higher than the surface finish of the plurality of protruding elements and the laterally extended protruding element.

65. The device of any of claims 61-64, wherein the surface finish of the plurality of protruding elements have an average surface roughness of about 0.15 m to about 0.70 pm.

66. The device of any of claims 62-65, wherein the surface finish of the laterally extended protruding element has an average surface roughness of about 0.15 pm to about 0.70 pm.

67. The device of any of claims 63-66, wherein the surface finish of the enlarged protruding element has an average surface roughness of about 0.012 pm to about 0.15 pm.

68. The device of any of claims 60-67, wherein the one or more fixed primary zones comprise one or more openings.

69. The device of any of claims 60-68, wherein the one or more fixed primary zones comprise two or more openings.

70. The device of any of claims 60-69, wherein the fixed primary zone comprises one or more secondary features wherein the secondary features are one or more protruding elements protruding from the surface of the laterally extended protruding element facing the first substrate.

71. The device of any of claims 1-70, wherein the device comprises one or more variable primary zones.

72. The device of any of claims 1-71, wherein the primary zones are in a pattern selected from the group consisting of a grid, a line, a non-grid, and a honeycomb pattern.

73. The device of any of claims 1-72, wherein edges of the plurality of protruding elements are flat edges.

74. The device of any of claims 1-73, wherein edges of the plurality of protruding elements are round edges.

75. The device of any of claims 1-74, wherein edges of the plurality of protruding elements have a protrusion.

76. The device of any of claims 1-75, wherein one or more of the plurality of protruding elements have serrations or grooves on one or more surfaces of the protruding elements.

77. The device of any claims 1-76, wherein one or more of the plurality of recessed elements have serrations or grooves on one or more surfaces of the recessed elements.

78. The device of claim 76 or 77, wherein the serration or grooves are in a pattern selected from the group consisting of waves, straight lines that are perpendicular or parallel to the edge of the pad, diagonal to the edge of the pad, cross or hatch pattern, and any combination thereof.

79. The device of any of claims 1-78, wherein one or more of the plurality of protruding elements have dimples or divots on one or more of the surfaces of the protruding elements.

80. The device of any of claims 1-79, wherein one or more of the plurality of recessed elements have dimples or divots on one or more surfaces of the recessed elements.

81. The device of claim 79 or 80, wherein the dimple or divots are concave, convex, or a combination thereof.

82. The device of any of claims 60-81, wherein the fixed primary zone comprises a hooked portion joined to a surface of the extended protruding element opposite the surface of the second substrate facing the first substrate.

83. The device of any of claims 60-81, wherein the fixed primary zone comprises a chamfered end.

84. The device of claim 83, wherein the chamfered end is crescent- shaped.

85. The device of any of claims 82-84, wherein the laterally extended protruding element comprises a raised beveled perimeter on the surface facing the first substrate.

86. The device of any of claims 82-84, wherein the laterally extended protruding element comprises a pinning wall about the perimeter of the extended protruding element on the surface facing the first substrate.

87. The device of any of claims 82-84, wherein the laterally extended protruding element comprises a recessed beveled edge about the perimeter of the extended protruding element on the surface facing the first substrate.

88. The device of any of claims 82-87, wherein the laterally extended protruding element comprises a plurality of ridges on the surface facing the first substrate.

89. The device of claims82-88, wherein the laterally extended protruding element comprises a plurality of raised pillars on the surface facing the first substrate.

90. The device of any of claims 1-89, wherein the device comprises a waste disposal region.

91. The device of claim 90, wherein the waste disposal region comprises an opening in the second substrate and a wedged portion extending from the second substrate into the central chamber.

92. The device of claims 90 or 91, wherein the waste disposal region is at an end of the sample processing region that is furthest away for the sample analysis region.

93. The device of any of claims 1-92, wherein the top substrate is made of a material selected from the group consisting of glass, silicon, ceramic, metal, polymethyl methacrylate (PMMA), polystyrene (PS), polycarbonate (PC), cyclic olefin copolymer (COC), cyclic olefin polymer (COP), polypropylene (PP), polyurethane (PU), polytetrafluoroethylene (PTFE), polyvinylchloride (PVC), polydimethylsiloxane (PDMS), acrylonitrile butadiene styrene (ABS), poly(lactic acid) (PLA), thermoplastic PU, clear resin, and polyethylene glycol diacrylate (PEGDA).

94. The device of any of claims 1-93, wherein the bottom substrate is made of a material selected from the group consisting of glass, silicon, ceramic, metal, polymethyl methacrylate (PMMA), polystyrene (PS), polycarbonate (PC), cyclic olefin copolymer (COC), cyclic olefin polymer (COP), polypropylene (PP), polyurethane (PU), polytetrafluoroethylene (PTFE), polyvinylchloride (PVC), polydimethylsiloxane (PDMS), acrylonitrile butadiene styrene (ABS), poly(lactic acid) (PLA), thermoplastic PU, clear resin, thin film, and polyethylene glycol diacrylate (PEGDA).

95. A device, comprising: a first substrate, and a second substrate positioned on the first substrate, wherein the first substrate comprises a sidewall about at least a portion of a periphery of the first substrate, wherein the first substrate, sidewall, and second substrate define a central chamber therebetween, wherein the second substrate comprises a surface facing the central chamber comprising a plurality of recessed elements, and a plurality of protruding elements; wherein primary zones are defined between a surface of the plurality of protruding elements facing the first substrate and a surface of the first substrate facing the second substrate;wherein secondary zones are defined between a surface of the plurality of recessed elements facing the first substrate and the surface of the first substrate facing the second substrate, wherein the second substrate has an opening in one or more of the primary zones, and wherein the first substrate and the second substrate have a difference in hydrophobicity or hydrophilicity.

96. The device of claim 95, wherein the at least a portion of the periphery comprises at least two, at least three, or at least four peripheral sides of the second substrate97. The device of claims 95 or 96, wherein the difference in hydrophobicity or hydrophilicity is a difference in the contact angle of the first substrate and the contact angle in the second substrate.

98. The device of claim 97, wherein the difference in the contact angle of the first substate and the contact angle of the second substrate is about a 10% to about a 60% difference in the contact angle.

99. The device of claim 98, wherein the difference in the contact angle of the first substate and the contact angle of the second substrate is about a 20% to about a 40% difference in the contact angle.

100. The device of claim 97, wherein the difference in the contact angle of the first substate and the contact angle of the second substrate is about a 5° to about a 60° difference in the contact angle.

101. The device of claim 100, wherein the difference in the contact angle of the first substate and the contact angle of the second substrate is about a 5° to about a 40° difference in the contact angle.

102. The device of any of claims 97, or 100-101, wherein the difference in the contact angle of the first substate and the contact angle of the second substrate is about a 5° to about a 30° difference in the contact angle.

103. The device of any of claims 95-102, wherein the first substrate is hydrophobic and the second substrate is hydrophilic.

104. The device of any of claims 95-102, wherein the first substrate is hydrophilic and the second substrate is hydrophobic.

105. The device of any of claims 95-102, wherein the first substrate is hydrophobic and the second substrate is hydrophobic.

106. The device of any of claims 95-102, wherein the first substrate is hydrophilic and the second substrate is hydrophilic.

107. The device of any of claims 95-106, wherein primary zones are discrete.

108. The device of any of claims 95-107, wherein secondary zones are connected.

109. The device of any of claim 95-108, wherein the second substrate has an opening in one or more of the secondary zones.

110. The device of any of claims 95-109, wherein the second substrate has an opening in each of the plurality of primary zones.

111. The device of any of claims 95-110, wherein the surface of the plurality of the protruding elements facing the first substrate and the surface of the first substrate facing the second substrate in the primary zones are separated by a first distance and the surface of the recessed element facing the first substrate and the surface of the first substrate facing the second substrate in the secondary zones are separated by a second distance.

112. The device of claim 111, wherein the first distance is less than the second distance.

113. The device of any of claims 95-112, wherein the protruding elements are a shape selected from the group consisting of a rectangle, a circle, a triangle, a pentagon, a hexagon, a heptagon, an octagon, a decagon, a dodecagon, an amoeboid, and a non-regular shape.

114. The device of any of claims 95-113, wherein the device comprises three or more primary zones separated by two or more secondary zones.

115. The device of any of claims 95-114, wherein the device comprises six or more primary zones separated by seven or more secondary zones.

116. The device of any of claims 95-115, wherein the device comprises nine or more primary zones separated by twelve or more secondary zones.

117. The device of any of claims 95-116, wherein the device comprises twelve or more primary zones separated by seventeen or more secondary zones.

118. The device of any of claims 95-117, wherein the device comprises fifteen or more primary zones separated by twenty-two or more secondary zones.

119. The device of any of claims 95-118, wherein the device comprises eighteen or more primary zones separated by twenty-seven or more secondary zones.

120. The device of any of claims 95-119, wherein the device comprises twenty-one or more primary zones separated by thirty-two or more secondary zones.

121. The device of any of claims 95-120, wherein the device comprises twenty-four or more primary zones separated by thirty-seven or more secondary zones.

122. The device of any of claims 95-121, further comprising a sample analysis region configured to analyze a sample.

123. The device of claim 122, wherein said sample analysis region is positioned adjacent to the central chamber on the first substrate.

124. The device of claims 122 or 123, wherein the sample analysis region is physically separated from the central chamber.

125. The device of claims 122 or 123, wherein the sample analysis region is not physically separated from the central chamber.

126. The device of claim 124, further comprising a transition zone wherein the transition zone fluidically connects the central chamber to the sample analysis region.

127. The device of any of claims 122-126, wherein the sample analysis region has a first end laterally separated from a second end and is defined by the surface of the second substrate facing the first substrate and the surface of the first substrate facing the second substrate.

128. The device of claim 127, wherein the surface of the second substrate facing the first substrate in the sample analysis region comprises an enlarged protruding element wherein: a surface of the enlarged protruding element facing the first substrate has a greater surface area than a surface of the plurality of protruding elements facing the first substrate, and the enlarged protruding element extends from the first end to the second end.

129. The device of claim 127 or 128, wherein the sample analysis region comprises a tertiary zone located at a midpoint between the first end and the second end of the of the sample analysis region wherein the tertiary zone is defined by: a) the surface of the second substrate facing the first substrate and the surface of the first substrate facing the second substrate, or b) the surface of the enlarged protruding element facing the first substrate and the surface of the first substrate facing the second substrate.

130. The device of claim 129, wherein the tertiary zone comprises at least one of selected from the group consisting of: one or more wells in the surface of the first substrate facing the second substrate, one or more nanopores, and one or more chambers.

131. The device of claim 130, wherein the one or more wells is an array of wells.

132. The device of any of claims 122-131, wherein the sample analysis region comprises two or more tertiary zones.

133. The device of claim 132, wherein the two or more tertiary zones are of the same type.

134. The device of claim 132, wherein the two or more tertiary zones are of different types.

135. The device of any of claims 95-134, wherein the plurality of primary zones are unbounded around the perimeter of each primary zone.

136. The device of any of claims 122-135, wherein the sample analysis region comprises a quaternary zone wherein the quaternary zone comprises a cylindrical opening spanning the second substrate and the quaternary zone is located at the second end of the sample analysis region.

137. The device of any of claims 122-136, wherein the sample analysis region comprises a quinary zone wherein the quinary zone is an opening spanning the second substrate and the quinary zone is located at the first end of the sample analysis region.

138. The device of claim 136, wherein the quaternary zone is a hydrophilic liquid well or reservoir.

139. The device of claim 137, wherein the quinary zone is a hydrophobic liquid well or reservoir.

140. The device of any of claims 136-139, wherein the quaternary zone comprises one or more substrate retention features.

141. The device of claim 140, wherein the one or more substrate retention features are selected from the group consisting of: quarter circle protrusions protruding from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate, horse shaped protrusions with tapered edges protruding from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate, horse shaped protrusions without tapered edges protruding from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the secondsubstrate, horse shaped grooves without tapered edges protruding from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate, rectangular protrusions protruding from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate, and circular protrusions protruding from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate.

142. The device of any of claims 137-141, wherein the sample analysis region comprises a substrate stopper feature comprising a ridge on the surface of the enlarged protruding element facing the first substrate that creates a difference in the height between the quinary zone and the quaternary zone.

143. The device of any of claims 128-142, wherein the enlarged protruding element comprises a pinning wall comprising a raised edge about the perimeter of the enlarged protruding element excluding the end comprising the quinary zone144. The device of any of claims 137-143, wherein the quinary zone comprise a barrier feature wherein the barrier feature is a half-circle that protrudes from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate.

145. The device of any of claims 95-144, wherein one or more of the primary zones comprises a fluid.

146. The device of claim 145, wherein the primary zone retains the fluid through surface tension and capillary forces.

147. The device of any of claims 95-146, wherein one or more secondary zones contains a fluid.

148. The device of any of claims 95-147, wherein the device comprises a pre-treatment region configured to pre-treat a sample prior to processing of the sample.

149. The device of claim 148, wherein the pre-treatment region comprises a plurality of elongated openings about a perimeter of the pre-treatment region wherein the elongatedopenings are separated from each other by a plurality of connected regions wherein the pretreatment region is defined by the surface of the second substrate facing the first substrate and the surface of the first substrate facing the second substrate enclosed by the plurality of elongated openings.

150. The device of claim 149, wherein the pre-treatment region comprises a plurality of openings in the second substrate.

151. The device of claims 149 or 150, wherein the pre-treatment region comprises a plurality of openings in the second substrate.

152. The device of any of claims 149-151, wherein the pre-treatment region comprises a raised edge about the perimeter of the pre-treatment region enclosed by the plurality of elongated openings.

153. The device of any of claims 148-152, wherein the pre-treatment region is at an end of the sample processing region that is furthest from the sample analysis region.

154. The device of any of claims 95-153, wherein the device comprises one or more fixed primary zones wherein the one or more fixed primary zones are formed from a laterally extended protruding element in the second substrate facing the first substrate wherein the laterally extended protruding element has a surface facing the first substrate that has a greater surface area than the surface of the plurality of protruding elements facing the first substrate.

155. The device of any of claims 95-154, wherein the plurality of protruding elements have a surface finish.

156. The device of any of claims 95-155, wherein the laterally extended protruding element has a surface finish.

157. The device of any of claims 95-156, wherein the enlarged protruding element has a surface finish.

158. The device of claim 157, wherein the surface finish of the enlarged protruding element is higher than the surface finish of the plurality of protruding elements and the laterally extended protruding element.

159. The device of any of claims 155-158, wherein the surface finish of the plurality of protruding elements have an average surface roughness of about 0.15 m to about 0.70 pm.

160. The device of any of claims 156-159, wherein the surface finish of the laterally extended protruding element has an average surface roughness of about 0.15 pm to about 0.70 pm.

161. The device of any of claims 157-160, wherein the surface finish of the enlarged protruding element has an average surface roughness of about 0.012 pm to about 0.15 pm.

162. The device of any of claims 154-161, wherein the one or more fixed primary zones comprise one or more openings.

163. The device of any of claims 154-162, wherein the one or more fixed primary zones comprise two or more openings.

164. The device of any of claims 154-163, wherein the fixed primary zone comprises one or more secondary features wherein the secondary features are one or more protruding elements protruding from the surface of the laterally extended protruding element facing the first substrate.

165. The device of any of claims 95-164, wherein the device comprises one or more variable primary zones.

166. The device of any of claims 95-165, wherein the primary zones are in a pattern selected from the group consisting of a grid, a line, a non-grid, and a honeycomb pattern.

167. The device of any of claims 95-166, wherein edges of the plurality of protruding elements are flat edges.

168. The device of any of claims 95-167, wherein edges of the plurality of protruding elements are round edges.

169. The device of any of claims 95-168, wherein edges of the plurality of protruding elements have a protrusion.

170. The device of any of claims 95-169, wherein one or more of the plurality of protruding elements have serrations or grooves on one or more surfaces of the protruding elements.

171. The device of any claims 95-170, wherein one or more of the plurality of recessed elements have serrations or grooves on one or more surfaces of the recessed elements.

172. The device of claim 170 or 171, wherein the serration or grooves are in a pattern selected from the group consisting of waves, straight lines that are perpendicular or parallel to the edge of the pad, diagonal to the edge of the pad, cross or hatch pattern, and any combination thereof.

173. The device of any of claims 95-172, wherein one or more of the plurality of protruding elements have dimples or divots on one or more of the surfaces of the protruding elements.

174. The device of any of claims 95-173, wherein one or more of the plurality of recessed elements have dimples or divots on one or more surfaces of the recessed elements.

175. The device of claim 173 or 174, wherein the dimple or divots are concave, convex, or a combination thereof.

176. The device of any of claims 154-175, wherein the fixed primary zone comprises a hooked portion joined to a surface of the extended protruding element opposite the surface of the second substrate facing the first substrate.

177. The device of any of claims 154-175, wherein the fixed primary zone comprises a chamfered end.

178. The device of claim 177, wherein the chamfered end is crescent- shaped.

179. The device of any of claims 176-178, wherein the laterally extended protruding element comprises a raised beveled perimeter on the surface facing the first substrate.

180. The device of any of claims 176-178, wherein the laterally extended protruding element comprises a pinning wall about the perimeter of the extended protruding element on the surface facing the first substrate.

181. The device of any of claims 176-178, wherein the laterally extended protruding element comprises a recessed beveled edge about the perimeter of the extended protruding element on the surface facing the first substrate.

182. The device of any of claims 176-181, wherein the laterally extended protruding element comprises a plurality of ridges on the surface facing the first substrate.

183. The device of claims 176- 182, wherein the laterally extended protruding element comprises a plurality of raised pillars on the surface facing the first substrate.

184. The device of any of claims 95-183, wherein the device comprises a waste disposal region.

185. The device of claim 184, wherein the waste disposal region comprises an opening in the second substrate and a wedged portion extending from the second substrate into the central chamber.

186. The device of claims 184 or 185, wherein the waste disposal region is at an end of the sample processing region that is furthest away for the sample analysis region.

187. The device of any of claims 95-186, wherein the top substrate is made of a material selected from the group consisting of glass, silicon, ceramic, metal, polymethyl methacrylate (PMMA), polystyrene (PS), polycarbonate (PC), cyclic olefin copolymer (COC), cyclic olefin polymer (COP), polypropylene (PP), polyurethane (PU), polytetrafluoroethylene (PTFE),polyvinylchloride (PVC), polydimethylsiloxane (PDMS), acrylonitrile butadiene styrene (ABS), poly(lactic acid) (PLA), thermoplastic PU, clear resin, and polyethylene glycol diacrylate (PEGDA).

188. The device of any of claims 95-187, wherein the bottom substrate is made of a material selected from the group consisting of glass, silicon, ceramic, metal, polymethyl methacrylate (PMMA), polystyrene (PS), polycarbonate (PC), cyclic olefin copolymer (COC), cyclic olefin polymer (COP), polypropylene (PP), polyurethane (PU), polytetrafluoroethylene (PTFE), polyvinylchloride (PVC), polydimethylsiloxane (PDMS), acrylonitrile butadiene styrene (ABS), poly(lactic acid) (PLA), thermoplastic PU, clear resin, thin film, and polyethylene glycol diacrylate (PEGDA).

189. A device, comprising: a first substrate, a spacer layer positioned on a surface of the first substrate wherein the spacer layer is disposed about at least a portion of a periphery of the first substrate, and a second substrate positioned on the spacer layer, wherein the first substrate, spacer layer, and second substrate define a central chamber therebetween, wherein the second substrate comprises a surface facing the central chamber comprising a plurality of recessed elements, and a plurality of protruding elements; wherein primary zones are defined between a surface of the plurality of protruding elements and the first substrate; and wherein the secondary zones are defined between a surface of the plurality of recessed elements and the surface of the first substrate facing the second substrate.

190. The device of claim 189, wherein the spacer layer is selected from the group consisting of: an adhesive layer, a shim layer, a first adhesive layer and a shim layer and a second adhesive layer, a raised feature layer, and a first adhesive layer and a raised feature layer and a second adhesive layer.

191. The device of claims 189 or 190, wherein the at least a portion of the periphery comprises at least two, at least three, or at least four peripheral sides of the second substrate192. The device of any of claims 189-191, wherein the difference in hydrophobicity or hydrophilicity is a difference in the contact angle of the first substrate and the contact angle in the second substrate.

193. The device of claim 192, wherein the difference in the contact angle of the first substate and the contact angle of the second substrate is about a 10% to about a 60% difference in the contact angle.

194. The device of claim 193, wherein the difference in the contact angle of the first substate and the contact angle of the second substrate is about a 20% to about a 40% difference in the contact angle.

195. The device of claim 192, wherein the difference in the contact angle of the first substate and the contact angle of the second substrate is about a 5° to about a 60° difference in the contact angle.

196. The device of claim 195, wherein the difference in the contact angle of the first substate and the contact angle of the second substrate is about a 5° to about a 40° difference in the contact angle.

197. The device of any of claims 192, or 195-196, wherein the difference in the contact angle of the first substate and the contact angle of the second substrate is about a 5° to about a 30° difference in the contact angle.

198. The device of any of claims 189-197, wherein the first substrate is hydrophobic and the second substrate is hydrophilic.

199. The device of any of claims 189-197, wherein the first substrate is hydrophilic and the second substrate is hydrophobic.

200. The device of any of claims 189-197, wherein the first substrate is hydrophobic and the second substrate is hydrophobic.

201. The device of any of claims 189-197, wherein the first substrate is hydrophilic and the second substrate is hydrophilic.

202. The device of any of claims 189-201, wherein primary zones are discrete.

203. The device of any of claims 189-202, wherein secondary zones are connected.

204. The device of any of claim 189-203, wherein the second substrate has an opening in one or more of the secondary zones.

205. The device of any of claims 189-204, wherein the second substrate has an opening in each of the plurality of primary zones.

206. The device of any of claims 189-205, wherein the surface of the plurality of the protruding elements facing the first substrate and the surface of the first substrate facing the second substrate in the primary zones are separated by a first distance and the surface of the recessed element facing the first substrate and the surface of the first substrate facing the second substrate in the secondary zones are separated by a second distance.

207. The device of claim 206, wherein the first distance is less than the second distance.

208. The device of any of claims 189-207, wherein the protruding elements are a shape selected from the group consisting of a rectangle, a circle, a triangle, a pentagon, a hexagon, a heptagon, an octagon, a decagon, a dodecagon, an amoeboid, and a non-regular shape.

209. The device of any of claims 189-208, wherein the device comprises three or more primary zones separated by two or more secondary zones.

210. The device of any of claims 189-209, wherein the device comprises six or more primary zones separated by seven or more secondary zones.

211. The device of any of claims 189-210, wherein the device comprises nine or more primary zones separated by twelve or more secondary zones.

212. The device of any of claims 189-211, wherein the device comprises twelve or more primary zones separated by seventeen or more secondary zones.

213. The device of any of claims 189-212, wherein the device comprises fifteen or more primary zones separated by twenty-two or more secondary zones.

214. The device of any of claims 189-213, wherein the device comprises eighteen or more primary zones separated by twenty- seven or more secondary zones.

215. The device of any of claims 189-214, wherein the device comprises twenty-one or more primary zones separated by thirty-two or more secondary zones.

216. The device of any of claims 189-215, wherein the device comprises twenty-four or more primary zones separated by thirty-seven or more secondary zones.

217. The device of any of claims 189-216, further comprising a sample analysis region configured to analyze a sample.

218. The device of claim 217, wherein said sample analysis region is positioned adjacent to the central chamber on the first substrate.

219. The device of claims 217 or 218, wherein the sample analysis region is physically separated from the central chamber.

220. The device of claims 217 or 218, wherein the sample analysis region is not physically separated from the central chamber.

221. The device of claim 219, further comprising a transition zone wherein the transition zone fluidically connects the central chamber to the sample analysis region.

222. The device of any of claims 217-221, wherein the sample analysis region has a first end laterally separated from a second end and is defined by the surface of the second substrate facing the first substrate and the surface of the first substrate facing the second substrate.

223. The device of claim 222, wherein the surface of the second substrate facing the first substrate in the sample analysis region comprises an enlarged protruding element wherein: a surface of the enlarged protruding element facing the first substrate has a greater surface area than a surface of the plurality of protruding elements facing the first substrate, and the enlarged protruding element extends from the first end to the second end.

224. The device of claim 222 or 223, wherein the sample analysis region comprises a tertiary zone located at a midpoint between the first end and the second end of the of the sample analysis region wherein the tertiary zone is defined by: a) the surface of the second substrate facing the first substrate and the surface of the first substrate facing the second substrate, or b) the surface of the enlarged protruding element facing the first substrate and the surface of the first substrate facing the second substrate.

225. The device of claim 224, wherein the tertiary zone comprises at least one of selected from the group consisting of: one or more wells in the surface of the first substrate facing the second substrate, one or more nanopores, and one or more chambers.

226. The device of claim 225, wherein the one or more wells is an array of wells.

227. The device of any of claims 217-226, wherein the sample analysis region comprises two or more tertiary zones.

228. The device of claim 227, wherein the two or more tertiary zones are of the same type.

229. The device of claim 227, wherein the two or more tertiary zones are of different types.

230. The device of any of claims 189-229, wherein the plurality of primary zones are unbounded around the perimeter of each primary zone.

231. The device of any of claims 217-230, wherein the sample analysis region comprises a quaternary zone wherein the quaternary zone comprises a cylindrical opening spanning thesecond substrate and the quaternary zone is located at the second end of the sample analysis region.

232. The device of any of claims 217-231, wherein the sample analysis region comprises a quinary zone wherein the quinary zone is an opening spanning the second substrate and the quinary zone is located at the first end of the sample analysis region.

233. The device of claim 231, wherein the quaternary zone is a hydrophilic liquid well or reservoir.

234. The device of claim 232, wherein the quinary zone is a hydrophobic liquid well or reservoir.

235. The device of any of claims 231-234, wherein the quaternary zone comprises one or more substrate retention features.

236. The device of claim 235, wherein the one or more substrate retention features are selected from the group consisting of: quarter circle protrusions protruding from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate, horse shaped protrusions with tapered edges protruding from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate, horse shaped protrusions without tapered edges protruding from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate, horse shaped grooves without tapered edges protruding from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate, rectangular protrusions protruding from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate, and circular protrusions protruding from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate.

237. The device of any of claims 232-236, wherein the sample analysis region comprises a substrate stopper feature comprising a ridge on the surface of the enlarged protruding element facing the first substrate that creates a difference in the height between the quinary zone and the quaternary zone.

238. The device of any of claims 223-237, wherein the enlarged protruding element comprises a pinning wall comprising a raised edge about the perimeter of the enlarged protruding element excluding the end comprising the quinary zone239. The device of any of claims 232-238, wherein the quinary zone comprise a barrier feature wherein the barrier feature is a half-circle that protrudes from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate.

240. The device of any of claims 189-239, wherein one or more of the primary zones comprises a fluid.

241. The device of claim 240, wherein the primary zone retains the fluid through surface tension and capillary forces.

242. The device of any of claims 189-241, wherein one or more secondary zones contains a fluid.

243. The device of any of claims 189-242, wherein the device comprises a pre-treatment region configured to pre-treat a sample prior to processing of the sample.

244. The device of claim 243, wherein the pre-treatment region comprises a plurality of elongated openings about a perimeter of the pre-treatment region wherein the elongated openings are separated from each other by a plurality of connected regions wherein the pretreatment region is defined by the surface of the second substrate facing the first substrate and the surface of the first substrate facing the second substrate enclosed by the plurality of elongated openings.

245. The device of claim 244, wherein the pre-treatment region comprises a plurality of openings in the second substrate.

246. The device of claims 244 or 245, wherein the pre-treatment region comprises a plurality of openings in the second substrate.

247. The device of any of claims 244-246, wherein the pre-treatment region comprises a raised edge about the perimeter of the pre-treatment region enclosed by the plurality of elongated openings.

248. The device of any of claims 243-247, wherein the pre-treatment region is at an end of the sample processing region that is furthest from the sample analysis region.

249. The device of any of claims 189-248, wherein the device comprises one or more fixed primary zones wherein the one or more fixed primary zones are formed from a laterally extended protruding element in the second substrate facing the first substrate wherein the laterally extended protruding element has a surface facing the first substrate that has a greater surface area than the surface of the plurality of protruding elements facing the first substrate.

250. The device of any of claims 189-249, wherein the plurality of protruding elements have a surface finish.

251. The device of any of claims 189-250, wherein the laterally extended protruding element has a surface finish.

252. The device of any of claims 189-251, wherein the enlarged protruding element has a surface finish.

253. The device of claim 252, wherein the surface finish of the enlarged protruding element is higher than the surface finish of the plurality of protruding elements and the laterally extended protruding element.

254. The device of any of claims 250-253, wherein the surface finish of the plurality of protruding elements have an average surface roughness of about 0.15 m to about 0.70 pm.

255. The device of any of claims 251-254, wherein the surface finish of the laterally extended protruding element has an average surface roughness of about 0.15 pm to about 0.70 pm.

256. The device of any of claims 252-255, wherein the surface finish of the enlarged protruding element has an average surface roughness of about 0.012 m to about 0.15 pm.

257. The device of any of claims 249-256, wherein the one or more fixed primary zones comprise one or more openings.

258. The device of any of claims 249-257, wherein the one or more fixed primary zones comprise two or more openings.

259. The device of any of claims 249-258, wherein the fixed primary zone comprises one or more secondary features wherein the secondary features are one or more protruding elements protruding from the surface of the laterally extended protruding element facing the first substrate.

260. The device of any of claims 189-259, wherein the device comprises one or more variable primary zones.

261. The device of any of claims 189-260, wherein the primary zones are in a pattern selected from the group consisting of a grid, a line, a non-grid, and a honeycomb pattern.

262. The device of any of claims 189-261, wherein edges of the plurality of protruding elements are flat edges.

263. The device of any of claims 189-262, wherein edges of the plurality of protruding elements are round edges.

264. The device of any of claims 189-263, wherein edges of the plurality of protruding elements have a protrusion.

265. The device of any of claims 189-264, wherein one or more of the plurality of protruding elements have serrations or grooves on one or more surfaces of the protruding elements.

266. The device of any claims 189-265, wherein one or more of the plurality of recessed elements have serrations or grooves on one or more surfaces of the recessed elements.

267. The device of claim 265 or 266, wherein the serration or grooves are in a pattern selected from the group consisting of waves, straight lines that are perpendicular or parallel to the edge of the pad, diagonal to the edge of the pad, cross or hatch pattern, and any combination thereof.

268. The device of any of claims 189-267, wherein one or more of the plurality of protruding elements have dimples or divots on one or more of the surfaces of the protruding elements.

269. The device of any of claims 189-268, wherein one or more of the plurality of recessed elements have dimples or divots on one or more surfaces of the recessed elements.

270. The device of claim 268 or 269, wherein the dimple or divots are concave, convex, or a combination thereof.

271. The device of any of claims 249-270, wherein the fixed primary zone comprises a hooked portion joined to a surface of the extended protruding element opposite the surface of the second substrate facing the first substrate.

272. The device of any of claims 249-270, wherein the fixed primary zone comprises a chamfered end.

273. The device of claim 272, wherein the chamfered end is crescent- shaped.

274. The device of any of claims 271-273, wherein the laterally extended protruding element comprises a raised beveled perimeter on the surface facing the first substrate.

275. The device of any of claims 271-273, wherein the laterally extended protruding element comprises a pinning wall about the perimeter of the extended protruding element on the surface facing the first substrate.

276. The device of any of claims 271-273, wherein the laterally extended protruding element comprises a recessed beveled edge about the perimeter of the extended protruding element on the surface facing the first substrate.

277. The device of any of claims 271-276, wherein the laterally extended protruding element comprises a plurality of ridges on the surface facing the first substrate.

278. The device of claims271-277, wherein the laterally extended protruding element comprises a plurality of raised pillars on the surface facing the first substrate.

279. The device of any of claims 189-278, wherein the device comprises a waste disposal region.

280. The device of claim 279, wherein the waste disposal region comprises an opening in the second substrate and a wedged portion extending from the second substrate into the central chamber.

281. The device of claims 279 or 280, wherein the waste disposal region is at an end of the sample processing region that is furthest away for the sample analysis region.

282. The device of any of claims 189-281, wherein the top substrate is made of a material selected from the group consisting of glass, silicon, ceramic, metal, polymethyl methacrylate (PMMA), polystyrene (PS), polycarbonate (PC), cyclic olefin copolymer (COC), cyclic olefin polymer (COP), polypropylene (PP), polyurethane (PU), polytetrafluoroethylene (PTFE), polyvinylchloride (PVC), polydimethylsiloxane (PDMS), acrylonitrile butadiene styrene (ABS), poly(lactic acid) (PLA), thermoplastic PU, clear resin, and polyethylene glycol diacrylate (PEGDA).

283. The device of any of claims 189-282, wherein the bottom substrate is made of a material selected from the group consisting of glass, silicon, ceramic, metal, polymethyl methacrylate (PMMA), polystyrene (PS), polycarbonate (PC), cyclic olefin copolymer (COC), cyclic olefin polymer (COP), polypropylene (PP), polyurethane (PU), polytetrafluoroethylene (PTFE), polyvinylchloride (PVC), polydimethylsiloxane (PDMS), acrylonitrile butadienestyrene (ABS), poly(lactic acid) (PLA), thermoplastic PU, clear resin, thin film, and polyethylene glycol diacrylate (PEGDA).

284. A method of mixing a sample, the method comprising: contacting the device of any of claims 1-94, 95-188, or 189-283 with a vibration source; and vibrating the vibration source, wherein the device comprises one or more fluids.

285. The method of claim 284, wherein the vibration source contacts the second substrate.

286. The method of claim 284, wherein the vibration source contacts the first substrate.

287. The method of claim 284, wherein the vibration source contacts the second and first substrate.

288. The method of claim 285, wherein the vibration source vibrates only the second substrate.

289. The method of claim 288, wherein the vibration source vibrates the second substrate vertically.

290. The method of claim 286, wherein the vibration source vibrates only the first substrate.

291. The method of claim 290, wherein the vibration source vibrates the first substrate vertically.

292. The method of claim 287, wherein the vibration source vibrates the second and first substrate.

293. The method of claim 292, wherein the vibration source vibrates the top and bottom substrate or second and first substrate vertically.

294. The method of claim 284, wherein the vibration source contacts the fixed primary zone.

295. The method of claim 294, wherein the vibration source vibrates the fixed primary zone vertically.

296. The method of claim 294, wherein the vibration source contacts the chamfered end of the fixed primary zone.

297. The method of claim 296, wherein the vibration source vibrates the chamfered end of the fixed primary zone vertically.

298. The method of any of claims 284-297, wherein the vibration source is selected from the group consisting of voice coil, a vibration motor, a piezo actuator, a motor, and a gas pump.

299. The method of claim 298, wherein the vibration source is the voice coil.

300. The method of claim 299, wherein the voice coil vibrates the device at frequency that is substantially resonant with the one or more fluids.

301. The method of any of claims 284-300, wherein the one or more fluids comprise microparticles.

302. A method of measuring or detecting a target analyte in a sample, the method comprising: a) depositing the sample comprising the target analyte in an opening of a primary zone of a device; b) depositing microparticles attached to a first specific binding partner that specifically binds to the target analyte in the opening of the primary zone comprising the sample thereby producing microparticles bound to the target analyte,c) subjecting the microparticles bound to the target analyte to a magnetic field to move the microparticles to a primary zone containing a second specific binding partner that is detectably labeled thereby producing microparticles bound to detectably labeled target analyte; d) subjecting the microparticles bound to the detectably labeled target analyte to a magnetic field to move the microparticles to one or more primary zones containing a wash buffer; e) subjecting the microparticles bound to the detectably labeled target analyte to a magnetic field to move the microparticles to a detection zone containing wells in the sample analysis region; f) adding a hydrophilic liquid to the wells that reacts with the detectably labeled second specific binding partner to produce a detectable signal prior to or after step e), g) sealing the wells using a hydrophobic liquid; and h) imaging the wells thereby detecting the detectable signal produced by the hydrophilic reacting with the second specific binding member; wherein the device comprises: a plurality of primary zones defined by a surface of a plurality of protruding elements on a surface of a second substrate facing a first substrate and a surface of the first substrate facing the second substrate, and a plurality of secondary zones defined by a surface of a plurality of recessed elements on the surface of the second substrate facing the first substrate and a surface of the first substrate facing the second substrate, wherein the second substrate has an opening in one or more of the primary zones and an opening in one or more of the secondary zones.

303. The method of claim 302, wherein the target analyte is a protein.

304. The method of claims 302 or 303, wherein the first and second specific binding members are antibodies that specifically bind to different epitopes on the target analyte.

305. The method of claims 302-304, wherein the detectable label is an enzyme that reacts with the hydrophilic liquid.

306. The method of any of claims 302-305, wherein the hydrophobic liquid is oil.

307. The method of any of claims 302-306, wherein the hydrophilic liquid is a substrate solution.

308. The method of any of claims 302-307, further comprising: i) depositing lysis buffer in the in the opening of the primary zone comprising the sample, wherein step i) occurs between steps a) and b)309. The method of any of claims 302-308, further comprising: j) mixing the primary zone comprising the sample and the microparticles, wherein step j) occurs between steps b) and c).

310. The method of any of claims 302-309, further comprising: k) subjecting the microparticles bound to the target analyte to a magnetic field to move the microparticles to one or more primary zones containing a wash buffer, wherein step k) occurs between steps b) and c).

311. The method of any of claims 302-310, wherein the one or more primary zones of step d) is two primary zones joined by a secondary zone each containing the wash buffer.

312. The method of any of claims 302-311, wherein the one or more primary zones of step j) is two primary zones joined by a secondary zone each containing the wash buffer.

313. The method of any of claims 302-312, wherein step b) further comprises depositing assisting particles.

314. The method of claim 313, wherein the assisting particles are configured to increase the influence of the magnetic force acting on the microparticles in the presence of assisting particles.

315. The method of claim 313 or 314, wherein the microparticles and the assisting particles are spherical and the diameter of the assisting solids supports is greater than the diameter of the assay solid supports.

316. The method of any of claims 313-315, wherein the microparticles and assisting particles includes between about 30,000 assisting particles and about 300,000 assisting particles.

317. The method of any of claims 313-316, wherein the microparticles and assisting particles contains more assisting particles than microparticles.

318. The method of any of claims 313-316, wherein the microparticles and assisting particles contains less assisting particles than microparticles.

319. The method of any of claims 313-317, wherein the assisting particle diameter is between about 5 pm and about 15 pm.

320. The method of any of claims 313-319, wherein the assisting particle diameter is about 10 pm.

321. The method of any of claims 313-320, wherein the microparticles and assisting particles comprise magnetic, paramagnetic, and / or superparamagnetic microparticles or beads.

322. The method of any of claims 313-321, wherein the assisting particles are configured to bind with one or more interferents within the sample.

323. The method of any of claims 313-322, wherein the microparticles and assisting particles moves across the detection zone at a speed of between about 0.35 mm / s and about 6.00 mm / s relative to the detection zone.

324. The method of any of claims 313-323, wherein the microparticles and assisting particles comprises a ratio of microparticles to assisting particles of between about 1: 1 and about 50: 1.

325. The method of any of claims 313-324, wherein the microparticles and assisting particles includes between about 30,000 assisting particles and about 300,000 assisting particles.

326. The method of any of claims 313-325, wherein the microparticles and assisting particles includes between about 30,000 assisting particles and about 80,000 assisting particles.

327. The method of any of claims 313-326, wherein the assisting particles include a negative surface charge.

328. The method of any of claims 302-326, wherein the magnetic field is produced by a magnet.

329. The method of claim 328, wherein the magnet has a first magnetic field along a bottom surface of the array of wells, the first magnetic field moving the microparticles and assisting particles and the target analyte across the bottom surface of the detection zone, wherein the magnet has a first magnet end and a second magnet end with a magnet axis defined there between, the magnet axis positioned at an angle of between about 0 degrees and about 80 degrees relative to the detection zone.

330. The method of claim 329, wherein the magnet axis is positioned at an angle of between about 10 degrees and about 30 degrees relative to the detection zone.

331. The method of claims 329 or 330, wherein the first magnet is moved along the detection zone at a first magnet distance defined between the detection zone and the magnet, the magnet distance being less than about 10mm.

332. The method of claims 329-331, wherein the first magnet is moved along the detection zone with the first magnet in contact with the bottom of the detection zone.

333. The method of any of claims 302-332, wherein the device is the device of any of claims 1-94, 95-188, or 189-283.

334. The method of any of claims 302-333, wherein the target analyte is anti-Mullerian Hormone (AMH), an autoantibody to CD25, Chemokine (C-X-C) motif ligand 13 (CXCL13), Dickkopf-3 (Dkk-3), IL-12p40, Interleukein 8 (IL-8), pl4 Endocan Fragment, SARS-CoV-2 IgA Antibody, SARS-CoV-2 IgG Antibody, SARS-CoV-2 IgM Antibody, Secretory Gelsolin (pGSN), Secretogranin II, ACE2, Albumin, Albuminuria, Alpha- Amylase, Apo H, B-2 Microglobulin, Brain natriuretic peptide (BNP) and derivatives thereof, CA 24-2, Carcinoembryonic Antigen (CEA), Cardiac myosin binding protein C, Ceruloplasmin,Cyclosporine, C-peptide, C-Reactive Protein (CRP), Dipeptidyl peptidase 4 (DPP-4), Digoxin, Fibrinogen Alpha Chain (FGA), Homocysteine, Interleukin 18 (IL- 18), Interleukin 6 (IL-6), Lactate Dehydrogenase (LD), Liver Fatty Acid Binding Protein (L-FABP), Lipase, Microalbuminuria, Neutrophil Gelatinase-Associated Lipocalin (NGAL), Osteopontin, Periostin, Peroxisome Proliferator-Activated Receptor Gamma Coactivator-1 Alpha (PGC-la), Proapoptotic Kinase R (PKR), Phosphorylated PKR (pPKR), Procalcitonin (PCT), Pepsinogen I, Pepsinogen II, Pro-SFTPB, PTH (Parathyroid Hormone), soluble Interleukin 2 (sIL-2), Sex Hormone-Binding Globulin (SHBG), Thioredoxin, TSH (Thyroid Stimulating Hormone), Vitamin D-Binding Protein, Alpha- synuclein, BARF1 (BamHl-A Reading Frame 1), Kidney Injury Molecule- 1 (KIM-1), Laminin gamma, LMP1 (Latent Membrane Protein 1), Neurofilament light chain (NF-L), Tau protein, Tau, UCH-L1 (Ubiquitin C-Terminal Hydrolase- Ll), Alkaline Phosphatase, Amylase, Aspartate Aminotransferase (AST), Calcium, Cholesterol, Creatine Kinase (CK), Carbon Dioxide (CO2), Creatinine, Direct Low-Density Lipoprotein (Direct LDL), Gamma-Glutamyl Transferase (GGT), High-Density Lipoprotein (HDL), Iron, Low-Density Lipoprotein (LDL), Magnesium, Potassium (K), Sodium (Na), Triglycerides, Uric Acid, Akt (Protein Kinase B), Amphiregulin, ANXA7 (Annexin A7), Androgen Receptor (AR), v-Raf Murine Sarcoma Viral Oncogene Homolog B (BRAF), Cyclin-Dependent Kinase Inhibitor IB (CDKN1B), MYC Proto-Oncogene (cMYC), Catenin Beta-1 (CTNNB1), Epidermal Growth Factor Receptor (EGFR), Ephrin Type-B Receptor 2 (EPHB2), Estrogen Receptor 1 (ESRI), Estrogen Receptor 2 (ESR2), Ferritin, Folate, Forkhead Box 03 (F0X03A), Mechanistic Target of Rapamycin Complex 1 (FRAP1), Fibroblast Growth Factor Receptor Substrate 2 (FRS2), GRB2-Associated Binding Protein 2 (Gab2), Glial Fibrillary Acidic Protein (GFAP), Growth Factor Receptor-Bound Protein 2 (Grb2), Growth Regulation By Estrogen In Breast Cancer 1 (GREB1), Hepatitis B e-antigen (HBeAg), Hepatitis B surface antigen (HBsAg), Hepatitis B core antigen (HBeAg), phosphorylated Hepatitis B core antigen (P- HBcAg), Hepatitis B core-related antigen (HBcrAg), Human Epidermal Growth Factor Receptor 2 (HER2), Human Epidermal Growth Factor Receptor 3 (HER3), Human Epidermal Growth Factor Receptor 4 (HER4), Insulin-Like Growth Factor 1 Receptor (IGF-IR), IL6R (Interleukin- 6 Receptor), Kruppel Like Factor 6 (KLF6), Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS), Leucine Zipper Tumor Suppressor 1 (LZTS1), Mitogen- Activated Protein Kinase Kinase 1 (MAP2K1), Mitogen- Activated Protein Kinase (MEK), Mitogen-Inducible Gene 6 Protein (MIG-6), Marker of Proliferation Ki-67 (MKI67), Mechanistic Target of Rapamycin (mTOR), Mucin 4, Cell Surface Associated (MUC4), Neural Precursor Cell Expressed Developmentally Down-Regulated Protein 4-1 (NEDD4-1), NK3 Homeobox 1 (NKX3-1),Neuregulin 1 (NRG1), Parkin, Parvovirus B19, 3-Phosphoinositide-Dependent Protein Kinase- 1 (PDK-1), Progesterone Receptor (PGR), PH Domain and Leucine-Rich Repeat Protein Phosphatase (PHLPP), Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha (PIK3CA), Paired-Like Homeodomain Transcription Factor 2 (PITX2), Protein Phosphatase 1 Regulatory Inhibitor Subunit IB (PPP1R1B), PRDX6 (Peroxiredoxin-6), Phosphatase and Tensin Homology (PTEN), Phosphatase and Tensin Homolog 1 (PTEN 1), PXN (Paxillin), Ribosomal Protein S6 Kinase (S6K), Src Homology 2 Domain-Containing Inositol Phosphatase (SHIP), Sirolimus, Src Proto-Oncogene Tyrosine-Protein Kinase Src (Src), Tacrolimus, Thyroxine, Triiodothyronine, Thyroglobulin, DNA Topoisomerase II (TOPO II), TRAb, Tuberous Sclerosis 1 (TSC1), Tuberous Sclerosis 2 (TSC2), Tumor necrosis factor-alpha receptors, Amyloid Beta-Protein 42 (Ap42), Creatine Kinase-MB (CK-MB), Anti-Cyclic Citrullinated Peptide (Anti-CCP), Anti-Thyroglobulin Antibody (Anti-Tg), Anti-Thyroid Peroxidase Antibody (Anti-TPO), Antistreptolysin O (ASO), Complement Component 3 (C3), Complement Component 4 (C4), D-Dimer, Rheumatoid Factor (RF), DJ-1, Leucine-rich repeat kinase 2, Mutated ATP13A2, Phenobarbital, Phenytoin, Prion protein, PTEN induced putative kinase 1, Alpha-Fetoprotein (AFP), CA 125 (MUC16), CA 15-3, CA 19-9, Cyclin I (CCNI), Cytomegalovirus (CMV), CYFRA21-1, Fibroblast Growth Factor 19 (FGF19), Gentamicin, Human Epididymis Protein 4 (HE-4), Neuron- Specific Enolase (NSE), Perinuclear AntiNeutrophil Cytoplasmic Antibody (p-ANCA), Protein Induced By Vitamin K Absence (PIVKA), Protein Induced By Vitamin K Absence-II (PIVKA-II), Pro-Surfactant B (Pro- SFTPB), Prostate Specific Antigen (PSA), Rubella, Squamous Cell Carcinoma Antigen (SCC), Toxo IgG, Toxo IgM, Beta Human Chorionic Gonadotropin (Beta-hcG), Botulinum toxins, Clostridium difficile toxins A and B, Dehydroepiandrosterone Sulfate (DHEA-S), Diphtheria toxin, E. coli enterotoxins, Fetuin-A, Follicle-Stimulating Hormone (FSH), Glycosylated hemoglobin (HbAlc), Hemoglobin Ale, Interleukin 1 alpha (ILla), Influenza HA antigen, Luteinizing Hormone (LH), Methotrexate, Myeloperoxidase (MPO), Neurofibromin 1 (NF-1), Plasma C-peptide, Placental Growth Factor (P1GF), Pro-GFP, Prolactin, SlOOp, Soluble Fms- Like Tyrosine Kinase-1 (sFlt-1), Testosterone, Tetanus toxin, Thymosin 1315, Alanine Aminotransferase (ALT), Bile Acids-Total, Bilirubin, Bilirubin-Direct, Bilirubin-Total, Calprotectin, Deoxyuridine Triphosphatase (DUTPase), Lactic Acid, Lactoferrin, Shiga toxin, Shiga-like toxin I, Shiga- like toxin II, Theophylline, Total Protein, Urea Nitrogen, Valproic acid, Vitamin B12, Voltage-Dependent Anion Channel 1 (VDAC1), Wilm's Tumor-1 protein, Amphetamines, Methamphetamines, Barbiturates, Benzodiazepines, Benzodiazepines (Serum), Cannabinoids, Ecstasy, Ethanol, Opiates, Phencyclidine (PCP) or any combination thereof.

335. The method of claim 334, wherein the target analyte is Hepatitis B core antigen (HBcAg), phosphorylated Hepatitis B core antigen (P-HBcAg) or a combination thereof.

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