Two-dimensional matrix droplet array

Abstract

This disclosure provides an apparatus for use in a range of different assays, such as immunoassays, nucleic acid analysis, metabolite analysis, clinical chemistry, and complete blood cell counts. The apparatus optionally includes a sample analysis region for analyzing samples processed within the apparatus. The apparatus includes a top substrate bonded to a bottom substrate, wherein the top substrate bonded to the bottom substrate forms two or more primary regions separated by one or more secondary regions, and wherein the top substrate has an opening in one or more of the primary regions.

Description

[0001] Cross-references to related applications This application claims priority to the filing dates of U.S. Provisional Patent Application Serial No. 63 / 601,393, filed November 21, 2023, and PCT Patent Application Serial No. PCT / US2024 / 036808, filed July 3, 2024, the disclosures of which are incorporated herein by reference in their entirety. Background Technology

[0002] Analytical analysis is typically performed by carrying out a sample preparation step, which is either performed manually or using sophisticated robotics. Following sample preparation, the determination of the analytes in the prepared sample further involves using expensive and complex systems to transport the prepared sample to a machine, which then performs the analysis on the analytes in the prepared sample.

[0003] In the field of analyte analysis, devices that can be used to prepare and measure samples for a variety of assays are highly desirable. Such devices would offer a low-cost option and significantly improve the convenience of performing analyte analyses, especially in clinical applications such as point-of-care testing.

[0004] Therefore, there is some interest in devices for sample preparation because they allow for reduced sample and reagent volumes, potentially higher sensitivity, and faster results. Summary of the Invention

[0005] This disclosure is defined by the appended claims.

[0006] This disclosure provides an apparatus that can be used for a range of different assays, such as immunoassays, nucleic acid analysis, metabolite analysis, clinical chemistry, and complete blood counts. The apparatus optionally includes a sample analysis region for analyzing samples processed within the apparatus.

[0007] In one aspect, the present disclosure provides an apparatus including a top substrate bonded to a bottom substrate, wherein the top substrate bonded to the bottom substrate forms two or more primary regions separated by one or more secondary regions, and wherein the top substrate has an opening in one or more of the primary regions.

[0008] In another aspect, this disclosure provides a sample processing apparatus comprising: a top substrate; a bottom substrate attached to the top substrate to form an internal volume, wherein the top substrate and / or the bottom substrate substantially defines a first plane; and a sample processing region within the internal volume, wherein the sample processing region comprises two or more primary regions and one or more secondary regions, wherein each primary region is separated from an adjacent primary region by one of the one or more secondary regions in a direction substantially along the first plane, wherein the top substrate includes an opening leading into a respective primary region of the two or more primary regions.

[0009] In other respects, this disclosure provides methods for using the discussed apparatus and sample processing apparatus.

[0010] An optional reagent delivery device is also provided for use with one type of the device. A pressure sample mixing device is also provided.

[0011] As will be further discussed below, the sample processing apparatus of this disclosure may include an internal volume formed by a top substrate and a bottom substrate when attached to each other. This internal volume may itself be divided into or comprise different regions connected to each other. For example, the apparatus may include a sample processing region located within or forming part of the internal volume. As microparticles move through the sample processing region, the sample processing region can be used to perform one or more processing steps on a sample attached to the microparticles.

[0012] The sample processing area can be connected to the sample analysis area, which can be used to analyze the processed sample. External devices can be used to perform this analysis, for example, to digitally image the sample analysis area. In this context, the connection between areas is intended to mean that microparticles can move between the areas, for example, that there are paths or channels for microparticles to move from one area to another. The sample processing area can be additionally or alternatively connected to the sample mixing area. For example, in use, microparticles can be able to travel from the sample mixing area to the sample processing area and then into the sample analysis area. Attached Figure Description

[0013] Figure 1 The illustration shows a top view of the sample processing apparatus according to one embodiment.

[0014] Figure 2 The illustration shows a top view of the sample processing apparatus according to another embodiment.

[0015] Figure 3 The illustration shows a top view of the sample processing apparatus according to another embodiment.

[0016] Figure 4The illustration shows a top view of the sample processing apparatus according to another embodiment.

[0017] Figure 5 The illustration shows a top view of the sample processing apparatus according to another embodiment.

[0018] Figure 6 The illustration shows a top view of the sample processing apparatus according to another embodiment.

[0019] Figure 7 An isometric view of the top of a sample processing apparatus according to one embodiment is illustrated.

[0020] Figure 8 An isometric view of the top of a sample processing apparatus according to another embodiment is illustrated.

[0021] Figure 9 The illustration shows a magnified view of the sample analysis area according to one embodiment.

[0022] Figure 10 An exemplary method of mixing samples using the apparatus according to one embodiment is illustrated.

[0023] Figure 11 An isometric view of the top of a sample processing apparatus according to another embodiment is illustrated.

[0024] Figure 12 The illustration shows a top view of the sample processing apparatus according to another embodiment.

[0025] Figure 13 The illustration shows a top view of the sample processing apparatus according to another embodiment.

[0026] Figure 14 The illustration shows a bottom view of the top (i.e., the top substrate) of a sample processing apparatus according to another embodiment.

[0027] Figure 15 The illustration shows a bottom view of the top (i.e., the top substrate) of a sample processing apparatus according to another embodiment.

[0028] Figure 16 The illustration shows cross-sections of two exemplary primary regions of the device according to an embodiment.

[0029] Figure 17 An exemplary sample processing path according to an embodiment is illustrated.

[0030] Figure 18 An exemplary sample processing path according to an embodiment is illustrated.

[0031] Figures 19A-19E An exemplary sample processing path according to an embodiment is illustrated.

[0032] Figure 20 An isometric view of a reagent delivery device according to an embodiment is illustrated.

[0033] Figure 21 An exploded view of a reagent delivery device according to an embodiment is shown.

[0034] Figure 22 The diagram illustrates an anatomical view of the components of a reagent delivery device.

[0035] Figure 23 The illustration shows a cross-section of an internal component of a reagent delivery device in an inactive position according to an embodiment.

[0036] Figure 24 The illustration shows a cross-section of an internal component of a reagent delivery device in the activated position according to an embodiment.

[0037] Figure 25 The illustration shows a cross-section of an internal component of a reagent delivery device according to an embodiment.

[0038] Figures 26A-26D The diagram shows a cross-section of the primary region of the sample in existence.

[0039] Figures 27A-27B An exemplary pad design is illustrated.

[0040] Figure 28A-28K An exemplary fluid retention feature is illustrated.

[0041] Figure 29 An exemplary sample mixing apparatus is illustrated.

[0042] Figures 30A-30C An exemplary sample mixing apparatus is illustrated.

[0043] Figure 31 The illustration shows an exemplary secondary feature for a fixed primary region.

[0044] Figures 32A-32B A illustrates an exemplary method of mixing samples using the apparatus according to one embodiment; B illustrates a bottom view of the top (i.e., top substrate) of a sample processing apparatus according to another embodiment.

[0045] Figure 33A-33O : AG illustrates an exemplary sample analysis region; HN illustrates an exemplary substrate retention feature; O illustrates an exemplary barrier feature.

[0046] Figure 34 The diagram shows Figure 8 and Figure 9The exemplary sample analysis area of ​​the embodiment depicted herein.

[0047] Figures 35A-35H The illustration depicts the use of magnets to move microparticles or microparticles and assist particle movement across an array of wells, according to aspects of the disclosed subject matter, and to load microparticles into the wells of the array.

[0048] Figure 36 It is a diagram illustrating the magnetic forces on multiple microparticles or microparticles and assisting particles.

[0049] Figures 37A-37C This is a cross-sectional view of an exemplary embodiment of the device.

[0050] Figures 38A-38B An exemplary embodiment of the device is illustrated.

[0051] Figure 39 An exemplary sample mixing region is illustrated.

[0052] Figures 40A-40B An exemplary embodiment of an apparatus containing a waste disposal area is illustrated.

[0053] Figures 41A-41B An exemplary preprocessing area is illustrated.

[0054] Figure 42 An exemplary method of mixing samples using the apparatus according to one embodiment is illustrated.

[0055] Figure 43 The illustration shows the features of an exemplary substrate stop portion.

[0056] Figures 44A-44D An exemplary feature of the sample mixing region is illustrated.

[0057] Figure 45 An exemplary sample analysis area is illustrated.

[0058] Figure 46 An exemplary sample processing path according to an embodiment is illustrated.

[0059] Figure 47 An exemplary embodiment of a device incorporating surface finish is illustrated.

[0060] Figure 48 An exemplary voice coil mixing method is illustrated.

[0061] Figure 49 An exemplary particle pattern generated by voice coil mixing is illustrated.

[0062] Figure 50 The illustration shows an exemplary particle mixing and movement generated by voice coil mixing.

[0063] Figure 51 The illustration shows an exemplary particle mixing produced by mixing voice coils at varying frequencies.

[0064] Figure 52 The illustration shows the use of beads in microwells to capture target nucleic acids in a sample prior to recombinase polymerase amplification (RPA), as described in Example 1. Specifically, Figure 52 A shows that when beads (such as those coated with polyethyleneimine (PEI)) are used in microwells containing target nucleic acids (positive samples), a time-dependent increase in fluorescence intensity is observed in some microwells, while almost no fluorescence is observed in microwells that do not contain any target nucleic acids (negative samples). Figure 52 B shows that a fluorescence well count of 5,000 cp is sufficient to distinguish microwells containing target DNA (positive samples) from microwells that do not contain any target DNA (negative samples; 0 cp in the assay).

[0065] Figure 53 The illustration shows that using beads in microwells to capture target nucleic acids in a sample before amplification (such as RPA) successfully shortens the amplification time for such target nucleic acids compared to using conventional PCR amplification, as described in Example 1.

[0066] Figure 54 An exemplary embodiment of the device is illustrated. Detailed Implementation

[0067] An apparatus is disclosed. The apparatus optionally includes a sample analysis region for analyzing samples processed within the apparatus. Exemplary methods for using the apparatus are also provided herein. An optional reagent delivery device for use in conjunction with the apparatus is also provided. Sample mixing apparatus and methods are also provided.

[0068] Before describing this disclosure in more detail, it will be understood that this disclosure is not limited to the specific embodiments described, and therefore variations are possible. It will also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive, as the scope of this disclosure will be limited only by the appended claims.

[0069] It must be noted that, unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” as used herein and in the appended claims include plural indicators. Thus, for example, a reference to “primary region” includes a plurality of such primary regions, and a reference to “the orifice” includes a reference to one or more orifices and their equivalents known to those skilled in the art, and so on.

[0070] All publications mentioned herein are incorporated by reference to disclose and describe these methods and / or materials in conjunction with the content cited in those publications. This disclosure has the authority to determine the extent to which there is conflict between this disclosure and the publications incorporated by reference.

[0071] Detailed description Embodiments of this disclosure relate to methods and apparatus for analyzing one or more analytes in a sample. The sample can be a variety of different samples, including but not limited to biological samples, environmental samples, food samples, water samples, 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 body fluids, blood, venous blood, capillary blood, serum, plasma, urine, saliva, sweat, sputum, semen, mucus, tears, skin fluid, lymph, amniotic fluid, interstitial fluid, intestinal fluid, gastrointestinal fluid, bronchoalveolar lavage fluid, cerebrospinal fluid, feces, nasal discharge, vaginal secretions, tissues, organs, etc. 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, cervical tissue, skin, etc. In some cases, the sample source may be an organ or tissue, such as a biopsy sample, which may be dissolved by tissue disintegration / cell lysis.

[0072] definition Before describing embodiments of this disclosure, it will be understood that this disclosure is not limited to the specific embodiments described, and therefore variations are naturally possible. It will also 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 “approximately” used in conjunction with quantities includes the stated value and has a meaning defined by the context. When used in the context of a range, the modifier “approximately” should also be considered to disclose a range defined by the absolute values ​​of its two endpoints. For example, a range “from approximately 2 to approximately 10” also discloses a range “from 2 to 10”. The term “approximately” can refer to plus or minus 10% of the indicated number. For example, “approximately 10%” can indicate a range of 9% to 11%, and “approximately 1” can mean from 0.9 to 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; that is, in a given orientation, the upper component is located at a higher elevation than the lower component, but these terms would change if the components were flipped. The terms “inlet” and “outlet” are relative to the fluid flowing through them in a given structure; for example, fluid flows into the structure through an inlet and out of the structure through an outlet.

[0075] The terms “horizontal” and “vertical” are used to indicate directions relative to an absolute reference (i.e., the Earth's plane). However, these terms should not be interpreted as requiring structures to be absolutely parallel or absolutely perpendicular to each other. For example, the first vertical structure and the second vertical structure are not necessarily parallel to each other. The terms “top” and “bottom” are used to refer to surfaces in which the top is always higher than the bottom relative to an absolute reference (i.e., the Earth's surface). The terms “upward” and “downward” are also relative to an absolute reference; upward always goes against Earth's gravity, while downward always goes towards Earth's gravity.

[0076] As used herein, the words “comprising,” “including,” “having,” “having,” “may,” “containing,” and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional actions or structures. The singular forms “an,” “a,” and “the” include plural references unless the context clearly specifies otherwise. This disclosure also contemplates other embodiments “including embodiments or elements presented herein,” “consisting of embodiments or elements presented herein,” and “consisting substantially of embodiments or elements presented herein,” whether or not explicitly stated.

[0077] In describing the ranges of numbers in this article, each intermediate number with the same precision is explicitly envisioned. For example, for the range of 6-9, the numbers 7 and 8 are envisioned in addition to 6 and 9, and for the range of 6.0-7.0, the numbers 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 envisioned.

[0078] The terms "microbeads" and "microparticles" are used interchangeably herein and refer to substantially spherical solid supports. Microbeads or microparticles are substantially spherical solid supports that are affected by a magnetic field such that the magnetic field can attract or repel the microparticles or magnetic particles. Microbeads or microparticles may occupy or settle within a pore array (such as, for example, in a pore array in a detection module). Microparticles and microbeads may contain at least one specific binding member that binds to an analyte of interest, and at least one detectable label. Alternatively, microparticles and microbeads may contain a first specific binding member that binds to an analyte and a second specific binding member that also binds to an analyte and contains at least one detectable label.

[0079] The terms “nonfunctional beads,” “heper beads,” and “assistant particles” are used interchangeably and refer to a substantially spherical assisting solid support that is larger in diameter than a microparticle and is configured to be chemically inert relative to other components of the analyte. As used herein, an assisting particle refers to a spherical particle that is magnetic or paramagnetic but does not typically interact chemically with other particles, including microparticles, conjugates, and / or reagents. In some exemplary embodiments, the assisting particles may be coated to chemically interact with interfering substances, i.e., any material that would interfere with the determination or analysis of the analyte of interest within the targeted sample. In such embodiments, the assisting particles may also improve the binding efficiency of microparticles, including, for illustrative and non-limiting purposes, by binding with interfering substances. Additionally and alternatively, the solid support may be substantially spherical in shape, but is not limited to this shape.

[0080] The term "adjacent" is used in relation to areas and regions of an apparatus and sample processing apparatus. The term "adjacent" can mean that adjacent entities are close to each other even if separated by another entity. For example, if two primary areas are adjacent to each other, they can be close to each other even if they are separated by, for example, intermediate secondary areas. Similarly, if two regions of an apparatus are close to each other even if separated by, for example, channels or openings, these two regions are described as adjacent. If the apparatus is planar, for example having a width and length significantly greater than its thickness (such that the width and length define a first plane), then adjacent can be understood to mean that the areas or regions in question are positioned close to each other along the width and / or length direction (e.g., along the plane of the apparatus), for example, they can be side-by-side along the width and / or length direction.

[0081] The term "height" is used in relation to areas and regions of a device. For example, "height" can be measured as, for instance, the shortest distance between the inner surfaces of the top substrate and the bottom substrate at a given location. For example, if the height of a given primary region is to be measured, it can be measured as the shortest distance between the inner surfaces of the top substrate and the bottom substrate at that primary region. If the device is planar, for example, having a width and length significantly greater than its thickness (such that the width and length define a first plane), the height can be measured in a direction substantially parallel to the thickness (e.g., perpendicular to the first plane).

[0082] The assisting solid support may be larger in diameter than other support media within the storage region and is configured to not chemically interact with any other components within the mixing region. Specifically, the diameter of the assisting solid support (e.g., auxiliary beads) may 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% larger than the diameter of other support media (e.g., microparticles).

[0083] The assisting particles can be configured such that they do not chemically bind or pair with other components of the target solution, such as microparticles, target conjugates, and / or target analytes. In some exemplary embodiments, both the microparticles and the assisting particles can be magnetic, paramagnetic, or superparamagnetic particles (or any combination thereof). In such exemplary embodiments, both the microparticles and the assisting particles can form chains of linked particles under the influence of a magnetic field or force, thereby promoting mixing within the target solution.

[0084] like Figure 36 As shown, this configuration can be achieved by including multiple cooperating particles within the sample. These cooperating particles are larger than microparticles. In some embodiments, the cooperating particles may have a diameter between about 5 µm and about 15 µm, and in some exemplary embodiments, the cooperating particles may have a diameter between about 8 µm and about 12 µm, preferably about 10 µm. In some exemplary embodiments, the cooperating particles do not affect the immune response or other interactions between the microparticles and the analyte of interest, antigen, antibody, or other particles. The cooperating particles may also be magnetic or paramagnetic, thus contributing to the strength of the effective magnetic force that acts to move the sample (which contains both cooperating particles and microparticles). In some exemplary embodiments, the cooperating particles may include a negative surface charge, for example, but not limited to, greater than or equal to -30 mV. The cooperating particles may also be sized so as not to interfere with the determination of the target microparticles. The combination of active microparticles and inactive cooperating particles can achieve the advantages of both: 1) strong magnetic coupling with the sample to enable movement across the measurement surface; and 2) high detection sensitivity due to the reduction in the (total) amount of microparticles in the sample.

[0085] For illustrative and non-limiting purposes, the particle and assisting particle ratio may include a particle-to-assisting particle ratio between approximately 1:1 and 100:1. In some embodiments, the particle and assisting particle ratio may include a particle-to-assisting particle ratio between approximately 1:1 and 50:1. In some embodiments, the particle and assisting particle ratio may include a particle-to-assisting particle ratio between approximately 1:1 and 25:1. In some embodiments, the particle and assisting particle ratio may include a particle-to-assisting particle ratio between approximately 1:1 and approximately 20:1. In some embodiments, the particle and assisting particle ratio may include a particle-to-assisting particle ratio between approximately 1:1 and approximately 15:1. In some embodiments, the particle and assisting particle ratio may include a particle-to-assisting particle ratio between approximately 1:1 and approximately 10:1. In some embodiments, the particle and assisting particle ratio may include a particle-to-assisting particle ratio between approximately 1:1 and approximately 5:1. In some embodiments, the particle and assisting particle ratio may include a particle-to-assisting particle ratio between approximately 5:1 and approximately 25:1. In some embodiments, the particle and assisting particle ratio may include a particle-to-assisting particle ratio between approximately 10:1 and approximately 20:1. In some embodiments, the particle and assisting particle ratio may include approximately 20:1. In some embodiments, the particle and assisting particle ratio may include approximately 10:1. In some embodiments, the particle and assisting particle ratio may include approximately 9:1. In some embodiments, the particle and assisting particle ratio may include approximately 8:1. In some embodiments, the particle and assisting particle ratio may include approximately 7:1. In some embodiments, the particle and assisting particle ratio may include approximately 6:1. In some embodiments, the particle and assisting particle ratio may include approximately 5:1. In some embodiments, the particle and assisting particle ratio may include approximately 4:1. In some embodiments, the particle and assisting particle ratio may include approximately 3:1. In some embodiments, the particle and assisting particle ratio may include approximately 2:1. In some embodiments, the particle and assisting particle ratio may include approximately 1:1.

[0086] "Component," "multiple components," or "at least one component" generally refers to capture antibodies, detection reagents or conjugates, calibrators, controls, sensitivity panels, containers, buffers, diluents, salts, enzymes, enzyme cofactors, detection reagents, pretreatment reagents / solutions, matrices (e.g., as solutions), stop solutions, etc., which may be included in a kit for the assay of test samples (such as patient urine, serum, whole blood, tissue aspirates, or plasma samples) according to the methods described herein and other methods known in the art. Some components may be in solution or reconstituted by lyophilization for use in the assay.

[0087] As used interchangeably herein, “label” or “detectable label” refers to a portion attached to a specific binding member or analyte to make the reaction between the specific binding member and the analyte detectable, and such a labeled specific binding member or analyte is referred to as “detectably labeled.” Labels can produce signals detectable by visual or instrumental means. Various labels include: (i) tags attached to a specific binding member or analyte by a cleavable linker; or (ii) substances that produce signals, such as chromophores, fluorescent compounds, enzymes, chemiluminescent compounds, radioactive compounds, and so on. Representative examples of labels include light-producing portions (e.g., acridine compounds) and fluorescence-producing portions (e.g., fluorescein). Other labels are described herein. In this respect, a portion itself may be undetectable but may become detectable upon reaction with another portion. The use of the term “detectably labeled” is intended to encompass such labels.

[0088] As used interchangeably in this document, “specifically binding partner” or “specifically binding member” refers to one of two distinct molecules that specifically recognizes the other molecule compared to its substantially less recognition of other molecules. This one of the two distinct molecules has a region on its surface or in a cavity that specifically binds to the particular spatial and polar organization of the other molecule and is thus defined as complementary to it. A molecule can be a member of a specific binding pair. For example, specific binding members can include, but are not limited to, proteins such as receptors, enzymes, antibodies and aptamers, peptides, nucleotides, oligonucleotides, nucleic acids, polynucleotides, and combinations thereof.

[0089] As used herein, "specific binding" or "specifically binding" can refer to the interaction of an antibody, protein, or peptide with a second chemical species, where the interaction depends on the presence of a specific structure on the chemical species (e.g., an antigenic determinant or epitope); for example, the antibody recognizes and binds to a specific protein structure, rather than the protein in general. If the antibody is specific for epitope "A," then in a reaction containing labeled "A" and an antibody, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A that binds to the antibody.

[0090] The terms “polynucleotide” and “nucleic acid”, used interchangeably herein, refer to polymers of nucleotides of any length, whether ribonucleotides or deoxynucleotides. Therefore, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The terms “polynucleotide” and “nucleic acid” should be understood to include single-stranded (e.g., sense or antisense) and double-stranded polynucleotides as applicable to the described embodiments.

[0091] In the context of "hybridizable," "complementary," or "substantially complementary," it means that nucleic acids (e.g., RNA, DNA) contain a nucleotide sequence that allows them to bind nonvalently (i.e., forming Watson-Crick base pairs and / or G / U base pairs) to another nucleic acid in a sequence-specific, antiparallel (i.e., one nucleic acid specifically binds to a complementary nucleic acid) manner, "annealing," or "hybridizing," under appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength. Standard Watson-Crick base pairings include: adenine / adenosine (A) paired with thymidine / thymidine (T), A paired with uracil / uridine (U), and guanine / guanosine (G) paired with cytosine / cytidine (C). Inosine (I) bases pair with cytosine / cytidine. Additionally, for hybridization between two RNA molecules (e.g., dsRNA), and for hybridization between DNA and RNA molecules (e.g., when a DNA target nucleic acid base pairs with a guide RNA base, etc.): G can also pair with U. For example, in the context of tRNA anticodon base pairing with codons in mRNA, G / U base pairing is partially responsible for the degeneracy (i.e., redundancy) of the genetic code. Therefore, in the context of this disclosure, G (e.g., G in the protein-binding segment (e.g., dsRNA duplex) of the guiding RNA molecule; G in the target nucleic acid (e.g., target DNA or RNA) that base pairs with the sensor RNA) is considered complementary to both U and C. For example, when a G / U base pair can be generated at a given nucleotide position in the protein-binding segment (e.g., dsRNA duplex) of the sensor RNA molecule, that position is not considered non-complementary, but rather complementary.

[0092] Hybridization requires two nucleic acids to contain complementary sequences, although base mismatches are possible. Suitable conditions for hybridization between two nucleic acids depend on their length and degree of complementarity (variables well known in the art). The greater the complementarity between the two nucleotide sequences, the higher the melting temperature (Tm) of the hybrid containing those sequences. Typically, hybridizable nucleic acids are 8 nucleotides or longer (e.g., 10 nucleotides or longer, 12 nucleotides or longer, 15 nucleotides or longer, 20 nucleotides or longer, 22 nucleotides or longer, 25 nucleotides or longer, or 30 nucleotides or longer).

[0093] It should be understood that a polynucleotide sequence does not need to be 100% complementary to its target nucleic acid sequence to be specifically hybridizing. Furthermore, polynucleotides can hybridize on one or more segments such that segments in the middle or adjacent segments do not participate in the hybridization event (e.g., loop structures or hairpin structures, 'bumps', etc.). Polynucleotides 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 with the target region within the target nucleic acid sequence to which it will hybridize. For example, the following antisense nucleic acid would represent 90% complementarity: in this antisense nucleic acid, 18 out of the 20 nucleotides of the antisense compound are complementary to the target region and will therefore specifically hybridize. The remaining non-complementary nucleotides can aggregate or disperse with complementary nucleotides and do not need to be linked to each other or to complementary nucleotides. The percentage complementarity between specific stretchers of nucleic acid sequences within a nucleic acid can be determined using any convenient method. Example methods include the BLAST program (Basic Local Alignment Search Tool) and the PowerBLAST program (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 for Unix Version 8, Genetics and Computational Research Group, University Research Park, Madison Wis.), for example, using the default settings, which use 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 polymer of amino acids of any length, which may include coding and non-coding amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having a modified peptide backbone.

[0095] As used interchangeably in this document, “analyte,” “target analyte,” and “analyte of interest” refer to a substance, material, or chemical component whose presence, absence, and / or amount is analyzed in a biological sample obtained from a subject. In some respects, an analyte is a biomolecule. Non-limiting examples of biomolecules include macromolecules such as proteins, lipids, and carbohydrates. In some cases, an analyte may be a hormone, antibody, growth factor, cytokine, enzyme, receptor (e.g., nerve, hormone, nutrient, and cell surface receptors) or its ligand, cancer markers (e.g., PSA, TNF-α), markers of myocardial infarction (e.g., troponin, creatine kinase-myocardial marker (CK-MB), B-type natriuretic peptide (also known as brain natriuretic peptide; BNP), N-terminal pro-brain natriuretic peptide (NT-proBNP), etc.), toxins, drugs (e.g., addictive drugs), metabolites (e.g., including vitamins), etc. Non-limiting examples of protein analytes include peptides, polypeptides, protein fragments, protein complexes, fusion proteins, recombinant proteins, phosphoproteins, glycoproteins, lipoproteins, etc.

[0096] As used herein, one or more “antibodies” refer to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies (such as, but not limited to, birds (e.g., ducks or geese), sharks, whales, and mammals including non-primates (e.g., cattle, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, etc.) or non-human primates (e.g., monkeys, chimpanzees, 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 their preparation methods are described in Wu, The terms C. et al. describe antibodies in Nature Biotechnology (25(11):1290-1297 (2007)) and PCT International Application WO 2001 / 058956, the contents of which are incorporated herein by reference, as well as epitope-binding fragments of the functional activity of any of the above. Antibodies comprise immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing analyte binding sites. Immunoglobulin molecules can belong to any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. For simplicity, antibodies against analytes are often referred to herein as “anti-analyte antibodies” or simply as “analyte antibodies.”

[0097] As used herein, “antibody fragment” refers to a portion of a complete antibody that includes the antigen-binding site or variable region. This portion does not include the constant heavy chain domain (i.e., CH2, CH3, or CH4, depending on the antibody isotype) of the Fc region of the complete 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, biantibodies, single-chain Fv (scFv) molecules, single-chain polypeptides containing only one light chain variable domain, single-chain polypeptides containing three CDRs of the light chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing three CDRs of the heavy chain variable region.

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

[0099] A capillary pressure barrier is a meniscus pinning structure that creates a capillary stop and enables meniscus alignment. Meniscus pinning occurs when energy must be applied to advance the meniscus past its pinned position. Typically, sharp ridges are used inside the channel or chamber to create a stable meniscus alignment feature that forces the meniscus to deform, making advance energy-unfavorable. The meniscus then tends to align along the resulting capillary pressure barrier unless additional energy is applied (in the form of, for example, an increase in fluid pressure).

[0100] A phase guide is defined as a capillary pressure barrier that spans the entire length of the advancing phase front, such that the advancing phase front aligns itself along the phase guide before intersecting with it. The primary function of the phase guide is to define and control the flow within a movable meniscus. Position, shape, advance, or some other physical characteristic can be influenced by the combined effects of the design of the stabilizing capillary pressure barrier and the energy (typically fluid pressure) applied to the fluid present on one or the other side of the meniscus. The primary and secondary regions of this disclosure differ from the phase guide in that they do not define a movable meniscus or control the flow within the advancing phase front. The primary and secondary regions pin the droplet in quiescent regions, and once deposited, it will not advance, flow, or move from these regions.

[0101] As used herein, “contact angle” refers to the angle at which a liquid surface meets a solid surface. The contact angle describes the shape of a droplet resting on a solid substrate surface and is the contact angle of the liquid on the solid substrate surface, measured at the contact line where the liquid, solid, and gas meet. Surfaces with a contact angle <90° are considered hydrophilic, and surfaces with a contact angle ≥90° are considered hydrophobic. As the contact angle of a surface increases, the surface is considered more hydrophobic. Unless otherwise stated, when the term contact angle is used, it refers to the 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, this document (including the definitions) shall prevail. Preferred methods and materials are described below; however, similar or equivalent methods and materials may be used in the practice or testing of this disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety to disclose and describe these methods and / or materials in conjunction with the content cited in the publications. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0103] 1. Overview This document provides an apparatus (also known as a sample processing apparatus) for sample processing and analyte detection. To provide organization for the description of the apparatus, firstly, an overall apparatus design will be disclosed. Secondly, the purpose of the apparatus will be disclosed. Thirdly, the benefits of the apparatus will be disclosed. After an overview of the basic design of the apparatus, a detailed disclosure of the design of the apparatus will be disclosed, including the arrangement of the sample processing area and, optionally, the arrangement of the sample analysis area. After a detailed disclosure of the apparatus design, an exemplary optional reagent delivery device will be disclosed. In some embodiments, bulk reagent delivery may be used instead of the optional reagent delivery device. After disclosing the exemplary optional reagent delivery device, an exemplary sample mixing apparatus and method will be disclosed.

[0104] I. Device Design The device includes a top substrate bonded to a bottom substrate, wherein the top substrate bonded to the bottom substrate forms two or more primary regions separated by two or more secondary regions. Each primary region contains an opening in the top substrate. In some embodiments, each secondary region contains an opening in the top substrate. In some embodiments, each secondary region does not contain an opening in the top substrate. A sample processing region is formed between the top substrate and the bottom substrate. The sample processing region is unbounded throughout the region (i.e., the interior of the device contains both primary and secondary regions). In some embodiments, the top substrate and the bottom substrate form a first distance between the top substrate and the bottom substrate within the primary regions, and a second distance between the top substrate and the bottom substrate within the secondary regions. In some embodiments, the first distance is smaller than the second distance.

[0105] The first distance can be smaller than the second distance because, in at least some primary regions, the pads in the primary regions protrude into the sample processing area. This means that, in the secondary regions, the distance from the pads to the inner surface of the bottom substrate is smaller than the distance from the inner surface of the top substrate to the inner surface of the bottom substrate.

[0106] The top substrate may be substantially planar, and 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 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 the same as the width and length of the bottom substrate, or the width and / or length may be different.

[0107] The device can be described as a "two-dimensional matrix droplet array" because it comprises multiple primary and secondary regions arranged substantially along two dimensions of a plane. At least some of the primary and secondary regions are configured to receive droplets of fluid.

[0108] In some embodiments, the device includes a first substrate and a second substrate positioned on the first substrate. The second substrate includes a sidewall surrounding at least a portion of its periphery, wherein the first substrate, the sidewall, and the second substrate define a central chamber therebetween. The second substrate includes a surface facing the central chamber, which includes a plurality of recessed elements and a plurality of protruding elements, wherein a primary region is defined between the surfaces of the plurality of protruding elements facing the first substrate and the surfaces of the first substrate facing the second substrate, and wherein a secondary region is defined between the surfaces of the recessed elements facing the first substrate and the surfaces of the first substrate facing the second substrate. The second substrate has an opening in one or more of the primary regions.

[0109] In some embodiments, the device includes a first substrate and a second substrate positioned on the first substrate. The first substrate includes a sidewall surrounding at least a portion of its periphery, wherein the first substrate, the sidewall, and the second substrate define a central cavity therebetween. The second substrate includes a surface facing the central cavity, which includes a plurality of recessed elements and a plurality of protruding elements, wherein a primary region is defined between the surfaces of the plurality of protruding elements facing the first substrate and the surfaces of the first substrate facing the second substrate, and wherein a secondary region is defined between the surfaces of the recessed elements facing the first substrate and the surfaces of the first substrate facing the second substrate. The second substrate has an opening in one or more of the primary regions.

[0110] In some embodiments, the device includes: a first substrate; a spacer layer positioned on a surface of the first substrate, wherein the spacer layer is disposed around at least a portion of a periphery of the first substrate; and a second substrate positioned on the first substrate. The first substrate includes a sidewall surrounding at least a portion of the periphery of the second substrate, wherein the first substrate, the sidewall, and the second substrate define a central cavity therebetween. The second substrate includes a surface facing the central cavity, comprising a plurality of recessed elements and a plurality of protruding elements, wherein a primary region is defined between the surfaces of the plurality of protruding elements facing the first substrate and the surfaces of the first substrate facing the second substrate, and wherein a secondary region is defined between the surfaces of the recessed elements facing the first substrate and the surfaces of the first substrate facing the second substrate. The second substrate has an opening in one or more of the primary regions. In some embodiments, the spacer layer is selected from the group consisting of: an adhesive layer, a gasket layer, and a raised feature layer. In some embodiments, the spacer layer is an adhesive layer. In some embodiments, the spacer layer is a gasket layer. In some embodiments, the spacer layer is a first adhesive layer, a gasket 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 an ultraviolet (UV) bonded adhesive.

[0111] 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".

[0112] In addition to the optional sample analysis area, the device includes a sample processing area. The sample processing area is configured to allow for modular sample processing. "Modular" means that, depending on the assay, the primary and / or secondary areas can be filled with specific reagents for a given assay (e.g., wash buffer, lysis buffer, coupling reagent, detection reagent, amplification reagent, etc.), and the device is compatible with different types of assays (e.g., immunoassay, nucleic acid analysis, metabolite analysis, clinical chemistry, etc.) and different ways of performing the assay (e.g., adjusting the number of washing steps, coupling steps, etc.). Microparticles are used to move the sample or components of the sample from one primary area to another. Generally, the sample is added to a primary area within the sample processing area. Microparticles are added simultaneously, before, or after the sample is added to the same primary and / or secondary area where the sample has been added or will be added. In some embodiments, microparticles are already present in the primary and / or secondary areas where the sample has been added. In some embodiments, the microparticles are microparticles and assisting particles. The sample or components of the sample are then bound to the microparticles, and the microparticles or microparticles and assisting particles move to different primary areas using a magnetic field. After sample processing is complete, microparticles or microparticles and assist particles move to the sample analysis area on the device or leave the device.

[0113] Figure 1 Illustrations of an embodiment of the device are disclosed. In this embodiment, device 100 includes a sample processing region 110. The sample processing region 110 is unbounded. By unbounded, it means that there is no physical barrier separating the primary region 101 and the secondary region 107. Although only a single element is marked for the primary region 101, each square (101) will represent a separate primary region. Although only a single element is marked for the secondary region 107, each space between the squares will represent a separate secondary region. The sample processing region includes the primary region 101, which has an opening 103. Although only a single element is marked for the opening 103 in the primary region, each opening (103) in the square (101) is intended to represent a separate opening in the primary region. The primary region is adjacent to the secondary region 107. The secondary region may include an opening 104. Although only a single element is marked for the opening in the secondary region 107, each opening (104) between the squares represents a separate opening in the secondary region. The sample processing area 110 may be connected to the tertiary area (i.e., the sample detection area) 112 by a transition area 113. The device may also include a quaternary area 114 (i.e., a hydrophilic liquid pore) connected to the tertiary area. The top and bottom substrates are bonded by an adhesive or clip 111. In some embodiments, the top and bottom substrates are bonded by an adhesive layer between the top and bottom substrates. In some embodiments, the adhesive is an ultraviolet (UV) bonded adhesive. In some embodiments, the top and bottom substrates are bonded using laser welding.

[0114] The following text was published Figure 1Alternative embodiments. In this embodiment, device 100 includes a sample processing region 110. Device 100 includes a second substrate positioned on a first substrate. The second substrate includes a surface facing a central chamber, which includes a plurality of recessed elements and a plurality of protruding elements. A primary region 101 is defined between the surfaces of the plurality of protruding elements facing the first substrate and the surfaces of the first substrate facing the second substrate. A secondary region 107 is defined between the surfaces of the plurality of recessed elements facing the first substrate and the surfaces of the first substrate facing the second substrate. The sample processing region 110 is unbounded. By unbounded, it means that there is no physical barrier separating the primary region 101 and the secondary region 107. Although only a single element is marked for the primary region 101, each square (101) will represent a separate primary region. In some embodiments, the primary regions are discrete. By “discrete,” it means that each primary region is physically separated from every other primary region. Although only a single element is marked for the secondary region 107, each space between the squares will represent a separate secondary region. In some embodiments, the secondary regions are connected. In terms of “connection,” it means that the secondary regions are physically associated with each other. The sample processing region includes a primary region 101 having an opening 103. Although only a single element is marked for the opening 103 in the primary region, each opening (103) in the square (101) is intended to represent a separate opening in the primary region. The primary region is adjacent to a secondary region 107. The secondary region may include an opening 104. Although only a single element is marked for the opening in the secondary region 107, each opening (104) between the squares represents a separate opening in the secondary region. The sample processing region 110 may be positioned adjacent to a sample analysis region including a tertiary region (i.e., a sample detection region) 112, a quaternary region 114 (i.e., a hydrophilic liquid pore) and a pentagonal region 115 (i.e., a hydrophobic liquid pore), wherein the tertiary region 112, the quaternary region 114, and the pentagonal region 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. A level 5 region 115 is located at the first end of the sample analysis region. The level 5 region includes an opening across the second substrate. A level 4 region 114 is located at the second end of the sample analysis region. The level 4 region includes a cylindrical opening across the second substrate. A level 3 region 112 is located at the midpoint between the first end (e.g., level 5 region 115) and the second end (e.g., level 4 region 115) of the sample analysis region. The sample analysis region may be connected to the sample processing region 110 by a transition region 113. A second substrate positioned above the first substrate is bonded together by an adhesive or clip 111. In some embodiments, the second substrate and the first substrate are bonded by an adhesive layer between the second substrate and the first substrate. In some embodiments, the adhesive is an ultraviolet (UV) bonded adhesive. In some embodiments, the second substrate and the first substrate are bonded using laser welding.

[0115] Figure 26B A schematic representation of the cross-section of the sample processing region in the embodiments is disclosed. Figure 26B In this design, the sample processing region 2605 includes a primary region 2608 and two secondary regions 2607. The sample processing region 2605 is formed by a top substrate 2609 bonded to a bottom substrate 2610. The primary region 2608 includes an opening 2603. The secondary regions include openings 2604. A sample or reagent 2602 can be added through the opening 2603 of the primary region 2608, wherein the sample or reagent 2602 is held in place by capillary forces generated by the opening 2603 and surface tension promoted by the edges of pads 2601 in the primary region 2608. Typically, the secondary regions include air and serve as hydrophobic regions assisting in holding the sample or reagent in the primary region. In some embodiments, the secondary regions also contain reagents or samples. In some embodiments, the secondary regions do not contain samples or reagents. In some embodiments, a portion of the secondary region contains samples or reagents, and a portion of the secondary region does not contain samples or reagents.

[0116] The following describes Figure 26B Alternative embodiments. Figure 26B A schematic representation of the cross-section of the sample processing region in the embodiments is disclosed. Figure 26BIn this embodiment, the sample processing region 2605 includes a primary region 2608 and two secondary regions 2607. The sample processing region 2605 is defined by a second substrate 2609 positioned on a first substrate 2610. The first substrate 2610 or the second substrate 2609 includes a sidewall surrounding at least a portion of the 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 includes a surface facing the central chamber (i.e., the space occupied by 2607 and 2608) (i.e., the inner portion of 2609), which includes a protruding element 2601 and a recessed element (i.e., the upper portion of 2607). Although only a single protruding element is shown, it will be understood that the device includes multiple protruding elements. Although only two recessed elements are shown, it will be understood that the device includes multiple recessed elements. A primary region 2608 is defined between the surface of the protruding element facing the first substrate 2610 (i.e., the inner portion of 2601) and the surface of the first substrate facing the second substrate (i.e., the inner portion of 2610). A secondary region 2607 is defined between the surface of the recessed element (i.e., the upper portion of 2607) and the surface of the first substrate facing the second substrate (i.e., the inner portion of 2610). A second substrate 2609 includes an opening 2603 in the primary region. A second substrate 2609 includes an opening 2604 in the secondary region. A sample or reagent 2602 can be added through the opening 2603 of the primary region 2608, wherein the sample or reagent 2602 is held in place by the capillary force generated by the opening 2603 and the surface tension promoted by the protruding element 2601 in the primary region 2608. Typically, the secondary region includes air and serves as a hydrophobic region to assist in holding the sample or reagent in the primary region. In some embodiments, the secondary region also contains a reagent or sample. In some embodiments, the secondary region does not contain a sample or reagent. In some embodiments, a portion of the secondary region contains a sample or reagent, and a portion of the secondary region does not contain a sample or reagent.

[0117] Figure 37A A schematic representation of a cross-section of the sample processing region of the apparatus in the embodiments is disclosed, wherein the second substrate includes sidewalls. Figure 37AIn this embodiment, device 3700 includes a first substrate 3702 and a second substrate 3701 positioned on the first substrate 3702. The second substrate 3701 includes a sidewall 3703 surrounding at least a portion of the 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 includes a surface facing the central chamber 3704, which includes a plurality of protruding elements (3705a, 3705b, and 3705c) and a plurality of recessed elements (3706a, 3706b, and 3706c). The boundary between the sidewall 3703 of the second substrate and the first substrate 3702 is defined by 3707 (black line). The primary regions (3710a, 3710b, and 3710c; bounded by thick black lines) are defined between the surfaces 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 (the inner surface of 3702). The secondary regions (3711a, 3711b, 3711c, and 3711d; bounded by thick black lines) are defined between the surfaces 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 (the inner surface of 3702). The second substrate 3701 has an opening 3708 in the primary regions. The second substrate 3701 has an opening 3709 in one or more of the secondary regions.

[0118] Figure 37B A schematic representation of a cross-section of the sample processing region of the apparatus in the embodiments is disclosed, wherein the first substrate includes sidewalls. Figure 37BIn this embodiment, device 3720 includes a first substrate 3722 and a second substrate 3721 positioned on the first substrate 3722. The first substrate 3722 includes a sidewall 3723 surrounding at least a portion of the periphery of the first substrate. The first substrate 3722, the sidewall 3723, and the second substrate 3721 define a central cavity 3724 therebetween. The second substrate includes a surface facing the central cavity, which includes a plurality of protruding elements (3725a, 3725b, and 3725c) and a plurality of recessed elements (3726a, 3726b, and 3726c). The boundary between the sidewall 3723 of the first substrate and the second substrate 3722 is defined by 3727 (black line). Primary regions (3730a, 3730b, and 3730c; bounded by thick black lines) are defined between the surfaces 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 (the inner surface of 3722). Secondary regions (3731a, 3731b, 3731c, and 3731d; bounded by thick black lines) are defined between the surfaces 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 (the inner surface of 3722). The second substrate 3721 has an opening 3728 in the primary regions. The second substrate 3721 has an opening 3729 in one or more of the secondary regions.

[0119] Figure 37C A schematic representation of a cross-section of the sample processing region of the apparatus in the embodiments is disclosed, wherein the apparatus includes a spacer layer. Figure 37CIn this embodiment, device 3740 includes a first substrate 3742 and a spacer layer 3743 positioned on the first substrate 3742, wherein the spacer layer is disposed around at least a portion of the periphery of the first substrate 3742. In some embodiments, the at least portion of the periphery includes at least two, at least three, or at least four peripheral sides of a second substrate. In some embodiments, the spacer layer is selected from the group consisting of adhesive layers, gasket layers, and raised feature layers. In some embodiments, the spacer layer is an adhesive layer. In some embodiments, the spacer layer is a gasket layer. In some embodiments, the spacer layer is a first adhesive layer, a gasket layer, and a second adhesive layer. A second substrate 3741 is positioned on the spacer layer 3743. In some embodiments, the adhesive is an ultraviolet (UV) bonded adhesive. The first substrate 3742, the spacer layer 3743, and the second substrate 3741 define a central chamber 3744 therebetween. The second substrate includes a surface facing the central cavity, which includes a plurality of protruding elements (3745a, 3745b, and 3745c) and a plurality of recessed elements (3746a, 3746b, and 3746c). The boundaries of the spacer layer 3743, the first substrate 3742, and the second substrate 3741 are defined by 3747 (black line). The primary region (3750a, 3750b, and 3750c; bounded by thick black lines) is defined between the surfaces 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 (the inner surface of 3742). The secondary regions (3751a, 3751b, 3751c, and 3751d; bounded by thick black lines) are defined between the surfaces 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 (the inner surface of 3742). The second substrate 3741 has an opening 3748 in the primary region. The second substrate 3741 has an opening 3749 in one or more of the secondary regions.

[0120] Can Figure 37A , Figure 37B and Figure 37C The schematic diagrams disclosed herein can be applied to any of the devices disclosed herein. Figure 37A , Figure 37B and Figure 37C The schematic diagram disclosed herein is applied to Figure 1-15 , Figure 17-1 The apparatus disclosed in Figures 9 and 32.

[0121] As discussed above, the device can use capillary forces and surface tension to retain fluid within its various zones. This effect arises from the small size of the device, such as the small size associated with the primary and secondary zones. In particular, the behavior of the contained fluid is dominated by capillary forces and surface tension rather than gravity. This means, for example, that an opening can provide capillary forces to retain fluid within a given zone, and the edges of a pad can provide surface tension to retain fluid within the primary zone.

[0122] The top and bottom substrates of this disclosure can be made of a range of different materials, such that these materials facilitate the methods and designs disclosed herein. The materials can be rigid or flexible. Stiffness and flexibility can be controlled by the materials used and the thickness of the materials in the top and bottom substrates. In some embodiments, the top and bottom substrates are made of the same material. In some embodiments, the top and bottom substrates are made of different materials. In some embodiments, the entire top substrate is made of the same material. In some embodiments, the top substrate is made of a combination of materials, wherein a portion of the top substrate is made of one material, and different portions of the top substrate are made of different materials. In some embodiments, the bottom substrate is made of a combination of materials, wherein a portion of the bottom substrate is made of one material, and different portions of the bottom substrate are made of different materials. For example, materials used in this disclosure include, but are not limited to, glass, silicon, ceramics, metals, polymethyl methacrylate (PMMA), polystyrene (PS), polycarbonate (PC), cyclic olefin copolymers (COC), cyclic olefin polymers (COP), polypropylene (PP), polyurethane (PU), polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC), polydimethylsiloxane (PDMS), acrylonitrile butadiene styrene (ABS), polylactic acid (PLA), thermoplastic PU, transparent resins, polyethylene glycol diacrylate (PEGDA), thin films, etc. In some embodiments, the top substrate (or second substrate) and the bottom substrate (or first substrate) have the same hydrophobicity. In some embodiments, the top substrate (second substrate) and the bottom substrate (or first substrate) have different hydrophobicities.

[0123] 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 polyvinyl chloride (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 polylactic 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 transparent resin. In some embodiments, all or a portion of the top substrate is made of polyethylene glycol diacrylate (PEGDA).

[0124] 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 the bottom 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 polyvinyl chloride (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 polylactic 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 transparent 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. Due to the deformability of the thin film, it provides the advantage of enhanced mixing of fluids in the device.

[0125] I(a). Sample processing area The sample processing area of ​​the apparatus disclosed herein has an arrangement of primary and secondary regions. In some embodiments, the primary regions are discrete. "Discrete" means that each primary region is physically separate from every other primary region. In some embodiments, the secondary regions are connected. "Connected" means that the secondary regions are physically associated with each other. There is a range in the number of primary and secondary regions, depending on the specific embodiment described. The arrangement of the primary and secondary regions is also variable and depends on the specific embodiment discussed.

[0126] The device includes a range in terms of the number of primary and secondary zones. For example, the device may include 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 include 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, twenty or more, twentieth or more, eleven or more, twentieth or more, thirteen ... Each adjacent primary region is separated by a secondary region, such that if there are 3 primary regions on a straight line, there will be a first secondary region between the first and second primary regions and a second secondary region between the second and third primary regions.

[0127] The primary and secondary zones can be arranged in several different patterns. For example, the pattern can be a grid, lines, or a non-grid pattern. When the pattern is a grid, the grid can 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 can be 1x3, 1x4, 1x5, 1x6, 1x7, 1x8, 1x9, or 1x10. When the pattern is a non-grid, it can be in the form of a honeycomb pattern, such as... Figure 11 As depicted in [the text]. When the pattern is non-grid, the non-grid can take the form of an irregular pattern, such as... Figure 27A As depicted in the text.

[0128] The pads or protruding elements in the primary zones can be separated by specific distances such that fluid contained in one primary zone does not bridge to another. The distance between the primary zones can be a range of values. For example, the distance between the primary zones can be approximately 1 mm to approximately 2 mm. In some embodiments, the distance between the primary zones can be greater than 2 mm. The range or specific distance can be any distance in between. For example, the distance between primary zones can be at least approximately 1.00 mm, at least approximately 1.01 mm, at least approximately 1.02 mm, at least approximately 1.03 mm, at least approximately 1.04 mm, at least approximately 1.05 mm, at least approximately 1.06 mm, at least approximately 1.07 mm, at least approximately 1.08 mm, at least approximately 1.09 mm, at least approximately 1.10 mm, at least approximately 1.11 mm, at least approximately 1.12 mm, at least approximately 1.13 mm, at least approximately 1.14 mm, at least approximately 1.15 mm, at least approximately 1.16 mm, at least approximately 1.17 mm, at least approximately 1.18 mm, at least approximately 1.19 mm, at least approximately 1.20 mm, at least approximately 1.21 mm, at least approximately 1.22 mm, at least approximately 1.23 mm, at least approximately 1.24 mm, at least approximately 1.25 mm, at least approximately 1.26 mm, at least approximately 1.27 mm, at least approximately 1.28 ... mm, at least approximately 1.29 mm, at least approximately 1.30 mm, at least approximately 1.31 mm, at least approximately 1.32 mm, at least approximately 1.33 mm, at least approximately 1.34 mm, at least approximately 1.35 mm, at least approximately 1.36 mm, at least approximately 1.37 mm, at least approximately 1.38 mm, at least approximately 1.39 mm, at least approximately 1.40 mm, at least approximately 1.41 mm, at least approximately 1.42 mm, at least approximately 1.43 mm, at least approximately 1.44 mm, at least approximately 1.45 mm, at least approximately 1.46 mm, at least approximately 1.47 mm, at least approximately 1.48 mm, at least approximately 1.49 mm, at least approximately 1.50 mm, at least approximately 1.51 mm, at least approximately 1.52 mm, at least approximately 1.53 mm, at least approximately 1.54 mm, at least approximately 1.55 mm, at least approximately 1.56 mm, at least approximately 1.57 mm, at least approximately 1.58 mm mm, at least approximately 1.59 mm, at least approximately 1.60 mm, at least approximately 1.61 mm, at least approximately 1.62 mm, at least approximately 1.63 mm, at least approximately 1.64 mm, at least approximately 1.65 mm, at least approximately 1.66 mm, at least approximately 1.67 mm, at least approximately 1.68 mm, at least approximately 1.69 mm, at least approximately 1.70 mm, at least approximately 1.71 mm, at least approximately 1.72 mm, at least approximately 1.73 mm, at least approximately 1.74 mm, at least approximately 1.75 mm, at least approximately 1.76 mm, at least approximately 1.77 mm, at least approximately 1.78 mm, at least approximately 1.79 mm, at least approximately 1.80 mm, at least approximately 1.81 mm, at least approximately 1.82 mm, at least approximately 1.83 mm, at least approximately 1.84 mm, at least approximately 1.85 mm, at least approximately 1.86 mm, at least approximately 1.87 mm, at least approximately 1.88 mm, at least approximately 1.89 mm, at least approximately 1.90 mm, at least approximately 1.91 mm, at least approximately 1.92 mm, at least approximately 1.93 mm, at least approximately 1.94 mm, at least approximately 1.95 mm, at least approximately 1.96 mm, at least approximately 1.97 mm, at least approximately 1.98 mm, at least approximately 1.99 mm, or at least approximately 2 mm.

[0129] A sample can be added to a fixed primary region or a variable primary region. A "fixed" primary region means that the primary region for sample addition is fixed in place, i.e., a specific primary region. In some embodiments, a fixed primary region has one or more openings. For example, one or more, two or more, three or more, four or more, or five or more openings may be present. A "variable" primary region means that a sample can be added to any of the primary regions present in the device. The terms "fixed primary region" and "variable primary region" are used only to refer to a primary region on which a sample is added and not to a primary region on which a sample is not directly added.

[0130] In some embodiments, the sample processing region includes a fixed primary region. When the sample processing region includes a fixed primary region, the fixed primary region may be larger than the primary region. In some embodiments, the fixed primary region is the same size as the primary region. In some embodiments, the fixed primary region is separate from the primary region. In some embodiments, the fixed primary region is a sample mixing region. In some embodiments, the fixed primary region has one or more openings. For example, one or more, two or more, three or more, four or more, or five or more openings may be present. In some embodiments, the sample processing region includes two or more fixed primary regions. The fixed primary region may have one or more secondary features that assist in mixing the fluid contained therein. A range of secondary features may exist in the fixed primary region. For example, 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 may be present. In some embodiments, the secondary features are based on Figure 31 In other words, secondary features may have any shape that facilitates mixing that can occur in a fixed primary region. Non-limiting examples of shapes include, but are not limited to, rectangles, circles, triangles, pentagons, hexagons, heptagons, octagons, decagons, dodecagons, amoebas, irregular shapes, etc.

[0131] In some embodiments, the fixed primary region is formed by laterally extending protruding elements in the second substrate facing the first substrate, wherein the laterally extending protruding elements have a surface facing the first substrate with a larger surface area than the surfaces of the plurality of protruding elements facing the first substrate. The laterally extending protruding elements may be referred to as third protruding elements. When the sample processing region includes the fixed primary region, the fixed primary region may be larger than the primary region. In some embodiments, the fixed primary region has a surface facing the first substrate with a larger surface area than the surfaces of the plurality of protruding elements facing the first substrate. In some embodiments, the fixed primary region is separate from the primary region. In some embodiments, the fixed primary region is a sample mixing region. In some embodiments, the fixed primary region is a cantilever, wherein only a portion of the fixed primary region is attached to the second substrate. In some embodiments, the fixed primary region has one or more openings. For example, one or more, two or more, three or more, four or more, or five or more openings may be present. In some embodiments, the sample processing region includes two or more fixed primary regions. The fixed primary region may have one or more secondary features that assist in mixing the fluids contained therein. A range of secondary features may exist in the fixed primary region. For example, 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 feature is based on Figure 31 In other words, secondary features may have any shape that facilitates mixing that can occur in a fixed primary region. Non-limiting examples of shapes include, but are not limited to, rectangles, circles, triangles, pentagons, hexagons, heptagons, octagons, decagons, dodecagons, amoebas, irregular shapes, etc.

[0132] In some embodiments, the sample processing region includes a variable primary region. This variable primary region can be any of the primary regions present in the sample processing region. The sample can be added to the variable primary region before, after, or simultaneously with any other reagent added to the device.

[0133] I(b). Sample analysis area The optional sample analysis region of the device disclosed herein includes one or more sample detection regions. 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 the device does not contain a sample analysis region, analysis of the sample or analyte contained therein is performed outside the device. There is a range in both the type and number of sample detection regions. Types of sample detection regions include, but are not limited to: pores or micropores, one or more chambers, nanopores, etc. In some embodiments, the sample analysis region includes hydrophobic liquid pores and hydrophilic liquid pores.

[0134] In some embodiments, the sample analysis region has a first end laterally separated from the second end and is defined by a surface of the second substrate facing the first substrate and a 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 includes an enlarged protruding element, wherein the surface of the enlarged protruding element facing the first substrate has a larger surface area than the surfaces 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.

[0135] In some embodiments, the sample analysis region includes a sample detection region, which includes wells. Wells are typically designed to accommodate one or more microparticles. In some embodiments, a well can accommodate only a single microparticle. In some embodiments, a well can accommodate two microparticles. In some embodiments, a well can accommodate three or more microparticles. A series of wells may exist within the sample detection region. For example, the sample detection region may contain approximately 100 or more, approximately 200 or more, approximately 500 or more, approximately 1000 or more, approximately 2000 or more, approximately 3000 or more, approximately 4000 or more, approximately 5000 or more, approximately 6000 or more, approximately 7000 or more, approximately 8000 or more, approximately 9000 or more, approximately 10000 or more, approximately 20000 or more, approximately 30000 or more, approximately 40000 or more, approximately 50 The sample detection area comprises 100,000 or more, approximately 60,000 or more, approximately 70,000 or more, approximately 80,000 or more, approximately 90,000 or more, approximately 100,000 or more, approximately 200,000 or more, approximately 300,000 or more, approximately 400,000 or more, approximately 500,000 or more, approximately 600,000 or more, approximately 700,000 or more, approximately 800,000 or more, approximately 900,000 or more, or approximately 1,000,000 or more pores or micropores. In some embodiments, the sample detection area comprises 100,000 or more pores or micropores. In some embodiments, the sample detection area comprises 300,000 or more pores or micropores. In some embodiments, the perforated portion of the sample detection area is optically transparent.

[0136] In some embodiments, the sample analysis area includes a sample detection area, which includes 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 both an imaging chamber and a reaction vessel. The chamber can be a range of different sizes and shapes, making the chamber suitable for detecting samples or analytes contained therein. The chamber can contain a reaction mixture having a volume from 1 μL to 1 mL. For example, the chamber can be sized to contain volumes from about 1 to about 10 μL, about 10 to about 50 μL, about 50 to about 100 μL, about 100 to about 200 μL, about 200 to about 300 μL, about 300 to about 400 μL, about 400 to about 500 μL, about 500 to about 600 μL, about 600 to about 700 μL, about 700 to about 800 μL, about 800 to about 900 μL, or from about 900 to about 100 μL. When the chamber is an imaging chamber, one or more of the sides of the chamber may be optically transparent. For example, the top of the chamber, the bottom of the chamber, the sides of the chamber, or any combination thereof may be optically transparent.

[0137] In some embodiments, the sample analysis region includes a sample detection region comprising a nanopore. When the sample detection region includes a nanopore, the sample includes nucleic acids. In some embodiments, the nanopore is designed to allow only a single nucleic acid translocation at a time. The nucleic acid can be single-stranded or double-stranded. When the nucleic acid is single-stranded, the nanopore has a diameter such that only a single-stranded nucleic acid can translocate. When the nucleic acid is single-stranded, the nanopore has a diameter such that only a single double-stranded nucleic acid can translocate. The nanopore can be any type of nanopore, including but not limited to biological nanopores, solid-state nanopores, etc.

[0138] The sample testing area disclosed herein may be a single sample testing area, multiple sample testing areas of the same type, or multiple sample testing areas of two or more different types.

[0139] When the sample detection region is a single sample detection region, it can be any of the sample detection regions described above or throughout this disclosure. In some embodiments, the sample detection region includes a pore. In some embodiments, the sample detection region includes a micropore. In some embodiments, the sample detection region includes a nanopore or nanochannel. In some embodiments, the sample detection region includes a chamber.

[0140] When the sample detection area includes wells or microwells, the wells or microwells may be specifically designed for electrochemical detection, imaging analysis, or absorbance-based measurements. In some embodiments, the wells or microwells are specifically designed for electrochemical detection. In some embodiments, the wells or microwells are specifically designed for image analysis. In some embodiments, the wells or microwells are specifically designed for absorbance-based measurements. In some embodiments, the wells or microwells are specifically designed for both electrochemical detection and image analysis. In some embodiments, the wells or microwells are specifically designed for both electrochemical detection and absorbance-based measurements. In some embodiments, the wells or microwells are specifically designed for both image analysis and absorbance-based measurements. In some embodiments, the wells or microwells are specifically designed for electrochemical detection, image analysis, and absorbance-based measurements.

[0141] When the sample detection area includes a pore or micropore, the pore or micropore may be housed in a chamber or an open area. In some embodiments, the pore or micropore is housed in a chamber. In some embodiments, the pore or micropore is located in an open area.

[0142] When the sample detection area includes a chamber, imaging chamber, or reaction vessel, the chamber, imaging chamber, or reaction vessel may be specifically designed for electrochemical detection, imaging analysis, or absorbance-based measurements. In some embodiments, the chamber or reaction vessel is specifically designed for electrochemical detection. In some embodiments, the chamber, imaging chamber, or reaction vessel is specifically designed for image analysis. In some embodiments, the chamber, imaging chamber, or reaction vessel is specifically designed for absorbance-based measurements. In some embodiments, the chamber or reaction vessel is specifically designed for both electrochemical detection and image analysis. In some embodiments, the chamber, imaging chamber, or reaction vessel is specifically designed for both electrochemical detection and absorbance-based measurements. In some embodiments, the chamber, imaging chamber, or reaction vessel is specifically designed for both image analysis and absorbance-based measurements. In some embodiments, the chamber, imaging chamber, or reaction vessel is specifically designed for electrochemical detection, image analysis, and absorbance-based measurements.

[0143] When the sample detection region includes nanopores or nanochannels, the nanopores or nanochannels may be housed in a chamber or an open region. In some embodiments, the nanopores or nanochannels are housed in a chamber. In some embodiments, the nanopores or nanochannels are located in an open region.

[0144] When a sample testing area contains multiple sample testing areas, it can be called a combined sample testing area. When a sample testing area contains multiple sample testing areas, it can contain multiple sample testing areas of the same type or multiple sample testing areas of different types.

[0145] In some embodiments, the sample detection area contains multiple sample detection areas of the same type, such as any of the sample detection areas described above. There may be a series of different numbers of sample detection areas of the same type that can be located in a combined sample detection area. For example, a combined sample detection area 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 areas. The sample detection areas in a combined sample detection area may be located in an open region or in discrete chambers linked in series, parallel, or in a grid-like manner. In some embodiments, the sample detection areas in a combined sample detection area are located in an open region. In some embodiments, the sample detection areas in a combined sample detection area are located in discrete chambers linked in series. In some embodiments, the sample detection areas in a combined sample detection area are located in discrete chambers linked in parallel. In some embodiments, the sample detection areas in a combined sample detection area are located in discrete chambers linked in a grid-like manner.

[0146] In some embodiments, the sample detection area contains multiple sample detection areas of the same type, such as any of the sample detection areas described above. There may be a series of different numbers of sample detection areas of the same type that can be located in a combined sample detection area. For example, a combined sample detection area 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 areas. The sample detection areas in a combined sample detection area may be located in an open region or in discrete chambers linked in series, parallel, or in a grid-like manner. In some embodiments, the sample detection areas in a combined sample detection area are located in an open region. In some embodiments, the sample detection areas in a combined sample detection area are located in discrete chambers linked in series. In some embodiments, the sample detection areas in a combined sample detection area are located in discrete chambers linked in parallel. In some embodiments, the sample detection areas in a combined sample detection area are located in discrete chambers linked in a grid-like manner.

[0147] In some embodiments, the sample detection region contains multiple sample detection regions of different types, such as any of the sample detection regions described above. There may be a series of different numbers of sample detection regions of different types that can be located within a combined sample detection region. For example, a combined sample detection region 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 regions. A combined sample detection region may contain multiple sample detection regions of the same or different types. For example, a combined sample detection region may contain two sample detection regions including pores or micropores and two sample detection regions including chambers or reaction vessels. In some embodiments, the combined sample detection region includes micropores or pores, and nanopores or nanochannels. In some embodiments, the combined sample detection region includes micropores or pores, and chambers, imaging chambers, or reaction vessels. In some embodiments, the combined sample detection region includes nanopores or nanochannels, and chambers or reaction vessels. The sample detection regions in a combined sample detection region may be located in an open region or in discrete chambers connected in series, parallel, or in a grid-like manner. In some embodiments, the sample detection area in the combined sample detection region is located in an open region. In some embodiments, the sample detection area in the combined sample detection region is located in discrete chambers linked in series. In some embodiments, the sample detection area in the combined sample detection region is located in discrete chambers linked in parallel. In some embodiments, the sample detection area in the combined sample detection region is located in discrete chambers linked in a grid-like manner.

[0148] In some embodiments, the sample analysis region includes hydrophobic liquid pores and hydrophilic liquid pores. When the sample analysis region includes hydrophobic liquid pores and hydrophilic liquid pores, the sample detection region includes pores or micropores. The hydrophilic liquid pores contain hydrophilic liquids and / or deliver hydrophilic liquids to the pores or micropores. The hydrophobic liquid pores contain hydrophobic liquids and / or deliver hydrophobic liquids to the pores or micropores. The hydrophobic liquid and / or hydrophilic liquid may be pre-filled in the device or added to the device. When adding the hydrophobic liquid and / or hydrophilic liquid to the device, the addition may be performed manually or robotically. Generally, the hydrophilic liquid is added before the hydrophobic liquid is added. When microparticles and analytes are present, the substrate typically interacts with the components in the pores, such that the interaction produces a detectable signal. In some embodiments, the hydrophilic liquid is a substrate solution. The substrate solution may be a substrate solution that reacts with a specific binding member (e.g., a second specific binding member or a detectably labeled second specific binding member) to produce a detectable signal. In some embodiments, the hydrophobic liquid is an oil. The oil may be any oil considered useful. In some cases, hydrophobic liquids are chosen based on their low affinity for water to reduce mixing with the substrate solution. In some cases, the hydrophobic liquid is an oil. In others, the hydrophobic liquid is 3M FC-40 oil, hydrocarbon oil, vegetable oil, or silicone liquid (e.g., silicone oil). In some cases, the oil is a fluorocarbon oil. In still others, the oil is Novec 7500, FC-40, or Galden HT200. The apparatus, sample analysis area, and method of use may be those disclosed in U.S. Provisional Patent Application Serial No. 63 / 601,654, and in International Patent Application Attorney No. ADDV-145WO, filed November 8, 2024, entitled “Sample Analysis Device and Methods,” and in U.S. Provisional Patent Application Attorney No. ADDV-155PRV, filed November 8, 2024, entitled “Automated Assay Processing Unit,” each of which is expressly incorporated herein by reference.

[0149] II. Uses of the device The apparatus disclosed herein can be used for several different types of assays, and these assays can be performed in different ways. The different types of assays and the different ways of performing them are based on the configuration of primary and secondary regions (e.g., the reagents contained in each region). Assays are performed using a sample path in which microparticles are moved from one primary region to another using a magnetic field. In some embodiments, the microparticles are microparticles and assisting particles. The different types of assays include, but are not limited to, immunoassays, nucleic acid analyses, metabolite analyses, clinical chemistry, complete blood counts (CBC), etc.

[0150] In some embodiments, the device can be used to perform an immunoassay. Any immunoassay can be used. For example, the immunoassay can be an enzyme-linked immunosorbent assay (ELISA), a competitive inhibition assay (such as a forward or reverse competitive inhibition assay), or a competitive binding assay. In some embodiments, a detectable label (e.g., one or more fluorescent labels, one or more tags attached via a cleavable linker that can be cleaved chemically or photoly) is attached to a capture antibody and / or a detection antibody.

[0151] In some embodiments, the device can be used to perform nucleic acid analysis. The device can employ various forms of nucleic acid analysis to detect analytes of interest (e.g., nucleic acids, non-nucleic acids tagged with nucleic acids, or nucleic acids derived from analytes), including but not limited to PCR, isothermal amplification, etc.

[0152] In some embodiments, the device can be used to perform metabolite analysis. In some cases, clinical chemistry panels include metabolic assays. Metabolite analysis helps assess, for example, the body's electrolyte balance and / or the status of several major organs. Examples of metabolite analyses that the device can perform include, but are not limited to, the Basal Metabolic Meal Plan (BMP), the Comprehensive Metabolic Meal Plan (CMP), electrolyte assays, lipid assays, liver assays, kidney function assays, and thyroid function assays. The Basal Metabolic Meal Plan (BMP) includes eight tests, all of which are found in the CMP. The BMP provides information on the current health status of the kidneys and respiratory system, as well as electrolyte and acid / base balance and blood glucose levels. The CMP measures liver and kidney health, blood glucose levels, blood acid / base balance, fluid and electrolyte balance, and important blood proteins. In some cases, the CMP measures glucose, calcium, total albumin and globulin, bilirubin, BUN (blood urea nitrogen), creatinine, albumin, sodium, potassium, bicarbonate, chloride, alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). Electrolyte tests are used to detect problems with fluid and electrolyte balance. For example, electrolyte tests measure levels of carbon dioxide, chloride, potassium, and sodium in the blood. Lipid tests are used to assess a subject's risk of cardiovascular disease. For example, lipid tests measure the amount of cholesterol and other fats in the blood, such as total cholesterol, LDL (low-density lipoprotein), HDL (high-density lipoprotein), and triglycerides. Liver tests (liver function packages) are used to screen for, detect, assess, and monitor acute and chronic liver inflammation (hepatitis), liver disease, and / or damage. Liver tests measure various enzymes, proteins, and other substances produced by the liver. For example, liver tests include albumin, total protein, ALP, ALT, AST, gamma-glutamyl transferase (GGT), bilirubin, lactate dehydrogenase (LD), and prothrombin time (PT). Kidney function tests (renal function tests) include tests such as albumin, creatinine, BUN, and eGFR to assess kidney function. Thyroid function tests are used to assess thyroid function and aid in the diagnosis of thyroid disease. Thyroid function tests measure thyroid hormones such as thyroxine (T4), triiodothyronine (T3), and thyroid-stimulating hormone (TSH). In some cases, high TSH levels indicate that the thyroid gland is not producing enough thyroid hormone (primary hypothyroidism). Conversely, low TSH levels usually indicate that the thyroid gland is producing too much thyroid hormone (hyperthyroidism). In other cases, elevated TSH and the presence of free T4 (FT4) or the free T4 index (FTI) indicate primary hypothyroidism due to disease in the thyroid gland.Low TSH and low FT4 or FTI indicate hypothyroidism due to problems involving the pituitary gland. Low TSH and elevated FT4 or FTI are found in individuals with hyperthyroidism. These clinical chemistry assays are well known in the art and are further described in the assays section of this disclosure.

[0153] In some embodiments, the device can be used to perform clinical chemistry. In certain cases, clinical chemistry may involve detecting electrochemical species or colorimetric reaction products generated by the 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 to the analyte and catalytically react with it to generate an electrochemical species or colorimetric reaction product. In other cases, clinical chemistry may involve using a first binding member to capture an analyte to generate a first complex comprising the analyte and the first binding member; contacting the complex with a second binding member (which 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 coupled to an enzyme that generates an electrochemical species or colorimetric reaction product upon exposure to a suitable substrate.

[0154] In some embodiments, the device can be used to perform a complete blood count of blood cells or blood cell types. Blood cells and blood cell types that can be detected by the device disclosed herein include, but are not limited to, red blood cells, hemoglobin, white blood cells (including neutrophils, lymphocytes, monocytes, eosinophils, and basophils), platelets, reticulocytes, and nucleated red blood cells. Various measurements can be performed on different blood components, including but not limited to cell count, cell size, cell complexity, granularity, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin level, and mean corpuscular hemoglobin concentration. In some embodiments, the measurements disclosed above can be performed using staining-independent methods in the absence of histological staining.

[0155] The primary zone may be filled with reagents to perform different types of assays or to perform assays in different ways. Reagents may be added to the primary zone manually by a user, robotically by a system employing the device, or by using a reagent delivery device. In some embodiments, reagents are also added to a secondary zone in addition to the primary zone.

[0156] Figures 26A-26D The illustration shows a cross-section of the sample processing area where samples and / or reagents are added. Figure 26A and Figure 26BThe illustration shows a simplified depiction of a sample processing area comprising a primary region and two secondary regions, wherein the primary region is filled with a sample or reagent. The primary region 2608 contains an opening 2603. The secondary regions 2607 contain openings 2604. A sample or reagent 2602 can be added through the opening 2603 of the primary region 2608, wherein the sample or reagent 2602 is held in place by capillary forces generated by the opening 2603 and surface tension facilitated by the edges of square pads 2601 in the primary region 2608.

[0157] Figure 26C The illustration shows a simplified depiction of the sample processing area, which contains three primary zones and four secondary zones, with two of the primary zones filled with samples or reagents.

[0158] Figure 26D The illustration shows a simplified depiction of a sample processing area comprising three primary zones and four secondary zones, with two of the primary zones and one of the secondary zones filled with a sample or reagent. In this embodiment, the sample or reagent is added through openings in two of the primary zones and one of the secondary zones. Adding a sample or reagent to three adjacent zones (i.e., two primary zones and one secondary zone) causes these zones to merge, resulting in an odd number of zones with increased volume.

[0159] The primary region of this disclosure is capable of accommodating a range of different volumes while maintaining those volumes within the primary region without overflowing into the secondary region or further. For example, the primary region can accommodate fluids of approximately 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, 45, 46, 47, 48, 49, 50, or more. In some embodiments, the primary region is capable of accommodating fluids between 10 and 24 μL.

[0160] When the primary region and adjacent secondary regions are filled with sample or fluid, they can accommodate a range of different volumes while maintaining those volumes within their respective regions without overflowing into the other region. For example, the primary and secondary regions can collectively accommodate approximately 10, approximately 11, approximately 12, approximately 13, approximately 14, approximately 15, approximately 16, approximately 17, approximately 18, approximately 19, approximately 20, approximately 21, approximately 22, approximately 23, approximately 24, approximately 25, approximately 26, approximately 27, approximately 28, approximately 29, approximately 30, approximately 31, approximately 32, approximately 33, approximately 34, approximately 35, approximately 36, approximately 37, approximately 38, approximately 39, approximately 40, approximately 41, approximately 42, approximately 43, approximately 44, approximately 45, approximately 46, approximately 47, approximately 48, approximately 49, approximately 50, approximately 51, approximately 52, approximately 53, and approximately... 54, approximately 55, approximately 56, approximately 57, approximately 58, approximately 59, approximately 60, approximately 61, approximately 62, approximately 63, approximately 64, approximately 65, approximately 66, approximately 67, approximately 68, approximately 69, approximately 70, approximately 71, approximately 72, approximately 73, approximately 74, approximately 75, approximately 76, approximately 77, approximately 78, approximately 79, approximately 80, approximately 81, approximately 82, approximately 83, approximately 84, approximately 85, approximately 86, approximately 87, approximately 88, approximately 89, approximately 90, approximately 91, approximately 92, approximately 93, approximately 94, approximately 95, approximately 96, approximately 97, approximately 98, approximately 99, approximately 100 or more μL of fluid. In some embodiments, the primary and secondary regions are capable of containing fluid between 20 and 50 μL.

[0161] When two primary zones and one secondary zone are filled with a sample or fluid, the two primary zones and one secondary zone can accommodate a range of different volumes while keeping the volume within the primary and secondary zones without overflowing into the other zone. For example, the two primary zones and one secondary zone can together accommodate approximately 15, approximately 16, approximately 17, approximately 18, approximately 19, approximately 20, approximately 21, approximately 22, approximately 23, approximately 24, approximately 25, approximately 26, approximately 27, approximately 28, approximately 29, approximately 30, approximately 31, approximately 32, approximately 33, approximately 34, approximately 35, approximately 36, approximately 37, approximately 38, approximately 39, approximately 40, approximately 41, approximately 42, approximately 43, approximately 44, approximately 45, approximately 46, approximately 47, and approximately 48. Approximately 49, Approximately 50, Approximately 51, Approximately 52, Approximately 53, Approximately 54, Approximately 55, Approximately 56, Approximately 57, Approximately 58, Approximately 59, Approximately 60, Approximately 61, Approximately 62, Approximately 63, Approximately 64, Approximately 65, Approximately 66, Approximately 67, Approximately 68, Approximately 69, Approximately 70, Approximately 71, Approximately 72, Approximately 73, Approximately 74, Approximately 75, Approximately 76, Approximately 77, Approximately 78, Approximately 79, Approximately 80, Approximately 81, Approximately 82, Approximately 83, Approximately 84, Approximately 85, Approximately 8 6, approximately 87, approximately 88, approximately 89, approximately 90, approximately 91, approximately 92, approximately 93, approximately 94, approximately 95, approximately 96, approximately 97, approximately 98, approximately 99, approximately 100, approximately 101, approximately 102, approximately 103, approximately 104, approximately 105, approximately 106, approximately 107, approximately 108, approximately 109, approximately 110, approximately 111, approximately 112, approximately 113, approximately 114, approximately 115, approximately 116, approximately 117, approximately 118, approximately 119, approximately Fluid of approximately 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, or more than approximately 150 μL. In some embodiments, the primary and secondary regions are capable of containing fluid between 30 and 150 μL.

[0162] The measurements using this device are performed using a sample path in which microparticles, or microparticles and assisting particles, move from one primary region to another using a magnetic field. Different embodiments of the device are described below. Figure 17-1 An exemplary sample path is disclosed in 9.

[0163] exist Figure 17 An exemplary sample path is illustrated. The sample and microparticles are present in a fixed primary region 1701 (e.g., a sample mixing region). In some embodiments, the microparticles are microparticles and assisting particles. Microparticles or microparticles and assisting particles move from the fixed primary region (black arrows) to a primary region 1702 containing reagents (e.g., a wash buffer). The microparticles or microparticles and assisting particles then move through two additional primary regions 1703 and 1704 containing reagents to a fourth primary region 1705 containing reagents (e.g., a conjugate). The microparticles or microparticles and assisting particles finally move from the fourth primary region 1705 to the sample analysis region 1706.

[0164] exist Figure 18 The present invention discloses short, medium, and long sample paths using the same exemplary apparatus. In the short sample path, the sample, along with microparticles or microparticles and cooperating particles, is present in a variable primary region (S), and the microparticles or microparticles and cooperating particles then move through a primary region 1801 containing a reagent (e.g., wash buffer) to a primary region containing a reagent (C; e.g., a conjugate). The microparticles or microparticles and cooperating particles then move from the primary region containing the reagent (C) through another primary region and finally terminate in a sample analysis region 1802. In the medium path, the sample, along with microparticles or microparticles and cooperating particles, is present in a variable primary region (S), and the microparticles or microparticles and cooperating particles move through four primary regions containing reagents (e.g., wash buffer) to a primary region containing a reagent (C; e.g., a conjugate). The microparticles or microparticles and cooperating particles then move through four additional primary regions containing reagents (e.g., wash buffer) and finally terminate in a sample analysis region. In a long sample path, the sample and microparticles reside in a variable primary region (S), and the microparticles move through seven primary regions containing reagents (e.g., wash buffer) to a primary region containing reagents (C; e.g., conjugates). The microparticles, or microparticles and cooperating particles, then move through an additional seven primary regions containing reagents (e.g., wash buffer) and finally terminate in the sample analysis region.

[0165] Figure 19 illustrates an exemplary path through an embodiment of the device. Figure 19A In this process, the sample, along with microparticles or microparticles and cooperating particles, is present in a fixed primary region 1901 (e.g., a mixing region) and passes through two merged primary regions and one secondary region (e.g., a merged region; speckled) 1902 containing reagents to reach a primary region 1903 containing reagents (dark streaks; e.g., a coupling agent). The microparticles or microparticles and cooperating particles then move through two more merged (spotted) primary regions and one secondary region containing reagents to finally terminate in the sample analysis region 1904. Figure 19BIn this process, the sample, along with microparticles or microparticles and cooperating particles, exists in a fixed primary region and moves through four primary regions containing reagents (dot-like, e.g., wash buffer) to a primary region containing reagents (dark streaks; e.g., conjugates). The microparticles or microparticles and cooperating particles then move through four primary regions containing reagents (dot-like) to finally terminate in the sample analysis region. Figure 19C In this process, the sample, along with microparticles or microparticles and cooperating particles, exists in a fixed primary region and moves through two merged regions containing reagents (dot-like, e.g., wash buffer) to a primary region containing reagents (dark streaks; e.g., conjugates). The microparticles or microparticles and cooperating particles then move through two merged regions (dot-like) containing reagents to finally terminate in the sample analysis region. Figure 19D In this process, the sample, along with microparticles or microparticles and cooperating particles, exists in a fixed primary region and moves through two primary regions containing reagents (dot-like, e.g., wash buffer) to a primary region containing reagents (dark streaks; e.g., conjugates). The microparticles or microparticles and cooperating particles then move through two primary regions containing reagents (dot-like) to finally terminate in the sample analysis region. Figure 19E In this process, the sample, along with microparticles or microparticles and cooperating particles, exists in a fixed primary region and moves through three primary regions containing reagents (dot-like, e.g., wash buffer) to a primary region containing reagents (dark stripes; e.g., conjugates). The microparticles or microparticles and cooperating particles then move through the three primary regions containing reagents (dot-like) to finally terminate in the sample analysis region.

[0166] Figure 46 An exemplary path through an embodiment of the device is disclosed. Figure 46 In step A, the sample, along with microparticles or microparticles and cooperating particles, is present in a fixed primary region 4601 (e.g., a mixing region) and moves through three primary regions containing reagents (e.g., a wash buffer) to a primary region 4602 containing reagents (dark stripes; e.g., a coupling agent). The microparticles or microparticles and cooperating particles then move through the three primary regions containing reagents (e.g., a wash buffer) to the sample analysis region. The microparticles or microparticles and cooperating particles then move from a quaternary region (e.g., a hydrophilic pore or reservoir) through a tertiary region (e.g., a sample detection region) and a pentadienary region (e.g., a hydrophobic pore or reservoir) 4605 away from the sample analysis region.

[0167] For illustrative and example purposes, and not for limitation, see reference. Figure 35AThe exemplary sample detection region 2750 is depicted herein. In an embodiment, the sample detection region 2750 includes a pore 2753 defined therein. The pore 2753 has a certain pore size. As described above and throughout, microparticles or an array of microparticles and assisting particles are movable across the pore 2753. In some embodiments, the microparticles include: (i) a plurality of microparticles, each having a microparticle diameter smaller than the pore size; and (ii) a plurality of assisting particles, each having an assisting particle diameter larger than the pore size. Specifically, the diameter of the assisting particles (e.g., assisting beads) may 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% larger than the diameter of the pore size. The array for moving microparticles or microparticles and helper particles across aperture 2753 also includes: seeding multiple microparticles or microparticles and helper particles into the apertures of the array.

[0168] As illustrated herein, the movement of microparticles or an array of microparticles and assisting particles across aperture 2753 includes: a magnet 2759 that moves along the aperture array. The magnet can move along the aperture array in any suitable configuration. For example, and not limited to, the magnet can be located above sample analysis region 2750. Additionally or alternatively, the magnet can be positioned adjacent to the sample analysis region. Additionally or alternatively, and as illustrated herein, the magnet can be located below sample analysis region 2750.

[0169] The position of the magnet relative to the sample analysis region 2750 and the array of holes 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 them across the array of holes 2753. For illustrative purposes, and as further described 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 them. For illustrative purposes, and as illustrated herein, the magnet can be moved along the array of holes at a magnet distance defined between the bottom surface 2754 of the array of holes and the magnet 2759. For example, and not limited to, the magnet distance can be between approximately 0 mm and approximately 10 mm. Additionally or alternatively, and as illustrated herein, the magnet can be moved along the array of holes 2753, wherein the magnet 2759 contacts the bottom surface 2754 of the array of holes.

[0170] As further illustrated herein, the shape and orientation of the magnet can also be chosen to achieve the desired magnetic field. For example, and not limitingly, the magnet 2759 can be angled relative to the bottom surface 2754 of the aperture array as it moves along the bottom surface of the aperture array. For illustrative and illustrative purposes, and not limiting, see references. Figure 35B The exemplary sample analysis region 2750 is depicted herein. A magnetic axis 2763 may be defined between a first magnet end 2761 and a second magnet end 2762, and is present when the magnet 2759 moves along the bottom surface 2754 of the aperture array. For illustrative purposes and not limiting, the magnetic axis 2763 may be positioned relative to the bottom surface of the aperture array at an angle 2760 between approximately 0 degrees and approximately 80 degrees. For example, and not limiting, the magnetic axis 2763 may be positioned at an angle 2760 between approximately 10 degrees and approximately 30 degrees. For example, and not limiting, and as illustrated herein, the magnetic axis 2763 may be positioned at an angle 2760 of approximately 20 degrees.

[0171] Additionally or alternatively, the magnetic element can be moved along the aperture array to achieve the desired movement and seeding of microparticles or microparticles and assisting particles within the aperture array. For illustrative purposes and as illustrated herein, magnet 2759 can be moved along a direction 2755 parallel to the bottom surface 2754 of the aperture array. Additionally or alternatively, the magnetic element can be moved along the aperture array at any suitable speed to achieve the desired movement and seeding of microparticles within the aperture array. For example, the speed of the magnet can be selected to achieve the desired processing time and microparticle loss during the movement of microparticles or microparticles and assisting particles across the array. For illustrative and not limiting purposes, magnet 2759 can be moved along a direction 2755 parallel to the bottom surface 2754 of the array of apertures 2753 at a speed between approximately 0.3 mm / s and approximately 10 mm / s. Alternatively or additionally, magnet 2759 may move along direction 2755 parallel to the bottom surface 2754 of the array of holes 2753 at a speed between approximately 0.3 mm / s and approximately 6 mm / s. Alternatively or additionally, magnet 2759 may move along direction 2755 parallel to the bottom surface 2754 of the array of holes 2753 at a speed between approximately 2 mm / s and approximately 6 mm / s. Alternatively or additionally, magnet 2759 may move along direction 2755 parallel to the bottom surface 2754 of the array of holes 2753 at a speed between approximately 4 mm / s and approximately 6 mm / s. For illustrative purposes and as illustrated herein, magnet 2759 may move along direction 2755 at an angle 2760 as described above, and direction 2760 may be selected to be aligned with an acute angle direction defined between magnetic axis 2763 and the bottom surface 2754 of the array of holes. Although reference is made to the movement of magnet 2759 along the aperture array, the relative motion between magnet 2759 and aperture array can be additionally or alternatively achieved by moving aperture array relative to magnet, as described above.

[0172] Additionally or alternatively, the type and shape of the magnet can be selected to provide the desired magnetic field. Magnet 2759 can be a permanent magnet or an electromagnet. Any suitable magnet shape can be selected. For example, and not limitingly, the magnet can define corners. Additionally or alternatively, and as illustrated herein, when magnet 2759 moves along the aperture array, the corners 2764 of the magnet can contact the bottom surface 2754 of the aperture array. For illustrative purposes and not limitingly, the magnet can have a cylindrical, triangular, square, spherical, or other suitable shape. Additionally or alternatively, and as illustrated herein, the magnet can have a rectangular shape.

[0173] As described above, the shape, orientation, and position of the magnetic element can be selected to provide the desired magnetic field. (Reference) Figures 35C-35HFor illustrative and not limiting purposes, the properties of the magnetic field in an exemplary detection region 2750 having a magnet 2759 are depicted, including multiple magnetic field lines emanating from the magnet. In the exemplary sample detection region 2750, the magnet 2759 is positioned such that its corner 2764 contacts the bottom surface 2754 of the aperture array, such that the magnetic field lines of the magnet 2759 extend through the bottom surface 2754 of the aperture array, with the magnetic field lines concentrated near the corner 2764 of the magnet 2759. Additionally, the exemplary magnet is positioned at an angle 2760 of approximately 20 degrees relative to the bottom surface 2754 with its magnetic axis 2763. Additionally or alternatively, the exemplary magnet may be positioned at an angle 2760 of approximately 0 degrees to 80 degrees with its magnetic axis 2763. Additionally or alternatively, the exemplary magnet may be positioned at an angle 2760 of approximately 10 degrees to 30 degrees with its magnetic axis 2763. Reference Figure 35B The array axis “X” can be defined along the top surface 2751 of the hole array, and the second axis “Y” can be defined perpendicular to the array axis. For illustration and as shown herein, the zero point on the array axis “X” is defined at the location where the corner 2764 of the magnet 2759 contacts the bottom surface 2754 of the hole array on the array axis “X”.

[0174] Additionally, the zero point on the second axis “Y” is defined at the top surface 2751 of the hole array.

[0175] refer to Figure 35D For the exemplary sample analysis region 2750 and magnet 2759 configuration described above, the magnetic force, measured in pN, is shown as a function of position along the array axis "X" and the second axis "Y". As shown, in the case where the corner 2764 of the magnet 2759 contacts the bottom surface 2754 of the hole array, a negative magnetic force is generated along the second axis Y. As illustrated herein, the negative magnetic force acts on microparticles or microparticles and assisting particles to pull the microparticles downward into the holes of the hole array, thereby loading or seeding the microparticles into the holes.

[0176] refer to Figure 35E and Figure 35FFor the exemplary detection region 2750 and magnet 2759 configuration described above, the magnetic force measured in pN is shown separately as a function of position along the array axis "X" for the second axis "Y" and the array axis "X". As illustrated herein, the magnetic force on the second axis "Y" can be defined as a generally parabolic shape when plotted, and may include sharp negative peaks in magnetic field strength in the case of the corner 2764 of magnet 2759 contacting the bottom surface 2754 of the array of holes. Additionally, as illustrated herein, the magnitude of the magnetic force on 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 microparticles to pull microparticles into the holes of the array, thereby seeding microparticles within the holes of the array. Additionally, and as illustrated herein, the magnetic field on the second axis "Y" can remain negative across the array axis "X", as shown in the figure. For example, and as illustrated herein, the magnetic field on the second axis “Y” can remain negative for at least 6 mm, as measured in the array axis “X” and as Figure 35E As depicted in the diagram, the negative magnetic field across the wide portion of the array of holes on the second axis "Y" can retain microparticles that have been seeded in the holes of the array within those holes.

[0177] refer to Figure 35G For the exemplary detection region 2750 and magnet 2759 configuration described above, the magnetic field strength, measured in H (AIM), is shown as a function of position along the array axis “X.” As illustrated herein, magnet 2759 can provide a strong magnetic field with a generally parabolic shape. As illustrated herein, in the case where the corner 2764 of magnet 2759 contacts the bottom surface 2754 of the hole array, the magnetic field strength can be approximately 400,000 H (AIM). The magnetic field strength can seed and retain microparticles within the holes of the hole array, as described herein.

[0178] refer to Figure 35H For the exemplary detection area 2750 and magnet 2759 configuration described above, the magnetic force measured in pN in the array axis “X” (Fx) and the second axis “Y” (Fy) is shown as a function of the position along the array axis “X” and the second axis “Y”.

[0179] Continue to refer to Figure 35B-35EThe achieved magnetic field can benefit the seeding and sealing of microparticles into the holes. For example, and not limited to, at the location where the corner 2764 of the magnet 2759 contacts the bottom surface 2754 of the hole array, the y-component of the magnetic force is high relative to the x-component. The relatively high magnitude of the y-component can help ensure that microparticles are loaded (i.e., seeded) into the holes and retained in the holes during the sealing process. Additionally, the magnetic field magnitude is high, and the magnetic field distribution has a high peak value. Making the magnetic field distribution have a high peak value can promote the aggregation of microparticles or microparticles and assisting particles as they move across the hole array. Increased aggregation of microparticles or microparticles and assisting particles during movement across the hole array results in less excess microparticles remaining on top of the array. Additionally, the magnitude of the x-component of the magnetic force can be large enough to continuously pull excess microparticles along the surface of the hole array. Increased magnetic force magnitude in the array axis “X” can help further remove excess microparticles remaining on top of the array.

[0180] The determinations that can be performed using the exemplary sample paths discussed above are further described in the section on methods for detecting target analytes in samples.

[0181] II. Benefits of the device The apparatus disclosed herein offers several advantages over apparatuses known in the art. These advantages include: a high level of modularity, allowing flexibility in sample processing workflows by permitting different regions to be filled with different reagents depending on the assay; the ability to use small volumes, thus allowing for scalability and faster reaction times; and the ability to simultaneously analyze multiple sample types. In terms of modularity, several exemplary embodiments of the apparatus disclosed herein provide a high level of configurability by using: multiple primary regions for sample or reagent addition; multiple secondary regions for hydrophobic separation (e.g., by the presence of air) or for merging to create a large sample or reagent area; and many different types of sample analysis regions configured to analyze proteins or antigens (e.g., immunoassays and clinical chemistry), nucleic acids (e.g., nucleic acid analysis), metabolites (e.g., clinical chemistry), or cells or cell types (e.g., immunoassays, clinical chemistry, nucleic acids, or CBC). The primary and secondary regions can be configured by the user to suit any type of assay or specific steps of a given assay. The ability to perform these assays using small volumes allows for reduced reagent costs and sample consumption, thereby allowing for the saving of hard-to-obtain samples. The modularity of the device also allows for customization of assays according to user preferences, and allows for the simultaneous or sequential execution of multiple assays on the same device for the same sample or multiple samples contained on a single device (e.g., immunoassay and nucleic acid analysis, or any combination of the assays disclosed above).

[0182] 2. Device Design The apparatus disclosed herein has a range of different layouts, including variations in the number of primary and secondary regions, different arrangements of the primary and secondary regions, the presence or absence of a sample mixing region, and the presence or absence of a fixed primary region. Figure 1-9 , Figure 11-1 9. Exemplary embodiments of the device are disclosed in Figures 32 and 38-41.

[0183] Figure 1 Illustrations of an embodiment of the device are disclosed. In this embodiment, device 100 includes a 3x8 grid of a primary region 101 and a secondary region 107. The device includes a sample processing region 110. The sample processing region 110 is unbounded. By unbounded, it means that there is no physical barrier separating the primary region 101 and the secondary region 107. The sample processing region includes the primary region 101, in which an opening 103 is provided. The primary region is adjacent to the secondary region 107. The secondary region may include an opening 104. The sample processing region 110 may be connected to a tertiary region (i.e., a sample detection region) 112 by a transition region 113. The device may also include a quaternary region 114 (i.e., a hydrophilic liquid pore) connected to the tertiary region. The device may also include a quinary region 115 (i.e., a hydrophobic liquid pore) connected to the tertiary region. A top substrate and a bottom substrate are bonded by an adhesive or clip 111. In some embodiments, the top substrate and the bottom substrate are bonded by an adhesive layer between the top substrate and the bottom substrate. In some embodiments, the adhesive is an ultraviolet (UV) bonded adhesive. In some embodiments, the top substrate and the bottom substrate are bonded together using laser welding.

[0184] The following text was published Figure 1Alternative embodiments are provided. In this embodiment, device 100 includes a 3x8 grid of primary region 101 and secondary region 107. Device 100 includes a second substrate positioned on a first substrate. The second substrate includes a surface facing a central chamber, which includes a plurality of recessed elements and a plurality of protruding elements. Primary region 101 is defined between the surfaces of the plurality of protruding elements facing the first substrate and the surfaces of the first substrate facing the second substrate. Secondary regions are defined between the surfaces of the plurality of recessed elements facing the first substrate and the surfaces of the first substrate facing the second substrate. The device includes a sample processing region 110. Sample processing region 110 is unbounded. By unbounded, it means that there is no physical barrier separating primary region 101 and secondary region 107. Although only a single element is marked for primary region 101, each square (101) will represent a separate primary region. In some embodiments, the primary regions are discrete. Although only a single element is marked for secondary region 107, each space between squares will represent a separate secondary region. In some embodiments, the secondary regions are connected. The sample processing region includes a primary region 101 having an opening 103. While only a single element is marked for the opening 103 in the primary region, each opening (103) within the square (101) is intended to represent a separate opening in the primary region. The primary region is adjacent to a secondary region 107. The secondary region may include openings 104. While only a single element is marked for the opening in the secondary region 107, each opening (104) between the squares represents a separate opening in the secondary region. The sample processing region 110 may be positioned adjacent to a sample analysis region including a tertiary region (i.e., a sample detection region) 112, a quaternary region 114 (i.e., a hydrophilic liquid pore), and a quinary region 115 (i.e., a hydrophobic liquid pore), wherein the tertiary region 112, quaternary region 114, and quinary region 115 are connected. The sample analysis region is defined by a surface of the second substrate facing the first substrate and a surface of the first substrate facing the second substrate. The quinary region 115 is located at a first end of the sample analysis region. The fifth-level region includes an opening across the second substrate. The fourth-level region 114 is located at the second end of the sample analysis region. The fourth-level region includes a cylindrical opening across the second substrate. The third-level region 112 is located at the midpoint between the first end (e.g., the fifth-level region 115) and the second end (e.g., the fourth-level region 115) of the sample analysis region. The sample analysis region may be connected to the sample processing region 110 by a transition region 113. The second substrate, positioned above the first substrate, is bonded together by an adhesive or clip 111. In some embodiments, the second substrate and the first substrate are bonded by an adhesive layer between the second substrate and the first substrate. In some embodiments, the adhesive is an ultraviolet (UV) bonded adhesive. In some embodiments, the second substrate and the first substrate are bonded using laser welding. [The last sentence appears to be incomplete and possibly refers to a different document.] Figure 1 The disclosed alternative embodiments are applied to Figure 2-6 This makes the above description describe Figure 2-6 The components of the device disclosed herein.

[0185] Figure 2 Illustrations of an embodiment of the device are disclosed. In this embodiment, device 200 includes a 2x4 grid of a primary region 201 and a secondary region 207. The device includes a sample processing region 210. The sample processing region 210 is unbounded. By unbounded, it means that there is no physical barrier separating the primary region 201 and the secondary region 207. The sample processing region includes the primary region 201, in which an opening 203 is provided. The primary region is adjacent to the secondary region 207. The secondary region may include an opening 204. The sample processing region 210 may be connected to a tertiary region (i.e., a sample detection region) 212 by a transition region 213. The device may also include a quaternary region 214 (i.e., a hydrophilic liquid pore) connected to the tertiary region. The device may also include a quinary region 215 (i.e., a hydrophobic liquid pore) connected to the tertiary region. A top substrate and a bottom substrate are bonded by an adhesive or clip 211. In some embodiments, the top substrate and the bottom substrate are bonded by an adhesive layer between the top substrate and the bottom substrate. In some embodiments, the adhesive is an ultraviolet (UV) bonded adhesive. In some embodiments, the top substrate and the bottom substrate are bonded together using laser welding.

[0186] Figure 3 Illustrations of an embodiment of the device are disclosed. In this embodiment, device 300 includes a 3x6 grid of a primary region 301 and a secondary region 307. The device includes a sample processing region 310. The sample processing region 310 is unbounded. By unbounded, it means that there is no physical barrier separating the primary region 301 and the secondary region 307. The sample processing region includes a primary region 301 having an opening 303. The primary region is adjacent to the secondary region 307. The secondary region may include an opening 304. The sample processing region 310 may be connected to a tertiary region (i.e., a sample detection region) 312 by a transition region 313. The device may also include a quaternary region 314 (i.e., a hydrophilic liquid pore) connected to the tertiary region. The device may also include a quinary region 315 (i.e., a hydrophobic liquid pore) connected to the tertiary region. A top substrate and a bottom substrate are bonded by an adhesive or clip 311. In some embodiments, the top substrate and the bottom substrate are bonded by an adhesive layer between the top substrate and the bottom substrate. In some embodiments, the top substrate and the bottom substrate are bonded together using laser welding.

[0187] Figure 3Illustrations of an embodiment of the device are disclosed. In this embodiment, device 300 includes a 3x6 grid of a primary region 301 and a secondary region 307. The device includes a sample processing region 310. The sample processing region 310 is unbounded. By unbounded, it means that there is no physical barrier separating the primary region 301 and the secondary region 307. The sample processing region includes the primary region 301, in which an opening 303 is provided. The primary region is adjacent to the secondary region 307. The secondary region may include an opening 304. The sample processing region 310 may be connected to a tertiary region (i.e., a sample detection region) 312 by a transition region 313. The device may also include a quaternary region 314 (i.e., a hydrophilic liquid pore) connected to the tertiary region. The device may also include a quinary region 315 (i.e., a hydrophobic liquid pore) connected to the tertiary region. A top substrate and a bottom substrate are bonded by an adhesive or clip 311. In some embodiments, the top substrate and the bottom substrate are bonded by an adhesive layer between the top substrate and the bottom substrate. In some embodiments, the adhesive is an ultraviolet (UV) bonded adhesive. In some embodiments, the top substrate and the bottom substrate are bonded together using laser welding.

[0188] Figure 4 Illustrations of an embodiment of the device are disclosed. In this embodiment, device 400 includes a 3x4 grid of a primary region 401 and a secondary region 407. The device includes a sample processing region 410. The sample processing region 410 is unbounded. By unbounded, it means that there is no physical barrier separating the primary region 401 and the secondary region 407. The sample processing region includes the primary region 401, in which an opening 403 is provided. The primary region is adjacent to the secondary region 407. The secondary region may include an opening 404. The sample processing region 410 may be connected to a tertiary region (i.e., a sample detection region) 412 by a transition region 413. The device may also include a quaternary region 414 (i.e., a hydrophilic liquid pore) connected to the tertiary region. The device may also include a quinary region 415 (i.e., a hydrophobic liquid pore) connected to the tertiary region. A top substrate and a bottom substrate are bonded by an adhesive or clip 411. In some embodiments, the top substrate and the bottom substrate are bonded by an adhesive layer between the top substrate and the bottom substrate. In some embodiments, the top substrate and the bottom substrate are bonded together using laser welding.

[0189] Figure 5Illustrations of an embodiment of the device are disclosed. In this embodiment, device 500 includes a 2x8 grid of primary region 501 and secondary region 507. The device includes a sample processing region 510. The sample processing region 510 is unbounded. By unbounded, it means that there is no physical barrier separating the primary region 501 and the secondary region 507. The sample processing region includes the primary region 501, in which an opening 503 is provided. The primary region is adjacent to the secondary region 507. The secondary region may include an opening 504. The sample processing region 510 may be connected to a tertiary region (i.e., a sample detection region) 512 by a transition region 513. The device may also include a quaternary region 514 (i.e., a hydrophilic liquid pore) connected to the tertiary region. The device may also include a quinary region 515 (i.e., a hydrophobic liquid pore) connected to the tertiary region. A top substrate and a bottom substrate are bonded by an adhesive or clip 511. In some embodiments, the top substrate and the bottom substrate are bonded by an adhesive layer between the top substrate and the bottom substrate. In some embodiments, the top substrate and the bottom substrate are bonded together using laser welding.

[0190] Figure 6 Illustrations of an embodiment of the device are disclosed. In this embodiment, device 600 includes a 2x6 grid of a primary region 601 and a secondary region 607. The device includes a sample processing region 610. The sample processing region 610 is unbounded. By unbounded, it means that there is no physical barrier separating the primary region 601 and the secondary region 607. The sample processing region includes the primary region 601, in which an opening 603 is provided. The primary region is adjacent to the secondary region 607. The secondary region may include an opening 604. The sample processing region 610 may be connected to a tertiary region (i.e., a sample detection region) 612 by a transition region 613. The device may also include a quaternary region 614 (i.e., a hydrophilic liquid pore) connected to the tertiary region. The device may also include a quinary region 615 (i.e., a hydrophobic liquid pore) connected to the tertiary region. A top substrate and a bottom substrate are bonded by an adhesive or clip 611. In some embodiments, the top substrate and the bottom substrate are bonded by an adhesive layer between the top substrate and the bottom substrate. In some embodiments, the adhesive is an ultraviolet (UV) bonded adhesive. In some embodiments, the top substrate and the bottom substrate are bonded together using laser welding.

[0191] Figure 7An isometric view of an embodiment of the device is disclosed. Dotted lines represent the internal portions of the device, while solid lines represent the external portions. In this embodiment, in addition to the fixed primary region 702, the device 700 also includes a 3x4 grid of primary region 703 and secondary region 705. In some, but not all, cases, the fixed primary region 702 is also a sample mixing region. The fixed primary region is physically connected to the hook-shaped portion 708. The boundary of the top outer portion of the fixed primary region is not attached to the rest of the device, except for the portion furthest from the primary and secondary regions. When a vertical force is applied to the hook-shaped portion 708, the top substrate (i.e., the roof) compresses and decompresses the fluid (e.g., the sample) that may be located in the fixed primary region 702, thereby mixing the fluid. The device includes a sample processing region 701. The sample processing region 701 is unbounded. By unbounded, it means that there is no physical barrier separating the primary region 703 and the secondary region 705. The sample processing area includes a primary region 703 having an opening 704. The primary region is adjacent to a secondary region (such as 705). The secondary region may include an opening 706. The sample processing area 701 may be connected to a tertiary region (i.e., the sample detection area) 710 by a transition region 707. The device may also include a quaternary region 711 (i.e., a hydrophilic liquid pore) connected to the tertiary region. The device may also include a quinary region 709 (i.e., a hydrophobic liquid pore) connected to the tertiary region.

[0192] The following text was published Figure 7Alternative embodiments are described. Dotted lines indicate internal portions of the device, while solid lines indicate external portions. In this embodiment, in addition to fixing the primary region 702, the device 700 also includes a 3x4 grid of primary region 703 and secondary region 705. The device 700 includes a second substrate positioned on a first substrate. The second substrate includes a surface facing a central chamber, which includes a plurality of recessed elements and a plurality of protruding elements. The primary region 701 is defined between the surfaces of the plurality of protruding elements facing the first substrate and the surfaces of the first substrate facing the second substrate. The secondary region is defined between the surfaces of the plurality of recessed elements facing the first substrate and the surfaces of the first substrate facing the second substrate. In some, but not all, cases, the fixed primary region 702 is also a sample mixing region. The fixed primary region 702 is formed by laterally extending protruding elements in the second substrate facing the first substrate, wherein the laterally extending protruding elements have a surface facing the first substrate that has a larger surface area than the surfaces of the plurality of protruding elements facing the first substrate. The primary region 707 includes a hook-shaped portion 708 attached to a surface of an extending protruding element, which faces the surface of the second substrate toward the first substrate. The boundary of the top outer portion of the primary region is not attached to the rest of the device, except for the portions furthest from the primary and secondary regions. When a vertical force is applied to the hook-shaped portion 708, the second substrate (i.e., the top plate) compresses and decompresses a fluid (e.g., a sample) that may be located in the primary region 702, thereby mixing the fluid. The device includes a sample processing region 701. The sample processing region 701 is unbounded. By unbounded, it means that there is no physical barrier separating the primary region 703 and the secondary region 705. The sample processing region includes the primary region 703, in which an opening 704 is provided. The primary region is adjacent to a secondary region (e.g., 705). The secondary region may include an opening 706. A sample processing region 701 can be positioned adjacent to a sample analysis region, which includes a tertiary region (i.e., a sample detection region) 710, a quaternary region 711 (i.e., a hydrophilic liquid pore), and a pentagonal region 709 (i.e., a hydrophobic liquid pore), wherein the tertiary region 707, quaternary region 711, and pentagonal region 709 are connected. The sample analysis region is defined by a surface of a second substrate facing the first substrate and a surface of the first substrate facing the second substrate. The pentagonal region 709 is located at a first end of the sample analysis region. The pentagonal region includes an opening across the second substrate. The quaternary region 711 is located at a second end of the sample analysis region. The quaternary region includes a cylindrical opening across the second substrate. The tertiary region 710 is located at the midpoint between the first end (e.g., pentagonal region 709) and the second end (e.g., quaternary region 711) of the sample analysis region. The sample analysis region can be connected to the sample processing region 701 by a transition region 707. Figure 7 The disclosed alternative embodiments are applied to Figure 15 This makes the above description describe Figure 15The components of the device disclosed herein.

[0193] Figure 8 An isometric view of an embodiment of the device is disclosed. Dotted lines represent the internal portions of the device, while solid lines represent the external portions. In this embodiment, in addition to an additional primary region and a fixed primary region 802, the device 800 also contains a 2x4 grid of primary region 803 and secondary region 806. In some, but not all, cases, the fixed primary region 802 is also a sample mixing region. The fixed primary region is physically connected to the hook-shaped portion 807. The boundary of the top outer portion of the fixed primary region is not attached to the rest of the device, except for the portions furthest from the primary and secondary regions. When a vertical force is applied to the hook-shaped portion 807, the top substrate (i.e., the top plate) compresses and decompresses the fluid (e.g., sample) that may be located in the fixed primary region 802, thereby mixing the fluid. The device includes a sample processing region 801. The sample processing region 801 is unbounded. By unbounded, it means that there is no physical barrier separating the primary region 803 and the secondary region 806. The sample processing area includes a primary region 803, in which an opening 804 is provided. The primary region is adjacent to a secondary region (such as 806). The secondary region may include an opening 805.

[0194] The following text was published Figure 8Alternative embodiments are described. Dotted lines indicate internal portions of the device, while solid lines indicate external portions. In this embodiment, in addition to an additional primary region and a fixed primary region 802, device 800 also includes a 2x4 grid of primary region 803 and secondary region 806. Device 800 includes a second substrate positioned on a first substrate. The second substrate includes a surface facing a central chamber, which includes a plurality of recessed elements and a plurality of protruding elements. The primary region is defined between the surfaces of the plurality of protruding elements facing the first substrate and the surfaces of the first substrate facing the second substrate. The secondary region is defined between the surfaces of the plurality of recessed elements facing the first substrate and the surfaces of the first substrate facing the second substrate. In some, but not all, cases, the fixed primary region 802 is also a sample mixing region. The fixed primary region 802 is formed by laterally extending protruding elements in the second substrate facing the first substrate, wherein the laterally extending protruding elements have a surface facing the first substrate that has a larger surface area than the surfaces of the plurality of protruding elements facing the first substrate. The primary region 802 includes a hook-shaped portion 807 attached to a surface of an extending protruding element, which faces the surface of the second substrate towards the first substrate. The boundary of the top outer portion of the primary region is not attached to the rest of the device, except for the portions furthest from the primary and secondary regions. When a vertical force is applied to the hook-shaped portion 807, the second substrate (i.e., the top plate) compresses and decompresses a fluid (e.g., a sample) that may be located in the primary region 802, thereby mixing the fluid. The device includes a sample processing region 801. The sample processing region 801 is unbounded. In this sense, unbounded means that there is no physical barrier separating the primary region 803 and the secondary region 806. The sample processing region includes the primary region 803, in which an opening 804 is provided. The primary region is adjacent to a secondary region (e.g., 806). The secondary region may include an opening 805. [The text abruptly ends here, likely due to an incomplete translation or missing information.] Figure 8 The disclosed alternative embodiments are applied to Figure 14 This makes the above description describe Figure 14 The components of the device disclosed herein.

[0195] Figure 9 It was made public. Figure 8The illustration depicts an exemplary sample analysis region of an embodiment. A sample processing region 800 may be connected to a sample analysis region 900 including a tertiary region (i.e., a sample detection region) 902. The device may also include a quaternary region 903 (i.e., a hydrophilic liquid pore) connected to the tertiary region. The quaternary region 903 (i.e., the hydrophilic liquid pore) may be surrounded by a substrate holding feature 904. The device may also include a quinary region 901 (i.e., a hydrophobic liquid pore) connected to the tertiary region. The sample analysis region may be a pad, such as a pad in a primary region. In some embodiments, when the sample analysis region is a pad, the pad has the same thickness as a pad in a primary region. In some embodiments, when the sample analysis region is a pad, the pad has a different thickness than a pad in a primary region. The substrate holding feature may be configured to assist in the removal of the hydrophilic liquid disclosed herein.

[0196] The following text was published Figure 9 Alternative embodiments. The sample processing region 800 may be connected to a sample analysis region 900 including a tertiary region (i.e., a sample detection region) 902. The sample analysis region includes an enlarged protruding element, wherein the surface of the enlarged protruding element facing the first substrate has a larger surface area than the surfaces of the plurality of protruding elements facing the first substrate, and the enlarged protruding element extends from a first end (e.g., a quinary region 901) to a second end (e.g., a quaternary region 903). The device may also include a quaternary region 903 (i.e., a hydrophilic liquid pore) connected to the tertiary region. The quaternary region 903 (i.e., a hydrophilic liquid pore) may be surrounded by a substrate holding feature 904. The quaternary region includes a cylindrical opening across a second substrate. The device may also include a quinary region 901 (i.e., a hydrophobic liquid pore) connected to the tertiary region. The quinary region includes an opening across a second substrate. The sample analysis region 900 is adjacent to the sample processing region 800. The sample analysis region is not physically separated from the sample processing region.

[0197] Figure 38A and Figure 38B Illustrations of embodiments of the device are disclosed. Figure 38A The embodiment shows the surface of the second substrate facing the central cavity or the first substrate. Figure 38BThe embodiment illustrates the surface of the second substrate facing away from the central chamber. Dotted lines indicate the internal portions of the device, while solid lines indicate the external portions. In this embodiment, device 3800 includes a 2x6 grid of primary region 3803 and secondary region 3806, and a fixed primary region 3802. Device 3800 includes a second substrate positioned on a first substrate. The second substrate includes a surface facing the central chamber, which includes a plurality of recessed elements and a plurality of protruding elements. The primary region is defined between the surfaces of the plurality of protruding elements facing the first substrate and the surfaces of the first substrate facing the second substrate. The secondary region is defined between the surfaces of the plurality of recessed elements facing the first substrate and the surfaces of the first substrate facing the second substrate. In some, but not all, cases, the fixed primary region 3802 is also a sample mixing region. The fixed primary region 3802 is formed by laterally extending protruding elements in the second substrate facing the first substrate, wherein the laterally extending protruding elements have a surface facing the first substrate that has a larger surface area than the surfaces of the plurality of protruding elements facing the first substrate. The laterally extending protruding elements may be referred to as third protruding elements. The fixed primary region 3802 includes a chamfered end 3812 laterally separated from the connecting end 3813. By "chamfered end," it means 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 boundary of the top outer portion of the fixed primary region is not attached to the rest of the device, except for the connecting end 3813. When a vertical force is applied to the chamfered end 3812, the second substrate (i.e., the top plate) compresses and decompresses a fluid (e.g., a sample) that may be located in the fixed primary region 3802, thereby mixing the fluid. The device includes a sample processing region 3801. The sample processing region 3801 is unbounded. By unbounded, it means that there is no physical barrier separating the primary region 3803 and the secondary region 3806. The sample processing region includes the primary region 3803, in which an opening 3804 is provided. The primary region is adjacent to a secondary region (e.g., 3806). The secondary region may include an opening 3805. The device 3800 includes a sample analysis region 3807. In some embodiments, the device 3800 includes a waste disposal region 3808. The waste disposal region 3808 is located at the end of the sample processing region 3801 furthest from the sample analysis region 3807. The waste disposal region 3808 includes an opening in a second substrate and has a wedge-shaped portion on the side of the opening closest to the edge of the device. In some embodiments, the device 3800 includes a gap port 3809. The gap port 3809 is an opening in the second substrate that is centrally separated from the waste disposal region 3808.

[0198] Figure 34 It was made public. Figure 8 and Figure 9The illustration depicts an exemplary sample analysis region of an embodiment. Fine dotted lines indicate internal portions of the device, while solid lines indicate external portions. The sample analysis region 3400 (bounded by thick dashed lines) is formed by bonding a top substrate 3401 to a bottom substrate 3402. A pad 3406 defines the sample analysis region. The sample analysis region 3400 includes a tertiary region 3403 (i.e., a sample detection region). The device may also include a quaternary region 3405 (i.e., a hydrophilic liquid pore) connected to the tertiary region. "Connected" means that the regions are adjacent to each other. The quaternary region 3405 (i.e., the hydrophilic liquid pore) may be held by a substrate region (not shown; e.g.) Figure 9 (As depicted in 904) Enclosed. The device may also include a quinary zone 3404 (i.e., a hydrophobic liquid orifice) connected to the tertiary zone.

[0199] Figure 45 An illustration of an exemplary sample analysis region of the embodiment depicted in FIG. 38 is disclosed. A sample processing region may be connected to a sample analysis region 4500 including a tertiary region (i.e., a sample detection region) 4501. The sample analysis region includes an enlarged protrusion 4502, wherein the surface of the enlarged protrusion facing a first substrate has a larger surface area than the surfaces of the plurality of protrusions facing the first substrate, and the enlarged protrusion extends from a first end (e.g., a quinary region 4504) to a second end (e.g., a quaternary region 4503). The quaternary region 4503 (i.e., a hydrophilic liquid pore) may be surrounded by a substrate holding feature 4505. The quaternary region includes a cylindrical opening across a second substrate. The quinary region 4504 includes an opening across the second substrate. The enlarged protrusion 4502 of the sample analysis region 4500 includes a pinned wall 4506 around the periphery of the enlarged protrusion 4502, which does not include the second end containing the quinary region 4504. Pinned wall 4406 is a raised edge that protrudes from enlarged protruding element 4502. The pinned wall assists in retaining the hydrophilic liquid in the sample analysis area when it is deposited or removed from the sample analysis area. Sample analysis area 4500 is adjacent to sample processing area. The sample analysis area is not physically separated from the sample processing area. The sample analysis area includes an anti-sinking feature 4507, which is a sloped recess in the surface of the second substrate facing away from the first substrate. This anti-sinking feature provides the benefit of producing a uniform second substrate thickness in the sample analysis area and providing increased optical clarity relative to a non-sloping recess. The uniform second substrate thickness increases the reproducibility of the device during the manufacturing process. Figure 45 The pinning wall in the device is used in any other embodiment of the device, such as Figure 1-9 , Figure 11-1 9. The embodiments disclosed in Figures 32 and 38-41. They can be... Figure 45 The sample analysis area depicted herein can be applied to any other embodiment of the device, such as Figure 1-9 , Figure 11-1 9. The embodiments disclosed in Figures 32 and 38-41.

[0200] Figure 33A -G discloses an illustration of an exemplary sample analysis region. Figure 33A It was made public. Figure 8 and Figure 9 The diagram depicts the sample analysis region. Dotted lines represent the internal portions of the device, while solid lines represent the external portions. The sample analysis region 3300 includes a tertiary region 3301 (e.g., a sample detection region) equal to the width of the sample analysis region 3300. The sample analysis region 3300 includes a quaternary region 3304 (i.e., hydrophilic liquid pores) having substrate holding features 3305a-d, which are quarter-circular and protrude into the quaternary region 3304. The sample analysis region includes a quintile region 3306 (e.g., a hydrophobic liquid pore). The left end 3302 has an elliptical shape with a smaller area than the right end 3303, which has a larger area and is circular.

[0201] Figure 33B An illustration of an exemplary sample analysis region is disclosed. The sample analysis region 3307 includes a tertiary region 3308 (e.g., a sample detection region) that is the width of the sample analysis region 3307. The sample analysis region 3307 includes quaternary regions 3311 (i.e., hydrophilic liquid pores) having substrate holding features 3312a-d, these substrate holding features being quarter-circular and protruding into the quaternary regions 3311. The sample analysis region includes a quintile region 3313 (e.g., a hydrophobic liquid pore). The left end 3309 has an elliptical shape, and the right end 3310 has an elliptical shape with the same dimensions as the left end.

[0202] Figure 33C An illustration of an exemplary sample analysis region is disclosed. The sample analysis region 3314 includes a tertiary region 3315 (e.g., a sample detection region), wherein the width of the sample analysis region is greater than that of the tertiary region. The sample analysis region 3314 includes a quaternary region 3318 (i.e., hydrophilic liquid pores) having substrate retention features 3319a-d, these substrate retention features being quarter-circular and protruding into the quaternary region 3318. The sample analysis region includes a pentagonal region 3320 (e.g., a hydrophobic liquid pore). The left end 3316 has an elliptical shape with a smaller area than the right end 3317, which has a larger area and is circular in shape.

[0203] Figure 33DAn illustration of an exemplary sample analysis region is disclosed. The sample analysis region 3321 includes a tertiary region 3322 (e.g., a sample detection region) that is the width of the sample analysis region 3321. The sample analysis region 3321 includes quaternary regions 3325 (i.e., hydrophilic liquid pores) having substrate holding features 3326a-d, these substrate holding features being quarter-circular and protruding into the quaternary regions 325. The sample analysis region includes a quintile region 3327 (e.g., hydrophobic liquid pores). The left end 3323 has an elliptical shape with a smaller area than the right end 3324, which has a larger area and is circular in shape.

[0204] Figure 33E An illustration of an exemplary sample analysis region is disclosed. The sample analysis region 3328 includes a tertiary region 3329 (e.g., a sample detection region), wherein the width of the sample analysis region 3321 at the edge of the tertiary region 3329 closest to the left end 3330 is smaller than the width at the edge of the tertiary region 3329 closest to the left end 3331. The sample analysis region 3328 includes quaternary regions 3332 (i.e., hydrophilic liquid pores) having substrate holding features 3333a-d, these substrate holding features being quarter-circular and protruding into the quaternary regions 3332. The sample analysis region includes a quintile region 3334 (e.g., hydrophobic liquid pores). The left end 3330 has an elliptical shape with a smaller area than the right end 3331, which has a larger area and is also elliptical.

[0205] Figure 33F An illustration of an exemplary sample analysis region is disclosed. The sample analysis region 3335 includes a tertiary region 3336 (e.g., a sample detection region) defining the width of the sample analysis region 3335. The sample analysis region 3335 includes quaternary regions 3339 (i.e., hydrophilic liquid pores) having substrate holding features 3340a-d, these substrate holding features being quarter-circular and protruding into the quaternary regions 3339. The sample analysis region includes a quintile region 3341 (e.g., a hydrophobic liquid pore). The left end 3337 has an elliptical shape with a smaller area than the right end 3338, which has a larger area and is circular, and its width decreases stepwise before the tertiary region 3336. In the sense of "stepwise width decrease," it means that there is an immediate, rather than gradual, symmetrical reduction in width.

[0206] Figure 33GAn illustration of an exemplary sample analysis region is disclosed. The sample analysis region 3342 includes a tertiary region 3343 (e.g., a sample detection region), wherein the width of the sample analysis region is greater than that of the tertiary region. The sample analysis region 3342 includes quaternary regions 3346 (i.e., hydrophilic liquid pores) having substrate retention features 3347a-d, these substrate retention features being quarter-circular and protruding into the quaternary regions 3346. The sample analysis region includes a pentagonal region 3348 (e.g., hydrophobic liquid pores). The left end 3344 has an elliptical shape with a smaller area than the right end 3345, which has a larger area and is circular. The left end has a stepped decrease in width before the tertiary region 3343.

[0207] Figure 33H -N illustrates exemplary substrate holding features. Figure 33H In the sample analysis region 3349, there is a quadrature region 3351 (i.e., a hydrophilic liquid pore) with substrate holding features 3351 and 3352. The substrate holding features 3351 and 3352 can protrude from the bottom substrate to the top substrate or from the top substrate to the bottom substrate. The substrate holding feature 3351 is centrally located on the right end 3354 and is quarter-circular. The substrate holding feature 3352 is located on the right end 3354 of the sample analysis region 3349 and is horseshoe-shaped with a gradually widening edge. Figure 33H The substrate retention features are applied to any sample analysis area disclosed herein.

[0208] exist Figure 33I In the sample analysis region 3356, there is a quadrature region 3359 (i.e., a hydrophilic liquid pore) with substrate holding features 3357 and 3358. The substrate holding features 3357 and 3358 can protrude from the bottom substrate to the top substrate or from the top substrate to the bottom substrate. The substrate holding feature 3357 is centrally located on the right end 3360 and is horseshoe-shaped without a tapered edge. The substrate holding feature 3358 is located on the right end 3360 of the sample analysis region 3356 and is also horseshoe-shaped without a tapered edge. The substrate holding features 3357 and 3358 are concentric, such that the substrate holding feature 3357 is nested within the substrate holding feature 3358. Figure 33I The substrate retention features are applied to any sample analysis area disclosed herein.

[0209] exist Figure 33JIn the sample analysis region 3361, there is a quadrature region 3362 (i.e., hydrophilic liquid pores) having substrate holding features 3364a-d. The substrate holding features 3364a-d are recesses in the top or bottom substrate of the sample analysis region 3361. The substrate holding features 3364a-d are located on the right end 3363 of the sample analysis region 3361 and are horseshoe-shaped. The substrate holding features 3364a-d are concentric, such that substrate holding feature 3364a is nested within substrate holding feature 3364b, substrate holding feature 3364b is nested within substrate holding feature 3364c, and substrate holding feature 3364c is nested within substrate holding feature 3364d. Figure 33I The substrate retention features are applied to any sample analysis area disclosed herein.

[0210] exist Figure 33K In the sample analysis region 3365, there is a quadrature region 3367 (i.e., a hydrophilic liquid pore) having substrate holding features 3368a-d and 3369. The substrate holding features 3368a-d are quarter-circular and protrude into the quadrature region 3367, located at the right end 3366. The substrate holding feature 3369 is located at the right end 3366 of the sample analysis region 3365 and is horseshoe-shaped. The substrate holding feature 3369 can protrude from the bottom substrate to the top substrate or from the top substrate to the bottom substrate. Figure 33K The substrate retention features are applied to any sample analysis area disclosed herein.

[0211] exist Figure 33L In the sample analysis region 3370, there is a quaternary region 3372 (i.e., a hydrophilic liquid pore) having substrate holding features 3373a-d and 3374a-b. The substrate holding features 3373a-d are quarter-circular and protrude into the quaternary region 3372, located at the right end 3371. The substrate holding features 3374a-b are located before the right end 3371 of the sample analysis region 3370 and are rectangular. The substrate holding features 3374a-b can protrude from the bottom substrate to the top substrate or from the top substrate to the bottom substrate. Figure 33L The substrate retention features are applied to any sample analysis area disclosed herein.

[0212] exist Figure 33MIn the sample analysis region 3375, there is a quaternary region 3377 (i.e., a hydrophilic liquid pore) having substrate holding features 3378a-d and 3379a-b. The substrate holding features 3378a-d are quarter-circular and protrude into the quaternary region 3377, located at the right end 3376. The substrate holding features 3379a-b are located before the right end 3376 of the sample analysis region 3375 and are circular in shape. The substrate holding features 3379a-b can protrude from the bottom substrate to the top substrate or from the top substrate to the bottom substrate. Figure 33M The substrate retention features are applied to any sample analysis area disclosed herein.

[0213] exist Figure 33N In the sample analysis region 3380, there is a quadrature region 3382 (i.e., a hydrophilic liquid pore) having substrate holding features 3383a-d and 3384. The substrate holding features 3383a-d are quarter-circular and protrude into the quadrature region 3382, located at the right end 3381. The substrate holding feature 3384 is located at the right end 3381 of the sample analysis region 3380 and is horseshoe-shaped. The substrate holding feature 3384 can protrude from the bottom substrate to the top substrate or from the top substrate to the bottom substrate. Figure 33K The substrate retention features are applied to any sample analysis area disclosed herein.

[0214] Figure 330 An illustration of an exemplary barrier feature is disclosed. The sample analysis region 3385 includes a five-level region 3386 (e.g., a hydrophobic liquid pore) on its left end 3387. The sample analysis region also includes a barrier feature 3388. The barrier feature 3388 is semi-circular. The barrier feature 3388 may protrude from a bottom substrate to a top substrate or from a top substrate to a bottom substrate. Figure 330 The barrier feature is applied to any sample analysis area disclosed herein. The barrier feature can be configured to assist in sealing the orifice using the hydrophobic liquid disclosed herein.

[0215] Figure 43Exemplary substrate stop features are disclosed. In some embodiments, the sample analysis region 4301 (dashed box) includes a substrate stop feature 4302. The sample analysis region includes an enlarged protruding element 4304, wherein the surface of the enlarged protruding element facing the first substrate has a larger surface area than the surfaces of the plurality of protruding elements facing the first substrate, and the enlarged protruding element extends from a first end (e.g., a level 5 region 4303) to a second end (e.g., a level 4305). The substrate stop feature includes a ridge that creates a height difference between the level 5 region 4303 (i.e., a hydrophobic liquid pore) and the level 4305 (i.e., a hydrophilic liquid pore). When a hydrophilic liquid is present in the sample analysis region, the substrate stop assists in retaining the hydrophilic liquid outside the level 5 region. The substrate stop feature may be present in any embodiment of a sample analysis region including an enlarged protruding element.

[0216] Figure 11 An isometric view illustration of an embodiment of the device is disclosed. Dotted lines represent the internal portions of the device, while solid lines represent the external portions. In this embodiment, in addition to a fixed primary region 1108, the device 1100 also includes a non-mesh pattern (i.e., a honeycomb pattern) of a primary region 1103 and a secondary region 1106. In some, but not all, cases, the fixed primary region 1108 is also a sample mixing region. The device includes a reagent / sample injection port 1109. The device includes a sample processing region 1105. The sample processing region 1105 is unbounded. By unbounded, it means that there is no physical barrier separating the primary region 1103 and the secondary region 1106. The sample processing region includes the primary region 1103, in which an opening 1104 is provided. The primary region is adjacent to the secondary region (such as 1106). The sample processing region 1105 may be connected to a tertiary region (i.e., a sample detection region) 1102. The device may also include a quaternary region 1107 (i.e., a hydrophilic liquid pore) connected to the tertiary region. The device may also include a quinary region 1101 (i.e., a hydrophobic liquid pore) connected to the tertiary region.

[0217] Figure 12 Depicting Figure 11 The diagram shows a top view of an embodiment of the device depicted. 1201 refers to the reagent / sample injection port. 1202 refers to the primary fixation region. 1206 refers to the primary region. 1207 refers to the secondary region. 1205 refers to the sample analysis region. 1203 refers to the hydrophilic liquid pore. 1204 refers to the hydrophobic liquid pore.

[0218] Figure 13 Depicting Figure 12An embodiment of the device features a reduced size and a primary region. 1301 refers to the reagent / sample injection port. 1302 refers to the stationary primary region. 1306 refers to the primary region. 1307 refers to the secondary region. 1305 refers to the sample analysis region. 1303 refers to the hydrophilic liquid pore. 1304 refers to the hydrophobic liquid pore.

[0219] Figure 14 Depicting according to Figure 8 This is a bottom view of the top substrate of the device. 1401 refers to the sample processing area. 1402 refers to the primary fixing area. 1403 refers to the primary area. 1404 refers to the opening in the primary area. 1407 refers to the secondary area. 1406 refers to the opening in the secondary area.

[0220] Figure 15 Depicting according to Figure 7 This is a bottom view of the top substrate of the device. 1501 refers to the sample processing area. 1502 refers to the fixed primary area. 1503 refers to the primary area. 1504 refers to the opening in the primary area. 1507 refers to the secondary area. 1506 refers to the opening in the secondary area. 1408 refers to the sample detection area.

[0221] Figure 32B Depicting according to Figure 32A This is a bottom view of the top substrate of the device. 3210 refers to the sample processing area. 3211 refers to the primary fixing area. 3212 refers to the primary area. 3213 refers to the opening in the primary area. 3214 refers to the secondary area. 3215 refers to an opening in the secondary area.

[0222] Figure 39 An illustration of an embodiment of the sample mixing region of the apparatus depicted in FIG38 is disclosed. Figure 39The embodiment illustrates the surface of the second substrate facing the central chamber or the first substrate. In this embodiment, the device 3900 includes a sample mixing region comprising a fixed primary region 3901 (red box). The fixed primary region 3901 is formed by laterally extending protruding elements 3902 in the second substrate facing the first substrate, wherein the laterally extending protruding elements have a surface facing the first substrate having a larger surface area than the surface of the plurality of protruding elements facing the first substrate. The fixed primary region 3901 includes an opening 3903 through which the sample can be deposited. The fixed primary region 3901 includes a chamfered end 3904 laterally separated from the connecting end 3905. In the sense of a “chamfered end,” it means 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 can be connected to a vibration source that vibrates vertically, causing the second substrate (i.e., the top plate) to compress and decompress a fluid (e.g., a sample) that may be located in the fixed primary region 3902, thereby mixing the fluid. The boundary of the top outer portion of the fixed primary region is not attached to the rest of the device, except for the connecting end 3905. In some embodiments, the laterally extending protruding element includes a raised beveled periphery 3907. "Raised beveled periphery" means that the periphery of the laterally extending protruding element has a raised beveled edge. The raised beveled edge assists in holding the sample in the sample mixing region during the mixing process. In some embodiments, the laterally extending protruding element includes a plurality of secondary features 3906a, 3906b, 3906c. The plurality of secondary features 3906a, 3906b, 3906c are raised supports that assist in mixing the sample that may be present in the sample mixing region. Figure 39 The sample mixing zone is applied to any other embodiment of the device, for example... Figure 1-9 , Figure 11-1 9. The embodiments disclosed in Figures 32 and 38-41.

[0223] Figure 40A and Figure 40B An illustration of an exemplary waste disposal area of ​​the apparatus depicted in Figure 38 is disclosed. Figure 40A and Figure 40BAn embodiment illustrates a waste disposal area on the surface of a second substrate facing away from the central chamber. The waste disposal area 4001 is located at the end of the sample processing area 4005 furthest from the sample analysis area 4006. The waste disposal area 4001 includes an opening located in the second substrate. The opening of the waste disposal area 4001 has a wedge-shaped portion 4002 extending into the central chamber. The wedge-shaped portion 4002 allows for wicking of waste 4003 from a probe that can dispense waste into a waste port. The wedge-shaped portion 4002 of the waste disposal area 4001 guides the waste 4003 into the central chamber. The waste disposal area provides the benefit of being able to dispose of waste directly into the device without the need for external disposal. Figure 40A and Figure 40B The waste disposal area is applied to any other embodiment of the device, such as... Figure 1-9 , Figure 11-1 9. The embodiments disclosed in Figures 32 and 38-41.

[0224] Figure 41A and Figure 41B An illustration of an exemplary preprocessing area of ​​the device is disclosed. Figure 41A The embodiment shows the surface of the second substrate facing away from the central cavity. Figure 41BThe embodiments illustrate a surface of the second substrate facing the central chamber or the first substrate. In some embodiments, the apparatus 4101 includes a pretreatment region 4102. This 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 square. The pretreatment region includes a plurality of elongated openings 4104a, 4104b, 4104c, and 4104d around the periphery of the pretreatment region 4102. The elongated openings 4104a, 4104b, 4104c, and 4104d around the periphery of the pretreatment region 4102 are separated from each other by a plurality of connecting regions 4107a, 4107b, 4107c, and 4107d. The plurality of connecting regions may be located at each corner of the pretreatment region 4102. The elongated openings serve as a hydrophobic barrier between the pretreatment region 4102 and the sample processing region 4103, and weaken the mechanical connection between the pretreatment region and the second substrate to enhance the mechanical vibration of the pretreatment region during mixing. The pretreatment region 4102 includes a raised edge 4106 surrounding a periphery enclosed by the plurality of elongated openings 4104a, 4104b, 4104c, and 4104d. The raised edge helps retain fluid deposited into the pretreatment region. The pretreatment region 4102 includes a plurality of openings 4105a, 4105b, and 4105c. The plurality of openings create a gas-liquid interface that assists in mixing any fluid deposited within the pretreatment region. In some embodiments, the pretreatment region is located at the end of the sample processing region 4103 furthest from the sample analysis region. In some embodiments, when the device includes a pretreatment region, the device does not include a sample mixing region. In some embodiments, when the device includes a pretreatment region, fluids on the device are mixed by vibrating the device, as described below. The pretreatment region allows for the pretreatment of a sample before it is introduced into the sample processing region. For example, the pretreatment region can be used to dilute a sample, lyse a sample, modify a sample or target analyte to allow for the capture of the target analyte, etc. In some embodiments, the pretreatment zone contains one or more pretreatment reagents, such as one or more detergents, surfactants, reducing agents, or any combination thereof. In yet another embodiment, the sample is contacted with the pretreatment reagents before being introduced into the sample treatment zone.

[0225] Figure 54Illustrations of an exemplary embodiment of an apparatus containing a pretreatment region are disclosed. In this embodiment, apparatus 5400 includes a 2x3 grid of primary region 5403 and secondary region 5406, and a fixed primary region 5402. Apparatus 5400 includes a second substrate positioned on a first substrate. The second substrate includes a surface facing a central chamber, which includes a plurality of recessed elements and a plurality of protruding elements. The primary region is defined between the surfaces of the plurality of protruding elements facing the first substrate and the surfaces of the first substrate facing the second substrate. The secondary region is defined between the surfaces of the plurality of recessed elements facing the first substrate and the surfaces of the first substrate facing the second substrate. In some, but not all, cases, the fixed primary region 5402 is also a sample mixing region. The fixed primary region 5402 is formed by laterally extending protruding elements in the second substrate facing the first substrate, wherein the laterally extending protruding elements have a surface facing the first substrate that has a larger surface area than the surfaces of the plurality of protruding elements facing the first substrate. The laterally extending protruding elements may be referred to as third protruding elements. The fixed primary region 5402 includes a chamfered end 5412 laterally separated from the connecting end 5413. By "chamfered end," it means 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 boundary of the top outer portion of the fixed primary region is not attached to the rest of the device, except for the connecting end 5413. When a vertical force is applied to the chamfered end 5412, the second substrate (i.e., the top plate) compresses and decompresses a fluid (e.g., a sample) that may be located in the fixed primary region 5402, thereby mixing the fluid. The device includes a sample processing region 5401. The sample processing region 5401 is unbounded. By unbounded, it means that there is no physical barrier separating the primary region 5403 and the secondary region 5406. The sample processing region includes the primary region 5403, in which an opening 5404 is provided. The primary region is adjacent to a secondary region (such as 5406). The secondary region may include an opening 5405. The device 5400 includes a sample analysis region 5407. In some embodiments, the device 5400 includes a waste disposal region 5408. The waste disposal region 5408 is located at the end of the sample processing region 5401 furthest from the sample analysis region 5407. The waste disposal region 5408 includes an opening in a second substrate and has a wedge-shaped portion on the side of the opening closest to the edge of the device.

[0226] In some embodiments, the device 5400 includes a pretreatment region 5416. The pretreatment region is formed between a surface of the second substrate facing the first substrate and a surface of the first substrate facing the second substrate. In some embodiments, the pretreatment region is square. The pretreatment region 5416 includes a raised edge 5414 around its periphery. This raised edge helps retain fluid deposited into the pretreatment region. The pretreatment region 5416 includes a plurality of openings 5415a, 5415b, and 5415c. The plurality of openings create a gas-liquid interface that assists in mixing any fluid deposited within the pretreatment region. In some embodiments, the pretreatment region is located at the end of the sample processing region 5401 furthest from the sample analysis region. In some embodiments, when the device includes the pretreatment region, fluids on the device are mixed by vibrating the device, as described below. The pretreatment region allows for the pretreatment of a sample before it is introduced into the sample processing region. For example, the pretreatment region can be used to dilute a sample, lyse a sample, modify a sample or target analyte to allow for the capture of the target analyte, etc. In some embodiments, the pretreatment zone contains one or more pretreatment reagents, such as one or more detergents, surfactants, reducing agents, or any combination thereof. In yet another embodiment, the sample is contacted with the pretreatment reagents before being introduced into the sample treatment zone.

[0227] I. Layout of the sample processing area The sample processing area disclosed herein has a range of different arrangements, such as those described above. The sample processing area disclosed herein also has a range in terms of the number of primary regions present. For example, the device may include 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 regions.

[0228] The primary region of this disclosure has a pad formed by a top substrate. The pad found in the primary region can have a wide range of shapes, including but not limited to rectangles, circles, triangles, pentagons, hexagons, heptagons, octagons, decagons, dodecagons, amoebas, irregular shapes, or pointed circular shapes. In some embodiments, the pad is rectangular or square, such as... Figure 1-7 The shape depicted in the image. In some embodiments, the pad is circular, such as... Figure 8 and Figure 14 The shape depicted in the image. In some embodiments, the pad is hexagonal, such as... Figure 11-13 The shape depicted in the image. In some embodiments, the pad is amoeba-like, such as... Figure 27A The shape depicted in the figure. In some embodiments, the pad is an irregular shape. In some embodiments, the pad is a round shape with a pointed tip, such as... Figure 27A The shape depicted. The pad in the primary region has edges. In some embodiments, the edges are sharp. In some embodiments, the edges are curved, such that the edges have a rounded shape. The pad in the primary region may have a specific 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... Figure 16 As depicted in the text.

[0229] One or more surfaces of a pad can be designed in such a way that they retain fluid. The term "pad surface" refers to any surface of the pad facing the interior of the device, such as the side of the pad facing another pad, the side of the pad facing the interior of the device, or the side of the pad facing the bottom substrate inside the device. The pad surface can also be referred to as the surface of the plurality of protruding elements facing the first substrate. Alternative terms for the pad surface are "leading face" and "side surface," where "leading face" refers to the surface of the pad facing the bottom substrate, and "side surface" refers to a surface other than the "leading face"; when the terminology is used and the device is planar, the "leading face" may be substantially parallel to the first plane, and the "side surface" may be substantially perpendicular to the first plane. For example, the one or more surfaces of the pad may contain serrations or grooves occupying all or part of a given pad. The one or more surfaces of the pad 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 the pad. The serrations or grooves can be in a range of different patterns, including but not limited to waves, straight lines perpendicular or parallel to the edge of the pad, straight lines diagonally opposite the edge of the pad, cross-shaped or shading patterns, and any combination thereof. In some embodiments, the serrations or grooves are in a wave pattern, such as... Figure 28G As depicted in the illustration. In some embodiments, the serrations or grooves are straight lines perpendicular to or parallel to the edge of the pad, such as... Figure 28A As depicted in -B. In some embodiments, the serrations or grooves are straight lines diagonally opposite to the edge of the pad, such as... Figure 28C As depicted in -D. In some embodiments, the serrations or grooves are in a cross shape or a shading pattern, such that two or more serrations or grooves intersect each other, such as... Figure 28EAs depicted in -F. In some embodiments, the serrations or grooves are evenly spaced. In some embodiments, the serrations or grooves are not evenly spaced. In some embodiments, all pads in the sample processing area contain serrations or grooves. In some embodiments, only a portion of the pads in the sample processing area contains grooves or serrations.

[0230] One or more of the surfaces of the device may have a surface finish. Surface finish provides the advantage of consistent surface roughness on surfaces with a finish, and provides consistent fluid behavior across multiple surfaces and devices sharing the same surface finish. In the context of "surface finish," it means that the surface of the pad may have a certain smoothness or polish. Surface finish can be described as Plastics Industry Association (SPI) finish or German Engineering Association (VDI) finish. VDI finish can also be referred to as VDI 3400. The surface of the pad may have any finish deemed useful. For example, the surface of the pad may have 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 VDI 340045. The surface of the pad may have SPI A1, A2, A3, B1, B2, B3, C1, C2, C3, D1, D2 or SPI D3. The surface finish may be a series of surface finishes. For example, the surface finish can be from SPI A1 to SPI A3, from SPI B1 to SPI B3, from SPI C1 to SPI C3, or from SPI D1 to D3. The surface finish can 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.

[0231] Surface finish can be defined by the average surface roughness (RA) measured in micrometers (μm). For example, SPI A1 may have an RA of approximately 0.012 to approximately 0.025 μm, SPI A2 may have an RA of approximately 0.025 to approximately 0.05 μm, SPI A3 may have an RA of approximately 0.05 to approximately 0.10 μm, SPI B1 may have an RA of approximately 0.05 to approximately 0.1 μm, SPI B2 may have an RA of approximately 0.1 to approximately 0.15 μm, SPI B3 may have an RA of approximately 0.28 to approximately 0.32 μm, SPI C1 may have an RA of approximately 0.35 to approximately 0.40 μm, SPI C2 may have an RA of approximately 0.45 to approximately 0.55 μm, SPI C3 may have an RA of approximately 0.63 to approximately 0.70 μm, SPI D1 may have an RA of approximately 0.80 to approximately 1.00 μm, SPI D2 may have an RA of approximately 1.00 to approximately 2.80 μm, and SPI... D3 can have an RA of approximately 3.20 to approximately 18.00 μm. VDI 3400 0 has an RA of approximately 0.100 μm, VDI 3400 1 has an RA of approximately 0.112 μm, VDI 3400 2 has an RA of approximately 0.126 μm, VDI 3400 3 has an RA of approximately 0.140 μm, VDI 3400 4 has an RA of approximately 0.160 μm, VDI 3400 5 has an RA of approximately 0.180 μm, VDI 3400 6 has an RA of approximately 0.200 μm, VDI 3400 7 has an RA of approximately 0.220 μm, VDI 3400 8 has an RA of approximately 0.250 μm, VDI 3400 9 has an RA of approximately 0.290 μm, and VDI 3400 10 has an RA of approximately 0.320 μm. VDI 3400 11 has an RA of approximately 0.350 μm, VDI 3400 12 has an RA of approximately 0.400 μm, VDI 3400 13 has an RA of approximately 0.450 μm, VDI 3400 14 has an RA of approximately 0.500 μm, VDI 3400 15 has an RA of approximately 0.560 μm, VDI 3400 16 has an RA of approximately 0.630 μm, VDI 3400 17 has an RA of approximately 0.700 μm, VDI 3400 18 has an RA of approximately 0.800 μm, VDI 3400 19 has an RA of approximately 0.900 μm, VDI 3400 20 has an RA of approximately 1.000 μm, and VDI 3400 21 has an RA of approximately 1.The VDI 3400 22 has an RA of approximately 1.260 μm, the VDI 3400 23 has an RA of approximately 1.400 μm, the VDI 3400 24 has an RA of approximately 1.600 μm, the VDI 3400 25 has an RA of approximately 1.800 μm, the VDI 3400 26 has an RA of approximately 2.000 μm, the VDI 3400 27 has an RA of approximately 2.200 μm, the VDI 3400 28 has an RA of approximately 2.500 μm, the VDI 3400 29 has an RA of approximately 2.800 μm, the VDI 3400 30 has an RA of approximately 3.200 μm, and the VDI 3400 31 has an RA of approximately 3.500 μm. The VDI 3400... VDI 3400 32 has an RA of approximately 4.000 μm; VDI 33 has an RA of approximately 4.500 μm; VDI 3400 34 has an RA of approximately 5.000 μm; VDI 3400 36 has an RA of approximately 6.300 μm; VDI 3400 37 has an RA of approximately 7.000 μm; VDI 3400 38 has an RA of approximately 8.000 μm; VDI 3400 39 has an RA of approximately 9.000 μm; VDI 3400 40 has an RA of approximately 10.000 μm; VDI 3400 41 has an RA of approximately 11.200 μm; VDI 3400 42 has an RA of approximately 12.600 μm; and VDI 3400 43 has an RA of approximately 14.000 μm. The RA (Radar Interaction) is approximately 16.000 μm for the VDI 3400 44 and approximately 18.000 μm for the VDI 3400 45.

[0232] In some embodiments, the surface of the pad has a surface finish. In some embodiments, the surfaces of the plurality of protruding elements facing the first substrate have a surface finish. In some embodiments, the surfaces of the enlarged protruding elements in the sample analysis region facing the first substrate have a surface finish. In some embodiments, the laterally extending protruding elements in the second substrate have a surface finish on the surface of the first substrate facing the sample mixing region. In some embodiments, the surfaces of the plurality of protruding elements facing the first substrate have a surface finish, and the surfaces of the enlarged protruding elements in the sample analysis region facing the first substrate have a surface finish. In some embodiments, the surfaces of the plurality of protruding elements facing the first substrate have a surface finish, the surfaces of the enlarged protruding elements in the sample analysis region facing the first substrate have a surface finish, and the laterally extending protruding elements in the second substrate have a surface finish on the surface of the first substrate facing the sample mixing region.

[0233] In some embodiments, the surface finish of the enlarged protruding elements in the sample analysis region has a higher surface finish than the surface finish of the surfaces of the plurality of protruding elements facing the first substrate. "Higher surface finish" means a lower average surface roughness (RA). For example, a surface finish of 0.012 μm is higher than a surface finish of 0.05 μm, and the surface finish of SPI A1 is higher than the surface finish of SPI A3. The higher surface finish of the enlarged protruding elements in the sample analysis region provides the additional benefit of increased optical clarity, thereby assisting in the detection of analytes in the sample detection region. In some embodiments, the surface finish of the enlarged protruding elements in the sample analysis region has a surface finish from approximately SPI A1 to approximately SPI A3. In some embodiments, the surfaces of the plurality of protruding elements facing the first substrate have a surface finish from approximately SPI B1 to approximately SPI C3. In some embodiments, the surfaces of the laterally extending protruding elements facing the first substrate have a surface finish from approximately SPI B1 to approximately SPI C3. In some embodiments, the surface finish of the enlarged protruding elements in the sample analysis region has a surface finish from about 0.012 μm to about 0.10 μm. In some embodiments, the surfaces of the plurality of protruding elements facing the first substrate have a surface finish from about 0.15 μm to about 0.70 μm. In some embodiments, the surfaces of the laterally extending protruding elements facing the first substrate have a surface finish from about 0.15 μm to about 0.70 μm.

[0234] Figure 47An illustration depicts an embodiment of a device 4700 having a surface finish on one or more surfaces. In this embodiment, the surfaces 4701a, 4701b, and 4701c of the plurality of protruding elements facing the first substrate have 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 laterally extending protruding element 4703 in the second substrate has a surface finish on the surface of the sample mixing region facing the first substrate. In some embodiments, the enlarged protruding element 4702 of the sample analysis region facing the first substrate has a higher surface finish than the surfaces 4701a, 4701b, and 4701c of the plurality of protruding elements facing the first substrate. 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 extending protruding element 4703 of 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 a substrate holding feature 4704. In some embodiments, the enlarged protruding element 4702 of the sample analysis region has a pinning wall 4705 surrounding the periphery of the enlarged protruding element 4702, the periphery excluding a second end containing a level 5 region (not shown). The pinning wall 4705 is a raised edge that protrudes from the enlarged protruding element 4702. In some embodiments, the substrate retaining feature 4704 has a surface finish. In some embodiments, the pinning wall has a surface finish.

[0235] Figure 28I The illustration depicts a pad with serrations or grooves in the primary region and a pad without serrations or grooves in the primary region. A top substrate 2801 and a bottom substrate 2802 define the interior of the device. The pad on the left has surfaces 2803a and 2803b, which are without serrations or grooves. The pad has three additional surfaces not depicted: one opposite surface 2803a; one opposite surface 2803b; and one facing the bottom substrate 2802. The pad on the right has serrations or grooves on surfaces 2804a and 2804b diagonally opposite the edge of the pad. The pad may have additional surfaces with serrations or grooves, such as the surface of the pad facing the bottom surface 2802 (e.g., Figure 28J (as depicted in the text), the surface of the pad opposite to surface 2804a, and the surface of the pad opposite to surface 2804b.

[0236] In some embodiments, one or more surfaces of the pad contain pits or dents, such as Figure 28HAs depicted in the figures. In some embodiments, the pit or dent is concave. In some embodiments, the pit or dent is convex (e.g., protrusion). In some embodiments, a portion of the pit or dent is convex and a portion of the pit or dent is concave. In some embodiments, the pit or dent is circular in shape. In some embodiments, the pit or dent is triangular in shape. In some embodiments, the pit or dent is rectangular in shape. In some embodiments, the pit or dent is pentagonal in shape. In some embodiments, the pit or dent is cylindrical in shape. In some embodiments, the pit or dent is hexagonal in shape. In some embodiments, the pit or dent is heptagonal in shape. In some embodiments, the pit or dent is octagonal in shape. In some embodiments, the pit or dent is decagonal in shape. In some embodiments, the pit or dent is dodecagonal in shape. In some embodiments, the pit or dent is amoeba-like in shape. In some embodiments, the pit or dent is irregular in shape. In some embodiments, the pits or dents are evenly spaced. In some embodiments, the pits or dents are not evenly spaced. In some embodiments, the serrations or grooves are not evenly spaced. In some embodiments, all pads in the sample processing area contain pits or dents. In some embodiments, only a portion of the pads in the sample processing area contains pits or dents. In some embodiments, a portion of the pads in the sample processing area contains pits or dents, and a portion of the pads contains serrations or grooves. In some embodiments, all or a portion of the pads in the sample processing area contains both serrations or grooves and pits or dents.

[0237] Figure 16 A diagram illustrating a cross-section of the primary region and pads in an embodiment of the device is shown. The primary region 1607 (between the dotted lines) is formed by a top substrate 1601 and a bottom substrate 1602. The primary region contains a pad 1604 or 1605. In some embodiments, the pad is flat (1604). In some embodiments, the pad has a protrusion 1605 around its entire edge, such that the protrusion 1605 is closer to the bottom substrate 1602 than the center of the pad 1606.

[0238] The pads disclosed herein have a range of thicknesses. For example, the pads may be 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 pads have a thickness between 0.3 mm and 0.8 mm.

[0239] The pad and the bottom substrate in the primary region may have specific patterning. This patterning can be designed to retain fluid within the primary region. In some embodiments, the pad and the bottom substrate have hydrophilic patterning. In some embodiments, the pad and the bottom substrate in the primary region have no patterning.

[0240] The sample processing region disclosed herein also has a range in terms of the number of secondary regions present. The device may include 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, twenty or more, twenty-one or more, twenty-two or more, twenty-three or more, twenty-four or more, twenty-five or more, twenty-six or more, twenty-seven or more, twenty-eight or more, twenty-nine or more, thirty or more, thirty-one or more, thirty-two or more, thirty-three or more, thirty-four or more, thirty-five or more, thirty-six or more, or thirty-seven or more secondary regions.

[0241] The secondary region of this disclosure is formed by a top substrate and a bottom substrate of the device. The secondary region exists between one or more primary regions. In some embodiments, the secondary region is located between two primary regions. The secondary region has 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 region. The height difference between the primary and secondary regions generates surface tension that can retain fluid in the primary region without overflowing into the secondary region. Figure 16 Figure 26 depicts the height difference between the primary and secondary regions. In some embodiments, the top and bottom substrates of the secondary region have hydrophobic patterned portions. In some embodiments, the top and bottom substrates of the secondary region do not have patterned portions. In some embodiments, the top and bottom substrates of the secondary region have uniform hydrophobicity.

[0242] Secondary regions can be designed in a way that allows them to retain fluid. For example, the top plate of a secondary region (i.e., the bottom surface of the top substrate facing the interior of the device) may contain serrations or grooves occupying all or part of a given top plate of the secondary region. In some embodiments, the serrations or grooves are in a wavy pattern, such as... Figure 28G and Figure 28K As depicted in [the text]. In some embodiments, the serrations or grooves are straight lines perpendicular or parallel to the edge of the pad adjacent to the secondary area, such as [examples]. Figure 28A As depicted in -B. In some embodiments, the serrations or grooves are straight lines diagonally aligned with the edge of the pad adjacent to the secondary area, such as... Figure 28C As depicted in -D. In some embodiments, the serrations or grooves are in a cross shape or a shading pattern, such that two or more serrations or grooves intersect each other, such as... Figure 28EAs depicted in -F. In some embodiments, the serrations or grooves are evenly spaced. In some embodiments, the serrations or grooves are not evenly spaced. In some embodiments, all top plates of the secondary region in the sample processing area contain serrations or grooves. In some embodiments, only a portion of the top plate of the secondary region in the sample processing area contains grooves or serrations.

[0243] In some embodiments, the top plate of the secondary region contains pits or indentations, such as Figure 28H As depicted in the figures. In some embodiments, the pit or dent is concave. In some embodiments, the pit or dent is convex (e.g., protrusion). In some embodiments, a portion of the pit or dent is convex and a portion of the pit or dent is concave. In some embodiments, the pit or dent is circular in shape. In some embodiments, the pit or dent is triangular in shape. In some embodiments, the pit or dent is rectangular in shape. In some embodiments, the pit or dent is pentagonal in shape. In some embodiments, the pit or dent is cylindrical in shape. In some embodiments, the pit or dent is hexagonal in shape. In some embodiments, the pit or dent is heptagonal in shape. In some embodiments, the pit or dent is octagonal in shape. In some embodiments, the pit or dent is decagonal in shape. In some embodiments, the pit or dent is dodecagonal in shape. In some embodiments, the pit or dent is amoeba-like in shape. In some embodiments, the pit or dent is irregular in shape. In some embodiments, the pits or dents are evenly spaced. In some embodiments, the pits or dents are not evenly spaced. In some embodiments, all top plates of the secondary region in the sample processing area contain pits or dents. In some embodiments, only a portion of the top plate of the secondary region in the sample processing area contains pits or dents. In some embodiments, a portion of the top plate of the secondary region in the sample processing area contains pits or dents, and a portion of the top plate of the secondary region contains serrations or grooves. In some embodiments, all or a portion of the top plate of the secondary region in the sample processing area contains both serrations or grooves and pits or dents.

[0244] In some embodiments, the top substrate has uniform hydrophobicity, such that the entire surface of the top substrate is hydrophobic, hydrophilic, or non-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 non-hydrophobic or hydrophilic. In some embodiments, the top substrate (e.g., the second substrate) is hydrophobic. In some embodiments, the top substrate (e.g., the second substrate) is hydrophilic. In some embodiments, the bottom substrate (e.g., the first substrate) is hydrophobic. In some embodiments, the bottom substrate (e.g., the first substrate) is hydrophilic. In some embodiments, the top substrate (e.g., the second substrate) is hydrophobic, and the bottom substrate (e.g., the first substrate) is hydrophobic. In some embodiments, the top substrate (e.g., the second substrate) is hydrophobic, and the bottom substrate (e.g., the first substrate) is hydrophilic. In some embodiments, the top substrate (e.g., the second substrate) is hydrophobic, and the bottom substrate (e.g., the first substrate) is hydrophilic. In embodiments where both the top substrate (e.g., the second substrate) and the bottom substrate (e.g., the first substrate) are hydrophobic, the top substrate (e.g., the second substrate) may have higher hydrophobicity than the bottom substrate (e.g., the first substrate). In embodiments where both the top substrate (e.g., the second substrate) and the bottom substrate (e.g., the first substrate) are hydrophobic, the bottom substrate (e.g., the first substrate) may have higher hydrophobicity than the top substrate (e.g., the second substrate). In embodiments where both the top substrate (e.g., the second substrate) and the bottom substrate (e.g., the first substrate) are hydrophilic, the top substrate (e.g., the second substrate) may have higher hydrophilicity than the bottom substrate (e.g., the first substrate). In embodiments where both the top substrate (e.g., the second substrate) and the bottom substrate (e.g., the first substrate) are hydrophilic, the bottom substrate (e.g., the first substrate) may have higher hydrophilicity than the top substrate (e.g., the second substrate).

[0245] In some embodiments, there is a hydrophobic or hydrophilic difference between the top substrate and the bottom substrate. When compared to a top substrate (e.g., the second substrate) and a bottom substrate (e.g., the first substrate) without a hydrophobic or hydrophilic difference, the hydrophobic or hydrophilic difference of the top substrate (e.g., the second substrate) or the bottom substrate (e.g., the first substrate) enhances fluid retention in the primary region. In some embodiments, there is a hydrophobic or hydrophilic difference between the second substrate and the first substrate. This hydrophobic or hydrophilic difference can be a difference in contact angle between the top substrate (e.g., the second substrate) and the bottom substrate (e.g., the first substrate). The difference in contact angle between the top substrate (e.g., the second substrate) and the bottom substrate (e.g., the first substrate) can exist within a certain range. For example, there may be a difference of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% between the contact angles of the top substrate (e.g., the second substrate) and the bottom substrate (e.g., the first substrate) or the bottom substrate (e.g., the second substrate) and the top substrate (e.g., the first substrate).

[0246] In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 5% to approximately 10%. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 10% to approximately 15%. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 15% to approximately 20%. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 20% to approximately 25%. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 25% to approximately 30%. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 30% to approximately 35%. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 35% to approximately 40%. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 40% to approximately 45%. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 45% to approximately 50%. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 50% to approximately 55%. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 55% to approximately 60%. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 60% to approximately 65%. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 65% ​​to approximately 70%. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 70% to approximately 75%.

[0247] In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 10% to approximately 20%. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 20% to approximately 30%. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 30% to approximately 40%. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 40% to approximately 50%. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 50% to approximately 60%. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 60% to approximately 70%.

[0248] In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 10% to approximately 70%. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 10% to approximately 60%. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 10% to approximately 50%. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 10% to approximately 40%. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 10% to approximately 30%. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 10% to approximately 20%.

[0249] In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 20% to approximately 70%. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 20% to approximately 60%. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 20% to approximately 50%. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 20% to approximately 40%. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 20% to approximately 30%.

[0250] The difference in contact angle between the top substrate (e.g., the second substrate) and the bottom substrate (e.g., the first substrate) can exist within a certain range. For example, the contact angles between the top substrate (or the second substrate) and the bottom substrate (first substrate) or the bottom substrate (or the first substrate) and the top substrate (second substrate) can vary to approximately 1°, approximately 2°, approximately 3°, approximately 4°, approximately 5°, approximately 6°, approximately 7°, approximately 8°, approximately 9°, approximately 10°, approximately 11°, approximately 12°, approximately 13°, approximately 14°, approximately 15°, approximately 16°, approximately 17°, approximately 18°, approximately 19°, approximately 20°, approximately 21°, approximately 22°, approximately 23°, approximately 24°, approximately 25°, approximately 26°, approximately 27°, approximately 28°, approximately 29°, approximately 30°, approximately 31°, approximately 32°, approximately 33°, approximately 34°, and more. Approximately 35°, approximately 36°, approximately 37°, approximately 38°, approximately 39°, approximately 40°, approximately 41°, approximately 42°, approximately 43°, approximately 44°, approximately 45°, approximately 46°, approximately 47°, approximately 48°, approximately 49°, approximately 50°, approximately 51°, approximately 52°, approximately 53°, approximately 54°, approximately 55°, approximately 56°, approximately 57°, approximately 58°, approximately 59°, approximately 60°, approximately 61°, approximately 62°, approximately 63°, approximately 64°, approximately 65°, approximately 66°, approximately 67°, approximately 68°, approximately 69°, approximately 70°, approximately 71°, approximately 72°, approximately 73°, approximately 74°, approximately 75°, or greater than 75°.

[0251] In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 5° to approximately 10°. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 10° to approximately 15°. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 15° to approximately 20°. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 20° to approximately 25°. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 25° to approximately 30°. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 30° to approximately 35°. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 35° to approximately 40°. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 40° to approximately 45°. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 45° to approximately 50°. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 50° to approximately 55°. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 55° to approximately 60°. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 60° to approximately 65°. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 65° to approximately 70°. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 70° to approximately 75°.

[0252] In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 10° to approximately 20°. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 20° to approximately 30°. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 30° to approximately 40°. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 40° to approximately 50°. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 50° to approximately 60°. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 60° to approximately 70°.

[0253] In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 5° to approximately 70°. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 5° to approximately 60°. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 5° to approximately 50°. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 5° to approximately 40°. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 5° to approximately 30°. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 5° to approximately 20°.

[0254] In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 10° to approximately 70°. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 10° to approximately 60°. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 10° to approximately 50°. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 10° to approximately 40°. In some embodiments, the contact angle of the first substrate or bottom substrate differs from the contact angle of the second substrate or top substrate by approximately 10° to approximately 30°.

[0255] In some embodiments, the contact angle of the first substrate is greater than a specific contact angle. For example, 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 example, 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°.

[0256] The primary region of this disclosure has an opening in the top substrate, through which air or fluid can be passively or actively added. When fluid is present in the primary region, the opening also generates capillary forces that retain the fluid within the primary region and prevent it from overflowing into the secondary region. The opening in the primary region has a range of different diameters. The diameter of the opening can be approximately 0.2, approximately 0.3, approximately 0.4, approximately 0.5, approximately 0.6, approximately 0.7, approximately 0.8, approximately 0.9, approximately 1.0, approximately 1.1, 1.2, approximately 1.3, approximately 1.4, approximately 1.5, or greater than approximately 1.5 mm.

[0257] In some embodiments, the secondary region has an opening in the top substrate. In some embodiments, the secondary region does not have an opening in the top substrate. When the secondary region has an opening in the top substrate, the opening allows air or fluid to be passively or actively added to the secondary region. The opening in the secondary region has a range of different diameters. The diameter of the 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.

[0258] In some embodiments, the sample processing area includes a sample mixing area. In some embodiments, the sample mixing area and the fixed primary area are the same. In some embodiments, the sample processing area includes two or more sample mixing areas. In some embodiments, the sample processing area includes a fixed primary area serving as a sample mixing area, and one or more additional sample mixing areas. In some embodiments, the sample processing area includes a fixed primary area that is not a sample mixing area, and a sample mixing area. Figure 7 , Figure 8 and Figure 10 An exemplary sample mixing region is disclosed.

[0259] Figure 10 An illustration of an exemplary sample mixing region is disclosed. In this embodiment, the sample mixing region includes a fixed primary region 1006 containing a sample 1005. The sample is added through an opening 1004 in the fixed primary region. The fixed primary region 1006 includes a hook-shaped portion 1002. The hook-shaped portion is capable of engaging with a vibration source 1001. The vibration source provides vertical motion 1003 to compress and decompress the sample 1005. The compression and decompression of the sample 1005 results in sample mixing. The fixed primary region 1006 is cantilevered because only this portion opposite the opening 1004 is attached to the top substrate. All other areas surrounding the fixed primary region are detached from the top substrate.

[0260] Figure 32A It was made public. Figure 10 An alternative embodiment in which the sample mixing region does not contain the hook-shaped portion. In this embodiment, the sample mixing region includes a fixed primary region 3200. The sample is added through an opening 3201 in the fixed primary region. In this embodiment, the fixed primary region contains two openings. In some embodiments, the fixed primary region contains one or more openings. In some embodiments, the fixed primary region contains three or more openings. The fixed primary region 3200 does not contain the hook-shaped portion. A vibration source contacts an end 3202 of the fixed primary region. The vibration source provides vertical motion to compress and decompress the sample. The compression and decompression of the sample results in sample mixing. The fixed primary region 3200 is cantilevered because only the portion opposite end 3202 is attached to the top substrate. All other areas surrounding the fixed primary region are detached from the top substrate.

[0261] Figure 42 An illustration of an exemplary sample mixing region is disclosed. The sample mixing region is based on... Figure 39The sample mixing section includes a fixed primary region 4200 containing laterally extending protruding elements 4205 facing the first substrate within the second substrate. These laterally extending protruding elements have a surface facing the first substrate with a larger surface area than the surfaces of the plurality of protruding elements facing the first substrate. The fixed primary region 4200 does not include hook-shaped portions. The fixed primary region 4200 includes a chamfered end 4201 laterally separated from the connecting end 4202. The sample is added through an opening 4206 in the fixed primary region. The chamfered end 4201 can be connected to a vibration source including a spool 4203. The vibration source provides vertical motion 4204 on the spool 4203 connected to the chamfered end 4201 to compress and decompress the sample. The compression and decompression of the sample results in sample mixing. The fixed primary region 4200 is cantilevered because only the portion opposite the opening 4202 is attached to the second substrate. All other areas surrounding the fixed primary region are detached from the second substrate.

[0262] Figure 44A An illustration of an exemplary sample mixing region is disclosed. The sample mixing region is illustrated from a view pointing towards the surface of the second substrate facing the first substrate. In this embodiment, the sample mixing region includes a fixed primary region 4400, which contains laterally extending protruding elements 4401 facing the first substrate in the second substrate, wherein the laterally extending protruding elements have a surface facing the first substrate having a larger surface area than the surface of the plurality of protruding elements facing the first substrate. The sample mixing region includes the fixed primary region 4400. The fixed primary region 4400 includes a hook-shaped portion (not shown). The fixed primary region 4400 includes an opening 4402 at an end laterally separated from the connecting end 4403. The sample is added through the opening 4402 in the fixed primary region. The fixed primary region 4400 includes a pinning wall 4404 around the periphery of the fixed primary region 4400. The pinning wall 4404 is a raised edge that protrudes from the laterally extending protruding element around the periphery of the laterally extending protruding element, the periphery of which does not include the connecting end 4403. When mixing samples, the pinned walls help hold the samples. The primary fixation region 4400 is cantilevered because only the connecting end 4403 is attached to the second substrate. All other areas surrounding the primary fixation region are detached from the second substrate.

[0263] Figure 44BAn illustration of an exemplary sample mixing region is disclosed. The sample mixing region is illustrated from a view pointing towards the surface of the second substrate facing the first substrate. In this embodiment, the sample mixing region includes a fixed primary region 4410 containing laterally extending protruding elements 4411 facing the first substrate in the second substrate, wherein the laterally extending protruding elements have a surface facing the first substrate having a larger surface area than the surface of the plurality of protruding elements facing the first substrate. The sample mixing region includes the fixed primary region 4410. The fixed primary region 4410 includes a hook-shaped portion (not shown). The fixed primary region 4410 includes an opening 4412 at an end laterally separated from the connecting end 4413. The sample is added through the opening 4412 in the fixed primary region. The fixed primary region 4400 contains a plurality of pillars 4414a, 4414b, and 4414c spaced apart around the surface of the laterally extending protruding elements facing the first substrate. The plurality of supports 4414a, 4414b, and 4414c are protruding cylinders extending from laterally extending protruding elements. The plurality of supports 4414a, 4414b, and 4414c assist in mixing the sample during compression and decompression of the fixed primary region and promote retention of liquid during mixing. The fixed primary region 4410 is cantilevered because only the connecting end 4413 is attached to the second substrate. All other areas surrounding the fixed primary region are detached from the second substrate.

[0264] Figure 44C An illustration of an exemplary sample mixing region is disclosed. The sample mixing region is illustrated from a view pointing towards the surface of the second substrate facing the first substrate. In this embodiment, the sample mixing region includes a fixed primary region 4420 containing laterally extending protruding elements 4421 facing the first substrate in the second substrate, wherein the laterally extending protruding elements have a surface facing the first substrate having a larger surface area than the surface of the plurality of protruding elements facing the first substrate. The sample mixing region includes the fixed primary region 4420. The fixed primary region 4420 includes a hook-shaped portion (not shown). The fixed primary region 4420 includes an opening 4422 at an end laterally separated from the connection end 4413. The sample is added through the opening 4422 in the fixed primary region. The fixed primary region 4420 contains a plurality of ridges 4424a, 4424b, and 4424c spaced apart around the surface of the laterally extending protruding elements facing the first substrate. The plurality of ridges 4424a, 4424b, and 4424c are raised edges, wherein the raised edges protrude from laterally extending protruding elements. The plurality of ridges 4424a, 4424b, and 4424c assist in mixing the sample during compression and decompression of the fixed primary region and promote retention of liquid during mixing. The fixed primary region 4420 is cantilevered because only the connecting end 4423 is attached to the second substrate. All other areas surrounding the fixed primary region are detached from the second substrate.

[0265] Figure 44D An illustration of an exemplary sample mixing region is disclosed. The sample mixing region is illustrated from a view pointing towards the surface of the second substrate facing the first substrate. In this embodiment, the sample mixing region includes a fixed primary region 4430 containing laterally extending protruding elements 4431 facing the first substrate in the second substrate, wherein the laterally extending protruding elements have a surface facing the first substrate with a larger surface area than the surfaces of the plurality of protruding elements facing the first substrate. The sample mixing region includes the fixed primary region 4430. The fixed primary region 4430 includes a hook-shaped portion (not shown). The fixed primary region 4430 includes an opening 4432 at an end laterally separated from the connecting end 4433. The sample is added through the opening 4432 in the fixed primary region. The fixed primary region 4430 contains a plurality of pillars 4434a, 4434b, and 4434c spaced apart around the surface of the laterally extending protruding elements facing the first substrate. The plurality of supports 4434a, 4434b, and 4434c are protruding cylinders projecting from laterally extending protruding elements. The fixed primary region 4430 includes a laterally extending protruding element 4431 with a recessed, beveled edge 4435 surrounding the entire periphery of the laterally extending protruding element 4431. The plurality of supports 4434a, 4434b, and 4434c assist in mixing the sample during compression and decompression of the fixed primary region and promote retention of liquid during mixing. The fixed primary region 4430 is cantilevered because only the connecting end 4433 is attached to the second substrate. All other areas surrounding the fixed primary region are detached from the second substrate.

[0266] II. Layout of the sample analysis area The optional sample analysis area of ​​the device disclosed herein contains several different areas, including but not limited to a transition area, a tertiary area (e.g., a sample detection area), a quaternary area (e.g., a hydrophilic liquid pore or reservoir), a pentad area (e.g., a hydrophobic liquid pore or reservoir), or any combination thereof.

[0267] In some embodiments, the sample analysis region includes a transition zone. The transition zone facilitates the transfer of microparticles, including 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 can be of any length that sufficiently separates the sample processing region from the sample analysis region such that little or no fluid transfers from the sample processing region to the sample analysis region.

[0268] In some embodiments, the sample analysis region includes a tertiary region. In some embodiments, the tertiary region is a sample detection region. The types of sample detection regions include, but are not limited to, pores or micropores, chambers, nanopores, etc. In some embodiments, the sample analysis region includes hydrophobic liquid pores and hydrophilic liquid pores.

[0269] In some embodiments, the sample analysis region includes a sample detection region comprising wells. Wells may have sub-felt volume, felt volume, sub-nanolital volume, nanoliter volume, submicroliter volume, or microliter volume. For example, a well may be a feltal well, a nanoliter well, or a microliter well. In some embodiments, all wells in the array may have substantially the same volume. The well array may have volumes up to 100 microliters, such as approximately 0.1 felt, approximately 1 felt, approximately 10 felt, approximately 25 felt, approximately 50 felt, approximately 100 felt, approximately 0.1 pL, approximately 1 pL, approximately 10 pL, approximately 25 pL, approximately 50 pL, approximately 100 pL, approximately 0.1 nL, approximately 1 nL, approximately 10 nL, approximately 25 nL, approximately 50 nL, approximately 100 nL, approximately 0.1 microliter, approximately 1 microliter, approximately 10 microliter, approximately 25 microliter, approximately 50 microliter, or approximately 100 microliter.

[0270] In some examples, a hole is an array of holes comprising multiple individual holes. The hole array may include multiple holes spaced 1 mm apart. 2 The quantity range can be from 10 9 Up to 10. In some cases, a coverage of approximately 12 mm can be manufactured. 2 An array of approximately 100,000 to 500,000 pores (e.g., fly-up pores) covering an area of ​​approximately [area missing]. Each pore is measured to be approximately 4.2 µm wide x 3.2 µm deep (a volume of approximately 50 fly-ups) and can potentially contain a single microparticle (approximately 3 µm in diameter). At this density, the fly-up pores are spaced apart from each other at a distance of approximately 7.4 µm. In some examples, the pore array can be fabricated as having individual pores with diameters ranging from 10 nm to 10,000 nm.

[0271] Placing a single microparticle bound to an analyte molecule in a well allows for either digital or analog readout. For example, for a low number of positive wells (<~70% positive), Poisson statistics can be used to quantify the analyte concentration digitally; for a high number of positive wells (>~70%), the relative intensity of the signal-carrying well is compared to the signal intensity generated from a single microparticle bound to an analyte molecule, and used to generate an analog signal. Digital signals can be used for lower analyte concentrations, while analog signals can be used for higher analyte concentrations. A combination of digital and analog quantification can be used, which extends the linear dynamic range. In some embodiments, the signal intensity of the well increases over time, indicating that more than one analyte is present in the well. In some embodiments, the signal intensity in the well remains constant, and the higher intensity of a well containing a single analyte indicates that more than one analyte is present in the well. As used herein, a “positive well” refers to a well that has a signal associated with the presence of a microparticle bound to an analyte molecule that exceeds a threshold. As used herein, a “negative well” refers to a well that may not have a signal associated with the presence of a microparticle bound to an analyte molecule. In some embodiments, the signal from the negative aperture may be at a background level, i.e., below a threshold.

[0272] The aperture can be any of a variety of shapes, such as a cylinder with a flat bottom surface, a cylinder with a circular bottom surface, a cube, a rectangle, a truncated cone, an inverted truncated cone, a pentagon, a triangle, a pyramid, or a cone. In some cases, the aperture may include sidewalls oriented to facilitate the reception and retention of microparticles present in a droplet that has moved across the aperture array. In some cases, the aperture may include sidewalls oriented to facilitate the reception and retention of microparticles not present in a droplet that has moved across the aperture array. In some examples, the aperture may include a first sidewall and a second sidewall, wherein the first sidewall may be opposite the second sidewall. In some examples, the first sidewall is oriented at an obtuse angle to the bottom of the aperture, and the second sidewall is oriented at an acute angle to the bottom of the aperture. In some embodiments, the movement of the droplet is parallel to the bottom of the aperture and from the first sidewall to the second sidewall. In some embodiments, the movement of the microparticles is parallel to the bottom of the aperture and from the first sidewall to the second sidewall.

[0273] In some embodiments, the sample analysis area includes a sample detection area, which includes a chamber. The chamber can be a range of different sizes and shapes, making it suitable for detecting the analyte. The chamber can contain a reaction mixture with a volume from 5 μL to 1 mL. For example, the reaction chamber can be sized to contain a reaction mixture with a volume from 5 μL to 500 μL. According to some embodiments, the reaction chamber is sized to contain a reaction mixture with a volume from 5 μL to 100 μL. In some embodiments, the fluid capacity of the chamber is about 1 mL or less, about 750 μL or less, about 500 μL or less, about 400 μL or less, about 300 μL or less, about 250 μL or less, about 200 μL or less, about 150 μL or less, about 100 μL or less, about 50 μL or less, or about 25 μL or less.

[0274] The chamber can be a series of different shapes to retain the analyte either within the reaction mixture or without it. In some embodiments, the chamber is cubic. In some embodiments, the chamber is cylindrical. In some embodiments, the chamber is hexagonal. In some embodiments, the chamber is spherical. In some embodiments, the chamber is octagonal. In some embodiments, the chamber is conical. In some embodiments, the chamber is cubic.

[0275] The bottom, sides, or top of the chamber may be optically transparent, allowing analytes to be detected by optical devices. In some embodiments, all sides of the chamber are optically transparent, allowing analytes to be detected by optical devices. In some embodiments, only the top and bottom of the chamber are optically transparent, allowing analytes to be detected by optical devices. 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.

[0276] In some embodiments, the chamber is a reaction vessel. As used herein, the term "reaction vessel" generally refers to a vessel in which amplification reactions, immunoassays, clinical chemistry, or whole blood component analysis are performed. Reaction vessels may be available from commercial sources (e.g., off-the-shelf components, such as a single microamule or multiple microamules linked together in a 96-well configuration) or may be custom-made. Reaction vessels useful for nucleic acid amplification reactions will typically be able to rapidly transfer heat across the vessel, for example, by using highly conductive materials (e.g., thermally conductive plastics) or by physically modifying the vessel (e.g., thin walls). Common reaction vessels include, but are not limited to, tubes, vials, multiwell plates, etc. Reaction vessels may be constructed from a variety of materials, including, but not limited to, polymeric materials.

[0277] The top opening of the reaction vessel can be any convenient shape. In some aspects, the top opening of the reaction chamber of the reaction vessel is circular. The shape of the reaction chamber can vary. According to some embodiments, the reaction chamber has a conical shape. The bottom of the reaction vessel can be flat. In other aspects, the reaction vessel has a circular bottom.

[0278] In some respects, the walls of the reaction chamber are straight, whereby "straight" means that the walls do not include "steps" (or "ridges"). In other respects, the walls of the reaction chamber include one or more steps (e.g., two or more, three or more, four or more, etc.). The one or more steps may be complementary to the shape of the cap used for the reaction vessel. For example, according to some embodiments, the reaction vessel includes steps forming an upper region and a lower region of the reaction vessel, wherein the shape of the upper region is complementary to the shape of the reaction vessel cap.

[0279] The volume of the reaction vessel can vary. In some aspects, the reaction chamber is sized to accommodate a reaction mixture having a volume ranging from 1 μL to 500 μL. For example, the dimensions of the reaction chamber may be sized to accommodate a reaction mixture having volumes ranging from 1 μL to 10 μL, 10 μL to 50 μL, 50 μL to 100 μL, 100 μL to 200 μL, 200 μL to 300 μL, 300 μL to 400 μL, 400 μL to 500 μL, etc. According to some embodiments, the reaction chamber is sized to accommodate a reaction mixture having a volume ranging from 1 μL to 200 μL.

[0280] In some respects, the fluid capacity of the reaction vessel is 500 mL or less, 450 μL or less, 400 μL or less, 350 μL or less, 300 μL or less, 250 μL or less, 200 μL or less, 150 μL or less, 100 μL or less, 50 μL or less, 25 μL or less, 20 μL or less, 15 μL or less, 10 μL or less, or 5 μL or less.

[0281] The outer surface of a reaction vessel can include various shapes and features. In some respects, the bottom surface of a reaction vessel is circular. In other respects, the bottom surface of a reaction vessel is flat.

[0282] The bottom, sides, or top of the reaction vessel may be optically transparent, allowing analytes to be detected by optical devices. In some embodiments, all sides of the reaction vessel are optically transparent, allowing analytes to be detected by optical devices. In some embodiments, only the top and bottom of the reaction vessel are optically transparent, allowing analytes to be detected by optical devices. In some embodiments, only the top of the reaction vessel is optically transparent, allowing analytes to be detected by optical devices. In some embodiments, only the bottom of the reaction vessel is optically transparent, allowing analytes to be detected by optical devices.

[0283] In some cases, the reaction vessel may be the reaction vessel described in U.S. Patent No. 10,648,018, which is expressly incorporated herein by reference.

[0284] In some embodiments, the sample analysis region contains nanopores. When the sample detection region includes nanopores, an analyte or analyte molecule (e.g., nucleic acid, non-nucleic acid with a nucleic acid tag, or nucleic acid generated by the analyte) is detected by allowing nucleic acid translocations through or across the nanopore. In some embodiments, analyte detection can be carried out by allowing nucleic acid translocations 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 nanopores are sized to allow no more than one nucleic acid translocation at a time. Therefore, in some embodiments, the size of the nanopore will generally depend on the size of the nucleic acid to be examined. Nucleic acids with double-stranded regions may require a larger nanopore size than is sufficient to allow translocations of fully single-stranded nucleic acids. Additionally, nucleic acids associated with microparticles (such as microparticle tags) may require a larger nanopore than oligomeric tags. Typically, a nanopore of about 1 nm in diameter may allow single-stranded polymers to pass through, while a nanopore size of 2 nm in diameter or larger will allow double-stranded nucleic acids to pass through. In some embodiments, the nanopore is selective for single-stranded tags (e.g., with a diameter from about 1 nm to less than 2 nm), while in other embodiments, the nanopore has a sufficient diameter to allow double-stranded polynucleotides (e.g., 2 nm or larger) to pass through. The selected nanopore size provides the optimal signal-to-noise ratio for the analyte of interest.

[0285] In some embodiments, the diameter of the nanopore may be between about 0.1 nm and about 1000 nm, 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 diameter of nanopores can be approximately 0.1 nm, approximately 0.2 nm, approximately 0.3 nm, approximately 0.4 nm, approximately 0.5 nm, approximately 0.6 nm, approximately 0.7 nm, approximately 0.8 nm, approximately 0.9 nm, approximately 1.0 nm, approximately 1.5 nm, approximately 2.0 nm, approximately 2.5 nm, approximately 3.0 nm, approximately 3.5 nm, approximately 4.0 nm, approximately 4.5 nm, approximately 5.0 nm, approximately 7.5 nm, approximately 10 nm, approximately 15 nm, approximately 20 nm, approximately 25 nm, approximately 30 nm, approximately 35 nm, approximately 40 nm, approximately 45 nm, approximately 50 nm, approximately 55 nm, approximately 60 nm, approximately 65 nm, approximately 70 nm, approximately 75 nm, approximately 80 nm, approximately 85 nm, approximately 90 nm, approximately 95 nm, approximately 100 nm, approximately 150 nm, approximately 200 nm, approximately 250 nm. nm, approximately 300 nm, approximately 3500 nm, approximately 400 nm, approximately 450 nm, approximately 500 nm, approximately 550 nm, approximately 600 nm, approximately 650 nm, approximately 700 nm, approximately 750 nm, approximately 800 nm, approximately 850 nm, approximately 900 nm, approximately 950 nm, or approximately 1000 nm.

[0286] Various types of nanopores can be used to analyze nucleic acids present in samples. These include, in particular, bio-nanopores employing biological nanopores embedded in a membrane. Another type of nanopore layer is the solid-state nanopore, in which the nanopores are wholly or partially made of fabricated or sculpted solid components (such as silicon). In some embodiments, the nanopores are solid-state nanopores generated using controlled dielectric breakdown. In some embodiments, the nanopores are solid-state nanopores generated by methods other than controlled dielectric breakdown.

[0287] In some embodiments, the length of the nanopores can be up to about 200 nm, for example, from about 0.1 nm 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 the nanopore layer can 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 30000, about 100000, about 300000 or more. The center-to-center distance between the nanopores in the layer can be approximately 100 nm to approximately 300 nm, approximately 300 nm to approximately 500 nm, approximately 500 nm to approximately 1000 nm, for example 100 nm, 150 nm, 200 nm or 300 nm.

[0288] In some embodiments, multiple nanopore layers (each containing one or more nanopores) may be arranged in series for the detection and / or counting of tags (e.g., polymers, aptamers, microparticles). In this case, nucleic acid detection and / or counting can be implemented by translocating nucleic acids through or across each nanopore layer. Thus, counting the number of nucleic acids translocated through or across nanopores in a layer / sheet / film refers to counting multiple nucleic acids translocated through or across one or more nanopores in one or more layers / sheets / films. In some embodiments, when more than one nanopore layer is present (e.g., one, two, three, four, five, six, or other technically feasible numbers of nanopore layers), they may optionally be present in series, wherein at least one nanopore in one layer is separated from or stacked on top of another nanopore in another layer (e.g., above or on top of it), etc. In the case of nanopore layers in series, at least two electrodes can be used to generate an electric field to drive the tag through the pores, and optionally, additional electrodes positioned between the nanopore layers can further provide a driving current.

[0289] Bio-nanopores For the detection and, optionally, counting of nucleic acids, any bio-nanopore with a channel size that permits nucleic acid translocation can be used. Two main classes of bio-nanopores are suitable for the methods disclosed herein. Non-voltage-gated nanopores allow molecules to pass through the nanopore without requiring a change in membrane potential to activate or open the channel. Voltage-gated nanopores, on the other hand, require a specific range of membrane potentials to activate the nanopore opening. Most studies on bio-nanopores have used α-hemolysin, a mushroom-shaped homooligomeric heptamer channel of approximately 10 nm in length found in Staphylococcus aureus. Each subunit contributes two β chains to form a 14-chain antiparallel β barrel. The nanopore formed by the β barrel-like structure has an entrance with a diameter of approximately 2.6 nm, which contains a lysine residue loop and leads to an internal cavity with a diameter of approximately 3.6 nm. The stem of the hemolysin nanopore, which penetrates the lipid bilayer, has an average inner diameter of approximately 2.0 nm and a 1.5 nm contraction between the vestibule and the stem. The size of the stem is sufficient to allow single-stranded nucleic acids to pass through, but insufficient to allow double-stranded nucleic acids to pass through. Therefore, α-hemolysin nanopores can be used as nanopores that are selective for single-chain polynucleotides and other polymers of similar size.

[0290] In other embodiments, the bioporous nanopores are sized sufficiently to allow polymers larger than single-stranded nucleic acids to pass through. An exemplary nanopore is mitochondrial porin, a voltage-dependent anion channel (VDAC) located in the outer mitochondrial membrane. Porins are obtained in purified form and, when recombined into artificial lipid bilayers, generate functional channels capable of allowing double-stranded nucleic acids to pass through (Szabo et al., 1998, F ASEB J. 12:495-502). Structural studies suggest that porins also possess a β-barrel structure with 13 or 16 chains (Rauch et al., 1994, BiochemBiophys Res Comm 200:908-915). Compared to nanopores formed from α-hemolysin, maltose porin (LamB), and short bacitracin, porins exhibit greater electrical conductivity. The greater conductivity of porins supports studies indicating that porin channels are sufficiently sized to allow double-stranded nucleic acids to pass through. The nanopore diameter of the porin molecule is estimated to be 4 nm. The diameter of the non-helical double-stranded nucleic acid is estimated to be approximately 2 nm.

[0291] Another type of bio-nanopore suitable for scanning double-stranded polynucleotides is the channel found in Bacillus subtilis (Szabo et al., 1997, Biol. Chem., 272:25275-25282). Plasma membrane vesicles made from Bacillus subtilis and incorporated into artificial membranes allow double-stranded DNA to be transferred across the membrane. The conductivity of nanopores formed from Bacillus subtilis membrane formulations is similar to that of mitochondrial porins. Although incomplete characterization of these nanopores exists (e.g., purified forms), purified forms are not necessary for the purposes of this paper. Individual nanopores can be isolated in detection devices by diluting plasma membrane formulations (either by dissolving them in a suitable detergent or by incorporating them into an artificial lipid membrane with sufficient surface area). Limiting the contact time between the membrane formulation (or protein formulation) and the artificial membrane by appropriately timed washing provides another method for incorporating individual nanopores into artificial lipid bilayers. Conductivity properties can be used to characterize nanopores incorporated into bilayer membranes.

[0292] In some cases, nanopores can be hybrid nanopores, in which biological nanopores are introduced into solid nanopores (e.g., nanopores fabricated in non-biological materials). For example, α-hemolysin nanopores can be inserted into solid nanopores. In some cases, nanopores can be hybrid nanopores as described in Hall et al., Nature Nanotechnology, November 28, 2010, Vol. 5, pp. 874-877.

[0293] Solid nanopores In other embodiments, nucleic acid analysis is performed by translocating tags through or across nanopores made of non-biological materials. Nanopores can be fabricated from a variety of solid-state materials using several different techniques, including but not limited to chemical deposition, electrochemical deposition, electroplating, electron beam engraving, ion beam engraving, nanolithography, chemical etching, laser ablation, focused ion beam deposition, atomic layer deposition, and other methods well known in the art (see, for example, Li et al., 2001, Nature 412:166-169; and WO 2004 / 085609).

[0294] In certain embodiments, the nanopores may be those described in WO13167952A1 or WO13167955A1. As described in WO13167952A1 or WO13167955A1, nanopores with accurate and uniform nanopore sizes can be formed by precisely enlarging the nanopores formed in the film. This method may involve: enlarging the nanopores by applying a high potential across them; measuring the current flowing through the nanopores; determining the size of the nanopores in part based on the measured current; and removing the potential applied to the nanopores when the size of the nanopores corresponds to the desired size. In some cases, the applied potential may have a pulsed waveform oscillating between high and low values, allowing the current flowing through the nanopores to be measured when the potential is applied to the nanopores at a low value.

[0295] For example, and not as a limitation, solid materials include any known semiconductor material, insulating material, and metal coated with an insulating material. Thus, at least a portion of (one or more) nanopores may include, but is not limited to, silicon, silicon dioxide, silicene, silicon oxide, graphene, silicon nitride, germanium, gallium arsenide, or metals, metal oxides, and metal colloids coated with an insulating material.

[0296] To fabricate nanopores at the nanoscale, various feedback procedures can be employed during the manufacturing process. In embodiments where ions pass through the pores, detecting the ion flow through the solid material provides a way to measure the pore size generated during manufacturing (see, for example, U.S. Publication No. 2005 / 0126905). In other embodiments, where electrodes define the pore size, the electron tunneling current between the electrodes provides information about the gap between them. An increase in the tunneling current indicates a decrease in the gap space between the electrodes. Other feedback techniques will be apparent to those skilled in the art.

[0297] In some embodiments, nanopores are fabricated using ion beam engraving, as described in Li et al., 2003, Nature Materials 2:611-615. In some embodiments, nanopores are fabricated using high current, as described in WO13167952A1 or WO13167955A1. In other embodiments, nanopores can be fabricated by a combination of electron beam lithography and high-energy electron beam engraving (see, for example, Storm et al., 2003, Nature Materials 2:537-540). A similar method for generating suitable nanopores by ion beam sputtering is described in Heng et al., 2004, Biophy J 87:2905-2911. Nanopores are formed on metal-oxide-semiconductor (CMOS) substrates using lithography performed with a focused high-energy electron beam, in conjunction with general techniques for fabricating ultrathin films. In other embodiments, the nanopores are constructed by sculpting silicon nitride, as provided in U.S. Patent Nos. 6,627,067; 6,464,842; 6,783,643; and U.S. Publication No. 2005 / 0006224.

[0298] In some embodiments, nanopores can be constructed as gold or silver nanopores. These nanopores are formed by using a template of a porous material (such as a polycarbonate filter prepared using a track etching method) and depositing gold or other suitable metals on the surface of the porous material. Track-etched polycarbonate films are typically formed by exposing a solid film material to high-energy nuclei (which create tracks in the film material). Chemical etching is then employed to convert the etched tracks into nanopores. The resulting nanopores have diameters of approximately 10 nm and larger. Adjusting the intensity of the nuclei controls the density of nanopores formed in the film. Nanopores are formed on the etched film by the following steps: 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 nanoporous material, which is then immersed in a solution containing Au(I) and a reducing agent. On the catalyst-containing surface, the reduction of Au(I) to metallic Au occurs. The amount of gold deposited depends on the incubation time, with increasing the incubation time reducing the inner diameter of the pores in the filter material. Therefore, the nanopore size can be controlled by adjusting the amount of metal deposited on the pores. The resulting nanopore size is measured using various techniques, such as by using the gas transport properties of simple diffusion or by measuring the ion flow through the pores using a patch-clamp system. The support material is either left intact or removed to leave the gold nanopores. Electroless plating techniques can form nanopore sizes ranging from less than about 1 nm to about 5 nm, or larger as needed. Gold nanopores with a diameter of about 0.6 nm appear to be distinguishable between Ru(bpy)2+2 and methyl viologen, thus confirming the selectivity of gold nanopores (Jirage et al., 1997, Science 278:655-658). Modification of the gold nanopore surface can be readily achieved by attaching thiol-containing compounds to the gold surface or by derivatizing the gold surface with other functional groups. This feature allows for the attachment of nanopore-modifying compounds as well as sensing labels, as discussed herein. Devices (such as the cis / trans devices for bio-nanopores described herein) can be used with gold nanopores to analyze individual encoded molecules.

[0299] In cases where the detection of an analyte (e.g., nucleic acid, non-nucleic acid tagged with nucleic acid, or nucleic acid generated from the analyte) involves a current (e.g., electron tunneling current) passing through the analyte, solid films can be metallized using various techniques. A conductive layer can be deposited on both sides of the film to create electrodes suitable for querying the tag along the length of the chain, such as longitudinal electron tunneling current. In other embodiments, a conductive layer can be deposited on one surface of the film to form electrodes suitable for querying the analyte across nanopores, such as lateral tunneling current. Various methods for depositing conductive materials are known, including sputtering deposition (i.e., physical vapor deposition), electroless deposition (e.g., colloidal suspensions), and electrolytic deposition. Other metal deposition techniques are filament evaporation, metal layer evaporation, electron beam evaporation, flash evaporation, and induction evaporation, and will be apparent to those skilled in the art.

[0300] In some embodiments, the detection electrode is formed by sputter deposition, wherein an ion beam bombards a metal block and vaporizes the metal atoms, which are then deposited on the wafer material as a thin film. Depending on the photolithography method used, the metal film is then etched by reactive ion etching or polished by chemical mechanical polishing. The metal film can be deposited on pre-formed nanopores or deposited prior to creating the pores.

[0301] In some embodiments, the detection electrode is fabricated by electrodeposition (see, for example, 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 No. 2003 / 0141189). This fabrication process is suitable for generating nanopores and corresponding detection electrodes positioned on one side of a solid film, such as for detecting lateral electron tunneling. Initially, a pair of facing electrodes are formed on a silicon dioxide layer supported on a silicon wafer using a conventional photolithography process. An electrolyte solution covers the electrodes, and metal ions are deposited on one of the electrodes by passing a current through the electrode pair. Over time, the deposition of metal on the electrodes reduces the gap distance between the electrodes, thereby creating not only a detection electrode but also a nanoscale gap for encoding molecular translocation. The gap distance between the electrodes can be controlled by several feedback processes.

[0302] In the case of detecting charge-induced field effects in imaging, semiconductors can be fabricated as described in U.S. Patent No. 6,413,792 and U.S. Publication No. 2003 / 0211502. The methods for fabricating these nanopore devices can use techniques similar to those employed to fabricate other solid-state nanopores.

[0303] The detection of analytes (such as polynucleotides) is performed as further described below. For the analysis of the analytes, nanopores can be configured in various formats. In some embodiments, the device includes a membrane (biological or solid membrane) containing nanopores held between two reservoirs (also referred to as cis and trans chambers) (see, for example, U.S. Patent No. 6,627,067). A conduit for electron migration between the two chambers allows electrical contact between the two chambers, and a voltage bias between the two chambers drives a tag to translocate through the nanopore. Variations of this configuration are used to analyze the current passing through the nanopore, as described in U.S. Patent Nos. 6,015,714 and 6,428,959; and Kasianowiscz et al., 1996, Proc Natl AcadSci USA 93:13770-13773, the disclosures of which are incorporated herein by reference.

[0304] A variation of the above device is disclosed in U.S. Patent Application Publication No. 2003 / 0141189. A pair of nanoelectrodes, fabricated by electrodeposition, are positioned on a substrate surface. The electrodes face each other and have a gap distance sufficient to allow a single nucleic acid to pass through. An insulating material protects the nanoelectrodes, thereby exposing only the tips of the nanoelectrodes for nucleic acid detection. The insulating material and the nanoelectrodes separate the chamber used as a sample reservoir from the chamber to which the polymer is delivered via translocation. Cathode and anode electrodes provide an electrophoretic electric field for driving the tag from the sample chamber to the delivery chamber.

[0305] The current bias used to drive the analyte through the nanopore can be generated by applying an electric field that guides it through the nanopore. In some embodiments, the electric field is a constant voltage or a constant current bias. In other embodiments, the movement of the tag is controlled by pulsed manipulation of the electrophoretic electric field parameters (see, for example, U.S. Patent Application No. 2003 / 141189 and U.S. Patent No. 6,627,067). Current pulses provide a method for precisely translocating one or only some bases of an oligonucleotide tag through the pore for a defined time period and briefly holding the tag within the pore, thereby providing greater resolution of the tag's electrical properties.

[0306] The nanopore device may also include an electric or electromagnetic field to restrict the orientation of the analyte as it passes through the nanopore. This holding field can be used to reduce the movement of the analyte within the nanopore. In some embodiments, an electric field orthogonal to the translocation direction is provided to restrict the movement of the tag molecule within the nanopore. This is illustrated in U.S. Application Publication No. 2003 / 0141189 by using two parallel conductive plates above and below a sample plate. These electrodes generate an electric field orthogonal to the translocation direction of the analyte molecule and thus hold the tag molecule to one of the sample plates. The negatively charged backbone of DNA or nucleic acids modified to have a negative charge on one strand will be oriented onto the anode plate, thereby restricting the movement of the tag molecule.

[0307] In other embodiments, control of the analyte's position is implemented by a method described in U.S. Patent Application Publication No. 2004 / 0149580, which employs an electromagnetic field generated within the nanopore via a series of electrode positions near or on the nanopore. In these embodiments, one set of electrodes applies a DC voltage and a radio frequency (RF) potential, while a second set of electrodes applies an opposite DC voltage and RF potential, the RF potential being phase-shifted by 180 degrees relative to the RF potential generated by the first set of electrodes. This RF quadrupole holds charged particles (e.g., nucleic acids) at the center of the field (i.e., the center of the pore).

[0308] In exemplary embodiments, the nanoporous membrane may be a multilayer stack of conductive and dielectric layers, wherein an embedded conductive layer or conductive layer gate provides a well-controlled and measurable electric field in and around the nanopore through which the tag translocates. In one aspect, the conductive layer may be graphene. Examples of stacked nanoporous membranes have been found, for example, in US20080187915 and US20140174927.

[0309] It should be understood that nanopores can be located in films, layers or other substrates, and these terms are used interchangeably to describe two-dimensional substrates that include nanopores.

[0310] In some embodiments, nanopores may be formed as part of an assay process used to detect and / or determine the concentration of an analyte using nanopores. Specifically, the apparatus for detecting and / or determining the concentration of an analyte using nanopores may initially be provided without nanopores formed in a membrane or layer. The apparatus may include a membrane separating two chambers (cis and trans) on opposite sides of the membrane. The cis and trans chambers may include a salt solution and may be connected to a power source. When a nanopore is to be formed in the membrane, a voltage is applied to the salt solution in the cis and trans chambers and the conductivity across the membrane is measured. Before the nanopore is formed, there is no current or only a minimal current measured across the membrane. After the nanopore is formed, the current measured across the membrane increases. The voltage may be applied for a sufficient amount of time to form a nanopore of the desired diameter. After the nanopore is formed, an analyte or label may be translocated through the nanopore, and the translocation event is detected. In some embodiments, the same salt solution may be used for both nanopore formation and for detecting the translocation of an analyte or label through the nanopore. Any suitable salt solution can be used for nanopore generation and / or the translocation of analytes or tags through the nanopores. Any salt solution that will not damage the counting tags can be used. Exemplary salt solutions include lithium chloride, potassium chloride, sodium chloride, calcium chloride, magnesium chloride, etc. The concentration of the salt solution can be selected based on the desired conductivity of the salt solution. In some embodiments, the salt solution may have a concentration ranging from about 1 mM to about 10 M, such as 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, or about 500 mM-1 M, 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.

[0311] In some embodiments, nanopores may become blocked, and blocked nanopores are cleared by adjusting the pattern of the voltage applied across the nanopore layer or membrane by the electrodes. In some cases, blocked nanopores are cleared by reversing the polarity of the voltage applied across the nanopore layer or membrane. In some cases, blocked nanopores are cleared by increasing the magnitude of the voltage applied across the nanopore layer or membrane. The increase in voltage may be a temporary increase lasting 10 seconds (s) or less, such as 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.

[0312] In some embodiments, the sample analysis region includes a quinary zone. In some embodiments, the quinary zone is a hydrophilic liquid pore. In some embodiments, the quinary zone is a hydrophilic liquid reservoir. When the quinary zone is a hydrophilic liquid pore, the hydrophilic liquid is added directly to the pore after microparticles or microparticles and helper particles are added to the sample detection region. In some embodiments, when the sample detection region includes pores or micropores, the hydrophilic liquid is added after microparticles are seeded into the pores or micropores. When the quinary zone is a hydrophilic liquid reservoir, the hydrophilic liquid can be extracted from the reservoir by means of a pump, gravity, suction, etc. In some embodiments, the hydrophilic liquid is a substrate solution. The substrate solution may be a substrate solution that reacts with a specific binding member (e.g., a second specific binding member or a detectably labeled second specific binding member) to generate a detectable signal.

[0313] In some embodiments, the fourth-level region includes one or more substrate holding features. The substrate holding features of this disclosure may be a series of different features and may be any combination of the substrate features described herein. In some embodiments, the substrate holding features are first, second, third, and fourth substrate holding features, wherein each substrate holding feature is quarter-circular and protrudes into the fourth-level region, such as... Figure 33G As depicted in the illustration. In some embodiments, the substrate retaining feature is centered at the upper right end of the sample analysis area, is quarter-circular in shape, and protrudes from the top or bottom substrate, as shown in the illustration. Figure 33H As depicted in the illustration. In some embodiments, the substrate holding feature is located at the right end of the sample analysis area, is horseshoe-shaped with a gradually widening edge, and protrudes from the top or bottom substrate, as shown in the illustration. Figure 33H As depicted in [the text]. In some embodiments, the substrate holding feature is a first substrate holding feature located at the right end of the sample analysis area, having a horseshoe shape without a tapered edge and concentric with a second substrate holding feature having a horseshoe shape without a tapered edge, wherein the first and second substrate holding features protrude from the top or bottom substrate, such as [example of a feature described in the text]. Figure 33I As depicted in the text.

[0314] In some embodiments, the substrate holding features are first, second, third, and fourth substrate holding features, wherein the first to fourth substrate holding features are grooves on the top or bottom substrate, located at the right end of the sample analysis area and concentric with each other, such as... Figure 33J As depicted in the illustration. In some embodiments, the substrate holding feature is located at the right end of the sample analysis area, is horseshoe-shaped without a tapered end, and protrudes from the top or bottom substrate, as shown in the illustration. Figure 33K As depicted in the description. In some embodiments, the substrate holding features are a first substrate holding feature and a second substrate holding feature, wherein the first substrate holding feature and the second substrate holding feature are located before the right end of the sample analysis region, are rectangular in shape, the first substrate holding feature is opposite to the second substrate holding feature, and the first substrate holding feature and the second substrate holding feature protrude from the top substrate or the bottom substrate, such as... Figure 33L As depicted in the description. In some embodiments, the substrate holding features are a first substrate holding feature and a second substrate holding feature, wherein the first substrate holding feature and the second substrate holding feature are located before the right end of the sample analysis region, are circular in shape, the first substrate holding feature is opposite to the second substrate holding feature, and the first substrate holding feature and the second substrate holding feature protrude from the top substrate or the bottom substrate, such as... Figure 33M As depicted in the text.

[0315] In some embodiments, the sample analysis region includes a quinary zone. In some embodiments, the quinary zone is a hydrophobic liquid pore. In some embodiments, the quinary zone is a hydrophobic liquid reservoir. When the quinary zone is a hydrophobic liquid pore, the hydrophobic liquid is added directly to the pore after the microparticles or microparticles and helper particles are added to the sample detection region and after the addition of the hydrophilic liquid. In some embodiments, when the sample detection region includes pores or micropores, the hydrophobic liquid is added to seal the pores or micropores for detection after the microparticles or microparticles and helper particles are seeded into the pores or micropores and after the addition of the hydrophilic liquid. When the quinary zone is a hydrophobic liquid reservoir, the hydrophobic liquid can be extracted from the reservoir by a pump, gravity, suction, etc. In some embodiments, the hydrophobic liquid is an oil. The oil can be any oil considered useful. In some cases, the hydrophobic liquid is selected based on its low affinity for water to reduce mixing of the hydrophobic liquid with the substrate solution. In some cases, the hydrophobic liquid is an oil. In some cases, the hydrophobic liquid is 3M FC-40 oil, hydrocarbon oil, vegetable oil, or silicone liquid (e.g., silicone oil). In some cases, the oil is a fluorocarbon oil. In other cases, the oil is Novec 7500, FC-40, or Galden HT200.

[0316] In some embodiments, the level 5 region includes a barrier feature. In some embodiments, the barrier feature is located at the upper left end of the sample analysis area, is semi-circular, and protrudes from the top or bottom substrate.

[0317] 3. Reagent delivery kit This disclosure also provides an optional reagent delivery device. The reagent delivery device includes a sample collection section, a reagent kit, a frame, a seal, and an integrated sample processing unit.

[0318] In some embodiments, a reagent delivery device is used to add one or more reagents to the device. In some embodiments, one or more reagents are added to the device by adding the one or more reagents individually to the device. In some embodiments, a bulk reagent delivery method is used to add one or more reagents to the device. As used herein, "bulk reagent delivery" refers to adding reagents to the device using a storage container containing a volume of reagents greater than the amount necessary to perform one of any of the assays disclosed herein. For example, the storage container contains a sufficient amount of reagents such that two or more of any of the assays disclosed can be performed without refilling the storage container. The storage container contains a sufficient quantity of reagent to allow for the performance of any of the disclosed assays two or more, three or more, four or more, five or more, ten or more, twenty or more, thirty or more, forty or more, fifty or more, one hundred or more, two hundred or more, three hundred or more, four hundred or more, five hundred or more, one thousand or more, two thousand or more, three thousand or more, four thousand or more, five thousand or more, six thousand or more, seven thousand or more, eight thousand or more, nine thousand or more, or one hundred thousand or more.

[0319] Figure 20 A diagram depicts a partially assembled reagent delivery device. The reagent delivery device includes a sample collection section 2002. The sample collection section includes an opening 2001 that connects to a capillary section for collecting samples. The sample collection section is connected to a frame 2003. The frame holds the reagent kit 2005. Within the kit is a separate plunger 2004. The reagent delivery device is connected to an exemplary sample processing device 2006 or 2007. The reagent delivery device can be connected to any device disclosed herein.

[0320] Figure 21A diagram depicting an exploded view of a reagent delivery device is provided. The reagent delivery device includes a sample collection section 2102. The sample collection section is connected to a hollow capillary section 2103. The capillary section is in contact with a sample to collect the sample within the hollow capillary section 2103. The reagent kit includes a plunger section 2104, a separate plunger 2105, and a reagent receiving section 2106. The reagent kit is connected to the interior of a frame 2107. The sample collection section 2102 is connected to the exterior of the frame 2107. A seal 2108 is connected to a sample processing device 2109. The seal 2108 and the sample processing section are connected to the interior of the bottom of the frame 2107.

[0321] Figure 22 An exemplary embodiment of the reagent kit is disclosed in an inverted view. The kit includes a seal 2201 connected to a reagent receiving portion 2203. The seal 2201 prevents reagent from leaving the reagent receiving portion 2203. A plunger portion includes one or more plungers physically connected such that pressing the plunger portion presses down the one or more plungers. In some embodiments, the plunger portion includes both independent plungers and connected plungers. The kit includes independent plungers fitted into openings 2205 and 2202. In some embodiments, the independent plungers dispense hydrophobic liquid from the reagent receiving portion 2203.

[0322] Figure 23 A diagram depicting a cross-section of the reagent delivery device in an inactive state is shown. The reagent delivery device has a sample collection section 2202 physically connected to a frame. The sample collection section contains an opening 2201 connected to a capillary section. When the capillary section 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 receiving section contains reagents 2208, which are dispensed into primary, secondary, quaternary, and / or pentathary zones after the plunger section is pressed. The reagent kit seal 2309 is punctured by a membrane puncture feature 2205, allowing reagent delivery to the sample processing device.

[0323] Figure 24 A diagram depicting a cross-section of the reagent delivery device in an activated state is shown. In the activated state, the plunger portion 2403 is depressed, and the membrane puncture feature 2407 thereby releases the reagent into the sample processing device 2406.

[0324] Figure 25 Alternative designs for the reagent kit are described. (Replacement) Figure 23 and Figure 24 The membrane puncture feature disclosed herein is that it is directly connected to the plunger portion and is present in the fluid chamber 2507. When pressed, the membrane puncture feature 2504 punctures the seal 2505 and allows reagent dispensing into the sample processing device.

[0325] 4. Methods and apparatus for mixing on or outside the apparatus. This disclosure provides a method and apparatus for mixing fluids in a device. The fluids for use in the device can be mixed within the device itself or mixed before being added to the device.

[0326] In some embodiments, fluids in the device are mixed by vibrating the entire device. In these embodiments, a vibration source contacts the device and causes the entire device to vibrate. In some embodiments, the vibration is vertical. In some embodiments, the vibration is horizontal. The vibration source can be a variety of different sources, including but not limited to voice coils, vibration motors, piezoelectric actuators, motors, gas pumps, etc.

[0327] In some embodiments, fluids in the device are mixed by vibrat...

Claims

1. An apparatus comprising: First substrate, and A second substrate positioned on the first substrate, wherein the second substrate includes a sidewall surrounding at least a portion of its periphery, wherein the first substrate, the sidewall, and the second substrate define a central cavity therebetween. The second substrate includes a surface facing the central cavity, which includes a plurality of recessed elements and a plurality of protruding elements; The primary region is defined between the surface of the plurality of protruding elements facing the first substrate and the surface of the first substrate facing the second substrate. The secondary region is defined between the surfaces of the plurality of recessed elements facing the first substrate and the surfaces of the first substrate facing the second substrate. Wherein, the second substrate has an opening in one or more of the primary regions, and The first substrate and the second substrate have differences in hydrophobicity or hydrophilicity.

2. The apparatus according to claim 1, wherein, At least a portion of the periphery includes at least two, at least three, or at least four peripheral sides of the second substrate.

3. The apparatus according to claim 1 or 2, wherein, The difference in hydrophobicity or hydrophilicity is the difference between the contact angle of the first substrate and the contact angle of the second substrate.

4. The apparatus according to claim 3, wherein, The difference between the contact angle of the first substrate and the contact angle of the second substrate is approximately 10% to approximately 60%.

5. The apparatus according to claim 4, wherein, The difference between the contact angle of the first substrate and the contact angle of the second substrate is approximately 20% to approximately 40%.

6. The apparatus according to claim 3, wherein, The difference between the contact angle of the first substrate and the contact angle of the second substrate is approximately 5° to approximately 60°.

7. The apparatus according to claim 6, wherein, The difference between the contact angle of the first substrate and the contact angle of the second substrate is approximately 5° to approximately 40°.

8. The apparatus according to any one of claims 3 or 6 to 7, wherein, The difference between the contact angle of the first substrate and the contact angle of the second substrate is approximately 5° to approximately 30°.

9. The apparatus according to any one of claims 1 to 8, wherein, The first substrate is hydrophobic, and the second substrate is hydrophilic.

10. The apparatus according to any one of claims 1 to 8, wherein, The first substrate is hydrophilic, and the second substrate is hydrophobic.

11. The apparatus according to any one of claims 1 to 8, wherein, The first substrate is hydrophobic, and the second substrate is hydrophobic.

12. The apparatus according to any one of claims 1 to 8, wherein, The first substrate is hydrophilic, and the second substrate is hydrophilic.

13. The apparatus according to any one of claims 1 to 12, wherein, The primary region is discrete.

14. The apparatus according to any one of claims 1 to 13, wherein, Secondary areas are connected.

15. The apparatus according to any one of claims 1 to 14, wherein, The second substrate has an opening in one or more of the secondary regions.

16. The apparatus according to any one of claims 1 to 15, wherein, The second substrate has an opening in each of the plurality of primary regions.

17. The apparatus according to any one of claims 1 to 16, wherein in the primary region, the surfaces of the plurality of protruding elements facing the first substrate and the surfaces of the first substrate facing the second substrate are separated by a first distance, and in the secondary region, the surfaces of the recessed elements facing the first substrate and the surfaces of the first substrate facing the second substrate are separated by a second distance.

18. The apparatus according to claim 17, wherein, The first distance is less than the second distance.

19. The apparatus according to any one of claims 1 to 18, wherein, The protruding element is a shape selected from the group consisting of: rectangle, circle, triangle, pentagon, hexagon, heptagon, octagon, decagon, dodecagon, amoeba-like and irregular shape.

20. The apparatus according to any one of claims 1 to 19, wherein, The device includes three or more primary regions separated by two or more secondary regions.

21. The apparatus according to any one of claims 1 to 20, wherein, The device includes six or more primary regions separated by seven or more secondary regions.

22. The apparatus according to any one of claims 1 to 21, wherein, The device includes nine or more primary regions separated by twelve or more secondary regions.

23. The apparatus according to any one of claims 1 to 22, wherein, The device includes twelve or more primary regions separated by seventeen or more secondary regions.

24. The apparatus according to any one of claims 1 to 23, wherein, The device includes fifteen or more primary regions separated by twenty-two or more secondary regions.

25. The apparatus according to any one of claims 1 to 24, wherein, The device includes eighteen or more primary regions separated by twenty-seven or more secondary regions.

26. The apparatus according to any one of claims 1 to 25, wherein, The device includes twenty-one or more primary regions separated by thirty-two or more secondary regions.

27. The apparatus according to any one of claims 1 to 26, wherein, The device includes twenty-four or more primary regions separated by thirty-seven or more secondary regions.

28. The apparatus according to any one of claims 1 to 27, further comprising a sample analysis region configured to analyze a sample.

29. The apparatus according to claim 28, wherein, The sample analysis area is positioned on the first substrate adjacent to the central chamber.

30. The apparatus according to claim 28 or 29, wherein, The sample analysis area is physically separated from the central chamber.

31. The apparatus according to claim 28 or 29, wherein, The sample analysis area is not physically separated from the central chamber.

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

33. The apparatus according to any one of claims 28 to 32, wherein, The sample analysis region has a first end that is laterally separated from the 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 apparatus according to claim 33, wherein, In the sample analysis area, the surface of the second substrate facing the first substrate includes enlarged protruding elements, wherein: The surface of the enlarged protruding element facing the first substrate has a larger surface area than the surfaces 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 apparatus according to claim 33 or 34, wherein, The sample analysis region includes a three-level region, which is located at the midpoint between the first and second ends of the sample analysis region, and is defined by the following: 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.

36. The apparatus according to claim 35, wherein, The three-level region includes at least one selected from the group consisting of: one or more holes, one or more nanopores, and one or more chambers in the surface of the first substrate facing the second substrate.

37. The apparatus according to claim 36, wherein, The one or more holes are a hole array.

38. The apparatus according to any one of claims 28 to 37, wherein, The sample analysis area includes two or more tertiary zones.

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

40. The apparatus according to claim 38, wherein, The two or more tertiary zones are of different types.

41. The apparatus according to any one of claims 1 to 40, wherein, The plurality of primary zones are unbounded around the perimeter of each primary zone.

42. The apparatus according to any one of claims 28 to 41, wherein, The sample analysis region includes a four-level region, wherein the four-level region includes a cylindrical opening across the second substrate, and the four-level region is located at the second end of the sample analysis region.

43. The apparatus according to any one of claims 28 to 42, wherein, The sample analysis region includes a five-level region, wherein the five-level region is an opening across the second substrate and is located at the first end of the sample analysis region.

44. The apparatus according to claim 42, wherein, The fourth level zone is a hydrophilic liquid pore or reservoir.

45. The apparatus according to claim 43, wherein, The fifth level zone is a hydrophobic liquid pore or reservoir.

46. ​​The apparatus according to any one of claims 42 to 45, wherein, The fourth-level region includes one or more substrate retention features.

47. The apparatus according to claim 46, wherein, The one or more substrates retain a feature selected from the group consisting of: a quarter-circular protrusion that protrudes from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate; Horseshoe-shaped protrusions with a gradually widening edge protruding from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate; horseshoe-shaped protrusions without a gradually widening edge protruding from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate; horseshoe-shaped grooves without a gradually widening edge protruding from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate. A rectangular protrusion protrudes 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 that protrude from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate.

48. The apparatus according to any one of claims 43 to 47, wherein, The sample analysis area includes a substrate stop feature, which includes a ridge on the surface of the enlarged protruding element facing the first substrate, the ridge creating a height difference between the fifth-level region and the fourth-level region.

49. The apparatus according to any one of claims 34 to 48, wherein, The enlarged protruding element includes a pinned wall, the pinned wall including a raised edge around the periphery of the enlarged protruding element, the periphery excluding the end containing the fifth-level zone.

50. The apparatus according to any one of claims 43 to 49, wherein, The fifth-level region includes a barrier feature, wherein the barrier feature is semi-circular and 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 apparatus according to any one of claims 1 to 50, wherein, One or more of the primary regions include fluid.

52. The apparatus according to claim 51, wherein, The primary region retains the fluid through surface tension and capillary force.

53. The apparatus according to any one of claims 1 to 52, wherein, One or more secondary zones contain fluid.

54. The apparatus according to any one of claims 1 to 53, wherein, The device includes a pretreatment area configured to pretreat the sample before processing it.

55. The apparatus according to claim 54, wherein, The pretreatment area includes a plurality of elongated openings around the periphery of the pretreatment area, wherein the elongated openings are separated from each other by a plurality of connecting areas, wherein the pretreatment area is defined by the surface of the second substrate facing the first substrate and the surface of the first substrate facing the second substrate, and is enclosed by the plurality of elongated openings.

56. The apparatus according to claim 55, wherein, The pretreatment area includes multiple openings in the second substrate.

57. The apparatus according to claim 55 or 56, wherein, The pretreatment area includes multiple openings in the second substrate.

58. The apparatus according to any one of claims 55 to 57, wherein, The pretreatment area includes a raised edge around the perimeter of the pretreatment area, the perimeter being enclosed by the plurality of elongated openings.

59. The apparatus according to any one of claims 54 to 58, wherein, The pretreatment area is located at the end of the sample processing area that is furthest from the sample analysis area.

60. The apparatus according to any one of claims 1 to 59, wherein, The device includes one or more fixed primary regions, wherein the one or more fixed primary regions are formed by laterally extending protruding elements in the second substrate facing the first substrate, wherein the laterally extending protruding elements have a surface facing the first substrate, the surface having a larger surface area than the surface of the plurality of protruding elements facing the first substrate.

61. The apparatus according to any one of claims 1 to 60, wherein, The plurality of protruding elements have a smooth surface finish.

62. The apparatus according to any one of claims 1 to 61, wherein, The laterally extending protruding element has a smooth surface finish.

63. The apparatus according to any one of claims 1 to 62, wherein, The enlarged protruding element has a smooth surface finish.

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

65. The apparatus according to any one of claims 61 to 64, wherein, The surface finish of the plurality of protruding elements has an average surface roughness of approximately 0.15 μm to approximately 0.70 μm.

66. The apparatus according to any one of claims 62 to 65, wherein, The surface finish of the laterally extending protruding element has an average surface roughness of approximately 0.15 μm to approximately 0.70 μm.

67. The apparatus according to any one of claims 63 to 66, wherein, The enlarged protruding element has an average surface roughness of approximately 0.012 μm to approximately 0.15 μm.

68. The apparatus according to any one of claims 60 to 67, wherein, The one or more fixed primary zones include one or more openings.

69. The apparatus according to any one of claims 60 to 68, wherein, The one or more fixed primary zones include two or more openings.

70. The apparatus according to any one of claims 60 to 69, wherein, The fixed primary region includes one or more secondary features, wherein the secondary features are one or more protruding elements that protrude from the surface of the laterally extending protruding element facing the first substrate.

71. The apparatus according to any one of claims 1 to 70, wherein, The device includes one or more variable primary regions.

72. The apparatus according to any one of claims 1 to 71, wherein, The primary area presents a pattern selected from the group consisting of: grid, line, non-grid, and honeycomb patterns.

73. The apparatus according to any one of claims 1 to 72, wherein, The edges of the plurality of protruding elements are flat edges.

74. The apparatus according to any one of claims 1 to 73, wherein, The edges of the plurality of protruding elements are rounded.

75. The apparatus according to any one of claims 1 to 74, wherein, The edges of the plurality of protruding elements have protrusions.

76. The apparatus according to any one of claims 1 to 75, wherein, One or more of the plurality of protruding elements have serrations or grooves on one or more surfaces of the protruding element.

77. The apparatus according to any one of claims 1 to 76, wherein, One or more of the plurality of recessed elements have serrations or grooves on one or more surfaces of the recessed element.

78. The apparatus according to claim 76 or 77, wherein, The serrations or grooves are in the form of a pattern selected from the group consisting of: waves, straight lines perpendicular or parallel to the edge of the pad, straight lines diagonally opposite to the edge of the pad, cross or shading patterns, and any combination thereof.

79. The apparatus according to any one of claims 1 to 78, wherein, One or more of the plurality of protruding elements have pits or dents on one or more of the surfaces of the protruding elements.

80. The apparatus according to any one of claims 1 to 79, wherein, One or more of the plurality of recessed elements have pits or dents on one or more surfaces of the recessed element.

81. The apparatus according to claim 79 or 80, wherein, The pit or indentation is concave, convex, or a combination thereof.

82. The apparatus according to any one of claims 60 to 81, wherein, The fixed primary region includes a hook-shaped portion connected to the surface of the extended protruding element, the surface of which is opposite to the surface of the second substrate facing the first substrate.

83. The apparatus according to any one of claims 60 to 81, wherein, The fixed primary region includes a chamfered end.

84. The apparatus according to claim 83, wherein, The chamfered end is crescent-shaped.

85. The apparatus according to any one of claims 82 to 84, wherein, The laterally extending protruding element includes a raised, beveled periphery on the surface facing the first substrate.

86. The apparatus according to any one of claims 82 to 84, wherein, The laterally extending protruding element includes a studded wall around the periphery of the extended protruding element on the surface facing the first substrate.

87. The apparatus according to any one of claims 82 to 84, wherein, The laterally extending protruding element includes a recessed, beveled edge around the periphery of the extended protruding element on the surface facing the first substrate.

88. The apparatus according to any one of claims 82 to 87, wherein, The laterally extending protruding element includes a plurality of ridges on the surface facing the first substrate.

89. The apparatus according to any one of claims 82 to 88, wherein, The laterally extending protruding element includes a plurality of raised struts on the surface facing the first substrate.

90. The apparatus according to any one of claims 1 to 89, wherein, The device includes a waste disposal area.

91. The apparatus according to claim 90, wherein, The waste disposal area includes an opening in the second substrate and a wedge-shaped portion extending from the second substrate into the central chamber.

92. The apparatus according to claim 90 or 91, wherein, The waste disposal area is located at the end of the sample processing area that is furthest from the sample analysis area.

93. The apparatus according to any one of claims 1 to 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), polyvinyl chloride (PVC), polydimethylsiloxane (PDMS), acrylonitrile butadiene styrene (ABS), polylactic acid (PLA), thermoplastic PU, transparent resin, and polyethylene glycol diacrylate (PEGDA).

94. The apparatus according to any one of claims 1 to 93, wherein, The bottom substrate is made of a material selected from the group consisting of: glass, silicon, ceramics, metals, polymethyl methacrylate (PMMA), polystyrene (PS), polycarbonate (PC), cyclic olefin copolymer (COC), cyclic olefin polymer (COP), polypropylene (PP), polyurethane (PU), polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC), polydimethylsiloxane (PDMS), acrylonitrile butadiene styrene (ABS), polylactic acid (PLA), thermoplastic PU, transparent resin, thin film, and polyethylene glycol diacrylate (PEGDA).

95. An apparatus comprising: First substrate, and A second substrate positioned on the first substrate, wherein the first substrate includes a sidewall surrounding at least a portion of the periphery of the first substrate, wherein the first substrate, the sidewall, and the second substrate define a central cavity therebetween. The second substrate includes a surface facing the central cavity, which includes a plurality of recessed elements and a plurality of protruding elements; The primary region is defined between the surface of the plurality of protruding elements facing the first substrate and the surface of the first substrate facing the second substrate. The secondary region is defined between the surfaces of the plurality of recessed elements facing the first substrate and the surfaces of the first substrate facing the second substrate. Wherein, the second substrate has an opening in one or more of the primary regions, and The first substrate and the second substrate have differences in hydrophobicity or hydrophilicity.

96. The apparatus according to claim 95, wherein, At least a portion of the periphery includes at least two, at least three, or at least four peripheral sides of the second substrate.

97. The apparatus according to claim 95 or 96, wherein, The difference in hydrophobicity or hydrophilicity is the difference between the contact angle of the first substrate and the contact angle of the second substrate.

98. The apparatus according to claim 97, wherein, The difference between the contact angle of the first substrate and the contact angle of the second substrate is approximately 10% to approximately 60%.

99. The apparatus according to claim 98, wherein, The difference between the contact angle of the first substrate and the contact angle of the second substrate is approximately 20% to approximately 40%.

100. The apparatus according to claim 97, wherein, The difference between the contact angle of the first substrate and the contact angle of the second substrate is approximately 5° to approximately 60°.

101. The apparatus according to claim 100, wherein, The difference between the contact angle of the first substrate and the contact angle of the second substrate is approximately 5° to approximately 40°.

102. The apparatus according to any one of claims 97 or 100 to 101, wherein, The difference between the contact angle of the first substrate and the contact angle of the second substrate is approximately 5° to approximately 30°.

103. The apparatus according to any one of claims 95 to 102, wherein, The first substrate is hydrophobic, and the second substrate is hydrophilic.

104. The apparatus according to any one of claims 95 to 102, wherein, The first substrate is hydrophilic, and the second substrate is hydrophobic.

105. The apparatus according to any one of claims 95 to 102, wherein, The first substrate is hydrophobic, and the second substrate is hydrophobic.

106. The apparatus according to any one of claims 95 to 102, wherein, The first substrate is hydrophilic, and the second substrate is hydrophilic.

107. The apparatus according to any one of claims 95 to 106, wherein, The primary region is discrete.

108. The apparatus according to any one of claims 95 to 107, wherein, Secondary areas are connected.

109. The apparatus according to any one of claims 95 to 108, wherein, The second substrate has an opening in one or more of the secondary regions.

110. The apparatus according to any one of claims 95 to 109, wherein, The second substrate has an opening in each of the plurality of primary regions.

111. The apparatus according to any one of claims 95 to 110, wherein, In the primary region, the surfaces of the plurality of protruding elements facing the first substrate and the surfaces of the first substrate facing the second substrate are separated by a first distance, and in the secondary region, the surfaces of the recessed elements facing the first substrate and the surfaces of the first substrate facing the second substrate are separated by a second distance.

112. The apparatus according to claim 111, wherein, The first distance is less than the second distance.

113. The apparatus according to any one of claims 95 to 112, wherein, The protruding element is a shape selected from the group consisting of: rectangle, circle, triangle, pentagon, hexagon, heptagon, octagon, decagon, dodecagon, amoeba-like and irregular shape.

114. The apparatus according to any one of claims 95 to 113, wherein, The device includes three or more primary regions separated by two or more secondary regions.

115. The apparatus according to any one of claims 95 to 114, wherein, The device includes six or more primary regions separated by seven or more secondary regions.

116. The apparatus according to any one of claims 95 to 115, wherein, The device includes nine or more primary regions separated by twelve or more secondary regions.

117. The apparatus according to any one of claims 95 to 116, wherein, The device includes twelve or more primary regions separated by seventeen or more secondary regions.

118. The apparatus according to any one of claims 95 to 117, wherein, The device includes fifteen or more primary regions separated by twenty-two or more secondary regions.

119. The apparatus according to any one of claims 95 to 118, wherein, The device includes eighteen or more primary regions separated by twenty-seven or more secondary regions.

120. The apparatus according to any one of claims 95 to 119, wherein, The device includes twenty-one or more primary regions separated by thirty-two or more secondary regions.

121. The apparatus according to any one of claims 95 to 120, wherein, The device includes twenty-four or more primary regions separated by thirty-seven or more secondary regions.

122. The apparatus according to any one of claims 95 to 121, further comprising a sample analysis region configured to analyze a sample.

123. The apparatus according to claim 122, wherein, The sample analysis area is positioned on the first substrate adjacent to the central chamber.

124. The apparatus according to claim 122 or 123, wherein, The sample analysis area is physically separated from the central chamber.

125. The apparatus according to claim 122 or 123, wherein, The sample analysis area is not physically separated from the central chamber.

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

127. The apparatus according to any one of claims 122 to 126, wherein, The sample analysis region has a first end that is laterally separated from the 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 apparatus according to claim 127, wherein, In the sample analysis area, the surface of the second substrate facing the first substrate includes enlarged protruding elements, wherein: The surface of the enlarged protruding element facing the first substrate has a larger surface area than the surfaces 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 apparatus according to claim 127 or 128, wherein, The sample analysis region includes a three-level region, which is located at the midpoint between the first and second ends of the sample analysis region, and is defined by the following: 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 apparatus according to claim 129, wherein, The three-level region includes at least one selected from the group consisting of: one or more holes, one or more nanopores, and one or more chambers in the surface of the first substrate facing the second substrate.

131. The apparatus according to claim 130, wherein, The one or more holes are a hole array.

132. The apparatus according to any one of claims 122 to 131, wherein, The sample analysis area includes two or more tertiary zones.

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

134. The apparatus according to claim 132, wherein, The two or more tertiary zones are of different types.

135. The apparatus according to any one of claims 95 to 134, wherein, The plurality of primary zones are unbounded around the perimeter of each primary zone.

136. The apparatus according to any one of claims 122 to 135, wherein, The sample analysis region includes a four-level region, wherein the four-level region includes a cylindrical opening across the second substrate, and the four-level region is located at the second end of the sample analysis region.

137. The apparatus according to any one of claims 122 to 136, wherein, The sample analysis region includes a five-level region, wherein the five-level region is an opening across the second substrate and is located at the first end of the sample analysis region.

138. The apparatus according to claim 136, wherein, The fourth level zone is a hydrophilic liquid pore or reservoir.

139. The apparatus according to claim 137, wherein, The fifth level zone is a hydrophobic liquid pore or reservoir.

140. The apparatus according to any one of claims 136 to 139, wherein, The fourth-level region includes one or more substrate retention features.

141. The apparatus according to claim 140, wherein, The one or more substrates retain a feature selected from the group consisting of: a quarter-circular protrusion that protrudes from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate; Horseshoe-shaped protrusions with widening edges protruding from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate; horseshoe-shaped protrusions without widening edges protruding from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate; horseshoe-shaped grooves without widening edges protruding from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate. A rectangular protrusion protrudes 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 that protrude from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate.

142. The apparatus according to any one of claims 137 to 141, wherein, The sample analysis area includes a substrate stop feature, which includes a ridge on the surface of the enlarged protruding element facing the first substrate, the ridge creating a height difference between the fifth-level region and the fourth-level region.

143. The apparatus according to any one of claims 128 to 142, wherein, The enlarged protruding element includes a pinned wall, the pinned wall including a raised edge around the periphery of the enlarged protruding element, the periphery excluding the end containing the level 5 zone.

144. The apparatus according to any one of claims 137 to 143, wherein, The fifth-level region includes a barrier feature, wherein the barrier feature is semi-circular and 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 apparatus according to any one of claims 95 to 144, wherein, One or more of the primary regions include fluid.

146. The apparatus according to claim 145, wherein, The primary region retains the fluid through surface tension and capillary force.

147. The apparatus according to any one of claims 95 to 146, wherein, One or more secondary zones contain fluid.

148. The apparatus according to any one of claims 95 to 147, wherein, The device includes a pretreatment area configured to pretreat the sample before processing it.

149. The apparatus according to claim 148, wherein, The pretreatment area includes a plurality of elongated openings around the periphery of the pretreatment area, wherein the elongated openings are separated from each other by a plurality of connecting areas, wherein the pretreatment area is defined by the surface of the second substrate facing the first substrate and the surface of the first substrate facing the second substrate, and is enclosed by the plurality of elongated openings.

150. The apparatus according to claim 149, wherein, The pretreatment area includes multiple openings in the second substrate.

151. The apparatus according to claim 149 or 150, wherein, The pretreatment area includes multiple openings in the second substrate.

152. The apparatus according to any one of claims 149 to 151, wherein, The pretreatment area includes a raised edge around the perimeter of the pretreatment area, the perimeter being enclosed by the plurality of elongated openings.

153. The apparatus according to any one of claims 148 to 152, wherein, The pretreatment area is located at the end of the sample processing area that is furthest from the sample analysis area.

154. The apparatus according to any one of claims 95 to 153, wherein, The device includes one or more fixed primary regions, wherein the one or more fixed primary regions are formed by laterally extending protruding elements in the second substrate facing the first substrate, wherein the laterally extending protruding elements have a surface facing the first substrate, the surface having a larger surface area than the surface of the plurality of protruding elements facing the first substrate.

155. The apparatus according to any one of claims 95 to 154, wherein, The plurality of protruding elements have a smooth surface finish.

156. The apparatus according to any one of claims 95 to 155, wherein, The laterally extending protruding element has a smooth surface finish.

157. The apparatus according to any one of claims 95 to 156, wherein, The enlarged protruding element has a smooth surface finish.

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

159. The apparatus according to any one of claims 155 to 158, wherein, The surface finish of the plurality of protruding elements has an average surface roughness of approximately 0.15 μm to approximately 0.70 μm.

160. The apparatus according to any one of claims 156 to 159, wherein, The surface finish of the laterally extending protruding element has an average surface roughness of approximately 0.15 μm to approximately 0.70 μm.

161. The apparatus according to any one of claims 157 to 160, wherein, The enlarged protruding element has an average surface roughness of approximately 0.012 μm to approximately 0.15 μm.

162. The apparatus according to any one of claims 154 to 161, wherein, The one or more fixed primary zones include one or more openings.

163. The apparatus according to any one of claims 154 to 162, wherein, The one or more fixed primary zones include two or more openings.

164. The apparatus according to any one of claims 154 to 163, wherein, The fixed primary region includes one or more secondary features, wherein the secondary features are one or more protruding elements that protrude from the surface of the laterally extending protruding element facing the first substrate.

165. The apparatus according to any one of claims 95 to 164, wherein, The device includes one or more variable primary regions.

166. The apparatus according to any one of claims 95 to 165, wherein, The primary area presents a pattern selected from the group consisting of: grid, line, non-grid, and honeycomb patterns.

167. The apparatus according to any one of claims 95 to 166, wherein, The edges of the plurality of protruding elements are flat edges.

168. The apparatus according to any one of claims 95 to 167, wherein, The edges of the plurality of protruding elements are rounded.

169. The apparatus according to any one of claims 95 to 168, wherein, The edges of the plurality of protruding elements have protrusions.

170. The apparatus according to any one of claims 95 to 169, wherein, One or more of the plurality of protruding elements have serrations or grooves on one or more surfaces of the protruding element.

171. The apparatus according to any one of claims 95 to 170, wherein, One or more of the plurality of recessed elements have serrations or grooves on one or more surfaces of the recessed element.

172. The apparatus according to claim 170 or 171, wherein, The serrations or grooves are in the form of a pattern selected from the group consisting of: waves, straight lines perpendicular or parallel to the edge of the pad, straight lines diagonally opposite to the edge of the pad, cross or shading patterns, and any combination thereof.

173. The apparatus according to any one of claims 95 to 172, wherein, One or more of the plurality of protruding elements have pits or dents on one or more of the surfaces of the protruding elements.

174. The apparatus according to any one of claims 95 to 173, wherein, One or more of the plurality of recessed elements have pits or dents on one or more surfaces of the recessed element.

175. The apparatus according to claim 173 or 174, wherein, The pit or indentation is concave, convex, or a combination thereof.

176. The apparatus according to any one of claims 154 to 175, wherein, The fixed primary region includes a hook-shaped portion connected to the surface of the extended protruding element, the surface of which is opposite to the surface of the second substrate facing the first substrate.

177. The apparatus according to any one of claims 154 to 175, wherein, The fixed primary region includes a chamfered end.

178. The apparatus according to claim 177, wherein, The chamfered end is crescent-shaped.

179. The apparatus according to any one of claims 176 to 178, wherein, The laterally extending protruding element includes a raised, beveled periphery on the surface facing the first substrate.

180. The apparatus according to any one of claims 176 to 178, wherein, The laterally extending protruding element includes a studded wall around the periphery of the extended protruding element on the surface facing the first substrate.

181. The apparatus according to any one of claims 176 to 178, wherein, The laterally extending protruding element includes a recessed, beveled edge around the periphery of the extended protruding element on the surface facing the first substrate.

182. The apparatus according to any one of claims 176 to 181, wherein, The laterally extending protruding element includes a plurality of ridges on the surface facing the first substrate.

183. The apparatus according to any one of claims 176 to 182, wherein, The laterally extending protruding element includes a plurality of raised struts on the surface facing the first substrate.

184. The apparatus according to any one of claims 95 to 183, wherein, The device includes a waste disposal area.

185. The apparatus according to claim 184, wherein, The waste disposal area includes an opening in the second substrate and a wedge-shaped portion extending from the second substrate into the central chamber.

186. The apparatus according to claim 184 or 185, wherein, The waste disposal area is located at the end of the sample processing area that is furthest from the sample analysis area.

187. The apparatus according to any one of claims 95 to 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), polyvinyl chloride (PVC), polydimethylsiloxane (PDMS), acrylonitrile butadiene styrene (ABS), polylactic acid (PLA), thermoplastic PU, transparent resin, and polyethylene glycol diacrylate (PEGDA).

188. The apparatus according to any one of claims 95 to 187, wherein, The bottom substrate is made of a material selected from the group consisting of: glass, silicon, ceramics, metals, polymethyl methacrylate (PMMA), polystyrene (PS), polycarbonate (PC), cyclic olefin copolymer (COC), cyclic olefin polymer (COP), polypropylene (PP), polyurethane (PU), polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC), polydimethylsiloxane (PDMS), acrylonitrile butadiene styrene (ABS), polylactic acid (PLA), thermoplastic PU, transparent resin, thin film, and polyethylene glycol diacrylate (PEGDA).

189. An apparatus comprising: First substrate, A spacer layer positioned on the surface of the first substrate, wherein the spacer layer is disposed around at least a portion of the periphery of the first substrate, and A second substrate positioned on the spacer layer, wherein the first substrate, the spacer layer, and the second substrate define a central cavity therebetween. The second substrate includes a surface facing the central cavity, which includes a plurality of recessed elements and a plurality of protruding elements; The primary region is defined between the surfaces of the plurality of protruding elements and the first substrate; and The secondary region is defined between the surfaces of the plurality of recessed elements and the surface of the first substrate facing the second substrate.

190. The apparatus according to claim 189, wherein, The spacer layer is selected from the group consisting of: an adhesive layer; a gasket layer; a first adhesive layer and a gasket 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 apparatus according to claim 189 or 190, wherein, At least a portion of the periphery includes at least two, at least three, or at least four peripheral sides of the second substrate.

192. The apparatus according to any one of claims 189 to 191, wherein, The difference in hydrophobicity or hydrophilicity is the difference between the contact angle of the first substrate and the contact angle of the second substrate.

193. The apparatus according to claim 192, wherein, The difference between the contact angle of the first substrate and the contact angle of the second substrate is approximately 10% to approximately 60%.

194. The apparatus according to claim 193, wherein, The difference between the contact angle of the first substrate and the contact angle of the second substrate is approximately 20% to approximately 40%.

195. The apparatus according to claim 192, wherein, The difference between the contact angle of the first substrate and the contact angle of the second substrate is approximately 5° to approximately 60°.

196. The apparatus according to claim 195, wherein, The difference between the contact angle of the first substrate and the contact angle of the second substrate is approximately 5° to approximately 40°.

197. The apparatus according to any one of claims 192 or 195 to 196, wherein, The difference between the contact angle of the first substrate and the contact angle of the second substrate is approximately 5° to approximately 30°.

198. The apparatus according to any one of claims 189 to 197, wherein, The first substrate is hydrophobic, and the second substrate is hydrophilic.

199. The apparatus according to any one of claims 189 to 197, wherein, The first substrate is hydrophilic, and the second substrate is hydrophobic.

200. The apparatus according to any one of claims 189 to 197, wherein, The first substrate is hydrophobic, and the second substrate is hydrophobic.

201. The apparatus according to any one of claims 189 to 197, wherein, The first substrate is hydrophilic, and the second substrate is hydrophilic.

202. The apparatus according to any one of claims 189 to 201, wherein, The primary region is discrete.

203. The apparatus according to any one of claims 189 to 202, wherein, Secondary areas are connected.

204. The apparatus according to any one of claims 189 to 203, wherein, The second substrate has an opening in one or more of the secondary regions.

205. The apparatus according to any one of claims 189 to 204, wherein, The second substrate has an opening in each of the plurality of primary regions.

206. The apparatus according to any one of claims 189 to 205, wherein, In the primary region, the surfaces of the plurality of protruding elements facing the first substrate and the surfaces of the first substrate facing the second substrate are separated by a first distance, and in the secondary region, the surfaces of the recessed elements facing the first substrate and the surfaces of the first substrate facing the second substrate are separated by a second distance.

207. The apparatus according to claim 206, wherein, The first distance is less than the second distance.

208. The apparatus according to any one of claims 189 to 207, wherein, The protruding element is a shape selected from the group consisting of: rectangle, circle, triangle, pentagon, hexagon, heptagon, octagon, decagon, dodecagon, amoeba-like and irregular shape.

209. The apparatus according to any one of claims 189 to 208, wherein, The device includes three or more primary regions separated by two or more secondary regions.

210. The apparatus according to any one of claims 189 to 209, wherein, The device includes six or more primary regions separated by seven or more secondary regions.

211. The apparatus according to any one of claims 189 to 210, wherein, The device includes nine or more primary regions separated by twelve or more secondary regions.

212. The apparatus according to any one of claims 189 to 211, wherein, The device includes twelve or more primary regions separated by seventeen or more secondary regions.

213. The apparatus according to any one of claims 189 to 212, wherein, The device includes fifteen or more primary regions separated by twenty-two or more secondary regions.

214. The apparatus according to any one of claims 189 to 213, wherein, The device includes eighteen or more primary regions separated by twenty-seven or more secondary regions.

215. The apparatus according to any one of claims 189 to 214, wherein, The device includes twenty-one or more primary regions separated by thirty-two or more secondary regions.

216. The apparatus according to any one of claims 189 to 215, wherein, The device includes twenty-four or more primary regions separated by thirty-seven or more secondary regions.

217. The apparatus according to any one of claims 189 to 216, further comprising a sample analysis region configured to analyze a sample.

218. The apparatus according to claim 217, wherein, The sample analysis area is positioned on the first substrate adjacent to the central chamber.

219. The apparatus according to claim 217 or 218, wherein, The sample analysis area is physically separated from the central chamber.

220. The apparatus according to claim 217 or 218, wherein, The sample analysis area is not physically separated from the central chamber.

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

222. The apparatus according to any one of claims 217 to 221, wherein, The sample analysis region has a first end that is laterally separated from the 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 apparatus according to claim 222, wherein, In the sample analysis area, the surface of the second substrate facing the first substrate includes enlarged protruding elements, wherein: The surface of the enlarged protruding element facing the first substrate has a larger surface area than the surfaces 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 apparatus according to claim 222 or 223, wherein, The sample analysis region includes a three-level region, which is located at the midpoint between the first and second ends of the sample analysis region, and is defined by the following: 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 apparatus according to claim 224, wherein, The three-level region includes at least one selected from the group consisting of: one or more holes, one or more nanopores, and one or more chambers in the surface of the first substrate facing the second substrate.

226. The apparatus according to claim 225, wherein, The one or more holes are a hole array.

227. The apparatus according to any one of claims 217 to 226, wherein, The sample analysis area includes two or more tertiary zones.

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

229. The apparatus according to claim 227, wherein, The two or more tertiary zones are of different types.

230. The apparatus according to any one of claims 189 to 229, wherein, The plurality of primary zones are unbounded around the perimeter of each primary zone.

231. The apparatus according to any one of claims 217 to 230, wherein, The sample analysis region includes a four-level region, wherein the four-level region includes a cylindrical opening across the second substrate, and the four-level region is located at the second end of the sample analysis region.

232. The apparatus according to any one of claims 217 to 231, wherein, The sample analysis region includes a five-level region, wherein the five-level region is an opening across the second substrate and is located at the first end of the sample analysis region.

233. The apparatus according to claim 231, wherein, The fourth level zone is a hydrophilic liquid pore or reservoir.

234. The apparatus according to claim 232, wherein, The fifth level zone is a hydrophobic liquid pore or reservoir.

235. The apparatus according to any one of claims 231 to 234, wherein, The fourth-level region includes one or more substrate retention features.

236. The apparatus according to claim 235, wherein, The one or more substrates retain a feature selected from the group consisting of: a quarter-circular protrusion that protrudes from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate; Horseshoe-shaped protrusions with a gradually widening edge protruding from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate; horseshoe-shaped protrusions without a gradually widening edge protruding from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate; horseshoe-shaped grooves without a gradually widening edge protruding from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate. A rectangular protrusion protrudes 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 that protrude from the surface of the second substrate facing the first substrate or the surface of the first substrate facing the second substrate.

237. The apparatus according to any one of claims 232 to 236, wherein, The sample analysis area includes a substrate stop feature, which includes a ridge on the surface of the enlarged protruding element facing the first substrate, the ridge creating a height difference between the fifth-level region and the fourth-level region.

238. The apparatus according to any one of claims 223 to 237, wherein, The enlarged protruding element includes a pinned wall, the pinned wall including a raised edge around the periphery of the enlarged protruding element, the periphery excluding the end containing the fifth-level zone.

239. The apparatus according to any one of claims 232 to 238, wherein, The fifth-level region includes a barrier feature, wherein the barrier feature is semi-circular and 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 apparatus according to any one of claims 189 to 239, wherein, One or more of the primary regions include fluid.

241. The apparatus according to claim 240, wherein, The primary region retains the fluid through surface tension and capillary force.

242. The apparatus according to any one of claims 189 to 241, wherein, One or more secondary zones contain fluid.

243. The apparatus according to any one of claims 189 to 242, wherein, The device includes a pretreatment area configured to pretreat the sample before processing it.

244. The apparatus according to claim 243, wherein, The pretreatment area includes a plurality of elongated openings around the periphery of the pretreatment area, wherein the elongated openings are separated from each other by a plurality of connecting areas, wherein the pretreatment area is defined by the surface of the second substrate facing the first substrate and the surface of the first substrate facing the second substrate, and is enclosed by the plurality of elongated openings.

245. The apparatus according to claim 244, wherein, The pretreatment area includes multiple openings in the second substrate.

246. The apparatus according to claim 244 or 245, wherein, The pretreatment area includes multiple openings in the second substrate.

247. The apparatus according to any one of claims 244 to 246, wherein, The pretreatment area includes a raised edge around the perimeter of the pretreatment area, the perimeter being enclosed by the plurality of elongated openings.

248. The apparatus according to any one of claims 243 to 247, wherein, The pretreatment area is located at the end of the sample processing area that is furthest from the sample analysis area.

249. The apparatus according to any one of claims 189 to 248, wherein, The device includes one or more fixed primary regions, wherein the one or more fixed primary regions are formed by laterally extending protruding elements in the second substrate facing the first substrate, wherein the laterally extending protruding elements have a surface facing the first substrate, the surface having a larger surface area than the surface of the plurality of protruding elements facing the first substrate.

250. The apparatus according to any one of claims 189 to 249, wherein, The plurality of protruding elements have a smooth surface finish.

251. The apparatus according to any one of claims 189 to 250, wherein, The laterally extending protruding element has a smooth surface finish.

252. The apparatus according to any one of claims 189 to 251, wherein, The enlarged protruding element has a smooth surface finish.

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

254. The apparatus according to any one of claims 250 to 253, wherein, The surface finish of the plurality of protruding elements has an average surface roughness of approximately 0.15 μm to approximately 0.70 μm.

255. The apparatus according to any one of claims 251 to 254, wherein, The surface finish of the laterally extending protruding element has an average surface roughness of approximately 0.15 μm to approximately 0.70 μm.

256. The apparatus according to any one of claims 252 to 255, wherein, The enlarged protruding element has an average surface roughness of approximately 0.012 μm to approximately 0.15 μm.

257. The apparatus according to any one of claims 249 to 256, wherein, The one or more fixed primary zones include one or more openings.

258. The apparatus according to any one of claims 249 to 257, wherein, The one or more fixed primary zones include two or more openings.

259. The apparatus according to any one of claims 249 to 258, wherein, The fixed primary region includes one or more secondary features, wherein the secondary features are one or more protruding elements that protrude from the surface of the laterally extending protruding element facing the first substrate.

260. The apparatus according to any one of claims 189 to 259, wherein, The device includes one or more variable primary regions.

261. The apparatus according to any one of claims 189 to 260, wherein, The primary area presents a pattern selected from the group consisting of: grid, line, non-grid, and honeycomb patterns.

262. The apparatus according to any one of claims 189 to 261, wherein, The edges of the plurality of protruding elements are flat edges.

263. The apparatus according to any one of claims 189 to 262, wherein, The edges of the plurality of protruding elements are rounded.

264. The apparatus according to any one of claims 189 to 263, wherein, The edges of the plurality of protruding elements have protrusions.

265. The apparatus according to any one of claims 189 to 264, wherein, One or more of the plurality of protruding elements have serrations or grooves on one or more surfaces of the protruding element.

266. The apparatus according to any one of claims 189 to 265, wherein, One or more of the plurality of recessed elements have serrations or grooves on one or more surfaces of the recessed element.

267. The apparatus according to claim 265 or 266, wherein, The serrations or grooves are in the form of a pattern selected from the group consisting of: waves, straight lines perpendicular or parallel to the edge of the pad, straight lines diagonally opposite to the edge of the pad, cross or shading patterns, and any combination thereof.

268. The apparatus according to any one of claims 189 to 267, wherein, One or more of the plurality of protruding elements have pits or dents on one or more of the surfaces of the protruding elements.

269. The apparatus according to any one of claims 189 to 268, wherein, One or more of the plurality of recessed elements have pits or dents on one or more surfaces of the recessed element.

270. The apparatus according to claim 268 or 269, wherein, The pit or indentation is concave, convex, or a combination thereof.

271. The apparatus according to any one of claims 249 to 270, wherein, The fixed primary region includes a hook-shaped portion connected to the surface of the extended protruding element, the surface of which is opposite to the surface of the second substrate facing the first substrate.

272. The apparatus according to any one of claims 249 to 270, wherein, The fixed primary region includes a chamfered end.

273. The apparatus according to claim 272, wherein, The chamfered end is crescent-shaped.

274. The apparatus according to any one of claims 271 to 273, wherein, The laterally extending protruding element includes a raised, beveled periphery on the surface facing the first substrate.

275. The apparatus according to any one of claims 271 to 273, wherein, The laterally extending protruding element includes a studded wall around the periphery of the extended protruding element on the surface facing the first substrate.

276. The apparatus according to any one of claims 271 to 273, wherein, The laterally extending protruding element includes a recessed, beveled edge around the periphery of the extended protruding element on the surface facing the first substrate.

277. The apparatus according to any one of claims 271 to 276, wherein, The laterally extending protruding element includes a plurality of ridges on the surface facing the first substrate.

278. The apparatus according to any one of claims 271 to 277, wherein, The laterally extending protruding element includes a plurality of raised struts on the surface facing the first substrate.

279. The apparatus according to any one of claims 189 to 278, wherein, The device includes a waste disposal area.

280. The apparatus according to claim 279, wherein, The waste disposal area includes an opening in the second substrate and a wedge-shaped portion extending from the second substrate into the central chamber.

281. The apparatus according to claim 279 or 280, wherein, The waste disposal area is located at the end of the sample processing area that is furthest from the sample analysis area.

282. The apparatus according to any one of claims 189 to 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), polyvinyl chloride (PVC), polydimethylsiloxane (PDMS), acrylonitrile butadiene styrene (ABS), polylactic acid (PLA), thermoplastic PU, transparent resin, and polyethylene glycol diacrylate (PEGDA).

283. The apparatus according to any one of claims 189 to 282, wherein, The bottom substrate is made of a material selected from the group consisting of: glass, silicon, ceramics, metals, polymethyl methacrylate (PMMA), polystyrene (PS), polycarbonate (PC), cyclic olefin copolymer (COC), cyclic olefin polymer (COP), polypropylene (PP), polyurethane (PU), polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC), polydimethylsiloxane (PDMS), acrylonitrile butadiene styrene (ABS), polylactic acid (PLA), thermoplastic PU, transparent resin, thin film, and polyethylene glycol diacrylate (PEGDA).

284. A method for mixing samples, the method comprising: The device according to any one of claims 1 to 94, 95 to 188, or 189 to 283 is brought into contact with the vibration source; as well as The vibration source is made to vibrate. The device includes one or more fluids.

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

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

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

288. The method according to claim 285, wherein, The vibration source causes only the second substrate to vibrate.

289. The method according to claim 288, wherein, The vibration source causes the second substrate to vibrate vertically.

290. The method according to claim 286, wherein, The vibration source causes only the first substrate to vibrate.

291. The method according to claim 290, wherein, The vibration source causes the first substrate to vibrate vertically.

292. The method according to claim 287, wherein, The vibration source causes the second substrate and the first substrate to vibrate.

293. The method according to claim 292, wherein, The vibration source causes the top substrate and the bottom substrate, or the second substrate and the first substrate, to vibrate vertically.

294. The method according to claim 284, wherein, The vibration source contacts the fixed primary region.

295. The method according to claim 294, wherein, The vibration source causes the fixed primary region to vibrate vertically.

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

297. The method according to claim 296, wherein, The vibration source causes the chamfered end of the fixed primary region to vibrate vertically.

298. The method according to any one of claims 284 to 297, wherein, The vibration source is selected from the group consisting of: voice coil, vibration motor, piezoelectric actuator, motor and gas pump.

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

300. The method of claim 299, wherein, The voice coil causes the device to vibrate at a frequency that substantially resonates with the one or more fluids.

301. The method according to any one of claims 284 to 300, wherein, The one or more fluids include microparticles.

302. A method for measuring or detecting a target analyte in a sample, the method comprising: a) Deposit a sample including the target analyte into an opening in the primary region of the device. b) Microparticles attached to a first specific binding partner that specifically binds to the target analyte are deposited in the opening of the primary region including the sample, thereby generating microparticles bound to the target analyte. c) Subjecting microparticles bound to the target analyte to a magnetic field to move the microparticles to a primary region containing a second specific binding partner with detectable labelling, thereby generating microparticles bound to the target analyte with detectable labelling; d) subjecting microparticles bound to the detectably labeled target analyte to a magnetic field to move the microparticles to one or more primary regions containing a washing buffer; e) subjecting microparticles bound to the detectably labeled target analyte to a magnetic field to move the microparticles to a pore-containing detection area in the sample analysis region; f) Adding a hydrophilic liquid to the pore before or after step e), the hydrophilic liquid reacting with the detectably labeled second specific binding partner to generate a detectable signal. g) Use a hydrophobic liquid to seal the orifice; as well as h) Image the pore to detect a detectable signal generated by the reaction of the hydrophilic member with the second specific binding member; The device includes: A plurality of primary regions are defined by both: the surfaces of a plurality of protruding elements located on the surface of the second substrate facing the first substrate; and the surface of the first substrate facing the second substrate. Multiple secondary regions are defined by both: the surfaces of multiple recessed elements located on the surface of the second substrate 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 regions and an opening in one or more of the secondary regions.

303. The method according to claim 302, wherein, The target analyte is a protein.

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

305. The method according to claims 302 to 304, wherein, The detectable marker is an enzyme that reacts with the hydrophilic liquid.

306. The method according to any one of claims 302 to 305, wherein, The hydrophobic liquid is oil.

307. The method according to any one of claims 302 to 306, wherein, The hydrophilic liquid is a substrate solution.

308. The method according to any one of claims 302 to 307, further comprising: i) Deposit lysis buffer into the opening of the primary region including the sample, wherein step i) occurs between steps a) and b).

309. The method according to any one of claims 302 to 308, further comprising: j) Mixing the primary region comprising the sample and the microparticles, wherein step j) occurs between steps b) and c).

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

311. The method according to any one of claims 302 to 310, wherein, The one or more primary regions in step d) are two primary regions connected by a secondary region, each primary region containing the washing buffer.

312. The method according to any one of claims 302 to 311, wherein, The one or more primary regions in step j) are two primary regions connected by a secondary region, each primary region containing the washing buffer.

313. The method according to any one of claims 302 to 312, wherein, Step B) also includes: depositing assisting particles.

314. The method according to claim 313, wherein, The assisting particle is configured to enhance the effect of the magnetic force acting on the microparticle in the presence of the assisting particle.

315. The method according to claim 313 or 314, wherein, The microparticles and the assisting particles are spherical, and the diameter of the assisting solid support is larger than the diameter of the measuring solid support.

316. The method according to any one of claims 313 to 315, wherein, The microparticles and the assisting particles comprise between approximately 30,000 and approximately 300,000 assisting particles.

317. The method according to any one of claims 313 to 316, wherein, The microparticles and assisting particles contain more assisting particles than microparticles.

318. The method according to any one of claims 313 to 316, wherein, The microparticles and assisting particles contain fewer assisting particles than microparticles.

319. The method according to any one of claims 313 to 317, wherein, The diameter of the assisting particles is between approximately 5 µm and approximately 15 µm.

320. The method according to any one of claims 313 to 319, wherein, The assisting particles have a diameter of approximately 10 µm.

321. The method according to any one of claims 313 to 320, wherein, The microparticles and assisting particles include magnetic, paramagnetic, and / or superparamagnetic microparticles or beads.

322. The method according to any one of claims 313 to 321, wherein, The assisting particles are configured to bind to one or more interfering substances within the sample.

323. The method according to any one of claims 313 to 322, wherein, The microparticles and assisting particles move across the detection zone at speeds between approximately 0.35 mm / s and approximately 6.00 mm / s.

324. The method according to any one of claims 313 to 323, wherein, The microparticles and assisting particles comprise a microparticle to assisting particle ratio between approximately 1:1 and approximately 50:

1.

325. The method according to any one of claims 313 to 324, wherein, The microparticles and assisting particles comprise between approximately 30,000 and approximately 300,000 assisting particles.

326. The method according to any one of claims 313 to 325, wherein, The microparticles and assisting particles comprise between approximately 30,000 and approximately 80,000 assisting particles.

327. The method according to any one of claims 313 to 326, wherein, The assisting particles include those with negative surface charge.

328. The method according to any one of claims 302 to 326, wherein, The magnetic field is generated by a magnet.

329. The method according to claim 328, wherein, The magnet has a first magnetic field along the bottom surface of the aperture array, the first magnetic field causing the microparticles and assisting particles and the target analyte to move across the bottom surface of the detection region, wherein the magnet has a first magnet end and a second magnet end and a magnetic axis defined therebetween, the magnetic axis being positioned relative to the detection region at an angle between approximately 0 degrees and approximately 80 degrees.

330. The method according to claim 329, wherein, The magnetic axis is positioned relative to the detection area at an angle between approximately 10 degrees and approximately 30 degrees.

331. The method according to claim 329 or 330, wherein, The first magnet moves along the detection area at a first magnet distance defined between the detection area and the magnet, the magnet distance being less than approximately 10 mm.

332. The method according to any one of claims 329 to 331, wherein, The first magnet moves along the detection area while in contact with the bottom of the detection area.

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

334. The method according to any one of claims 302 to 333, wherein, The target analytes are: anti-Müllerian hormone (AMH), CD25 autoantibody, chemokine (CXC) motif ligand 13 (CXCL13), Dickkopf-3 (Dkk-3), IL-12p40, interleukin-8 (IL-8), p14 endothelial cell-specific molecular fragment, SARS-CoV-2 IgA antibody, SARS-CoV-2 IgG antibody, SARS-CoV-2 IgM antibody, secretory glucosolvan (pGSN), secretory granulin II, ACE2, albumin, albuminuria, α-amylase, ApoH, β-2 microglobulin, brain natriuretic peptide (BNP) and its derivatives, and 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 α 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, periosteal protein, peroxisome proliferator-activated receptor gamma coactivator-1 α (PGC-1a), pro-apoptotic kinase R (PKR), 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, α-synuclein, BARF1 (BamH1-A reading frame 1), kidney injury molecule-1 (KIM-1), laminin γ, LMP1 (latent membrane protein 1), neurofilament light chain (NF-L), Tau protein, Tau, UCH-L1 (ubiquitin C-terminal hydrolase-L1), 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), bimodalin, ANXA7 (annexin A7), androgen receptor (AR), v-Raf murine sarcoma virus oncogene homolog B (BRAF), cyclin-dependent kinase inhibitor 1B (CDKN1B), MYC proto-oncogene (cMYC), catalin β-1 (CTNNB1), epidermal growth factor receptor (EGFR), liver glycoside-β receptor 2 (EPHB2), estrogen receptor 1 (ESR1), estrogen receptor 2 (ESR2), ferritin, folic acid,Forkhead box protein O3 (FOXO3A), rapamycin mechanotarget protein complex 1 (FRAP1), fibroblast growth factor receptor substrate 2 (FRS2), GRB2-associated binding protein 2 (Gab2), glial fibrillary acidic protein (GFAP), growth factor receptor binding protein 2 (Grb2), breast cancer estrogen regulatory protein 1 (GREB1), hepatitis B e antigen (HBeAg), hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg), phosphorylated hepatitis B core antigen (P-HBcAg), hepatitis B core-associated 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 (KLF6), Kirsten rat sarcoma virus oncogene homolog (KRAS), leucine zipper tumor suppressor gene 1 (LZTS1), mitogen activator protein Kinase 1 (MAP2K1), mitogen-activated protein kinase (MEK), mitogen-induced gene 6 protein (MIG-6), proliferation marker Ki-67 (MKI67), rapamycin mechanotarget protein (mTOR), mucin 4 cell surface association (MUC4), neural progenitor cell expression developmental downregulated protein 4-1 (NEDD4-1), NK3 homeobox protein 1 (NKX3-1), neuroregulatory protein 1 (NRG1), perkinin, parvovirus B19, phosphoinositol-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 α (PIK3CA), paired-like homology domain transcription factor 2 (PITX2), protein phosphatase 1 regulatory inhibitor subunit 1B (PPP1R1B), PRDX6 (peroxidase-6), phosphatase and tensin homology (PTEN), phosphatase and tensin homology 1 (PTEN) 1) PXN (pile protein), ribosomal protein S6 kinase (S6K), inositol phosphatase containing the Src homolog 2 domain (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-α receptor, amyloid-β protein 42 (Aβ42), creatine kinase-MB (CK-MB), anti-cyclic citrullinated peptide (anti-CCP), anti-thyroglobulin antibody (anti-Tg), anti-thyroid peroxidase antibody (anti-TPO), anti-streptolysin O (ASO), complement component 3 (C3).Complement component 4 (C4), D-dimer, rheumatoid factor (RF), DJ-1, leucine-rich repeat kinase 2, mutant ATP13A2, phenobarbital, phenytoin, prions, PTEN-induced putative kinase 1, alpha-fetoprotein (AFP), CA125 (MUC16), CA 15-3, CA 19-9, Cyclin I (CCNI), Cytomegalovirus (CMV), CYFRA21-1, Fibroblast Growth Factor 19 (FGF19), Gentamicin, Human Epididymal Protein 4 (HE-4), Neuron-Specific Enolase (NSE), Perinuclear Anti-Neutrophic Cytoplasmic Antibody (p-ANCA), Vitamin K Deficiency Induced Protein (PIVKA), Vitamin K Deficiency Induced Protein-II (PIVKA-II), Pro-Surfural Protein B (Pro-SFTPB), Prostate-Specific Antigen (PSA), Rubella Virus, Squamous Cell Carcinoma Antigen (SCC), Toxoplasma gondii Antibody IgG, Toxoplasma gondii Antibody IgM, β-Human Chorionic Gonadotropin (β-hCG), Botulinum Toxin, Clostridium difficile Toxin A and B, Dehydroepiandrosterone Sulfate (DHEA-S), Diphtheria Toxin, Escherichia coli Enterotoxin, Fetoglobulin-A, Follicle-Stimulating Hormone (FSH), Glycosylated Hemoglobin (HbA1c), Hemoglobin A1c, Interleukin IL-1α, influenza HA antigen, luteinizing hormone (LH), methotrexate, myeloperoxidase (MPO), neurofibromatosis protein 1 (NF-1), plasma C-peptide, placental growth factor (PlGF), Pro-GFP, prolactin, S100β, soluble Fms-like tyrosine kinase-1 (sFlt-1), testosterone, tetanus toxin, thymosin 1315, alanine aminotransferase (ALT), total bile acids, bilirubin, direct bilirubin, total bilirubin Calprotectin, deoxyuridine triphosphatase (DUTPase), lactate, lactoferrin, shiga toxin, shiga-like toxin I, shiga-like toxin II, theophylline, total protein, blood urea nitrogen, valproic acid, vitamin B12, voltage-dependent anion channel 1 (VDAC1), Wilms' tumor-1 protein, amphetamine, methamphetamine, barbiturates, benzodiazepines, benzodiazepines (serum), cannabinoids, ecstasy, ethanol, opioids, cyclohexylpiperidine (PCP), or any combination thereof.

335. The method according to 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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