WO2025137358A1

WO2025137358A1 - Devices, systems, and methods for magnetic-microparticle manipulation

Info

Publication number: WO2025137358A1
Application number: PCT/US2024/061141
Authority: WO
Inventors: Tomohiro Ikeda; Keisuke Yamaguchi; Ryotaro Chiba; Toru Yoshimura; Yousuke OMOKAWA
Original assignee: Abbott Laboratories
Current assignee: Abbott Laboratories
Priority date: 2023-12-21
Filing date: 2024-12-19
Publication date: 2025-06-26
Anticipated expiration: 2026-06-21

Abstract

The present disclosure provides assay cartridges, sliding magnet arrays, system, and methods that may be used for a range of different assays such as immunoassays, nucleic acid analysis, metabolite analysis, clinical chemistry, and complete blood cell count. The assay cartridges or systems optionally contain a sample analysis region to analyze the samples processed in the device. Assay cartridges comprise a first substrate having a first face comprising a pattern of grooves; a second substrate having a first face, wherein the first substrate is positioned in relation to the second substrate such that the first face of the first substrate faces the first face of the second substrate; and a first region and a second region, wherein the first and second regions are defined by the pattern of grooves and are located between the first and second substrates.

Description

DEVICES, SYSTEMS, AND METHODS FOR MAGNETIC-MICROPARTICLE MANIPULATION

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application No. 63/613,628, filed December 21, 2023, which application is incorporated herein by reference in its entirety.

INTRODUCTION

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

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

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

SUMMARY

The present disclosure provides assay cartridges, sliding magnet arrays, and systems that may be used for a range of different sample preparation techniques or assays such as immunoassays, nucleic acid analysis, metabolite analysis, clinical chemistry, and complete blood cell count.

Magnetic bead manipulation techniques are useful in the context of devices used for sample preparation. Existing techniques are limited, however, insofar as single-magnet and single-axis processing requires a relatively longer-size test chip because microparticle pellets can move along only one-axis (i.e., the x-axis). That is, such techniques require longer processing path lengths in the x-axis, which is not desirable in the context of low-cost chip fabrication. Further, while techniques utilizing multiple magnets on multiple-axis actuator processing could realize XY-flexible movement in a small/lower-cost test chip. However, such two-axis movement results in a larger body-size and more frequent maintenance of the processing unit/apparatus. Embodiments of the present invention address these issues with existing techniques providing a magnet-array driven by a single actuator (along the x-axis) that is nonetheless configured to generate movement of microparticles in the XY -plane without a second additional actuator. Embodiments of the present invention provide techniques for the advanced manipulation of the magnetic microparticles. Such are useful for assay processing via magnetsliding which is a sample preparation technique. Advanced microparticle manipulation of the present invention contributes to both (i) miniaturization of an assay processing unit/apparatus and (ii) realization of a low-cost test chip.

Embodiments of the present invention provide novel and useful techniques for use with, for example, sample preparation, including in the field of the bead-based biomarker assay. In embodiments, a magnet attracts magnetic microparticles locally in a manner that causes them to form a pellet. Such pellet can follow the sliding magnet on the thin solid surface which separates the magnet and microparticles. When liquid droplets align on the trajectory line of sliding magnet, the surface of microparticles can be treated sequentially with various droplet solutions by repeating events of (1) extraction of the pellet to an outside of the droplet and (2) re-dispersion into the same or another droplet.

Embodiments of the present invention contribute to techniques for (1) quick washing of the microparticle surface, and (2) utilizing small sample volumes (such as, for example, sample volumes of about 10 pL) in assays. In some cases, embodiments of the present invention may be applied in the context of nucleic acid extraction (purification/isolation of analyte) and one or more associated assays.

As described herein, embodiments of the present invention provide new techniques for realizing the advanced manipulation of magnetic microparticles by magnet sliding. In certain conventional techniques, microparticles move on one-axis by following the trajectory of a single sliding magnet during assay processing. However, in embodiments of the present invention, microparticles can be transferred among multiple magnets in a sliding magnet array via switching the dominant magnet with respect to the microparticles. As a result, the action area is expanded to not only the line along an x-axis but also along a second axis, the y-axis. Additionally, embodiments of the invention do not require any additional actuator in the second dimension (y-axis) allowing the processing unit/hardware to be kept compact.

Advantages of embodiments of the present invention include, among other things, that embodiments can realize manipulation of magnetic microparticles in two axes (XY plane) using only a single actuator system (of only X-axis). As a result, designs for smaller test chips and more compact/robust assay processing units can be achieved. In existing techniques in which microparticle pellets are moved in only a single line, such test chips become relatively long because the reagent drops must make a line along the sliding axis, which design constraints are not preferred for low-cost fabrication. In existing techniques that offer movement in two dimensions using multiple actuators, the processing unit is necessarily relatively larger and requires more frequent maintenance for multiple actuators such that cost for quality control relatively increases.

Embodiments of the present invention find use in a number of contexts. For example, embodiments find use (i) in biomarker assays using the magnetic microparticle manipulation on a solid surface by multiple magnets; (ii) as microfluidic device/apparatus using the magnetic- microparticle manipulation on a solid surface by multiple magnets; (iii) in method to manipulate magnetic microparticles via switching the dominant magnet; and (iv) in assays involving the combination of multiple-magnet processing of the present invention with detection using a nanowell array (fL-chamber, Digital assay).

As described herein, when a magnet is located in proximity to magnetic microparticles, microparticles are capable of moving along a solid substrate or film or plate (i.e., separating such magnet and microparticles) and to be drawn along, following the attraction the magnetic field of the magnet. As described herein, embodiments of the present invention are configured such that microparticles can: (i) escape out from a droplet into air (i.e., an air gap surrounding a liquid droplet); (ii) pass the air-gap between adjacent droplets by sliding along the surface of a substrate; and (iii) entering into a new droplet. Such cycles can be repeated in a sequential microparticle-surface treatment using a droplet array, which embodiments can be used in connection with processing rapid sample preparation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates isometric views of systems according to another embodiment.

FIG. 2 illustrates side views of assay cartridges according to another embodiment and certain techniques for locating liquid droplets within assay cartridges.

FIG. 3 illustrates top, side and isometric views of an assay cartridge according to another embodiment and potential trajectories of microparticle pellets.

FIG. 4 illustrates top and side views according to another embodiment as well as the implications of configurations of boundaries of liquid droplets on microparticle pellet movement.

FIG. 5 illustrates top views of a system according to another embodiment and demonstrates the movement of microparticle pellets between liquid droplets.

FIG. 6 illustrates top views of sliding magnet arrays according to other embodiments. FIG. 7 illustrates side and top views of a system according to another embodiment and demonstrates the movement of microparticles pellets along two axes.

FIG. 8 illustrates top views of aspects of a system according to one embodiment and further illustrates the movement of microparticle pellets within and among liquid droplets.

FIG. 9 illustrates top views of aspects of a system according to one embodiment and further illustrates the movement of microparticle pellets within and among liquid droplets.

FIGS. 10A-B illustrates top views of systems according to another embodiment.

FIGS. 11A-B illustrates top views of systems according to another embodiment and show the arrangement of a plurality of regions in rows and columns defined by a pattern of grooves and comprising liquid droplets.

In the figures, elements having the same or similar reference numerals have the same or similar features, unless explicitly stated otherwise.

DETAILED DESCRIPTION OF THE INVENTION

Assay cartridges are disclosed. Assay cartridges optionally contain a sample analysis region to analyze samples processed in the assay cartridge. Also provided are sliding magnet arrays. Also provided are systems for moving microparticles in an assay cartridge. Also provided herein are exemplary methods for using the assay cartridge, sliding magnet array, and systems.

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

In instances, one or more samples may be obtained from a single subject. In other instances, a plurality of samples may be obtained from a plurality of subjects. Samples may comprise any convenient substance associated with any number of types of subjects, including subjects that are human, as described herein.

In some instances, the sample is a biological sample. The term “biological sample” is used in its conventional sense to refer to a whole organism, plant, fungi or a subset of animal tissues, cells or component parts which may in certain instances be found in blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen. As such, a “biological sample” refers to both the native organism or a subset of its tissues as well as to a homogenate, lysate or extract prepared from the organism or a subset of its tissues, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, sections of the skin, respiratory, gastrointestinal, cardiovascular, and genitourinary tracts, tears, saliva, milk, blood cells, tumors or organs. Biological samples may be any type of organismic tissue, including both healthy and diseased tissue (e.g., cancerous, malignant, necrotic, etc.). In certain embodiments, the biological sample is a liquid sample, such as blood or derivative thereof, e.g., plasma, tears, urine, semen, etc., where in some instances the sample is a blood sample, including whole blood, such as blood obtained from venipuncture or fingerstick (where the blood may or may not be combined with any reagents prior to assay, such as preservatives, anticoagulants, etc.).

In certain embodiments the source of the sample is a “mammal” or “mammalian,” where these terms are used broadly to describe organisms which are within the class mammalia, including the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees and monkeys). In some instances, the subjects are humans. Embodiments of the present invention may be applied to samples obtained from human subjects of both genders and at any stage of development (i.e., neonates, infant, juvenile, adolescent, adult), where in certain embodiments the human subject is a juvenile, adolescent, or adult. While the present invention may be applied to samples from a human subject, it is to be understood that the methods may also be carried-out on samples from other animal subjects (that is, in “non-human subjects”) such as, but not limited to, birds, mice, rats, dogs, cats, livestock and horses.

Definitions:

Before the present invention is described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. The modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context. When used in the context of a range, the modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the range of from about “2 to about 10” also discloses the range “from 2 to 10.” The term “about” may refer to plus or minus 10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9 to 1.1.

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

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

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

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

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

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

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

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

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

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

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

“Label” or “detectable label” as used interchangeably herein refers to a moiety attached to a specific binding member or analyte to render the reaction between the specific binding member and the analyte detectable, and the specific binding member or analyte so labeled is referred to as “detectably labeled.” A label can produce a signal that is detectable by visual or instrumental means. Various labels include: (i) a tag attached to a specific binding member or analyte by a cleavable linker; or (ii) signal-producing substance, such as chromagens, fluorescent compounds, enzymes, chemiluminescent compounds, radioactive compounds, and the like. Representative examples of labels include moieties that produce light, e.g., acridinium compounds, and moieties that produce fluorescence, e.g., fluorescein. Other labels are described herein. In this regard, the moiety, itself, may not be detectable but may become detectable upon reaction with yet another moiety. Use of the term “detectably labeled” is intended to encompass such labeling. “Specific binding partner” or “specific binding member” as used interchangeably herein refer to one of two different molecules that specifically recognizes the other molecule compared to substantially less recognition of other molecules. The one of two different molecules has an area on the surface or in a cavity, which specifically binds to and is thereby defined as complementary with a particular spatial and polar organization of the other molecule. The molecules may be members of a specific binding pair. For example, a specific binding member may include, but not limited to, a protein, such as a receptor, an enzyme, an antibody and an aptamer, a peptide, a nucleotide, oligonucleotide, a nucleic acid, a polynucleotide and combinations thereof.

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

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

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

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

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

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

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

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

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

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

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention.

All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

While the apparatus and method has or will be described for the sake of grammatical fluidity with functional explanations, it is to be expressly understood that the claims, unless expressly formulated under 35 U.S.C. § 112, are not to be construed as necessarily limited in any way by the construction of "means" or "steps" limitations, but are to be accorded the full scope of the meaning and equivalents of the definition provided by the claims under the judicial doctrine of equivalents, and in the case where the claims are expressly formulated under 35 U.S.C. § 112 are to be accorded full statutory equivalents under 35 U.S.C. § 112.

In further describing various aspects of the invention, assay cartridges and components thereof are described first in greater detail. Following this, sliding magnet arrays and components thereof are described. Following this, moving microparticles in an assay cartridge are described. Following this a review of methods of using the assay cartridges, sliding magnet arrays and systems for practicing the subject methods are described.

Assay Cartridge:

Assay cartridges are provided. Aspects of assay cartridges include: a first substrate having a first face comprising a pattern of grooves; a second substrate having a first face, wherein the first substrate is positioned in relation to the second substate such that the first face of the first substrate faces the first face of the second substrate; and a first region and a second region, wherein the first and second regions are defined by the pattern of grooves and are located between the first and second substrates.

In embodiments, the first and second substrates are arranged such that the first face of the first substrate is directly opposed to the first face of the second substrate. In other embodiments, the first and second substrates are spaced apart. For example, some embodiments further comprise an air gap between the first and second substrates. In some cases, a spacer is present between the first and second substrates. In embodiments, the first substrate is a lid, and the second substrate is a bottom-sheet.

In embodiments, the first region comprises a first opening in the first substrate. In such cases, the first opening may be configured for loading fluid into the first region. In other embodiments, the second region comprises a second opening in the first substrate. In such cases, the second opening is configured for loading fluid to the second region.

In embodiments, the pattern of grooves is configured to retain fluid between the first and second substrates within an area bounded by the pattern of grooves. In some cases, the pattern of grooves is configured such that fluid is not retained outside an area bounded by the pattern of grooves. In other cases, the pattern of grooves is configured such that fluid is excluded from an area bounded by the pattern of grooves. In embodiments, the first region and the second region comprise straight edges and angled comers. In some cases, the angled corners are configured to allow a microparticle (as used in this detailed description of embodiments, unless indicated otherwise or when such is clear from the context, microparticle may refer to microparticle or microbead or microparticle pellet or assigning beads or pellet of assisting beads or combinations thereof) to escape from liquid present at the angled comer. In some cases, the straight edges are configured to prevent a microparticle from escaping from liquid present at the angled corner. In certain cases, the angled corners comprise one or more: 90-degree angled corners. In other cases, the angled corners comprise one or more: acute-angled corners, 90-degree angled corners or obtuse-angled comers. In embodiments, the pattern of groves comprises a plurality of groves with width of 0.3 mm to 1.0 mm. In embodiments, the pattern of groves is configured such that liquid droplets (i.e., first and second regions, when loaded with fluid) are separated by about 0.1 mm to about 2.0 mm, such as about 0.1 mm or about 0.2 mm or about 0.3 mm or about 0.4 mm or about 0.5 mm or about 0.6 mm or about 0.7 mm or about 0.8 mm or about 0.9 mm or about 1.0 mm or about 1.1 mm or about 1.2 mm or about 1.3 mm or about 1.4 mm or about 1.5 mm or about 1.6 mm or about 1.7 mm or about 1.8 mm or about 1.9 mm or about 2.0 mm or more.

Some embodiments further comprise a third region, a fourth region, a fifth region, a sixth region, a seventh region, an eighth region, a ninth region, and a tenth region. In such cases, the third through tenth regions may be defined by the pattern of grooves.

In embodiments, one or more of the regions comprise hydrophilic patterning. In other embodiments, one or more of the regions comprise a hydrophilic coating on the first face of the first substrate. In certain embodiments, the first face of the first substrate comprises a hydrophobic coating on the first face of the first substrate. In some cases, one or more of the regions comprise a hydrophobic coating on the first face of the first substrate. In embodiments, the pattern of grooves comprises a pattern of angled rectangles in a row. In some embodiments, an angled comer of the first region is coaxial with an angled corner of the second region. In other embodiments, the pattern of grooves comprises a pattern of angled rectangles in rows and columns and an angled comer of the first region is coaxial with an angled comer of the second region.

In embodiments, one or more of the regions comprise a fluid. In other embodiments, one or more of the regions comprise microparticles. In such cases, the microparticles may comprise a material that is attracted to a magnet. In such cases, the first and second substrates may comprise a material that is not attracted to a magnet.

In embodiments, each region is the same size. In other embodiments, one or more of the regions are different sizes.

Embodiments of the present invention may further comprise a sample analysis region.

As described herein, embodiments of assay cartridges (the device) comprise a first (top) substrate bound to a second (bottom) substrate where a pattern of grooves on the top substrate bound to the bottom substrate define first and second regions. Each region contains an opening in the top substrate. In some embodiments, space between the regions does not contain an opening in the top substrate. A sample processing region is formed between the top and the bottom substrate, i.e., within and between liquid droplets present between the top and bottom substrates. The sample processing region is unbounded throughout the region (i.e., the interior of the device).

In embodiments, first and second regions may comprise a sample processing region in addition to an optional sample analysis region. The sample or components of the sample are moved from one region to another region using microparticles. In general, a sample is added to a first region, e.g., a first region in the sample processing region. Microparticles are added at the same time, before, or after the sample is added to the same first and/or second region that the sample is added to or will be added to. In some embodiments, the microparticles are already present in the first and/or second regions to which the sample is added. In some embodiments, the microparticles are microparticles and assisting particles. The sample or component of the sample is then bound to the microparticles and the microparticles or microparticles and assisting particles are moved using a magnetic field to a different region. After sample processing is complete, the microparticles or microparticles and assisting particles are moved to a sample analysis region on the device or off the device.

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

In some embodiments, all or a portion of the first (also referred to herein as 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 polypropylene (PP). In some embodiments, all or a portion of the top substrate is made of polyurethane (PU). In some embodiments, all or a portion of the top substrate is made of polytetrafluoroethylene (PTFE). In some embodiments, all or a portion of the top substrate is made of polyvinylchloride (PVC). In some embodiments, all or a portion of the top substrate is made of poly dimethylsiloxane (PDMS). In some embodiments, all or a portion of the top substrate is made of acrylonitrile butadiene styrene (ABS). In some embodiments, all or a portion of the top substrate is made of poly(lactic acid) (PLA). In some embodiments, all or a portion of the top substrate is made of thermoplastic PU. In some embodiments, all or a portion of the top substrate is made of clear resin. In some embodiments, all or a portion of the top substrate is made of polyethylene glycol diacrylate (PEGDA).

In some embodiments, all or a portion of the second (also referred to herein as 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 polypropylene (PP). In some embodiments, all or a portion of the bottom substrate is made of polyurethane (PU). In some embodiments, all or a portion of the bottom substrate is made of polytetrafluoroethylene (PTFE). In some embodiments, all or a portion of the bottom substrate is made of polyvinylchloride (PVC). In some embodiments, all or a portion of the bottom substrate is made of polydimethylsiloxane (PDMS). In some embodiments, all or a portion of the bottom substrate is made of acrylonitrile butadiene styrene (ABS). In some embodiments, all or a portion of the bottom substrate is made of poly(lactic acid) (PLA). In some embodiments, all or a portion of the bottom substrate is made of thermoplastic PU. In some embodiments, all or a portion of the bottom substrate is made of clear resin. In some embodiments, all or a portion of the bottom substrate is made of polyethylene glycol diacrylate (PEGDA).

In embodiments of the assay cartridge (the device), the first and second regions defined by the pattern of grooves, liquid droplets present therein, and, when the device is loaded with liquid droplets into each of the first and second regions, space, i.e., an air gap, between the first and second regions, together, comprise a sample processing region. Generally, the space between first and second regions (when such first and second regions are loaded with liquid droplets, as described herein) comprises air and serve as, for example, a zone, such as a hydrophobic zone, that assists in the holding of liquid droplets that in some cases include the sample or reagents in the first and second regions.

The sample processing region of the devices disclosed herein comprise a plurality of regions, including the first and second regions, and have an arrangement of such regions. There is a range in the number of regions that is dependent on the particular embodiment being described. The arrangement of such regions is also variable and dependent on the particular embodiment discussed. The device comprises a range in the number of regions. For example, the device may comprise about 2 or more, about 3 or more, about 4 or more, about 5 or more, about 6 or more, about 7 or more, about 8 or more, about 9 or more, about 10 or more, about 11 or more, about 12 or more, about 13 or more, about 14 or more, about 15 or more, about 16 or more, about 17 or more, about 18 or more, about 19 or more, or about 20 or more. Such regions are defined by the pattern of grooves present on the first substrate, as described herein. Each adjacent region, when such are loaded with liquid droplets, is separated by an air gap.

The plurality of regions, including the first and second regions, may be arranged in a number of different patterns. For instance, the pattern may be a grid or lines. In some cases, the regions may be arranged in rows and columns. In some cases, the regions comprise angled comers, which may optionally be arranged such that vertices of angled corners align. When the pattern is a grid, the grid may be 2x4, 2x5, 2x6, 2x7, 2x8, 2x9, or 2x10 where the first number indicates the number of columns, and the second number indicates the number of rows. When the pattern is a line, the line may be 1x3, 1x4, 1x5, 1x6, 1x7, 1x8, 1x9, or 1x10.

The assay cartridges, sliding magnet arrays, systems, and methods disclosed herein may be used for a number of different assay types and the assays may be performed in different ways. The different types of assays and the different ways that the assays are performed are based on, among other things, how the first and second or more regions of the assay cartridge are configured (e.g., the reagents contained in each region). The assays are conducted using a sample path where the microparticles are moved within one region (i.e., within one liquid droplet of a region) and/or from one region (i.e., from one liquid droplet of a region to another liquid droplet of another region) to another using one or more magnetic fields (i.e., as present on the sliding magnet array). In some embodiments, the microparticles are microparticles and assisting particles. The different types of assays include, without limitation, immunoassays, nucleic acid analysis, metabolite analysis, clinical chemistry, complete blood count (CBC), etc.

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

In some embodiments, the assay cartridges, sliding magnet arrays, systems, and methods may be used to perform nucleic acid analysis. The assay cartridges, sliding magnet arrays, systems, and methods may employ various forms of nucleic acid analysis to detect analytes of interest, e.g., a nucleic acid, a non-nucleic acid containing a nucleic acid tag, or a nucleic acid produced from the analyte, including, without limitation, PCR, isothermal amplification, etc.

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

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

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

In order to perform different types of assays or perform the assays in different ways, the plurality of regions of the assay cartridge may be filled with reagents. The reagents may be added to the plurality of regions manually by the user, robotically by a system employing the assay cartridges, sliding magnet arrays, systems, and methods, or through the use of a reagent delivery device.

The first and second or more regions of the present disclosure are capable of holding a range of different volumes of fluid while retaining the volume within the boundary or boundaries defined by the pattern of grooves without spilling into air gaps between regions or beyond. The assays used with the assay cartridge, sliding magnet array, system, and methods are conducted using a sample path where the microparticles or microparticles and assisting particles are moved with a region or from one region to another region using a magnetic field. Exemplary sample paths are disclosed in FIGS. 8-10 using different embodiments of the device.

As described above and throughout, microparticles or microparticles and assisting particles can be moved with a region comprising a liquid droplet or among regions comprising liquid droplets. In some embodiments, the microparticles include (i) a plurality of microparticles, and (ii) a plurality of assisting particles.

As embodied herein, moving the microparticles or microparticles and assisting particles across regions of the assay cartridge includes moving a sliding magnet array across the assay cartridge. The magnet array can be moved along the assay cartridge in any suitable configuration. For example and not limitation, the magnet array can be located below the assay cartridge.

The position of the magnet relative to the assay cartridge can be selected based on the desired strength of the magnetic field to be applied to the microparticles or microparticles and assisting particles to move the microparticles or microparticles and assisting particles within the assay cartridge. For purpose of example, and as described further herein, the distance between the magnet and the microparticles or microparticles and assisting particles can be selected based on the desired strength of the magnetic field to be applied to the microparticles or microparticles and assisting particles. For purpose of example and as embodied herein, the magnet can be moved along the regions of the assay cartridge at a magnet distance defined between a bottom surface of the assay cartridge and the sliding magnet array. For example and not limitation, the magnet distance can be between about 0mm and about 10mm. Additionally or alternatively, and as embodied herein, the magnet can be moved along the assay cartridge with the sliding magnet array in contact with a bottom surface of the assay cartridge.

As further embodied herein, the shape and orientation of the sliding magnet array and magnets thereof can also be selected to achieve the desired magnetic field. For example and not limitation, the sliding magnet array or magnets thereof can be angled relative to the bottom surface of the assay cartridge as the magnet is moved along the bottom surface of the assay cartridge.

Additionally or alternatively, the sliding magnet array or magnets thereof can be moved along the assay cartridge to achieve desired movement and seeding of microparticles or microparticles and assisting particles in the assay cartridge. For purpose of example and as embodied herein, the sliding magnet array can be moved in a direction parallel to the bottom surface of the assay cartridge. Additionally or alternatively, the sliding magnet array can be moved along the assay cartridge at any suitable speed to achieve desired movement and seeding of microparticles in the sliding magnet array. For example, the speed of the magnet can be selected to achieve desired process times and microparticle loss during movement of the microparticles or microparticles and assisting particles across the assay cartridge. For purpose of example and not limitation, sliding magnet array can be moved in a direction parallel to a bottom surface of the assay cartridge at a speed of between about 0.3 mm/s and about 10 mm/s. Additionally or alternatively, sliding magnet array can be moved in a direction parallel to a bottom surface of the assay cartridge at a speed of between about 0.3 mm/s and about 6 mm/s. Additionally or alternatively, sliding magnet array can be moved in a direction parallel to a bottom surface of the assay cartridge at a speed of between about 2 mm/s and about 6 mm/s. Additionally or alternatively, sliding magnet array can be moved in a direction parallel to a bottom surface of the assay cartridge at a speed of between about 4 mm/s and about 6 mm/s. Although reference is made to the sliding magnet array moving along the assay cartridge, relative motion between the sliding magnet array and the assay cartridge can additionally or alternatively be achieved by moving the assay cartridge relative to the sliding magnet array.

Additionally or alternatively, the type and shape of one or more magnets of the sliding magnet array can be selected to provide the desired magnetic field. Sliding magnet array can comprise a permanent magnet or an electromagnet. Any suitable magnet shape can be selected. For example and not limitation, the magnet can define a corner. Additionally or alternatively, and as embodied herein, the corner of a magnet can be in contact with the bottom surface of the assay cartridge as the sliding magnet array moves along the assay cartridge. For purpose of example and not limitation, one or more magnets of the sliding magnet array can have a cylindrical, triangular, square, spherical, or other suitable shape. Additionally or alternatively, and as embodied herein, a magnet of the sliding magnet array can have a rectangular shape.

The assay cartridges, sliding magnet arrays, systems, and methods of the present disclosure provide certain benefits over devices known in the art. The benefits of the devices of the present disclosure include the ability to translate microparticles or microparticle pellets in two dimensions using an actuator configured to move in only a single dimension as well as high levels of modularity, the ability to use small volumes, and provide the ability to analyze multiple sample types simultaneously. In terms of the modularity, the multiple exemplary embodiments of the devices disclosed herein provide high levels of configurability through the usage of multiple regions for sample or reagent addition or for hydrophobic separation (e.g., through the presence of air) or for merging to create large sample or reagent zones, many different types of sample analysis regions configured to analyze proteins or antigens (e.g., immunoassays and clinical chemistry), nucleic acids (e.g., nucleic acid analysis), metabolites (e.g., clinical chemistry), or cells or cells types (e.g., immunoassays, clinical chemistry, nucleic acids, or CBC). The two or more regions can be configured by the user to fit any type of assay or the particular steps of a given assay. The ability to move microparticles or microparticle pellets in two dimensions using a single actuator (i.e., actuator on a single dimension) allows for more compact and cost effective designs. The ability to perform these assays using small volumes allows for reduced costs of reagents and reduced consumption of sample which allows for the conservation of difficult to obtain samples. The modularity of the device also allows assays to be customized to the user’s preference and also allows multiple assays (e.g., immunoassays and nucleic acid analysis, or any combination of the assays disclosed above) to be performed simultaneously or sequentially on the same device for the same sample or multiple samples contained on a single device.

Sliding Magnet Array:

Sliding magnet arrays are provided. Aspects of sliding magnet arrays include: a first magnet; and a second magnet, wherein the first magnet and the second magnet are separated from each other at a diagonal orientation, and the separation of the first magnet and the second magnet creates a border between the first and second magnets having a first side and a second side.

In embodiments, the border is defined by a line that is equidistant between the first and second magnets. In some embodiments, the first and second magnets are of substantially the same magnetic strength. In some embodiments, the first and second magnets comprise magnetic fields of substantially the same magnitude. In certain cases, on the first side of the border, the first magnet creates a first dominate magnetic field, and on the second side of the border, the second magnet creates a second dominate magnetic field.

In embodiments, the first magnet and the second magnet are configured to translate. In some embodiments, the first magnet and the second magnet are configured to translate in a single dimension. In other embodiments, the first magnet and the second magnet are configured to translate using a single actuator. In certain embodiments, the single actuator is configured to translate the first and second magnets in a single dimension. In still other embodiments, the first and second magnets are physically connected.

Some embodiments further comprise a third magnet that is separated from the first and second magnets. In some such cases, the separation of the first magnet, the second magnet and the third magnet creates borders defining the first side, the second side and a third side. In embodiments, on the third side of the border, the third magnet creates and third dominate magnetic field. In embodiments, the first magnet, the second magnet and the third magnet are configured to translate. In some embodiments, the first magnet, the second magnet and the third magnet are configured to translate in a single dimension. In other embodiments, the first magnet, the second magnet, and the third magnet are configured to translate using a single actuator. In still other embodiments, the single actuator is configured to translate the first, second and third magnets in a single dimension.

In embodiments, the first magnet, the second magnet, and the third magnet are physically connected.

Systems for moving microparticles in an assay cartridge:

Systems for moving microparticles in an assay cartridge are provided. Aspects of systems for moving microparticles in an assay cartridge include: an assay cartridge of the present invention; and a sliding magnet array of the present invention, wherein the first region and the second region comprise two or more straight edges and two or more angled comers, the second substrate having a second face that does not face the first face of the first substrate, and the sliding magnet array having a first face positioned in relation to the second substrate such that the first face of the sliding magnet array faces the second face of the second substrate.

In embodiments, the assay cartridge and the sliding magnet array are positioned such that the sliding magnet array can mechanically influence magnetic particles present in the assay cartridge. In some embodiments, the first region comprises microparticles. In certain embodiments, the system is configured such that the two or more straight edges of the first region prevent the sliding magnet array from moving the microparticles from the first region to the second region. In some cases, the sliding magnet array is able to move the microparticles from the first region to the second region at the two or more angled corners. Some systems of the present invention further comprise a first liquid present in the first region and a second liquid present in a second region.

Further details regarding aspects of embodiments of systems, assay cartridges and sliding magnet arrays of the present invention, as well as methods of using such aspects in connection with, for example, nucleic acid processing, are found in: Berry SM, Pezzi HM, LaVanway AJ, Guckenberger DJ, Anderson MA, Beebe DJ. AirJump: Using Interfaces to Instantly Perform Simultaneous Extractions. ACS Appl Mater Interfaces. 2016 Jun 22;8(24): 15040-5. doi: 10.1021/acsami.6b02555. Epub 2016 Jun 10. PMID: 27249333; PMCID: PMC5058634, as well as “Parallel RNA extraction using magnetic beads and a droplet array” Xu Shi, et al. Lab Chip, 2015,15, 1059-1065; https://doi.org/10.1039/C4LC01111B, the disclosures of which are incorporated herein by reference. Exemplary Embodiments of Assay Cartridges, Sliding Magnetic Arrays and Systems:

As described herein, embodiments of the present invention are configured for use in connection with sample preparation techniques. For example, embodiments are configured for use in connection with bead-based biomarker assays. In some cases, embodiments are configured for sample preparation for bead-based biomarker assays. Further, as described herein, embodiments of the present invention are configured for use in connection with beadbased manipulation techniques. Specifically, embodiments are configured for use with magnetic bead-based manipulation techniques. That is, embodiments of the invention utilize magnetic forces to mechanically influence microparticles. Microparticles capable of being attracted to a magnet may be influenced by the presence of a magnet to aggregate together to form a microparticle pellet. Embodiments of the present invention may further manipulate microparticles by utilizing the movement of a magnet to move a micro pellet. Embodiments of the present invention comprise utilizing the movement of a plurality of magnets to draw a micro pellet through a plurality of regions. In embodiments, regions may comprise one or more fluids and/or one or more reagents relevant to sample preparation.

FIG. 1 depicts a schematic of aspects of an embodiment of a system the present invention 100. Aspects of system 100 are illustrated, in part, in contrast with a schematic of aspects of a system according to a conventional technique 199. Aspects of system 100 are configured to enable microparticle pellet 180 to travel in a first dimension 151 consistent with trajectory 151 of motion of magnet array 150. Similarly, aspects of a conventional system 199 are configured to enable microparticle pellet 180 to travel in a first dimension 151 consistent with trajectory 151 of motion of magnet 198.

System 100 comprises magnet array 150 comprising two individual magnets 150a, 150b connected together by a common substrate such that when magnet array 150 is translated 151, magnets 150a, 150b of magnet array 150 remain at a fixed distance from each other. Magnets 150a, 150b are oriented at a diagonal relative to the trajectory 151 of magnet array 150; i.e., a diagonal relative to the x-axis. In such diagonal orientation, magnet 150b leads magnet 150a as magnet array 150 is moved along trajectory 151 (i.e., from left to right). Magnet array 150 is positioned such that it faces a second surface (not shown) of second substrate 110; i.e., magnet array 150 is located beneath second substrate 110. In other words, magnet array 150 is positioned on the opposite side of second substrate 110 than microparticle pellet 180.

System 100 is further configured such that microparticle pellet 180 can be moved in first dimension 151 as well as second dimension 152; i.e., both along the x-axis and along the y-axis. As described herein, system 100 is configured to translate microparticle pellet 180 in two dimensions 151, 152 notwithstanding that magnet array 150 is configured to move in only a single dimension 151. That is, system 100 is capable of utilizing a displacement protocol, e.g., a translation stage, an actuator or a motor, of conventional system 199, i.e., that moves in only a single axis.

System 100 provides a new methodology to realize the advanced manipulation of magnetic microparticles (uPs) (as seen in microparticle pellet 180) by magnet-sliding. In the conventional way seen in system 199, magnetic microparticle 180 moves on one-axis 151, just following trajectory 151 of the sliding magnet 198 during assay processing. In contrast, in system 100, microparticle pellet 180 can be transferred among multiple-magnets-in-array (i.e., magnets 150a, 150b of magnet array 150) via switching the dominant magnet, as described herein. The action area is expanded to not only the line on X-axis (magnet- sliding) 151 but also second axis 152, the Y-axis. Additionally, system 100 does not require any additional actuator. Instead, it can utilize an actuator for use with conventional system 199 and thereby can keep the processing unit/hardware compact.

FIGS. 2 depict aspects of embodiments of assay cartridges of the present invention.

As described below, assay cartridge 204 in FIG. 2 is a hybrid approach referred to as an “air-wall droplet” type or technique. In assay 204, 204 have no physical wall to block the passage of microparticle droplets, 204 enable liquid droplets to take a large footprint (i.e., a small amount of fluid may correspond to a large area of liquid droplets on first face 220a of second substrate 220) and such a relatively reduced exposed surface helps mitigate the effects of evaporation; i.e., helps prevent liquid droplets 290 from evaporating.

FIG. 2 depicts assay cartridge 204, in each case according to an embodiment of the present invention. Assay cartridge 204 includes first substrate 220 in which pattern of grooves 221 comprises a hydrophilic coating 221. Such hydrophilic coating 221 in assay cartridge 204 similarly fixes liquid droplets 290 in place by attracting liquid droplets 290 to specific, fixed locations between first and second substrates 220, 220. Alternatively, assay cartridge 204 may be configured such that areas of first face 220a of first substrate 220 outside pattern of grooves 221 also include a hydrophobic coating, such that liquid droplets 290 are urged to remain in fixed locations by discouraging their movement into regions of first face 220a of first substrate outside pattern of grooves 221. Alternatively, each groove of pattern of grooves 221 in first face 220a of first substrate of assay cartridge 204 may include either a hydrophilic or hydrophobic coating and such may differ as desired, depending on the characteristics of the liquid droplet 290 to be held in place in different areas of the pattern of grooves 221.

In assay cartridge 204, liquid droplets 290 are urged to remain, i.e., “held,” in fixed positions relative to second substrate 220 such that microparticle pellets (not shown) can be translated through each liquid droplet 290. Further, by “holding” liquid droplets 290 in place, the location and shape of liquid droplets 290 are more easily retained as microparticle pellets are moved into, out of, or within each liquid droplet 290. As described herein, certain borders of liquid droplets 290 are configured, e.g., shaped, to allow a microparticle pellet to move through such that microparticle pellet can exit the droplet, i.e., move from inside the droplet to outside the droplet. Other borders of liquid droplets 290 are configured, e.g., shaped, to inhibit or prevent microparticle pellet from moving through or exiting the droplet. In cases, a microparticle pellet may be inhibited from exiting a droplet due in part to surface tension effects related to the shape of the border of the liquid droplet, as described herein.

As described, assay cartridge 204 are a hybrid approach, in which (i) assay cartridges 204 have no physical wall to block the passage of microparticle, and (ii) assay cartridge 204 enable liquid droplets to take a large footprint (i.e., a small amount of fluid may correspond to a large area of liquid droplets on first face 220a of second substrate 220) and such a relatively reduced exposed surface helps mitigate the effects of evaporation; i.e., helps prevent liquid droplets 290 from evaporating.

These aspects of assay cartridge, 204 are illustrated in FIG. 3. Assay cartridge 300 is an embodiment of an assay cartridge according to the present invention that utilizes an “air-wall droplet” type or technique discussed above in connection with assay cartridge 204. FIG. 4 provides top, side and isometric views of assay cartridge 300 with a plurality of liquid droplets 390 present on first face 320a of second substrate 320; i.e., between first face 320a of second substrate 320 and first face 330a of first substrate 330. Microparticle pellet 380 is present within a liquid droplet 390. Because assay cartridge 300 does not include spacers, microparticle pellet is able to be moved along a plurality of different trajectories into different liquid droplets 390 present on assay cartridge 300. That is, the routes available for microparticle pellet 380 to translate among liquid droplets 390 is not constrained by, for example, a spacer configured to fix liquid droplets in place. Since assay cartridge 300 comprises a pattern of grooves (not shown) for fixing liquid droplets in place, no such spacer need be present in assay cartridge 300. The side view of assay cartridge illustrates, by contrast with a “full-open” type or technique (as in assay cartridge 301), how liquid droplet 390 in assay cartridge 300 is held between first and second substrates 320, 330 such that liquid droplet comprises a larger footprint (i.e., takes up a larger area on first face 320a of second substrate 320), which enables flexibility in designing assay cartridge 300 and, since liquid droplet is covered by first substrate 330 (i.e., first substrate 330 acts as a lid over the top of liquid droplet 390), evaporation of liquid droplet 390 are mitigated in assay cartridge 300.

FIG. 4 presents a schematic view of an embodiment of a system 400 according to the present invention to illustrate the movement, or inhibition of movement, of a microparticle pellet. That is, system 400 comprises two different illustrative configurations of liquid droplet 490, as described below. Microparticle pellet 480 is present within liquid droplet 490.

Liquid droplet 490 is present in system 400 between first and second substrates 420, 430. Liquid droplet 490 is fixed in place in system 400, relative to second substrate 420, by a pattern of grooves (not shown) on first face 430a of first substrate 430. Magnet 450 is located in system 400 opposite second face 420b of second substrate 450 (i.e., opposite second substrate 420 from first substrate 430). Magnet 450 may be a magnet array, for example. Magnet is configured to translate along trajectory 451. Magnet 450 may be caused to translate along trajectory 451 based on any convenient displacement protocol, such as an actuator or the like ( not shown). Microparticle pellet 480 is influenced by magnet 450; i.e., influence of magnet 450 causes microparticle pellets to aggregate into microparticle pellet 450, and, further, microparticle pellet 450 is attracted to magnet 450. System 400 is configured such that movement of magnet 450 along trajectory 451 causes microparticle pellet 480 to be urged to follow the movement of magnet 450 along trajectory 541.

An interface between the border of liquid droplet 490 and air (i.e., external to liquid droplet 490) is referred to as wall 491 or wall interface. Wall interface 491 is defined by the pattern of groves present on first substrate 430. That is, the shape of liquid droplet, including its borders, such as wall 491, are defined by the pattern of grooves present on first substrate 430. Walls of liquid droplets 490 can take the form of angled corner walls or flat walls. Both such configurations appear substantially the same when viewed from the side view. However, the distinction between such configurations is seen when liquid droplet 490 of system 400 is viewed from a top view. The pattern of grooves (not shown) of first substrate 430 may be shaped such that liquid droplet 490a comprises angled corner wall 491a, as seen in the top view of system 400. Alternatively, the pattern of grooves (not shown) of first substrate 430 may be shaped such that liquid droplet 490b comprises flat wall 491b, as seen in the top view of system 400.

Angled comer wall 491a of liquid droplet 490a is shaped such that as magnet 450 is translated along trajectory 451, microparticle pellet 480 follows magnet 450, and, further, microparticle pellet 480 is able to exit liquid droplet 490a at angled corner wall 491a. That is, internal forces, e.g., surface tension forces, of liquid droplet 490a do not inhibit microparticle pellet 480 passing through angled comer wall 491a.

In contrast, flat wall 491b of liquid droplet 490b is shaped such that as magnet 450 is translated along trajectory 451, microparticle pellet 480 follows magnet 450 while microparticle pellet 450 is inside liquid droplet 490b but does not, i.e., cannot, follow magnet 450 outside of liquid droplet 490b. That is, while magnet 450 is able to translate along trajectory 451 beneath second substrate 420, microparticle pellet 480 is not able to exit liquid droplet 490b at flat wall 491b. In such configuration, internal forces, e.g., surface tension forces, of liquid droplet 490b inhibit microparticle pellet 480 from passing through flat wall 491b. In such embodiments, microparticle pellet 480 remains in a fixed position at flat wall 491b while magnet 450 continues to move along trajectory 451. In system 450, angled comer wall 491a allows microparticle pellet 480 to exit or to be pulled out of liquid droplet 490 by magnet 440 (i.e., “pullout”), whereas flat wall 491b prevents microparticle pellet 480 from exiting or being pulled out of liquid droplet 490 by magnet 440 (i.e., “stop”).

In system 400, magnet 450 attracts magnetic microparticles (uPs) and make microparticle pellet 480. Also, in system 400, microparticle pellet 480 chases the moving/sliding of magnet 440 by magnetic attraction, as magnet 450 is moved along trajectory 451. However, such moving/sliding of microparticle pellet 480 can be limited in droplet 490. For example, in cases where the number of the loaded microparticles of microparticle pellet 480 is sufficient to overcome the surface tension of wall 491 of liquid droplet 490, microparticle pellet 480 can be moved outside of droplet 490 (i.e., “magnetic pull-out” can occur). Under such circumstances, microparticle pellets continue to chase magnet 450 as it is moved along trajectory 451 (as seen with respect to droplet 490a and wall 491a). On the other hand, if the amount of the loaded microparticles of microparticle pellet 480 is not sufficient, the chasing microparticle pellet 480 is forced to stop at wall/interface 491 between inside and outside of droplet 490 (as seen with respect to droplet 490b and wall 491b). Additionally, it is known that flat wall 491b can stop the chasing more easily than a wall with a cornered top (i.e., angled comer wall 491a), when the number of loaded microparticles of microparticle pellet 480 is optimal. As described here, in system 400, such (i) “stop” or (ii) “pullout” conditions may be effectively used for (i) switching the dominant magnet when magnet 450 comprises a magnet array with two or more magnets, and (ii) transferring microparticle pellet 480 to another liquid droplet present in system 400.

FIG. 5 presents pictures and schematics of system 500 according an embodiment of the present invention and further shows, in steps 1 through 4, the operation of transferring microparticle pellet from first liquid droplet 590a to second liquid droplet 590b via angled comer wall 591a of first liquid droplet 590a. Top views of system 500 are shown with different positions of magnet 550 and microparticle pellet 580.

In system 500, pattern of grooves 570 present on first substrate (not identified in figure because first substrate is otherwise transparent and the figure shows system 500 from a top view). Pattern of grooves 535 defines boundaries of first and second liquid droplets 590a, 590b with liquid present within liquid droplets 590a, 590b, and air present between them. Microparticle pellet 580 is present in system 500. First substrate comprises openings 533. Openings 533 are configured such that fluid can be loaded within pattern of grooves 535 to form first and second liquid droplets 590a, 590b, for example. That is, openings 533 provide access to a space between first and second substrates of system 500.

In step 1, magnet 550 is present below first liquid droplet 590a and microparticle pellet 550 is present within first liquid droplet 590a. As magnet 550 is translated along a trajectory form the left side of the figure to the right side of the figure, microparticle pellet 580 is urged to follow or slide along second substrate in the same trajectory as magnet 550. Step 2 shows magnet 550, having moved further to the right, relative to step 1, such that microparticle pellet 580 is drawn closer to angled comer wall 591a of first liquid droplet 590a. As magnet continues to move to the right in step 3, microparticle pellet 580 escapes from first liquid droplet 590a and enters an air gap between first and second liquid droplets 590a, 590b. Due to the shape of first liquid droplet 590a, in particular angled wall 591a of first liquid droplet, surface tension of first liquid droplet 590a does not prevent microparticle 580 from escaping first liquid droplet 590a and moving in the air gap between first and second liquid droplets 590a, 590b. As magnet continues to move to the right in step 4, microparticle pellet 580 continues to be drawn to magnet 550 such that microparticle pellet 580 is drawn to the right of the figure, eventually entering second liquid droplet 590b. Steps 1 through 4 show how the translation of magnet present underneath a second substrate of system 500 causes microparticle pellet 580 to be transferred from first liquid droplet 590a to second liquid droplet 590b via an air gap between such droplets. That is, steps 1 through 4 illustrate a trans-droplet migration process according to an embodiment of the present invention. The sliding of magnet 550 attracts the magnetic particle pellet 580 (where such microparticle pellet 580 may comprise, for example, Dyanbeads M270 carboxyl with, for example, 800,000 particles) in order to achieve “trans-droplet migration,” i.e., migration of microparticle 580 from first to second liquid droplets 590a, 590b. Microparticle pellet 580 is transferred from first to second liquid droplets 590a, 590b via passing through air-gap separating such droplets and defined by pattern of grooves 535.

FIG. 6 presents three different embodiments of magnet arrays of an embodiment of a system according to the present invention. Magnet array 610 includes only one magnet 610a. Magnetic microparticle beads 615a near magnet 610a are attracted towards (i.e., a magnetic force urges microparticles 615a towards) magnet 610a. In an embodiment of a system of the invention, magnet array 610 would be positioned on a second face of a second substrate (i.e., bottom sheet), and microparticles 615a are positioned on a first face of such second substrate (i.e., bottom sheet) where the magnet array 610 and microparticles 615a are on opposite sides of such second substrate. That is, magnet array 610 is beneath a bottom sheet (second substrate) and microparticles 615a rest on a top surface of such bottom sheet). Since magnet array 610 comprises only a single magnet 610a, all microparticles 615a are attracted to magnet 610a.

Magnet array 620 comprises first and second magnets 620a, 620b. Magnets 620a, 620b are held at a fixed distance away from each other. Border line or boundary line 629 bisects the distance between two magnets 620a, 620b. Border line or boundary line 629 demarcates first and second regions 629a, 629b on either side of boundary line 629. Microparticles 625a present within first region 629a are primarily attracted to first magnet 620a, and microparticles 625b present within second region 629b are primarily attracted to second magnet 620b. That is, like different watershed regions, in which water present in an area flows downward towards different drainage regions, microparticle 625a is closer (i.e., less distance) to first magnet 620a, such that microparticle 625 a is more strongly attracted to first magnet 620a. Similarly, microparticle 625b is closer (i.e., less distance) to second magnet 620b, such that microparticle 625b is more strongly attracted to second magnet 620b. Microparticles 625a present within first region 629a on one side of boundary or border 629 are closer to, and therefore more strongly attracted to, first magnet 620a. Microparticles 625b present in second region 629b on another side of boundary or border 629 are closer to, and therefore more strongly attracted to, second magnet 620b.

Magnet array 630 comprises first, second and third magnets 630a, 630b, 630c. Magnets 630a, 630b, 630c are held at a fixed distance away from each other. Border line or boundary line 639 bisects the distance between each pair of two magnets of magnet array 630. Border line or boundary line 639 demarcates first, second and third regions 639a, 639b, 639c on each side of boundary line 639. Microparticles 635a present within first region 639a are primarily attracted to first magnet 630a; microparticles 635b present within second region 639b are primarily attracted to second magnet 630b; and microparticles 635c present within third region 639c are primarily attracted to third magnet 630c. As described above, like different watershed regions, in which water present in an area flows downward towards different drainage regions, microparticle 635a is closer (i.e., less distance) to first magnet 630a, such that microparticle 635a is more strongly attracted to first magnet 630a. Similarly, microparticle 635b is closer (i.e., less distance) to second magnet 630b, such that microparticle 635b is more strongly attracted to second magnet 630b. Microparticle 635c is closer (i.e., less distance) to third magnet 630c, such that microparticle 635c is more strongly attracted to third magnet 630c. Microparticles 635a present within first region 639a on one side of boundary or border 639 are closer to, and therefore more strongly attracted to, first magnet 630a. Microparticles 635b present in second region 639b on another side of boundary or border 639 are closer to, and therefore more strongly attracted to, second magnet 630b. Microparticles 635c present in third region 639c on another side of boundary or border 639 are closer to, and therefore more strongly attracted to, third magnet 630c.

As described herein, embodiments of magnet arrays according to the present invention comprise a plurality of magnets, of which distance between such magnets has been fixed. Embodiments of the invention are configured such that magnets can be focused, as described herein. While in certain embodiments, a magnet array comprises only one magnet, in embodiments in which a magnet array comprises a plurality of magnets, such magnet array can induce a new action or motion of a microparticle pellet caused by interactions between/among the magnets (such actions or motion may be referred to as “trans-pole migration” or “TPM”). In embodiments in which the magnet array comprises a plurality of magnets, individual magnetic territories/borders exist like watersheds, as described above. In such cases, travel directions of the microparticle pellets are determined by the territory or region in which the microparticle pellet is located (where different regions correspond to different areas closer to one or another magnet of the magnet array); i.e., in which regions relative to a border or boundary between magnets. The microparticle pellet is most strongly attracted to a dominant magnet (i.e., the closest magnet) until the microparticle is moved such that the dominant magnet is changed, as described herein. In other words, a single magnet of the magnet array remains the dominate magnet until the microparticle pellet exceeds or crosses over a border-line or boundary-line into another region.

Other embodiments of magnet arrays according to the present invention may comprise still more magnets, such as four or more magnets. Magnet arrays comprising a plurality of magnets may utilize any convenient substrate to keep magnets spaced apart from each other at a fixed distance and a fixed orientation (i.e., with respect to individual magnets or with respect to other aspects of systems of the invention, such as liquid droplets). In particular, such substrates used to hold magnets at a fixed distance may be configured to keep magnets at a fixed distance away from each other and at a fixed orientation as the magnet array is translated.

FIG. 7 presents an illustration of trans-pole migration in connection with an embodiment of a system 700 of the present invention. System 700 includes second substrate 720 with first face 720a and second face 720b. Present on first face 720a of second substrate 720 are liquid droplet 790, and within liquid droplet 790 is microparticle pellet 770. Liquid droplet 790 is present between second substrate 720 and first substrate 730 and, as described above, exhibits a large footprint, meaning it is relatively spread out between first and second substrates 720, 730. Liquid droplet 790 includes interface 791 at a liquid-air boundary or interface. Interface 791 is a wall interface or wall boundary, meaning, when viewed from the top view, interface 791 of liquid droplet 790 is substantially flat and does not include, for example an angled corner. Wall boundary 791 is configured, e.g., shaped, as such so that internal forces of liquid droplet 790 cause microparticle pellet 770 to stop when brought into contact with wall interface 791. That is, wall interface 790 prevents microparticle pellet 770 from existing liquid droplet 790 as microparticle pellet 770 is drawn towards and meets with wall interface 791.

System 700 also includes sliding magnet array 750. Magnet array 750 is present beneath second substrate 720. That is, magnet array 750 is directly opposed to second face or surface 720b of second substrate. Magnet array 750 is configured to translate or slide along trajectory 751. Trajectory 751 is a single axis, meaning magnet array 750 is configured to move only in a single dimension, i.e., left to right or right to left in the figure.

Magnet array 750 includes two magnets, first magnet 750a and second magnet 750b. First and second magnets 750a, 750b are held at a fixed distance from each other that does not change as magnet array 750 is translated along trajectory 751. First and second magnets 750a, 750b are oriented in a relatively diagonal orientation relative to trajectory 751 with second magnet 750b leading, closer to the foreground in the figure and first magnet 750a following, further from the foreground in the figure. In embodiments any convenient fixed magnet or electromagnet may be employed. Both first and second magnets 750a, 750b are or approximately the same size and apply substantially the same magnitude magnetic force. Therefore, border line or boundary line 759 is shown as a dotted line that bisects the distance between first and second magnets 750a, 750b.

As described herein, microparticles (including microparticle pellet 770) when present on one side of border 759 are more strongly attracted to first magnet 750a, and microparticles (including microparticle pellet 770) when present on the other side of border 759 are more strongly attracted to second magnet 750b. That is, for microparticles (including microparticle pellet 770) present on one side of border 759, first magnet 750a is dominate, and for microparticles (including microparticle pellet 770) present on the other side of border 759, second magnet 750b is dominate.

Steps 1 through 6 shown in the top view of system 700 illustrate the steps of trans-pole migration of microparticle pellet 770. That is, steps 1 through 6 show how system 700 causes microparticle pellet 770 to translate in two dimensions (i.e., along both x- and y-axes), notwithstanding that magnet array 750 translates in only a single dimension (i.e., along only the x-axis).

In step 1, magnet array 750 is moving along trajectory 751 (i.e., left to right in the figure). As magnet array 750 moves along trajectory 751, second magnet 750b is dominate with respect to microparticle pellet 770. That is, microparticle pellet 770 is closer to and therefore microparticle pellet 770 is more strongly attracted to second magnet 750b. As magnet array 750 moves along trajectory 751, microparticle pellet 770 follows, i.e., is pulled, through liquid droplet 790 along first face 720a of second substrate 720. Microparticle pellet follows its trajectory 771a. Microparticle pellet 770 is pulled through liquid droplet 790 from left to right until microparticle pellet 770 reaches wall interface 791 of liquid droplet 790. When microparticle pellet 770 reaches wall interface 791 , wall interface 791 prevents microparticle pellet 770 from moving further along trajectory 751 of magnet array 750. That is, microparticle pellet 770 is prevented from moving further right in the figure. Instead, as shown in step 2, microparticle pellet 770 remains in a fixed position at wall 791 as magnet array 750 continues to translate to the right along trajectory 751.

As magnet array 750 continues to translate along trajectory 751, border 759 between first and second magnets 750a, 750b crosses over microparticle pellet 770, as shown in steps 3 and 4. When border 759 between first and second magnets 750a, 750b crosses over microparticle pellet 770, second magnet 750b is no longer dominate with respect to microparticle pellet 770, and, instead, first magnet 750a becomes dominate with respect to microparticle pellet 770. That is, microparticle pellet 770 was initially primarily attracted to (i.e., more strongly attracted to) second magnet 750b (i.e., second magnet 750b exerts a greater magnitude force on microparticle pellet 770 than first magnet 750a does) and, upon crossing border 759, is thereafter primarily attracted to first magnet 750a. Microparticle pellet 770 effectively crosses border 759 due to the movement of magnet array 750, even though microparticle pellet 770 itself remains static. Microparticle pellet’s fixed position relative to liquid droplet 790 is shown as trajectory 771b.

In steps 5 and 6, microparticle pellet 770, which is now primarily attracted to first magnet 750a, is drawn towards first magnet 750a. Since first magnet 750a is displaced along the y-axis from the position of microparticle pellet 770, microparticle pellet 770 moves in a second dimension, along the y-axis, closer to the position of first magnet 750a. Since microparticle pellet 770, even after transitioning to being more strongly attracted to first magnet 750a, is unable to exit liquid droplet 790 at wall boundary 791, microparticle pellet 770 moves along or follows wall boundary 791 as it moves in the y-axis towards first magnet 750a. Since microparticle pellet 770 remains on the same side of border 759, microparticle pellet 770 will thereafter be drawn toward or be driven by first magnet 750a.

In the steps of trans-pole migration described above, the “stop” condition, described herein (e.g., in which microparticle pellet 770 is stopped at wall interface 791) is utilized in order to effectively move microparticle pellet 770 across border 759. Under the “stop” condition, the positional relation of microparticle pellet 770 and the dominant magnet changes as magnet array 750 continues to slide along trajectory 751. By the movement of magnet array 750, microparticle pellet 770 approaches border 791 and crosses over it, and then simultaneously, the dominate magnet is switched (i.e., microparticle pellet 770 is thereafter more strongly attracted to first magnet 750a than second magnet 750b). Such is an illustration of how “trans-pole migration” in embodiments of the present invention provides new techniques for manipulating microparticles, e.g., microparticle pellets, in two dimensions, i.e., in XY-plane, even using a simple system based on a single-actuator unit (e.g., an actuator of existing techniques, configured to move in only one dimension).

FIG. 8 presents embodiments of aspects of system 800 according to the present invention and further illustrates movement of microparticle pellet 890 within and between liquid droplets 890. FIG. 8 shows, in subpanel A, components of system 800 used in connection with demonstrating the movement of microparticle pellet 890 using system 800. FIG. 8 shows, in subpanel B, behavior of magnetic microparticle pellet 890. In particular, microparticle pellet 890 can move to the direction indicated by arrows via two modes: sliding (i.e., movement substantially in the x-axis following a magnet across or through droplets 890) and TPM (trans- pole migration) (i.e., movement within droplet 890 substantially in the y-axis as microparticle pellet 890 moves towards a newly dominant magnet). FIG. 8 shows, in subpanel (C) pictures of movie frames collected and corresponding schematic illustrations for three kinds of elemental actions: movement associated with a “TPM event” in which microparticle pellet 890 moves from bottom- to upper-magnet; movement associated with a “TPM event” in which microparticle pellet 890 moves from upper- to bottom-magnet; and movement associated with a “pullout” event in which microparticle pellet 890 moves from left- to right-droplet 890.

In FIG. 8, subpanel A, microparticle pellet 890 is present in liquid droplet 890 and is present on one side of border 858 such that first magnet 850a is dominate with respect to microparticle pellet 890. Magnet array 850 comprises first and second magnets 850a, 850b and is moved along one-dimensional trajectories 851a, 85 lb, i.e., left and right in the figure. Liquid droplets 890 are arranged on system 800 such that vertices of angled comers 891a of liquid droplets 890 are aligned with each other.

FIG. 8, subpanel B presents a schematic of trajectories, in which microparticle pellet 890 is pulled out of liquid droplet 890 at angled corers 891a. As described herein, translating a microparticle pellet through an angled corner of liquid droplet 890 does not prevent microparticle pellet from escaping or exiting the liquid droplet. On the other hand, wall interfaces or wall boundaries 891b prevent microparticle pellets from exiting liquid droplet 890. As described herein, wall boundaries 891b may be utilized in connection with performing trans- pole migration “TPM” to move microparticle pellets in a second dimension, i.e., along the y- axis. System 800 used in connection with collecting information about trajectories of microparticles shown in FIG. 8 is as follows. The assay cartridge of system 800 comprises three pieces: (i) a second substrate comprising a cyclo-olefine polymer (COP) bottom-sheet; (ii) a first substrate comprising an acrylic lid; and (iii) a spacer between first and second substrates comprising 145 pm double-sided spacer tape configured to connect first and second substrates at the edges of assay cartridge. Such a three-piece structure forms a 145 pm-thin space (i.e., volume between first and second substrates), in which liquid droplets 890 locate. The shape (e.g. cornered-tops and flat-walls) of liquid droplets 880 is designated by the patterning (i.e., the pattern of grooves 835) on the first substrate (i.e., on the lid). Individual liquid droplets 890 are surrounded by an air gap and are isolated from each other. In addition, (i) microparticle pellet 890 comprises of magnetic microparticles that are Dynabeads M270 carboxylic acid with 800,000 particles; (ii) liquid droplets comprise two aqueous droplets; and (iii) magnet array 850 is a two-magnet array comprising two 6mm diameter Neodymium magnets. First and second magnets 850a, 850b of magnet array 850 are arranged at a diagonal orientation. Magnet array 850 was driven in the X-axis only (i.e., left and/or right in the figure) relative to assay cartridge of system 800 by a single actuator at a maximum velocity of 0.3 mm/sec.

The output behavior of the magnetic microparticle pellet 890 is shown in FIG. 8, subpanels A-C. Microparticle pellet 890 can move in the direction indicated by the various arrows via (i) sliding (i.e., in which microparticle pellet 890 is drawn by or follows first or second magnet 850a, 850b of magnet array 850) and (ii) TPM (trans-pole migration) (in which the dominant magnet with respect to microparticle pellet 890 is changed and microparticle pellet 880 is drawn in the y-axis towards the newly dominant magnet). Utilizing TPM can generate more complex action using XY-plane than sliding only. FIG. 8, subpanel C shows schematic illustrations for three kinds of elemental actions corresponding to different trajectories of microparticle pellet. Two directions (to the relatively upper magnet (first magnet 850a) and to the relatively bottom magnet (second magnet 850b)) of TPM events were confirmed. “Pullout” to the other droplet is observed also when microparticle pellet 890 is present at an angled corner 891a droplet 890 and escaped out of or exited such droplet 890 without staying at wall 891b of air-water interface of droplet 890.

Embodiments of the present invention can be configured to control the movement of microparticle pellets 890 in XY-plane (i.e., in two dimensions), in part, by configuring (i) the shape of liquid droplets 890 as well as (ii) the dimension, size, orientation or position of an array of multiple magnets.

System 800 further comprises opening 835 in first substrate configured to allow liquid to be added to form liquid droplet 890. FIG. 9 presents illustrations of how a system 900 that is an embodiment of the present invention is utilized to perform each of intra-droplet and inter-droplet movement of a microparticle pellet. System 900 comprises a plurality of liquid droplets 990 arranged in rows and columns. System 900 further comprises magnet array 950 with first and second magnets 950a, 950b. Liquid droplets 990 comprise a plurality of angled comers 991a configured to allow microparticle pellets to exit liquid droplets, i.e., to be pulled out of liquid droplets. Liquid droplets 990 further comprise a plurality of wall interfaces 991b configured to prevent microparticle pellets from exiting liquid droplets 990, i.e., such that microparticle pellets can be fixed or pinned to wall interface 991b which effect may be utilized in connection with trans-pole migration movement of microparticle pellets (i.e., movement of microparticle pellets in a second dimension, i.e., y-axis). The microparticle pellet trajectories shown in the figure illustrate how microparticle pellets can utilize wall boundaries 991b to be moved within a single droplet 990 (intra-droplet action) and can utilize angled comers 991a and wall boundaries 991b to be moved across a series of liquid droplets, i.e., through a plurality of different liquid droplets and air gaps separating each boundary between liquid droplets 990.

FIGS. 10A-C show assay cartridge 1000 according to embodiments of the present invention. Pattern of grooves 1035 in first substrate of assay cartridge 1000 is configured such that liquid droplets 1090 are present substantially in a row of rectangles in which vertices of angled comers 1091a are aligned. That is, pattern of grooves 1035 defines angled comers 1091a. Angled corners 1091a are configured to permit microparticle droplets to exit liquid droplets, i.e., to be dragged by a sliding magnet array, between different liquid droplets. First substrate comprises openings 1033 for loading liquid into a space between first and second substrates, bounded by patterning 1035.

FIG. 10A shows assay cartridge 1000 with “groove-patterned acrylic plate”, dimensions 26 mm by 76 mm and with 0.8 mm-width lines/groove lays on the surface. In FIG. 10 A, assay cartridge 1000a comprises 12 cells (i.e., 12 regions of patterning 1035 where liquid droplets form) that are aligned (i.e., vertices of angled corners align across the row) and where each cell comprises a hole 1033 of diameter 2.0 mm configured as an inlet for liquid to be added to form liquid droplets.

FIG. 10B shows assay cartridge 1000 that is an air- wall droplet array loaded with aqueous solution that comprises a dye for clarity. The shape of the liquid droplets 1090 formed is determined by the groove-pattern 1035. Each liquid droplet 1090 is surrounded by air (i.e., there is an air gap between each liquid droplet 1090).

Assay cartridge 1000 comprises an “air-wall droplet array,” as described herein. Assay cartridge 1000 comprises a groove-pattered acrylic plate (i.e., grooved to form pattern of grooves 1035), then used as a lid (first substrate) of flowthrough device 1000. The groove-line of pattern of grooves 1035 was patterned with 0.8 mm width and 0.8 mm depth, on an acrylic plate (first substrate). Assay cartridge comprises a COP bottom sheet (second substrate), which, together with the patterned acrylic lid (first substrate), is assembled with a double-side tape (2 mm width line type and approximately 145 pm thickness) at the longer-ends of rectangle of flowthrough device 1000. 7 pL of aqueous solution (i.e., 0.25 mM DEA buffer, pH 9.25 containing 0.05% Tween20) is loaded in each cell. Such aqueous solution forms the droplet array shown in FIG. 10B.

FIG. 11 A shows assay cartridge 1100. Assay cartridge 1100 is a high density array of cells. Assay cartridge comprises ten cells in a small chip of dimensions 26 mm by 38 mm. Loaded assay cartridge 1100 demonstrates that there is no leaking or fusion of the droplet array. The aqueous solution loaded into assay cartridge 1100 had been colored with some levels of blue-dye concentrations to demonstrate the no-leak, no-fusion aspects of the assay cartridge 1100.

FIG. 1 IB shows results of testing pushing the water-droplet by oil-loading to show that both hydrophilic and hydrophobic fluids can be made available in a single cell (i.e., area defined by the pattern of grooves). That is, FIG. 1 IB shows the operation of pushing water-droplet by hydrophobic oil. The grooved acrylic lid (i.e., the patterning 1135 of first substrate) can hold both hydrophilic and hydrophobic fluid. Loaded oil makes the water-droplet run away. This air-wall droplet embodiment is configured for liquid exchange without overflow. Such is also configured to work as a microfluidic device as well as “in a tunnel”-type flowthrough, as described herein.

Methods:

Assay cartridges, sliding magnet arrays and systems of the invention find use in a variety of applications. In some instances, assay cartridges, sliding magnet arrays and systems find use in the manipulation of the magnetic microparticles. In some cases, such would be useful for assay processing via magnet- sliding as a sample preparation method. Embodiments of the present invention are useful in sample preparation methods, such as in the field of the beadbased biomarker assay. Embodiments facilitate techniques with (1) washing of the surface of the microparticles and (2) utilizing small sample volume assays (such as, for example, assays utilizing 10 pL sample volume). Embodiments of the present invention are utilized in connection with methods of biomarker assay using the magnetic-microparticle manipulation on a solid surface by multiple magnets; utilizing microfluidic device/apparatus using the magnetic- microparticle manipulation on a solid surface by multiple magnets; techniques for manipulating magnetic-microparticles via switching a dominant magnet; and performing assays utilizing a combination of multiple-magnet processing according to embodiments of the present invention in combination with the detection using a nanowell array (e.g., fL-chamber, Digital assay).

In some instances, the various embodiments of the assay cartridges, sliding magnet arrays and systems described herein are employed in methods of moving microparticles in an assay cartridge. Aspects of such methods comprise a) adding microparticles to the first region of a system of the present invention, and b) translating the sliding magnet array in a single dimension. In some cases, translating the sliding magnet array comprises translating the sliding magnet array across a boundary of the first region or translating the sliding magnet array within the boundary of the first region. In other cases, translating the sliding magnet array comprises drawing the microparticles across the first face of the second substrate.

Embodiments of the methods of the present invention further comprise drawing the microparticles across an angled corner of the first region. Other embodiments further comprise drawing the microparticles to a straight edge of the first region. Still other embodiments further comprise drawing the microparticles from the first region to the second region.

In some cases, methods of the present invention are methods of manipulating microparticles or sample preparation or performing a biomarker assay.

The present invention provides methods of detecting a target analyte in a sample using the devices disclosed herein. The samples that may be used with the methods will be discussed first followed by the types of analytes followed by the specific types of assay (e.g., digital analysis, immunoassays, nucleic acid analysis, clinical chemistry, etc.) to be used in the methods.

Samples:

Aspects of the present disclosure devices that may be used to process and analyze different analytes or different types of analytes present in a biological sample and methods of use thereof. In some embodiments, the devices of the present disclosure comprise regions where the sample may be deposited. In order to analyze a sample, more specifically analytes in a sample, the sample is derived from one or more of various sample sources described in this section.

Sample Type:

As used herein, "sample", "test sample", or "biological sample" refers to a sample containing or suspected of containing an analyte. For example, International Publication No. WO 2016/161400 are incorporated by reference herein and samples of the present disclosure are further described below. In some embodiments, a sample of the present disclosure is derived from any suitable source. In other embodiments, the sample comprise a liquid, fluent particulate solid, or fluid suspension of solid particles. In certain embodiments, the sample may be a liquid sample or a liquid extract of a solid sample. In some cases, the sample may be processed prior to the analysis described herein. For example, the sample may be separated or purified from its source prior to analysis; however, in certain embodiments, an unprocessed sample containing the analyte may be assayed directly.

In some embodiments, the source of the analyte molecule may be synthetic (e.g., produced in a laboratory), the environment (e.g., air, soil, etc.), fluid samples, e.g., water supplies, etc.), an animal, e.g., a mammal, a plant, or any combination thereof.

In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and/or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. Tn the claims, 35 U.S.C. §1 12(f) or 35 U.S.C. §112(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase “means for” or the exact phrase “step for” is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 112 (f) or 35 U.S.C. §112(6) is not invoked.

Claims

What is claimed is:

1. An assay cartridge, comprising: a first substrate having a first face comprising a pattern of grooves; a second substrate having a first face, wherein the first substrate is positioned in relation to the second substate such that the first face of the first substrate faces the first face of the second substrate; and a first region and a second region, wherein the first and second regions are defined by the pattern of grooves and are located between the first and second substrates.

2. The assay cartridge of claim 1, wherein the first and second substrates are arranged such that the first face of the first substrate is directly opposed to the first face of the second substrate.

3. The assay cartridge of any of the previous claims, wherein the first and second substrates are spaced apart.

4. The assay cartridge of any of the previous claims, further comprising an air gap between the first and second substrates.

5. The assay cartridge of any of the previous claims, further comprising a spacer present between the first and second substrates.

6. The assay cartridge of any of the previous claims, wherein the first substrate is a lid, and the second substrate is a bottom-sheet.

7. The assay cartridge of any of the previous claims, wherein the first region comprises a first opening in the first substrate.

8. The assay cartridge of claim 7, wherein the first opening is configured for loading fluid into the first region.

9. The assay cartridge of any of the previous claims, wherein the second region comprises a second opening in the first substrate.

10. The assay cartridge of claim 9, wherein the second opening is configured for loading fluid to the second region.

11. The assay cartridge of any of the previous claims, wherein the pattern of grooves is configured to retain fluid between the first and second substrates within an area bounded by the pattern of grooves.

12. The assay cartridge of any of the previous claims, wherein the pattern of grooves is configured such that fluid is not retained outside an area bounded by the pattern of grooves.

13. The assay cartridge of any of the previous claims, wherein the pattern of grooves is configured such that fluid is excluded from an area bounded by the pattern of grooves.

14. The assay cartridge of any of the previous claims, wherein the first region and the second region comprise straight edges and angled comers.

15. The assay cartridge of claim 14, wherein the angled corners are configured to allow a microparticle to escape from liquid present at the angled comer.

16. The assay cartridge of any of claims 14 to 15, wherein the straight edges are configured to prevent a microparticle from escaping from liquid present at the angled comer.

17. The assay cartridge of any of claims 14 to 16, wherein the angled comers comprise one or more: 90-degree angled corners.

18. The assay cartridge of any of claims 14 to 17, wherein the angled comers comprise one or more: acute-angled corners, 90-degree angled corners or obtuse-angled comers.

19. The assay cartridge of any of the previous claims, wherein the pattern of groves comprises a plurality of groves with width of 0.3 mm to 1.0 mm.

20. The assay cartridge of any of the previous claims, further comprising a third region, a fourth region, a fifth region, a sixth region, a seventh region, an eighth region, a ninth region, and a tenth region.

21. The assay cartridge of claim 20, wherein the third through tenth regions are defined by the pattern of grooves.

22. The assay cartridge of any of any of the previous claims, wherein one or more of the regions comprise hydrophilic patterning.

23. The assay cartridge of any of the previous claims, wherein one or more of the regions comprise a hydrophilic coating on the first face of the first substrate.

24. The assay cartridge of any of the previous claims, wherein the first face of the first substrate comprises a hydrophobic coating on the first face of the first substrate.

25. The assay cartridge of any of the previous claims, wherein one or more of the regions comprise a hydrophobic coating on the first face of the first substrate.

26. The assay cartridge of any of the previous claims, wherein the pattern of grooves comprises a pattern of angled rectangles in a row.

27. The assay cartridge of any of the previous claims, wherein an angled comer of the first region is coaxial with an angled corner of the second region.

28. The assay cartridge of any of the previous claims, wherein the pattern of grooves comprises a pattern of angled rectangles in rows and columns and an angled comer of the first region is coaxial with an angled corner of the second region.

29. The assay cartridge of any of the previous claims, wherein one or more of the regions comprise a fluid.

30. The assay cartridge of any of the previous claims, wherein one or more of the regions comprise microparticles.

31. The assay cartridge of claim 30, wherein the microparticles are attracted to a magnet.

32. The assay cartridge of any of claims 30 to 31, wherein the first and second substrates are not attracted to a magnet.

33. The assay cartridge of any of the previous claims, wherein each region is the same size.

34. The assay cartridge of any of the previous claims, where one or more of the regions are different sizes.

35. The assay cartridge of any of the previous claims, further comprising a sample analysis region.

36. A sliding magnet array, comprising a first magnet; and a second magnet, wherein the first magnet and the second magnet are separated from each other at a diagonal orientation, and the separation of the first magnet and the second magnet creates a border between the first and second magnets having a first side and a second side.

37. The sliding magnet array of claim 36, wherein the border is defined by a line that is equidistant between the first and second magnets.

38. The sliding magnet array of any of claims 36 to 37, wherein the first and second magnets arc of substantially the same magnetic strength.

39. The sliding magnet array of any of claims 36 to 38, wherein the first and second magnets comprise magnetic fields of substantially the same magnitude.

40. The sliding magnet array of claim 36, wherein on the first side of the border, the first magnet creates a first dominate magnetic field, and on the second side of the border, the second magnet creates a second dominate magnetic field.

41. The sliding magnet array of claims 36 or 40, wherein the first magnet and the second magnet are configured to translate.

42. The sliding magnet array of claims 36 or 41, wherein the first magnet and the second magnet are configured to translate in a single dimension.

43. The sliding magnet array of claims 36 or 42, wherein the first magnet and the second magnet are configured to translate using a single actuator.

44. The sliding magnet array of claims 36 or 43, wherein the single actuator is configured to translate the first and second magnets in a single dimension.

45. The sliding magnet array of any of claims 36 to 44, wherein the first and second magnets are physically connected.

46. The sliding magnet array of any of claims 36 to 45, further comprising a third magnet that is separated from the first and second magnets.

47. The sliding magnet array of claim 46, wherein the separation of the first magnet, the second magnet and the third magnet creates borders defining the first side, the second side and a third side.

48. The sliding magnet array of claims 46 or 47, wherein on the third side of the border, the third magnet creates and third dominate magnetic field.

49. The sliding magnet array of any of claims 46 to 48, wherein the first magnet, the second magnet and the third magnet are configured to translate.

50. The sliding magnet array of any of claims 46 to 49, wherein the first magnet, the second magnet and the third magnet are configured to translate in a single dimension.

51. The sliding magnet array of any of claims 46 to 50, wherein the first magnet, the second magnet, and the third magnet are configured to translate using a single actuator.

52. The sliding magnet array of claims 46 to 51, wherein the single actuator is configured to translate the first, second and third magnets in a single dimension.

53. The sliding magnet array of any of claims 46 to 52, wherein the first magnet, the second magnet, and the third magnet are physically connected.

54. A system for moving microparticles in an assay cartridge, the system comprising: an assay cartridge of any of claims 1 to 35; and a sliding magnet array of claims 36 to 53; wherein the first region and the second region comprise two or more straight edges and two or more angled corners, the second substrate having a second face that does not face the first face of the first substrate, and the sliding magnet array having a first face positioned in relation to the second substrate such that the first face of the sliding magnet array faces the second face of the second substrate.

55. The system of claim 54, wherein the assay cartridge and the sliding magnet array are positioned such that the sliding magnet array can mechanically influence magnetic particles present in the assay cartridge.

56. The system of any of claims 54 to 55, wherein the first region comprises microparticles.

57. The system of any of claims 54 to 56, wherein the system is configured such that the two or more straight edges of the first region prevent the sliding magnet array from moving the microparticles from the first region to the second region.

58. The system of any of claims 54 to 57, wherein the sliding magnet array is able to move the microparticles from the first region to the second region at the two or more angled comers.

59. The system of any of claims 54 to 58, further comprising a first liquid present in the first region and a second liquid present in a second region.

60. A method of moving microparticles in an assay cartridge, the method comprising: a) adding microparticles to the first region of the system of any of claims 54 to 59, b) translating the sliding magnet array in a single dimension.

61. The method of claim 60, wherein translating the sliding magnet array comprises translating the sliding magnet array across a boundary of the first region or translating the sliding magnet array within the boundary of the first region.

62. The method of any of claims 60 to 61, wherein translating the sliding magnet array comprises drawing the microparticles across the first face of the second substrate.

63. The method of any of claims 60 to 62, further comprising drawing the microparticles across an angled comer of the first region.

64. The method of any of claims 60 to 63, further comprising drawing the microparticles to a straight edge of the first region.

65. The method of any of claims 60 to 64, further comprising drawing the microparticles from the first region to the second region.

66. The method of any of claims 60 to 65, wherein the method is a method of manipulating microparticles.

67. The method of any of claims 60 to 66, wherein the method is a method of sample preparation.

68. The method of any of claims 60 to 67, wherein the method is a method of performing a biomarker assay.