Stable interface system and method

Abstract

1. A device for isolating, collecting, and / or transporting target molecules to an assay region, the device comprising an enclosure having an upper portion adapted to receive a sample, a central hollow body portion or other region or cavity suitable for receiving a magnet or magnetic field(s) or magnetic force(s), at least one aqueous phase or layer and at least one oil / wax phase or phase or gas phase or phase stabilized in proximity to one another within the enclosure by a target-permeable structural material associated with the aqueous phase / layer or gas phase / layer or oil / wax phase / layer, or both, and a lower portion containing or adapted to contain one or more reagents for performing an assay.

Description

[Technical Field]

[0001] Declarations regarding related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 423,670, filed November 8, 2022, the entire contents of which are incorporated herein by reference for all purposes.

[0002] Declaration of government support This invention was made with government support under R43 OD023021-01A1 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Incorporation by Reference All U.S. patents, U.S. patent applications, publications, foreign patents, foreign and PCT published applications, literature and other documents, references and publications mentioned herein, and all listed as references cited in any patent or patents issued therefrom, are incorporated herein by reference in their entirety. The incorporated information is a part of this application as if all text and other content were repeated in this application, and is treated as part of the text and content of this application as filed.

[0004] The present invention relates generally to the separation, isolation, purification, identification, detection and quantification of substances.

[0005] Systems, devices, compositions, and methods for positioning and / or processing targets are provided herein. Targets can be positioned in a number of ways, including positively (e.g., by displacing or isolating the target for detection or measurement) and negatively (by positioning or removing one or all non-targets). Targets or materials to which targets are attached can be displaced, separated, isolated, detected, identified, analyzed, screened, quantified, or purified using the systems, devices, and methods of the invention. The systems, devices, and methods of the invention include systems, devices, and methods for isolating and / or detecting targets or analytes (including cells, proteins, DNA, RNA, or pathogens or portions of pathogens, e.g., proteins, nucleic acids, etc.) in a sample. In particular, systems and devices are provided herein that include one or more oils and / or one or more aqueous phases and / or one or more gas phases stabilized in close contact with each other. The systems, devices, and methods of the invention have many applications. For example, they can be used to isolate, separate, transfer, purify, mix, bind, and / or subsequently detect the presence or amount of a target or target analyte from a sample or other mixture. Target positioning can be achieved by isolating, separating, or transferring the target or by isolating, separating, or transferring a substance bound to the target using the methods, devices, or systems of the invention, and can be done positively or negatively. In some aspects, provided herein are systems and devices comprising one or more stabilized oils and / or one or more stabilized aqueous phases and / or one or more gas phases that can be used to transfer or purify a target or analyte from a sample or mixture that contains, may contain, or is suspected or suspected of containing the target or analyte using magnetic, electric, or acceleration-based forces (e.g., via gravity or via a centrifuge) to pull the target or analyte through one or more phases or layers. In some embodiments, the systems and devices include reagents for detecting, identifying, analyzing, isolating, or quantitating the target or analyte. Quantitation may be positive-negative, semi-quantitative, or quantitative relative to the target. Isolation or purification may be complete or partial.One or more or all of the reagents for target or analyte detection, identification, analysis, isolation or quantification may be contained in one or more parts or portions of the system or device, one or more aqueous and / or oil phases or layers of the system or device, a base phase or layer of the system or device, a lower phase, layer or tier of the system or device, or a terminal phase, layer or tier of the system or device (in vertical or latitudinal embodiments), or a seam, junction or junction (in horizontal or longitudinal or other phase / layer directions in non-vertical or non-latitudinal embodiments). [Background technology]

[0006] The following contains information that may be useful in understanding the present invention. No admission is made that any of the information is prior art or relevant to any invention described or claimed herein, or that any publication or document specifically or implicitly mentioned is prior art or a reference that may be used in assessing the patentability of the invention described or claimed.

[0007] The ability to transfer, isolate, purify, separate, identify, quantify, or otherwise manipulate targets or analytes (e.g., nucleic acids, proteins, whole cells, or contaminants) from a complex background is a critical requirement for many common analytical or other processes in diagnostics, biological research, biomarker discovery, forensics, and the like. However, conventional processes, including, for example, analyte purification processes, can be or are time-consuming, expensive, laborious, etc., and often represent a bottleneck in such processes, e.g., analytical processes. Furthermore, some methodologies damage the sample or cause undesirable loss or inconsistent yield of sample. Thus, there is a need, and provided herein, for improved systems, methods, and devices for target manipulation, including isolation, separation, and purification of targets or target analytes, and subsequent rapid detection, identification, and quantification of analytes or other targets from samples. Summary of the Invention [Problem to be solved by the invention]

[0008] (Brief summary) The invention described and claimed herein has many features and embodiments, including but not limited to those set forth, described, or referenced in this Summary of the Invention, which is not intended to be exhaustive, and the invention described and claimed herein is not limited to or restricted by the features or embodiments identified in this introduction, which are included for purposes of illustration only, and not limitation.

[0009] The present invention includes multi-layer systems and devices that provide autonomous operation of processing steps through manipulation of target positioning forces. The multi-layer system may be within a vessel. [Means for solving the problem]

[0010] In one aspect, the invention provides autonomous sample preparation (e.g., lysis, washing, and / or solid phase target binding) and testing (e.g., PCR, LAMP, etc.) performed in a single device requiring only the addition of sample and the application of force (e.g., magnetic force) to obtain and / or read results. In some embodiments, the invention optionally includes means for communicating results for viewing, analysis, and / or storage (e.g., to a computer or phone).

[0011] In some aspects, provided herein are systems, methods, and apparatus, as well as compositions, for isolating or positioning a target from a sample and processing the target. In some embodiments, the target of interest is the target itself. In some embodiments, the target of interest is a target bound to a solid phase or the solid phase itself.

[0012] In some embodiments, the system, method, or apparatus includes at least one aqueous phase or layer and at least one oil phase or layer stabilized adjacent to each other in a container. In some embodiments, the at least one aqueous phase or layer and the at least one oil phase or layer are stabilized in the container by a hydrophilic porous material associated with the at least one aqueous phase and / or a hydrophobic porous material associated with the at least one oil phase. In some embodiments, the at least one aqueous phase and the at least one oil phase are stabilized in the container by adjusting geometric characteristics or one or more chemical or physical material characteristics.

[0013] In some embodiments where the system, method, or device includes at least one aqueous phase or layer and at least one oil phase or layer, only at least one aqueous phase or layer and at least one oil phase or layer are stabilized. In some embodiments where the system, method, or device includes more than one aqueous phase or layer and one or more oil phases or layers, only one of the aqueous phases or layers is stabilized. In some embodiments where the system, method, or device includes more than one aqueous phase or layer and one or more oil phases or layers, more than one or all of the aqueous phases or layers are stabilized. For example, in embodiments of the present invention having four aqueous phases or layers, one, two, three, or all four may be stabilized. In some embodiments where the system, method, or device includes more than one aqueous phase or layer and one or more oil phases or layers, only one of the oil phases or layers is stabilized. In some embodiments where the system, method, or device includes more than one oil phase or layer and one or more aqueous phases or layers, more than one or all of the oil phases or layers are stabilized. For example, in an embodiment of the present invention having four oil phases or layers, one, two, three or all four may be stabilized.

[0014] In another embodiment of the invention where the device, system or method comprises an aqueous phase or layer and / or multiple oil phases or layers, for example, 1 to 6 aqueous phases or layers and 1 to 6 oil phases or layers, 1 to 6 of the aqueous phases or layers and / or 1 to 6 of the oil phases or layers may be stabilized.

[0015] In some embodiments, the device, system, or method includes at least one stabilized aqueous phase or layer. In some embodiments, the at least one aqueous phase or layer is stabilized by a hydrophilic porous material associated with the at least one aqueous phase or layer. In some of these embodiments, the device, system, or method including at least one stabilized aqueous phase or layer does not include an oil phase or layer or a stabilized oil phase or layer. In some embodiments, the device, system, or method including at least one stabilized aqueous phase or layer also includes a gas phase or layer. In some embodiments, the gas phase or layer includes, for example, air or an inert gas. In some embodiments, the gas layer includes helium, neon, argon, krypton, xenon, radon, oganesson, or the like. In some of these embodiments, the device, system, or method including at least one stabilized aqueous phase or layer includes at least one oil phase or layer and / or at least one stabilized oil phase or layer. In some of these embodiments, the device, system, or method comprising at least one stabilized aqueous phase or layer comprises at least one oil phase or layer and / or at least one stabilized oil phase or layer and at least one gas phase or layer. In some embodiments, the device, system, or method comprises at least two stabilized aqueous phases or layers. In some embodiments, the device, system, or method comprising at least one stabilized aqueous phase is in a container or vessel.

[0016] In some embodiments, the device, system, or method includes at least one stabilized oil phase or layer. In some embodiments, the at least one oil phase or layer is stabilized by a hydrophobic porous material associated with the at least one oil phase or layer. In some of these embodiments, the device, system, or method including at least one stabilized oil phase or layer does not include an aqueous phase or layer or a stabilized aqueous phase or layer. In some embodiments, the device, system, or method including at least one stabilized oil phase or layer also includes a gas phase or layer. In some embodiments, the gas phase or layer includes, for example, air or an inert gas. In some embodiments, the gas layer includes helium, neon, argon, krypton, xenon, radon, oganesson, or the like. In some of these embodiments, the device, system, or method including at least one stabilized oil phase or layer includes at least one aqueous phase or layer and / or at least one stabilized aqueous phase or layer. In some of these embodiments, the device, system, or method comprising at least one stabilized oil phase or layer comprises at least one aqueous phase or layer and / or at least one stabilized aqueous phase or layer and at least one gas phase or phase. In some embodiments, the device, system, or method comprises at least two stabilized oil phases or layers. In some embodiments, the device, system, or method comprising at least one stabilized oil phase is in a container or vessel.

[0017] In some embodiments, the least one aqueous phase or layer or at least one oil phase or layer is stabilized in a container or vessel using a porous material. The material is selected to allow desired material movement through the device or system. The porous material may be a mesh. In some embodiments, one or more of the least one aqueous phase or layer is stabilized with at least one hydrophilic porous material or mesh. In some embodiments, one or more of the least one oil phase or layer is stabilized with at least one hydrophobic porous material or mesh. In one embodiment, the porous material and / or the hydrophobic and / or hydrophilic mesh has at least one predetermined pore size, set of pore sizes, or range of pore sizes. In some embodiments, the aqueous phase or layer and the oil phase or layer are stabilized in close proximity to each other in a vessel.

[0018] In some embodiments, one or more of the phases or layers is stabilized, and the phases or layers contain fluids with multiple densities and / or density gradients.

[0019] In some embodiments, one or more phases or layers, when present in the system, may be stabilized within a system or device, e.g., a container, by adjusting one or more chemical or physical material characteristics selected from the geometric characteristics of the material or the density, surface chemistry, and porosity of the hydrophilic / hydrophobic porous material.

[0020] In some embodiments, systems, devices, and methods are designed and used for target positioning. By way of example, one or more targets can be transferred, separated, isolated, detected, identified, analyzed, screened, quantified, or purified using the systems, devices, or methods of the present invention. The systems, devices, and methods of the present invention include systems, devices, and methods for isolating and / or detecting analytes in a sample. In some embodiments, the systems, methods, or devices include one or more oils and / or one or more aqueous phases and / or one or more gas phases stabilized in close contact with each other. These systems and devices have many uses. For example, they can be used to isolate, separate, transfer, purify, mix, bind, and / or subsequently detect the presence or amount of targets or target analytes from a sample or other mixture.

[0021] A target can be positively or negatively positioned in a number of ways. A target can be positively positioned, for example, by isolating the target (e.g., for detection or measurement). A target can be negatively positioned, for example, by positioning or removing one or more or all non-targets.

[0022] In some embodiments, positioning of a target can be done positively, for example, by isolating, separating, transferring, or binding the target or a substance bound to the target using a method, device, or system of the invention.

[0023] In some embodiments, positioning a target can be done negatively, for example, by isolating, separating, moving, or binding the target or a substance bound to the target using a method, device, or system of the invention.

[0024] In some aspects, provided herein are systems, devices, and methods that include one or more stabilized oils and / or one or more stabilized aqueous phases and / or one or more gas phases that can be used to transfer or purify targets or analytes from samples or mixtures that contain, may contain, or are suspected or suspected of containing the targets or analytes using magnetic, electric, or acceleration-based forces (e.g., via gravity or via a centrifuge) to pull the targets or analytes through one or more layers. In some embodiments, the systems and devices include reagents for target or analyte detection, identification, analysis, isolation, or quantification. Quantification may be positive-negative, semi-quantitative, or quantitative relative to the target. Isolation may be complete or partial. One or more or all of the reagents for target or analyte detection, identification, analysis, isolation, or quantification may be contained within the system or device.

[0025] In some embodiments, the systems, devices, compositions, and methods of the present invention include autonomously operated processing steps. In some embodiments, the present invention provides for the processing / exposing / modification of any solid phase (e.g., paramagnetic particles) that can be transported through a layer / interface. In one aspect, each step functions as a purification / separation step, e.g., as paramagnetic particles pass through a phase, layer, or interface. In other aspects, other functionality can occur when paramagnetic particles are present, e.g., within a phase, layer, or interface (e.g., chemical modification of the solid phase, elution of the solid phase, etc.). In some embodiments, the solid phase is a solid support to which a target is attached (e.g., immobilized, bound, restrained, or sequestered, whether directly or indirectly). However, anything to which a target is attached can function as a "solid phase." Semi-solids can also function as solid phases. Solid phases include paramagnetic particles. Meshes or other porous solid support structures used to stabilize phases or layers of the present invention can be solid phases. In some embodiments, the target can be a solid phase, e.g., a cell. In other embodiments, each step functions as a purification / separation step, e.g., non-target components of the sample pass through a phase, layer, or interface, and the target (on a solid, semi-solid, or solid phase, e.g., attached to paramagnetic particles) remains, while the non-target components are released. This is an example of negative selection.

[0026] In some embodiments, the at least one aqueous phase and the at least one oil phase are stabilized in the container by a hydrophilic porous material associated with the at least one aqueous phase or layer, a hydrophobic porous material associated with the at least one oil phase or layer, and by adjusting the surface chemistry or surface energy of the phases or layers such that the buoyancy force of either the one oil phase or layer or the at least one aqueous phase or layer is overcome and is less than the surface tension between the at least one oil phase and the hydrophobic porous material or the at least one aqueous phase and the hydrophilic porous material.

[0027] In some embodiments, multiple aqueous phases or layers and multiple oil and / or gas phases or layers are present in the system. In some embodiments, the system includes a first aqueous phase or layer, a second aqueous phase or layer, a first oil phase or layer, and a second oil phase or layer, with or without one or more gas phases or layers. In some embodiments, the phases or layers are stacked alternately within the container such that the first and second aqueous phases or layers do not directly contact each other, and the first and second oil phases or layers do not directly contact each other.

[0028] In some embodiments, the container comprises a top opening that allows for the addition of a sample to the container. In some embodiments, the aqueous phase is closest to the top opening of the container. In some embodiments, the oil phase is closest to the top opening of the container. In some embodiments, a device or system of the present invention allows for the addition of a sample to a device without a top or bottom, e.g., an insert containing a system of the present invention. The phases and / or layers are positioned as desired in such embodiments, and the sample can be added to a layer or layer designated as the "first" phase or layer or the "sample-receiving" phase or layer.

[0029] In some embodiments, at least one aqueous phase comprises a lysis buffer. In some embodiments, at least one aqueous phase comprises a wash buffer.

[0030] In some embodiments, the system further comprises paramagnetic particles (PMPs). In some embodiments, the PMPs are contained within a container. The PMPs may be lyophilized or in liquid form. In some embodiments, the PMPs are contained within at least one aqueous phase. In some embodiments, the PMPs bind to a target or target analyte and may be referred to as a "target-binding" PMP (or other target-capture solid phase). In some embodiments, the target-binding PMPs or other target-binding solid phases bind to a target or target analyte and may be referred to as a "target-binding" PMP (or other target-capture solid phase). In some embodiments, the target-binding PMPs or other target-binding solid phases are conjugated with a target-binding agent, e.g., an antibody, antibody fragment, single-chain Fv, etc., and directed to the target and used as a PMP targeting agent. Other useful target-binding agents include oligonucleotides. In some embodiments, the target-binding oligonucleotides comprise a sequence that targets mRNA (e.g., a poly-dT sequence that binds to a poly-A tail on mRNA) or a specific sequence of RNA or DNA.

[0031] In some embodiments, provided herein is a system for isolating a target analyte from a sample, comprising a first aqueous phase or layer, a second aqueous phase or layer, a first oil phase or layer, and a second oil phase or layer. In some embodiments, the phases or layers are stacked alternately within a container such that the first and second aqueous phases or layers are not in direct contact with each other, and the first and second oil phases or layers are not in direct contact with each other. In some embodiments, the phases or layers are stabilized within the container by a hydrophilic porous material associated with the first aqueous phase or layer, a hydrophilic porous material associated with the second aqueous phase or layer, a hydrophobic porous material associated with the first oil phase or layer, and a hydrophobic porous material associated with the second oil phase or layer. In some embodiments, the phases or layers are further stabilized within the container by adjusting the surface chemistry so that fluid retention forces associating the fluid layers with the support structure dominate other forces (e.g., buoyancy or momentum changes) that might otherwise disrupt functional layer organization or phase order.

[0032] In some embodiments, the container comprises a top opening that allows for the addition of a sample to the container. In some embodiments, the first aqueous phase or layer is closest to the top opening of the container. In some embodiments, the first oil phase or layer is closest to the top opening of the container. In some embodiments, the first aqueous phase or layer comprises a lysis buffer. In some embodiments, the second aqueous phase or layer comprises a wash buffer. In some embodiments, a device or system of the present invention allows for the addition of a sample to a container or device that does not have a top or bottom, thus, for example, an insert containing a system of the present invention that does not contain a bottom integral with its sides. The first and second aqueous phases and the first and second oil phases or layers are positioned as desired in such embodiments, and the sample can be added to the layer or layers designated as the "first" phase or layer or the "sample-receiving" phase or layer.

[0033] The system may further include paramagnetic particles (PMPs). The PMPs may be contained in a container. In some embodiments, the PMPs are lyophilized. In some embodiments, the PMPs are in liquid form. In some embodiments, the PMPs are contained in a first aqueous phase.

[0034] In any of the embodiments described herein, the system may further include a magnet. The container may include a multi-well plate. The system may further include a sample. The sample may be a biological sample or a sewage sample. In some embodiments, the biological sample comprises a nasopharyngeal sample, an oropharyngeal sample, an oral swab sample, an oral sponge sample, a nasal swab sample, a middle turbinate sample, or a saliva sample.

[0035] The systems described herein can be used in methods for isolating any desired target or substance. In some embodiments, the target is a nucleic acid. In some embodiments, the target is a viral nucleic acid. For example, the target may be a SARS-CoV-2 nucleic acid. In some embodiments, the target is a protein (e.g., a hormone or any other protein), a carbohydrate, a glycolipid, a cell, a circulating tumor cell, etc. In some embodiments, any substance that can bind to a "solid phase" described herein, which may be, for example, a PMP (attached either directly or indirectly), may be a target in one or more of the systems, devices, compositions, and methods of the invention.

[0036] One or more or all of the reagents for target detection, identification, analysis, isolation, or quantification may be contained in one or more parts or portions of a system or device. In some embodiments, one or more or all of the reagents for target detection, identification, analysis, isolation, or quantification may be contained in one or more aqueous and / or oil phases or layers of a system or device. In some embodiments, the systems and devices described herein further include a reagent for target detection contained in a base phase or layer or bottom surface of a container. In some embodiments, one or more or all of the reagents for target detection, identification, analysis, isolation, or quantification are contained in a lower phase, layer, or tier of a system or device, but above the base layer. In some embodiments, one or more or all of the reagents for target detection, identification, analysis, isolation, or quantification are contained in a terminal phase, layer, or tier (in vertical or latitudinal embodiments), or a seam, junction, or joint (in horizontal, longitudinal, or other phase / layer orientation in non-vertical or non-latitudinal embodiments) of a system or device.

[0037] The reagents for detecting the target may include reagents for a loop-mediated isothermal amplification (LAMP) or reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay. In some embodiments, the LAMP or RT-LAMP assay is a colorimetric or fluorescent assay. In other embodiments, the reagents for detecting the target include reagents for PCR, RT-PCR, qPCR, qtPCR, multiplex PCR, assembly PCR, asymmetric PCR, or the like. In other embodiments, the reagents for detecting the target include reagents for an immunoassay, which may use antibodies and / or antibody fragments to detect or measure the target or target analyte. In some embodiments, the immunoassay is an enzyme immunoassay, ELISA (enzyme-linked immunosorbent assay, including direct ELISA, indirect ELISA, sandwich ELISA, and competitive ELISA), IEMA (enzyme-linked immunosorbent assay), radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay (CLIA), and enumeration immunoassay (CIA). The vessel containing the PCR reaction fluid can be, for example, a simple cup-shaped vessel at the bottom of the device, or in some embodiments, the geometric features of the vessel containing the reaction fluid can be shaped to make thermal cycling more efficient. For example, in some embodiments, the vessel containing the reaction fluid can have a high aspect ratio to facilitate more rapid transfer of heat (i.e., reducing the distance that temperature must be conducted to facilitate thermocycling of the reaction). In some embodiments, the vessel containing the reaction fluid comprises, consists essentially of, or consists of microfluidic channels. In some embodiments, the vessel containing the reaction fluid is made of a different material than the rest of the device. In some embodiments, the vessel containing the reaction fluid has a heating element integrated therein.

[0038] The devices, systems, methods and compositions of the invention can be used to test or assay any molecular target, including biomolecules, proteins, hormones, nucleic acids, drugs, etc. In some embodiments, the devices, systems, methods and compositions of the invention are used to transfer, separate, isolate, purify, identify, detect and / or quantify targets, including, but not limited to, those described or referenced herein.

[0039] In some embodiments, the present invention provides a disposable cartridge comprising a flow-through assay for determining the presence or amount of a target in a sample, the disposable cartridge comprising a sample application space, a cartridge top, a cartridge bottom, reagents for target detection or quantification, and an enclosure, characterized in that the cartridge comprises, together with or within the enclosure, target-binding paramagnetic particles (or other target-binding carrier material), at least one aqueous phase or layer and at least one gas phase or layer or oil phase or layer stabilized in close proximity to each other by the inclusion of a porous (e.g., target-permeable) structural material associated with the aqueous phase / layer or gas phase / layer or oil phase / layer, or both, and optionally utilizing a magnet. Other and / or alternative phases may be used or included (e.g., two oil phases with or without aqueous phases or layers).

[0040] In some embodiments, the present invention provides a flow assay (e.g., lateral flow, vertical flow) device or cartridge comprising a sample application portion, a conjugate portion, a test portion, and pre-immobilized reagents in different portions of the flow assay device or cartridge, characterized by the inclusion of target-binding paramagnetic particles (or other target-binding carrier material), at least one aqueous phase or layer and at least one gas or oil phase or layer stabilized in close proximity to each other by the inclusion of a porous (e.g., target-permeable) structural material associated with the aqueous phase, gas phase, or oil phase, or both, and a magnet. Other and / or alternative phases may be used or included (e.g., two oil phases with or without an aqueous phase or layer). In some embodiments, the improved flow device is designed and / or configured for use as a disposable point-of-care cartridge or device.

[0041] In some embodiments, the present invention provides immunometric assays for determining the presence, concentration, or amount of a target substance in a sample, comprising forming a ternary complex of a first labeled binding agent, the target substance, and a second binding agent bound to a solid support, wherein the presence or amount of the substance in the sample is determined by measuring either the amount of labeled binding agent bound to the solid support or the amount of unreacted labeled binding agent. The immunometric assays are characterized by utilizing target-binding solid-phase particles (or other target-binding support materials), at least one aqueous phase or layer and at least one gas phase or oil phase or layer stabilized in close proximity to each other by the inclusion of a porous structure material associated with the aqueous phase or layer, gas phase or layer, oil phase or layer, or both. Other and / or alternative phases may be used or included (e.g., two oil phases with or without an aqueous phase or layer). In some embodiments, the solid phase is a paramagnetic particle, and improved assays include or use a magnet. In some embodiments, one or more of the binding agents is an antibody, an antibody fragment, an oligonucleotide, an aptamer, a peptide, a peptidomimetic, a natural or chemically modified antisense oligonucleotide, or other suitable agent that aids in target capture. In some embodiments, the immunometric assay is contained in a single container.

[0042] In some embodiments, the present invention provides a nucleic acid amplification test for determining the presence or amount of a target substance in a sample, including nucleic acid sequence amplification and sequence detection, characterized in that the test comprises at least one aqueous phase or layer and at least one gas or oil phase or layer stabilized in close proximity to each other by the inclusion of target-binding paramagnetic particles (or other target-binding carrier material), a porous structural material associated with the aqueous phase or layer, or a gas or oil phase or layer, or both, and optionally a magnet. In some embodiments, the nucleic acid amplification test is PCR or RT-PCR. In some embodiments, the nucleic acid amplification test is isothermal. In some embodiments, the isothermal nucleic acid amplification test is reverse transcription polymerase chain reaction (RT-PCR), nicking endonuclease amplification reaction (NEAR), transcription-mediated amplification (TMA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), clustered regularly interspaced short palindromic repeats (CRISPR), or strand displacement amplification (SDA). In some embodiments, the nucleic acid amplification test is contained in a single container.

[0043] In some embodiments of the present invention useful for performing one or more steps of an assay for detecting or measuring a target or target analyte, one or more of the phases or layers of a device or system may contain one or more of several different buffers. In some embodiments, one or more phases or layers include a coating buffer, a blocking buffer, a stabilizing buffer, a wash buffer, or act as or contain a sample diluent. In some embodiments, an antibody or antibody fragment is used to generate a detection signal. In some embodiments, an assay performed using a device, system, or method of the present invention includes a magnetically actuated immunoassay, in which movement or positioning of the target or target analyte is achieved using magnetic separation using magnetic particles. In some embodiments, the particles used in these embodiments are made from magnetite cores that are chemically modified by the attachment of antibodies or antibody fragments. In some embodiments, one or more or all components of the assay are used to isolate or purify a target or target analyte.

[0044] In some aspects, provided herein are methods for isolating targets from a sample. In some embodiments, provided herein are methods for isolating targets from a sample, comprising adding a sample to a system described herein and applying a magnetic force to the system. In some embodiments, the sample is contacted with paramagnetic particles before applying the magnetic force to the system. In some embodiments, PMPs are contacted with a biological sample before adding the biological sample to the system. Contacting the sample with the paramagnetic particles produces one or more target-PMP complexes, and applying the magnetic force to the system attracts the target-PMP complexes through the phases in the system toward the bottom surface of the container.

[0045] In some embodiments, the method further includes detecting a target in the biological sample. In some embodiments, the system further includes a reagent for detecting the target contained in the bottom surface of the container, and detecting the target includes attracting a complex of the target and the PMP to the reagent for detecting the target through the plurality of porous materials. In some embodiments, the reagent includes a reagent for a loop-mediated isothermal amplification (LAMP) or a reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay. In some embodiments, detecting the target includes detecting a signal generated during a LAMP or RT-LAMP assay. In some embodiments, the LAMP or RT-LAMP assay is a colorimetric assay or a fluorescent assay.

[0046] In some embodiments, the sample is a biological sample or a sewage sample. For example, the biological sample can be a nasopharyngeal sample, an oropharyngeal sample, an oral swab sample, an oral sponge sample, a nasal swab sample, a middle turbinate sample, or a saliva sample. In other embodiments, the sample is blood, cerebrospinal fluid, urine, tissue, biopsy tissue, or the like. Any type of sample containing or suspected of containing a target of interest is contemplated for use in the systems and methods of the present invention. In some embodiments, the biological sample is obtained from a subject suspected of having an infectious disease. In some embodiments, the subject is suspected of having a viral infection. For example, the subject may be suspected of having a viral upper respiratory tract infection. In some embodiments, the subject is suspected of having an infectious disease such as SARS-CoV2, SARS, coronavirus, rhinovirus, influenza virus, or respiratory syncytial virus. In some embodiments, the target comprises viral nucleic acid. For example, the target may comprise SARS-CoV-2 nucleic acid.

[0047] In some embodiments, the present invention provides stabilized interfacial systems, which include (a) at least two fluids, (b) at least one associated structural material having at least one pore, and (c) at least one fluid phase, layer, or interface stabilized by the associated structural material.

[0048] In some embodiments, the present invention provides compositions comprising a fluid phase, layer, or interface stabilized by a target-permeable structural material associated with and favoring the fluid phase, layer, or interface. In some embodiments, the structural material is selected to allow passage of one or more targets. In some embodiments, the structural material is selected to allow movement or positioning of one or more targets. In some embodiments, the present invention provides stabilized interface systems used to move or position targets.

[0049] In some embodiments, the present invention provides a method of stabilizing a fluid phase, layer, or interface in a system or device for positioning a target, comprising associating a target permeable stabilization structure with the fluid phase, layer, or interface.

[0050] In some embodiments, the present invention provides miscible interface systems, methods, and related compositions. In some embodiments, the miscible interface systems include compositions comprising two or more fluid domains with different properties within a phase or layer, where the fluid domains are stabilized relative to one another using a solid or semi-solid structure or material with at least one pore that allows mass transport of the fluid components by diffusion to dominate bulk fluid motion.

[0051] In some embodiments, the miscible interface system composition comprises two or more fluid domains with different properties within a phase or layer, the fluid domains being stabilized relative to one another using a solid or semi-solid structure or material having one or more pores, the stabilization allowing mass transport of the fluid components by diffusion to prevail over bulk fluid movement.

[0052] In some embodiments of the miscible interface systems, methods, and related compositions, the structural materials are selected to permit passage of one or more targets. In some embodiments, the structural materials are selected to permit movement or positioning of one or more targets. In some embodiments, the present invention provides miscible interface systems used to increase or position targets.

[0053] In some embodiments, for example, positioning using stabilized interfacial systems and / or miscible interfacial systems and / or related compositions can be achieved using active positioning (e.g., using external forces, including magnetic forces, rotational forces, acceleration forces, assisted gravity, etc.) In some embodiments, positioning can be passive and occur without the use of external forces (e.g., by diffusion, simple gravity, osmosis, etc.).

[0054] In some embodiments, the sample is a sample used to determine paternity, hi some embodiments, the sample is for use in prenatal or postnatal screening.

[0055] In some embodiments, a system or device of the present invention is Bluetooth enabled or enabled with another communication capability (e.g., WiFi, NFC, etc.). In some embodiments, system or device results or results from the methods described herein are transmitted to another device (e.g., phone, tablet, CPU, computer, etc.) via Bluetooth or other communication capability. [Brief explanation of the drawings]

[0056] [Figure 1]1 illustrates one embodiment of the system described herein. The system includes reagents for LAMP-based detection of a target contained on the bottom surface of a vessel. The system includes a plurality of (2) porous materials. The system includes a lysis buffer and a wash buffer. As shown in the figure, the system is layered from top to bottom in the following order: (1) coconut oil, (2) lysis buffer (with associated glass mesh), (3) coconut oil (with associated porous ("Porex") material), (4) wash buffer (with associated glass mesh), (5) coconut oil (with associated porous ("Porex") material), and (6) reagents for the LAMP reaction (with associated glass mesh). Each porous material may be a hydrophilic glass mesh. Alternatively, one porous material may be a glass mesh and the other porous material may be a synthetic hydrophobic polymer mesh. The biological sample may be mixed with PMP and then added to the vessel. A magnet is applied to the bottom of the vessel, thereby drawing the target-PMP complex through the layer and into contact with the LAMP reagent. The vessel is incubated at an appropriate temperature (e.g., 65°C) to perform the LAMP assay, after which the resulting signal can be measured. In this embodiment, the resulting signal is colorimetric. In some embodiments, the PMP is conjugated to a target binding agent, such as an antibody, antibody fragment, single-chain Fv, etc., and directed to the target and used as a PMP targeting agent. Embodiments of the present invention can include as many phases or layers as desired, each with or without associated structural materials (e.g., materials with desired porosity), including those structural materials favoring the phase or layer. [Figure 2A]FIG. 2A shows a side view of one embodiment of the system described herein. The vessel comprises a multi-well plate. One well contains stacked porous materials associated with oil (yellow). In the figure, the porous materials are a synthetic hydrophobic polypropylene polymer mesh and a hydrophilic glass mesh, referred to herein as "Porex." The system contains seven mesh materials. From top to bottom, the layers are as follows: (1) lysis buffer (blue aqueous layer) (stabilized by hydrophilic glass mesh), (2) Porex (stabilizes the oil phase), (3) wash buffer (red aqueous layer) (stabilized by glass mesh), (4) "Porex," (5) wash buffer (blue aqueous layer) (stabilized by hydrophilic glass mesh), (6) "Porex," and (7) LAMP reagents (red aqueous layer) (stabilized by hydrophilic glass mesh). [Figure 2B] Figure 2B shows bottom and top views of the system described in Figure 1 after application of paramagnetic particles and magnetic pulldown. All three of the systems shown contain oil (yellow) and glass mesh at the bottom of the well. A comparison is shown of three systems: (1) a synthetic polypropylene polymer mesh associated with oil (referred to as a "Porex pad"), (2) no porous material (e.g., aqueous or oil phase only; "+ control"), and a glass mesh and synthetic polypropylene polymer mesh ("glass mesh + Porex pad"). As shown, both the synthetic polypropylene polymer mesh (e.g., "Porex" pad) and the combination of the Porex pad and glass mesh allow beads to pass through the porous material. The "+ control" condition shows what 100% bead penetration would look like. [Figure 3] Bottom and top views after magnetic bead pulldown are shown when small holes are created in the glass mesh material. 1 mm holes resulted in significantly faster pulldown and larger bead clumps. 0.5 mm holes resulted in faster pulldown. [Figure 4]1 illustrates another embodiment of the system described herein. As in FIG. 1 , the system includes reagents for LAMP-based detection of a target contained in a final or terminal layer, e.g., a layer on or toward or at the bottom surface of the container. In this particular embodiment, the system includes a polytetrafluoroethylene (PTFE) O-ring to retain the LAMP reagents at the bottom of the container and to provide a film surface for the porous material to rest on. In this embodiment, the porous material includes a hydrophobic polypropylene (PP) mesh. The system includes multiple porous materials. In this example, two porous materials are shown (e.g., two layers). Each porous material can consist essentially of or consist of PP mesh, including PP mesh. Alternatively, for example, one material can include PP mesh (hydrophobic) and the other material can include glass mesh (hydrophilic). The system in this embodiment also includes a lysis buffer and a wash buffer. As shown in the figure, the system is composed of layers, from top to bottom, in the following order: (1) mineral oil, (2) lysis buffer (hydrophilic glass mesh / cloth), (3) porous material (hydrophobic PP mesh), (4) wash buffer (hydrophilic glass mesh), (5) porous material (hydrophobic PP mesh), and (6) PTFEO-rings and reagents for LAMP or other reactions. A biological sample is mixed with the target-binding PMP and then added to the vessel. A magnet is applied to the bottom of the vessel, thereby attracting the target-PMP complex through the layers and bringing it into contact with, for example, the LAMP reagents. The vessel is incubated at an appropriate temperature (e.g., 65°C) to perform the LAMP assay, after which the resulting signal can be measured. In this embodiment, the resulting signal is colorimetric. [Figure 5]Images of the system described in Figure 4 are shown, containing a PTFE O-ring holding LAMP reagents on the bottom surface of the vessel. As shown, the system labeled "+PP mesh + glass mesh" is composed of the following layers, from top to bottom: (1) mineral oil with a polypropylene mesh, (2) water with a glass mesh, (3) mineral oil with a polypropylene mesh, and (4) a PTFE O-ring holding LAMP reagents. As shown, the system labeled "+PP mesh" is composed of the following layers, from top to bottom: (1) mineral oil with a polypropylene mesh, and (2) a PTFE O-ring holding LAMP reagents. As shown, the system labeled "control" is composed of the following layers, from top to bottom: (1) a PTFE O-ring holding LAMP reagents. Images after bead pulldown are shown, showing that the target-PMP complex was pulled down to the center of the O-ring, thereby contacting the LAMP reagents. Red indicates the location of the LAMP reagents. [Figure 6]Another embodiment of the system of the present invention is shown. In this embodiment, the system includes a custom-made glycol-modified polyethylene terephthalate (PETG) insert at the bottom of the vessel to hold the LAMP reagents. The system includes multiple porous materials. In this example, two porous materials are shown (e.g., two layers). Each porous material may include, for example, a PP mesh. Alternatively, one material may include a PP mesh and the other a nylon mesh. The system includes a lysis buffer and a wash buffer. As shown in the figure, the system is composed of layers, from top to bottom, as follows: (1) mineral oil, (2) aqueous lysis buffer (stabilized by a hydrophilic nylon mesh), (3) porous material (e.g., a PP mesh) associated with mineral oil, (4) aqueous wash buffer (stabilized by a nylon mesh), (5) porous material (e.g., a PP mesh) associated with mineral oil, and (6) reagents for the LAMP reaction held within the PETG insert. A sample, e.g., a biological sample, is mixed with PMP, which binds the target if present in the sample, and then added to a container. A magnet is applied to the bottom of the container, thereby attracting the target-PMP complex through the layer and contacting it with the LAMP reagent. The container is incubated at an appropriate temperature (e.g., 65°C) to perform the LAMP assay, and the resulting signal can then be measured to determine the amount of target, if any. In this embodiment, the target is present and the resulting signal is colorimetric. [Figure 7] 1 shows the results of a colorimetric LAMP assay after magnetic bead pulldown using an embodiment of the system described herein. The biological sample was mixed with PMP and added to the system (referred to as "bead-derived template"). Data are compared to a control and a control plus PETG insert. As shown, the system enabled sufficient pulldown of the target-PMP complex (visible as a brown spot in the well) and successful colorimetric LAMP-based detection of the target. [Figure 8]1 shows the results of a colorimetric LAMP assay to determine the limit of detection (LOD) using primers for SARS-CoV-2. [Figure 9] Images showing successful target-PMP complexation from a saliva sample are shown. The system was equipped with a PETG insert. The porous material comprised polypropylene and nylon mesh. The saliva sample was diluted, and lysis buffer was added directly to the sample. The sample was heated to 55°C for 15 minutes, followed by heating to 98°C for 3 minutes. The sample was cooled to room temperature, mixed with PMP, and added to a vessel (e.g., a well of a multi-well plate containing the porous material and wash buffer). A magnet was applied to the bottom of the vessel to attract the target-PMP complex through the purification layer. [Figure 10]1 is a schematic diagram outlining one embodiment of the system and method of the present invention described and claimed herein. A vessel containing reagents for LAMP-based detection of a desired target housed in the bottom surface of the vessel can be prepared. The reagents can be secured to the bottom surface by suitable means, including inserts (e.g., PETG inserts) or O-rings. The system includes a wash buffer and multiple stacked porous materials within the vessel. From top to bottom, the system includes: (1) a polypropylene mesh associated with mineral oil, (2) a wash buffer associated with nylon mesh, (3) a polypropylene mesh associated with mineral oil, (4) a wash buffer associated with nylon mesh, (5) a polypropylene mesh associated with mineral oil, and (6) a PETG insert with LAMP reagents. The vessel may be prepackaged in a multiwell plate, with each well of the plate containing the contents of a single vessel. This multiwell plate can be packaged in a kit. The system further includes a magnet. In this case, the magnets are in an array such that each magnet in the array aligns with a single well in the multiwell plate. The biological sample is lysed, mixed with paramagnetic particles, and added to a multi-well plate. A magnetic array is placed in a suitable location adjacent to the bottom of the plate, and the target-PMP complex is drawn through the purification layer (e.g., through the porous material and wash buffer) and contacted with the LAMP reagents. The plate is incubated at 65°C, and the signal (e.g., colorimetric, fluorescent, etc.) is measured. [Figure 11A]

[0043] Figure 11A shows an embodiment of a system of the present invention for isolating and detecting an analyte. Figure 11A shows paramagnetic particles in an aqueous phase (i). Application of a magnetic force under the system pulls paramagnetic particles (e.g., target-PMP complexes) from the oil phases (ii, iii, and iv) toward the bottom surface of the system. Figure 11B shows a vessel holding the system. The bottom surface of the vessel contains reagents for analyte detection (shown in red). The aqueous and oil phases are stabilized by forces including buoyancy and fluid retention forces (e.g., wetting, surface tension, capillary action). Figure 11C shows an exemplary process for isolating and detecting an analyte using an embodiment of the system of the present invention described and claimed herein. [Figure 11B]

[0043] Figure 11A shows an embodiment of a system of the present invention for isolating and detecting an analyte. Figure 11A shows paramagnetic particles in an aqueous phase (i). Application of a magnetic force under the system pulls paramagnetic particles (e.g., target-PMP complexes) from the oil phases (ii, iii, and iv) toward the bottom surface of the system. Figure 11B shows a vessel holding the system. The bottom surface of the vessel contains reagents for analyte detection (shown in red). The aqueous and oil phases are stabilized by forces including buoyancy and fluid retention forces (e.g., wetting, surface tension, capillary action). Figure 11C shows an exemplary process for isolating and detecting an analyte using an embodiment of the system of the present invention described and claimed herein. [Figure 11C]

[0043] Figure 11A shows an embodiment of a system of the present invention for isolating and detecting an analyte. Figure 11A shows paramagnetic particles in an aqueous phase (i). Application of a magnetic force under the system pulls paramagnetic particles (e.g., target-PMP complexes) from the oil phases (ii, iii, and iv) toward the bottom surface of the system. Figure 11B shows a vessel holding the system. The bottom surface of the vessel contains reagents for analyte detection (shown in red). The aqueous and oil phases are stabilized by forces including buoyancy and fluid retention forces (e.g., wetting, surface tension, capillary action). Figure 11C shows an exemplary process for isolating and detecting an analyte using an embodiment of the system of the present invention described and claimed herein. [Figure 12]A diagram of a point-of-care (POC) single-use system described herein is shown. This embodiment includes reagents for LAMP-based (or RT-LAMP) detection housed in the bottom surface of a container. In this embodiment, the bottom surface of the container includes a septum that divides the bottom of the container into multiple wells that can be filled with reagents for LAMP-based detection, thereby assaying for different portions of the target, i.e., spatially separated multiplexes. In this embodiment, from top to bottom, the system includes a lysis / binding buffer with PMPs ("extraction buffer + PMPs"), solidified wax with a polypropylene mesh, and LAMP reagents. In this embodiment, the sample is a biological sample, such as saliva, containing the target or suspect. In this embodiment, the sample is added to the lysis / binding buffer containing PMPs and mixed. Upon heating above the melting temperature of the wax and applying a magnetic field to the bottom of the container, the wax melts, allowing the target PMPs to be drawn down and contact the multiple LAMP reagents. The container can be incubated at an appropriate temperature (e.g., 65°C) and the LAMP assay can be performed. [Figure 13]A photograph (left) and cutaway view (right) of one embodiment of the system described herein are shown. This embodiment of a single-use point-of-care (POC) system contains reagents for LAMP-based (or RT-LAMP) detection (green) contained on the bottom surface of a container. The system includes a meltable wax layer (yellow) and multiple porous polypropylene materials ("mesh") (gray). The system contains a lysis / binding buffer with PMP ("PMP / sample mixture"). As shown in the figure, the system is composed of layers in the following order from top to bottom: lysis / binding buffer with PMP, porous materials, and reagents for the LAMP reaction. Each porous material can be a polypropylene mesh or the like. Alternatively, one porous material can be a nylon mesh and the other porous material can be a synthetic hydrophobic polymer mesh or the like. A sample, e.g., a biological sample, can be added to the container. A magnet is applied to the bottom of the container, thereby drawing the target-PMP complex through the layers and into contact with the LAMP reagents. The vessel can be incubated at an appropriate temperature (eg, 65° C.) to perform the LAMP assay. [Figure 14]13 illustrates another embodiment of the system described herein. Similar to FIG. 13, in this embodiment, which is a POC, a single-use system is shown with the corresponding workflow from sample acquisition to LAMP reconstitution. In this embodiment, lyophilized LAMP (or RT-LAMP) is contained on the bottom surface of a container, either in bead form or dispensed on the bottom surface of the container. The system includes a meltable wax layer (yellow), a LAMP reconstitution buffer, a plurality of porous polypropylene materials ("mesh") (gray), and a lysis / binding buffer with PMPs ("PMP / sample mixture"). In some embodiments, PMPs and the salt component of the lysis / binding buffer are frozen into the meltable wax layer, and the sample reconstitution buffer is separately contained in the device such that upon melting of the wax, the reconstitution buffer, salt component, and PMPs are combined. In some embodiments, a pierceable membrane is attached to the top of the container. In this embodiment of the workflow, biological samples (e.g., saliva, sputum, urine, blood, etc.) are collected in separate tubes. The tube and vessel (containing the LAMP reagents and the plurality of porous materials) are then attached together, and the puncturable membrane membrane is pierced, allowing the lysis / binding buffer ("sample buffer") and PMP to mix with the biological sample. Upon inversion of the system and heating the system above the melting temperature of the wax, the LAMP mixture and LAMP reconstitution buffer mix. [Figure 15]1 is a schematic diagram outlining one embodiment of the systems and methods of the present invention described and claimed herein. In this embodiment, the system contains reagents for detecting a target in a biological sample (e.g., saliva, sputum, urine, blood, cell culture medium, etc.) using an enzyme-linked immunosorbent assay (ELISA). From top to bottom, the system comprises a primary antibody binding buffer consisting in part of an antibody-conjugated PMP with a nylon porous material, mineral oil with a polypropylene porous material, a secondary conjugated antibody binding buffer (or buffer) containing a secondary antibody conjugated to an enzyme (e.g., horseradish peroxidase), a solidified wax layer with a polypropylene porous material, and a substrate solution containing an enzyme substrate (e.g., 3,3',5,5'-tetramethylbenzidine). In this embodiment, the biological sample is added to the top of the system and mixed with the primary antibody binding buffer, allowing the target to bind to the primary antibody. A magnetic field is applied to the bottom of the container, which draws the target-PMP complex through the mineral oil layer into the secondary conjugated antibody binding buffer. When the temperature is above the freezing temperature of the secondary conjugate antibody binding buffer and below the melting temperature of the wax layer, the target-PMP complex remains in the secondary conjugate antibody binding buffer until the temperature is raised above the wax melting temperature. Incubation in this layer allows the secondary conjugate antibody to bind to the target-PMP complex. In this embodiment, when the temperature is raised above the melting point of the wax, the conjugate secondary antibody-target-PMP complex is drawn into the substrate solution. Once in the substrate solution, the conjugate secondary antibody-target-PMP complex can catalyze an enzymatic reaction on the substrate, thereby allowing the target to be detected. [Figure 16]1 is a schematic diagram outlining one embodiment of the systems and methods of the present invention described and claimed herein. In this embodiment, the system contains reagents for isolating and detecting cell-based targets (e.g., circulating tumor cells (CTCs), neutrophils, T cells, mesenchymal stem cells, etc.) in a biological sample (e.g., saliva, sputum, urine, blood, cell culture medium, etc.). In this embodiment, the cell-based targets are CTCs. From top to bottom, the system includes a CTC binding buffer consisting in part of an antibody-conjugated PMP with a nylon porous material, mineral oil with a polypropylene porous material, a fluorescent antibody binding buffer consisting in part of an antibody conjugated to a fluorophore (e.g., green fluorescent protein (GFP), red fluorescent protein (RFP), etc.), a solidified wax layer with a polypropylene porous material, and an aqueous solution (e.g., phosphate-buffered saline, etc.). In this embodiment, a biological sample containing target cells is added to the top of the system and mixed with the CTC binding buffer, allowing the target cells to bind to the antibody-PMP. A magnetic field is applied to the bottom of the container, which draws the target-PMP complex through the mineral oil layer and into the fluorescent antibody binding buffer. If the temperature is above the freezing temperature of the fluorescent antibody binding buffer and below the melting temperature of the wax layer, the target-PMP complex will remain in the fluorescent antibody binding buffer until the temperature is raised above the wax melting temperature. Incubation in this layer allows the fluorescent antibody to bind to the target-PMP complex. In this embodiment, when the temperature is raised above the melting point of the wax, the fluorescent antibody-target-PMP complex is drawn into the water. Once in aqueous solution, the target cells can be counted using a fluorescent microscope. [Figure 17]Figure 17a shows the phases and layers in the tube and the mesh-stabilized interface. Figure 17a shows an oil-stabilized phase or layer (yellow) contained within a test tube, depicted as a "mesh-stabilized interface," above an unstabilized water phase or layer (blue). When a second aqueous fluid ("water (dyed red)") (red), which is a denser fluid than oil, is added to the system, the oil and "red" water phases invert but do not mix with the bottom water layer due to the presence of the stabilized oil layer. In some embodiments, another stabilized oil layer can be added to the system of Figure 17a, trapping the bulk of the oil in the test tube between the two oil layers, as depicted in Figure 17b. [Figure 18] 1 shows an embodiment of the system described herein. The system includes a stabilized hydrophobic layer (yellow) in a sealed container with a charge-coupled device (CCD) or other optical detector in close proximity to a porous structural material. The porous structural material, immersed in a hydrophobic fluid, has regularly arranged circular pores. By adjusting the volume of hydrophobic fluid in the system, a complex set of optical lenses can be formed. The magnification of light can be increased or decreased depending on the volume of hydrophobic fluid in the system. [Figure 19]

[0023] Figures 1A-1C show examples of different SIFT configuration embodiments, including single and multiple stabilizing aqueous or oil phases or layers. These figures are shown as a single cross section, in other words, the cross section does not have to remain constant in dimensions on and off the page. Twists or bends of cross-sectional elements, for example, or changes in the position or dimensions of cross-sectional elements, can occur at different cross-sectional locations. [Figure 20]This section describes a fluid-loving porous structural material immersed in a volume of preferred fluid that is unsaturated, saturated, and supersaturated. If the volume of preferred fluid is insufficient to associate with the material throughout the material, it is considered an unsaturated volume. When unsaturated, the preferred fluid typically remains supported by the porous structural material, while unpreferred fluid can potentially fill the remaining space within the porous support structure. If the preferred fluid can associate with the material throughout the material, the volume of preferred fluid is considered saturated. A volume of preferred fluid that exceeds the saturated volume is considered a supersaturated volume, resulting in fluid not being completely contained and / or supported by the porous structural material. The region of preferred fluid above or below the porous structural material is generally less stable than the supported volume of preferred fluid and is subject to convective or turbulent forces or bulk mixing. [Figure 21A] 1 shows one embodiment of a device described herein that includes a container containing one or more stabilized phases or layers (blue planar mesh configuration) useful for performing the assays described herein, above an external movable magnet or magnetic device, a space or cavity for receiving a magnetic field or force. [Figure 21B] 1 shows one embodiment of a device described herein that includes an external movable magnet or magnetic device, a container that includes one or more stabilized phases or layers (blue torus mesh configuration) that surround a space or cavity for receiving a magnetic field or force. [Figure 22]FIG. 22a shows one embodiment of a device comprising a screw-on cartridge (FIG. 22a) including a body / enclosure with a top and bottom, paramagnetic particles in a sample buffer, a SIFT mesh (hydrophobic), a space or cavity or other central portion for application of magnetic force or insertion of a movable magnet, a hydrophobic phase (e.g., oil, wax, etc.), and reagents for performing an assay (e.g., RT-LAMP reagents or RT-qPCR reagents for target amplification and identification). FIG. 22b shows a magnet (e.g., a permanent magnet) inserted into the magnet cavity by an electromagnet. FIG. 22c and FIG. 22d show paramagnetic particles accumulating on the SIFT mesh (FIG. 20c) around the central dome-shaped feature (FIG. 20d). FIG. 22e shows the electromagnet initiating the magnet's fall, pulling the beads toward the SIFT mesh. FIG. 22f shows the paramagnetic particle packet flattening after contact with the stabilized interface (SIFT) mesh, and the bead packet moving through the stabilized interface(s). Figures 22g and 22h show the permanent magnet descending and continuing to pull the PMP packet through the mesh. [Figure 23A] One embodiment of the device with multiplex capabilities is shown, including a screw cap and magnetic cavity for inserting the sample, in this case a swab, directly into a buffer that may also contain paramagnetic particles. [Figure 23B] 24 shows the bottom of the device of FIG. 23, which is a six-zone multiplex (hexaplex) device, and the opening of the cavity for inserting the magnet. [Figure 24] A premixed (non-lyophilized) embodiment of the magnet cavity device shown in Figure 23 is shown. "Premixed embodiment" refers to an aqueous RT-LAMP assay format that, in this particular embodiment, includes all the reagents (i.e., primers, salts, polymerase, etc.) needed to perform the assay, provided and already in solution. The bottom-most aqueous fraction, consisting of the RT-LAMP assay, is already premixed and requires only the target nucleic acid to proceed. [Figure 25]23 shows an embodiment of the lyophilized reagents in the device, in which the RT-LAMP assay is divided into an aqueous fraction called "LAMP reconstitution buffer" and a "lyophilized RT-LAMP" consisting of lyophilized reaction components. The two components are separated by a single hydrophobic wax layer, and the RT-LAMP assay reagents are reconstituted at the time of use of the device. [Figure 26] 26 illustrates an embodiment of a magnet cavity-containing device of the present invention that is sonicated and heated by another device, e.g., a reader or other device having sonication and temperature control systems and capabilities. The first image in FIG. 26 illustrates a device receiving ultrasound within the device, e.g., a reader. The second image in FIG. 26 illustrates a device receiving heat by conduction and / or convection. Heat can be generated using any desired means, including, e.g., infrared, electricity, ultrasound, etc. The third image illustrates the use of a force (e.g., electromagnetic force) to control the position of a magnet (e.g., a permanent magnet) within a cavity, hollow, space, etc., that is open to the outside at the bottom of the device. In this third image, the cavity of the device can extend to the bottom of the stabilized interface and / or SIFT mesh. After a dwell time during which a magnet, e.g., a permanent magnet, draws the magnetic bead / target (e.g., PMP / target) complex across the interface and, in this particular embodiment, into the wax / oil phase, the force (e.g., electromagnetic force) is adjusted to allow the magnet to fall, in this embodiment attracting the PMP / target complex and / or allowing the PMP / target complex to fall or move into the RT-LAMP reaction components. [Figure 27] 21B is a graph of the results of an experiment showing that PMP+ targets are translocated and evenly divided in a multiplex device of the invention (here, a hexaplex device) after bead pulldown through a SIFT-stabilized interface (in this case, a donut / torus mesh embodiment according to FIG. 21B) using fluorescent RT-LAMP against SARS-CoV-2 as readout. [Figure 28]Colorimetric RT-LAMP in a multiplex (hexaplex) device of the present invention (non-doughnut / torus embodiment according to Figure 21A) is shown. Colorimetric RT-LAMP primers targeting either SARS-CoV-2 (positive control) or RNaseP (negative control) were added to alternating wells of the hexaplex device. SARS-CoV-2 virions were added as sample (no RNaseP template was present), and the SARS-CoV-2 target was delivered by PMP bead jumping. The "before" photograph shows the device and wells before the RT-LAMP reaction. The "after" photograph shows wells after the RT-LAMP reaction, with "-" indicating results from a negative control RT-LAMP assay (primers targeting RNaseP) and "+" indicating results from a positive control RT-LAMP assay (primers targeting SARS-CoV-2). [Figure 29] 21A shows results from a PMP "jumping" test in a vacuum thermoformed hexaplex Multipex assay device of the present invention (donut / torus embodiment according to FIG. 21B). [Figure 30] 1 illustrates an embodiment of a device having a core that can receive or generate a magnetic field or force. DETAILED DESCRIPTION OF THE INVENTION

[0057] definition As used herein, the term "container" refers to any device, receptacle, or vessel capable of holding a system of the present invention, including any device, receptacle, or vessel in which a method of the present invention may be performed. In some embodiments, the vessel is a cylinder. In some embodiments, the vessel is portable. Containers and vessels include, for example, any vessel, receptacle, receptacle, holder, carrier, cartridge, bottle, plate(s), well(s), or storage device capable of holding a described system. In some embodiments, the vessel is a label or an injection-molded vessel with embossed lettering at the time of manufacture to eliminate some or all of the need for an external label. In some embodiments, the vessel is a disposable or single-use vessel. The vessels and vessels may be cooled or heated, or capable of being cooled or heated, either externally or by built-in or added internal means. The vessels and vessels may provide stability and maintenance of fluids, including one or more phases and layers, of the present invention during manufacture, storage, and transportation. The vessels and vessels may provide for the movement of fluids during use of the systems and methods of the present invention. Receptacles and vessels that can house the systems of the invention or that can be used to perform the methods of the invention include reaction plates and microtiter plates, including 24-well PCR plates, 96-well plates, and 384-well plates, as well as other plate formats. The receptacles and vessels can provide utilities, including the use of electrical, optical, mechanical, and liquid interfaces, as well as detection and quantification reagents. In some embodiments, the vessels include a top opening to allow the addition of a sample to the vessel. In some embodiments, the first aqueous phase is closest to the top opening of the vessel. In some embodiments, the first oil phase is closest to the top opening of the vessel. In some embodiments, the first aqueous phase comprises a lysis buffer. In some embodiments, the second aqueous phase comprises a wash buffer. In some embodiments of the systems or devices of the invention, the vessel or construct containing the system does not have an integral bottom. In some embodiments, the vessel has only sides and is open at both ends. In one such embodiment, the vessel is an insert, an example of which is shown in Figures 11B and 11C.In such embodiments, the sample may be added to a layer or layer designated as the "first" or "sample-receiving" phase or layer. In the example insert shown in FIG. 11C, the terminal layer is a porous plastic screen. The terminal layer of a device or system of the invention that allows for sample addition to a container or device that does not contain a bottom integral with its sides can be a mesh or any porous material that holds the system and allows it to be performed as described. In some embodiments, the open end is used to allow removal of targets or target-binding particles (e.g., PMPs) from the system (e.g., using a magnet). In some embodiments, the targets or target-binding particles are removed to another container or vessel (e.g., a multi-well plate), or to or onto a detector (e.g., a reader, Bluetooth-enabled reader or instrument, etc.) that can accept the targets or target-binding particles, or to or onto a surface or porous material (e.g., a spot card for drying and transport of samples for subsequent analysis, etc.).

[0058] As used herein, the terms "phase" or "layer" are used interchangeably and refer to a region of material (stabilized or unstabilized, as described herein) bound by one or more other materials. Phases or layers include aqueous layers, oil layers, gas layers, emulsion layers, particle suspension layers, and stabilized versions of such layers or other layers used in systems, devices, or methods. An example of a phase or layer would be a volume of air surrounded by water. Water and air are miscible (per Henry's law), but the liquid form of water and the gaseous form of air are generally not considered substantially miscible. The stabilized phases or layers described herein are compositions of the present invention. Stabilized phases or layers include the devices and systems of the present invention and are used in the methods of the present invention.

[0059] As used herein, the term "oil" refers to any of a number of substances, usually liquid or semi-solid, that are insoluble in water. This substance is sometimes greasy, sometimes derived from plant, animal, or mineral sources, but can also be non-greasy. Oils include carbon and silicone-based polymer compounds, mineral oil, silicone oil, paraffin wax, and fluorinated oils, etc. Oils also include mixtures of oils (e.g., waxes with different melting temperatures; polymer oils with different chain lengths; mineral oil and silicone oil; etc.). Oils also include oil-oil emulsions.

[0060] As used herein, the term "oil layer" or "oil phase" refers to a layer in the system of the present invention that contains oil, is substantially hydrophobic, and is substantially immiscible with the aqueous layer. Suitable oil layers in the system of the present invention include, for example, mineral oil, coconut oil, and vegetable oil. As mentioned above, other oils include carbon and silicone-based polymer compounds, mineral oil, silicone oil, paraffin wax, and fluorine-based oils.

[0061] As used herein, the term "interface" refers to a surface that forms a common boundary or transition between adjacent regions, bodies, materials, phases, or layers. In some embodiments, an interface refers to a point or transition where separate phases or layers in the systems, devices, and methods of the present invention meet one another. For example, a transition exists between water and air due to the miscibility of water with air (i.e., due to Henry's law and diffusion-based mixing), transitioning from liquid water to air saturated with water to air with some level of non-saturation of water. An example of a transition within a phase or layer is water with different levels of salinity, where differences in density allow for regions with different properties with a transition between regions with intermediate levels of salinity.

[0062] As used herein, the term "aqueous" means water-based, containing water, or using or having water as an ingredient. In some embodiments, an aqueous medium or region contains water and other ingredients. Lysis buffers, wash buffers, and the like can comprise the aqueous phase described in embodiments of the systems and methods of the present invention. In some embodiments, the water-based medium contains various concentrations (e.g., 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, 99.9%) of other water-soluble substances, such as salts or ionic liquids (e.g., ammonium sulfate, guanidine isothiocyanate, cyanine dyes, tetraethylammonium, and tetrabutylammonium), polar solvents (e.g., ethanol, phenol, methanol, acetonitrile), acids / bases (e.g., sulfuric acid, acetic acid, sodium hydroxide), sugars (e.g., sucrose, glucose, mannose), or polymers (e.g., polyethylene glycol, hyaluronic acid, chitin, collagen I, etc.).

[0063] In some embodiments, the phase(s), layer(s) or region(s) in the devices, systems, methods and compositions of the present invention may be an emulsion, e.g., a dispersion of droplets of one liquid in another liquid in which it is not soluble or miscible.

[0064] In some embodiments, non-oily liquid phases or layers may be used that are not water-based, for example, 100% ethanol, phenol, acetonitrile, or other compatible solvents. These may be referred to as "non-oily / non-aqueous" phases or layers.

[0065] An aqueous layer, phase, or region is a water-based layer, phase, or region. An aqueous layer comprises water. As used herein, in some embodiments, the term "aqueous layer" or "aqueous phase" refers to an aqueous region bounded or surrounded by any "non-aqueous" material, such as device plastic, atmospheric / gaseous material, oil, wax, etc. In some constructions, the aqueous layer need not be homogeneous and can have transitions between multiple aqueous regions with different properties within a single aqueous layer; for example, a layer may contain two aqueous medium regions with different densities.

[0066] As used herein, the term "porous" means having pores or other narrow spaces capable of retaining gas or liquid, or allowing targets (or non-targets) as defined herein (whether bound or not to a solid phase or other support or portion thereof) to pass or not pass as desired. Reference to a "porous material" or structure or a "porous mesh" or "porous layer" refers to a material that includes empty space, i.e., space not occupied by the main backbone of atoms that make up the structure of the material. A material through which targets (whether bound or not to a solid phase) pass is an example of a porous material. A material that is not a target (whether bound or not to a solid phase) and through which non-target materials do not pass is also an example of a porous material. A porous material or structure, porous mesh, or porous layer need not be constructed of or consist of a single material, i.e., it need not be homogeneous. A porous material or structure, porous mesh, or porous layer for use in the systems, devices, methods, and compositions of the present invention may comprise different materials, i.e., it may be heterogeneous or non-homogeneous (e.g., in one embodiment, it comprises polystyrene and nylon or a spatially variable mixture).

[0067] As used herein, the terms "detect," "detecting," or "detection" can describe either the general act of discovery or identification or the specific observation of a detectably labeled composition. The term "detecting," when used in reference to a target in a sample, refers to detecting either the presence or absence of the target in the sample. In some embodiments, the term "detecting" a target in a sample refers to determining that the target is present in the sample. In some embodiments, "detecting" a target in a sample refers to determining that the target is not present in the sample or is not present in a sufficient amount in the sample to be detected.

[0068] As used herein, the term "biological sample" is used in the broadest sense and encompasses many sample types that can be obtained from a subject. Biological samples can be obtained from animals (including humans) and can include fluids (e.g., urine, blood, blood products, sputum, saliva, etc.), solids, tissues (including biopsy tissue, tumor tissue, bone marrow, etc.), and gases. Biological samples include blood products such as saliva, plasma, and serum. In some embodiments, the biological sample is a nasopharyngeal sample, an oropharyngeal sample, an oral swab or sponge sample, a nasal swab sample, a middle turbinate sample, or a saliva sample. In some embodiments, the biological sample is a saliva sample. As used herein, the term "saliva sample" includes, for example, a saliva sample collected from a subject. In some embodiments, the biological sample is a nasopharyngeal (NP) sample. A "nasopharyngeal sample" refers to a sample collected from the nasopharyngeal cavity of a subject, including, for example, a specimen collected using a swab inserted into the nasal cavity or nasopharynx of a subject. The biological sample may be subjected to various pretreatment steps before undergoing the methods described herein. For example, the biological sample may be frozen, heated, mixed with various denaturants (e.g., guanidinium thiocyanate), mixed with viscosity-reducing reagents (e.g., DTT), mixed with inhibitors of target degradation (e.g., protease inhibitors, RNAse inhibitors, etc.), mixed with various buffers, or subjected to other suitable pretreatment steps. Any of the substances added to the biological sample (e.g., denaturants, viscosity-reducing reagents, inhibitors of target degradation, buffers, etc.) may be added to the biological sample or may be present in a storage buffer present in the container from which the sample is collected (e.g., present in a storage buffer in a sample collection tube or other collection device or container). In some embodiments, the sample contains or is suspected of containing a microorganism (e.g., a live or attenuated pathogenic or disease-causing microorganism).

[0069] As used herein, the term "sample" is used in the broadest sense and encompasses many sample types. In some embodiments, a "sample" is a "biological sample," as described above. In other embodiments, a sample may be an environmental sample, such as a sewage sample, which is useful for environmental and wastewater-based epidemiology. Thus, in some embodiments, a "sample" refers to a portion of a substance taken or selected from a larger volume of material. In some embodiments, a sample refers to any substance that contains or is suspected of containing a target. In some embodiments, a sample is a whole volume of a substance, such as blood. In some embodiments, a sample is blood, cerebrospinal fluid, urine, tissue, biopsy tissue, etc. Any type of sample that contains or is suspected of containing a target of interest is contemplated for use in the systems and methods of the present invention.

[0070] As used herein, the term "preference" in the context of two fluids interacting with a substrate, e.g., a mesh, can be defined using the contact angle of the interfacial fluid with the substrate. For example, the association of a fluid with a solid surface substrate at an interface is determined by the surface properties of the substrate and the chemical properties of the two fluids, which may be liquid-liquid, liquid-gas, or gas-gas. The contact angle for a fluid-fluid-material combination is routinely used to quantify the equilibrium of this interaction and can be affected by many factors, such as temperature, pressure, and surface charge. The contact angle is the angle between the surface of the substrate and the tangent to the fluid interface where the fluid interface intersects the substrate. A "preferred" or preferred fluid is one with a contact angle of less than 90°. In some cases, these preferences are hydrophobic and hydrophilic in nature. Some can be classified based on contact angle: for example, (i) superhydrophilic (0°≦θ<10°), (ii) hydrophilic (10°≦θ<90°), (iii) hydrophobic (90°≦θ<150°), and (iv) superhydrophobic (150°<θ≦180°). Generally, superhydrophobic surfaces exhibit water contact angles greater than 150° and sliding angles less than 5°. These preferences are also relevant to other embodiments of the present invention, which provide stabilized interface systems and methods and related compositions. Substrates, support materials, meshes, and porous substrates and porous support materials and meshes can be selected based on preferred interfaces and associated with one or more fluids. This includes fluid preference selection, which affects whether the mesh and fluid can be associated to achieve functional performance. Depending on the conditions, which are the nature of the fluid or material, the preference of the material for the fluid(s) may also change or be altered (e.g., materials may be selected that have different preferences for different layers or conditions, such as, for example, PP-water-mineral oil vs. PP-water-silicone oil, PP-water-oil at 20° C. vs. PP-water-oil at 65° C.). In some embodiments, fluids (e.g., oil added to displace air) or conditions (e.g., temperature) may be changed, swapped out, or interchanged during or as part of the use of the methods of the invention. For example, an aqueous phase or layer may be replaced with an oil phase or layer, or the temperature may be changed for performance or ease of use.

[0071] Some preferred substrates are porous materials. Some preferred substrates are porous structural materials. Some preferred substrates are meshes. Some porous materials, porous structural materials, and meshes are hydrophilic. Some porous materials, porous structural materials, and meshes are selected for their degree of hydrophilicity. Some porous materials, porous structural materials, and meshes are hydrophobic. Some porous materials, porous structural materials, and meshes are selected for their hydrophobicity.

[0072] As used herein with respect to components of a system or device described herein, the term "stabilized" indicates that the components maintain functionality for their intended purpose(s) or remain immiscible with one another over the course of transportation, storage, and / or use of the system. "Immiscible" refers to components that do not naturally or typically form a homogeneous mixture. Generally, a stabilized component (e.g., a phase or layer) remains functionally separate from another component (e.g., another phase or layer). Functionally separate refers to a phase or layer that itself performs or continues to perform a function in the system, method, or device of the invention. For example, the aqueous and oil phases or layers of the systems described herein are typically immiscible with one another (e.g., the phases or layers remain substantially separate from one another and do not form a homogeneous mixture). "Stabilized" can also be used to indicate that this immiscibility of the aqueous and oil and / or gas phases or layers remains throughout the life of the system or device or during the performance of a method. For example, the support structure used to stabilize a layer may substantially dissolve during use (e.g., sucrose is first dried onto a mesh structure after wetting with the addition of an aqueous medium), thereby altering the fluid's association with the structure in a desirable manner and potentially altering the fluid's preferences. An interface may be a common boundary between phases or layers, a transition between phases or layers, a transition within a phase or layer, etc. Thus, a "stabilized" region within a phase or layer need not be "immiscible." A region within a phase or layer may also be considered stabilized if the components form a stable transition within the layer or phase.

[0073] As used herein, a "stabilized layer or phase" refers to a layer or phase (e.g., an aqueous layer or phase, an oil layer or phase, or a gas layer or phase) associated with a support structure that favors the fluid of that layer or phase relative to at least one other fluid. The structure's preference for an associated layer or phase stabilizes the layer or phase (e.g., fluid) in association with the support structure and helps prevent the phase or phase from becoming disrupted during interactions with the other phase(s) or layer(s). Thus, other potentially disruptive phase(s) or layer(s) or fluid(s) need not be part of the layer or phase system or be located immediately adjacent to the layer or phase, but only occasionally associate with the layer or phase (e.g., stabilization may be used as a safety measure against unintended interactions with fluids not typically in the system or when adding foreign substances, e.g., a sample, to the system). In some embodiments, the support structure of a stabilized layer is inherently porous, thereby allowing at least some substances, e.g., more than one desired substance, to pass through the structure. The support structure does not necessarily have to be fixed or bound in orientation or position, but serves primarily to facilitate the association of a preferred fluid with the structure. Furthermore, the support structure need not be permanently associated with a particular phase or layer, but can be repositioned for removal from the system or associated with a different phase or layer. A phase or layer can be repositioned, for example, by manipulating the solid substrate, so that it is forcibly removed from the phase or layer with which it was associated. This can be done, for example, to isolate a negatively selected target. Similarly, the stability provided by the support structure allows the stabilized phase or layer to be repositioned or reoriented, or to pass through other fluids, if necessary.

[0074] As used herein, "associated" means, in whole or in part, involved, combined, or linked. The term "associated" includes functionally associated or linked. In some embodiments, for example, a substrate, solid phase, or structural material (e.g., a mesh) is associated with a phase or layer if it provides a stabilizing function to the phase or layer wherever the substrate, solid phase, or structural material is disposed or positioned within a system or device of the invention in association with the phase or layer. A substrate, solid phase, or structural material need not be immersed within a phase or layer to be associated with or to provide a stabilizing function to the phase or layer.

[0075] As used herein, the term "immersed" means entirely or partially below the surface of a liquid. Thus, for example, a mesh may be entirely immersed in or within a liquid phase or layer, or may be at, near, or on the surface of, or only partially within, a phase or layer, but not entirely. A phase or layer may comprise or essentially consist of a porous substrate, solid phase, or structural material. A phase or layer may be a porous substrate, solid phase, or structural material that comprises or essentially consists of an oil phase or layer, or an oil layer or phase may comprise or essentially consist of a porous substrate, solid phase, or structural material. In some embodiments, for example, a mesh (an example of a porous substrate, solid phase, or structural material) may be entirely or partially submerged in oil, or an oil phase or layer may comprise or consist essentially of oil within the mesh. In some embodiments, for example, a mesh (or other porous substrate, solid phase, or structural material) may be entirely or partially submerged in an aqueous liquid phase or layer, or an aqueous phase or layer may comprise an aqueous liquid within the mesh.

[0076] As used herein, the term "stack" or "stacked" refers to materials (e.g., aqueous phase or layer, oil phase or layer, gas phase or layer, porous material, hydrophobic mesh, hydrophilic mesh, etc.) in a system disclosed herein that are axially aligned (or not aligned) with one another along an axis, e.g., the Y axis within a vessel (e.g., in a vertical manner) or the X axis within a vessel (e.g., in a horizontal manner). The layers may be in any desired 3D orientation. The layers need not be planar, and the arrangement can be as desired. For example, a system may include multiple porous materials that are "stacked" within a vessel. This term does not necessarily indicate that the porous materials are in direct contact with one another within the stack. Rather, the porous materials may be spaced apart or in direct contact in some areas and spaced apart in other areas. The porous material may be associated with or separated by an aqueous phase or layer (e.g., wash buffer, lysis buffer) and / or associated with or separated by an oil phase or layer (e.g., mineral oil, coconut oil).

[0077] As used herein, the term "subject" refers to the entity from which a biological sample is obtained. The subject may be a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is an inanimate object other than a mammal. In some embodiments, the subject is the environment.

[0078] The term "target" is used in the broadest sense and refers to any desired substance, including any substance within a sample. In one embodiment, a target includes any substance that can be bound, either directly or indirectly, to a paramagnetic particle or other solid phase, e.g., via a conjugated antibody or antibody fragment, and can be pulled from a sample by application of magnetic force. In some embodiments, a target is a protein (e.g., an antibody, hormone, etc.), a carbohydrate (e.g., glycogen, chitin, etc.), a whole cell, a cellular component (e.g., mitochondria, exosomes, nucleus, etc.), or a nucleic acid (e.g., DNA, RNA, etc.). In some embodiments, a target is a metabolite, carbohydrate, glycopeptide, or lipid. A target includes an analyte. A target can also include a substance that is not of interest but is instead removed (e.g., in negative selection) to enrich for a substance of interest. In some embodiments, a target is a substance to be, for example, transferred, separated, isolated, detected, identified, analyzed, screened, quantified, or purified.

[0079] The term "analyte" or "target analyte" refers to any substance being identified or measured.

[0080] A "magnet" for use in the systems, devices, or methods of the present invention refers to a means for generating a magnetic force. As used herein, magnets include permanent magnets, temporary magnets, and electromagnets. Magnets also include switchable magnets. Permanent magnets and temporary magnets are materials whose magnetic field is maintained by the material itself. In some embodiments, the permanent magnet is a neodymium magnet (e.g., N52 or other neodymium magnets such as N35, N42, or N55 magnets). Temporary magnets are generally made from soft metals that are magnetized only when exposed to a permanent magnetic field or electric current. They become magnetized when in contact with a magnetic field. When the magnetic field is removed, they gradually lose their magnetism. An electromagnet is a type of magnet whose magnetic field is generated by an electric current. A switchable magnet is a device whose generated magnetic field can be changed between at least two configurations. This includes, for example, an electromagnet that has the ability to be turned "on" or "off," or even continuously adjustable between a fully "on" state and a fully "off" state. Other examples include devices in which permanent magnets can be oriented relative to one another to variably cancel each other's magnetic fields or combine the magnetic fields to create at least two magnetic field configurations. Permanent magnets that can be turned on and off are also switchable magnets.

[0081] With regard to materials for the electromagnet, examples include a coil of copper wire wound around a core typically made of iron, nickel, or cobalt, or alloys containing these metals (e.g., various types of steel and stainless steel). However, the core is not required, but does enhance the performance of the electromagnet. Similarly, alternative core materials that are uncommon or enhance the performance of the electromagnet may be used as part of the electromagnet used in the present invention. In some embodiments, beneficial properties of the electromagnet selected for use in the devices and readers of the present invention are temperature stability and resistance to rust / corrosion that may result from use in certain applications (e.g., high humidity conditions).

[0082] Detailed Description The present invention includes multi-layer and multi-phase systems within a vessel that provide autonomous operation of processing steps through manipulation of forces to position targets.

[0083] In one aspect, the present invention provides a self-contained system and device for sample preparation and target testing (e.g., PCR, LAMP, etc.) in a single vessel, requiring only the addition of sample, application of force (e.g., magnetic force), and in some embodiments, reading of the result.

[0084] In some aspects, provided herein are systems, methods, and devices for isolating or positioning a target from a sample and processing the target. In some embodiments, the target of interest is the target itself. In some embodiments, the target of interest is a target bound to a solid phase or is the solid phase itself. In some embodiments, the target is isolated and detected. In some embodiments, the target is purified. In some embodiments, the target is quantified. In some aspects, provided herein are systems, devices, compositions, and methods for positioning and / or processing a target. A target or a substance to which a target is bound can be positioned by the present invention in a number of ways, including positive (e.g., by isolating the target or removing the target from the sample for detection or measurement or disposal) and negative (e.g., by positioning or removing one or more or all non-targets). Using the systems, devices, and methods of the present invention, targets can be transferred, separated, isolated, detected, identified, analyzed, screened, quantified, or purified using the systems, devices, and methods of the present invention. The systems, devices, and methods of the present invention include systems, devices, and methods for isolating and / or detecting targets or analytes (including pathogens or parts of pathogens, e.g., proteins, nucleic acids, etc.) in a sample. In particular, systems and devices are provided herein that include one or more oils and / or one or more aqueous phases and / or one or more gas phases stabilized in close contact with each other. The systems, devices, and methods of the present invention have many uses. For example, they can be used to isolate, separate, transfer, purify, mix, bind, and / or subsequently detect the presence or amount of targets or target analytes from a sample or other mixture.

[0085] In some aspects, provided herein are systems and devices comprising one or more stabilized oils and / or one or more stabilized aqueous phases and / or one or more gas phases that can be used to transfer or purify targets or analytes from samples or mixtures that contain, may contain, or may be suspected of containing the targets or analytes using forces. Forces include any force, including, for example, magnetic forces, electric forces, or acceleration-based forces (e.g., via gravity or via a centrifuge) to pull the targets or analytes through one or more phases or layers.

[0086] In some embodiments, systems and devices include reagents for the detection, identification, analysis, isolation, or quantification of a target or analyte. Quantification may be positive-negative, semi-quantitative, or quantitative for the target. Isolation or purification may be complete or partial. One or more or all of the reagents for the detection, identification, analysis, isolation, or quantification of a target or analyte may be contained in one or more aqueous and / or oil phases or layers of the system or device, e.g., in a base phase or layer of the system or device, in one or more parts or portions of the system or device. In some embodiments, one or more or all of the reagents for the detection, identification, analysis, isolation, or quantification of a target are contained in a lower phase, layer, or tier of the system or device, but above the base layer. In some embodiments, one or more or all of the reagents are in a lower phase, layer, or tier of the system or device, or in a terminal phase, layer, or tier of the system or device (in vertical or latitudinal embodiments). In some embodiments, one or more or all of the reagents are at a seam, junction, or junction (in non-vertical or non-latitudinal embodiments, in the horizontal or longitudinal or other phase / layer direction). In some embodiments, one or more or all of the reagents for target detection, identification, analysis, isolation, or quantification are contained in a terminal phase, layer, or stratum or terminal phase, layer, or stratum (in vertical or latitudinal embodiments) of a system or device, or at a seam, junction, or junction (in the horizontal or longitudinal or other phase / layer direction, in non-vertical or non-latitudinal embodiments). These are examples of reagent placements, but are not inclusive of all possible placements, and may be any desired or appropriate placement in light of the structure or desired performance of the system or device.

[0087] The systems described herein can be used in methods for isolating any desired target or substance. In some embodiments, the target is a nucleic acid. In some embodiments, the target is a viral nucleic acid. For example, the target can be a SARS-CoV-2 nucleic acid. In some embodiments, the target is a protein (e.g., a hormone or any other protein), a carbohydrate, a glycolipid, a cell, a circulating tumor cell, etc. Any substance that can bind (either directly or indirectly) to a PMP can be a target in one or more of the systems, devices, compositions, and methods of the present invention.

[0088] In some embodiments where the system, method, or apparatus comprises at least one aqueous phase or layer and at least one oil phase or layer, only the at least one aqueous phase or layer and the at least one oil phase or layer are stabilized.

[0089] In some embodiments where the system, method, or device includes more than one aqueous phase or layer and one or more oil phases or layers, only one of the aqueous phases or layers is stabilized. In some embodiments where the system, method, or device includes more than one aqueous phase or layer and one or more oil phases or layers, more than one or all of the aqueous phases or layers are stabilized. For example, in embodiments of the invention having four aqueous phases or layers, one, two, three, or all four may be stabilized. In some embodiments where the system, method, or device includes more than one aqueous phase or layer and one or more oil phases or layers, only one of the oil phases or layers is stabilized. In some embodiments where the system, method, or device includes more than one oil phase or layer and one or more aqueous phases or layers, more than one or all of the oil phases or layers are stabilized. For example, in embodiments of the invention having four oil phases or layers, one, two, three, or all four may be stabilized.

[0090] In another embodiment where the device, system, or method includes an aqueous phase or layer and / or multiple oil phases or layers, e.g., 1 to 6 aqueous phases or layers and 1 to 6 oil phases or layers, 1 to 6 of the aqueous phases or layers and / or 1 to 6 of the oil phases or layers may be stabilized.

[0091] In some embodiments, the device, system, or method includes at least one stabilized aqueous phase or layer. In some embodiments, the at least one aqueous phase or layer is stabilized by a hydrophilic porous material immersed on, immersed in, or otherwise associated with the at least one aqueous phase or layer. In some of these embodiments, the device, system, or method including at least one stabilized aqueous phase or layer does not include an oil phase or layer or a stabilized oil phase or layer. In some embodiments, the device, system, or method including at least one stabilized aqueous phase or layer also includes a gas phase or layer. In some embodiments, the gas phase or layer includes, for example, air or an inert gas. In some embodiments, the gas phase includes helium, neon, argon, krypton, xenon, radon, oganesson, or the like. The gas can be a mixture of gases (e.g., air; air with volatiles; helium and neon, or the like). The gas can be in the form of a plasma. In some of these embodiments, the device, system, or method comprising at least one stabilized aqueous phase or layer comprises at least one oil phase or layer and / or at least one stabilized oil phase or layer. In some of these embodiments, the device, system, or method comprising at least one stabilized aqueous phase or layer comprises at least one oil phase or layer and / or at least one stabilized oil phase or layer and at least one gas phase or layer. In some embodiments, the device, system, or method comprises at least two stabilized aqueous phases or layers. In some embodiments, the device, system, or method comprising at least one stabilized aqueous phase is in a container or vessel. In some embodiments, the device, system, or method comprises at least one stabilized aqueous phase or layer and at least one gas phase or layer, but does not comprise an oil phase or layer.

[0092] In some embodiments, the device, system, or method includes at least one stabilized oil phase or layer. In some embodiments, the at least one oil phase or layer is stabilized by a hydrophobic porous material associated with the at least one oil phase or layer. In some of these embodiments, the device, system, or method including at least one stabilized oil phase or layer does not include an aqueous phase or layer or a stabilized aqueous phase or layer. In some embodiments, the device, system, or method including at least one stabilized oil phase or layer also includes a gas phase or layer. In some embodiments, the gas phase or layer includes, for example, air or an inert gas. In some embodiments, the gas layer includes helium, neon, argon, krypton, xenon, radon, oganesson, or the like. In some of these embodiments, the device, system, or method including at least one stabilized oil phase or layer includes at least one aqueous phase or layer and / or at least one stabilized aqueous phase or layer. In some of these embodiments, the device, system, or method comprising at least one stabilized oil phase or layer comprises at least one aqueous phase or layer and / or at least one stabilized aqueous phase or layer and at least one gas phase or layer. In some embodiments, the device, system, or method comprises at least two stabilized oil phases or layers. In some embodiments, the device, system, or method comprising at least one stabilized oil phase is within a container or vessel. In some embodiments, the device, system, or method comprises at least one stabilized oil phase or layer and at least one gas phase or layer, but does not comprise an aqueous phase or layer.

[0093] In some embodiments, the least one aqueous phase or layer or at least one oil phase or layer is stabilized in a container or vessel using a porous material. The material is selected to allow desired material movement through the device or system. The porous material may be a mesh. In some embodiments, one or more of the least one aqueous phase or layer is stabilized with at least one hydrophilic porous material(s) or mesh(es). In some embodiments, one or more of the least one oil phase or layer is stabilized with at least one hydrophobic porous material(s) or mesh(es). In one embodiment, the porous material and / or the hydrophobic and / or hydrophilic mesh has at least one predetermined pore size, set of pore sizes, or range of pore sizes. In some embodiments, the aqueous phase or layer and the oil phase or layer are stabilized in close proximity to each other in a vessel.

[0094] In some embodiments, one or more phases or layers, when present in the system, may be stabilized in the container by adjusting one or more chemical or physical material characteristics selected from material geometry or density, surface chemistry, and porosity of the hydrophilic / hydrophobic porous material.

[0095] In another aspect, the present invention provides miscible interfacial systems and methods and related compositions, referred to herein as "MIFT." In some MIFT embodiments, one or more phases or layers, when present in the system, can be stabilized within a container by adjusting the material geometry or one or more chemical or physical material characteristics selected from, for example, density, interfacial chemistry, viscosity, electrical or surface charge properties, magnetic properties, and porosity of hydrophilic / hydrophobic porous materials. In some embodiments, the materials include natural and synthetic porous matrices (e.g., sea sponges, nylon foams, polypropylene sponges, agarose gels, etc.), natural and synthetic porous materials (e.g., polysaccharide meshes, nylon meshes, etc.). Examples of adjusting material geometry include material size and thickness, pore size(s), pore distribution(s), porosity(s), and the degree or uniformity of pore connectivity. Examples of adjusting interfacial chemistry include oxygen plasma treatment (i.e., the addition of hydroxyl groups to the material surface) and glycosylation. Examples of adjusting viscosity include the use of different concentrations of methylcellulose or polyethylene glycol. Examples of adjusting surface charge include different levels of treatment with oxygen plasma or piranha solution, or adjusting the pH or salinity concentration or gradient or charge-shielding molecules such as surfactants in associated fluids. Examples of adjusting the electrical properties of a material include using materials with different dielectric properties or applying an electrical charge or current to the material. An example of adjusting the magnetic properties of a material would be to include different levels of ferromagnetic, paramagnetic, or diamagnetic material by changing the concentration of the material included or the potential porosity (i.e., ratio of pore volume to material volume) of the material at a given material concentration within the material.

[0096] In some embodiments of MIFT, one or more regions of a phase or layer are stabilized. In some embodiments, a phase or layer contains a fluid with multiple regions with different properties or property gradients (e.g., a fluid with different densities and / or a transition between density gradients). When one fluid is denser than the other within an aqueous layer containing a transition, for example, if the size of the container is large enough, attractive, convective, or turbulent forces induce homogenization / bulk mixing between the two fluids and rapid separation of the transition. As the size of the container decreases, hydrodynamic resistance increases, which can reduce the velocity resulting from any source of pressure difference (e.g., advection, convection, etc.) such that particle or molecule transport via diffusion can dominate or surpass transport resulting from bulk fluid motion (e.g., convection, advection, etc.). In some embodiments, diffusion-dominated or diffusion-dominated conditions can be described using the Peclet number. At diffusion-dominated or diffusion-dominated dimensions, dissolution of the transition occurs more slowly, e.g., over minutes to hours or days. In some aspects, systems, devices, and compositions are provided herein for stabilizing an aqueous layer containing at least one transition such that transport is diffusion-dominated or diffusion-dominated. In some embodiments, this is accomplished using a porous solid or semi-solid structure or material (e.g., a mesh, gel, or aggregate of solid or semi-solid particles). The same can be done for other types of phases or layers, such as oil or gas phases. This embodiment is referred to as a miscible interface system and can be used to stabilize the properties of different fluid domains and / or transitions within a phase or layer. A MIFT miscible interface system, as described, may be stabilized overall and need not contain one or more structural supports associated with the phase or layer. In some embodiments, stability of the fluid domain or transition can be achieved by sufficient reduction of the phase or layer dimensions or the inclusion of an emulsion within the phase or layer. In some embodiments of a miscible interface system, a substance (e.g., a target-binding particle) can be contained in one fluid region of a phase or layer and positioned in another fluid region (e.g., using a magnet or utilizing gravity and particle density to position the PMP).One example of such an approach is to create an aqueous phase or layer deep enough to hold both a volume of aqueous wash buffer and a volume of aqueous sample containing another substance (e.g., PMP). In this example, wash buffer can be added to a porous support structure, and gravity can position the bulk of the fluid in the lower half of the support structure. In this example, an aqueous sample containing PMPs, for example, can then be added to the porous support structure. The result is a phase or layer with two predominantly aqueous regions with different properties. Subsequent application of a force (e.g., a magnetic field in the case of PMPs) can then be used to attract the substance (e.g., target-bound PMPs) to the wash buffer region without significant contaminant or carryover from the sample region. A particular advantage of this system is that the stabilized, miscible aqueous fluid provides a means for isolating, washing, or concentrating a sample with little carryover and little or no energy barriers that can impede target migration or positioning (e.g., compared to positioning target-bound PMPs through an aqueous-oil interface). Such miscible interface systems and compositions can be integrated with one or more stabilized interface systems to create new functionality. In some embodiments, the present invention provides miscible interface compositions in combination with stabilized interface systems. The present invention includes multiple MIFT-SIFT combinations for use as compositions and in the devices and systems of the present invention.

[0097] The miscible interface systems and compositions of the present invention can be assembled and manufactured as described herein. To create a stabilized miscible layer containing two miscible fluids of different densities, for example, a first fluid is added to a container. A porous support material that favors the two fluids is cut to appropriate dimensions (e.g., diameter, thickness, etc.) so that the material can be compressed into the container and immersed in the first fluid. A second fluid is then added to the container along with another porous support material that favors the two fluids and cut to appropriate dimensions (e.g., diameter, thickness, etc.). In some embodiments, no support material is used.

[0098] In another aspect, the present invention provides stabilized interface systems and methods and related compositions, sometimes referred to herein as SIFT. In some embodiments, the compositions of the systems and methods include at least two fluids capable of forming a stable fluid-fluid interface and at least one associated structural material having at least one fluid-loving pore. In some embodiments, at least one of the at least two fluids is an aqueous fluid. In some embodiments, the structural material having at least one fluid-loving pore is associated with an aqueous fluid. In some embodiments, at least one of the at least two fluids is an oil. In some embodiments, the structural material having at least one fluid-loving pore is associated with an oil. In some embodiments, one of the at least two fluids is a gas. The structural material consists essentially of, or consists of, natural and / or synthetic material(s). In some embodiments, one or more or all of the characteristics of the structural material may be uniform. In some embodiments, one or more or all of the characteristics of the structural material may be heterogeneous; for example, the structural material may undergo a phase change from solid to liquid (e.g., a porous structural material comprised of wax that melts upon heating), or they may dissolve or change upon the addition of a reagent (e.g., a mesh loaded with dried sucrose to reduce the pore size of the material after the addition of an aqueous fluid as the sucrose dissolves, allowing the pore size to then re-expand, or to expose a more hydrophobic material underlying the hydrophilic sucrose as the sucrose dissolves, or alternatively, a porous structural material comprised of proteins that degrade upon the addition of trypsin). In some embodiments of the stabilized interface systems and methods, the pore or pores in the structural material each comprise an opening that allows the passage of at least one fluid and at least one entity of interest (e.g., target) through the material. In some embodiments, the structural support can stabilize at least one interface between fluids. In some embodiments, the material comprises natural and synthetic components (e.g., chitin, collagen I, polypropylene, nylon, etc.).

[0099] In some embodiments of the stabilized interfacial systems and methods / compositions, the structural material is a mesh. In some embodiments, materials include natural and synthetic porous matrices (e.g., sea sponges, nylon foams, polypropylene sponges, agarose gels, etc.), natural and synthetic porous materials (e.g., polysaccharide meshes, nylon meshes, etc.). Examples of adjusting material geometry include material size and thickness, pore size(s), pore distribution(s), porosity(s), and the degree or uniformity of pore connectivity. Examples of adjusting surface chemistry include oxygen plasma treatment (i.e., the addition of hydroxyl groups to the material surface) and glycosylation. Examples of adjusting viscosity include the use of different concentrations of methylcellulose or polyethylene glycol. Examples of adjusting surface charge include different levels of treatment with oxygen plasma or piranha solution, or adjusting the pH or salinity concentration or gradient or charge-shielding molecules such as surfactants in associated fluids. Examples of adjusting the electrical properties of a material include the use of materials with different dielectric properties or the application of an electrical charge or current to the material. An example of adjusting the magnetic properties of a material would be the inclusion of different levels of ferromagnetic, paramagnetic, or diamagnetic material by varying the concentration of the included material or the potential porosity (i.e., the ratio of pore volume to material volume) of the material at a given material concentration within the material. In some embodiments of the stabilized interfacial systems, compositions, and methods, the dimensions of the structural material are the same as or approximately the same as the dimensions of the fluid phase or layer with which it is associated. In some embodiments of the stabilized interfacial systems, compositions, and methods, the dimensions of the structural material are less than the dimensions of the fluid phase or layer with which it is associated. In some embodiments of the stabilized interfacial systems, compositions, and methods, the dimensions of the structural material are greater than the dimensions of the fluid phase or layer with which it is associated (e.g., the extension of a phase or layer or folds / bends within the phase or layer). In some embodiments, two or more different structural materials can be used within a single phase or layer to create a stabilized phase or layer (e.g., nylon and polystyrene can be used together to stabilize an aqueous phase or layer or two meshes of the same molecular composition but different geometries or pore sizes, etc.).

[0100] In some embodiments of the stabilized interfacial systems and methods / compositions, the structural material may be a film. In other embodiments, the structural material may be flexible or stretchable. For example, a porous mesh used to stabilize a phase or layer may be flexible or rigid, or may transition between rigid and flexible (e.g., via application of different temperatures).

[0101] In some embodiments of the stabilized interfacial systems, compositions, and methods, the structural material prefers oil over another fluid. In some embodiments, the porous structural material associated with the oil is hydrophobic. The degree of hydrophobicity in the stabilized interfacial systems and methods of the present invention is selected based on the contact angle between the porous structural material, the oil, and the second fluid. In some embodiments, additional criteria for selecting the degree of hydrophobicity in the stabilized interfacial systems and methods of the present invention include optimizing the surface tension across the interface so that the target-PMPs are easier or harder to pull across the interface, for example, magnetically, and / or considering overall device stability for transportation and storage. In some embodiments, the structural material(s) in the stabilized interfacial systems or methods are immersed in an oil layer.

[0102] In some embodiments of the stabilized interfacial systems, compositions, and methods, the structural material favors aqueous fluids. In some embodiments, the porous structural material associated with the aqueous fluid is hydrophilic. The degree of hydrophilicity in the stabilized interfacial systems and methods of the present invention is selected based on the contact angle between the porous structural material, the oil, and the second fluid. In some embodiments, additional criteria for selecting the degree of hydrophobicity in the stabilized interfacial systems and methods of the present invention include optimizing the surface tension across the interface so that the target-PMPs are, for example, easier or more difficult to magnetically pull across the interface and / or taking into account overall device stability for transportation and storage. In some embodiments, the structural material(s) in the stabilized interfacial systems or methods are immersed in an aqueous layer.

[0103] In some embodiments of stabilized interface systems and methods / compositions, compositions comprising at least two fluids capable of forming a stable fluid-fluid interface comprise fluid layers having at least one associated structural material comprising at least one fluid-loving pore. In some embodiments, the layers are aqueous and oil. In some embodiments, more than one layer is associated with a structural material comprising at least one fluid-loving pore in its associated fluid (e.g., a system having a stabilized aqueous layer and a stabilized oil layer).

[0104] In some embodiments, the stabilized interfacial system / composition is protected, backed, or bound by an upper and / or lower material comprising sufficient material to protect the one or more stabilized interfacial systems for subsequent storage, use, processing, transformation, etc. In some embodiments, the bound one or more stabilized interfacial systems form a stable or protected layer or structure. In some embodiments, the system is manufactured, stored, or transported in the form of a protected layer (e.g., a rolled layer). In some embodiments, the upper and / or lower bound comprises a porous substrate that allows the entity of interest to migrate through the substrate (e.g., a stabilized gas layer such as a polypropylene mesh protective layer on one or both sides of the stabilized aqueous layer). The entity of interest may be a target or any entity having mass. Examples of entities of interest may include photons, electrons, atoms, molecules, proteins, protein complexes, or anything with mass, such as particles generated by thermodynamic processes such as chemical reactions, mechanical breakdown, radioactive decay, or biological processes before entering or at some point within the system. In some embodiments, a protective layer is not required for storage or packaging (e.g., a two-layer system including an aqueous layer and an oil layer rolled onto itself may provide protection for the inner layer from evaporation, contamination, etc.).

[0105] In some embodiments, one or more stabilized interface systems are located within a container (e.g., a multi-well plate, an Eppendorf tube, an injection-molded container, an insert, a cartridge, etc.) or on a surface of a device. In some embodiments, the stabilized interface composition or system comprises a container. In some embodiments, the stabilized interface composition or system is rolled up for storage prior to use.

[0106] In some embodiments, one or more stabilized interface systems are incorporated as part of a lateral or vertical flow assay (i.e., a "flow assay"). For example, a lateral flow assay having a hydrophilic sample pad positioned below a second hydrophobic pad creates a stabilized interface system when aqueous sample fluid is added to the sample pad. For example, the sample can contain target-bound PMPs, and a magnet can be used to isolate the PMPs from the aqueous sample fluid through the hydrophobic upper layer, thereby allowing sample washing. In some embodiments, more than two material layers can be used to add functionality. For example, a system can include an upper aqueous sample layer, a middle hydrophobic gas layer, and a lower hydrophilic gas layer in conjunction with components for standard lateral flow detection. Target-bound PMPs in the sample layer can be repositioned to the lower hydrophilic layer using a magnet. Aqueous fluid can then be added to the hydrophilic gas layer, thereby replacing some or all of the gas phase with a more preferred aqueous fluid. The aqueous fluid can contain an acid / base / buffer (or other physical or chemical release mechanism) that allows for the elution of the target from the target-bound PMP for subsequent detection by the coupled lateral flow component. Similarly, the target PMP itself may be used as the detection particle in the coupled lateral flow component, thereby eliminating the need for target release. Also, in this example, the lateral flow component need not be coupled to the stabilized interface system(s) first, but can be coupled later. These embodiments are also applicable to MIFT systems. For example, a miscible interface system can be constructed to largely isolate fluid flow from passing through a region of the lateral flow assay, thereby allowing diffusion to dominate over transport into the bulk fluid flow. Such a system can be useful for extending the introduction of reagents into the bulk fluid flow within the LFA via diffusion. This can be achieved, for example, by adding a reagent-containing material to the top of a portion of the lateral flow assay where the added material has a much smaller pore size compared to the lateral flow component.Thus, fluidic resistance in the loaded material significantly impedes bulk fluid transport of reagents into the lateral flow component compared to diffusion-based transport. Similarly, fluidic components in the lateral flow component can be selected by particle size to diffuse into the loaded component and perform dialysis in lateral flow assays. The use of MIFT and SIFT systems can be used for other assay formats or sample processing steps as well. For example, a wick designed to deliver a sample to a downstream processing step can include a two-layer MIFT system, using a first layer to stabilize a buffer solution while allowing bulk fluid flow of the sample through a second layer. In this example, buffer can slowly diffuse into the sample stream through the second layer over time to condition the sample or aid in subsequent sample processing (e.g., adjust / maintain pH or introduce a blocking agent).

[0107] Structural materials and compositions thereof, including stabilized interface systems and methods, inherently have no limitations or boundaries with respect to overall shape, size, thickness, uniformity, or orientation (e.g., the material can be porous particulate, fabric-type, quilted or multi-layer / laminate designs monolithically constructed from smaller patches (e.g., 10s, 100s, or 1000s) of different materials or hundreds of yards of edges bonded or sewn together, amorphous sintered porous blocks of plastic roughly the size of a golf ball, pipette tips with a single orifice / hole, or any combination thereof). Regardless of the overall geometry of the material, the pore geometry defines the relevant boundaries of the interface(s) and the effective diameter of the pore(s) for assessing the magnitude of the stabilizing fluid-material interaction forces relative to the destabilizing force(s). If more than one pore is present in a material, there are no inherent limitations on the relative location of the pores or the uniformity or non-uniformity of the pores with respect to size, geometry, or surface properties. Nor are there any restrictions on the boundary lines of a hole boundary being contiguous or spaced apart. For example, a dumbbell-shaped structure may effectively define two holes despite having a boundary line of one shape. Similarly, a crenellated structure may define a single hole with many separate supporting boundary line segments (i.e., similar to dashed lines) that act together to produce a single hole boundary. There are no inherent restrictions as to whether the entities of interest can or should be able to pass through any or all of the fluid-fluid interface(s). If there is more than one entity of interest, there are no inherent restrictions on the similarity or dissimilarity of the entities of interest or their distribution of characteristics.

[0108] Unless otherwise specified, limitations on the characteristics of the components of the system are determined solely by the particular application of the invention and are considered to be within the scope of the invention.

[0109] The ratio X (Equation 1) can be defined similarly to other dimensionless quantities intended to assess the relative magnitude of surface tension to other forces affecting a fluid-fluid interface. However, because these forces act on the shared interfacial area between the fluids, X is defined as the ratio of pressures instead of forces (i.e., pressure = force / area), where P 表面張力 is the pressure resulting from the curvature of the fluid interface, and P 他 is the pressure arising from any other source.

[0110]

number

[0111] For example, for a quiescent volume of water (HO) of depth L supported by a porous hydrophobic membrane of uniform pore radius r, surrounded by air, X is given by Equation 2, where γ is the surface tension of water with air.

[0112]

number

[0113] In some embodiments, the size of the pore(s) in the structural material is selected to achieve a desired level of interface stability for a particular application. In some embodiments, pore size is a design factor used to adjust the stability of the interface when more or less stability is desired. For example, if an application requires that the interface be stable under static conditions but unstable when further destabilizing pressure is applied, the value of X is set to <1 under static conditions and >1 when further destabilizing pressure is applied, thereby limiting the range of allowable characteristic surface tension pressure(s) of the system.

[0114] Other sources of pressure that can potentially destabilize an interface (e.g., an aqueous-air interface) include the pressure head (e.g., P = ρgh) resulting from the depth of the fluid, the additional acceleration experienced when placed in a centrifuge, or when a collision induces rapid acceleration, or when pouring a fluid into a container. Indeed, some destabilizing pressures are spatially nonuniform, such as acoustic waves or hydrodynamics arising during vigorous fluid mixing, which can cause spatial and time-dependent fluctuations in pressure at the interface. Similarly, spatially nonuniform pressures can be applied to an interface by paramagnetic particles in a magnetic field at the fluid boundary. In such situations, localized destabilization of the interface can occur, acting on length scales shorter than those defined by the interface boundary. In any geometry, situation, or system, the stability of an interface can be assessed by considering the relative magnitude of the destabilizing pressure (Pi) to the magnitude of the stabilizing surface tension pressure (P). In general, an interface is considered stable when the characteristic pressure of surface tension is sufficiently greater than the destabilizing pressure (i.e., X < 1) to provide the stabilizing function described herein. This amount can be used to establish the applicable range of parameters in different systems to which the present invention applies.

[0115] In some embodiments of the present invention, a fluid-loving porous structural material is immersed in a volume of the preferred fluid. A volume of fluid can be considered saturated if the preferred fluid can associate with the material throughout or throughout the material. Typically, there is a range of volumes that can saturate a given porous structural material. If a volume is insufficient to associate with the material throughout or throughout the material, it is considered to be unsaturated. When unsaturated, the preferred fluid typically remains supported by the porous structural material, while the unpreferred fluid can potentially associate with the remaining space within the porous support structure. A volume exceeding the saturated volume is considered a supersaturated volume, which can also be used. While in some cases this may result in some fluid not being fully contained and / or fully supported by the porous structural material, it may be advantageous in manufacturing, etc. Thus, by way of example, the upper fluid layer can be intentionally overfilled or supersaturated (see, e.g., FIG. 17). In other regions, the portions of the fluid-overfilled region above or below the porous structural material are generally less stable or supported than the supported region or volume of the preferred fluid and are more susceptible to bulk fluid movement or turbulent forces or bulk mixing. In this example, the overfilled region may be useful (e.g., easier or more tolerant to manufacture), but may not be optimal without impeding function. In some embodiments, the stabilized layer or phase is under-filled, filled, or over-filled. See Figure 20.

[0116] In some embodiments, stabilized interface systems and methods and related compositions include (1) at least one structural material having at least one pore, (2) at least two fluids, and (3) at least one stabilized interface, phase, or layer. In some embodiments, the size of the pores allows a target, or a target bound to another material (e.g., a solid phase or other carrier material), to pass through without the application of an external force. In other embodiments, the target, or a target bound to another material (e.g., a solid phase or other carrier material), is moved through the pores using an external force (e.g., magnetic force, centrifugation).

[0117] In some embodiments, MIFT (Miscible Interface System) or SIFT (Stabilized Interface System) devices, methods, and compositions can be used to move or position a substance, e.g., a target. In some embodiments, movement or positioning using MIFT and / or SIFT systems, devices, methods, and compositions can be active movement or positioning (e.g., using external forces including magnetic forces, rotational forces, acceleration forces, assisted gravity, etc.). In some embodiments, movement or positioning using MIFT and / or SIFT systems, devices, methods, and compositions can be passive movement or positioning and can occur without the use of external forces (e.g., by diffusion, simple gravity, osmosis, etc.).

[0118] System performance can be tailored by altering fluid preferences to provide benefits in some applications (e.g., increased stability during handling or transportation of devices utilizing structural material(s) to stabilize the interface). Accordingly, in some embodiments of the present invention, it is advantageous to include at least one system fluid that is preferred or energetically favorable to interact with the system structural material relative to at least one other system fluid. Indeed, the more different the energetics (i.e., relative preferences) of the interactions, the more stable the interface. For example, the preference of untreated polystyrene for water relative to oil can be altered by treating the polystyrene with oxygen plasma, further increasing its preference for water and improving its stability. Additionally, for example, surfactants can be added to the fluid before or during use of the present invention to differentially affect the fluid-material interactions, thereby altering the stability of the interface(s). Similarly, for example, temperature can also be used to alter the energetics of the interactions.

[0119] Furthermore, in some embodiments, some or all of the fluids may undergo one or more transitions between solid and fluid forms (i.e., are "switchable"). For example, in some embodiments, it is advantageous to have at least one fluid solid at room temperature and fluid at the operating temperature for a particular application. Solidification of one of the fluids allows the interface to become ultrastable in the presence of destabilizing pressures, while the fluid form allows for the application of forces and pressures to destabilize the interface(s) as needed for different applications. In some embodiments, the transition between solid and fluid forms is achieved using temperature. In other embodiments, it is achieved using chemistry (e.g., liquefaction agents that can potentially change the chemistry of the original fluid), hydration (e.g., rehydration of dried sucrose), or mechanical means (e.g., ultrasonic liquefaction of dense colloids). Therefore, we refer to such interfaces as "switchable" fluids that can be used to generate switchable interfaces. In some embodiments, the switchable fluid enables switchable operation of the system. For example, a temperature-sensitive wax can be used to generate a stable interface with water and interact with a polypropylene structural material to stabilize the wax-aqueous interface. In such a system, the wax is solid at room temperature, providing much greater stability to the interface than if it were a fluid. In its solid form, the interface is robust to handling and transportation, thereby preventing the passage of entities of interest through the pores of the structural material. Upon application of heat, the wax becomes fluid, enabling applications in which one or more entities of interest must pass through pores in the structural material. For example, in its fluid form, paramagnetic particles in a magnetic field can be used to overcome the stabilizing influence of surface tension and transport the paramagnetic particles and any substances bound to them through the pores of the structural material. Thus, the use of switchable fluids and interfaces can provide additional functionality and flexibility to the present invention.

[0120] In some applications, a consistent level of stability of the fluid interface(s) is maintained. In other applications, the level of stability of the fluid interface(s) is maintained until the interface(s) is / are intentionally destabilized. In other applications, the stability of the interface(s) is / are maintained or destabilized in a controllable and reversible manner. In any of the above applications, the fluid-fluid interface(s) can be established or created at some point in the system or potentially deestablished or disassembled (e.g., by adding or removing fluids to reconfigure the system). In some embodiments, switchable fluids can be used for any of the fluids in the above scenarios to generate a combinatorial set of possible configurations. Further permutations of the above configurations can be generated using different relative positions of the system components and the timing of the establishment or transition between configurations.

[0121] The topology and surface properties of the support structure can be tailored so that any externally applied forces, including but not limited to gravity, centripetal, magnetic, and electrical forces, are negligible. With this invention, the art is no longer constrained to microchannels and small tubes, but instead, using solid or semi-solid, porous support structures associated with phases or layers or at interfaces, nearly any desired fluid system can be created across any dimension. In system / composition embodiments, topology and surface chemistry are the primary defining features for the support structure (to alter liquid surface tension). If a fluid system is desired that has all of the fluids in contact with each other, the support structure contains at least one opening through which the fluids can flow / interact. This opening is large enough to allow the passage of targets or target-binding particles, and, at the other end of the size spectrum, small enough to ensure an interface that overcomes surface tension for the application, as desired. These size constraints depend on the fluid, material properties, and system conditions, such as temperature and pressure. The invention allows for an unlimited number of openings in the support structure, as long as the association with the desired fluid is maintained throughout or across the support structure as needed during or for the application of the invention. Furthermore, the shape of the opening or hole is not important so long as association with the preferred fluid is maintained throughout or across the support structure opening or hole as required during or for the application of the present invention.

[0122] In another aspect, the present invention provides a mesh-reinforced system for particle isolation. The particles include target-binding particles, but need not be other particles, i.e., particles that are not target-binding particles. In the case of target-bound particles (e.g., target-bound PMPs), when particles are transferred from fluid A to fluid B through a fluid-fluid interface, a small amount of fluid A typically transfers through the interface with the target-bound particles, which may be referred to as carryover fluid. Carryover fluid is typically undesirable during the target isolation process (e.g., causing reduced sensitivity and specificity in qPCR), and therefore, systems for reducing carryover are of significant value. The second graph in Figure 11A shows that the inclusion of a porous support structure in the stabilized interface at the point of isolation (SIFT) significantly reduces carryover, particularly when compared to isolation using an unstabilized interface. These graphs are based on validated data.

[0123] Typically, as shown in the first graph in FIG. 11A, as particles are brought through a fluid-fluid interface, a focal point occurs at the interface (in this case, the aqueous-oil interface), deforming the interface as particles aggregate to create a single "valley" where large aggregates are formed that can overcome the interfacial barrier and enter Fluid B. When a stabilized interface is used, as shown in the second graph in FIG. 11A, there are at least two very important and useful effects. First, many smaller "valleys" are formed at the interface instead of a single large valley. Thus, smaller aggregates are formed to pass through the interface. This reduces the average carryover fluid associated with the aggregates. This is because larger aggregates have a smaller surface-to-volume ratio than smaller aggregates. Particles at the surface of the aggregates are associated with less carryover fluid, and therefore the larger surface-to-volume ratio for the aggregates results in an overall reduction in carryover. Second, as aggregates push out and separate from the bulk of fluid A, smaller valleys have greater surface tension pressure to pinch the tails of fluid A connecting the aggregates to the bulk of fluid A before they break away from the bulk fluid. This is supported by previous observations that fluids with more stable interfaces (without stabilizing structures) similarly reduce carryover fluid.

[0124] Thus, the performance of the mesh-reinforced system shown (or other systems using alternative structural supports with desired porosity, e.g., SIFT systems) for isolating target-bound particles with respect to fluid carryover can be adjusted through adjustments to the support structure material (e.g., geometry, pore size, porosity, distribution and heterogeneity of pore geometry, larger-scale curvature or topology of the stabilized interface, etc.) or the fluid utilized (e.g., aqueous-oil, aqueous-gas, water-silicone oil, water-fluorinated oil, water-mineral oil, addition of surfactants, etc.). Similarly, the overall thickness of the stabilized layer can be increased, or additional layers of support material within the stabilized fluid phase or layer can also be used to reduce carryover. If the stabilized layer is too thin, aggregates brought through the stabilized layer may create temporary (or potentially permanent) fluid bridges through the stabilized layer. In some cases (e.g., some applications with high bead densities), this can be advantageous, allowing a steady flow of high-density particles to result in rapid isolation of particles with a high target-to-carryover ratio. Fluid bridges may have other purposes, such as enabling electrical conduction through the stabilized layer. In other cases (e.g., some applications with lower bead densities), such fluid bridges may be considered a disadvantage, in which case increasing the thickness of the stabilized layer can reduce or eliminate the likelihood of fluid bridge formation. Additional layers or depth to the porous support structure may also provide other benefits. For example, as pore sizes become smaller, if multiple layers of support material are used, large aggregates that form upon entry into Fluid B may be captured by subsequent layers of the support structure, thereby isolating smaller aggregates from larger aggregates and providing a secondary isolation step that further reduces carryover. Enhanced particle isolation by such meshes (with or without target binding) is useful across multiple industries, from medical diagnostics and sample preparation to agricultural and large-scale biochemical engineering applications.

[0125] This system and related devices, methods, and compositions allow target positioning to proceed more quickly and efficiently. See FIG. 11A. Furthermore, contaminants are essentially eliminated instead of being washed away in the traditional sense, while carryover of contaminants, which is typically limited to the intermediate volume between beads, is reduced and minimized using these systems, devices, methods, and compositions. The stabilized interface systems and compositions of the present invention can be assembled and manufactured as described herein. To generate a stabilized layer, a first of two immiscible fluids is added to a container. A porous support material that prefers the first fluid is cut to appropriate dimensions (e.g., diameter, thickness, etc.) so that the material is pressed into position and immersed in the first fluid as needed within the container. A second fluid is then added to the container. Optionally, a porous support material that prefers the second fluid, cut to appropriate dimensions (e.g., diameter, thickness, etc.), is associated (e.g., immersed) in the second fluid.

[0126] In some embodiments, systems, devices, and methods are designed and used for target positioning. By way of example, one or more targets can be transferred, separated, isolated, detected, identified, analyzed, screened, quantified, or purified using the systems, devices, or methods of the present invention. The systems, devices, and methods of the present invention include systems, devices, and methods for isolating and / or detecting analytes in a sample. In some embodiments, the system, method, or device includes one or more oils and / or one or more aqueous phases and / or one or more gas phases stabilized in close contact with each other. These systems and devices have many uses. For example, they can be used to isolate, separate, transfer, purify, mix, combine, and / or subsequently detect the presence or amount of a target or target analyte from a sample or other mixture.

[0127] Targets can be positively or negatively positioned in a number of ways. Targets can be positively positioned, for example, by displacing, isolating, or removing the target from the sample (e.g., for detection or measurement, or disposal, etc.). Targets can be negatively positioned, for example, by positioning or removing one or more or all non-targets.

[0128] In some embodiments, the methods, devices, or systems of the invention can be used to position a target positively, for example, by isolating, separating, moving, or binding the target or a substance bound to the target.

[0129] In some embodiments, the methods, devices, or systems of the invention can be used to negatively position a target, for example, by isolating, separating, moving, or binding the target or a substance bound to the target.

[0130] In some aspects, provided herein are systems, devices, and methods that include one or more stabilized oils and / or one or more stabilized aqueous phases and / or one or more gas phases that can be used to transfer or purify targets or analytes from samples or mixtures that contain, may contain, or are suspected or suspected of containing the targets or analytes using magnetic, electric, or acceleration-based forces (e.g., via gravity or via a centrifuge) to pull the targets or analytes through one or more layers. In some embodiments, the systems and devices include reagents for detection, identification, analysis, isolation, or quantification of the targets or analytes. Quantification can be positive-negative, semi-quantitative, or quantitative relative to the target. Isolation can be complete or partial. One or more or all of the reagents for detection, identification, analysis, isolation, or quantification of the targets or analytes can be contained in the system or device.

[0131] In some embodiments, the systems, devices, compositions, and methods of the present invention are autonomous operations of processing steps. In some embodiments, the present invention provides for the processing / exposing / modification of any solid phase (e.g., paramagnetic particles) that can be transported through a layer / interface. In one aspect, each step functions as, for example, a purification / separation step, where paramagnetic particles pass through a phase, layer, or interface. In other aspects, other functionality can occur when paramagnetic particles are present, for example, within a phase, layer, or interface (e.g., chemical modification of the solid phase, elution from the solid phase, etc.). As mentioned, a solid phase is a solid support to which a target is bound, attached, or immobilized (whether directly or indirectly). A solid phase includes paramagnetic particles. A semi-solid can function as a solid phase. In some embodiments, anything to which a target is attached can function as a "solid phase." Porous solid support structures other than meshes are generally not considered solid phases. In some embodiments, a target can be a solid phase, for example, a cell.

[0132] One embodiment of the present invention is to apply the systems, devices or methods described herein to a specific target or analyte in a specific matrix.

[0133] In some aspects, systems and methods for isolating a target from a sample are provided herein. In some embodiments, the systems and methods of the present invention are used to detect and / or quantify a target in a sample. In some embodiments, the systems and methods of the present invention are used to determine the presence or amount of a target in a sample. In some embodiments, the systems include a vessel containing at least one aqueous phase (e.g., an aqueous layer) and at least one oil phase (e.g., an oil layer). The aqueous phase or layer and the oil phase or layer are stabilized within the vessel, and in some embodiments, are stabilized in close or otherwise functional proximity to each other within the vessel, with a distance between them as desired or required for the function of the system, method, or device.

[0134] The system may include any suitable or desirable number of aqueous and oil and / or gas phases or layers to facilitate isolation of target analytes. In some embodiments, the system includes one aqueous phase. In some embodiments, the system includes more than one aqueous phase. In some embodiments, the system includes one oil phase. In some embodiments, the system includes more than one oil phase. In some embodiments, the system includes one aqueous phase and one oil phase. In some embodiments, the system includes more than one aqueous phase and more than one oil phase. In some embodiments, the system includes at least two aqueous phases and at least two oil phases. In some embodiments, the aqueous and oil phases are stacked alternately such that the two aqueous phases are not in direct contact with each other and the two oil phases are not in direct contact with each other. In some embodiments, the phase closest to the top of the container (e.g., the phase that first contacts the sample) is the aqueous phase. In other embodiments, the phase closest to the top of the container is the oil phase. For example, in some embodiments, the aqueous and oil phases are stacked alternately such that the system comprises, from top to bottom, a first aqueous phase, a first oil phase, a second aqueous phase, and a second oil phase. In other embodiments, the system comprises, from top to bottom, a first oil phase, a first aqueous phase, a second oil phase, and a second aqueous phase. In some embodiments, the system comprises at least three aqueous phases and at least three oil phases, at least four aqueous phases and at least four oil phases, at least five aqueous phases and at least five oil phases, etc. In some embodiments, one or more of the aqueous phases are stabilized. In some embodiments, one or more of the oil phases are stabilized. In some embodiments, one or more of the aqueous phases and one or more of the oil phases are stabilized.

[0135] In some embodiments, at least one aqueous phase comprises a lysis buffer. In some embodiments, the lysis buffer is the first aqueous phase (e.g., the aqueous phase closest to the top of the container). An appropriate lysis buffer is selected based on the properties of the sample and the target. Thus, the sample can be added to the system such that the sample contacts the lysis buffer before coming into contact with any other components of the system. For example, the lysis buffer can be contained in the container above the multiple porous materials and above any oil phase present in the container, such that the biological sample contacts the lysis buffer before passing through the multiple porous materials. This promotes lysis of cells contained within the sample, thereby promoting the release of target analytes contained therein prior to isolation, purification, or evaluation of the presence or amount of the target. In other embodiments, the first aqueous phase is below the first oil phase (e.g., the first oil phase is closest to the top of the container). In such embodiments, the first oil phase helps remove potential contaminants from the sample before lysing the sample and releasing the target analytes.

[0136] In some embodiments, the lysis buffer may be contained between one or more layers of the plurality of porous materials (e.g., between one porous material and another porous material). In some embodiments, the lysis buffer is contained above the plurality of porous materials and / or between one or more layers of the plurality of porous materials. In other words, the first aqueous phase and the second aqueous phase may comprise a lysis buffer. In some embodiments, for example, in isolating nucleic acids, a first lysis step is performed, followed by a wash step using a lysis or other buffer. In some embodiments, the lysis buffer is added to the biological sample before adding the sample to the system. For example, the lysis buffer may be added to the biological sample as part of one or more pretreatment steps performed in preparation for adding the sample to the system.

[0137] Any suitable lysis buffer may be used. In some embodiments, the lysis buffer comprises a salt (e.g., NaCl, KCl, (NH4)2SO4, etc.). In some embodiments, the lysis buffer comprises a detergent. For example, the biological sample may contain an ionic detergent (e.g., sodium dodecyl sulfate, deoxycholate, cholate, etc.), a non-ionic detergent (e.g., Triton X-100, DDM, digitonin, Tween 20, Tween 40, NP-40, Pluronic F-127), a zwitterionic detergent, or a chaotropic detergent. In some embodiments, the lysis buffer comprises 0-5% detergent (v / v). For example, a biological sample can contain 0%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, or about 5% detergent. Detergents are most widely used for lysing mammalian cells. To lyse bacterial cells, the cell wall must be disrupted to access the cell membrane, and detergents are often used in conjunction with lysozyme. Agents for lysing viruses for downstream assays are virus-dependent and known in the art. The lysis buffer can be made up to the appropriate volume for subsequent use by adding an appropriate buffer. For example, lysis can be made up to the appropriate volume by adding phosphate-buffered saline (PBS), Tris-HCl, saline, etc. The lysis buffer may contain one or more enzymes or chemical agents to aid in the degradation of the contents therein to facilitate the release of desired targets. For example, the lysis buffer may further contain one or more enzymes, such as one or more proteolytic enzymes. In certain embodiments, the lysis buffer may include proteinase K. The lysis buffer may further contain one or more appropriate reagents to prevent degradation of targets within the sample. For example, appropriate reagents and / or inhibitors (e.g., RNase inhibitors, nuclease inhibitors, etc.) may be added to the lysis buffer prior to use in the systems described herein.

[0138] In some embodiments, at least one aqueous phase comprises a wash buffer. The purpose of the wash buffer is generally to dilute undesired components of the sample that are carried by, for example, a PMP to the wash buffer layer, where the PMP is used before transferring the PMP to the next phase or layer. Another purpose is, for example, to facilitate the release of undesired sample components bound to the PMP before transferring them to the next layer. A wash solution or wash buffer can be used to "mitigate" (e.g., chemically) undesired sample components that are carried to the wash layer before transferring them to the next layer.

[0139] In some embodiments, the aqueous phase comprising the wash buffer is not the first aqueous phase (e.g., it is not the aqueous phase closest to the top of the container). For example, the wash buffer may be the second aqueous phase, the third aqueous phase, the fourth aqueous phase, etc. In some embodiments, multiple aqueous phases comprise a wash buffer. For example, the first aqueous phase may comprise a lysis buffer, and the second and third aqueous phases may comprise wash buffers. In some embodiments, the aqueous phase comprising the wash buffer is below the aqueous phase comprising the lysis buffer and above the reagents for detecting the target. In some embodiments, the wash buffer comprises water. In some embodiments, the wash buffer comprises ethanol. In some embodiments, the wash step or wash buffer is performed using a lysis buffer or a mixture of a wash buffer and a lysis buffer.

[0140] In some embodiments, the system further comprises paramagnetic particles. In some embodiments, one or more aqueous phases further comprise paramagnetic particles (PMPs). In some embodiments, the first aqueous phase further comprises paramagnetic particles. The paramagnetic particles bind to the target analytes, thereby generating one or more target-PMP complexes. In some embodiments, the PMPs bind to the target or target analytes and are referred to as "target-binding" PMPs (or other target-capture solid phases). In some embodiments, the target-binding PMPs or other target-binding solid phases are conjugated with target binding agents, such as antibodies, antibody fragments, single-chain Fvs, oligonucleotides, aptamers, peptidomimetics, etc., directed to the target and used as the described PMP targeting agents, "target-binding" PMPs. Any suitable paramagnetic particles may be used. In some embodiments, paramagnetic particles may be purchased from a commercial vendor. The specific type of paramagnetic particles used depends on the target to be isolated from the sample. For example, particles with a relatively large surface area may be preferred for binding nucleic acids, such as viral RNA. In some embodiments, as noted, one or more paramagnetic particles can be functionalized to aid in target capture / purification. For example, some or all of the paramagnetic particles can be functionalized with one or more antibodies, antigen-binding fragments (e.g., F(ab')2, Fab, Fab', Fv, etc., generated from the variable regions of IgG and IgM, which can vary in size, valency, and Fc content), single-chain variable fragments (scFV), recombinant antibody fragments (rAbF), aptamers, peptides and peptidomimetics, natural and chemically modified antisense oligonucleotides, or other suitable agents to aid in target capture. In some embodiments, different paramagnetic particles are functionalized for different targets, such that one group of paramagnetic particles can function to indicate successful interaction with and / or isolation from a sample (e.g., particles targeting human RNaseP RNA / DNA in saliva as a means to indicate successful lysis of the sample and / or successful mixing and subsequent isolation of PMP and the sample). In some embodiments, different sets of paramagnetic particles can serve as positive or negative controls.In some embodiments, the paramagnetic particles may be functionalized with one or more spike protein antibodies to aid in the capture of SARS, coronavirus, SARS-CoV-2, and related targets. As used herein, reference to paramagnetic particles or PMPs includes functionalized paramagnetic particles.

[0141] The paramagnetic particles may be lyophilized or dried. The PMPs may be contained in a lyophilized or dried mixture or solution. In other embodiments, the paramagnetic particles may be in a liquid formulation. The paramagnetic particles are contacted with a sample, thereby generating complexes of the multiple targets and the PMPs. In some embodiments, the paramagnetic particles are contained within a container holding the multiple porous materials. For example, the PMPs may be part of the first aqueous phase. Alternatively, the paramagnetic particles may be contained separately (e.g., in a container separate from the multiple porous materials). When contained separately, the paramagnetic particles may be added to the container containing the multiple porous materials before, after, or simultaneously with the addition of the sample to the container. For example, the PMPs may be added to the first aqueous phase present in the container. In some embodiments, the first aqueous phase also contains a lysis buffer, such that addition of the sample to the container results in lysis of the cells contained therein and binding of the targets or analytes to the PMPs present in the aqueous phase. Alternatively, paramagnetic particles can be mixed with the sample to produce a composition containing complexes of multiple targets and PMPs, and the composition can be added to the vessel.

[0142] Any suitable amount of PMP can be contacted with the sample. In embodiments in which the PMP is contained in a liquid formulation, any suitable volume of a liquid composition containing paramagnetic particles can be contacted with the sample. In some embodiments, the volume of the liquid composition containing PMP can be equal to or greater than the volume of the sample. For example, the volume of the liquid composition containing PMP can be at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, or at least 500% of the volume of the sample.

[0143] Any suitable concentration of PMP can be used to ensure sufficient binding of PMP to the target (e.g., formation of a sufficient number of target-PMP complexes). For lyophilized PMP formulations, any suitable weight of lyophilized product can be used to ensure the appropriate concentration of PMP to be contacted with the sample. For liquid formulations, the liquid composition containing PMP can contain any suitable concentration of PMP to ensure sufficient binding of PMP to the target (e.g., formation of a sufficient number of target-PMP complexes). For example, PMP can be present in the liquid composition at about 1-20% (v / v). For example, PMP can be present in the liquid composition in an amount of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or about 20% (v / v).

[0144] In some embodiments, a liquid composition comprising a PMP contains other suitable reagents for sample processing / handling. For example, a liquid composition comprising a PMP may contain one or more detergents, reducing agents, buffers, inhibitors, enzymes (e.g., proteases), denaturants, etc. Any additional reagents present in the sample may also be present in a liquid composition comprising a PMP. For example, a liquid composition may further include one or more reagents for reducing the viscosity of the sample. For example, a liquid composition may comprise a PMP and DTT. A liquid composition may include other suitable buffers, inhibitors, etc. to prevent degradation of targets (e.g., target nucleic acids, target proteins, etc.) during sample processing. Suitable inhibitors that may be present in a liquid composition comprising a PMP include, for example, RNase inhibitors, protease inhibitors, nuclease inhibitors, etc. A lyophilized PMP formulation may contain other suitable reagents commonly used in the lyophilization process, including bulking agents, stabilizers, and other suitable excipients.

[0145] In some embodiments, the systems described herein further include at least one oil phase. The oil phase can be any suitable hydrophobic liquid. In some embodiments, the oil phase can include mineral oil, coconut oil, vegetable oil, etc. In some embodiments, the oil phase (e.g., a layer of light mineral oil, coconut oil, etc.), if present in the system, resides above the multiple porous materials and above the wash buffer and / or lysis buffer. Thus, the sample passes through the oil layer before contacting the lysis buffer.

[0146] The aqueous and oil phases may be stabilized within the container by one or more factors. In some embodiments, the aqueous and oil phases are at least partially stabilized by the use of a porous material. For example, the systems described herein may include multiple porous materials. The multiple porous materials are stacked within the container such that the target (e.g., target-PMP complex) passes through the multiple porous layers during the purification process. In some embodiments, the porous materials do not directly contact each other within the stack. For example, one or more porous materials may be separated by an aqueous phase (e.g., wash buffer, lysis buffer) or an oil phase (e.g., mineral oil, coconut oil). In some embodiments, the one or more porous materials directly contact each other.

[0147] Any suitable porous material may be used. In some embodiments, the porous material is hydrophilic. In some embodiments, the hydrophilic porous material is within or includes an aqueous phase or layer. In some embodiments, the porous material is hydrophobic. In some embodiments, the hydrophobic porous material is within or includes an oil phase or layer. In some embodiments, the porous material is a fibrous glass material. For example, the porous material may be a fibrous hydrophilic glass mesh. In some embodiments, the porous material is a synthetic mesh material. For example, the porous material may include a polypropylene mesh, a polyethylene mesh, a polyester mesh, a nylon mesh, or a polyetheretherketone (PEEK) mesh. In some embodiments, the synthetic mesh material is hydrophobic. In some embodiments, the synthetic mesh material is hydrophilic. For example, nylon-6 is an exemplary synthetic mesh material that is hydrophilic. Nylon-6 and nylon-6 capillary-channeled polymer (C-CP) fibers are hydrophilic.

[0148] In some embodiments, each of the porous materials is the same. In other words, the system includes a plurality of porous materials stacked in a container, and each layer in the stack includes the same porous material. In other embodiments, one or more of the porous materials is different from one or more other porous materials. In other words, the system includes a plurality of porous materials stacked in a container, and one or more layers in the stack are different from another layer.

[0149] In various embodiments, the porous materials are selected based on the size of the pores or openings in the material and the size of the targets or analytes in the sample, the size of the targets or analytes bound to the carrier or solid phase (e.g., PMPs) and / or the size of the elements desired to be excluded during the method. In some embodiments, the size of the pores or openings in one or more of the porous materials is different from that in one or more other porous materials. In some embodiments, the size of the pores or openings in one or more of the porous materials is the same as that in one or more other porous materials in the system.

[0150] In some embodiments, a hydrophilic porous material is associated with at least one aqueous phase or layer, and a hydrophobic porous material is associated with at least one oil phase or layer. For example, a hydrophilic porous material (e.g., glass mesh, nylon) may be associated with one or more aqueous phases or layers within a container, and a synthetic hydrophobic mesh material may be associated with at least one oil phase or layer. In some embodiments, each aqueous phase or layer comprises or consists essentially of a hydrophilic porous material, and each oil phase or layer comprises or consists essentially of a hydrophobic porous material. In some embodiments, a first aqueous phase or layer comprises or consists essentially of a dissolution buffer and is stabilized by a hydrophilic porous material (e.g., glass mesh, nylon) associated with the first aqueous phase or layer. In some embodiments, the first aqueous phase or layer and at least one additional aqueous phase or layer comprise or consist essentially of a hydrophilic porous material. For example, the first aqueous phase or layer can comprise or consist essentially of a lysis buffer, and the second aqueous phase or layer (and potentially the third aqueous phase or layer, the fourth aqueous phase or layer, etc.) comprises or consists essentially of a wash buffer and a hydrophilic porous material. In some embodiments, at least one oil layer is stabilized by a hydrophobic mesh (e.g., a hydrophobic synthetic mesh). For example, the first oil layer, the second oil layer, the third oil layer, etc. may comprise or consist essentially of a hydrophobic synthetic mesh associated with the oil layer.

[0151] In some embodiments, the aqueous phase or layer and the oil phase or layer are at least partially stabilized by adjusting one or more chemical or physical material characteristics. For example, the aqueous phase or layer and the oil phase or layer, when present in the system, can be stabilized by adjusting the geometry or one or more chemical or physical material characteristics, including the density, interfacial chemistry, surface free energy, fluid retention, and / or porosity, of the hydrophilic or hydrophobic porous material. In some embodiments, the aqueous phase or layer and the oil phase or layer are stabilized in close contact with each other under appropriate conditions so that the fluid retention force that associates the support structure with the fluid layer dominates other forces (e.g., buoyancy) that would otherwise disrupt the functional layer organization or phase order. For example, in a simple two-phase system in which water is introduced into a container over the top of oil, which has a lower density than water under gravity, buoyancy forces reconfigure the system so that the oil forms a layer over the top of the aqueous phase, relative to gravity. If a hydrophobic stabilized porous material is present on the top surface of the oil and adheres to the walls of the container before introducing water, the fluid retention forces in the porous material will prevent the forces / pressures resulting from buoyancy and the pouring action from relocating the oil layer to the top position, thereby maintaining the oil below the aqueous layer.

[0152] In some embodiments, oil phase stabilization is adjusted, modified, or selected by using oils of different densities. The oil phase may also be adjusted or modified by creating phases or layers where surface tension and / or capillary forces dominate over forces resulting from density and / or acceleration (e.g., gravity).

[0153] The density and / or surface properties of aqueous phases or layers, or potentially associated supporting structures, can be adjusted to adjust the stability of the layer or its association with the device or supporting structure. In some embodiments, the density and / or surface properties of one or more aqueous phases or layers are adjusted or modified by using aqueous phases or layers containing different salts or different amounts of salt, surfactants, etc. The surface properties of structures associated with fluids can be similarly adjusted, for example, through oxygen plasma treatment of polystyrene mesh to increase its preference for fluids such as water. Aqueous phases or layers can also be modified by creating phases or layers containing heavy liquids. Heavy liquids include sodium polytungstate, sodium metatungstate, and lithium metatungstate. All of these are inorganic compounds based on the [H2W12O40]6- polyanion that dissolve in water to form very dense solutions and can be diluted to form aqueous phases or layers that are not dense, but are denser than pure water-based phases or layers. In some embodiments, an aqueous phase or layer is modified by creating a phase or layer in which surface tension and / or capillary forces dominate over forces resulting from density and / or acceleration (e.g., gravity).

[0154] The density, mechanical properties, and / or surface properties of a phase or layer in the systems, devices, and methods of the present invention can also be adjusted using a phase change of the material, e.g., melting, boiling, sublimation, etc. Phase change materials useful for adjusting or modifying the buoyancy and / or surface tension of a phase or layer include polymeric compounds such as polyethylene glycol and methoxypolyethylene glycol. In some embodiments, the phase change material can be a paraffin wax or the like having an operating temperature above the melting temperature of the wax.

[0155] The porosity of the support structure can be adjusted or selected by using specific materials with varying pore sizes or different pore size ranges. Some useful porous materials (e.g., nitrocellulose) are made with different pore sizes. Pore size can also be adjusted or modified in situ (e.g., using porous materials containing swelling or degrading hydrogels or dry sugars).

[0156] In some embodiments, targets are positively or negatively positioned. Targets can be positively positioned, for example, by positioning or isolating the target (e.g., for detection or measurement) or removing the target from a sample or substance. Targets can be negatively positioned, for example, by positioning or removing one or more or all non-targets. In some embodiments, positioning a target is positively achieved by positioning the target or a substance linked to the target away from other substances, for example, substances in a biological or other sample, for example, by isolating, separating, moving, or binding the target or a substance bound to the target using a method, device, or system of the invention.

[0157] In some embodiments, positioning a target is done negatively by moving target-bound substances away from the target and removing non-target substances, e.g., by isolating, separating, moving, or binding the target or target-bound substances using a method, device, or system of the invention.

[0158] In some embodiments of the present invention using antibody-based cell isolation, for example, either positive or negative selection can be used. Cells that can be isolated using the methods, devices, or systems of the present invention include, for example, stem cells, circulating fetal cells, circulating tumor cells, etc. The present invention can also be used to isolate rare cells that may be masked by variability within a larger, more diverse background of cells (e.g., bloodstream, biopsy tissue, etc.) or between patients. In particular, the methods, devices, and systems of the present invention provide a means to separate rare target cells from background, either positively or negatively.

[0159] Positive selection can utilize antibodies to capture cells in an antigen-dependent manner, resulting in a captured population specific to a selected cell marker (via antibodies, carbohydrate receptors, etc.). While accurate, positive selection requires that the marker be specific to the target population and known a priori. Negative selection can be used when the distinguishing marker is unknown or expressed at different levels but not distinct (i.e., shared by adjacent cell populations). Negative selection embodiments of the present invention utilize known non-target markers to deplete background populations. In this approach, target cells remain uncaptured, allowing for isolation by a negative approach. In negative selection, the target is positioned away from other materials by displacing them away from the target, rather than displacing the target itself (or the material to which it is bound).

[0160] The systems described herein include a vessel housing various components of the system (e.g., at least one aqueous phase (e.g., aqueous layer), at least one oil phase (e.g., oil layer), multiple porous materials, etc.). Any suitable vessel may be used. An appropriate vessel may be selected based on the desired application of the system. Examples include, but are not limited to, test tubes, microcentrifuge tubes, dishes, slides, plates, multi-well plates (e.g., 4-well, 8-well, 12-well, 96-well, 384-well, etc.), flasks, vials, channels, etc. In some embodiments, the vessel is a multi-well plate so that multiple samples can be processed simultaneously.

[0161] The vessels may be of any suitable size. In some embodiments, small vessels (e.g., multi-well plates) may be tailored for the isolation of analytes from biological samples. In other embodiments, larger vessels may be tailored for the isolation of analytes from environmental samples, such as sewage samples. Any suitable volumes of aqueous and oil phases may be used, depending on the vessel selected. For example, for multi-well plates, relatively small volumes of aqueous and oil phases may be used (e.g., less than 0.5 ml). However, it will be understood that as the size of the vessel increases, the volumes of the aqueous and oil phases will be adjusted appropriately.

[0162] In some embodiments, the system further includes a reagent for detecting the target. In some embodiments, the reagent for detecting the target is contained relative to, at, or on the bottom surface of the container, such that the sample passes through a plurality of porous materials before contacting the reagent for detecting the target. For example, the reagent for detecting the target can be stabilized on the bottom surface of the container by a suitable porous material. For example, the reagent for detecting the target can be associated with a hydrophilic porous material (e.g., glass mesh, nylon) and positioned or stabilized below an oil phase or layer. The reagent can be stabilized above or at the bottom surface of the container by a structural porous material, for example, in some embodiments, a hydrophilic porous material (e.g., glass mesh, nylon). In some embodiments, the reagent for detecting the target does not need to be associated with a support structure. For example, the reagent can be positioned below a stabilized oil phase or layer to hold it in place. In some embodiments, the reagents are associated with a support structure (e.g., a non-porous material device such as a porous glass mesh or a nylon O-ring) and positioned below the stabilized oil phase or layer. For example, a reagent for detecting a target or multiple targets is held at the bottom of a container by adding an appropriate material to the top of the reagent to hold it in place. In some embodiments, a reagent for detecting a target or multiple targets can be held against, at, or on the bottom surface of the container by placing a fibrous material (e.g., polypropylene mesh) on top of the reagent. In some embodiments, the reagent is held on the bottom surface of the container by a non-mesh or non-porous material device, such as an O-ring (e.g., a PTFE O-ring).

[0163] In other embodiments, the reagents for detecting the target may be present in a container separate from the containers containing at least one aqueous phase and at least one oil phase. For example, the container containing at least one aqueous phase, at least one oil phase, and stacked porous material may be placed on top of or within a separate container holding the reagents for detecting the target. For example, the container containing at least one aqueous phase, at least one oil phase, and porous material may be used as an insert and placed within a separate container holding the reagents for detecting the target. A magnet may be placed below the container holding the reagents for detecting the target so that the complex of the target and PMP is attracted through the material held within the insert and comes into contact with the reagents for detecting the target. Note that in some described embodiments, the system contains at least one aqueous phase or layer and at least one gas phase or layer, but no oil phase or layer; in other embodiments, the system contains at least one oil phase or layer and at least one gas phase or layer, but no aqueous phase or layer.

[0164] In some embodiments of the system, the magnet is part of the container. In some embodiments, including embodiments for single-use applications, for example, the magnet may be contained within or affixed to the container (e.g., to the side of the container, the bottom of the container, etc.). In other embodiments of the system, the magnet may be attached to or affixed to the outside bottom or side of the container. In other embodiments of the system, the bottom of the container or a portion of the bottom of the container includes the magnet. In other embodiments of the system, the side of the container or a portion of the side of the container includes the magnet.

[0165] In other embodiments, the magnet used is part of a fixture, instrument, holder, tool, etc. used to position the magnet relative to the PMP.

[0166] In some embodiments, the reagents for detecting a target include reagents for nucleic acid amplification (e.g., PCR, isothermal amplification, etc.) and / or sequencing. In some embodiments, the reagents for detecting a target include reagents for RT-PCR, qPCR, qtPCR, multiplex PCR, assembly PCR, asymmetric PCR, etc. In other embodiments, the reagents for detecting a target include reagents for an immunoassay, which may use antibodies and / or antibody fragments to detect or measure a target or target analyte. In some embodiments, the immunoassay is an enzyme immunoassay, ELISA (enzyme-linked immunosorbent assay, including direct ELISA, indirect ELISA, sandwich ELISA, and competitive ELISA), IEMA (enzyme-linked immunosorbent assay), radioimmunoassay (RIA), fluorescent immunoassay, chemiluminescence immunoassay (CLIA), and enumeration immunoassay (CIA).

[0167] In some embodiments, the present invention provides a disposable cartridge comprising a flow-through assay for determining the presence or amount of a target in a fluid sample, comprising a sample application space, a cartridge top, a cartridge bottom, reagents for target detection or quantification, and an enclosure, wherein the disposable cartridge comprises at least one aqueous phase and at least one oil phase stabilized in close proximity to each other by the inclusion of target-binding paramagnetic particles, a porous structural material associated with the aqueous phase, the oil phase, or both, within the enclosure, and utilizing a magnet. Other and / or alternative phases may be used or included.

[0168] In some embodiments, the present invention provides a flow assay device (e.g., lateral flow, vertical flow) comprising a sample application portion, a conjugate portion, a test portion, and pre-immobilized reagents in different portions of the device, characterized by utilizing at least one aqueous phase and at least one gas or oil phase stabilized in close proximity to each other by the inclusion of target-binding paramagnetic particles, a porous structural material associated with the aqueous phase, gas phase, or oil phase, or both, and a magnet. Other and / or alternative phases may be used or included. In some embodiments, the improved flow assay device is designed for use as a disposable point-of-care device or cartridge.

[0169] In some embodiments, the present invention provides an immunometric assay for determining the presence or concentration of a target substance in a sample, comprising forming a ternary complex of a first labeled binding agent, the target substance, and a second binding agent bound to a solid support, wherein the presence or amount of the substance in the sample is determined by measuring either the amount of labeled binding agent bound to the solid support or the amount of unreacted labeled binding agent. The immunometric assay comprises target-binding paramagnetic particles, at least one aqueous phase and at least one oil phase stabilized in close proximity to each other by the inclusion of a porous structural material associated with the aqueous phase, the oil phase, or both, and a magnet. Other and / or alternative phases may be used or included. In some embodiments, the solid phase is a paramagnetic particle. In some embodiments, one or more of the binding agents is an antibody, antibody fragment, oligonucleotide, aptamer, peptide, peptidomimetic, natural or chemically modified antisense oligonucleotide, or other suitable agent to aid in target capture. In other embodiments, the assay improved with the use of at least one aqueous phase and at least one oil phase stabilized in close proximity to each other by the inclusion of target-binding paramagnetic particles, a porous structural material associated with the aqueous phase or the oil phase, is an IEMA, RIA, CIA, CLIA, or fluorescent immunoassay.

[0170] In some embodiments, the reagents for detecting a target include reagents for identifying one aspect of the target. In some embodiments, the reagents for detecting a target include reagents for identifying more than one aspect of the target. Aspects of a target include, for example, peptides, proteins, glycoproteins, epigenetic modifications of nucleic acids, nucleic acid sequences, cell surface receptors, cell types, etc. In some embodiments, the reagents for detecting a target include reagents for identifying more than one target or one or more aspects of one or more targets. In some embodiments, the reagents for detecting more than one target are contained in different physically separate parts of a system or device or in different parts of a container comprising a system or device of the invention. In some embodiments, multiple targets are isolated and multiple types of reagents for detecting these targets are contained within a single device or system. See, e.g., Example 13.

[0171] In some embodiments of the present invention useful for performing one or more steps of an assay for detecting or measuring a target or target analyte, one or more of the phases or layers of a device or system may contain one or more of several different buffers. In some embodiments, one or more phases or layers include a coating buffer, a blocking buffer, a stabilizing buffer, a wash buffer, or act as or contain a sample diluent. In some embodiments, an antibody or antibody fragment is used to obtain a detection signal. In some embodiments, an assay performed using a device, system, or method of the present invention includes a magnetically actuated immunoassay in which target or target analyte movement or positioning is achieved using magnetic separation using magnetic particles. In some embodiments, the particles used in these embodiments consist of a magnetite core that has been chemically modified with the attachment of an antibody or antibody fragment. In some embodiments, one or more or all components of the assay are used to isolate or purify a target or target analyte.

[0172] In some embodiments, the reagents for target detection include reagents for loop-mediated isothermal amplification (LAMP)-based detection of the target. Generally, a LAMP reaction mixture contains a DNA polymerase with strong strand displacement activity and high temperature tolerance, and up to six DNA oligonucleotides of specific structure. An RT-LAMP reaction mixture further contains a reverse transcriptase. A sample containing potential template molecules is added to the reaction and incubated at a constant temperature (e.g., 65°C) for 20 to 60 minutes. The oligonucleotides serve as primers for the reverse transcriptase, and additional oligonucleotides for the DNA polymerase are designed to loop back the DNA product at its ends. These, in turn, serve as self-priming templates for the DNA polymerase. In the presence of several RNA template molecules, a chain reaction is initiated and continues until the added reagents (particularly deoxynucleotide triphosphates) are exhausted.

[0173] LAMP or RT-LAMP assays are particularly useful embodiments due to their rapid nature, one-tube processing, and easy visualization of results without the need for expensive equipment or additional materials. In certain embodiments, the reagents for target detection include reagents for a colorimetric assay to detect the abundance of the target. Such embodiments allow for easy visualization of whether a sample contains the target of interest. In some embodiments, the sample collection device contains reagents for a colorimetric loop-mediated isothermal amplification (LAMP) assay. In embodiments in which the nucleic acid is RNA, the sample collection device may contain reagents for a colorimetric RT-LAMP assay. In some embodiments, the reagents for a colorimetric LAMP assay (or colorimetric RT-LAMP assay) further include an indicator that allows for evaluation of a color change in the sample in the presence of sufficient nucleic acid (e.g., the target nucleic acid that the LAMP or RT-LAMP reagents are designed to detect). Suitable indicators include pH-sensitive indicators and metal-sensitive indicators. In some embodiments, a pH-sensitive indicator (e.g., phenol red) may be used because it is easily visualized with the naked eye. The signal detection approach that is most suitable for a particular application (e.g., enzymatic (fluorometric, calorimetric, chemiluminescent, enhanced chemiluminescent), radiometric, direct fluorescence, time-resolved fluorescence, direct chemiluminescence, phosphorescence, etc.) will be determined by the user. Signal amplification techniques and strategies can also be used in the systems, devices, compositions and methods of the invention, as well as multiplexing techniques.

[0174] In some embodiments, the reagents for target detection include reagents for a fluorescent assay for detecting or determining the amount of a target either quantitatively, semi-quantitatively, or at a predetermined threshold amount. For example, the sample collection device may contain reagents for a fluorescent LAMP or fluorescent RT-LAMP assay. Any suitable fluorescent dye may be used in a fluorescent LAMP or fluorescent RT-LAMP assay to enable a fluorescent signal to be generated in the presence of sufficient nucleic acid.

[0175] In some embodiments, the reagents for detection of the target include reagents for a "yes / no" assay.

[0176] In some embodiments, the reagents comprise oligonucleotides (e.g., primers) designed for detection of bacterial nucleic acid or nucleic acid from any life form or replicative unit, including nucleic acid from eukaryotic cells, mitochondria, chloroplasts, etc. In some embodiments, the nucleic acid is bacterial nucleic acid. In some embodiments, the nucleic acid is viral nucleic acid. In some embodiments, the nucleic acid is nucleic acid from any source, including synthetic or genetically engineered sources.

[0177] In some embodiments, the reagents comprise oligonucleotides designed for the detection of viral RNA, e.g., SARS-CoV2, coronavirus, rhinovirus, influenza virus, respiratory syncytial virus, adenovirus, parainfluenza virus, human immunodeficiency virus, human papillomavirus, rotavirus, hepatitis virus (including hepatitis A, B, C, D, and / or E), Zika virus, Ebola virus, Mycobacterium tuberculosis, Borrelia burgdorferi, Borrelia mayonii, Staphylococcus bacteria, Aspergillus fungi (including Aspergillus niger), or Streptococcus (including Streptococcus pyogenes). For example, the reagents may comprise oligonucleotides designed for the detection of a viral upper respiratory tract infection selected from SARS-CoV2, SARS, coronavirus, rhinovirus, influenza virus, respiratory syncytial virus, etc. In some embodiments, the reagents comprise oligonucleotides for the detection of SARS-CoV-2 RNA or fragments thereof.

[0178] In some embodiments, the system further comprises a magnet. The magnet is used to attract the target-PMP complexes through the stacked porous material and bring them into contact with the reagents for analyte detection, and thus may be referred to herein as a "purification magnet." The purification magnet may result in partial or complete purification. The purification magnet may be of appropriate strength and / or located at the bottom of the container or in an appropriate vicinity within the container to attract some, most, substantially all, or all of the target-PMP complexes through the stacked porous material. For example, the purification magnet may be located below the container. In some embodiments, the system comprises multiple purification magnets (e.g., arranged in an array). For example, multiple purification magnets may be used to simultaneously or sequentially serve multiple containers (e.g., a multi-well plate containing multiple samples for which analyte isolation and detection are desired). In some embodiments, a second set of magnets is used to affect or adjust the uniformity and strength of the purification magnets. For example, when purification magnets are arranged in an array pattern, the second set of magnets may be positioned around the perimeter of the array to reduce edge effects and thereby maintain a more consistent magnetic field for each purification magnet in the array. Thus, the second set of magnets may be referred to herein as "magnetic field stabilizing magnets." After binding, the magnets may be applied to a system, such as the base of a container containing multiple porous materials, thereby drawing the target-PMP complex through the stacked porous materials to purify or substantially isolate the target from other components within the sample. The pore size of the porous material should be sufficient to allow the target-PMP complex to pass through the pores while preventing other undesired contaminants from passing through. Undesired contaminants may come from any source, such as undesired components of the original sample, the environment, assay reagents, equipment, etc.

[0179] The pore size of the porous material can be optimized depending on the target to be isolated. In some embodiments, the pore size can range from 0.5 μM to 0.5 mm.

[0180] The systems described herein can be used to isolate targets from any desired sample. In some embodiments, the sample is a biological sample. In some embodiments, the biological sample is a nasopharyngeal sample, an oropharyngeal sample, an oral swab or sponge sample, a nasal swab sample, a middle turbinate sample, or a saliva sample. In certain embodiments, the biological sample is a saliva sample. In other embodiments, the biological sample is an NP sample. In some embodiments, the sample is an environmental sample. For example, the sample may be a sewage sample. In some embodiments, the environmental or biological sample is a crude sample and / or one or more target molecules are not purified or amplified from the sample prior to application of the methods or manipulation by the devices, systems, methods, or compositions of the invention.

[0181] In some embodiments, the biological sample is first mixed with a solid phase or lysis / binding buffer and a lysis / binding buffer with or containing a solid phase (e.g., PMP), and then added to a system or device, which may be in a container. In some embodiments, the system or device already contains a lysis / binding buffer and a PMP, and the biological sample is added thereto. In some embodiments involving biological samples obtained via swabs (e.g., nasopharyngeal samples, oropharyngeal samples, oral swab samples, oral sponge samples, nasal swab samples, middle turbinate samples, etc.), the swab is immersed and mixed in the lysis / binding buffer (and PMP, if already contained in the container). In some embodiments, the biological sample is obtained using a separate device or container, which is then coupled with a container already containing the lysis / binding buffer and a solid phase, e.g., PMP. The coupling / joining of the two containers / devices facilitates the introduction of the biological sample into the system or device. In some embodiments, the biological sample undergoes certain processing or pre-treatment steps before being added to the system or device.

[0182] The sample may be collected and / or stored in a suitable container (e.g., a sample collection container) prior to adding the sample to the systems described herein. Any type of sample collection container suitable for receiving and storing a sample may be used. Examples of sample collection containers include, but are not limited to, tubes containing a reversibly removable cap, bags, syringes, droppers, etc. In some embodiments, the sample is pre-processed before use in the systems described herein. For example, the sample may be pre-processed in the sample collection container. As another example, the sample may be transferred to a suitable second container and pre-processed in the second container.

[0183] In some embodiments, a sample may be pretreated to inactivate potential pathogens (e.g., viruses, bacteria) within the sample. For example, a sample may be pretreated before use in a system described herein. In some embodiments, a sample may be pretreated to lyse cells within the sample, thereby releasing targets (e.g., nucleic acids) for subsequent detection. In such embodiments, the pretreatment step achieves both cell lysis (e.g., release of nucleic acids) and inactivation of potential pathogens within the sample. In some embodiments, a sample may be pretreated by freezing, heating, and / or adding a denaturing agent to the sample. For example, a sample may be pretreated by heating to a temperature sufficient for an appropriate time to inactivate potential pathogens within the sample. For example, a sample may be heated to about 40°C or above. For example, a biological sample may be heated to about 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 100°C, or greater than 100°C. The sample may be maintained at the heated temperature for a suitable period of time, such as 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, or more than 1 hour. In certain embodiments, the sample may be heated to 98°C-100°C for 5 minutes to achieve both cell lysis and viral inactivation in a single heat treatment step. In some embodiments, sample pretreatment includes adding a denaturant to inactivate potential pathogens in the sample. For example, the denaturant may be present in the lysis buffer with which the sample is contacted. Suitable denaturants include guanidine-based denaturants (e.g., guanidine hydrochloride, guanidine thiocyanate, etc.) and detergents (e.g., Triton X-100, Tween 20). In some embodiments, the sample does not contain a denaturant. For example, in some embodiments, the sample (e.g., a saliva sample) may not contain a guanidine-based denaturant. In some embodiments, the sample (e.g., a saliva sample) contains less than 0.3 M of a guanidine-based denaturant. For example, the sample (eg, a saliva sample) may contain less than 0.3M, less than 0.25M, less than 0.2M, less than 0.15M, less than 0.1M, or less than 0.5M of the guanidine-based denaturant.

[0184] The viscosity of certain samples (e.g., saliva) can make sample handling difficult. Furthermore, the viscosity of samples collected from different individuals varies, posing potential problems for inter-subject sample collection variability. For example, a saliva sample with a high viscosity may result in a lower volume of saliva being successfully pipetted into the desired container (e.g., for subsequent detection of pathogens in the sample) compared to saliva with a low viscosity. This can lead to potential downstream problems with variable or inaccurate results, including false-negative results. In some embodiments, samples may be pretreated to reduce their viscosity, thereby improving sample handling in subsequent processing steps. In certain embodiments, a pretreatment step may be performed to inactivate pathogens in the sample and reduce the viscosity of the sample in one step. In some embodiments, one or more agents for reducing viscosity may be added to the sample before using it in the systems described herein. In some embodiments, the agent for reducing viscosity is a reducing agent. Suitable reducing agents include, for example, dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP) or 2-mercaptoethanol.

[0185] Any suitable amount of reducing agent may be added to the sample (or present in a storage buffer in which the sample is placed upon collection). In some embodiments, the reducing agent is present in a lysis buffer with which the sample is contacted. In some embodiments, a suitable concentration of reducing agent may range from 0 to 500 mM. For example, a suitable concentration of DTT or TCEP can be in the range of 0 to 250 mM (e.g., 0 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, or about 250 mM). For example, dithiothreitol (DTT) may be added at an appropriate concentration to a biological sample (e.g., a saliva sample) to reduce the viscosity of the sample. In some embodiments, DTT may be added to achieve a 1× concentration in the saliva sample. As another example, a suitable concentration of 2-mercaptoethanol can be in the range of 0 to 500 mM (e.g., 0 mM, about 25 mM, about 50 mM, about 75 mM, about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM, about 275 mM, 300 mM, about 325 mM, about 350 mM, about 375 mM, about 400 mM, about 425 mM, about 450 mM, about 475 mM, or about 500 mM).

[0186] In some embodiments, a viscosity-reducing agent (e.g., DTT) is added to a sample before heating the sample (e.g., to inactivate pathogens and / or induce cell lysis). In some embodiments, the viscosity-reducing agent may be present in a sample storage buffer to which the sample is added after collection. In some embodiments, the viscosity-reducing agent is added to a sample after heating the sample. In some embodiments, the viscosity-reducing agent is present in a lysis buffer to which the sample is contacted. In some embodiments, freezing of the sample may be performed to reduce the viscosity of the sample. Any suitable pretreatment step or combination of pretreatment steps may be performed to achieve the desired result (e.g., cell lysis, pathogen inactivation, and / or reduction of sample viscosity).

[0187] The sample may further contain an appropriate detergent. For example, the sample may contain an ionic detergent (e.g., sodium dodecyl sulfate, deoxycholate, cholate, etc.), a non-ionic detergent (e.g., Triton X-100, DDM, digitonin, Tween 20, Tween 40, Pluronic F-127), a zwitterionic detergent, or a chaotropic detergent. In some embodiments, the sample contains 0-5% detergent (v / v). For example, the sample may contain 0%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, or about 5% detergent. The detergent may be added to the sample (eg, contacted with the sample as part of a lysis buffer) and / or may be present in a sample storage buffer to which the sample is added upon collection.

[0188] In some embodiments, the sample contains a non-ionic detergent (e.g., Triton X-100). For example, the sample may contain 0.001 to 0.1% Triton X-100. The sample can be brought to an appropriate volume for subsequent use by the addition of an appropriate buffer. For example, the sample can be brought to an appropriate volume by the addition of phosphate-buffered saline (PBS), universal transport medium (UTM), saline, etc. Such a buffer may be present in a sample storage buffer that is added to the sample or to which the sample is added upon collection. The sample may contain one or more enzymes or chemical agents to aid in the degradation of the contents therein to facilitate the release of desired targets. For example, the sample may further contain one or more enzymes, such as one or more proteolytic enzymes. In certain embodiments, the sample may contain proteinase K. The sample may further contain one or more appropriate reagents to prevent degradation of targets within the sample. For example, appropriate buffers and / or inhibitors (e.g., RNase inhibitors, nuclease inhibitors, etc.) can be added to the sample before use in the systems described herein.

[0189] The systems, devices, compositions, and methods described herein can be used for the isolation, detection, identification, or quantification of any desired target from any sample or source. The devices, compositions, and methods of the present invention can be used to locate any desired target from any sample or source for any purpose, including detection, quantification, etc.

[0190] In some embodiments, the devices, systems, and / or methods are used for the isolation and subsequent detection of a desired target. In some embodiments, the target is a cell. In some embodiments, the target is a nucleic acid (e.g., DNA, RNA, or various subtypes thereof, including mRNA and rRNA), a protein, a metabolite, a carbohydrate, a glycopeptide, or a lipid. For example, the target may be DNA or RNA. In some embodiments, the target may be a nucleic acid or protein (e.g., an antibody, a hormone, etc.) obtained from a pathogen infecting one or more subjects from which the sample was obtained. For example, the target may be bacterial nucleic acid (e.g., bacterial DNA or RNA) or viral nucleic acid (e.g., viral DNA or RNA). As another example, the target may be an antibody produced by a subject in response to infection with a pathogen.

[0191] In some embodiments, the devices, systems, and / or methods are used to determine identity or paternity by sample analysis, hi some embodiments, the sample is prepared for use in the devices, systems, and / or methods of the invention for prenatal or postnatal screening.

[0192] In some embodiments, a sample is obtained from a subject suspected of having an infectious disease. For example, a biological sample may be obtained from a subject suspected of having an infectious disease. In some embodiments, an environmental sample may be obtained from an area where one or more members of a population are suspected of having an infectious disease. For example, sewage may be collected and used to determine whether one or more members of the surrounding population have the infectious disease of the subject. The subject or one or more members of the population may be suspected of having any infectious disease caused by a pathogen that can be detected in the sample or an infectious disease that causes the individual to produce antibodies that can be detected in the sample. In some embodiments, the subject from whom the biological sample is obtained or one or more individuals in a population proximate to the area from which the environmental sample is taken is suspected of having SARS-CoV2, coronavirus, rhinovirus, influenza virus, respiratory syncytial virus, adenovirus, parainfluenza virus, human immunodeficiency virus, human papillomavirus, rotavirus, hepatitis virus (including hepatitis A, B, C, D, and / or E), Zika virus, Ebola virus, Mycobacterium tuberculosis, Borrelia burgdorferi, Staphylococcus, Aspergillus fungi (including Aspergillus niger), or Streptococcus (including Streptococcus pyogenes). In some embodiments, the subject or member of the population may be suspected of having a bacterial or viral infection. For example, the subject or member of the population may be suspected of having an upper respiratory tract infection. For example, a subject or member of a population may be suspected of having a viral upper respiratory tract infection, including infection with SARS-CoV-2, coronavirus, rhinovirus, influenza, respiratory syncytial virus, etc.

[0193] The systems described herein find use in methods for positioning or isolating a target from a sample and / or detecting, identifying, quantifying, or purifying (partially or completely) a target. In some embodiments, the systems described herein find use in methods for isolating and subsequently detecting a target in a sample. For example, in some aspects, provided herein are methods for isolating a target from a sample, comprising adding the sample to a system described herein. In some embodiments, the sample is lysed by contacting it with a lysis buffer, thereby releasing the target analytes. The method further comprises applying a magnetic force to the bottom or side of the container, depending on the orientation of the phases or layers, mesh, etc., thereby drawing the target analytes through the plurality of porous materials, thereby purifying the target from other possible contaminants present in the sample. In some aspects, provided herein are methods for isolating and detecting a target in a sample. The method comprises adding the sample to a system comprising a container containing the plurality of porous materials described herein and reagents for target detection. The method further includes applying a magnetic force to the bottom or side of the container, thereby drawing the targets through the plurality of porous materials and bringing them into contact with reagents for detecting the targets contained on the bottom surface of the container.

[0194] In some embodiments of the methods described herein, a sample is contacted with paramagnetic particles or functionalized paramagnetic particles (PMPs) described herein before applying a magnetic force to the system. Contacting the sample with the paramagnetic particles results in one or more target-PMP complexes, and applying a magnetic force to the system attracts the target-PMP complexes to the bottom surface of the container through the porous materials. In some embodiments, the sample is contacted (e.g., mixed) with the paramagnetic particles in a separate container to obtain a composition containing one or more target-PMP complexes, which is then placed (e.g., pipetted) into a container containing the porous materials. In other embodiments, the paramagnetic particles are contained in a container containing the porous materials and the aqueous and oil phase(s). For example, lyophilized paramagnetic particles may be present in the container. The paramagnetic particles may be present in the container in liquid form (e.g., as part of a lysis buffer). In such embodiments, adding the sample to the container contacts the sample with the PMPs, thereby obtaining target-PMP complexes within the container itself.

[0195] In some embodiments, the sample is contacted with a lysis buffer described herein. As described above, the lysis buffer can be contacted with the sample before adding it to the system, or the lysis buffer can be present in a container containing multiple porous materials. Contacting the sample with the lysis buffer allows for the release of targets from various components of the sample, thereby facilitating subsequent isolation and / or detection of the targets.

[0196] In some embodiments, the sample is contacted with a wash buffer.As described above, the wash buffer can be present in a container that contains multiple porous materials.By applying magnetic force to the bottom surface of the container, the target (for example, the target-PMP complex) is attracted through the wash buffer present in the container, thereby facilitating further purification of the target.

[0197] In some embodiments, the method further includes detecting the target after partial, complete, or substantially complete removal or isolation (as desired or required for the purpose of the method) from the sample. In such methods, the system includes a reagent for detecting the target contained at, near, or on the bottom (or side) surface of a container described herein. The target and PMP complex is drawn from the aqueous and oil phases and through the porous material and contacted with the reagent for target detection. In some embodiments, an appropriate incubation time is allowed to pass at an appropriate temperature (e.g., 65°C for 20-60 minutes), and the signal resulting from the contact is measured. For example, a colorimetric signal (e.g., a color change) or a fluorescent signal can be measured to determine whether the well contains the target. Measurement of the signal (e.g., a color change, a fluorescent signal) can occur, for example, by visualization (e.g., by the naked eye). Alternatively, the signal can be measured using an instrument such as a plate reader. For example, a fluorescent signal can be measured using a plate reader. In some embodiments, the isolated target-PMP complex is contacted with a reagent for LAMP-based detection of the target, and the signal resulting from the contact is measured. For example, the signal may be a colorimetric signal (e.g., a signal from a colorimetric RT-LAMP assay) or a fluorescent signal (e.g., a signal from a fluorescent RT-LAMP assay).

[0198] In some embodiments, the methods described herein are performed on a single sample. In other embodiments, the methods are performed on multiple samples simultaneously. In some embodiments, samples may be pooled and then used in the systems and methods described herein. For example, biological samples may be collected from multiple separate individuals, pooled together, and used in the methods described herein to determine whether a population has a case of infection with a pathogen (e.g., SARS-CoV2). As another example, multiple biological samples may be collected from an individual, and multiple biological samples from separate individuals may be pooled to increase the amount of sample available to be used in the methods described herein. Such embodiments may be useful, for example, when an individual may not be able to provide a sufficient volume of saliva during a single collection or when multiple tests may be performed using the same sample.

[0199] In some embodiments, the method steps described herein are automated. In some embodiments, the sample preparation steps described herein are automated. In some embodiments, the detection steps described herein are automated. In some embodiments, the acquisition of results is automated. In some embodiments, the transmission of results to another device or a non-user third party is automated. In some automated embodiments, the steps and / or methods described herein are performed by a computer, and the computer includes a processor and memory. The memory may contain software that causes the processor to perform certain tasks. For example, the memory may contain software that instructs the processor to attach pipette tips to a multi-channel pipette, aspirate a sample, mix a biological sample with PMPs to form a composition comprising one or more target-PMP complexes, aspirate the composition into a system described herein, bring a magnet into proximity with the bottom surface or other surface (e.g., side) of a vessel containing the sample, or turn on electromagnets on or near the surface of the vessel and in the plurality of porous materials, as well as other functions necessary to carry out the claimed methods.

[0200] The invention described herein includes a system for isolating a target from a sample, the system comprising at least one aqueous phase and at least one oil phase stabilized in proximity to each other in a container. In some embodiments of the system, the at least one aqueous phase and the at least one oil phase are stabilized in the container by a hydrophilic porous material immersed in the at least one aqueous phase and / or a hydrophobic porous material immersed in the at least one oil phase. In some embodiments of the system, the at least one aqueous phase and the at least one oil phase, when present in the system, are stabilized in the container by adjusting one or more chemical or physical material characteristics selected from buoyancy, surface chemistry, and porosity of the hydrophilic / hydrophobic porous material. In some embodiments, the at least one aqueous phase and the at least one oil phase are stabilized in the container by a hydrophilic porous material immersed in the at least one aqueous phase, a hydrophobic porous material immersed in the at least one oil phase, and by adjusting the surface chemistry so that the buoyancy of the at least one oil phase is less than the surface tension of the at least one aqueous phase. In any of these embodiments, the system may include a first aqueous phase, a second aqueous phase, a first oil phase, and a second oil phase. In any of these embodiments of the systems described herein, the phases may be stacked alternately within the container such that the first and second aqueous phases do not directly contact each other and the first and second oil phases do not directly contact each other. In any of these embodiments, the system is provided in an apparatus including a container, where the container may have a top opening to allow for the addition of a sample to the container. In any of these embodiments, the system is provided in an apparatus including an insert, where the insert may have a top opening to allow for the addition of a sample to the insert. In some embodiments, at least one aqueous phase is closest to the top opening of the container. In some embodiments, at least one aqueous phase is closest to the portion of the insert to which the sample is added. In some embodiments, at least one oil phase is closest to the top opening of the container. In some embodiments, at least one oil phase is closest to the portion of the insert to which the sample is added.In any of these embodiments, at least one aqueous phase may comprise, consist essentially of, or consist of a lysis buffer. In any of these embodiments, at least one aqueous phase may comprise, consist essentially of, or consist of a wash buffer. In any of these embodiments, the system, device, container, or insert may include paramagnetic particles (PMPs). In some embodiments, the PMPs are contained within a container. In the claimed systems, the PMPs are lyophilized, dried, or in liquid form. In some embodiments, the PMPs are contained within at least one aqueous phase. In some embodiments with more than one aqueous phase or layer, the PMPs are contained within more than one or all of the aqueous phases or layers. In some embodiments, the PMPs are contained within at least one oil phase or layer. In some embodiments with more than one oil phase or layer, the PMPs are contained within more than one or all of the oil phases or layers. In any of these embodiments, the system further comprises a magnet or other device for providing a magnetic force.

[0201] The invention described herein includes a system for isolating a target analyte from a sample, the system comprising a first aqueous phase, a second aqueous phase, a first oil phase, and a second oil phase, (a) the phases are stacked alternately in a container such that the first and second aqueous phases are not in direct contact with each other and the first and second oil phases are not in contact with each other, and (b) the phases are stabilized in the container by (i) a hydrophilic porous material immersed in the first aqueous phase, (ii) a hydrophilic porous material immersed in the second aqueous phase; (iii) a hydrophobic porous material immersed in the first oil phase; and (iv) a hydrophobic porous material immersed in the second oil phase.

[0202] In some embodiments of this system, the phases are further stabilized within the container by adjusting the surface chemistry so that the buoyancy of each oil phase is less than the surface tension of each aqueous phase and / or so that the buoyancy of each oil phase is less than the water-retaining capacity of each hydrophilic porous material. In some embodiments, the container comprises a top opening to allow addition of a sample to the container. Either the first aqueous phase or layer or the first oil phase or layer may be closest to the top opening of the container. The first aqueous phase or layer may comprise or consist essentially of a lysis buffer. In some embodiments of this system, the second aqueous phase comprises a wash buffer. In some embodiments of this system, the system further comprises a PMP. In some embodiments of this system, the PMP is contained within the container. In some embodiments, the PMP may be lyophilized or in dry or liquid form. In some embodiments, the PMP is contained within the first aqueous phase, the second aqueous phase, or both. In some embodiments, the PMP is contained within the first oil phase, the second aqueous phase, or both. In any of these embodiments, the system further comprises a magnet or other device for providing a magnetic force. In some embodiments, the container comprises a multi-well plate. In some embodiments, the container comprises an insert. In some embodiments, the insert is insertable into the multi-well plate. In some embodiments, the container comprises a single-use device.

[0203] The systems, devices, methods, and compositions of the invention can be used (and may include reagents therefor) to transfer, isolate (in whole or in part), purify (in whole or in part), detect, and / or quantify targets, including, for example, small molecules, proteins, peptides, immunoglobulins (e.g., IgA, IgM, IgG, IgE, lambda light chain, kappa light chain), enzymes, lipids, receptors (e.g., Her2 receptor), nucleic acids (e.g., DNA, introns, exons, non-coding elements, RNA, rRNA, mRNA, microRNA), circulating tumor DNA (ctDNA), orphan non-coding RNA (oncRNA), circulating pathogen DNA and circulating pathogen RNA, antigens (e.g., PSA), hormones (e.g., testosterone), and cancer and other cells, including circulating tumor cells (e.g., circulating tumor cells of epithelial origin associated with metastatic breast, prostate, and colorectal cancer), circulating endothelial cells, cellular vesicles, exosomes, and bacterial quorum-sensing molecules. Targets and analytes include biomarkers, including molecular and histological biomarkers, screening markers (primary, secondary and targeted), diagnostic biomarkers, prognostic biomarkers, predictive biomarkers, pharmacodynamic / response biomarkers, susceptibility / risk biomarkers, monitoring biomarkers and safety biomarkers.

[0204] Any bacteria, virus, or other pathogen can be tested, isolated, separated, purified, identified, detected, or quantified using the systems, devices, methods, or compositions of the present invention. In some embodiments, the viral target for testing, isolation, separation, purification, detection, or quantification is a virus of the Coronaviridae family, the Picornaviridae family, the Caliciviridae family, the Flaviviridae family, the Togaviridae family, the Bornaviridae family, the Filoviridae family, the Paramyxoviridae family, the Pneumoviridae family, the Rhabdoviridae family, the Arenaviridae family, the Bunyaviridae family, the Orthomyxoviridae family, or a Deltavirus. In other embodiments, the virus is a coronavirus, SARS, poliovirus, rhinovirus, hepatitis A virus, Norwalk virus, yellow fever virus, West Nile virus, hepatitis C virus, dengue virus, Zika virus, rubella virus, Ross River virus, Sindbis virus, chikungunya virus, Borna disease virus, Ebola virus, Marburg virus, measles virus, mumps virus, Nipah virus, Hendra virus, Newcastle disease virus, human respiratory syncytial virus, rabies virus, Lassa virus, hantavirus, Crimean-Congo hemorrhagic fever virus, influenza virus, or hepatitis D virus. In some embodiments, the virus is one or more of the above viruses (or another virus) that has evolved or mutated into a new lineage. In some embodiments, the virus is a human-generated, mutated, or engineered virus.

[0205] In some embodiments, the invention provides methods of monitoring or assessing viral disease outbreaks and / or viral evolution using the systems, devices, methods, or related compositions of the invention.

[0206] In some embodiments, the devices, systems, methods and compositions of the invention are used in methods of screening samples for viral antigens, viral nucleic acids and / or virus-specific antibodies, bacterial and / or other pathogen-specific antigens, nucleic acids and / or antibodies.

[0207] In some embodiments, a nuclease inactivation step is performed in or with a device, system, method, or composition of the invention in assaying, testing, screening, separating, isolating, purifying, identifying, detecting, and / or quantitating target nucleic acids. Some embodiments include heat inactivation, chemical inactivation, ultrasonic inactivation, etc. In some embodiments, targets or other protocols for testing or assaying using the systems, devices, methods, and compositions of the invention include tumor markers (e.g., alpha-fetoprotein (AFP), beta-2-microglobulin (B2M), beta-human chorionic gonadotropin (β-hCG), bladder tumor antigen (BTA), chromogranin A (CcA, neuroendocrine tumors), gastrin (gastrinoma), 5-HIAA (carcinoid tumors), ALK gene rearrangements and overexpression, BCL2 gene rearrangements, BRCA1 and BRCA2 gene mutations); cancer genes and partial sequences Cancer markers include, for example, programmed death ligand 1; ER / PR, CA15-3, and CA27.29 (breast cancer); EGFR, KRAS, and UGT1A1 (colorectal cancer); HER-2 / neu (breast cancer and gastric cancer); c-KIT / CD117 gastrointestinal stromal tumor, mucosal melanoma, acute myeloid leukemia, and mast cell disease; CD20, CD30, FIP1L1-PDGFRalh, Philadelphia chromosome, PML / RAR-alpha, TPMT, UGT1A1 (leukemia, lymphoma); EML4 / ALK, EGFR, KRAS (lung cancer), BRAF (melanoma), CA125, CA125 II and HE4 (ovarian cancer); BRAF V600 mutations (e.g., cutaneous melanoma, colorectal cancer, and non-small cell lung cancer); CA19-9 and CA19-9 XR (pancreatic cancer, gallbladder cancer, bile duct cancer, and gastric cancer); calcitonin (medullary thyroid cancer); carcinoembryonic antigen (CEA) (colorectal cancer and other cancers); CD19 and CD22 (B-cell lymphoma and leukemia); CD20 (non-Hodgkin's lymphoma); CD25 (non-Hodgkin's (T-cell) lymphoma); CD30 (classical Hodgkin's lymphoma, B-cell and T-cell lymphoma); CD33 (acute myeloid leukemia); chromosome 17p deletion (chronic lymphocytic leukemia); chromosomes 3, 7, 17, and 9p2 (bladder cancer); nuclear matrix protein 22, fibrin / fibrinogen (bladder cancer);Cytokeratin fragment 21-1 (lung cancer); cyclin D1 (CCND1) gene rearrangement or expression (lymphoma, myeloma); des-gamma-carboxyprothrombin (DCP) (hepatocellular carcinoma); gene mutations (e.g., DPD, EGFR, FGFR2, FGFR, FLT3, IDH1, IDH2, JAK2, KRAS, and MYD88 gene mutations); gene rearrangements (e.g., IRF4 gene, ROS1 gene, and T-cell receptor gene rearrangements); gene fusions (e.g., NTRK gene fusions and PML / RARα fusion genes); PCA3 mRNA, PSA free and total PSA (prostate cancer); HER2 / neu gene amplification or protein overexpression (breast cancer, ovarian cancer, bladder cancer, pancreatic cancer, and gastric cancer); lactate dehydrogenase (germ cell tumors, lymphoma, leukemia, melanoma, and neuroblastoma); MYC gene expression, myeloperoxidase (MPO), terminal transferase (TdT) (lymphoma, leukemia); neuron-specific enolase (NSE) (neuroblastoma); and tumor suppressors lost in cancer (e.g., BRCA1, BRCA2), such as prostatic acid phosphatase (PAP) (metastatic prostate cancer), cardiovascular and cardiometabolic markers (e.g., C-reactive protein (CRP), troponins, including high-sensitivity cardiac troponin I and cardiac troponin T (e.g., cTnI and cTnT), B-type troponin (B-type troponin), and cytoplasmic reticulocyte colony stimulating factor (CRP). thiuretic peptides (e.g., BNP and NT-proBNP), D-dimer, tetranectin, serum cyclin-dependent kinase 9, CK-MB, galectin-3, adiponectin, adipocyte fatty acid-binding protein, heart-type fatty acid-binding protein, lipocalin-2, fibroblast growth factors 19 and 21, retinol-binding protein 4, plasminogen activator inhibitor 1, 25-hydroxyvitamin D and proprotein convertase subtilisin / kexin type 9 (PSCK9), lipocalin-2, H-FABP, A-FABP), triglycerides, high-density lipoprotein (HDL)-cholesterol and low-density lipoprotein (LDL)-cholesterol; growth factors (e.g., TGFβ, FGF-19, FGF-21, EGF, PDGF);and inflammatory biomarkers (e.g., interferons and cytokines (e.g., TNFα, IL-1, IL-6 and other interleukins), alpha-1 antitrypsin, alpha-1 glycoprotein, anti-CCP, ASO (antistreptolysin), complement C3, complement C4, CRP, IgA, IgE, IgG, IgM, procalcitonin, PCT (BRAHMS), rheumatoid factor), chemokines (e.g., G-CSF, GM-CSF), RPB-4, PAI-1, 25-hydroxyvitamin D, etc.). In some embodiments, targets include hormone, amyloid, and other receptors (e.g., IFN receptors, IL-6 receptors, IL-10 family receptors, TGFβ family receptors, chemokine receptors); protein signatures (e.g., 5-protein signature (OVA1)), and gene signatures (e.g., 17-, 21-, 46-, and 70-gene signatures). In other embodiments, targets include disease vectors, including bacteria, viruses, and fungi. In some embodiments, targets include bacterial, viral, and / or fungal nucleic acids, alone, together, or in multiplexed formats;

[0208] Other targets include active B-12, B12, ferritin, folate, haptoglobin, homocysteine, iron, transferrin, and UIBC (unsaturated iron binding capacity). These targets may be used, for example, in assays or tests for anemia.

[0209] Other targets include active alkaline phosphatase, calcium, intact PTH, magnesium, phosphorus, and vitamin D. These targets can be used in assays or tests for bone diseases or disorders, including, for example, assessing bone remodeling and identifying disorders involving mineral pathways that affect bone formation.

[0210] Other targets include targets for use in tests or assays to test for many types of cancer, including breast cancer, colon cancer, gastrointestinal cancer, liver cancer, ovarian cancer, pancreatic cancer, testicular cancer, and prostate cancer, as well as others listed. Additional cancer-related targets include CYFRA 21-1 (cytokeratin 19 fragment), pepsinogen I and pepsinogen II, and PIVKA-II (circulating precursor of prothrombin and hepatocellular carcinoma marker), proGRP (progastrin-releasing peptide), and SCC (squamous cell carcinoma-associated antigen).

[0211] Other targets include targets for use in tests or assays to test for metabolic diseases that affect glucose function, including diabetes, including C-peptide, creatinine, creatinine (enzyme), fructosamine, glucose, hemoglobin A1c, insulin, and microalbumin.

[0212] Other targets include targets for use in tests or assays to test for the presence of drugs of abuse and toxic levels of prescription drugs, including acetaminophen, amphetamine / methamphetamine, barbiturates, benzodiazepines, cannabinoids, cocaine, ecstasy, methadone, ethanol, methanol, sedatives, PCP (phencyclidine), salicylates, and antidepressants, including tricyclic antidepressants.

[0213] Other targets include targets for use in reproductive endocrinology tests or assays to assess fertility and / or pregnancy status, including DHEA-S, estradiol, FSH, hCG (including total beta-hCG), LH (luteinizing hormone), progesterone, prolactin, SHBG (sex hormone binding globulin), and testosterone (free testosterone, bound testosterone, and / or total testosterone). Assays that can be performed using the systems, devices, methods, and compositions of the present invention include second-generation testosterone assays.

[0214] Other targets include targets for use in infectious disease tests or assays, including CMV IgG, CMV IgM, CMV IgG avidity, rubella IgG, rubella IgM, Toxoplasma IgG, Toxoplasma IgM, and Toxoplasma IgG avidity.

[0215] Targets also include hepatitis targets, including anti-HAV IgG, anti-HAV IgM, anti-HBc IgM, anti-HBe, anti-HBs, anti-HCV, HBeAg (including HBsAg quantification and qualification), and HCV Antibody Antibody (HCV Antibody) Ag. Other infectious disease targets include Chagas (caused by the parasite Trypanosoma cruzi), EBV EBNA-1-IgG, EBV VCA IgG, EBV VCA IgM, and syphilis TOP. Other targets include anti-HTLV-I / HTLV-II (retroviruses).

[0216] Other targets include targets for use in tests or assays using the systems, devices, methods, and compositions of the present invention to assess or diagnose liver function and / or liver disease. These targets include albumin (BCB and BCP), alkaline phosphatase, alpha-1 antitrypsin, ALT (alanine aminotransferase), ALT, activated (alanine aminotransferase), ammonia, AST (aspartate aminotransferase), AST, activated (aspartate aminotransferase), bile acids, cholinesterase, cholinesterase / dibucaine, direct bilirubin, total bilirubin, GGT (gamma-glutamyltransferase), lactate dehydrogenase, and PIVKA-II (des-gamma-carboxyprothrombin).

[0217] Other targets include targets for use in tests or assays using the systems, devices, methods and compositions of the invention to assess or diagnose traumatic brain injury (mTBI [UCH-L1+GFAP]).

[0218] Other targets include targets for use in tests or assays using the systems, devices, methods, and compositions of the invention to assess or diagnose thyroid disorders, including anti-thyroid peroxidase (anti-TPO) and anti-thyroglobulin (anti-TG) antibodies, free T3 (triiodothyronine), total T3, free T4 (thyroxine), total T4, TSH (thyroid stimulating hormone), and T uptake (thyroid hormone uptake, which provides information about the number of thyroid hormone binding sites, primarily consisting of thyroid-binding globulin, thyroxine-binding prealbumin, and albumin).

[0219] Other targets include targets for use in tests or assays using the systems, devices, methods and compositions of the present invention to assess or diagnose kidney diseases or disorders. These targets include beta-2-microglobulin, creatine, creatine (enzyme), cystatin C, microalbumin, NGAL (neutrophil gelatinase-associated lipocalin), protein (urine / CSF), urinary nitrogen, and uric acid.

[0220] Other targets include targets for use in tests or assays using the systems, devices, methods and compositions of the present invention to help prevent rejection and reduce toxicity in transplant patients. Targets include cyclosporine, sirolimus and tacrolimus.

[0221] Other targets include the proteins apolipoprotein A1, apolipoprotein B, transferrin, ceruloplasmin, haptoglobin, Lp(a), and prealbumin.

[0222] Other targets include any therapeutic agent(s) for treatment monitoring and precision medicine, e.g., tailoring medical decisions, treatments, practices, or products to subgroups of patients. Targets for use in a subject, monitoring or evaluation of therapeutic activity in a subject, or suitability for a subject include any commercially available therapeutic agent or therapeutic agent candidate (including clinical trial candidate). These include, for example, amikacin, digitoxin, digoxin, lithium, methotrexate, steroids (e.g., progesterone), phenytoin, quinidine, theophylline, anticonvulsants (e.g., valproic acid), antifungals, antivirals, and antibiotics (e.g., tobramycin, vancomycin).

[0223] In some embodiments, the devices, systems, methods, or compositions are used in (or as) diagnostic methods, including, but not limited to, diagnostic methods, assays, and tests for targets including pathogens, cardiovascular and neurological phenomena, and diseases, disorders, and conditions, including cancer (e.g., early detection of cancer), and other methods, assays, and tests directed to any of the targets disclosed or referenced herein.

[0224] The devices, systems, methods, and compositions of the invention can be used in or with any assay format or device. Formats include direct, indirect, and sandwich assays performed manually or semi-automatically, such as in multiwell plates (e.g., 8-, 24-, 48-, 96-, and 384-well plates) in which samples are measured in duplicate. They include any immunoassay, including any of the immunoassays described or referenced herein. They include the use of the devices, systems, methods, and compositions of the invention in or for any ligand binding assay that measures binding between a ligand and a receptor, any immunoassay that detects antibody-antigen binding, and any bioassay that measures biological activity in response to a stimulus.

[0225] In some embodiments, the present invention includes any assay or assay device or assay format characterized in that it comprises a fluid-fluid interface and / or fluid phase or layer stabilized with at least one fluid-loving associated support structure (e.g., a porous mesh).

[0226] In some embodiments, reagents for detecting or quantifying a target may be contained at, near, or on the bottom surface of the container. In some embodiments, the reagents for detecting a target include reagents for a loop-mediated isothermal amplification (LAMP) or reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay. In some embodiments, the LAMP or RT-LAMP assay is a colorimetric or fluorescent assay.

[0227] The present invention includes the use of a system or device in a method for isolating a target from a sample. Another embodiment of the present invention includes a method for isolating a target from a sample, the method comprising: (a) adding the sample to a system comprising at least one aqueous phase and at least one oil phase stabilized in close proximity to each other in a container; and (b) applying a magnetic force to the system, wherein the sample is contacted with paramagnetic particles (PMPs) prior to applying the magnetic force to the system, whereby one or more target-PMP complexes are generated by contacting the sample with the paramagnetic particles, and applying the magnetic force to the system attracts the target-PMP complexes through the at least one aqueous phase and the at least one oil phase toward the bottom surface of the container. In some embodiments of the method, the at least one aqueous phase and the at least one oil phase are stabilized in the container by a hydrophilic porous material immersed in the at least one aqueous phase and / or a hydrophobic porous material immersed in the at least one oil phase, and / or by adjusting one or more chemical or physical material characteristics selected from buoyancy, surface chemistry, and porosity of the hydrophilic / hydrophobic porous material, if present in the system. In some embodiments of the method, the method involves the use of a system of two or more aqueous phases or layers and two or more oil phases or layers, which may or may not be stacked alternately within the vessel, e.g., such that the first and second aqueous phases do not directly contact each other and the first and second oil phases do not directly contact each other. Other embodiments regarding vessels, inserts, PMPs, wash buffers, lysis buffers, etc., as well as samples and reagents, are as described above.

[0228] In another method embodiment of the present invention for isolating a target from a sample, the method includes: (a) adding the sample to a system comprising a first aqueous phase, a second aqueous phase, a first oil phase, and a second oil phase, the phases or layers being stacked alternately in a container such that the first and second aqueous phases are not in direct contact with each other and the first and second oil phases are not in direct contact with each other, and the phases or layers are stabilized in the container by a hydrophilic porous material immersed in the first aqueous phase, a hydrophilic porous material immersed in the second aqueous phase, a hydrophobic porous material immersed in the first oil phase, and a hydrophobic porous material immersed in the second oil phase; and (b) applying a magnetic force to the system, the sample being contacted with paramagnetic particles (PMPs) before applying the magnetic force to the system, contacting the sample with the paramagnetic particles to generate one or more target-PMP complexes, and applying the magnetic force to the system attracts the target-PMP complexes through the phases toward the bottom surface of the container. In some embodiments of this method, the phases are further stabilized in the container by adjusting the surface chemistry so that the buoyancy of each oil phase is less than the surface tension of each aqueous phase and / or less than the water-retaining capacity of each hydrophilic porous material. In some embodiments of this method, the container comprises a top opening to allow for the addition of a sample. In some embodiments of this method, the first aqueous phase or layer or the first oil phase or layer is closest to the top opening of the container. In some embodiments of this method, the first aqueous phase or layer or the second aqueous phase or layer, or both, comprises a lysis buffer or consists essentially of a lysis buffer. In some embodiments of this method, the first aqueous phase or layer or the second aqueous phase or layer, or both, comprises a wash buffer or consists essentially of a wash buffer. In some embodiments of this method, the first aqueous phase or layer or the second aqueous phase or layer comprises a lysis buffer or consists essentially of a lysis buffer, and the first aqueous phase or layer or the second aqueous phase or layer comprises a wash buffer or consists essentially of a wash buffer. In some embodiments of this method, all of the PMPs are contained within the container. In some embodiments of this method, all of the PMPs are contained within one or more aqueous and / or oil phases or layers.In other embodiments of this method, some PMPs are contained within a container, and some are added to the sample or the container, or both, during the method. In some embodiments, the sample is a biological sample, an environmental sample (e.g., a sewage sample), a saliva sample, a swab sample, or a sample obtained from a subject suspected of having an infectious disease. In some embodiments, the subject is suspected of having a viral infection, a viral upper respiratory tract infection, or an infection selected from, for example, SARS-CoV-2, SARS, coronavirus, rhinovirus, influenza, and respiratory syncytial virus. In certain embodiments, the target comprises viral nucleic acid. In some embodiments, the target comprises SARS-CoV-2, hepatitis B, hepatitis C, HIV, West Nile virus, herpes, and / or influenza nucleic acid.

[0229] In some embodiments, the fluid phases and layers, including aqueous, gas, and oil phases and layers, stabilizing structures, and other components described herein, are designed and incorporated together in a support (e.g., container, vessel, insert, etc.) to form systems, devices, and methods using certain predefined design guidelines. Design guidelines for each component may depend on one or more factors such as, for example, support design (i.e., single-piece body, multi-piece body, modular body, single read chamber, multi-read chamber, etc.), manufacturing process (e.g., injection molding, blow molding, hot stamping, casting, machining, etc.), phases and layers (e.g., aqueous, oil, gas, blends, mixtures, emulsions, etc.), structural material (e.g., polypropylene mesh, nylon mesh, glass mesh, porous plastic screen, PVDF, polystyrene, or other stabilizing structure), material porosity, functional requirements (e.g., sample size, reagent volume, detection technology, time to result, incubation, heating, etc.), safety / handling requirements (e.g., self-containment, regulatory approval, ease of use, etc.), and, in the case of assays, assay requirements (e.g., binding assay, competitive binding assay, single-step assay, two-step assay, etc.).

[0230] An embodiment of the present invention, depicted in Figure 15 for use in a sandwich ELISA assay, includes, for example, materials and methods for the following construction. This embodiment uses the following materials: a container (e.g., a 96-well microtiter plate, an injection-molded article, etc.), a hydrophobic porous structural material (e.g., polypropylene mesh, etc.), a hydrophilic porous structural material (e.g., nylon mesh, etc.), paraffin wax having a melting temperature of approximately 35°C, mineral oil, a primary antibody binding buffer (containing buffering components, salt components, detergent, protein components, etc.), paramagnetic particles conjugated to an antibody against a target, a secondary conjugated antibody binding buffer (containing a secondary antibody conjugated to an enzyme, e.g., HRP, alkaline phosphatase, etc.), buffering components, salt components, detergent, protein components, etc.), and a substrate solution (e.g., TMB, para-nitrophenyl phosphate, etc.). The construction and establishment of the stabilized layer and other assay components is as follows: the substrate solution is first added to the bottom (surface) of the container. The container is then heated to above 35°C, and liquid paraffin wax is added. The hydrophobic porous structural material is cut to an appropriate size (e.g., diameter, thickness, etc.) so that the material can be pressed into a container, and added to liquid paraffin. The container is then brought to room temperature (e.g., 22°C), which allows the paraffin to solidify into wax. The hydrophilic porous structural material (e.g., nylon) is cut to an appropriate size (e.g., diameter, thickness, etc.), first immersed in a secondary conjugate antibody binding buffer, and then placed in the container. Mineral oil is added to the container along with the hydrophobic porous structural material, which is then cut to an appropriate size (e.g., diameter, thickness, etc.). Finally, a primary antibody binding buffer and paramagnetic particles conjugated to an antibody against the target are then added to the container along with the hydrophilic porous structural material (e.g., nylon) cut to an appropriate size (e.g., diameter, thickness, etc.).

[0231] In other embodiments, the assay is an RT-LAMP assay, etc. In some embodiments, reagents for RT-LAMP are dried or lyophilized onto the bottom surface of a container. In some embodiments, stabilized phases are established by first immersing an appropriate permeable material (e.g., based on contact angle, pore size, porosity, etc.) in a desired fluid, which is then placed in a container. In some embodiments, layers are constructed in a dry format, whereby an appropriate porous material associated with a phase or layer is immersed in a desired fluid, removed from the fluid, and then frozen (e.g., wax immersion, water freezing, etc.). These components are then added to a container in layers. In some embodiments, excess fluid is added to a container, and porous structural material with and without associated fluid is added to the fluid. In some embodiments, the porous structural material is first placed in a container, and fluid is added. In some embodiments, the temperature is altered to adjust the fluid phase for construction purposes. In some embodiments, the ambient pressure is altered to adjust the fluid phase for construction purposes. In some embodiments, the ambient gas composition is adjusted for construction purposes. In some embodiments, the reagents are dried or lyophilized in a container. In some embodiments, solid components (e.g., salt crystals, PMP, etc.) are added to the stabilized phase before an additional stabilized phase is layered on top. In some embodiments, the buildup of the stabilized phase is performed in an automated manner.

[0232] In some embodiments, a device or system of the invention, including a disposable device or assay or cartridge and / or a point-of-care device or assay or cartridge, is equipped with Bluetooth® functionality (e.g., a chip with a Bluetooth® radio) to allow for transmission of results to a Bluetooth® equipped device (e.g., a phone or computer). In some embodiments, the results of the system or device or results from the methods described herein are transmitted to another device (e.g., a phone, tablet, CPU, computer, imaging device, storage device, etc.) via Bluetooth® or other communication functionality (e.g., Wifi, near-field communication, cellular network, etc.).

[0233] In some embodiments, a computer system is programmed or otherwise configured (or associated with or comprising an apparatus or system of the invention) to perform a method of the present disclosure. In some embodiments, a CPU or computer can execute a sequence of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location. The instructions may be directed to a CPU, which can then be programmed or otherwise configured to perform a method of the present disclosure. Examples of operations performed by a CPU can include sample addition, addition of PMPs (or other target-binding solid phase material(s)), movement of a stabilizing interface structure, application of magnetic or other forces to (or in) a device or system, heating, cooling, or thermal cycling. The CPU can be part of a circuit, such as an integrated circuit. One or more other components of the system can be included in the circuit. In some cases, the circuit is an application-specific integrated circuit.

[0234] A computer system may also include memory or memory locations (e.g., random access memory, read-only memory, flash memory), electronic storage (e.g., hard disk), communication interfaces for communicating with one or more other systems, and peripheral devices such as cache, other memory, data storage devices, and / or electronic display adapters. The storage device may be a data storage device (or data repository) for storing data. The computer system may be operably coupled to a computer network with the aid of a communication interface. The network may be the Internet, an Internet and / or extranet, or an intranet and / or extranet in communication with the Internet. The network may include one or more computer servers, enabling distributed computing such as cloud computing. In some cases, with the aid of a computer system, a network may implement a peer-to-peer network, allowing devices coupled to the computer system to act as clients or servers.

[0235] Devices and functions having magnet receiving areas, ring magnets, electromagnets, magnetic force and / or magnetic field receiving areas, etc. In another embodiment, the invention provides a device or vessel for use in separating, isolating, purifying, identifying, detecting, and quantifying substances, including transporting, processing, and assaying target molecules to determine the presence or amount of the target in a sample, the device or vessel comprising one or more stabilized layers useful for performing the assays described herein and an area for receiving an external movable magnet (e.g., a permanent magnet) or a switchable magnet or magnetic force or field (e.g., an electromagnet). Permanent magnets, switchable magnets, and electromagnets are all known in the art.

[0236] In some embodiments, the device or container includes a central hollow body portion for receiving an external movable magnet or conductive magnetizable implement (e.g., a rod) or other device capable of providing a magnetic force.

[0237] In some embodiments, the device or container includes a central hollow body portion for receiving an external conductive, magnetizable insert (e.g., a rod), electromagnet, etc., or other device capable of providing a magnetic force.

[0238] In some embodiments, the insert (e.g., rod, core, probe, etc.) is an electromagnet, and the portion of the insert that resides within the cavity does not necessarily have a coil itself. The coil may be configured to reside outside the cavity, where the coil is merely at the base of the electromagnet insert. The magnetic field will largely follow the core (e.g., metal core), bringing the magnetic field within the cavity close to the stabilizing mesh, layer, etc., as desired. This is advantageous for space-constrained devices, as the coil takes up space and the magnetic field strength can be supplemented with additional current.

[0239] In some embodiments, a portion of the device is magnetizable and the magnetic field is movable relative to the interior of the device so that the magnetic field partially or completely fills a portion of the device (e.g., the core or central hollow body portion) when target molecules are harvested, and the magnetic field is partially or completely removed when particles are released.

[0240] In some embodiments, the device or vessel comprises a multi-layer geometric device, e.g., a cylinder or other polygon, that includes one or more stabilized layers useful for performing the assays described herein, a central portion for receiving a magnetic force. In some embodiments, the magnetic force comprises an electromagnet. In some embodiments, the magnetic force comprises magnetic or magnetizable segments, e.g., electromagnetic segments or switchable magnets, that can be switched on or off (in whole or in part) to provide a magnetic force (including a magnetic gradient) that draws magnetic particle-target molecule complexes through the device, including through one or more stabilized layers and any other layers present in the device.

[0241] In some embodiments, the device or container includes a central portion for receiving or generating a magnetic force. In some embodiments, the central portion is solid. In some embodiments, the central portion is hollow. In some embodiments, the central portion is honeycomb-shaped. In other embodiments, the central portion is in the form of a parallelepiped, such as a cube, rhombohedron, or parallelepiped, that can receive or generate a magnetic field or force.

[0242] Referring to Figure 30, a core containing electromagnets is shown, which may be engaged in series, for example, from top to bottom. In other embodiments of the device of Figure 30, the electromagnets are replaced with a central portion or core capable of receiving a magnetic field or force. The coating may be made of any desired material, including, for example, iron or iron alloys such as steel, nickel, cobalt, and silica, and may be constructed as desired to provide the required magnetic force. See, for example, the solid and other constructions described in the preceding paragraph.

[0243] In some embodiments, the central portion of the device of the present invention is integral with the device and comprises a magnetizable portion, rod, or other geometric shape capable of receiving a magnetic force or generating a magnetic field, including temporary magnets and switchable magnets.

[0244] Both the central hollow body portion and the central portion can have any shape. In some embodiments, the shape of the central hollow body portion and / or central portion for receiving a magnetic force or generating a magnetic field is cylindrical. In some embodiments, the shape of the central hollow body portion and / or central portion is polygonal, such as rectangular, hexagonal, star-shaped, etc.

[0245] In some embodiments, the movable magnet, rod, etc. and / or external conductive magnetizable implement (e.g., rod) or other device capable of providing a magnetic force is cylindrical. In some embodiments, the rod is another shape. In some embodiments, the rod is, for example, rectangular, pentagonal, hexagonal, or any other polygonal shape. The rod need only have a shape that fits the desired length within the device, for example, to provide a magnetic force or field up to or through one or more of the stabilizing layers or meshes.

[0246] In some embodiments, the central hollow body portion is in the form of a circular or polygonal bore and can be of any desired shape or length suitable for attracting magnetic particle-target molecule complex(es) within the device upon use of an inserted magnet, a switchable magnet, or application of a magnetic force or magnetic field.

[0247] In some embodiments, the central hollow body portion passes through one or more or all of the stabilized layers or mesh, hi some embodiments, the central hollow body portion does not pass through any of the stabilized layers or mesh.

[0248] In some embodiments, the magnetic force or magnetic field is applied to the entire central hollow body portion of the device. In some embodiments, the magnetic force or magnetic field is applied to a portion (or portions) of the central hollow body portion of the device. For example, the central hollow body portion of the device may have two, three, four, five, six, or more portions constructed to be capable of separately receiving magnetic forces, e.g., using electromagnetic coils. In some embodiments, the separate portions capable of being magnetized (e.g., sequentially magnetized) can surround all or a portion or portions of the central hollow body portion of the device. In some embodiments, the separate portions capable of being magnetized can form portions of the walls of the central hollow body portion of the device, if desired for magnetic particle-target molecule movement, e.g., pull-down.

[0249] In some embodiments, the magnetic force or field is applied by a permanent magnet, a temporary magnet, a switchable magnet, or an electromagnet.

[0250] In some embodiments, the magnetic force or field is applied by an electric current.

[0251] In some embodiments, a magnet inserted into the central hollow body portion passes through one or more or all of the stabilized layers or mesh, hi some embodiments, a magnet inserted into the central hollow body portion does not pass through any of the stabilized layers or mesh.

[0252] In some embodiments, the central portion or upper portion of the central hollow body portion of the device is in the form of a dome, cone, or other desired shape. Useful dome shapes include, for example, hemispheres or hemispherical domes, onion domes, elliptical domes, saucer domes, etc. Cone shapes include, for example, triangular, square, pentagonal, hexagonal, and other polygonal pyramids. For example, dome and cone springing or other shape springing may start from any desired point on the central portion or central hollow body portion of the device. The angle of the dome is set by adjusting the height of the dome. The angle of the cone may also be set by adjusting the height of the cone so that the base angle is any angle of 1 degree or greater, e.g., 10-80 degrees, 20-70 degrees, 25-75 degrees, 30-60 or 70 degrees, 40-50 degrees, e.g., 30 degrees, 33 degrees, 35 degrees, 40 degrees, 45 degrees, 48 ​​degrees, 49 degrees, etc., or any number of degrees within or between any of these ranges. In some embodiments, the apex of the top of the central portion or central hollow body portion of the device is above or below one or more or all of the stabilized layers or meshes. The dimensions of the length, width, height, and overall shape of the central portion or central hollow body portion of the device, including the top portion (e.g., flat, dome, cone-shaped, etc.), are suitable for attracting and moving magnetic particle-target molecule complex(es) throughout the device upon use of an inserted magnet (e.g., in the case of a device having a central hollow portion), a switchable magnet, or an applied magnetic force or magnetic field (e.g., in the case of a device having a central portion capable of receiving a magnetic field or magnetic force).

[0253] In some embodiments and methods of the present invention, the magnet is moved the entire length of the central hollow body portion. In some embodiments and methods of the present invention, the magnet is moved a length less than the entire length of the central hollow body portion. The amount of movement and residence time of the magnet in the central hollow body portion is determined based on the assay to be performed and the force desired to attract and move the magnetic particle-target molecule complex(es) through the device with the inserted magnet.

[0254] In some embodiments and methods of the invention, a magnetic force or magnetic field is applied to a central body portion of the device, which may or may not be hollow. In some embodiments and methods of the invention, the magnetic force or magnetic field is applied to a length less than the entire length of the central body portion. The strength, location, and amount (time) of the applied magnetic force or applied magnetic field is based on the assay to be performed and the force desired to attract and move the magnetic particle-target molecule complex(es) through the device upon use of an inserted magnet, a switchable magnet, or an applied magnetic force or magnetic field.

[0255] In some embodiments and methods of the present invention, the device does not use a moving magnet. In some embodiments and methods of the present invention, the device includes an electromagnet. In some embodiments and methods of the present invention, the device including an electromagnet is an internal electromagnet. In some embodiments and methods of the present invention, the device includes an electromagnet on or including a surface of the device. In some embodiments and methods of the present invention, the device includes ring magnet(s) or ring electromagnet(s).

[0256] In other embodiments and methods of the invention, a ring magnet or ring electromagnet moves up and down the outside of the device to move the target-bound magnetic particles through the device to an assay zone, region, or well. In still other embodiments and methods of the invention, the device of the invention moves in and out of the ring magnet or ring electromagnet to move the target-bound magnetic particles through the device to an assay zone, region, or well where, in one embodiment, all or part of the assay may be performed.

[0257] In some embodiments, the device or container includes a cavity or other opening for inserting a magnet used to attract and pull down target molecules attached to a magnetic substance, i.e., a substance that responds to an applied magnetic field. Useful magnetic substances include ferromagnetic, paramagnetic, and diamagnetic substances. In some embodiments, the magnetic substance is, comprises, or consists essentially of paramagnetic particles. In some embodiments, the magnetic substance is, comprises, or consists essentially of magnetic nanoparticles, including, for example, ferrite nanoparticles. In some embodiments, the magnetic substance is, comprises, or consists essentially of superparamagnetic nanoparticles. In some embodiments, the magnetic substance is conjugated to or functionalized with another target-binding substance, such as an antibody, antibody fragment, scFv, etc., as described.

[0258] In some embodiments, portions of the device are magnetizable and the magnetic field is movable relative to the interior of the device such that when target molecules are harvested, the magnetic field partially or completely fills the central or hollow body portion of the device (or portions thereof), and when particles are moved to a desired location and / or released, the magnetic field(s) are partially or completely removed.

[0259] In some embodiments, the device is a single-assay device, hi some embodiments, the device is a multiplexed device, constructed to include two or more, e.g., three, four, five, six, or more, wells and / or channels or other reagent, substrate, and / or assay zones useful for measuring multiple targets simultaneously.

[0260] In some embodiments, the well or assay zone functions as a temporary collection zone or elution zone, hi some embodiments, the temporary collection zone is removable or has a removable, pierceable, fracturable or destroyable surface(s) or feature to provide access to the collected material.

[0261] In other embodiments, reagents for use in determining the presence or amount of multiple different targets are provided in a single well or channel. In some embodiments, the device is a multiplex device and is used to identify the presence or amount of multiple targets in a single well or channel. Multiplex devices of the invention are useful for collecting multiple data sets from the same sample.

[0262] In some embodiments, the device or container includes a cavity or other opening or feature (e.g., a central solid or partially solid portion) adapted for application of a magnetic or magnetic force, e.g., an electromagnetic force, within (or across) the cavity or other opening or feature. In some embodiments, the electromagnetic force is applied and removed (or conversely, is not present and is later applied). In some embodiments, the electromagnetic force is gradually applied and removed (or conversely, is not present and is later gradually applied) from the top to or toward the bottom of the cavity or other opening or feature (e.g., a central solid or partially solid portion adapted for application of a magnetic or magnetic force).

[0263] In some embodiments, the device or vessel includes a cavity or other opening or feature (e.g., a central solid or partially solid portion) that contains a magnetic or magnetic field gradient. In some embodiments, the magnetic or magnetic field gradient is established using gradient coils.

[0264] In some embodiments, the device or vessel includes a core or other portion within the device that includes a magnetized or magnetizable element, a magnetic gradient, or a field gradient. In some embodiments, the magnetizable element is an electromagnet. In some embodiments, the electromagnet can be activated automatically or manually. In some embodiments, the magnetic or field gradient is established using a gradient coil, which can be activated manually or automatically, e.g., via software, a switch, etc., in an external device, e.g., a reader, including the readers described herein.

[0265] In some embodiments, target molecules are moved to a collection or assay region. In some embodiments, magnetic particles are associated with one target molecule or multiple target molecules. In some embodiments, the target molecule(s) are in a sample, e.g., a biological sample. In some embodiments, the target molecules are moved by using a magnetic field. Magnetotransport methods useful for moving target molecules are disclosed.

[0266] In some embodiments, the container comprises a body, a space or region within the body for introducing a substance containing or suspected of containing a target, a space or appropriately sized hollow, cavity, recess, or crevice within the container body for inserting a movable device or other device useful for attracting particles, including targets, e.g., magnetic particles, including the described paramagnetic particles, ferromagnetic particles, etc. Some embodiments of devices having regions for receiving magnets are shown in Figures 21-25 and 26.

[0267] In some embodiments, the device includes a container having a sample receiving portion including an enclosure including a space or opening for sample application or insertion, a top, a bottom, and a central hollow body portion or other space for receiving a magnet or magnetic force, and the container further includes at least one aqueous phase or layer and at least one oil / wax phase or layer or gas phase or layer stabilized in close proximity to each other by the inclusion of a porous (e.g., permeable to target-magnetic particle complexes, beads, and / or solid phases) structural material associated with the aqueous phase / layer or gas phase / layer or oil / wax phase / layer, or both. Other phases or layers and / or alternative phases or layers may be used or included (e.g., two oil phases with or without an aqueous phase or layer). In some embodiments, the structural material includes a SIFT composition, system, or device. In some embodiments, the structural material includes a MIFT composition, system, or device. One embodiment of the present invention including a SIFT structure is shown in FIG. 22. Other embodiments of the present invention including a SIFT structure and a magnetic cavity are shown in FIGS. 24 and 25. The present invention also provides methods for performing the described assays and other functions using the device. See, for example, Figures 23 and 26.

[0268] In some embodiments, the container further comprises target-binding paramagnetic particles (or another target-binding carrier substance). In other embodiments, the paramagnetic particles (or another target-binding carrier substance) are mixed with the sample before introduction into the device or container. In other embodiments, some paramagnetic particles (or another target-binding carrier substance) are provided in the device or container, and some are mixed with the sample before introduction into the device or container.

[0269] In some assay embodiments, a magnet is moved into the magnet-receiving region of the vessel by any desired means, including, for example, mechanically, electromechanically (e.g., using a switch), or electromagnetically, all of which can be controlled, for example, manually, using software, or an integrated circuit-style assembly, such that the magnet moves into the device, attracts magnetic particles and / or target / magnetic particle complexes, e.g., PMPs and / or target-bound PMPs, and lowers, e.g., via gravity or by controlled lowering, gravitationally, mechanically, or automatically, at a desired or predetermined pace. In some embodiments, as described above, magnetizable cores, electromagnets, etc., are fabricated to provide controlled movement or gradient magnetic forces for movement of magnetic particle-target molecule complexes.

[0270] In some embodiments, the assay is provided in a device or container and includes one or more (or all) reagents for target detection or quantitation. See, for example, the embodiment of Figure 24. In some embodiments, the assay (or one or more reagents for the assay) is external to a container, which is connected to an assay-containing device that includes one or more or all of the reagents for performing the assay.

[0271] In some embodiments, the sample is a nasopharyngeal sample, an oropharyngeal sample, an oral swab sample, an oral sponge sample, a nasal swab sample, a middle turbinate sample, or a saliva sample. In certain embodiments, the biological sample is an oral swab or a nasal swab, or a saliva sample. Nasopharyngeal (NP) swabs are considered the highest yielding samples in diagnostic testing for respiratory viruses, including SARS-CoV-2. In some embodiments, the sample is an environmental sample. For example, the sample may be a sewage sample.

[0272] In some embodiments, the environmental or biological sample is a crude sample, i.e., a sample in which the target molecule has not been wholly or partially isolated, purified, or amplified from the sample prior to application of the device of the present invention. In some embodiments, the environmental or biological sample is a sample that has been enriched or amplified for the target (e.g., by prior purification / isolation / extraction methods, etc.).

[0273] In some embodiments, as described above, the biological or other sample may be first mixed with a lysis / binding buffer or other buffer with or without a solid phase or lysis / binding buffer and a solid phase (e.g., PMPs), and then added to the device or container. In some embodiments, the container or device already contains a lysis / binding buffer, other buffer, etc., and a magnetic material with or without another solid phase (e.g., PMPs), and the biological sample is added thereto. In some embodiments involving a biological sample obtained via a swab, the swab is immersed and mixed with a lysis / binding buffer, etc. (and, if already contained in the device or container, e.g., PMPs). In some embodiments, the biological sample is obtained using a separate device or container, and then coupled with a container already containing a lysis / binding buffer and a solid phase, e.g., PMPs. See, e.g., FIG. 23. The coupling / joining of two containers / devices provides for the introduction of the biological or other sample into the container or device. In some embodiments, for example, the container includes threads or other means (e.g., snap fits and tabs, snap-ons, snap points, push-in rivets (including clinch, countersink, snap and barbed push-in rivets), snap rivets, quarter-turn fasteners, clips, adhesives, etc.) for receiving a device, tool, or instrument containing a sample containing or suspected of containing a target (e.g., a swab). Other attachment means include a molded top that includes a shoulder-washer type opening for receiving the sample or sample-containing device. See, e.g., Figures 22 and 23. Any means or mechanism for connecting a sample-containing device to the devices of the invention may be used. Such connection means are well known.

[0274] In some embodiments, the threads or other attachment means are located on the outside of the container. See FIGS. 22 and 23. In some embodiments, the device has internal threads designed to receive a sample-containing device having external threads. In some embodiments, the threads or other attachment means are embedded into or comprise a portion of the top of the container that is adapted to receive a sample-containing device (e.g., a swab). Such inserts include features (e.g., threads) that are embedded into the container (e.g., top) by any number of processes, including injection molding, ultrasonic welding, heat staking, and press-fitting. In some embodiments, these features (e.g., threads) are molded into the container. In other words, in some embodiments, the devices of the present invention may be externally threaded, in some embodiments, the devices of the present invention may be internally threaded, and in some embodiments, a portion of the top of the device of the present invention may include a threaded opening or may alternatively be constructed in other formats with an opening designed to receive a sample-containing device, e.g., with a tab, snap point, snap-on, etc. In some embodiments, the cap can contain a sealed reagent(s) (e.g., functionalized magnetic particles, lysis binding buffer, extraction buffer, etc.), and the act of placing the cap on the device pierces or breaks a seal or other form of barrier to release the reagent(s). In some embodiments, the device can contain a sealed reagent(s), and the act of placing the cap on the device pierces or breaks a seal or other form of barrier to release the reagent(s) contained within the device.

[0275] In some embodiments, the sample application space or the top of the device further comprises one or more of a buffer (e.g., reverse transcription buffer, lysis binding buffer, etc.), an extraction buffer or extractant, magnetic particles (e.g., paramagnetic particles), a stabilizing agent, an antimicrobial agent or other sample modifying reagent, and magnetic particles (e.g., paramagnetic particles) conjugated to or functionalized with a target binding agent.

[0276] In some embodiments, one or more of the stabilized phases or layers in the devices of the invention further comprise one or more of the following, as needed or desired: a buffer (e.g., a reverse transcription buffer, a lysis binding buffer, etc.), an extraction buffer or extractant, one or more paramagnetic particles conjugated to a target binding agent.

[0277] In some embodiments, as described above, the devices of the present invention are configured for multiplex assays, allowing for simultaneous measurement of multiple analytes. See Figures 23A and 23B, which illustrate a hexaplex assay. A multiplex assay can be derived from an ELISA format, an amplification format, or the like, but instead of performing a single signal measurement, multiple signal measurements are performed as described above. Assays using the multiplex devices of the present invention or assays using the devices of the present invention in a multiplexed manner such that many targets (e.g., proteins, antibodies, nucleic acids, cells, etc.) can be measured simultaneously. In a multiplex configuration, target / bead complexes are dispersed throughout the mesh and moved or pulled in roughly equal or somewhat similar proportions (or other proportions, as desired) to physically distinct compartments, including wells or channels, each containing unique amplification / detection chemistry for a specific target.

[0278] In some embodiments, the wells or assay zones function as temporary collection or elution zones. In some embodiments, the bottom of the device may be removable or may have removable or pierceable surface(s) or other structures to provide access to the collected material, such as crushable, rupturable, or destructible surface(s) or surface(s) or port(s) that provide access to other wells or collection zones. The collected material can then be used for any downstream processing (e.g., detection, quantitation, manipulation, analysis, etc.) or can be removed and transferred to another device (e.g., a thermocycler).

[0279] In some embodiments, one or more wells may be at the bottom of a channel contained within the device. In some embodiments, the opening(s) of the channel(s) contained within the device may begin at or just below the final stabilized phase or layer, or may begin at or below the final phase or layer in the device. For example, referring to Figures 23B and 28, each shows six wells, one or more of which may comprise a length of channel that extends into the device and terminates at a point below the last or terminal phase or layer or stabilized phase or layer.

[0280] In some embodiments, the device or container is a cartridge. In some embodiments, the device is disposable. In some embodiments, the device container or cartridge is a point-of-care container or cartridge and is provided with or without a point-of-care reader. In some embodiments, the device or container, e.g., cartridge, is reusable. In some embodiments, the point-of-care reader is a single device or cartridge reader. In some embodiments, the point-of-care reader is capable of reading more than one device (e.g., cartridge).

[0281] Point-of-care testing devices provide results within a short time of taking the test, allowing for rapid diagnosis and quicker decisions regarding patient care. Point-of-care testing, also known as near-patient testing, using point-of-care devices and readers such as those described herein, enables physicians and medical teams to obtain laboratory-quality, real-time results in minutes instead of hours.

[0282] In one embodiment, the device, which may be in the form of a cartridge, may be cylindrical or may be formed in another geometric shape, for example, square, rectangular, triangular, or pentagonal, hexagonal, heptagonal, octagonal, nonagonal, decagonal, etc., or a mixture of geometric shapes, for example, square top and cylindrical bottom, rectangular top and cylindrical bottom, cylindrical top and rectangular bottom, square top, cylindrical in the middle and rectangular bottom, etc., as desired. In some embodiments, the cartridge (or the bottom of the cartridge) is formed to fit a protrusion or cavity on a plate or reader containing a moving magnet or magnetic rod or other structure capable of receiving or generating a magnetic field or force.

[0283] Figure 21A shows one embodiment of the device with a configuration for the magnet insertion cavity, and Figure 21B shows a different magnet cavity configuration with a minimum distance between the top of the magnet and the top of the mesh, which can provide improved or enhanced bead jumping through the mesh / phase / layer.

[0284] Another embodiment of the device of the present invention is shown in Figure 22. Figure 22a shows a screw-type cartridge with a central hollow section for receiving a magnet. In this embodiment, the cartridge contains a main body, paramagnetic particles (PMPs) in a sample buffer, a SIFT mesh (hydrophobic), a hydrophobic phase (e.g., oil, wax, etc.), and reagents for performing an assay (e.g., RT-LAMP (loop-mediated isothermal amplification) reagents or RT-qPCR reagents for amplifying and identifying a target (e.g., a pathogen transcript)). Figure 22b shows the permanent magnet inserted into the cavity by the electromagnet. Figures 22c and 22d show that PMPs accumulate on the SIFT mesh (Figure 20c) around the middle dome-shaped feature (Figure 20d). Figure 22e shows that the electromagnet is released and the permanent magnet begins to fall, pulling the beads toward the SIFT mesh. Figure 22f shows that upon contact with the SIFT mesh (stabilized interface), the PMP packet flattens along the mesh, allowing the bead packet to move through the mesh pores. Figures 22g and 22h show that the permanent magnet continues to fall (by gravity in this embodiment) and continues to pull the PMP packet through the mesh. Once through the mesh, gravity takes over and continues to pull the PMPs into the RT-LAMP reaction reagents, regardless of whether the permanent magnet is still coupled to the device.

[0285] Figure 23 shows an example of one embodiment of a device of the invention with an external thread for direct insertion of the device containing a sample, in this case a swab, with an internal thread into a buffer (e.g., lysis binding buffer) with or without target-binding paramagnetic particles. The bottom of the device, which is a multiplex device, displays a cavity for inserting a magnet.

[0286] Figure 24 shows a premix embodiment of the device shown in Figure 23. Premix embodiment is used here to refer to an aqueous format of the RT-LAMP assay where all reagents needed to perform the assay (i.e., primers, salts, polymerase, etc.) are already in solution / dissolved.

[0287] FIG. 25 shows an embodiment of the freeze-dried reagent of the device shown in FIG.

[0288] Figure 26 illustrates an embodiment of the present invention referred to as an "internal reader." In this embodiment, when a device of the present invention is inserted into another instrument for the device, e.g., a reader, an ultrasonic sonotrode within the device (e.g., reader) couples to the device (e.g., exterior) and transmits ultrasonic energy through the device or a portion thereof (e.g., body or portion thereof) to the sample / buffer / PMP mixture, lysing any microorganisms that may be present. The device and its contents further receive heat from the reader through a combination of conduction and convection. As the contents of the tube heat up, in this embodiment, the wax melts, causing phase inversion at the bottom of the tube. This phase inversion facilitates reconstitution of the lyophilized RT-LAMP pellet with LAMP reconstitution buffer. Once the wax melts and the RT-LAMP reaction solution is reconstituted, an electromagnet repels the magnet upward into a magnet-receiving cavity within the device, opening to the exterior at the bottom of the device; in some embodiments, the cavity can extend to the bottom of the stabilized interface. A magnet, e.g., a neodymium permanent magnet, pulls the PMP / target complex across the interface and into the wax / oil phase. After a dwell time, the electromagnet is turned off, allowing the permanent magnet to fall and pull the PMP / target complex into the RT-LAMP reaction solution.

[0289] In some embodiments, the magnet, e.g., a neodymium permanent magnet, is plated to protect the magnet from corrosion. In some embodiments, the magnet is plated with Ni-Cu-Ni. Plating to protect magnets from oxidation and other damage is well known in the art. The present invention also provides groups of vessels or multiple vessels, e.g., cartridges, e.g., groups of 6, 12, 24, 48, 96, 384, or 1536 vessels or caps or removable components, which may be attached or arranged in a matrix and designed to fit into corresponding 6, 12, 24, 48, 96, 384, or 1536 instruments (e.g., plate readers). In such embodiments, multiple samples may be processed simultaneously.

[0290] In some embodiments, any component or surface (or portion of a surface) of the device, including, for example, the cap, body, removable component, mesh, swab, or other sample collection device(s), or any wall, cavity, etc., can comprise an exclusively liquid-repellent surface. See Li, C., Hite, Z, Warrick, JW, Li, J., Geller, SH, Trantow, VG, McClean, MN, and Beebe, DJ, Under oil open-channel microfluidics empowered by exclusive liquid repellency Science Advances 6:16 (April 17, 2020); Li C, Yu J, Schehr J, Berry SM, Leal TA, Lang JM, Beebe DJ. Exclusive Liquid Repellency: An Open Multi-Liquid-Phase Technology for Rare Cell Culture and Single-Cell Processing. ACS Appl Mater Interfaces. 2018 May 23;10(20). See also U.S. Patent Application Publication No. 16 / 623,194, "Systems and Methods for Undermedia Repellency." Other durable liquid repellent surfaces now known in the art or later developed may also be used.

[0291] Reader devices for devices having magnet receiving areas, magnetic force and / or magnetic field receiving areas, ring magnets, electromagnets, etc. The present invention also provides an instrument or reader configured to accept a single vessel / cartridge or a bundle of vessels / cartridges, the instrument or reader including electromagnetic or mechanical means for elevating a movable magnet, e.g., a permanent magnet, into a central hollow body portion of the vessel / cartridge. The instrument or reader may also be configured with a rod or other structure capable of receiving or generating a magnetic field or force within the device of the present invention, as described. The reader may also be configured to sonicate the vessel(s) / cartridge(s) via an ultrasonic node element and to provide heat as needed for phase melting and assay reactions via a heating element. In some embodiments, the movable magnet in the instrument / reader is free-floating. In some embodiments, the movable magnet in the instrument / reader is attached to a rod or switch that is engaged to manually or automatically elevate the movable magnet into the device. In some embodiments, the movable magnet in the instrument / reader is a permanent magnet, a temporary magnet, a switchable magnet, or an electromagnet. In some embodiments, the instrument / reader is configured to read assay results, such as fluorometric, calorimetric, luminescent, chemiluminescent, enhanced chemiluminescent, radiometric, direct fluorescence, time-resolved fluorescence, direct chemiluminescent, and / or phosphorescent assay results. Additionally, the reader may be configured with means for short-range transfer of assay result data. In some embodiments, the means for short-range data transfer is Bluetooth, near-field communication (NFC), Wi-Fi, or direct Wi-Fi.

[0292] In some embodiments, the reader, which may be a point-of-care (POC) reader, a portable reader, or a laboratory-based reader, includes one or a combination of the following systems: a magnetic actuation module (for remote actuation or wireless control of magnet insertion into the magnet-receiving cavity of the device; or for the introduction of magnetic force into the cavity of the device), a temperature control system (for providing the appropriate temperature for assays requiring a specific temperature or temperature range), an ultrasound module (for lysing microorganisms, etc.), an optical reading module (for reading, monitoring, and providing assay results), a barcode scanner (for identifying and inventorying test devices, samples, etc., e.g., cartridges), a computing system (consisting of a logic unit (CPU) or integrated circuit, a memory unit, a connectivity unit (e.g., Bluetooth, WiFi, NFC, etc.), and an electronic display), and a power control system. Each of these modules and systems, including the magnetic actuation module, temperature control module, ultrasound and optical reading module, and barcode scanner, are known and available in the art.

[0293] In some embodiments, the reader interfaces with a disposable test device, e.g., a cartridge, upon insertion of a test cartridge into the reader. In some embodiments, insertion of a test cartridge into the reader automatically initiates the test / assay. In some embodiments, the reader is designed to initiate the test / assay upon successful scanning of a barcode on the test cartridge. In some embodiments, the test / assay is initiated by triggering a button. In some embodiments, the test / assay is initiated by triggering an electronic switch (inductive circuit, capacitive switch).

[0294] Once initiated, in some embodiments, a barcode scan is performed.

[0295] In some embodiments, the temperature control unit is already on and begins heating the test / assay device / cartridge as soon as it is inserted into the reader. In some embodiments, insertion of the device into the reader activates the temperature control unit. In some embodiments, there is an incubation period during which the device is heated and wax, if present inside the device, is allowed time to melt.

[0296] In some embodiments, insertion of the device, e.g., cartridge, activates the ultrasonic module. In some embodiments, the ultrasonic module transmits power through a sonotrode that fits around the outside of the center of the test cartridge like a cuff, and transmits power through the body (e.g., plastic) of the test device (e.g., assay cartridge) to the portion of the device containing the sample. In some embodiments, the sonotrode is a simple probe-type instrument that contacts a portion of the exterior of the device. In some embodiments, the ultrasonic power lasts for a period of time. In other embodiments, the ultrasound is pulsed for a period of time.

[0297] In some embodiments, the magnetic actuation module is turned on after a period of heating up the device or otherwise bringing it to the appropriate temperature. In some embodiments, the magnetic actuation module is turned on after sonication of a sample in the device. In some embodiments, the magnetic actuation module is powered via a digital signal (on / off). In some embodiments, the magnetic actuation module is powered via an analog signal to precisely control the raising and lowering of the permanent magnet component of the magnetic actuation module.

[0298] In some embodiments, the optical read module is comprised of a light source having a defined spectrum of output light, an optical filter specific to a particular wavelength of light emitted from the test cartridge, and a photoresistor. In some embodiments, the optical read module is comprised of a light source having a defined spectrum of output light, an optical filter specific to a particular wavelength of light emitted from the test cartridge, and a CCD camera. In some embodiments, fluorescence is measured from a test device, e.g., a cartridge. In some embodiments, color is measured from an assay device. In some embodiments, absorbance is measured from a device. In some embodiments, the output signal from the assay device is continuously monitored. In some embodiments, the output signal from the device is measured periodically according to a set interval. In some embodiments, an endpoint measurement is performed.

[0299] In some embodiments, the computing system processes the raw data from the optical reading module and outputs the test results directly to the user on an electronic display (e.g., computer, phone, tablet, app, etc.). In some embodiments, the computing system processes the raw data from the optical reading module and outputs the test results to the user via Bluetooth or other communication means. In some embodiments, the computing system processes the raw data from the optical reading module and outputs the test results to the user or to a storage device via WiFi or other communication systems, e.g., NFC or direct WiFi.

[0300] The foregoing description of exemplary embodiments of the present disclosure has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosure to the precise form disclosed, as modifications and variations are possible in light of the above teachings or may be acquired from practice of the present disclosure. The embodiments were chosen and described as practical applications of the present disclosure to explain the principles of the present disclosure and to enable those skilled in the art to utilize the present disclosure, with various modifications, in various embodiments suited to the particular uses contemplated. It is intended that the scope of the present disclosure be defined by the claims appended hereto and their equivalents. [Example]

[0301] Example 1: Systems and methods for LAMP-based detection of SARS-CoV-2 RNA The system, apparatus, and method described in this example include reagents for LAMP-based detection of a target contained on the bottom surface of a vessel. The system includes two porous materials, a lysis buffer, and a wash buffer. As shown in Figure 1, in this embodiment, the system is layered from top to bottom as follows: (1) coconut oil, (2) lysis buffer with glass mesh, (3) coconut oil with Porex mesh, (4) wash buffer with glass mesh, (5) coconut oil with Porex mesh, and (6) reagents for the LAMP reaction with glass mesh.

[0302] Each porous material may be a hydrophilic glass mesh, or one porous material may be a glass mesh and the ...

Claims

1. 1. A device for transporting target molecules to an area for isolation, collection, and / or assay, the transport device comprising: (a) an upper portion configured for introducing a sample containing or suspected of containing a target molecule or multiple target molecules; (b) a bottom portion having an area containing one or more reagents for performing an assay or multiple assays; (c) an enclosure including a central hollow body portion suitable for receiving a magnet; and (d) at least one aqueous phase or layer and at least one oil / wax phase or layer or gas phase or layer stabilized in proximity to each other by a target-permeable structural material associated with the aqueous phase / layer or gas phase / layer or oil / wax phase / layer, or both.

2. 10. The device of claim 1, wherein the target permeable structural material comprises or consists essentially of a SIFT composition, system, or device.

3. 10. The device of claim 1, wherein the target permeable structural material comprises or consists essentially of a MIFT composition, system, or device.

4. 10. The device of claim 1, wherein the target permeable structural material comprises or consists essentially of a SIFT composition, system or device and a MIFT composition, system or device.

5. 10. The device of claim 1, wherein a portion of the device's enclosure includes at least one ferromagnetic material, such as a plate or tube or material that is highly susceptible to magnetization, the strength of which depends on the strength of the applied magnetic field, and which ferromagnetic material ceases after the applied magnetic field is removed.

6. 10. The device of claim 1, wherein the magnet is movable relative to the central hollow body portion of the enclosure such that the magnet can be moved partially or completely within the central hollow body portion when target molecules are collected and partially or completely outside the device when particles are released.

7. The device of claim 1 , wherein the device comprises a reagent for determining the presence or amount of a target molecule.

8. 8. The device of claim 7, wherein the reagent is lyophilized.

9. 8. The device of claim 7, wherein the reagents include one or more reagents for performing a loop-mediated isothermal amplification assay or a reverse transcriptase loop-mediated isothermal amplification assay.

10. 8. The device of claim 7, wherein the reagents comprise one or more reagents for performing an assay selected from the group consisting of PCR, RT-PCR, qPCR, qtPCR, multiplex PCR, assembly PCR, and asymmetric PCR.

11. The device of claim 1 , wherein the reagents for determining the presence or amount of a target molecule comprise immunoassay reagents.

12. 12. The device of claim 11, wherein the immunoassay reagents comprise one or more reagents for performing an assay selected from the group consisting of enzyme immunoassay, ELISA, direct ELISA, indirect ELISA, sandwich ELISA, competitive ELISA, enzyme-linked immunosorbent assay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, and enumeration immunoassay.

13. The device of claim 1 , wherein the top of the device includes an opening for introducing the sample.

14. 10. The device of claim 1, further comprising: (e) an internally threaded portion; and (f) an externally threaded sample collection device that can be removed from the device for sample collection and reconnected to the device for assaying the sample for one or more target molecules.

15. 15. The device of claim 14, wherein the externally threaded sample collection device comprises a swab.

16. The device of claim 1 , wherein the target molecule is selected from the group consisting of a nucleic acid, a protein, and a cell.

17. 10. The device of claim 1, wherein the device is a multiplex device.

18. 18. The device of claim 17, wherein the multiplex device is a hexaplex device.

19. 10. The device of claim 1, wherein the device is capable of simultaneously determining the presence or amount of different target molecules in a single sample.

20. 10. The device of claim 1, wherein the central hollow body portion is lined with a coating to facilitate magnet penetration.

21. 21. The device of claim 20, wherein the coating is a polymer coating.

22. 10. The device of claim 1, which is a single-use device.

23. 10. A reader for use with the device of claim 1, the reader including an apparatus configured to receive the device, an electromagnet, and a movable magnet, wherein engagement of the electromagnet causes the movable permanent magnet to be raised to the device on the reader.

24. 24. The reader of claim 23, wherein the moving magnet is free-floating.

25. 24. The reader of claim 23, wherein the moving magnet is a permanent magnet.

26. 24. The reader of claim 23, wherein the movable magnet is a temporary magnet.

27. 24. The reader of claim 23, wherein the moving magnet is an electromagnet.

28. 10. A reader for use with the device of claim 1, wherein the reader includes a movable magnet attached to an instrument, rod, or switch configured to receive the device, and wherein engagement with the rod or switch causes the movable magnet to be lifted to the device placed on the reader.

29. 24. The reader of claim 23, further comprising a temperature regulating element.

30. 24. The reader of claim 23 configured to read fluorometric, calorimetric, luminescent, chemiluminescent, enhanced chemiluminescent, radiometric, direct fluorescence, time-resolved fluorescence, direct chemiluminescent and / or phosphorescent assay results.

31. 24. The reader of claim 23, further comprising means for short-distance data transfer of assay results.

32. 24. The reader of claim 23, wherein the means for short-range data transfer is selected from the group consisting of Bluetooth, near-field communication, Wi-fi and direct Wi-fi.

33. 10. A method for determining the presence or amount of a target molecule, comprising: (a) introducing a sample containing or suspected of containing at least one target molecule into the device of claim 1; (b) associating the sample with target-specific magnetic particles before or after introducing the sample into the device; (c) bringing a magnet into a central hollow body portion of the device to attract the target-specific magnetic particles; (d) lowering the magnet to pull the magnetic particles through a target-permeable structural material, at least one aqueous phase or layer, and at least one oil / wax or gas phase or layer, bringing the magnetic particles into contact with an assay reagent for the target molecule; and (e) assaying the target-specific magnetic particles to determine the presence or amount of the target molecule.

34. 34. The method of claim 33, wherein the magnet is a permanent magnet.

35. 34. The method of claim 33, wherein the assay is an amplification assay.

36. 34. The method of claim 33, wherein the assay is a reverse transcriptase loop-mediated isothermal amplification assay.

37. 34. The method of claim 33, wherein the device is disposable.

38. 34. The method of claim 33, further comprising transmitting the assay results to a viewing or storage device.

39. 39. The method of claim 38, wherein the viewing device or storage device is a CPU, computer, phone, or tablet.

40. 39. The method of claim 38, wherein the results are transmitted via Bluetooth, near field communication, Wi-Fi or Wi-Fi Direct.

41. 39. The method of claim 38, wherein the results are transmitted over a wired or cellular network.

42. 10. A method for manufacturing the device of claim 1, comprising integrating a container having an upper portion adapted to receive a sample, a bottom portion adapted to receive one or more reagents for performing an assay, a space for receiving a magnet or core for receiving a magnetic field or magnetic force, at least one aqueous phase or layer and at least one oil / wax phase or layer or gas phase or layer, and at least one target permeable structural material, wherein the at least one target permeable structural material is associated with at least one aqueous phase / layer or at least one gas phase / layer or oil / wax phase / layer or both.

43. 43. A method for manufacturing the device of claim 42, wherein the device is manufactured to include a detachable sample collection device.

44. 44. A method for manufacturing a device as recited in claim 43, wherein the removable sample collection device has external fastening threads attached to mating internal threads contained in the top of the device.

45. 45. A method for manufacturing a device according to claim 44, wherein the removable sample collection device is made in the form of a screw cap.

46. 45. A method for manufacturing the device of claim 44, wherein the detachable sample collection device is fabricated to include a swab.

47. 1. A device for transporting target molecules to a region for isolation, collection, and / or assay, the transport device comprising: (a) an upper portion configured for introducing a sample; (b) a bottom portion including a collection zone; (c) a central body portion suitable for providing a magnetic force for attracting and moving target-bound magnetic particles; and (d) at least one aqueous phase or layer and at least one oil / wax phase or layer or gas phase or layer stabilized in close proximity to each other by a target-permeable structural material associated with the aqueous phase / layer or gas phase / layer or oil / wax phase / layer, or both.

48. 48. The device of claim 47, wherein the collection zone further comprises one or more reagents for performing an assay.

49. 48. The device of claim 47, comprising more than one collection zone.

50. 48. The device of claim 47, wherein the collection zone comprises a well.

51. 48. The device of claim 47, wherein the collection zone comprises a channel.

52. The device of claim 1 , wherein one or more portions of the device comprise an exclusively liquid-repellent surface.

53. 52. The apparatus of claim 51 , wherein one or more portions of the apparatus comprise a three-phase system comprising a solid surface, a liquid dispersed phase, and a liquid continuous phase, which results in complete repellency of the dispersed droplets by the liquid continuous phase about the solid surface.

54. 48. The device of claim 47, wherein the central body portion includes a core adapted to receive a magnetic field or force.

55. 55. The device of claim 54, wherein one or more distinct portions of the core are adapted to receive a magnetic field or force.

56. 56. The apparatus of claim 55, wherein one or more separate portions of the core are adapted to receive a magnetic field or force in series from top to bottom.

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