Systems and methods for isolating targets from a fluid sample - Patents.com

Abstract

Provided herein are systems and methods for isolating, extracting or separating at least one target or component from a sample or a sample-containing fluid. In some aspects, provided herein are systems and methods for isolating at least one target or component from a sample or a sample-containing fluid by diffusion of particles from the sample or sample-containing fluid to a target fluid. In some aspects, provided herein are systems and methods for isolating at least one target from a fluid sample by attracting particles (e.g., targets, contaminants) bound to magnetic particles (e.g., paramagnetic particles) to a target fluid through application of magnetic force. In some aspects, provided herein are systems and methods for transferring targets or sample components, or complexes comprising magnetic particles and targets or sample components, to another or different device or reagent mixture or reagent for further processing and / or analysis, identification or quantification.

Description

[Technical field]

[0001] <Statement of government support> This invention was made with Government support under Grants R43 OD023021-01A1 and R44 GM119881 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0002] <Statement Regarding Related Applications> This application claims priority to U.S. Provisional Patent Application No. 63 / 324,924, filed March 29, 2022, the entire contents of which are incorporated herein by reference for all purposes.

[0003] <Incorporated by reference> All U.S. patents, U.S. patent applications, publications, foreign patents, foreign and PCT published applications, articles and other documents, references and publications described herein, as well as all that are listed as references cited in any patent(s) issuing from this specification, are incorporated herein by reference in their entirety. The incorporated information is part of this application to the same extent as if all of the 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] <Technical field> The present invention generally relates to extraction, separation, and isolation of materials. Provided herein are systems and methods for extracting, separating, or isolating targets. In some aspects, provided herein are systems and methods for isolating targets from a sample or a fluid containing a sample by natural transport processes (e.g., diffusion, convection, etc.) that move targets from the sample or fluid containing the sample to a target fluid. In some aspects, provided herein are systems and methods for extracting or isolating targets from a sample, e.g., a fluid sample, by using active forces (e.g., centrifugation, magnetism, etc.), which may include drawing one or more targets and / or one or more contaminants bound to magnetic particles into a target fluid through application of magnetic force. [Background technology]

[0005] The ability to extract, separate or isolate targets (e.g., nucleic acids, proteins, whole cells) from a complex background is a key prerequisite for many common analytical processes in diagnostics, biological research, biomarker discovery, forensics, etc. However, traditional target isolation processes are time consuming, expensive, laborious and often represent a bottleneck in the analytical process. Summary of the Invention

[0006] The invention described and claimed herein has many attributes and embodiments, including, but not limited to, those described or illustrated or referenced in this brief summary. The brief summary is not intended to be comprehensive, and the invention described and claimed herein is not limited to or by the features or embodiments identified in this introduction, which are included for purposes of example only and not limitation.

[0007] Some conventional methods for extracting or separating or isolating targets can cause damage to the sample or result in unwanted loss or inconsistent yield of the sample. Also, some methodologies involve significant levels of unwanted carryover during the isolation of the target, resulting in contamination that can jeopardize downstream applications, such as assays for the detection of analytes. Therefore, improved methods and systems for extracting, separating and / or isolating targets from samples are needed and are provided herein.

[0008] Other conventional methodologies involving the use of beads (e.g., paramagnetic particles) to extract, separate and / or isolate targets from a sample result in incomplete bead transfer and / or capture for analysis. Provided herein are improved methods and systems for extracting, separating and / or isolating targets from a sample where no beads or bead packets remain when performing one or more of the methods, including extraction / separation / isolation methods that allow all beads and bead packets to be transferred, inoperable and counted.

[0009] The methods and devices and systems of the present invention can be used to extract, separate or isolate targets from a sample for further processing and / or transfer to another device or reagent mixture or reagent for further analysis, identification, quantification or use.

[0010] In some aspects, methods and systems are provided herein for extracting and / or isolating at least one target from a fluid sample or a fluid comprising a sample. In some aspects, methods and systems are provided for extracting and / or isolating at least one target from a fluid sample or a fluid comprising a sample for processing. In some aspects, methods and systems are provided for extracting and / or isolating at least one target from a fluid sample or a fluid comprising a sample and transferring the target to another device for processing. In some embodiments, the target is an analyte and the processing or device for processing is an assay. In some embodiments, the assay is used to qualitatively evaluate or quantitatively measure the presence, amount, or functional activity of the target. In some aspects, methods and systems are provided for processing and presenting at least one target from a fluid sample or from a fluid comprising a sample for an assay. In some embodiments, the method or system of the invention comprises an assay for detecting, identifying, and / or determining the presence or amount of a target or analyte.

[0011] In some embodiments, the method includes placing a container containing the sample in a target fluid. In some embodiments, the container includes an opening that allows movement of particles from the sample to the target fluid. In some embodiments, the sample is sufficiently miscible in the target fluid. In some embodiments, the particles moving from the sample to the target fluid are targets (e.g., cells, analytes, etc.). In some embodiments, the opening allows movement of at least one target from the sample to the target fluid. In some embodiments, the particles moving from the sample to the target fluid are contaminants. In some embodiments, the opening allows movement of at least one contaminant from the sample to the target fluid. In some embodiments, the movement of particles from the sample to the target fluid (e.g., movement of at least one target, movement of at least one contaminant, or both) occurs naturally, i.e., without the application of forces (e.g., through diffusion, osmosis, gravity, random walk, etc.), or occurs by other means (e.g., acceleration, filtration, centrifugation, dynamic dialysis, electrophoresis, etc.). In some embodiments, a combination of one or more methods of movement due to natural forces and / or applied forces may be utilized, either sequentially or simultaneously.

[0012] In some embodiments, the method further includes removing fluid from the destination fluid into the container after the container is placed in the destination fluid (e.g., by aspiration, capillary action, etc.). For example, fluid may be aspirated or otherwise transferred into the container before, during, and / or after transfer of the particles from the container to the destination fluid.

[0013] In some embodiments, the at least one target, if present in the sample, binds to at least one paramagnetic particle (PMP) to form one or more target-PMP complexes. In some embodiments, the opening allows for the transfer of the one or more target-PMP complexes from the sample to the destination fluid. In some embodiments, the transfer of the one or more target-PMP complexes from the sample to the destination fluid occurs by application of a magnetic force to attract the one or more target-PMP complexes from the sample to the destination fluid.

[0014] In some embodiments, the at least one contaminant, if present in the sample, binds to one or more paramagnetic particles (PMPs) to form one or more contaminant-PMP complexes. In some embodiments, the opening allows for the transfer of the one or more contaminant-PMP complexes from the sample to the target fluid. In some embodiments, the transfer of the one or more contaminant-PMP complexes from the sample to the target fluid occurs through application of a magnetic force to attract the one or more contaminant-PMP complexes from the sample to the target fluid.

[0015] In some embodiments, the method includes applying a magnetic force to attract one or more target-PMP complexes or one or more contaminant-PMP complexes from the sample to a fluid of interest, and the method further includes aspirating the fluid into a container before, during, and / or after applying the magnetic force.

[0016] In some embodiments, the method further comprises creating a pocket of air, oil, or gas proximate the opening of the container prior to inserting the container into the target fluid.

[0017] In some embodiments, at least one target is a cell. In some embodiments, at least one target is an analyte. In some embodiments, the sample is a biological fluid. In some embodiments, the vessel is a pipette tip, a straw, or a capillary tube.

[0018] In some embodiments, the target fluid is a wash buffer, an elution buffer, or a reaction mixture containing reagents for detecting the target. The target fluid can be any that is sufficiently miscible with the sample or fluid in the container, including, for example, an extraction buffer, water, saline, precipitation buffer, etc.

[0019] In some embodiments, the method further comprises placing the container in a cleaning fluid to reduce potential contaminants on an exterior surface of the container prior to placing the container in the target fluid.

[0020] In some aspects, a system for isolating at least one target from a fluid sample is provided herein. In some embodiments, the system optionally includes a fluid sample comprising at least one target. In some embodiments, the system includes a vessel for receiving the fluid sample, the vessel comprising an opening. In some embodiments, the system includes a collection device comprising a fluid of interest. In some embodiments, the sample or a fluid comprising the sample is fully miscible with the fluid of interest.

[0021] In some embodiments, the fluid sample comprises at least one target bound to one or more paramagnetic particles, thereby forming one or more target-PMP complexes. In some embodiments, the fluid sample comprises at least one contaminant bound to one or more paramagnetic particles, thereby forming one or more contaminant-PMP complexes. In some embodiments, the system further comprises a magnet.

[0022] In some embodiments, the system further comprises a washing fluid. In some embodiments, the washing fluid is contained within the collection device. In some embodiments, the washing fluid is contained within the collection device and is above the target fluid such that the vessel containing the sample passes through the washing fluid before contacting the target fluid. In some embodiments, the washing fluid is an oil or other layer that is above the target fluid. In some embodiments, the washing fluid and the target fluid are immiscible. In some embodiments, the washing fluid is not contained within the collection device.

[0023] In some embodiments, at least one target is a cell. In some embodiments, at least one target is an analyte. In some embodiments, the sample is a biological fluid. In some embodiments, the vessel is a pipette tip, a straw, or a capillary tube. In some embodiments, the target fluid is a wash buffer, an elution buffer, or a reaction mixture that includes a reagent for detecting the target.

[0024] In a method of isolating at least one target from a fluid sample, the method includes placing a container containing the sample in a target fluid, the container includes an opening that allows for the movement of particles from the sample to the target fluid, and the sample is sufficiently miscible with the target fluid. In some embodiments of the method, the opening allows for the movement of at least one target from the sample to the target fluid. In some embodiments of the method, the opening allows for the movement of at least one contaminant from the sample to the target fluid. In some embodiments of these methods, the movement of particles from the sample to the target fluid occurs through diffusion, gravity, or acceleration. In some embodiments of these methods, the method further includes aspirating fluid into the container after the container is placed in the target fluid, and the fluid may be aspirated into the container before, during, and / or after the movement of particles from the container to the target fluid. In some embodiments of these methods, the at least one target, if present in the sample, binds to at least one paramagnetic particle (PMP) to form one or more target-PMP complexes. In some embodiments, the at least one contaminant, if present in the sample, binds to one or more paramagnetic particles (PMPs) to form one or more contaminant-PMP complexes. In some embodiments, the opening allows for the movement of one or more target-PMP complexes and / or contaminant-PMP complexes from the sample to the destination fluid. In some embodiments, the movement of one or more target-PMP complexes and / or contaminant-PMP complexes from the sample to the destination fluid occurs by application of a magnetic force to attract one or more target-PMP complexes from the sample to the destination fluid. In some embodiments of these methods, the method further comprises aspirating fluid into the container before, during, and / or after application of the magnetic force. In any of these methods, the method may further comprise generating a pocket of air, oil, or gas proximate the opening of the container prior to inserting the container into the destination fluid. In some embodiments of these methods, the at least one target is a cell. In some embodiments of these methods, the at least one target is an analyte. In some embodiments of these methods, the sample is a biological fluid, a biological sample, or an environmental sample.In some embodiments of these methods, the container is a pipette tip, a straw, or a capillary tube or similar device. In some embodiments of these methods, the destination fluid is or comprises a wash buffer, an elution buffer, or a reaction mixture containing reagents for detecting the target and / or contaminants. In any of these methods, the method may further comprise placing the container in a wash fluid to reduce potential contaminants on an exterior surface of the container prior to placing the container in the destination fluid.

[0025] Also included are systems, including systems for isolating at least one target from a fluid sample or a fluid containing the sample, the system comprising a vessel containing the fluid sample, the vessel comprising an opening and a collection device containing a target fluid, the target fluid being sufficiently miscible with the fluid sample or the fluid containing the sample. In some embodiments, the system may further comprise a fluid sample containing at least one target (or suspected of containing at least one target). In some embodiments, the fluid sample comprises at least one target bound to one or more paramagnetic particles, thereby forming one or more target-PMP complexes. In some embodiments, the fluid sample comprises at least one contaminant bound to one or more paramagnetic particles, thereby forming one or more contaminant-PMP complexes. In some embodiments, the system further comprises a magnet or other means for generating a magnetic force. In some embodiments, the system further comprises a washing fluid (and / or a means for holding the washing fluid). In some embodiments, the washing fluid is contained within the collection device and is above the target fluid such that the vessel containing the sample passes through the washing fluid before contacting the target fluid. In some embodiments, the washing fluid and the target fluid are immiscible. In some embodiments of the systems, at least one target is a cell or an analyte. In some embodiments of the systems, the sample is a biological fluid, a biological sample, or an environmental sample. In some embodiments of the systems, the container is a pipette tip, a straw, or a capillary tube or similar device. In some embodiments of these systems, the target fluid is or includes a wash buffer, an elution buffer, or a reaction mixture that includes reagents for detecting targets and / or contaminants. [Brief description of the drawings]

[0026] [Figure 1]FIG. 1 shows a schematic diagram of one embodiment of the method described herein. The schematic diagram illustrates isolating a target bound to a paramagnetic particle to generate one or more target-PMP complexes. The target-PMP complexes are contained within a vessel that includes an opening (1). The vessel is placed in a target fluid, which in this example is a wash solution (2). A magnetic force is applied, thereby drawing the target-PMP complexes out of the vessel (3). In addition, the sample diffuses into the target fluid due to the miscibility of the two fluids. The diffusion distance around the vessel can be calculated (4). The volume of diffusion can be calculated based on the diffusion distance, and this volume can be moved (e.g., aspirated) back into the vessel (5), thereby significantly reducing contamination within the target fluid. [Diagram 2] FIG. 2 shows a schematic diagram of another embodiment of the method described herein. In some embodiments, an air pocket (e.g., an "air bubble") may be generated in a vessel housing a fluid sample. The air bubble is generated prior to inserting the vessel into the target fluid. After inserting the vessel into the target fluid (in this case, the wash fluid), the wash fluid is transferred (e.g., by aspiration) into the vessel. The aspiration causes the air pocket (e.g., air bubble) to rise in the vessel, and the air bubble is initially positioned at the interface of the two fluids, thereby allowing contact between the fluid sample and the target fluid, but limiting the area of ​​the initial interface between the two fluids. Following contact of the two fluids, transfer of the target from the sample to the target fluid can occur around the air bubble through the fully miscible interface between the two fluids. This can be used, for example, to limit diffusion-based transport of contaminants or other undesirable substances. [Diagram 3]FIG. 3 shows the results of an experiment to quantify contamination resulting from carryover of contaminants from the exterior surface of a pipette tip into a target fluid (e.g., "carryover contamination"). A solution of tartrazine (a yellow dye) and paramagnetic particles (PMPs) was aspirated into a pipette tip, which was then forced into a well containing double-distilled water (ddH2O) with a magnet underneath it. The beads were allowed to move from the pipette tip into the well. Once all of the beads had moved into the well, the tip was removed. The beads in the well were then removed (via magnet) and the absorbance of tartrazine in the target fluid was measured (i.e., measured by spectrophotometry). The % carryover of tartrazine was then calculated. [Figure 4]Figures 4 and 5 show results from two experiments comparing carryover contamination of various techniques. "Tube-It" refers to mimicking washing of beads in a tube where the beads are held by a magnetic force to the side of the tube while fluids are added and removed. Similarly, "Tube-It" refers to holding the beads by a magnet to the bottom of a 96-well plate while fluids are added and removed. "Fuse-It" refers to a minimal approach where a device suitable for containing and manipulating the sample (e.g., a pipette tip) is brought to the destination well and removed. "Pre-wash-aspirate" and "Post-wash-aspirate" refer to (1) aspirating a wash solution into a device suitable for containing and manipulating the sample (e.g., a pipette tip) before transferring the targets / beads from the sample to the destination fluid (pre-aspirate) or (2) aspirating a wash solution into a device suitable for containing and manipulating the sample (e.g., a pipette tip) after transferring the targets / beads from the sample to the destination fluid (post-aspirate). An example of an improved Fuse-It procedure includes "Bubble-It", which refers to (3) pre-aspiration of air into the pipette tip before inserting the tip into the destination fluid, as described in FIG. 2. In some embodiments, the steps included in Bubble-It and Fuse-It may be used together in combination (see FIG. 2, last panel). The phrase "Tip-Dip" refers to the brief immersion of a device suitable for containing and manipulating a sample (e.g., a pipette tip) into, for example, a sacrificial well of a wash solution, before entering the final destination fluid, as a means of reducing carryover of surface contamination. In particular, FIG. 4 compares the performance of Tube-It with the variation of Fuse-It. The data demonstrate that Fuse-It with both pre-aspiration and post-aspiration can provide the same cleaning benefits as a single Tube-It wash, regardless of whether the beads are, for example, pre-concentrated to the end of the pipette tip before entering the wash solution. The data also demonstrate the considerable benefits provided when Fuse-It is augmented by Bubble-It. With the Bubble-It procedure included in Fuse-It, cleaning is over four times better than with Tube-It alone.After two washes (i.e., performing Fuse-It and Bubble-It twice on one sample), Fuse-It with Bubble-It is about 16 times better (i.e., 4 x 4) than two Tube-It washes. Figure 5 shows the benefit of removing surface carryover when performing Fuse-It in a separate experiment, again confirming the relative benefit of Fuse-It compared to Tube-It (Figure 5, bar 1 vs. bar 2). About 30% of the contamination from Fuse-It with Bubble-It is due to surface carryover outside the tip, as shown by the results with and without tip-dip (Figure 5, bar 2 vs. bar 3). In both Figures 4 and 5, "Carryover (%)" refers to the carryover percentage of tartrazine measured by spectrophotometry. [Diagram 5]Figures 4 and 5 show results from two experiments comparing carryover contamination of various techniques. "Tube-It" refers to mimicking washing of beads in a tube where the beads are held by a magnetic force to the side of the tube while fluids are added and removed. Similarly, "Tube-It" refers to holding the beads by a magnet to the bottom of a 96-well plate while fluids are added and removed. "Fuse-It" refers to a minimal approach where a device suitable for containing and manipulating the sample (e.g., a pipette tip) is brought to the destination well and removed. "Pre-wash-aspirate" and "Post-wash-aspirate" refer to (1) aspirating a wash solution into a device suitable for containing and manipulating the sample (e.g., a pipette tip) before transferring the targets / beads from the sample to the destination fluid (pre-aspirate) or (2) aspirating a wash solution into a device suitable for containing and manipulating the sample (e.g., a pipette tip) after transferring the targets / beads from the sample to the destination fluid (post-aspirate). An example of an improved Fuse-It procedure includes "Bubble-It", which refers to (3) pre-aspiration of air into the pipette tip before inserting the tip into the destination fluid, as described in FIG. 2. In some embodiments, the steps included in Bubble-It and Fuse-It may be used together in combination (see FIG. 2, last panel). The phrase "Tip-Dip" refers to the brief immersion of a device suitable for containing and manipulating a sample (e.g., a pipette tip) into, for example, a sacrificial well of a wash solution, before entering the final destination fluid, as a means of reducing carryover of surface contamination. In particular, FIG. 4 compares the performance of Tube-It with the variation of Fuse-It. The data demonstrate that Fuse-It with both pre-aspiration and post-aspiration can provide the same cleaning benefits as a single Tube-It wash, regardless of whether the beads are, for example, pre-concentrated to the end of the pipette tip before entering the wash solution. The data also demonstrate the considerable benefits provided when Fuse-It is augmented by Bubble-It. With the Bubble-It procedure included in Fuse-It, cleaning is over four times better than with Tube-It alone.After two washes (i.e., performing Fuse-It and Bubble-It twice on one sample), Fuse-It with Bubble-It is about 16 times better (i.e., 4 x 4) than two Tube-It washes. Figure 5 shows the benefit of removing surface carryover when performing Fuse-It in a separate experiment, again confirming the relative benefit of Fuse-It compared to Tube-It (Figure 5, bar 1 vs. bar 2). About 30% of the contamination from Fuse-It with Bubble-It is due to surface carryover outside the tip, as shown by the results with and without tip-dip (Figure 5, bar 2 vs. bar 3). In both Figures 4 and 5, "Carryover (%)" refers to the carryover percentage of tartrazine measured by spectrophotometry. [Figure 6] Figure 6 compares the timing breakdown of running the same protocol for extracting nucleic acids with the KingFisher magnet in the sheath technique and the method of the invention, in this case Fuse-It with Pipetmax, and shows that the method of the invention is twice as fast as the KingFisher technique (i.e., the "technique" portion of the total time). In the figure legend, chip management refers to loading and ejecting the chip. Fluid handling refers to well mixing. Technique refers to bead collection and transfer. [Figure 7] FIG. 7 shows several variables that improve bead movement from a pipette tip with Fuse-It and its effect on carryover. [Figure 8] Figure 8 shows a purity comparison between the KingFisher magnet in sheath technology and the TSP platform using the Macherey Nagel Nucleomag Tissue kit. TSP refers to chip-based sample preparation and encompasses the methods of the present invention including Chip-It, Fuse-It and Bubble-It. The 24uL TSP method is roughly equivalent to the KingFisher in carryover. The 10μL TSP platform using the method of the present invention results in 10 times less carryover. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] <Definition> To facilitate the understanding of this disclosure, several terms and phrases are defined below.

[0028] As used herein, the term "container" refers to any device, receptacle, or vessel capable of holding material, including a sample, and includes any device, receptacle, or vessel that can contain, store, or hold a sample and can be used in the methods of the invention. In some embodiments, the container is a device that can be used to perform a pre-aspiration step, a post-aspiration step, or a Bubble-It procedure, as described herein. In some embodiments, the container is a pipette or a pipette-type device. In some embodiments, the container is portable. In some embodiments, the container is a disposable or single-use container. In some embodiments, the container is reusable. In some embodiments, the container is part of an automated or semi-automated system that includes elements for accomplishing the methods of the invention and operating the system. The vessels and containers include, for example, any vessel, container, receptacle, holder, carrier, cartridge, or storage device that can hold material, including a sample. The container provides for fluid transfer during use of the systems and methods of the invention. In some embodiments, the container comprises a top opening that allows for the addition of a sample to the container. In some embodiments, the container contains material, including, for example, a lysis buffer, a wash buffer, or an extraction buffer, or other material. In some embodiments, the vessel may comprise a filter, mesh, or any useful porous or other material to provide pre-filtration of sample contaminants prior to or during insertion of the vessel into a target fluid when performing a method or operating a system of the invention. In some embodiments, the vessel comprises one or more openings that allow for removal of targets or target-binding particles (e.g., PMPs) from the sample (e.g., using a magnet).In some embodiments, the contaminant-binding particles (e.g., PMPs) are removed from the sample through one or more openings (e.g., using a magnet) and the targets or target-binding particles remaining in the container are moved to another vessel or destination (e.g., a multi-well plate), or moved into or onto a detector (e.g., a reader, a Bluetooth®-enabled reader or instrument, etc.) that can accept the targets or target-binding particles, or onto or into a surface or porous material (e.g., a spot card, etc. for drying and transport of the sample for subsequent analysis).

[0029] As used herein, the term "carryover" is used to describe the movement of unwanted material from one location to another (e.g., from one container or mixture to another). Carryover, when used in reference to the removal of contaminants or unwanted (unwanted or undesired or undesirable) materials, refers to the movement of unwanted (unwanted or undesired or undesirable) material from one location to another (e.g., from one container to another, or from one fluid to another, or from one fluid to a container, or from a container to a fluid, or from one area to another, etc.). Carryover may be used, for example, in reference to an action to "reduce the carryover of unwanted material" or the unwanted material itself (e.g., "concentration of carryover in the final reaction").

[0030] As used herein, the term "destination vessel" refers to any device, receptacle, or vessel capable of holding a fluid of interest and receiving a vessel. In some embodiments of the method and / or system, the destination vessel is initially empty and later receives the fluid of interest when, for example, a vessel containing a sample is brought into proximity with a surface and a small amount of fluid is passively or actively dispensed from the vessel to produce the fluid of interest on the surface.

[0031] As used herein, the term "target fluid" refers to a fluid that is sufficiently miscible with the fluid in the vessel (e.g., the fluid containing the sample). In some embodiments, the target fluid comprises one or more of a wash buffer, an elution buffer, a precipitation buffer, water, saline, and a reaction mixture containing a reagent for detecting a target. In some embodiments, the target fluid is heated to a higher temperature than the sample-containing materials in the vessel (e.g., to aid in movement by convection). In some embodiments, the target fluid is selected or configured to allow movement of the sample-containing materials down a pressure gradient (e.g., a pressure gradient from a vessel containing the sample to and / or through the target fluid in the vessel).

[0032] As used herein, the term "magnetic particle" refers to a particulate material (e.g., micro- or nano-material) that exhibits magnetic properties when exposed to a magnetic field (e.g., an external or internal magnetic field). In some embodiments, the magnetic particle comprises two components: a magnetic material (e.g., iron, nickel, cobalt, etc.) and a functional chemical component (e.g., an antibody or an antibody binding fragment). In some embodiments, the magnetic particle is polymer-functionalized, amine-functionalized, aldehyde-functionalized, surfactant-functionalized, ligand-functionalized, etc. For use in the methods and systems of the present invention, the magnetic particles are selected to have a desired particle size or average particle size (e.g., depending on the size of the target, the size of the vessel opening, the inclusion and use of a prefilter of a particular size, etc.). Magnetic particles include particles that include a paramagnetic material (e.g., paramagnetic particles (PMPs), paramagnetic spheres, etc.). Paramagnetic materials include materials that include paramagnetic atoms (e.g., aluminum, iron oxide, etc.). Magnetic particles include particles that include a ferromagnetic material (e.g., ferromagnetic particles).

[0033] A "magnet" for use in the systems, devices or methods of the invention refers to a means for generating a magnetic force. As used herein, magnets include permanent magnets, temporary magnets and electromagnets.

[0034] As used herein, the term "washing fluid" refers to any fluid useful for removing or isolating contaminating fluids or undesirable materials from the surface of a vessel to prevent them (in whole or in part) from entering or contaminating the target fluid. For example, "washing fluid" includes a separate wash buffer well for immersing the vessel, an oil overlay over the target fluid that helps displace or remove contaminating fluids from the surface of the vessel, and the like. The wash fluid may be a fluid that mimics the target fluid, may be in the target fluid, or may be maintained separately elsewhere in the system of the invention.

[0035] As used herein, the term "oil phase" refers to a phase in the system of the present invention that compromises oil, is substantially hydrophobic, and is substantially immiscible with the aqueous phase (e.g., the target fluid). Suitable oil phases in some systems of the present invention include, for example, mineral oil, coconut oil, and vegetable oil. Other oils include, for example, carbon-based and silicone-based polymeric compounds, mineral oil, silicone oil, paraffin wax, and fluorinated oil. As used herein, the term "oil" refers to any of a number of substances that are not soluble in water, usually liquid or semi-solid substances. Oil also includes mixtures of oils (e.g., waxes with different melting points, polymeric oils with different chain lengths, mineral oil and silicone oil, etc.). Oil also includes oil-oil emulsions.

[0036] As used herein, the term "interface" refers to a surface that forms a common boundary or transition area between adjacent regions, bodies, materials, phases or layers (e.g., the boundary between an oil layer and a target fluid).

[0037] As used herein, the term "porous" means having pores or other small spaces that can allow targets (or non-targets, e.g., contaminants), whether bound or unbound to or part of a solid phase or other carrier, to pass or not pass as desired. Reference to a "porous material" or structure, or a "porous mesh" or a "porous layer" means a material that includes void space, i.e., space that is not occupied by the backbone of atoms that make up the structure of the material. A material through which targets (whether bound or unbound to a solid phase) pass is an example of a porous material. A material through which non-target materials that are not targets (whether bound or unbound to a solid phase) do not pass is also an example of a porous material. A porous material or structure, a porous mesh, or a porous layer need not be constructed from or consist of a single material, i.e., it does not need to be homogenous. Porous materials or structures, porous meshes or layers for use in the systems, devices, methods and compositions of the invention may comprise different materials, i.e., may be heterogeneous or inhomogeneous (e.g., in one embodiment, comprises polystyrene and nylon or a spatially variable mixture).

[0038] As used herein, the terms "detect", "detecting", or "detection" may describe either the general act of finding or identifying a detectably labeled composition or the specific observation. The term "detecting", when used in reference to a target, refers to detecting either the presence, absence, or amount of a target in a sample. In some embodiments, detecting may be qualitative, semi-quantitative, or quantitative. In some embodiments, "detecting" a target in a sample refers to determining that a target is present in a sample. In some embodiments, "detecting" a target in a sample refers to determining that a target is not present in the sample or is not present in an amount sufficient to be detected in the sample. In some embodiments, "detecting" a target in a sample refers to determining the amount of a target present in a sample, or that the amount of a target present in a sample is equal to or greater than a threshold amount.

[0039] The term "fluid" is used herein in the broadest sense and refers to any substance that flows. In some embodiments, the fluid is a liquid. For example, a "fluid sample" can refer to a liquid sample. In other embodiments, the fluid is a gas.

[0040] As used herein, the term "sample" is used in the broadest sense and includes many sample types. In some embodiments, the sample is a "biological sample." In other embodiments, the sample may be an "environmental sample." In some embodiments, the sample refers to a portion of a material collected or selected from a larger volume of material. In some embodiments, the sample refers to any material that contains or is suspected of containing a target. In some embodiments, the sample is a whole volume of a substance, such as blood. In some embodiments, the sample is blood, cerebrospinal fluid, urine, tissue, biopsy tissue, etc. Any type of fluid, including samples that contain or are suspected of containing a target (and / or contaminant) of interest, is contemplated for use in the systems and methods of the present invention.

[0041] In some embodiments, the fluid sample is a biological sample. As used herein, the term "biological sample" is used in the broadest sense and includes many sample types that can be obtained from a subject. Biological samples may be obtained from animals (including humans) and include fluids (e.g., urine, blood, blood products, sputum, saliva, cerebrospinal fluid, etc.), solids, tissues, and gases. Biological samples include saliva, blood products, such as plasma, serum, etc. 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 blood sample, a serum sample, or a plasma sample. In some embodiments, the biological sample is a saliva sample. The term "saliva sample" as used herein refers to a sample of saliva derived from or collected from a subject. In some embodiments, the biological sample is a nasopharyngeal (NP) sample. A "nasopharyngeal sample" refers to a specimen collected using a swab inserted into the nasopharyngeal cavity of a subject.

[0042] The biological sample may be subjected to various pretreatment steps prior to carrying out 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 (e.g., denaturants, viscosity-reducing reagents, inhibitors of target degradation, buffers, etc.) added to the biological sample may be added to the biological sample or may be present in a storage buffer present in the container in which the sample is collected (e.g., present in a storage buffer in a sample collection tube). In some embodiments, the sample contains or is suspected of containing a microorganism (e.g., any type of pathogenic or disease-causing microorganism).

[0043] In some embodiments, the fluid sample is an environmental sample. The term "environmental sample" refers to any sample obtained from the environment. Common environmental samples include air samples, water samples, soil samples, samples of biological materials, and samples of waste (liquid, solid, or sludge). For example, an environmental sample can be a sewage, soil, water, or air sample. Environmental samples also include sampling of inanimate objects and surfaces (e.g., samples and swab samples collected from buildings, etc.), as well as sampling and swab sampling of inanimate environments in facilities (e.g., health care facilities) that include environmental surfaces such as floors, walls, fixtures and equipment, furniture, and other parts of the physical infrastructure, including air filters, HEPA filters, water filters, and other filters.

[0044] The term "fully miscible" as used herein refers to the ability of two fluids to interact with each other to remove any significant barriers (e.g., fluid-fluid interface barriers, such as oil-aqueous fluid interfaces) to the movement of a target from one fluid to the other. For example, a fluid sample that is fully miscible with a target fluid allows the movement of a target from the fluid sample to the target fluid. Being fully miscible does not necessarily mean that the two fluids become a homogenous mixture. Rather, in some cases, the two fluids may remain mostly separate but still interact or otherwise allow the movement of a target from one fluid to the other. For example, a layer of a sucrose density gradient is fully miscible. Similarly, a biological sample containing a target may be fully miscible with a wash buffer or elution buffer, thereby allowing the movement of the target from the biological sample to the buffer. As a counter example, air is partially miscible with water, but is not sufficiently miscible to promote interactions at the air-water interface that allow the movement of particles. Thus, air and water are not referred to herein as "fully miscible". Similarly, materials such as silicone oil and water do not create a sufficiently miscible interface to facilitate transfer (e.g., complete or near complete target-PMP complex transfer) from a vessel containing a sample. In some embodiments, the methods and systems of the invention provide complete target transfer (e.g., including complete target-PMP complex transfer) from a sample or vessel containing a sample to a location within a destination fluid or vessel. In some embodiments, the methods and systems of the invention provide complete target-contaminant transfer (e.g., including complete contaminant-PMP complex transfer) from a sample or vessel containing a sample to a location within a destination fluid or vessel.

[0045] The term "subject" as used herein refers to the entity from which a sample is obtained. The subject may be a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a plant. In some embodiments, the subject is an inanimate object.

[0046] As used herein, the term "target" is used in the broadest sense and refers to any desired material to be isolated from and / or detected in a sample. In some embodiments, the target is bound to a paramagnetic particle and is isolated from the sample via application of a magnetic force. In other embodiments, the target is a particle of sufficient density to be isolated from the sample without application of a magnetic force (e.g., via gravity) or via application of another force (e.g., centrifugation, etc.). In some embodiments, the target is a protein (e.g., an antibody), a whole cell, or a nucleic acid (e.g., DNA, RNA). In some embodiments, the target is a metabolite, a carbohydrate, a glycopeptide, or a lipid.

[0047] The term "analyte" or "target analyte" refers to any substance to be extracted, isolated, identified, or measured.

[0048] Detailed Description In some aspects, provided herein are systems and methods for extracting and / or isolating at least one target from a fluid sample. In some embodiments, provided herein are systems and methods for isolating at least one target from a biological sample.

[0049] The methods described herein have many advantages over currently used sample purification techniques and techniques for extracting and / or isolating at least one target from a sample (e.g., a biological sample). Current chip-based sample purification techniques rely on the use of fluid-air interfaces or fluid-oil interfaces or immiscible fluid interfaces as "barriers" to limit carryover of contaminants. These processes can be difficult and difficult to culture, requiring precise distances, magnetic field strengths, and immiscible fluids such as oil. Similarly, these barriers create a fundamental problem for achieving 100% transfer of all particles. In contrast, the methods described herein rely on the creation of traversable restrictions that minimize contamination while allowing transfer of targets from the sample to the destination fluid.

[0050] The methods described herein rely on the use of various techniques to allow the passage of desired particles (e.g., targets or analytes) from the sample to the destination fluid while reducing the passage of undesired particles (e.g., contaminants). As used herein, the term "particle(s)" is meant to include both "target" particles (e.g., targets or analytes) and "contaminant" particles (e.g., contaminants). The terms "contaminant" or "contaminant particles" are used interchangeably in the broadest sense and include solids (e.g., undesired nucleic acids, proteins, cellular debris, etc.) and / or liquids (e.g., buffers, sample fluids, etc.). In some embodiments, allowing the passage of desired particles may include allowing the passage of target particles into the destination fluid. In some embodiments, reducing the passage of contaminant particles may include reducing the passage of liquids into the destination fluid (e.g., liquid samples using pre-wash and / or post-wash aspiration, air bubbles-It, reducing the interfacial area or interaction time between fluids, etc.), while the target particles themselves can pass through to the destination fluid. In other embodiments, allowing the passage of desired particles includes reducing the passage of desired particles (e.g., target particles) into the destination fluid while allowing the passage of contaminants into the destination fluid.

[0051] In some embodiments, the method relies on the passage of desired targets from the sample to the destination fluid while preventing / minimizing the passage of contaminants to the destination fluid. In some embodiments, the passage of contaminants is restricted by restricting the flow of liquid containing the potential contaminants. For example, in some embodiments, preventing / minimizing the passage of contaminants includes allowing the passage of desired targets (e.g., desired target particles) contained within the liquid sample to the destination fluid while restricting the flow of the liquid sample itself. Thus, the method restricts the flow of liquid containing the potential contaminants, thereby minimizing the passage of contaminants into the destination fluid.

[0052] In some embodiments, the methods described herein include the passage of contaminants from the sample to the destination fluid. While the methods described herein include the passage of targets from the sample to the destination fluid (e.g., positive selection), it is understood that the methods and techniques described herein can also be designed to allow contaminants to pass from the sample to the destination fluid, leaving the desired target behind (e.g., negative selection). In both positive and negative selection, the target is considered to be "isolated" from the fluid sample in that the target is substantially separated from potential contaminants. As used herein, the terms "isolated" or "isolating" or "purified" or "purifying" refer to the act of separating a target from one or more or all contaminants. These terms are meant to include isolation methods that involve the transfer of targets from a sample to a destination fluid (e.g., positive selection of targets), and isolation methods that involve the retention of targets in a container while contaminants pass from the sample to the destination fluid (e.g., negative selection).

[0053] In some embodiments, the method includes placing a container containing the sample in the target fluid. In some embodiments, the method includes placing a container containing the sample on a surface. In some embodiments, placing the container containing the sample on the surface results in the transfer of a volume of fluid from the container to the surface, thereby generating the target fluid on the surface. In some embodiments, the surface includes a hydrophobic material. In other embodiments, the surface includes a hydrophilic material. The surface may be glass or plastic, such as a well or dish. In other embodiments, the surface may include a porous material. The porous material may be wet or dry. In other embodiments, the surface may include a magnet. In other embodiments, the surface may include an assay material (e.g., a lateral flow assay sample pad, etc.). In some embodiments, the surface includes a pad (e.g., an absorbent pad such as a cotton pad, a blood spotting disk, etc.). In some embodiments, the container containing the sample is brought into proximity with the surface and a small amount of fluid is passively or actively dispensed from the container to generate the target fluid on the surface. Such methods may be advantageous when the surface is rigid and / or impact between the container and the surface is undesirable. In some embodiments, after production of the target fluid, a volume of the target fluid can be aspirated back into the sample container to minimize carryover of background contaminants.

[0054] In some embodiments, the container comprises an opening that allows for the transfer of particles from the sample to the destination fluid. In some embodiments, the particles are target particles. In some embodiments, the particles are contaminant particles. In some embodiments, the container comprises an opening that allows for the transfer of potential contaminants from the sample to the destination fluid, thereby leaving the targets in the initial container. In some embodiments, the sample is sufficiently miscible with the destination fluid. Thus, transfer of targets or transfer of potential contaminants from the sample to the destination fluid can occur.

[0055] The systems and methods described herein may be used to isolate any desired target from a sample. For example, the target may be a nucleic acid (e.g., DNA, RNA, or various subtypes thereof, including mRNA), a protein, a metabolite, a carbohydrate, a glycopeptide, or a lipid. In some embodiments, the target may be a nucleic acid or a protein (e.g., an antibody) resulting from a pathogen infecting the subject from which the biological sample was obtained. For example, the target may be a bacterial nucleic acid (e.g., bacterial DNA or RNA) or a 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 by a pathogen. In some embodiments, the target may be a cell.

[0056] In some embodiments, a particle (e.g., a target or a contaminant) is bound to a capture moiety. In some embodiments, a target is bound to at least one capture moiety. In some embodiments, a contaminant is bound to at least one capture moiety. In some embodiments, a capture moiety is a paramagnetic particle (PMP). In some embodiments, a particle is bound to a capture moiety to form a complex. For example, in some embodiments, a particle is bound to a PMP to form a particle-PMP complex. A particle-PMP complex can be a target-PMP complex or a contaminant-PMP complex. In some embodiments, a target is bound to at least one paramagnetic particle (PMP) to form one or more target-PMP complexes. In some embodiments, a method includes mixing a fluid sample (e.g., a biological sample) with a capture moiety (e.g., a PMP). Mixing the sample with the PMP allows the PMP to bind to the particle in the sample, thereby generating one or more complexes. For example, in some embodiments, mixing the sample with the PMP allows the PMP to bind to the target, if present in the sample, thereby generating one or more target-PMP complexes.

[0057] The PMP may be contained in a liquid formulation. Alternatively, the PMP may be a lyophilized formulation. The lyophilized PMP formulation may contain other suitable reagents that are typically used in the lyophilization process, including bulking agents, stabilizers, and other suitable excipients. For example, the lyophilized PMP may be present in a container (e.g., a pipette tip) that contains a fluid sample. As another example, the PMP may be added to the sample either before the sample-PMP mixture is aspirated into the container or after the sample is in the container.

[0058] Any suitable paramagnetic particle can be used. In some embodiments, the paramagnetic particles can be purchased from a commercial supplier. The particular type of paramagnetic particle used depends on the target to be isolated from the biological sample. For example, particles with a relatively large surface area may be preferred for binding to nucleic acids such as viral RNA. In some embodiments, the paramagnetic particles can be functionalized to aid in the capture / purification of the target. For example, the paramagnetic particles can be functionalized with one or more antibodies, aptamers, or other suitable agents to aid in the capture of the target. In some embodiments, the paramagnetic particles can be functionalized with one or more spike protein antibodies to aid in the capture of SARS, coronavirus, SARS-CoV-2, and related targets. In some embodiments, the paramagnetic particles can be functionalized with a suitable moiety to facilitate the capture of the target antibody. For example, the paramagnetic particles can be functionalized with spike proteins or synthetic variants thereof to facilitate the capture of antibodies produced as a result of infection with SARS, coronavirus, SARS-CoV-2, or other related pathogens.

[0059] Any suitable amount of PMP may be present in the sample. In embodiments in which the PMP is included in a liquid, any suitable volume of liquid composition including the paramagnetic particles may be present in the sample. Any suitable concentration of PMP may be used to ensure sufficient binding of the PMP to the target (e.g., formation of a sufficient number of target-PMP complexes). In the case of a lyophilized PMP formulation, any suitable weight of lyophilized product may be used to ensure that an appropriate concentration of PMP is present in the fluid sample. In the case of a liquid formulation, the liquid composition including PMP may include any suitable concentration of PMP to ensure sufficient binding of the PMP to the target (e.g., formation of a sufficient number of target-PMP complexes). The appropriate amount of PMP may depend on many factors, including the volume of the liquid sample, the size of the beads, the target intended to be isolated, etc. For example, the PMP may be present at about 0.01 to 98% (v / v). For example, PMP may be present in an amount of about 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 98% (v / v).

[0060] In some embodiments, the fluid sample contains other suitable reagents for sample processing / handling. For example, the fluid sample may contain one or more detergents, reducing agents, buffers, inhibitors, enzymes (e.g., proteases), denaturants, etc. For example, the sample may further include one or more reagents for reducing the viscosity of the sample. For example, the sample may include PMP and DTT. The sample 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 the sample include, for example, RNase inhibitors, protease inhibitors, nuclease inhibitors, etc.

[0061] The target fluid can be any suitable fluid, including the same or similar fluid that comprises the sample. In some embodiments, the methods described herein are used to transfer a target of interest from a sample to the target fluid. In other embodiments, the target can be a contaminant, and the methods described herein are used to transfer the contaminant from the sample while leaving the target of interest (e.g., protein, nucleic acid, etc.) in the vessel. In any of the methods described herein, the target fluid can be a liquid. For example, the target fluid can be a wash buffer. As another example, the target fluid can be an elution buffer. In some embodiments, the target fluid comprises water. In some embodiments, the target fluid comprises ethanol. In some embodiments, the target fluid comprises substantially the same fluidic material as the fluid sample (including, for example, embodiments in which the target fluid is generated on the surface by placing a vessel containing the sample fluid on the surface or by dispensing some of the fluid sample on the surface).

[0062] The fluid sample and the destination fluid may be any suitable fluids that are sufficiently miscible with each other to allow passage of the target from the sample to the destination fluid or passage of the contaminant from the sample to the destination fluid. In some embodiments, the passage of the particles (e.g., the passage of the target or the passage of the contaminant) occurs through diffusion. In some embodiments, the passage of the particles occurs due to gravity or other forms of acceleration (e.g., via a centrifuge). In some embodiments, the movement of the particle-PMP complexes is induced. For example, in some embodiments, the movement of the particle-PMP complexes is induced, such as by application of a magnetic force to the system. For example, a magnetic force may be applied, thereby drawing the particle-PMP complexes through an opening and into the destination fluid. As another example, in some embodiments, a vessel containing the sample is placed on a surface and a destination fluid is generated on the surface. The particle-PMP complexes may aggregate at the interface between the vessel and the surface, and a magnetic force may be applied to hold the beads on the surface while the vessel is removed. Thus, the target-PMP complexes are transferred from the vessel to the destination fluid generated on the surface. In some embodiments, the process of contacting such a container with a surface may be performed multiple times without refilling the container with sample, thereby generating multiple spots of target-PMP complexes in succession. In some embodiments, a method of isolating a target from a fluid sample includes placing a container containing a sample including one or more target-PMP complexes in a destination fluid and applying a magnetic force to attract the target-PMP complexes from the sample to the destination fluid. In other embodiments, the method includes placing a container containing a sample including one or more contaminant-PMP complexes in a destination fluid and applying a magnetic force to attract the contaminant-PMP complexes from the sample to the destination fluid.

[0063] The systems and methods described herein rely on the use of a passable restriction to facilitate target purification while minimizing contamination. As used herein, the term "passable restriction" refers to any suitable mechanism that allows the movement (e.g., passage) of a desired particle (e.g., the passage of a target or the passage of a contaminant) from a fluid sample to a destination fluid, while also restricting (e.g., restricting) the passage of an undesired particle from the fluid sample to the destination fluid. For example, in some embodiments, a "passable restriction" refers to a suitable mechanism, including those described above, that allows the movement of a target from a fluid sample to a destination fluid while minimizing the amount of contamination that occurs before, during, and / or after the movement of the target. This process is referred to herein as "positive selection." In other embodiments, a "passable restriction" refers to a mechanism that allows the passage of a contaminant from a fluid sample to a destination fluid, while the desired target remains in a vessel holding the fluid sample. This process is referred to herein as "negative selection."

[0064] The claimed system and method are designed to use a passable restriction to minimize contamination that would otherwise occur during transfer between fluids. Undesirable contamination can occur in a variety of ways. In the context of the claimed method, contamination generally occurs when the fluid in the chip contacts the wash solution by: (a) contaminated surface carryover; (b) diffusion; (c) convection (e.g., due to pressure, density differences, or temperature differences); (d) sedimentation (e.g., background cells settling downward during isolation of target cells); (e) association with beads (e.g., on the surface or in bulk via direct or indirect connections); or (f) surface tension-based forces (e.g., Marangoni). Typically, surface carryover, diffusion, and convection contamination dominate. Thus, various passable restrictions can be employed to reduce contamination, such as contamination from any of the sources mentioned above.

[0065] In some embodiments, at least one passable restriction is employed to minimize diffusion-based contamination. Diffusion-based carryover is affected by several parameters: (1) geometry (e.g., cross-sectional area of ​​the opening), (2) diffusion coefficient, and (3) time, any or all of which may be manipulated, set, or adjusted in the methods and systems of the present invention as desired.

[0066] In some embodiments, at least one passable restriction is employed to minimize convection-based contamination. Convection carryover is essentially any non-diffusion-based movement of fluids and can result from many different sources, including: (1) density differences between fluids that induce convection in the presence of gravity / acceleration (e.g., induced by temperature or salinity), (2) momentum (e.g., container movement or vibration), (3) pressure (e.g., applied by a pipette pressure mechanism), and (4) entrainment (e.g., movement of objects within a fluid that induces convection).

[0067] In some embodiments, the permeable restriction is achieved by controlling the size of the magnetic particles used to capture the target analytes and / or target contaminants.

[0068] In some embodiments, a permeable restriction is achieved by controlling the size and / or nature of the magnetic particles used to capture the target analytes and / or target contaminants. In some embodiments, for example, a permeable restriction is achieved by using ferromagnetic particles for the capture and transfer of one target or contaminant complex and paramagnetic particles for the capture and transfer of another target or contaminant complex. A permeable restriction can also be created by controlling the size of the magnetic particles (e.g., the size of the ferromagnetic and / or paramagnetic particles) used to capture the target analytes and / or target contaminants.

[0069] In some embodiments, the passable restriction is achieved by controlling the geometry of the container and its opening for containing the sample. For example, the passable restriction can be achieved by controlling the size and / or shape of the container and the size and / or shape of the opening of the container. The container can be any suitable container with an opening, including, but not limited to, a pipette tip, a tube, a capillary, a straw, and the like. Generally speaking, a larger opening (e.g., a larger pipette tip orifice) allows more diffusion to occur in the same unit of time compared to diffusion for a smaller opening. Thus, a larger opening allows for increased passage of potential contaminants into the container (e.g., into the pipette tip) compared to a smaller opening. In other words, a larger opening allows for increased diffusion-based contamination compared to a smaller opening. However, a larger opening also facilitates movement of the target out of the container. For example, a larger opening can help avoid blockages that may otherwise occur during movement of the target out of a smaller pipette tip. Thus, in some embodiments, a passable restriction for minimizing diffusion-based contamination involves selecting an appropriately sized opening to facilitate proper movement of targets from the container while minimizing the number of contaminants that can exit the opening by diffusion into the target fluid.

[0070] In some embodiments, the opening is generally circular. In some embodiments, the opening is generally circular with an average inner diameter of about 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2 mm, 2.1 mm, 2.2 mm, 2.3 mm, 2.4 mm, or 2.5 mm, 2.6 mm, 2.7 mm, 2.8 mm, 2.9 mm, 3.0 mm. In some embodiments, larger openings may be required. For example, for sewage samples containing aggregated (e.g., clumped) contaminants, the diameter of the opening may be greater than 3.0 mm. For example, the diameter may be 3.0mm, 3.5mm, 4mm, 4.5mm, 5mm, 5.5mm, 6mm, 6.5mm, 7mm, 7.5mm, 8mm, 8.5mm, 9mm, 9.5mm, 10mm, or more.

[0071] In some embodiments, the container comprises multiple openings. For example, the container may include an array of openings. Such an array of openings may provide many small areas where a flow rate or flow can be achieved. For example, such an array of openings provides many small areas where targets (or, in the case of negative selection, contaminants) can pass through the openings, but the viscous resistance increases approximately proportional to 1 / r^4, where r is the radius of the opening. Thus, the use of many small holes strongly resists unintended convective transport of contaminants, but allows the passage of targets even if one or more other openings are blocked. In contrast, if only a single opening is used, convective contamination is much easier, and blockage would prevent any subsequent transfer.

[0072] In some embodiments, the opening may be in close proximity to a surface of a vessel containing the destination liquid. For example, the opening may be brought into contact with a surface of a vessel containing the destination liquid, effectively reducing the cross-sectional area over which diffusion or convection would otherwise occur. In some embodiments, the opening may be brought into contact with a surface of a vessel containing the destination fluid, thereby closing the vessel opening (e.g., a pipette tip), thereby preventing diffusion of contaminants through the opening while targets are agglutinated proximate the opening in the vessel containing the sample. After sufficient agglutination of the targets has occurred, the vessel opening (e.g., a pipette tip) may be removed from the destination liquid, thereby leaving the targets in the destination liquid while minimizing diffusion-based carryover. In other embodiments, the opening may be brought into contact with a surface of a vessel containing the destination fluid, thereby closing the vessel opening (e.g., a pipette tip), thereby preventing diffusion of the targets through the vessel opening while contaminants are agglutinated proximate the opening in the vessel containing the sample. After sufficient coagulation of the contaminants has occurred, the container (e.g., pipette and / or pipette tip) can be removed from the target liquid, thereby retaining the target within the pipette tip (e.g., within the container) while depositing the contaminants within the target liquid.

[0073] In some embodiments, the passable restriction is achieved by using pressure. For example, in some embodiments, pressure is applied to induce convection of the fluid into the opening (e.g., by aspirating the fluid with the container), thereby causing a flow (i.e., backflow) in the opposite direction to the analyte (e.g., target or contaminant) being transferred. Backflow before, during, or after transfer can prevent diffusion-based contamination. Backflow may be used to prevent undesired pass-through contaminants when the target is the analyte being transferred. In other embodiments, backflow may be used to prevent undesired pass-through of the target from the container when a negative selection process is employed.

[0074] The Peclet number is used to describe the relative balance of convection / advection. As used herein, the term "convection" applies to the movement of fluids, while "advection" refers to the movement of particles dissolved or suspended within the fluid. When the Peclet number is 1, diffusion and convection / advection are approximately balanced. When it is greater than 1, convection / advection dominates diffusion, preventing contamination. Movement within the flow (e.g., movement of a target) may be affected by external forces such as acceleration / gravity or magnetic forces, which are not considered by the Peclet number. Thus, in this example, the Peclet number refers to the transport of a contaminant, which is essentially determined by the flow of the fluid and the diffusion of the contaminant within that fluid. Sufficient backflow (e.g., Pe>>1) prevents diffusion-based movement of the contaminant from a container (e.g., a pipette or pipette tip) to the target fluid, but external forces can move the target despite the backflow. However, if the fluid flow is too fast, the attractive forces on a moving target may be able to impede the target (e.g., a target particle) from moving. The fluid velocity at the tip that would impede movement can be approximated using Stokes' equation for the attractive forces on a particle in a flow that would balance any forces (e.g., gravity or magnetic) that aid in movement.

[0075] In some embodiments, the method includes aspirating the fluid into the container after the container is placed in the destination fluid. In some embodiments, the fluid is aspirated into the container before, during, and / or after transfer (e.g., transfer of the target or transfer of the contaminant, or both) from the container to the destination fluid. In some embodiments, the fluid is aspirated into the container before transferring the desired entity (e.g., the target and / or contaminant). The initial aspirate draws the fluid into the container so that any potential particles that move by diffusion must first move through the aspirated portion of the fluid before they can escape. Aspirating the fluid so that multiple millimeters of fluid are in the container (e.g., pipette tip) can provide a few minutes of time before significant diffusion-based transfer of particles (i.e., targets or contaminants) can occur from the sample in the container to the destination fluid. In some embodiments, diffusion-based contamination can be estimated using the following formula:

[0076]

number

[0077] In some embodiments, staggering the timing of the pulling and transmitting forces simplifies operation and provides robust performance. In some embodiments, the method includes aspirating the fluid into a container following transfer from the sample to the fluid of interest (e.g., target transfer, contaminant transfer, or both). Aspiration after transfer may be performed to retrieve potential carryover contaminants (see FIG. 1). Contaminants carried over via diffusion diffuse slowly, and the distance they diffuse can be conservatively estimated using the following equation based on one dimension (versus a potentially more accurate 3D model): L = sqrt(2 * D * t) where L is the diffusion distance, D is the diffusion coefficient, and t is the time that diffusion has progressed (see step 4 in Figure 1). This distance then informs how much of the volume surrounding the tip should be aspirated to recapture the diffusion-based contaminant. For example, the volume can be calculated using the following formula: v=4 / 3 * pi * L 3 where v is the volume of diffusion and L is the diffusion distance.

[0078] In some embodiments, aspiration may occur both before (e.g., pre-aspiration) and after (e.g., post-aspiration) movement of the target. In some embodiments, both pre-aspiration and post-aspiration can be performed to provide superior protection against contamination.

[0079] In some embodiments, the passable restriction is achieved by creating a pocket of air, oil, or gas proximate the opening of the container. In some embodiments, the passable restriction is achieved by creating a pocket of air (e.g., "air bubbles") or oil (e.g., "oil bubbles") proximate the opening of the container before inserting the container into the target fluid. In some embodiments, the passable restriction is achieved by creating an air pocket proximate the opening of the container before inserting the container into the target fluid. Such an embodiment is shown in FIG. 3. In some embodiments, an air pocket may be preferred or advantageous over an oil pocket. For example, when using a standard pipette tip as the container, air may be advantageous because standard polypropylene pipette tip material is oleophilic. Pre-aspiration with oil would create a barrier that interacts strongly with the pipette because it is oleophilic, creating a barrier to target movement. In other words, the use of oil in such embodiments may represent a non-passable restriction. In contrast, air has a weaker interaction with polypropylene than oil, allowing the bubble to separate from the pipette wall more easily and allowing fluid to merge around the sides of the bubble. This can be enhanced by surface treatments or cleaning additives that enhance the interaction between the sample / sample and the pipette tip (e.g., in the case of aqueous solutions, pre-coating with a surfactant such as BSA or a detergent, or via oxygen plasma treatment, as well as detergents or surfactants in the sample or cleaning solution).

[0080] In some embodiments, a method for isolating a target from a fluid sample includes generating an air pocket proximate an opening of a container, inserting the container into a target solution, and aspirating the fluid into the container. In such embodiments, aspirating the fluid in the container causes an air pocket (e.g., an air bubble) to rise in the container, with the air bubble initially positioned at the interface of the two fluids, thereby allowing contact between the fluid sample and the target fluid, but limiting the area of ​​the initial interface between the two fluids. Following contact of the two fluids, migration of the target from the sample to the target fluid can occur around the air bubble through the fully miscible interface between the two fluids. Alternatively, migration of a contaminant from the sample to the target fluid can occur around the air bubble while the target remains held in the container.

[0081] In some embodiments, the walls of the container (e.g., pipette) are tapered. Thus, as fluid is aspirated and air moves upward, it will eventually form a bubble that becomes sufficiently spherical to allow the fluid to merge around the bubble, depending on the relative affinity / hydrophobicity of the sample / wash fluid, the tip material, and what is used for the "bubble" (in some embodiments, it is, for example, air, but it can also be a hydrophobic liquid such as oil or special gas). The volume of air should be optimized to facilitate proper transfer of the desired portion from the sample to the target fluid. For example, if too much air is aspirated, the bubble will become approximately cylindrical, separating the fluids by a large distance and preventing fusion or interfacial contact of the two fluids. In contrast, if too little air is aspirated, the area of ​​the interface between the two fluids will not be significantly reduced to prevent contamination. If successfully performed, the bubble will either remain in the container "between" the two fluids (essentially stuck to one side of the pipette wall) or it will be dislodged and rise up. In either case, mixing of the fluids during merging occurs within the pipette tip, a diffusion / convection barrier is established, and the beads are allowed to pass through the wash fluid within the pipette tip before entering the destination fluid.

[0082] The volume of the air pocket may vary depending on the geometry of the container and / or the opening of the container. In addition, the volume of the air pocket may vary depending on whether the inner surface of the container is treated with a suitable agent / additive. Furthermore, the volume of the air pocket may vary depending on whether the sample fluid contains additional agents, such as detergents, that may affect the interaction between the sample and the container.

[0083] In some embodiments, the volume of the air pocket is about 1 μl to about 100 μl. In some embodiments, the volume of the air pocket is about 1 μl, about 5 μl, about 10 μl, about 15 μl, about 20 μl, about 25 μl, about 30 μl, about 35 μl, about 40 μl, about 45 μl, about 50 μl, about 55 μl, or about 60 μl. In some embodiments, the volume of the air pocket depends on the dimensions of the container (e.g., pipette tip) that holds the air pocket. For example, in some embodiments, the air pocket is of sufficient volume that the distance from the container (e.g., pipette tip) opening to the top of the air pocket (e.g., the portion of the air pocket furthest from the pipette tip opening) is approximately equal to the diameter of the container (e.g., pipette tip). In some embodiments, the air pocket is of sufficient volume such that the distance from the container (e.g., pipette tip) opening to the top of the air pocket is about 50% to about 300% of the diameter of the container (e.g., pipette tip) opening (e.g., about 1 / 2 the diameter of the opening to about 3 times the diameter of the container (e.g., pipette tip) opening). For example, this distance can be about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, or about 300% of the diameter of the container (e.g., pipette tip) opening.

[0084] In some embodiments, the interaction (e.g., fusion) between the fluid sample and the target fluid can be initiated or assisted by adding a surfactant to the fluid sample. For example, the addition of a surfactant can help promote the interaction between the fluid sample and the target fluid when an air pocket is created in the container. As another example, the interaction between the fluid sample and the target fluid can be initiated or assisted by inducing movement within the container, especially in aspects where an air pocket (or oil pocket, or gas pocket) is created. For example, flicking or otherwise disturbing the container can induce movement of the air pocket, thereby allowing fusion of the fluid sample and the target fluid to occur within the container.

[0085] In some embodiments, the geometry of the vessel can be selected to optimize its use as an air, oil, or gas pocket as a passable restriction. For example, the geometry of the vessel can be selected to help maintain the integrity of the air pocket during fluid fusion (e.g., during interaction of the fluid sample with the fluid of interest). In some embodiments, a serrated ridge on the inner surface of the pipette tip forming a ring shape can help create and maintain a more effective passable restriction. For example, this radial restriction helps prevent air bubbles from rising within the pipette tip after fluid fusion, thereby allowing the bubble restriction to be maintained during bead transfer. Alternatively, surface patterning or geometry can be used to create preferential pinning points within the pipette, such that multiple fluids can be aspirated with air bubbles between them. For example, pinning points can be created such that a first wash fluid and a second wash fluid can be aspirated. As additional wash fluid is aspirated, the conical nature of the container (e.g., pipette tip) induces a greater vertical displacement of the lower interface than the upper interface, causing them to break away from their pinned positions first and bring the wash solution into communication with the sample fluid (e.g., merging it).

[0086] In some embodiments, at least one technique is used to reduce carryover contamination from contaminated surfaces. For example, at least one technique is used to reduce potential contamination from the surface of the container when placed in the target fluid. For example, in some embodiments, the method of isolating a target includes placing a portion of the container (e.g., a pipette tip) in a wash fluid to remove contaminants from the exterior surface of the container. For example, a portion of the container proximal to the opening of the container may be placed in the wash fluid to remove contaminants from the exterior surface of the container prior to placing the container in the target fluid. Such a step may be referred to herein as a "pre-rinse" of the container. In the case of a pipette tip, for example, a portion of the pipette tip may be placed in the wash fluid prior to placing the pipette tip in the target fluid, and may be referred to as a "tip-dip". The placement in the wash fluid thereby minimizes the passage of potential contaminants from the exterior surface of the container (e.g., the pipette tip) into the target fluid (e.g., when the pipette tip is placed into the target fluid).

[0087] Another suitable technique that can be used to minimize carryover contamination is to limit the surface area in contact with the sample fluid. This can be done in multiple ways. One way to do this is to track the height of the sample fluid during aspiration into the container. In liquid handling terminology, this is sometimes called "liquid following". This provides a way to aspirate fluid to the bottom of the container without having to unnecessarily submerge the pipette tip in the sample. Another method would be to use microstructures on the container (e.g., pipette tip). For example, sharp-edged grooves could be added along the length of a hydrophobic pipette tip. When submerged in an aqueous sample, the sample will avoid wetting inside the grooves, thereby reducing the wetted / contaminated surface area and thus reducing carryover of surface contaminants. Nanostructures or textures used to create superhydrophobic surfaces would function similarly, reducing surface-based carryover contamination. Thus, such methods can also be viewed as traversable restrictions that allow target movement while minimizing potential carryover contamination from the surface of the container (e.g., the surface of the pipette tip).

[0088] In some embodiments, multiple techniques are used to reduce contamination. For example, in some embodiments, a pre-rinse step and at least one passable restriction are used. In some embodiments, multiple passable restrictions are employed. For example, the method may include creating multiple passable restrictions to minimize contamination, including any combination of the above passable restrictions.

[0089] In some embodiments, the method further includes pre-concentrating the target analyte proximate the opening of the container. For example, pre-concentration can be performed by applying a first magnetic force to the container to generate a concentration of target-PMP complexes proximate the opening of the container. After generating the concentration of target-PMP complexes proximate the opening of the container, the container can be placed in a destination solution, and the target can be transferred from the container to the destination fluid, such as by diffusion or application of a magnetic force to attract the target-PMP complexes to the destination fluid. In such embodiments, aggregation of the target-PMP complexes themselves serves as a restriction to the passage of potential contaminants. For example, aggregation of the target-PMP complexes can serve as a physical blockage to minimize the passage of potential contaminants from the pipette tip to the destination fluid, as the potential contaminants are trapped above the complexes (e.g., trapped in the pipette tip, farther from the destination fluid compared to the complexes).

[0090] In other embodiments, the target fluid may be contained within a suitable container that further comprises a cleaning fluid. For example, the target fluid and the cleaning fluid may be present as separate layers, such that the cleaning fluid is above the target fluid. Thus, the container passes through the cleaning fluid before contacting the target fluid, thereby facilitating the removal of potential contaminants on the exterior surface of the container by the cleaning fluid before contacting the target fluid. Suitable cleaning fluids include, for example, liquids or oils. For example, the cleaning fluid may be a liquid with a lower density than the target fluid. As another example, the cleaning fluid may be a hydrophobic liquid. For example, the cleaning fluid may be a layer of oil, such as mineral oil, suspended above the target fluid. In some embodiments, the cleaning fluid and the target fluid are not sufficiently miscible, thereby preventing the diffusion of contaminants from the cleaning fluid to the target fluid. For example, oil and water are not sufficiently miscible, thereby representing effective cleaning fluids (e.g., oil) and target fluids (e.g., water), respectively. In some embodiments, the wash fluid (e.g., oil) acts as a barrier (e.g., a passable restriction) that pushes residual fluid / contaminants back out of the pipette tip as the tip is immersed in the oil. For example, when a pipette tip is immersed in a layer of oil and then immersed in a target fluid, the oil layer pushes potential contaminants outside the pipette tip up the pipette tip, thereby preventing these carryover contaminants from contacting the target fluid.

[0091] The systems and methods described herein may be used to isolate targets from any suitable fluid sample (e.g., target analytes and / or target contaminants). In some embodiments, the sample is an environmental sample. In some embodiments, the sample is a biological sample. The sample may be collected and / or stored in a suitable container (e.g., a sample collection container) prior to processing the sample by the methods described herein. Any type of sample collection container suitable for receiving and storing the sample until performing the described methods for detection of the target can be used. Examples of sample collection containers include, but are not limited to, tubes containing a reversibly removable cap, bags, syringes, droppers, and the like. In some embodiments, the sample is pre-processed prior to performing the methods described herein.

[0092] In some embodiments, the sample may be pretreated to inactivate potential pathogens (e.g., viruses, bacteria) within the sample. In some embodiments, the sample may be pretreated to lyse cells within the sample, thereby releasing targets (e.g., nucleic acids) for subsequent isolation. 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, the sample may be pretreated by freezing, heating, and / or adding a denaturing agent to the sample. For example, the sample may be pretreated by heating to a temperature sufficient to inactivate potential pathogens within the sample for an appropriate time. For example, the sample may be heated to about 40° C. or higher. For example, the 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 heating temperature for a suitable duration, 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 5-10 minutes to achieve both cell lysis and viral inactivation in a single heat treatment step. In some embodiments, pretreating the sample includes adding a denaturant to inactivate potential pathogens within the sample. For example, suitable denaturants include guanidine-based denaturants (e.g., guanidine hydrochloride, guanidine thiocyanate, etc.) and detergents (e.g., Triton X-100, tween 20).

[0093] The sample may further comprise a suitable detergent. For example, the sample may comprise 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 comprises 0-5% (v / v) detergent. For example, the sample may comprise 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. 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.

[0094] The sample may further comprise a suitable buffer. For example, in some embodiments, the sample may comprise phosphate buffered saline (PBS), universal transport medium (UTM), saline, etc. The sample may comprise one or more enzymes or chemicals that help degrade the contents in the sample to facilitate the release of the desired target. For example, the sample may comprise one or more enzymes, such as one or more proteases. In certain embodiments, the sample may comprise proteinase K. The sample may further comprise one or more suitable reagents to prevent degradation of the target in the sample. For example, suitable buffers and / or inhibitors (e.g., RNase inhibitors, nuclease inhibitors, etc.) may be present in the sample.

[0095] In some embodiments, the inner surface of the vessel (e.g., pipette tip) that contains the fluid sample is patterned. In some embodiments, this includes hydrophilic / hydrophobic and / or oleophilic / oleophobic patterning. Patterning can be achieved through structural patterning (inclusion / exclusion of edges or boundaries and scaling of features to increase or decrease the dominance of surface tension effects), surface texturing (use of micropillars on hydrophobic surfaces to make them superhydrophobic), or modification of surface chemistry (e.g., chemical or oxygen plasma treatment of polymers). In some embodiments, such patterning methods are used to help stabilize or facilitate the positioning of air pockets (or oil pockets, or gas pockets). Patterning can be used to prevent wetting of the surface as well for certain vessel (e.g., tip) designs. In some embodiments, patterning is used to prevent the fluid sample and / or targets contained therein from adhering to the vessel (e.g., pipette) material.

[0096] In some embodiments, the target fluid is held in a suitable collection device. Any desired collection device or target vessel may be used. 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. Other suitable examples include porous materials, such as filter paper, and assay surfaces. "Attracting" particles or complexes containing particles to a collection device is meant to encompass any means of contacting the particles with the sample collection device or its surface. For example, attracting particles onto a piece of filter paper, onto a plate, etc. is meant to encompass by "attracting" the particles to the sample collection device. For automated high-throughput systems and methods, multi-well plates used in combination with a multi-pipette array are particularly useful.

[0097] In some embodiments, the target fluid retained in the collection device comprises a wash buffer. For example, in certain embodiments, separate wells on a multi-well plate comprise the wash buffer. In some embodiments, the wash buffer comprises water. In some embodiments, the wash buffer comprises ethanol. In some embodiments, a layer of oil (e.g., light mineral oil) may be present on top of the wash buffer. In some embodiments, the methods described herein further comprise aspirating the target fluid (e.g., wash buffer) (and potentially a layer of oil, if present) from the sample collection device (e.g., well) while the device is positioned over the magnet such that the target-PMP complexes remain, and drying the target-PMP complexes, if desired. In some embodiments, the target-PMP complexes are not dried prior to detecting the target. In some embodiments, an additional wash step or overlay with a reagent may be performed.

[0098] In some embodiments, the target fluid is removed. In some embodiments, following removal of the target fluid (e.g., a wash buffer), reagents for detection of the target may be added to the collection device or target container, or a suitable method for detection of the target may be performed.

[0099] In some embodiments, the container, destination container, and / or sample collection device may contain reagents for detection of a target.

[0100] In some embodiments, reagents for detection of the target are added after removal of some or all of the original target fluid.

[0101] In other embodiments that rely on negative selection, the targets are retained in the vessel while potential contaminants are passed into the destination fluid, In such embodiments, after removal of the contaminants, the targets may be contacted with a suitable wash buffer, elution buffer, or reagent for detection of the targets.

[0102] Reagents suitable for target detection include, for example, reagents for nucleic acid amplification (e.g., PCR, isothermal amplification, etc.) and / or sequencing. In some embodiments, the reagents for target detection include reagents for loop-mediated isothermal amplification (LAMP)-based detection of targets. In general, LAMP reactions include a DNA polymerase with strong strand displacement activity and resistance to high temperatures and up to six DNA oligonucleotides of specific structure. RT-LAMP reactions further include a reverse transcriptase. A sample with 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 act as primers for the reverse transcriptase and additional oligonucleotides for the DNA polymerase are designed so that the DNA products loop back at their ends. These then serve as self-priming templates for the DNA polymerase. In the presence of a small number of RNA template molecules, the chain reaction is allowed to proceed and then run until the added reagents (e.g., deoxynucleotide triphosphates) are exhausted.

[0103] LAMP or RT-LAMP assays may be preferred 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 detecting the target include reagents for a colorimetric assay for detecting the target. Suitable colorimetric assays include, for example, colorimetric loop-mediated isothermal amplification assays. Such embodiments allow for easy visualization of whether a sample contains a target of interest. In some embodiments, the sample collection device contains reagents for a colorimetric loop-mediated isothermal amplification (LAMP) assay. In embodiments where 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 preferred embodiments, pH-sensitive indicators (e.g., phenol red) may be used as they are easily visualized by the naked eye.

[0104] In some embodiments, the reagents for detecting the target include reagents for a fluorescent assay for detecting the target. For example, the sample collection device may contain reagents for a fluorescent LAMP or fluorescent RT-LAMP assay. Any suitable fluorescent dye can be used in the fluorescent LAMP or fluorescent RT-LAMP assay to generate a fluorescent signal in the presence of sufficient nucleic acid.

[0105] In some embodiments, the reagents include oligonucleotides (e.g., primers) designed for detection of bacterial nucleic acids. In some embodiments, the reagents include oligonucleotides designed for detection of viral RNA. For example, the reagents may include oligonucleotides designed for detection of viral upper respiratory tract infections selected from SARS-CoV2, SARS, coronavirus, rhinovirus, influenza, respiratory syncytial virus, and the like. In some embodiments, the reagents include oligonucleotides for detection of SARS-CoV-2 RNA.

[0106] Reagents suitable for detecting targets also include, for example, reagents for immunodetection of targets and / or contaminants. In some embodiments, the reagents for detecting targets and / or contaminants include reagents for immunoassays, which may use antibodies and / or antibody fragments to detect or measure targets or target analytes. In some embodiments, the immunoassays are enzyme immunoassays, ELISAs (enzyme-linked immunosorbent assays, including direct ELISAs, indirect ELISAs, sandwich ELISAs, and competitive ELISAs), IEMAs (immunoenzymatic assays), radioimmunoassays, fluoroimmunoassays, chemiluminescent immunoassays (CLIA), and enumeration immunoassays (CIA).

[0107] In some embodiments, the sample is a biological sample obtained from a subject suspected of having an infection. In some embodiments, the subject is suspected of having SARS-CoV2, coronavirus, rhinovirus, influenza, respiratory syncytial virus, adenovirus, parainfluenza, human immunodeficiency virus, human papillomavirus, rotavirus, hepatitis C virus, Zika virus, Ebola virus, tuberculosis, Borrelia borreliae, Staphylococcus aureus, Aspergillus, or Streptococcus pyogenes. In some embodiments, the subject may be suspected of having a bacterial or viral infection. For example, the subject may be suspected of having an upper respiratory tract infection. For example, the subject may be suspected of having a viral upper respiratory tract infection, including infection with SARS-CoV-2, coronavirus, rhinovirus, influenza, respiratory syncytial virus, etc.

[0108] In some embodiments, the methods described herein are automated. For example, the methods may be performed by a computer including a processor and a memory. The memory may include software that instructs the processor to perform a given task. To facilitate automation of some or all of the steps of the method, an instrument including one or more robotic components that allow the movement of system components can be used. In some embodiments, the systems and methods of the present invention (e.g., Fuse-It) and approaches to improve performance (e.g., Bubble-It, Chip-It) can all be achieved using automation as demonstrated in preliminary data (see, e.g., Figures 6, 7, and 8) using the methods of the present invention, for example, using a Gilson PIPETMAX liquid handling robot and associated Trilution and Protocol Builder software.

[0109] 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, and modifications and variations are possible in light of the above teachings or may be acquired from practice of the present disclosure. The embodiments have been chosen and described to explain the principles of the present disclosure and as practical applications of the present disclosure to enable those skilled in the art to utilize the present disclosure in various embodiments and with various modifications suitable for the particular use contemplated. It is intended that the scope of the present disclosure be defined by the appended claims and their equivalents. EXAMPLES

[0110] [Example 1] The performance of various embodiments of the methods described herein was evaluated using absorbance measurements of tartrazine (yellow food coloring) concentration. In this example, the "sample" was added to water containing 0.05% Triton X100. The target fluid (e.g., wash buffer) was water. Concentrations were measured relative to the initial sample concentration using a DeNovix ds-11 spectrophotometer set to measure absorbance at 430 nm (i.e., absorbance near the tartrazine peak). Legend nomenclature is as follows: "Tube-It" refers to mimicking the washing of beads in a tube, where the beads are held to the side of the tube while fluid is added and removed. Instead, a 96-well plate is used here to mimic this process, with beads held to the bottom of the 96-well plate with a magnet while fluid is added and removed. "Fuse-It" refers to a method of extracting or isolating at least one target from a fluid comprising a sample having multiple components, comprising placing a container containing the fluid comprising the sample in a destination fluid that is sufficiently miscible with the fluid comprising the sample, the container comprising an opening that allows movement of particles from the sample to the destination fluid, and extracting or isolating the target by selectively transferring particles from the fluid comprising the sample to the destination fluid. In some embodiments, the target is extracted or isolated by selectively transferring the target from the fluid comprising the sample to the destination fluid. In some embodiments, the target is extracted or isolated by selectively transferring contaminants (e.g., contaminant particles) from the fluid comprising the sample to the destination fluid. In this example, the tip is a pipette tip, and refers to a minimal approach where the tip is brought to a destination well and removed according to the method of the invention. Pre-aspiration and post-aspiration refer to pre-aspiration and / or post-aspiration of a wash buffer (e.g., water). "Bubble-It" refers to pre-aspiration of air into the pipette tip, another method of the invention described herein. The phrase "tip-dip" refers to another variation of the method of the present invention in which the pipette tip is briefly immersed in a sacrificial well of wash fluid before entering the final destination fluid as a means of carrying over surface contamination.

[0111] FIG. 4 compares the performance of Tube-It with the variation of Fuse-It. The data demonstrated that Fuse-It with both pre-aspiration and post-aspiration can provide the same washing benefit as a single Tube-It wash, whether or not the beads are pre-concentrated to the end of the tip before entering the wash fluid. The data also demonstrated the considerable benefit provided when Fuse-It is enhanced with Bubble-It. With Bubble-It, washing is over 4 times better than Tube-It. After two washes, Fuse-It with Bubble-It is about 16 times (i.e., 4×4) better than two Tube-It washes.

[0112] Figure 5 shows, in a separate experiment, the benefit of eliminating surface carryover when performing Fuse-It, again confirming the relative benefit of Fuse-It compared to Tube-It. Approximately 30% of the contamination from Fuse-It with Bubble-It is due to surface carryover outside the chip. The inventors envision that replacing the chip-dip procedure with an oil overlay in the sample well will also be advantageous for some future applications.

[0113] Taken together, these data demonstrate the functionality and benefits of all the methods of the present invention, as well as multiple approaches to improve performance with and without Bubble-It, and with and without Tip-Dip.

[0114] [Example 2] This example compares the timing breakdown of running the same protocol for extracting nucleic acids using KingFisher technology and Fuse-It run using Pipetmax.

[0115] Sixteen samples of HeLa cells at various concentrations were processed using Macherey Nagel Nucleomag Tissue kits. The KingFisher utilized a magnet in sheath technique for extraction and the Pipetmax utilized Fuse-It.

[0116] Each step of the protocol was timed and grouped into three parts: "Chip management" refers to loading and unloading the chip; "Fluid handling" refers to thorough mixing; and "Technique" refers to bead collection and transfer.

[0117] The Fuse-It technology proved to be twice as fast as the KingFisher magnet in sheath technology, as shown in Figure 6. Importantly, an additional benefit that the Fuse-It offers over other technologies such as KingFisher is the ability to prepare plates for running extractions or PCR.

[0118] [Example 3] The performance of various embodiments of the methods described herein was evaluated using several variables that improve bead transfer from a pipette tip with Fuse-It. These improvements were evaluated using absorbance measurements of tartrazine (yellow food coloring), as shown in FIG.

[0119] Samples were added to PBS with 0.05% Triton containing Nucleomag paramagnetic particles. Figure 7 shows several variables to improve Fuse-It bead transfer from the pipette tip and shows its effect on carryover. The higher the absorbance, the higher the carryover.

[0120] Migration time refers to how long the pipette remains in the well while the PMPs move out of the tip. A slight increase in carryover was observed with a 5 s increase in migration time. This is due to an increase in bead movement within 5 s. There is no significant difference between 5 and 20 s.

[0121] Pipette movement speed refers to the speed at which the pipette enters and exits the well. A slower pipette entry movement speed results in higher carryover compared to a faster movement speed.

[0122] Pipette tapping refers to the action of moving the pipette tip, which is located at the bottom of the well, up and down 1 mm. Pipette tapping can be utilized for PMPs with low magnetic response. There is no significant increase in carryover with increasing number of taps.

[0123] [Example 4] This example provides a purity comparison between KingFisher and "TSP" platforms using the Macherey Nagel Nucleomag Tissue kit (i.e., "Extraction Kit"). TSP refers to "tip-based sample preparation" and encompasses the methods of the present invention, including Tip-It (using a pipette tip), Fuse-It, and Bubble-It.

[0124] Samples were tartrazine reconstituted in the kit buffer provided. TSP data was normalized to KingFisher set to 1. Carryover of individual steps refers to the carryover from the well where the beads are collected to the well where they are transferred. Sample carryover refers to how much of the original sample is carried to the elution well. This TSP protocol used a combination of Fuse-It and Bubble-It as the transfer mechanism. 24μL and 10μL refer to the volume of PMP used with the TMP and extraction kit, KingFisher ran 24μL of PMP (i.e. 24μL and 10μL refer to the volume of bead solution added to the sample).

[0125] As shown in Figure 8, the 24uL TSP method is nearly equivalent to the KingFisher in carryover. The 10uL TSP method of the present invention is superior, yielding 10 times less carryover.

Claims

1. A method for extracting or isolating at least one target or component from a fluid containing a sample having a plurality of components, comprising placing a container containing the fluid containing the sample in a target fluid that is sufficiently miscible with the fluid containing the sample, the container having an opening that allows the transfer of particles from the sample to the target fluid.

2. 10. The method of claim 1, wherein the target or component is extracted or isolated by selectively transferring particles from the fluid containing the sample to the target fluid.

3. The method of claim 1 or claim 2, wherein the opening allows the transfer of at least one target or component from the sample to the target fluid.

4. The method of claim 1 or claim 2, wherein the opening allows for the transfer of at least one contaminant from the sample to the target fluid.

5. The method of any one of claims 1 to 4, wherein the movement of the particles from the sample to the target fluid occurs by diffusion, gravity, or acceleration.

6. The method of any one of claims 1 to 5, further comprising aspirating fluid into the container after the container is placed in the target fluid.

7. The method of claim 6 , wherein the fluid is aspirated into the vessel before, during, and / or after transfer of the particles from the vessel to the destination fluid.

8. 8. The method of claim 1, wherein the at least one target or component, if present in the sample, binds to at least one paramagnetic particle (PMP) to form one or more target-PMP complexes or component-PMP complexes.

9. The method of claim 8, wherein the opening allows for the transfer of the one or more target-PMP complexes and / or the one or more component-PMP complexes from the sample to the target fluid.

10. The method of claim 8 or claim 9, wherein the movement of the one or more target-PMP complexes and / or the one or more component-PMP complexes from the sample to the target fluid occurs by application of a magnetic force to attract the one or more target-PMP complexes from the sample to the target fluid.

11. 5. The method of claim 4, wherein the at least one contaminant, if present in the sample, binds to one or more paramagnetic particles (PMPs) to form one or more contaminant-PMP complexes.

12. The method of claim 11, wherein the opening allows for the transfer of the one or more contaminant-PMP complexes from the sample to the target fluid.

13. The method of claim 11 or 12, wherein the movement of the one or more contaminant-PMP complexes from the sample to the target fluid occurs by application of a magnetic force to attract the one or more contaminant-PMP complexes from the sample to the target fluid.

14. 14. The method of claim 10 or claim 13, further comprising aspirating the fluid into the container before, during and / or after application of the magnetic force.

15. 15. The method of any one of claims 1 to 14, further comprising creating a pocket of air, oil, or gas proximate the opening of the vessel prior to inserting the vessel into the target fluid.

16. The method of claims 1 to 15, wherein said at least one target or component is a cell.

17. The method according to claims 1 to 15, wherein said at least one target or component is an analyte.

18. The method of any one of claims 1 to 17, wherein the sample comprises a biological fluid.

19. The method of any one of claims 1 to 18, wherein the vessel is a pipette tip, a straw, or a capillary tube.

20. 20. The method of any one of claims 1 to 19, wherein the target fluid comprises a wash buffer, a precipitation buffer, a lysis buffer, an extraction buffer, an elution buffer, or a reaction mixture containing a reagent for detecting the target.

21. 21. The method of any one of claims 1 to 20, further comprising placing the container in a cleaning fluid to reduce potential contaminants on an exterior surface of the container prior to placing the container in the target fluid.

22. 1. A system for isolating or extracting at least one target or component from a sample, comprising: a. a container for containing a sample, the container having an opening; b. a collection device containing the target fluid; and c. Optionally, a sample containing said at least one target or component. wherein the sample is sufficiently miscible with the target fluid.

23. The system of claim 22, wherein the sample is a fluid and comprises the at least one target or component bound to one or more paramagnetic particles, thereby forming one or more target-PMP complexes and / or one or more component-PMP complexes.

24. 23. The system of claim 22, wherein the sample is a fluid and comprises at least one contaminant bound to one or more paramagnetic particles, thereby forming one or more contaminant-PMP complexes.

25. The system of any one of claims 22 to 24, further comprising a magnet.

26. The system of any one of claims 22 to 25, further comprising a cleaning fluid.

27. 27. The system of claim 26, wherein the washing fluid is contained within the collection device and is above the target fluid such that the vessel containing the sample passes through the washing fluid before contacting the target fluid.

28. 28. The system of claim 27, wherein the wash fluid and the target fluid are immiscible.

29. The system of any one of claims 22 to 28, wherein the at least one target or component or contaminant is a cell.

30. The system of any one of claims 22 to 28, wherein the at least one target or component is an analyte.

31. The system of any one of claims 22 to 30, wherein the sample comprises a biological fluid.

32. The system of any one of claims 22 to 31, wherein the container is a pipette tip, a straw, or a capillary tube.

33. The system according to any one of claims 22 to 32, wherein the target fluid is a wash buffer, an elution buffer, or a reaction mixture comprising a reagent for detecting the target.

34. 22. The method of any one of claims 1 to 21, further comprising the step of transferring the at least one target, the at least one component, the at least one contaminant, the one or more target-PMP complexes, the one or more component-PMP complexes and / or the one or more contaminant-PMP complexes to another or different device or reagent mixture or reagent for further processing and / or analysis, identification, quantification or use.

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