Methods, compositions and kits for concentrating target analyte from large number of fluid samples

Abstract

The present disclosure relates to methods, compositions and kits for concentrating and purifying one or more target analytes from a large number of fluid samples. In some embodiments, the methods involve at least two first aqueous two-phase system (ATPS) compositions. Some embodiments provide kits comprising at least two ATPS compositions and a binding buffer. Other embodiments provide methods of treating bladder cancer in a patient in need thereof.

Description

[0001] Cross - reference to related applications

[0002] This application claims priority and the benefit of U.S. Provisional Application Serial No. 63 / 381,932, filed on November 2, 2022. The entire content of the foregoing application is hereby incorporated by reference in its entirety for all purposes. Technical field

[0003] This application relates to methods, compositions, and kits for using aqueous two - phase systems (ATPS) to improve the concentration and purification of target analytes, particularly methods, compositions, and kits for improving the concentration and purification of target analytes in large fluid samples. Background art

[0004] Effectively concentrating and separating target analytes from large biological samples for downstream applications (such as diagnostic tests) is a challenging task. Therefore, there is a need for improved methods that are simpler, cheaper, and can rapidly process large numbers of samples to provide high - quality target analytes. Summary of the invention

[0005] Disclosed herein are novel methods, compositions, and kits for separating, concentrating, and / or purifying target analytes (such as nucleic acids), wherein an aqueous two - phase system (ATPS) and a purification system (such as an extraction column) are employed without the need for complex equipment.

[0006] In some embodiments, provided is a method for concentrating and purifying one or more target analytes from a large fluid sample, the method comprising the steps of: (a) dividing the large fluid sample containing the one or more target analytes into at least two aliquots of sample solution; (b) preparing at least two first aqueous two - phase system (ATPS) compositions, wherein each first ATPS composition comprises a polymer, a salt, a surfactant, or any combination thereof dissolved in an aqueous solution to form a first - phase solution and a second - phase solution; (c) adding each aliquot of the sample solution prepared from the large fluid sample containing the one or more target analytes to each first ATPS composition such that the one or more target analytes partition into each first - phase solution; (d) further processing each first - phase solution to form a final - phase solution; (e) mixing the final - phase solution with at least one purification composition to form a mixed solution; (f) contacting the mixed solution with a purification system configured to selectively separate the one or more target analytes; and (g) collecting the one or more target analytes from the purification system to obtain a final solution containing the concentrated and purified one or more target analytes.

[0007] In some embodiments, a method for concentrating and purifying one or more target analytes from a large volume of fluid sample is provided, the method comprising the steps of: (a) dividing the large volume of fluid sample containing the one or more target analytes into at least two aliquots of sample solution; (b) preparing at least two aliquots of a first aqueous two-phase system (ATPS) composition, wherein the first ATPS composition comprises a polymer, a salt, a surfactant, or a combination thereof dissolved in an aqueous solution to form a first-phase solution and a second-phase solution; (c) adding each aliquot of the sample solution containing the one or more target analytes to each aliquot of the first ATPS composition such that the one or more target analytes partition into the first-phase solution; (d) collecting the first-phase solutions of the at least two aliquots of the first ATPS composition and mixing the first-phase solutions with a second ATPS composition, wherein the second ATPS composition comprises a polymer, a salt, a surfactant, or a combination thereof dissolved in an aqueous solution to form a third-phase solution and a fourth-phase solution, such that the one or more target analytes partition into and are concentrated in the third-phase solution; (e) collecting the third-phase solution and mixing the third-phase solution with a binding buffer to form a mixed solution, wherein the binding buffer comprises at least one dissociating agent; (f) loading the mixed solution onto an extraction column configured to selectively extract and purify the one or more target analytes; (g) eluting and collecting the one or more target analytes from the extraction column.

[0008] Another embodiment provides an ATPS composition comprising one or more polymers, salts, surfactants, or combinations thereof as described herein.

[0009] In some embodiments, an ATPS composition selected from the group consisting of A1, A2, A3, A4, AA1, AA2, AA3, and AA4 is provided.

[0010] In some embodiments, a kit is provided that includes a first ATPS composition selected from the group consisting of A1, A2, A3, and A4; a second ATPS composition selected from the group consisting of AA1, AA2, AA3, and AA4; and a binding buffer selected from the group consisting of B1, B2, and B3.

[0011] In some embodiments, a method for concentrating and purifying one or more target analytes from a large volume of fluid sample is provided, the method comprising the steps of:

[0012] (a) preparing a first aqueous two-phase system (ATPS) composition, wherein the first ATPS composition comprises a polymer, a salt, a surfactant, or a combination thereof dissolved in an aqueous solution to form a first-phase solution and a second-phase solution;

[0013] (b) Add a sample solution prepared from the large volume of fluid sample containing one or more target analytes to the first ATPS composition such that the one or more target analytes are partitioned into the first phase solution;

[0014] (c) Collect the first phase solution and mix the first phase solution with a second ATPS composition, wherein the second ATPS composition comprises a polymer, a salt, a surfactant, or a combination thereof dissolved in an aqueous solution to form a third phase solution and a fourth phase solution, such that the one or more target analytes are partitioned into the third phase solution and concentrated in the third phase solution;

[0015] (d) Collect the third phase solution and mix the third phase solution with a binding buffer to form a mixed solution, wherein the binding buffer comprises at least one dissociating agent;

[0016] (e) Load the mixed solution onto an extraction column configured to selectively extract and purify the one or more target analytes;

[0017] (f) Elute and collect the one or more target analytes from the extraction column to obtain a final solution containing the concentrated and purified one or more target analytes.

[0018] In some embodiments, provided is a method for concentrating and purifying one or more target analytes from a large volume of fluid sample, the method comprising the following steps:

[0019] (a) Divide the large volume of fluid sample containing the one or more target analytes into at least two aliquots of sample solution;

[0020] (b) Prepare at least two aliquots of a first aqueous two-phase system (ATPS) composition, wherein the first ATPS composition comprises a polymer, a salt, a surfactant, or a combination thereof dissolved in an aqueous solution to form a first phase solution and a second phase solution;

[0021] (c) Add each aliquot of the sample solution containing the one or more target analytes to each aliquot of the first ATPS composition such that the one or more target analytes are partitioned into the first phase solution;

[0022] (d) Collect the first phase solution of the at least two aliquots of the first ATPS composition and mix the first phase solution with a second ATPS composition, wherein the second ATPS composition comprises a polymer, a salt, a surfactant, or a combination thereof dissolved in an aqueous solution to form a third phase solution and a fourth phase solution, such that the one or more target analytes are partitioned into and concentrated in the third phase solution;

[0023] (e) Collect the third phase solution and mix the third phase solution with a binding buffer to form a mixed solution, wherein the binding buffer comprises at least one dissociating agent;

[0024] (f) Load the mixed solution onto an extraction column configured to selectively extract and purify the one or more target analytes;

[0025] (g) Elute and collect the one or more target analytes from the extraction column to obtain a final solution containing the concentrated and purified one or more target analytes.

[0026] In some embodiments, corresponding kits can advantageously be used in conjunction with and for performing the methods according to various aspects of the present invention. In some embodiments, the kits can include the components described in the various embodiments, but can additionally include containers accessible by syringes or pipettes for storage, packaging, and / or reaction and optionally devices for manipulating aqueous solutions. Such containers and devices can include columns, test tubes, capillaries, plastic tubes, falcon tubes, culture tubes, microtiter plates, pipettes, cuvettes, and the like.

[0027] Other example embodiments are discussed herein.

[0028] Advantages

[0029] The various embodiments of the present disclosure have many advantages.

[0030] In some embodiments, the methods, compositions, and kits of the present disclosure are surprisingly effective at concentrating and separating target analytes from large volumes of liquid biological samples. These methods, compositions, and kits are particularly effective at separating target analytes present at very low concentrations in biological samples, such as cell-free DNA (cfDNA). These methods, compositions, and kits allow for more accurate detection and identification of target analytes from large volumes of liquid, which is particularly useful for diagnostic purposes. These methods, compositions, and kits are particularly suitable for biological samples that typically occur in large volumes, such as urine, saliva, blood, and the like.

[0031] In some embodiments, the methods, compositions, and kits of the present disclosure provide simple, inexpensive, and effective means for purifying target analytes from different clinical / biological samples of different volumes, particularly when dealing with large-volume or large quantities of fluid samples to provide high-quality target analytes. The methods, compositions, and kits disclosed herein relate to aqueous two-phase systems (ATPS) in upstream processes, providing great flexibility for use in various downstream processes.

[0032] In some embodiments, for various reasons, it is desirable to divide a large-volume or large quantity of fluid sample into smaller aliquots. For example, multiple smaller aliquots (also referred to as "parallel ATPS" in some embodiments) can be processed in parallel to save time, minimize reagent use, make the extreme volume ratios in the ATPS smaller, and accommodate the sample size limitations of available instruments.

[0033] In some embodiments, it has surprisingly been found that although the disclosed methods include additional steps (such as dividing a large quantity of fluid sample into at least two aliquots, performing phase separation using multiple parallel ATPSs, etc.) (which are potential sources of target analyte loss (e.g., due to incomplete target partitioning in the ATPS)), the recovery efficiency of the target analyte using the disclosed methods is not significantly different from the recovery efficiency of methods using a single ATPS to process a large quantity of fluid sample.

[0034] Accordingly, those skilled in the art can adapt the methods and kits of the present disclosure in different laboratory and equipment settings to achieve comparable DNA recovery from large-volume or large quantities of fluid samples while minimizing errors and sample losses associated with sample handling and processing.

[0035] In some embodiments, a large clinical / biological sample volume results in a large target-rich phase in the first ATPS. In some embodiments, a second ATPS is used to concentrate the large target-rich phase of the first ATPS into a more concentrated and smaller-volume target-rich phase in the second ATPS for ease of downstream processing.

[0036] In some embodiments, having a second ATPS after the first ATPS can further concentrate the DNA for detection.

[0037] These and other features and characteristics, as well as the operating methods and functions of the related components, will become more apparent upon consideration of the following detailed description and the appended claims in reference to the accompanying drawings, all of which form a part of this specification, where like reference numerals represent corresponding parts in the various figures. However, it should be clearly understood that these figures are for illustrative and descriptive purposes only and are not intended as a definition of the limitations of the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1AIt is a graph showing the average CT values of 145bp DNA recovered from urine using a centrifugal column with and without a pre-ATPS step according to an exemplary embodiment.

[0040] Figure 1B It is a graph showing the average CT values of 2000bp DNA recovered from urine using a centrifugal column with and without a pre-ATPS step according to an exemplary embodiment.

[0041] Figure 1C It is a graph showing the average CT values of 145bp DNA recovered from urine using magnetic beads with and without a pre-ATPS step according to an exemplary embodiment.

[0042] Figure 1D It is a graph showing the average CT values of 2000bp DNA recovered from urine using magnetic beads with and without a pre-ATPS step according to an exemplary embodiment.

[0043] Figure 2A It is a graph showing the average CT values of 145bp DNA recovered from urine under different extraction conditions according to Table 3.

[0044] Figure 2B It is a graph showing the average CT values of 145bp DNA recovered from urine under different extraction conditions according to Table 6.

[0045] Figure 3A It is a graph showing the average CT values of 145bp DNA recovered from urine samples of three individual donors using the first ATPS with different top phase:bottom phase volume ratios according to Table 9.

[0046] Figure 3B It is a graph showing the average CT values of 145bp DNA recovered from 0.25x PBS as a sample matrix using the first ATPS with different top phase:bottom phase volume ratios according to Table 9.

[0047] Figure 3C It is a graph showing the average CT values of 145bp DNA recovered from urine samples of three individual donors using the second ATPS with different top phase:bottom phase volume ratios according to Table 10.

[0048] Figure 3D It is a graph showing the average CT values of 145bp DNA recovered from 0.25x PBS as a sample matrix using the second ATPS with different top phase:bottom phase volume ratios according to Table 10.

[0049] Figure 4AIt is a graph showing the recovery of 145bp DNA spiking (copy number / μL) using Conditions A - F according to Table 15.

[0050] Figure 4B It is a graph showing the average concentration (copy number / μL) of DNA recovered using the kits and conditions according to Table 16. Detailed Description of the Invention

[0051] Unless otherwise specified, the terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and detailed descriptions of well-known functions and configurations that may obscure the gist of the invention are omitted.

[0052] As used herein and in the claims, "comprising" and "including" mean including the following elements but not excluding other elements.

[0053] As used herein and in the claims, the term "comprising" (or any related form, such as "comprise" and "comprises"), "including" (or any related form, such as "include" or "includes"), "containing" (or any related form, such as "contain" or "contains"), or "having" (or any related form, such as "have" or "has") means including the following elements but not excluding other elements. It should be understood that for each embodiment in which the term "comprising" (or any related form, such as "comprise" and "comprises"), "including" (or any related form, such as "include" or "includes"), or "containing" (or any related form, such as "contain" or "contains") is used, this disclosure / application also includes alternative embodiments in which the term "comprising", "including", "containing", or "having" is replaced by "consisting essentially of" or "consisting of". These alternative embodiments using "consisting of" or "consisting essentially of" are understood to be embodiments of a smaller scope of the "including", "comprising", or "containing" embodiments.

[0054] For example, alternative embodiments of "a solution comprising A, B, and C" would be "a solution consisting of A, B, and C" and "a solution consisting essentially of A, B, and C". Even if the latter two embodiments are not explicitly written out, this disclosure / application includes those embodiments. In addition, it should be understood that the scopes of the three embodiments listed above are different.

[0055] For clarity, "comprising", "including", and "containing" and any related forms are open - ended terms that allow additional elements or features in addition to the specified essential elements, while "consisting of" is a closed - ended term that is limited to the elements recited in the claim and does not include any elements, steps, or components not specified in the claim.

[0056] "Consisting essentially of" limits the scope of the claim to specific materials, components, or steps ("essential elements") that do not materially affect the (multiple) essential features of the claimed invention. In some embodiments, the essential features are one or more of the basic and novel features of the claimed invention.

[0057] As used herein, the singular forms "a / an" and "the" are intended to also include the plural forms, unless the context clearly indicates otherwise. When a range is recited in the specification, the range is understood to include at least each discrete point within the recited range. For example, in some embodiments, 1 - 7 refers to 1, 2, 3, 4, 5, 6, and 7. Unless otherwise stated, a range is intended to include all values within the recited range, including integers, fractions, parts, etc. For example, the range 1 - 7 recited in a claim refers to a range that includes values and sub - ranges such as 1, 1.5, 2 - 3, 6, and 7.

[0058] As used herein, the term "about" is understood to be within the normal tolerances in the art and not more than ±10% of the stated value. By way of example only, about 50 means from 45 to 55, including all values therebetween. As used herein, the phrase "about" a particular value also includes the particular value, e.g., about 50 includes 50.

[0059] As used herein, "aqueous" refers to a characteristic property of a solvent / solute system in which the solvating substance predominantly has hydrophilic characteristics. Examples of aqueous solvent / solute systems include those in which water or an aqueous composition is the major solvent. Polymer and / or surfactant components (the use of which is described in the embodiments) are "aqueous" because they form an aqueous phase when combined with a solvent such as water. In addition, as understood by one of ordinary skill in the art, herein, the term liquid "mixture" refers only to the combination of the components as defined herein.

[0060] As used herein, an aqueous two-phase system (ATPS) refers to a liquid-liquid separation system in which the separation or concentration of an analyte can be achieved by partitioning, where two phases, portions, regions, components, etc. interact with at least one analyte to which they are exposed and optionally dissolved in different ways. When two immiscible phases at a certain concentration form components, such as salts and polymers, or two incompatible polymers (e.g., PEG and dextran) are mixed in an aqueous solution, an ATPS is formed. ATPS methods are relatively inexpensive and scalable because they use two-phase partitioning to separate analytes (e.g., nucleic acids) from contaminants.

[0061] As used herein, the term "isolated" means that the analyte is removed from its original environment and thus altered from its original environment. For example, an isolated nucleic acid provided typically has fewer non-nucleic acid components (e.g., proteins, lipids) compared to the amount of the component present in the source sample. A composition comprising an isolated analyte (e.g., a sample nucleic acid) can be substantially isolated (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% free of non-analyte components (e.g., non-nucleic acid components)).

[0062] As used herein, "concentrate" means that the mass ratio of the analyte in question to the solution in which the analyte is suspended is higher than the mass ratio of the analyte in its pre-concentrated solution. For example, it can be slightly higher, or more preferably at least two-fold, ten-fold or one-hundred-fold higher.

[0063] As used herein, the term "downstream purification system" refers to a device, method or process for purifying and selectively separating a target analyte by chemical or physical means. In some embodiments, the downstream purification system comprises a solid-phase medium, where the solid-phase medium is a solid-phase extraction column. In some embodiments, the solid-phase extraction column is a centrifugal column. In some embodiments, the solid-phase medium is a plurality of beads, silica resin, silica membrane, silica gel, alumina gel, size exclusion resin or ion exchange resin. In some embodiments, the downstream purification system is a method or process that includes a step of precipitating the target analyte from a purified composition. In some embodiments, the purified composition comprises an alcohol.

[0064] As used herein, the terms "flow-through", "flow-by" and "supernatant" all refer to a liquid or solution that passes through or is separated from a solid-phase medium, which can be removed or separated from the solid-phase medium. In some embodiments, the supernatant refers to the flow-through passing through a column.

[0065] As used herein, the terms "perturbing" or "perturbation" refer to the process of introducing a physical force and interference into a given system. In some embodiments, perturbing a solid phase extraction complex introduces a centrifugal force, a magnetic force, or a combination thereof, which causes one or more target analytes to separate from or into the solid phase medium or the supernatant. In some embodiments, examples of perturbing or perturbation include, but are not limited to, centrifugation, evacuation, magnetization, vortexing, rotation, eddy current, spinning, oscillation, stirring, shaking, and combinations thereof. In some embodiments, centrifugation or vortexing is achieved using a centrifuge or a vortexer. In some embodiments, evacuation refers to contacting the solid phase extraction complex with a vacuum manifold to produce a flow-through or a supernatant. In some embodiments, perturbing such as magnetization, rotation, vortexing, spinning, oscillation, stirring, and shaking is achieved manually or with a suitable instrument. In some embodiments, centrifugation and magnetization are performed simultaneously.

[0066] As used herein, "cell-free DNA" (cfDNA) is DNA that exists extracellularly, such as DNA present in a sample obtained from a subject (e.g., blood, plasma, serum, or urine).

[0067] As used herein, the term "polymer" refers to any polymer comprising at least one substituted or unsubstituted monomer. Examples of polymers include, but are not limited to, homopolymers, copolymers, terpolymers, random copolymers, and block copolymers. Block copolymers include, but are not limited to, block, graft, dendrimer, and star polymers. As used herein, a copolymer refers to a polymer derived from two monomer species; similarly, a terpolymer refers to a polymer derived from three monomer species. Polymers also include various morphologies, including but not limited to linear polymers, branched polymers, random polymers, crosslinked polymers, and dendrimer systems. In some embodiments, polymers also include their chemically modified equivalents, such as hydrophobically modified or silicone-modified. For example, a polyacrylamide polymer refers to any polymer comprising at least one substituted or unsubstituted acrylamide unit, such as a homopolymer, copolymer, terpolymer, random copolymer, block copolymer, or terblock copolymer of polyacrylamide; the polyacrylamide can be a linear polymer, branched polymer, random polymer, crosslinked polymer, or dendrimer of polyacrylamide; the polyacrylamide can be a hydrophobically modified polyacrylamide or a silicone-modified polyacrylamide.

[0068] In some embodiments, examples of polymers include, but are not limited to, polyethers, polyimines, polyalkylene glycols, vinyl polymers, alkoxylated surfactants, polysaccharides, polyether-modified silicones, polyacrylamides, polyacrylic acids, and their copolymers. In some embodiments, the polymer is hydrophobically modified or silicone-modified.

[0069] Examples of polyalkylene glycols (also referred to as "PAG" or "poly(alkylene oxide)" or "poly(ethylene oxide)") include, but are not limited to, hydrophobically modified polyalkylene glycols, poly(alkylene oxide) polymers, poly(alkylene oxide) copolymers, hydrophobically modified poly(alkylene oxide) copolymers, dipropylene glycol, tripropylene glycol, polyethylene glycol (also referred to as "PEG"), and polypropylene glycol (also referred to as "PPG"). In some embodiments, examples of copolymers of PAG include, but are not limited to, poly(ethylene glycol-propylene glycol) (also referred to as "PEG-PPG" or "UCON") and poly(ethylene glycol-random-propylene glycol) (also referred to as "PEG-random-PPG"). In some embodiments, PEG-PPG comprises a random copolymer, a block copolymer, or a combination thereof. In some embodiments, PEG-PPG comprises a random copolymer and a block copolymer. In some embodiments, PEG-PPG is PEG-random-PPG.

[0070] As used herein, "vinyl polymer" refers to a group of polymers derived from substituted vinyl (H 2 C=CHR) monomers. Examples of vinyl polymers include, but are not limited to, polyvinylpyrrolidone, polyvinyl alcohol, polyvinylcaprolactam, and polyvinyl methyl ether.

[0071] Examples of polysaccharides include, but are not limited to, dextran, carboxymethyl dextran, dextran sulfate, hydroxypropyl dextran, starch, carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl hydroxyethyl cellulose, and maltodextrin. In some embodiments, the polysaccharide is alkoxylated starch, alkoxylated cellulose, or alkyl hydroxyalkyl cellulose.

[0072] Examples of polyacrylamides include, but are not limited to, poly N-isopropylacrylamide.

[0073] Examples of polyimines include, but are not limited to, polyethyleneimine.

[0074] Examples of alkoxylated surfactants include, but are not limited to, carboxylates, sulfonates, petroleum sulfonates, alkylbenzene sulfonates, naphthalene sulfonates, olefin sulfonates, alkyl sulfates, sulfates, sulfated natural oils, sulfated natural fats, sulfated esters, sulfated alkanolamides, sulfated alkylphenols, ethoxylated alkylphenols, sodium N-lauroyl sarcosinate (NLS), ethoxylated fatty alcohols, polyoxyethylene surfactants, carboxylic esters, polyethylene glycol esters, sorbitan esters, fatty acid diol esters, carboxamides, single alkanolamine condensates, and polyoxyethylene fatty acid amides.

[0075] In some embodiments, the polymer has an average molecular weight of about 200 - 1,000 Da, 200 - 35,000 Da, 300 - 35,000 Da, 400 - 2,000 Da, or 400 - 35,000 Da. Examples thereof include, but are not limited to, polyalkylene glycols (PAGs) having an average molecular weight of about 400 Da, 500 Da, 600 Da, 700 Da, 800 Da, 900 Da, 1,000 Da, 2,000 Da, 3,000 Da, 4,000 Da, 5,000 Da, 6,000 Da, 7,000 Da, 8,000 Da, 9,000 Da, 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, and 35,000 Da. In some embodiments, the PAG has an average molecular weight within the range between any two of the above molecular weights.

[0076] Examples of PAGs include, but are not limited to, PEG 200, PEG 300, PEG 400, PEG 500, PEG 600, PEG 700, PEG 800, PEG 900, PEG 1000, PEG 2000, PEG 3000, PEG 4000, PEG 5000, PEG 6000, PEG 7000, PEG 8000, PEG 9000, PEG 10000, PEG 15000, PEG 20000, PEG 25000, PEG 30000, PEG 35000, PPG 425, PPG 725, PPG 900, PPG 1000, and PPG 2000. In some embodiments, the PEG has an average molecular weight within the range between any two of the above PEG molecular weights. In some embodiments, the PPG has an average molecular weight within the range between any two of the above PPG molecular weights.

[0077] In some embodiments, the polymer comprises ethylene oxide (EO) and propylene oxide (PO) units, and the ethylene oxide:propylene oxide (EO:PO) ratio is 90:10 to 10:90. In some embodiments, the polymer has an EO:PO ratio of 10:90, 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, or 90:10. In some embodiments, the polymer has an EO:PO ratio within the range between any two of the above ratios.

[0078] In some embodiments, the polymer is a PAG having an average molecular weight of about 980 - 12,000 Da and an EO:PO ratio of 50:50 to 75:25. Examples thereof include, but are not limited to, PEG-PPGs having an average molecular weight of about 980 Da, 1,230 Da, 1,590 Da, 2,470 Da, 2,660 Da, 3,380 Da, 3,930 Da, 6,950 Da, and 12,000 Da. In some embodiments, the PEG-PPG has an average molecular weight within the range between any two of the above PEG-PPG molecular weights. In some embodiments, the PEG-PPG contains an EO:PO ratio of 50:50 or 75:25. In some embodiments, the polymer is a PEG-random-PPG having an average molecular weight of about 2,500 or 12,000 Da and an EO:PO ratio of about 75:25.

[0079] In some embodiments, the polymer is a vinyl polymer having an average molecular weight of about 2,500 - 2,500,000 Da. Examples thereof include, but are not limited to, polyvinylpyrrolidones having an average molecular weight of about 2,500 Da, 10,000 Da, 40,000 Da, 100,000 Da, and 2,500,000 Da. In some embodiments, the vinyl polymer has an average molecular weight within the range between any two of the above molecular weights.

[0080] In some embodiments, the polymer is a polysaccharide and has an average molecular weight of about 6,000 - 5,000,000 Da. Examples thereof include, but are not limited to, dextrans having an average molecular weight of about 6,000 Da, 12,000 Da, 25,000 Da, 60,000 Da, 70,000 Da, 80,000 Da, 150,000 Da, 270,000 Da, 410,000 Da, 450,000 Da, 550,000 Da, 650,000 Da, 670,000 Da, 1,500,000 Da, 2,000,000 Da, 2,800,000 Da, 4,000,000 Da, and 5,000,000 Da. In some embodiments, the dextran has an average molecular weight within the range between any two of the above molecular weights.

[0081] In some embodiments, the polymer is a polyether and has an average molecular weight of about 200 - 35,000 Da. Examples thereof include, but are not limited to, silicone-modified polyethers (or “polyether-modified silicones”) having an average molecular weight of about 200 - 35,000 Da.

[0082] In some embodiments, the polymer is polyacrylamide and has an average molecular weight of 1,000 - 5,000,000 Da. Examples thereof include, but are not limited to, polyacrylamide or poly(N-isopropylacrylamide) having an average molecular weight of 1,000 Da, 2,000 Da, 5,000 Da, 10,000 Da, 40,000 Da, 85,000 Da, 5,000,000 Da. In some embodiments, the polyolefin has an average molecular weight within the range between any of the above two molecular weights.

[0083] In some embodiments, the polymer is polyacrylic acid and has an average molecular weight of about 1,250 - 4,000,000 Da. Examples thereof include, but are not limited to, polyacrylic acid having an average molecular weight of 1,200 Da, 2,100 Da, 5,100 Da, 8,000 Da, 8,600 Da, 8,700 Da, 16,000 Da, and 83,000 Da. In some embodiments, the polyolefin has an average molecular weight within the range between any of the above two molecular weights.

[0084] As used herein, the term "salt" refers to a substance having at least one cation and at least one anion. Examples of salts include, but are not limited to, salts wherein the cation is sodium, potassium, calcium, ammonium, lithium, magnesium, aluminum, cesium, barium, straight-chain or branched trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium, or tetrabutylammonium, and / or wherein the anion is phosphate, hydrogen phosphate, dihydrogen phosphate, sulfate, sulfide, sulfite, bisulfate, carbonate, bicarbonate, acetate, nitrate, nitrite, sulfite, chloride, fluoride, chlorate, perchlorate, chlorite, hypochlorite, bromide, bromate, hypobromite, iodide, iodate, cyanate, thiocyanate, isothiocyanate, oxalate, formate, chromate, dichromate, permanganate, hydroxide, hydrogen ion, citrate, borate, or tris(hydroxymethyl)aminomethane. In some embodiments, the salt is a lyophilic salt, a discrete salt, or an inorganic salt.

[0085] As used herein, examples of "surfactant" include, but are not limited to, anionic surfactants, nonionic surfactants, cationic surfactants, zwitterionic surfactants, or amphoteric surfactants.

[0086] Examples of anionic surfactants include, but are not limited to, carboxylates, sulfonates, petroleum sulfonates, alkylbenzene sulfonates, naphthalene sulfonates, olefin sulfonates, alkyl sulfates, sulfates, sulfated natural oils, sulfated natural fats, sulfated esters, sulfated alkanolamides, sulfated alkylphenols, ethoxylated alkylphenols, and sodium N-lauroylsarcosinate (NLS).

[0087] Examples of nonionic surfactants include, but are not limited to, ethoxylated fatty alcohols, polyoxyethylene surfactants, carboxylic acid esters, polyethylene glycol esters, sorbitan esters, fatty acid diol esters, carboxamides, single-chain alkanolamine condensates, and polyoxyethylene fatty acid amides.

[0088] Examples of cationic surfactants include, but are not limited to, quaternary ammonium salts, amines with amide bonds, polyoxyethylene alkylamines, polyoxyethylene alicyclic amines, N,N,N',N'-tetrasubstituted ethylenediamines, and 2-alkyl-1-hydroxyethyl-2-imidazolines.

[0089] Examples of amphoteric surfactants include, but are not limited to, N-cocoyl-3-aminopropionic acid or its sodium salt, N-tallow-3-iminodipropionate or its disodium salt, N-carboxymethyl-N,N-dimethyl-N-9-octadecenyl ammonium hydroxide, N-cocoylamidoethyl-N-hydroxyethyl glycine or its sodium salt, and sodium N-lauroylsarcosinate (NLS).

[0090] In some embodiments, the surfactant comprises a polymer such as PAG. In some embodiments, the surfactant has an EO x -PO y -EO x structure, where EO refers to an ethylene oxide unit, PO refers to a propylene oxide unit, and x and y are the number of monomers, respectively. In some embodiments, x = 2 - 136. In some embodiments, y = 16 - 62. In some embodiments, examples of the surfactant include, but are not limited to, (C 2 H 4 O) n C 14 H 22 O, where n = 4 - 10 (e.g., Triton X-100, Triton X-114, Triton X-45, Tween 20, Igepal CA630), Brij58, Brij O10, Brij L23, EO x -PO y -EO x , where x = 2 - 136 and y = 16 - 62 (e.g., Pluronic L-61, Pluronic F-127), UCON, sodium dodecyl sulfate, sodium cholate, sodium deoxycholate, sodium N-lauroylsarcosinate (NLS), cetyltrimethylammonium bromide, or span 80.

[0091] In some embodiments, the target analyte is nucleic acid, protein, antigen, biomolecule, sugar moiety, lipid, sterol, exosome, or any combination thereof. In some embodiments, examples of the target analyte include, but are not limited to, genomic DNA (gDNA), cDNA, plasmid DNA, mitochondrial DNA, cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), circulating fetal DNA, microbial cell-free DNA, microRNA (miRNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), circular RNA, long non-coding RNA (lncRNA), or a combination thereof.

[0092] As used herein, "biological sample" refers to any tangible material obtained directly or indirectly from an organism (e.g., virus, bacterium, plant, animal, or human). Examples of biological samples include, but are not limited to, nucleic acids, proteins, cells, organelles, tissue extracts, tissues, organs, biological fluids (e.g., blood, plasma, urine, saliva, feces, cerebrospinal fluid (CSF), lymph fluid, serum, sputum, peritoneal fluid, sweat, tears, nasal swabs, vaginal swabs, endocervical swabs, semen, breast milk, and other body fluids).

[0093] As used herein, "clinical sample" refers to any sample obtained directly or indirectly from a subject (e.g., a human). In some embodiments, the subject is a human patient. Examples of clinical samples include, but are not limited to, blood, plasma, urine, saliva, feces, cerebrospinal fluid (CSF), lymph fluid, serum, sputum, peritoneal fluid, sweat, tears, nasal swabs, vaginal swabs, endocervical swabs, semen, breast milk, and other body fluids.

[0094] When referring to a liquid sample in this disclosure, the terms "large volume", "substantial amount", "high volume" or "substantial fluid", "substantial fluid sample" refer to a biological sample having a volume of at least 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 200 mL, 300 mL, 400 mL, or 500 mL. In some embodiments, the volume of the sample is 1 - 5 mL, 1 - 10 mL, 15 - 20 mL, 10 - 20 mL, 20 - 30 mL, or 30 - 40 mL. In some embodiments, the volume of the sample is at least 40 mL. In some embodiments, the sample has the following volume ranges: 10 mL–40 mL, 10 mL–50 mL, 10 mL–100 mL; 40 mL–50 mL, 40 mL–60 mL, 40 mL–100 mL, 40 mL–160 mL, 40 mL–200 mL, 50 mL–100 mL, 50 mL–200 mL, or 50 mL–300 mL. In some embodiments, the sample has the following volumes: at least 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, or 100 mL; and at most 100 mL, 200 mL, 300 mL, 400 mL, or 500 mL.

[0095] As used herein, the terms "Ct", "CT", "Ct value", or "CT value" refer to the cycle threshold and represent the cycles of a PCR amplification assay where the signal (e.g., fluorescence) indicating the generation of an amplicon from a reporter first becomes detectable above the background level. In some embodiments, the CT value is an indirect indication of the amount of target nucleic acid detected in a particular sample. Generally, a lower CT value indicates a higher amount of target nucleic acid in the sample, while a higher CT value indicates a lower amount of target nucleic acid in the sample.

[0096] As used herein, the term "disrupting agent" refers to a substance that disrupts the hydrogen bond network between water molecules in a solution. In some embodiments, the disrupting agent is thiocyanate, isothiocyanate, perchlorate, acetate, trichloroacetate, trifluoroacetate, chloride, or iodide. Examples of disrupting agents include, but are not limited to, guanidine hydrochloride (GHCl), guanidine thiocyanate, guanidine isothiocyanate (GITC), sodium thiocyanate, sodium iodide, sodium perchlorate, sodium trichloroacetate, sodium trifluoroacetate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2 - propanol, thiourea, urea, etc.

[0097] Embodiments of the present invention

[0098] Example 1

[0099] On the one hand, a method for concentrating and purifying one or more target analytes from a large volume of fluid sample is provided, the method comprising the following steps:

[0100] (a) Prepare a first aqueous two-phase system (ATPS) composition, wherein the first ATPS composition comprises a polymer, a salt, a surfactant, or a combination thereof dissolved in an aqueous solution to form a first-phase solution and a second-phase solution;

[0101] (b) Add a sample solution prepared from the large volume of fluid sample containing one or more target analytes to the first ATPS composition such that the one or more target analytes partition into the first-phase solution;

[0102] (c) Collect the first-phase solution and mix the first-phase solution with a second ATPS composition, wherein the second ATPS composition comprises a polymer, a salt, a surfactant, or a combination thereof dissolved in an aqueous solution to form a third-phase solution and a fourth-phase solution, such that the one or more target analytes partition into the third-phase solution and are concentrated in the third-phase solution;

[0103] (d) Collect the third-phase solution and mix the third-phase solution with a binding buffer to form a mixed solution, wherein the binding buffer comprises at least one dissociating agent;

[0104] (e) Load the mixed solution onto an extraction column configured to selectively extract and purify the one or more target analytes;

[0105] (f) Elute and collect the one or more target analytes from the extraction column to obtain a final solution containing the concentrated and purified one or more target analytes.

[0106] In some embodiments, the sample solution is prepared by dividing the large volume of fluid sample containing the one or more target analytes into at least two aliquots of the sample solution, and the first ATPS composition is divided into at least two aliquots, wherein step (b) further comprises the following steps:

[0107] (i) Add each aliquot of the sample solution prepared from the large volume of fluid sample containing the one or more target analytes to each aliquot of the first ATPS composition such that the one or more target analytes partition into the first-phase solution;

[0108] (ii) Collect and combine the first-phase solutions of the at least two aliquots of the first ATPS composition to form the first-phase solution for step (c).

[0109] In some embodiments, the extraction column is a centrifugal column, and wherein step (e) further comprises the steps of:

[0110] (i) Loading a portion of the mixed solution onto the extraction column;

[0111] (ii) Centrifuging the extraction column and discarding the flow-through (also referred to as the "supernatant"); and

[0112] (iii) Repeating steps (i) and (ii) above until the entire mixed solution has passed through the extraction column.

[0113] In some embodiments, the method further comprises the step of:

[0114] (g) Performing a diagnostic assay on the final solution to detect and quantify the one or more target analytes.

[0115] In another aspect, there is provided a method for concentrating and purifying one or more target analytes from a large volume of fluid sample, the method comprising the steps of:

[0116] (a) Dividing the large volume of fluid sample containing the one or more target analytes into at least two aliquots of sample solution;

[0117] (b) Preparing at least two aliquots of a first aqueous two-phase system (ATPS) composition, wherein the first ATPS composition comprises a polymer, a salt, a surfactant, or a combination thereof dissolved in an aqueous solution to form a first-phase solution and a second-phase solution;

[0118] (c) Adding each aliquot of the sample solution containing the one or more target analytes to each aliquot of the first ATPS composition such that the one or more target analytes partition into the first-phase solution;

[0119] (d) Collecting the first-phase solutions of the at least two aliquots of the first ATPS composition and mixing the first-phase solutions with a second ATPS composition, wherein the second ATPS composition comprises a polymer, a salt, a surfactant, or a combination thereof dissolved in an aqueous solution to form a third-phase solution and a fourth-phase solution, such that the one or more target analytes partition into the third-phase solution and are concentrated in the third-phase solution;

[0120] (e) Collecting the third-phase solution and mixing the third-phase solution with a binding buffer to form a mixed solution, wherein the binding buffer comprises at least one dissociating agent;

[0121] (f) Loading the mixed solution onto an extraction column configured to selectively extract and purify the one or more target analytes;

[0122] (g) Elute and collect the one or more target analytes from the extraction column to obtain a final solution containing the concentrated and purified one or more target analytes.

[0123] In another aspect, a method for concentrating and purifying one or more target analytes from a large volume of fluid sample is provided, the method comprising the steps of:

[0124] (a) Divide the large volume of fluid sample containing the one or more target analytes into at least two aliquots of sample solution;

[0125] (b) Prepare at least two aliquots of a first aqueous two-phase system (ATPS) composition, wherein the first ATPS composition comprises a polymer, a salt, a surfactant, or a combination thereof dissolved in an aqueous solution to form a first-phase solution and a second-phase solution;

[0126] (c) Add each aliquot of the sample solution containing the one or more target analytes to each aliquot of the first ATPS composition such that the one or more target analytes partition into the first-phase solution;

[0127] (d) Collect the first-phase solutions of the at least two aliquots of the first ATPS composition and mix the first-phase solutions with a second ATPS composition, wherein the second ATPS composition comprises a polymer, a salt, a surfactant, or a combination thereof dissolved in an aqueous solution to form a third-phase solution and a fourth-phase solution, such that the one or more target analytes partition into the third-phase solution and are concentrated in the third-phase solution;

[0128] (e) Collect the third-phase solution and mix the third-phase solution with a binding buffer to form a mixed solution, wherein the binding buffer comprises at least one dissociating agent;

[0129] (f) Load a portion of the mixed solution onto an extraction column configured to selectively extract and purify the one or more target analytes;

[0130] (g) Centrifuge the extraction column and discard the flow-through;

[0131] (h) Repeat steps (f) and (g) above until all of the mixed solution has passed through the extraction column;

[0132] (i) Elute and collect the one or more target analytes from the extraction column; obtain a final solution containing the concentrated and purified one or more target analytes; and

[0133] (j) Perform a diagnostic assay on the final solution to detect and quantify the one or more target analytes.

[0134] In some embodiments, the bulk fluid sample is selected from the group consisting of blood, plasma, serum, cerebrospinal fluid, urine, saliva, feces, tears, sputum, nasopharyngeal mucus, vaginal secretions, and penile secretions. In some embodiments, the bulk fluid sample is a sample matrix dissolved in a suitable preparation buffer, e.g., fecal matter dissolved in an appropriate volume (e.g., 500 mL) of water.

[0135] In some embodiments, the bulk fluid sample is urine.

[0136] In some embodiments, the volume of the bulk fluid sample is 40 mL or greater, such as 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 200 mL, 300 mL, 400 mL, 500 mL or greater.

[0137] In some embodiments, the volume of each aliquot of the sample solution is at most 25 ml, 26 mL, 27 mL, 28 mL, 29 mL, 30 mL, 31 mL, 32 mL, 33 mL, 34 mL, 35 mL, 36 mL, 37 mL, 38 mL, 39 mL or 40 mL.

[0138] In some embodiments, the target analytes are selected from the group consisting of nucleic acids, proteins, antigens, biomolecules, sugar moieties, lipids, sterols, and combinations thereof.

[0139] In some embodiments, the target analytes are DNA.

[0140] In some embodiments, the target analytes are cell-free DNA or circulating tumor DNA.

[0141] In some embodiments, the polymer is dissolved in an aqueous solution at a concentration of 4% - 84% (w / w).

[0142] In some embodiments, the salt is dissolved in an aqueous solution at a concentration of 1% - 55% (w / w). In some embodiments, the salt is dissolved in an aqueous solution at a concentration of 8% - 55% (w / w).

[0143] In some embodiments, the surfactant is dissolved in an aqueous solution at a concentration of 0.05% - 10% (w / w). In some embodiments, the surfactant is dissolved in an aqueous solution at a concentration of 0.05% - 9.8% (w / w).

[0144] On the other hand, there is provided an ATPS composition selected from the group consisting of A1, A2, A3, A4, AA1, AA2, AA3, and AA4 in Table 1a.

[0145] On the other hand, a kit is provided, which comprises a first ATPS composition selected from the group consisting of A1, A2, A3, and A4 in Table 1a; a second ATPS composition selected from the group consisting of AA1, AA2, AA3, and AA4 in Table 1a; and a binding buffer selected from the group consisting of B1, B2, and B3 in Table 1a.

[0146] In some embodiments, the kit further comprises an extraction column.

[0147] Various ATPS systems useful in various embodiments of the present invention include, but are not limited to, polymer-polymer, polymer-salt, polymer-surfactant, salt-surfactant, surfactant, surfactant-surfactant, or polymer-salt-surfactant.

[0148] In one embodiment, the first and / or second ATPS composition comprises a polymer. In some embodiments, polymers that can be used include, but are not limited to, polyalkylene glycols, such as hydrophobically modified polyalkylene glycols, poly(oxyalkylene) polymers, poly(oxyalkylene) copolymers, such as hydrophobically modified poly(oxyalkylene) copolymers, polyvinylpyrrolidone, polyvinyl alcohol, polyvinylcaprolactam, polyvinyl methyl ether, alkoxylated surfactants, alkoxylated starches, alkoxylated celluloses, alkyl hydroxyalkyl celluloses, silicone-modified polyethers, and poly(N-isopropylacrylamide) and its copolymers. In another embodiment, the first-phase-forming polymer comprises polyethylene glycol (PEG), polypropylene glycol (PPG), or dextran. In some embodiments, the polymer is selected from the group consisting of polyethers, polyimines, polyalkylene glycols, vinyl polymers, alkoxylated surfactants, polysaccharides, alkoxylated starches, alkoxylated celluloses, alkyl hydroxyalkyl celluloses, polyether-modified silicones, polyacrylamides, polyacrylic acids, and their copolymers. In some embodiments, the polymer is selected from the group consisting of dipropylene glycol, tripropylene glycol, polyethylene glycol, polypropylene glycol, poly(ethylene glycol-propylene glycol), poly(ethylene glycol-random-propylene glycol), polyvinylpyrrolidone, polyvinyl alcohol, polyvinylcaprolactam, polyvinyl methyl ether, dextran, carboxymethyl dextran, dextran sulfate, hydroxypropyl dextran, starch, carboxymethyl cellulose, polyacrylic acid, hydroxypropyl cellulose, methyl cellulose, ethyl hydroxyethyl cellulose, maltodextrin, polyethyleneimine, poly(N-isopropylacrylamide), and its copolymers. In some embodiments, the polymer is selected from the group consisting of dipropylene glycol, tripropylene glycol, polyethylene glycol, polypropylene glycol, poly(ethylene glycol-propylene glycol), poly(ethylene glycol-random-propylene glycol), polyvinylpyrrolidone, polyvinyl alcohol, polyvinylcaprolactam, polyvinyl methyl ether, and poly(N-isopropylacrylamide). In some embodiments, the polymer is selected from the group consisting of polyacrylamides, polyacrylic acids, and their copolymers. In some embodiments, the polymer is selected from the group consisting of dextran, carboxymethyl dextran, dextran sulfate, hydroxypropyl dextran, and starch. In some embodiments, the polymer has an average molecular weight in the range of 200-1,000 Da, 200-35,000 Da, 425-2,000 Da, 400-35,000 Da, 980-12,000 Da, or 3,400-5,000,000 Da. In some embodiments, the polymer comprises ethylene oxide and propylene oxide units, and the polymer has an EO:PO ratio of 90:10 to 10:90.

[0149] In one embodiment, the polymer concentration of the first and / or second ATPS composition ranges from about 4% to about 84% (w / w) by weight of the total weight of the aqueous solution. In various embodiments, the polymer solution is selected from about 4% w / w, about 4.5% w / w, about 5% w / w, about 5.5% w / w, about 6% w / w, about 6.5% w / w, about 7% w / w, about 7.5% w / w, about 8% w / w, about 8.5% w / w, about 9% w / w, about 9.5% w / w, about 10% w / w, about 10.5% w / w, about 11% w / w, about 11.5% w / w, about 12% w / w, about 12.5% w / w, about 13% w / w, about 13.5% w / w, about 14% w / w, about 14.5% w / w, about 15% w / w, about 15.5% w / w, about 16% w / w, about 16.5% w / w, about 17% w / w, about 17.5% w / w, about 18% w / w, about 18.5% w / w, about 19% w / w, about 19.5% w / w, about 20% w / w, about 20.5% w / w, about 21% w / w, about 21.5% w / w, about 22% w / w, about 22.5% w / w, about 23% w / w, about 23.5% w / w, about 24% w / w, about 24.5% w / w, about 35% w / w, about 35.5% w / w, about 36% w / w, about 36.5% w / w, about 37% w / w, about 37.5% w / w, about 38% w / w, about 38.5% w / w, about 39% w / w, about 39.5% w / w, about 40% w / w, about 40.5% w / w, about 41% w / w, about 41.5% w / w, about 42% w / w, about 42.5% w / w, about 43% w / w, about 43.5% w / w, about 44% w / w, about 44.5% w / w, about 45% w / w, about 45.5% w / w, about 46% w / w, about 46.5% w / w, about 47% w / w, about 47.5% w / w, about 48% w / w, about 48.5% w / w, about 49% w / w, about 49.5% w / w, about 50% w / w, about 50.5% w / w, about 51% w / w, about 51.5% w / w, about 52% w / w, about 52.5% w / w, about 53% w / w, about 53.5% w / w, about 54% w / w, about 54.5% w / w, about 55% w / w, about 55.5% w / w, about 56% w / w, about 56.5% w / w, about 57% w / w, about 57.5% w / w, about 58% w / w, about 58.5% w / w, about 59% w / w, about 59.5% w / w, about 60% w / w, about 60.5% w / w, about 61% w / w, about 61.5% w / w, about 62% w / w, about 62.5% w / w, about 63% w / w, about 63.5% w / w, about 64% w / w, about 64.5% w / w, about 65% w / w, about 65.Polymer solutions of 5% w / w, about 66% w / w, about 66.5% w / w, about 67% w / w, about 67.5% w / w, about 68% w / w, about 68.5% w / w, about 69% w / w, about 69.5% w / w, about 70% w / w, about 70.5% w / w, about 71% w / w, about 71.5% w / w, about 72% w / w, about 72.5% w / w, about 73% w / w, about 73.5% w / w, about 74% w / w, about 74.5% w / w, about 75% w / w, about 75.5% w / w, about 76% w / w, about 76.5% w / w, about 77% w / w, about 77.5% w / w, about 78% w / w, about 78.5% w / w, about 79% w / w, about 79.5% w / w, about 80% w / w, about 80.5% w / w, about 81% w / w, about 81.5% w / w, about 82% w / w, about 82.5% w / w, about 83% w / w, about 83.5% w / w and about 84% w / w.

[0150] In one embodiment, the first and / or second ATPS composition contains a salt and thus forms a salt solution. In some embodiments, the salt includes but is not limited to lyotropic salts, discrete salts, inorganic salts containing cations and anions, the cations being, for example, linear or branched trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium and tetrabutylammonium, and the anions being, for example, phosphate, sulfate, nitrate, chloride and bicarbonate. In another embodiment, the salt contains NaCl, Na 3 PO 4 、K 3 PO 4 、Na 2 SO 4 、potassium citrate, (NH 4 ) 2 SO 4 、sodium citrate, sodium acetate or a combination thereof. Other salts can also be used, such as ammonium acetate. In another embodiment, the salt can be selected from magnesium salts, lithium salts, sodium salts, potassium salts, cesium salts, zinc salts and aluminum salts. In some embodiments, the salt can be selected from bromide salts, iodide salts, fluoride salts, carbonates, sulfates, citrates, carboxylates, borates and phosphates. In some embodiments, the salt contains potassium phosphate. In some embodiments, the salt contains ammonium sulfate.

[0151] In one embodiment, the total salt concentration ranges from about 0.01% to about 90%. Those skilled in the art will understand that the amount of salt required to form the aqueous two-phase system will be affected by the molecular weight, concentration and physical state of the polymer.

[0152] In various embodiments, the salt concentration is from about 1% - 55% w / w. In various embodiments, the salt concentration is about 1% w / w, about 1.5% w / w, about 2% w / w, about 2.5% w / w, about 3% w / w, about 3.5% w / w, about 4% w / w, about 4.5% w / w, about 5% w / w, about 5.5% w / w, about 6% w / w, about 6.5% w / w, about 7% w / w, about 7.5% w / w, about 8% w / w, about 8.5% w / w, about 9% w / w, about 9.5% w / w, about 10% w / w, about 10.5% w / w, about 11% w / w, about 11.5% w / w, about 12% w / w, about 12.5% w / w, about 13% w / w, about 13.5% w / w, about 14% w / w, about 14.5% w / w, about 15% w / w, about 15.5% w / w, about 16% w / w, about 16.5% w / w, about 17% w / w, about 17.5% w / w, about 18% w / w, about 18.5% w / w, about 19% w / w, about 19.5% w / w, about 20% w / w, about 20.5% w / w, about 21% w / w, about 21.5% w / w, about 22% w / w, about 22.5% w / w, about 23% w / w, about 23.5% w / w, about 24% w / w, about 24.5% w / w, about 35% w / w, about 35.5% w / w, about 36% w / w, about 36.5% w / w, about 37% w / w, about 37.5% w / w, about 38% w / w, about 38.5% w / w, about 39% w / w, about 39.5% w / w, about 40% w / w, about 40.5% w / w, about 41% w / w, about 41.5% w / w, about 42% w / w, about 42.5% w / w, about 43% w / w, about 43.5% w / w, about 44% w / w, about 44.5% w / w, about 45% w / w, about 45.5% w / w, about 46% w / w, about 46.5% w / w, about 47% w / w, about 47.5% w / w, about 48% w / w, about 48.5% w / w, about 49% w / w, about 49.5% w / w, about 50% w / w, about 50.5% w / w, about 51% w / w, about 51.5% w / w, about 52% w / w, about 52.5% w / w, about 53% w / w, about 53.5% w / w, about 54% w / w, about 54.5% w / w, about 55% w / w, about 55.5% w / w, about 56% w / w, about 56.5% w / w, about 57% w / w, about 57.5% w / w, about 58% w / w, about 58.5% w / w, about 59% w / w, about 59.5% w / w, about 60% w / w, about 60.5% w / w, about 61% w / w, about 61.5% w / w, about 62% w / w, about 62.5% w / w, about 63% w / w, about 63.5% w / w, about 64% w / w, about 64.5% w / w, about 65% w / w, about 65.5% w / w, about 66% w / w, about 66.5% w / w, about 67% w / w, about 67.5% w / w, about 68% w / w, about 68.5% w / w, about 69% w / w, about 69.5% w / w, about 70% w / w, about 70.5% w / w, about 71% w / w, about 71.5% w / w, about 72% w / w, about 72.5% w / w, about 73% w / w, about 73.5% w / w, about 74% w / w, about 74.5% w / w, about 75% w / w, about 75.5% w / w, about 76% w / w, about 76.5% w / w, about 77% w / w, about 77.5% w / w, about 78% w / w, about 78.5% w / w, about 79% w / w, about 79.5% w / w or about 80% w / w.

[0153] In one embodiment, the first and / or second ATPS composition comprises a surfactant. In some embodiments, possible surfactants that can be used include, but are not limited to, Triton-X, Triton-114, Igepal CA-630 and Nonidet P-40, anionic surfactants (such as carboxylates, sulfonates, petroleum sulfonates, alkylbenzene sulfonates, naphthalene sulfonates, olefin sulfonates, alkyl sulfates, sulfates, sulfated natural oils, sulfated natural fats, sulfated esters, sulfated alkanolamides, sulfated alkylphenols, ethoxylated alkylphenols), nonionic surfactants (such as ethoxylated fatty alcohols, polyoxyethylene surfactants, carboxylic esters, polyethylene glycol esters, sorbitan esters, fatty acid ethylene glycol esters, carboxylic acid amides, monoalkanolamine concentrates, polyoxyethylene fatty acid amides), cationic surfactants (such as quaternary ammonium salts, amines with amide bonds, polyoxyethylene alkyl and cycloaliphatic amines, n,n,n',n' tetra-substituted ethylenediamine, 2-alkyl-1-hydroxyethyl-2-imidazoline), and amphoteric surfactants (such as n-cocoyl 3-aminopropionic acid and its sodium salt, n-tallowyl 3-imino dipropionate and its disodium salt, n-carboxymethyl n-dimethyl n-9-octadecenyl ammonium hydroxide, n-cocoylamidoethyl n-hydroxyethyl glycine and its sodium salt).

[0154] In one embodiment, the surfactant concentration of the first ATPS composition ranges from about 0.05% w / w to about 10% w / w. In various embodiments, the surfactant concentration is about 0.05% w / w, 0.1% w / w, about 0.2% w / w, about 0.3% w / w, about 0.4% w / w, about 0.5% w / w, about 0.6% w / w, about 0.7% w / w, about 0.8% w / w, about 0.9% w / w, about 1% w / w, 1.1% w / w, about 1.2% w / w, about 1.3% w / w, about 1.4% w / w, about 1.5% w / w, about 1.6% w / w, about 1.7% w / w, about 1.8% w / w, about 1.9% w / w, about 2% w / w, about 2.1% w / w, about 2.2% w / w, about 2.3% w / w, about 2.4% w / w, about 2.5% w / w, about 2.6% w / w, about 2.7% w / w, about 2.8% w / w, about 2.9% w / w, about 3% w / w, 3.1% w / w, about 3.2% w / w, about 3.3% w / w, about 3.4% w / w, about 3.5% w / w, about 3.6% w / w, about 3.7% w / w, about 3.8% w / w, about 3.9% w / w, about 4% w / w, about 4.1% w / w, about 4.2% w / w, about 4.3% w / w, about 4.4% w / w, about 4.5% w / w, about 4.6% w / w, about 4.7% w / w, about 4.8% w / w, about 4.9% w / w, about 5% w / w, about 5.1% w / w, about 5.2% w / w, about 5.3% w / w, about 5.4% w / w, about 5.5% w / w, about 5.6% w / w, about 5.7% w / w, about 5.8% w / w, about 5.9% w / w, about 6% w / w, 6.1% w / w, about 6.2% w / w, about 6.3% w / w, about 6.4% w / w, about 6.5% w / w, about 6.6% w / w, about 6.7% w / w, about 6.8% w / w, about 6.9% w / w, about 7% w / w, about 7.1% w / w, about 7.2% w / w, about 7.3% w / w, about 7.4% w / w, about 7.5% w / w, about 7.6% w / w, about 7.7% w / w, about 7.8% w / w, about 7.9% w / w, about 8% w / w, about 8.1% w / w, about 8.2% w / w, about 8.3% w / w, about 8.4% w / w, about 8.5% w / w, about 8.6% w / w, about 8.7% w / w, about 8.8% w / w, about 8.9% w / w, about 9% w / w, 9.1% w / w, about 9.2% w / w, about 9.3% w / w, about 9.4% w / w, about 9.5% w / w, about 9.6% w / w, about 9.7% w / w, about 9.8% w / w, about 9.9% w / w or about 10% w / w.

[0155] In one embodiment, the binding buffer contains a chaotropic agent. In some embodiments, possible chaotropic agents include, but are not limited to, n-butanol, ethanol, guanidine chloride, guanidine thiocyanate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea.

[0156] In one embodiment, the concentration of the chaotropic agent in the binding buffer ranges from about 0.1 M to 8 M. In various embodiments, the concentration of the chaotropic agent is about 0.1 M, about 0.2 M, about 0.3 M, about 0.4 M, about 0.5 M, about 0.6 M, about 0.7 M, about 0.8 M, about 0.9 M, about 1 M, about 1.1 M, about 1.2 M, about 1.3 M, about 1.4 M, about 1.5 M, about 1.6 M, about 1.7 M, about 1.8 M, about 1.9 M, about 2 M, about 2.1 M, about 2.2 M, about 2.3 M, about 2.4 M, about 2.5 M, about 2.6 M, about 2.7 M, about 2.8 M, about 2.9 M, about 3 M, about 3.1 M, about 3.2 M, about 3.3 M, about 3.4 M, about 3.5 M, about 3.6 M, about 3.7 M, about 3.8 M, about 3.9 M, about 4 M, about 4.1 M, about 4.2 M, about 4.3 M, about 4.4 M, about 4.5 M, about 4.6 M, about 4.7 M, about 4.8 M, about 4.9 M, about 5 M, about 5.1 M, about 5.2 M, about 5.3 M, about 5.4 M, about 5.5 M, about 5.6 M, about 5.7 M, about 5.8 M, about 5.9 M, about 6 M, about 6.1 M, about 6.2 M, about 6.3 M, about 6.4 M, about 6.5 M, about 6.6 M, about 6.7 M, about 6.8 M, about 6.9 M, about 7 M, about 7.1 M, about 7.2 M, about 7.3 M, about 7.4 M, about 7.5 M, about 7.6 M, about 7.7 M, about 7.8 M, about 7.9 M, or about 8 M.

[0157] In one embodiment, possible extraction columns that can be used include, but are not limited to, Epoch life science - EconoSpin silica membrane mini spin column - 1920 - 250, HiBinds RNAmini - RNACOL - 02, Corbition silica spin column - PC0054, PuroSpin micro silica spin column - Luna Nano USP003, Purospin nano silica spin column - Lunonano USP002, Qiagen RNEasy minElute, Qiagen minElute - 700384Qiagen GMBH, and Qiagen mini.

[0158] Example 2

[0159] In some embodiments, provided is a method for concentrating and purifying one or more target analytes from a large volume of fluid sample, the method comprising the steps of: (a) dividing the large volume of fluid sample containing the one or more target analytes into at least two aliquots of sample solution; (b) preparing at least two first aqueous two-phase system (ATPS) compositions, wherein each first ATPS composition comprises a polymer, a salt, a surfactant, or any combination thereof dissolved in an aqueous solution to form a first phase solution and a second phase solution; (c) adding each aliquot of the sample solution prepared from the large volume of fluid sample containing the one or more target analytes to each first ATPS composition such that the one or more target analytes partition into each first phase solution; (d) further processing each first phase solution to form a final phase solution; (e) mixing the final phase solution with at least one purification composition to form a mixed solution; (f) contacting the mixed solution with a downstream purification system configured to selectively separate the one or more target analytes; and (g) collecting the one or more target analytes from the downstream purification system to obtain a final solution containing the concentrated and purified one or more target analytes.

[0160] In some embodiments, the further processing of step (d) comprises collecting and combining each first phase solution to form the final phase solution.

[0161] In some embodiments, the further processing of step (d) comprises the steps of: (i) collecting each first phase solution; (ii) mixing each first phase solution with a second ATPS composition, wherein the second ATPS composition comprises a polymer, a salt, a surfactant, or a combination thereof dissolved in an aqueous solution to form a third phase solution and a fourth phase solution, such that the one or more target analytes partition into the third phase solution and are concentrated in the third phase solution; (iii) collecting and combining the third phase solutions to form the final phase solution.

[0162] In some embodiments, the further processing of step (d) comprises the steps of: (i) collecting and combining each first phase solution to form a combined first phase solution; (ii) mixing each combined first phase solution with a second ATPS composition, wherein the second ATPS composition comprises a polymer, a salt, a surfactant, or a combination thereof dissolved in an aqueous solution to form a third phase solution and a fourth phase solution, such that the one or more target analytes partition into the third phase solution and are concentrated in the third phase solution; (iii) collecting the third phase solution to form the final phase solution.

[0163] In some embodiments, the purification composition is a binding buffer comprising at least one discrete agent; the downstream purification system comprises a solid-phase medium; and step (f) further comprises the steps of: (i) contacting a portion of the mixed solution with the solid-phase medium such that the one or more target analytes bind to the solid-phase medium to form a solid-phase extraction complex; (ii) disturbing the solid-phase extraction complex and discarding the flow-through or supernatant; and (iii) optionally repeating steps (i) and (ii).

[0164] In some embodiments, the solid-phase medium is a solid-phase extraction column.

[0165] In some embodiments, the solid-phase extraction column is a centrifugal column.

[0166] In some embodiments, a method is provided wherein the solid-phase medium is a plurality of beads.

[0167] In some embodiments, the plurality of beads are magnetic beads, silica-based beads, carboxyl beads, hydroxyl beads, amine-coated beads, or any combination thereof.

[0168] In some embodiments, a method is provided that further comprises the step of: (h) performing a diagnostic assay on the final solution to detect, quantify, characterize, or any combination thereof, the one or more target analytes.

[0169] In some embodiments, provided is a method for concentrating and purifying one or more target analytes from a large volume of fluid sample, the method comprising the steps of: (a) dividing the large volume of fluid sample containing the one or more target analytes into at least two aliquots of sample solution; (b) preparing at least two aliquots of a first aqueous two-phase system (ATPS) composition, wherein the first ATPS composition comprises a polymer, a salt, a surfactant, or a combination thereof dissolved in an aqueous solution to form a first-phase solution and a second-phase solution; (c) adding each aliquot of the sample solution containing the one or more target analytes to each aliquot of the first ATPS composition such that the one or more target analytes partition into the first-phase solution; (d) collecting the first-phase solutions of the at least two aliquots of the first ATPS composition and mixing the first-phase solutions with a second ATPS composition, wherein the second ATPS composition comprises a polymer, a salt, a surfactant, or a combination thereof dissolved in an aqueous solution to form a third-phase solution and a fourth-phase solution, such that the one or more target analytes partition into the third-phase solution and are concentrated in the third-phase solution; (e) collecting the third-phase solution and mixing the third-phase solution with a binding buffer to form a mixed solution, wherein the binding buffer comprises at least one dissociating agent; (f) loading the mixed solution onto an extraction column configured to selectively extract and purify the one or more target analytes; (g) eluting and collecting the one or more target analytes from the extraction column.

[0170] In some embodiments, the large volume of fluid sample is selected from the group consisting of blood, plasma, serum, cerebrospinal fluid, urine, saliva, feces, tears, sputum, nasopharyngeal mucus, vaginal secretions, and penile secretions.

[0171] In some embodiments, the large volume of fluid sample is urine.

[0172] In some embodiments, the large volume of fluid sample has a volume of at least 10 mL.

[0173] In some embodiments, the large volume of fluid sample has a volume of 40 mL or greater.

[0174] In some embodiments, each aliquot of the sample solution has a volume of up to 40 ml.

[0175] In some embodiments, each aliquot of the sample solution has a volume of 10 to 40 mL.

[0176] In some embodiments, the one or more target analytes are selected from the group consisting of nucleic acids, proteins, antigens, biomolecules, sugar moieties, lipids, sterols, and any combination thereof.

[0177] In some embodiments, one or more target analytes are DNA.

[0178] In some embodiments, one or more target analytes are genomic DNA (gDNA), complementary DNA (cDNA), plasmid DNA, mitochondrial DNA, cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), circulating fetal DNA, microbial cell-free DNA, microRNA (miRNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), circular RNA, long non-coding RNA (lncRNA), or combinations thereof.

[0179] In some embodiments, one or more target analytes are cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA).

[0180] In some embodiments, the polymer is dissolved in an aqueous solution at a concentration of 0.5% - 80% (w / v).

[0181] In some embodiments, the polymer is selected from the group consisting of polyethers, polyimines, polyalkylene glycols, vinyl polymers, alkoxylated surfactants, polysaccharides, alkoxylated starches, alkoxylated celluloses, alkyl hydroxyalkyl celluloses, polyether-modified silicones, polyacrylamides, polyacrylic acids, and copolymers thereof. In some embodiments, the polymer is hydrophobically modified or silicone-modified.

[0182] In some embodiments, the polymer is dipropylene glycol, tripropylene glycol, polyethylene glycol, polypropylene glycol, poly(ethylene glycol - propylene glycol), poly(ethylene glycol - random - propylene glycol), polyvinylpyrrolidone, polyvinyl alcohol, polyvinylcaprolactam, polyvinyl methyl ether, dextran, carboxymethyl dextran, dextran sulfate, hydroxypropyl dextran, starch, carboxymethyl cellulose, polyacrylic acid, hydroxypropyl cellulose, methyl cellulose, ethyl hydroxyethyl cellulose, maltodextrin, polyethyleneimine, poly(N - isopropylacrylamide), or copolymers thereof.

[0183] In some embodiments, the polymer is dipropylene glycol, tripropylene glycol, polyethylene glycol, polypropylene glycol, poly(ethylene glycol - propylene glycol), poly(ethylene glycol - random - propylene glycol), polyvinylpyrrolidone, polyvinyl alcohol, polyvinylcaprolactam, polyvinyl methyl ether, or poly(N - isopropylacrylamide).

[0184] In some embodiments, the polymer is polyacrylamide, polyacrylic acid, or copolymers thereof. In some embodiments, the polymer is dextran, carboxymethyl dextran, dextran sulfate, hydroxypropyl dextran, or starch.

[0185] In some embodiments, the polymer has an average molecular weight in the range of 200 - 1,000 Da, 200 - 35,000 Da, 425 - 2,000 Da, 400 - 35,000 Da, 980 - 12,000 Da, or 3,400 - 5,000,000 Da. In some embodiments, the polymer comprises ethylene oxide and propylene oxide units. In some embodiments, the polymer has an EO:PO ratio of 90:10 to 10:90.

[0186] In some embodiments, the salt is dissolved in an aqueous solution at a concentration of 0.1% to 80% (weight / volume).

[0187] In some embodiments, the salt includes cations selected from the group consisting of sodium, potassium, calcium, ammonium, lithium, magnesium, aluminum, cesium, barium, straight-chain or branched trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium, and tetrabutylammonium.

[0188] In some embodiments, the salt includes anions selected from the group consisting of phosphate, hydrogen phosphate, dihydrogen phosphate, sulfate, sulfide, sulfite, bisulfate, carbonate, bicarbonate, acetate, nitrate, nitrite, sulfite, chloride, fluoride, chlorate, perchlorate, chlorite, hypochlorite, bromide, bromate, hypobromite, iodide, iodate, cyanate, thiocyanate, isothiocyanate, oxalate, formate, chromate, dichromate, permanganate, hydroxide, hydrogen ion, citrate, borate, and tris(hydroxymethyl)aminomethane.

[0189] In some embodiments, the salt is selected from the group consisting of aluminum chloride, aluminum phosphate, aluminum carbonate, magnesium chloride, magnesium phosphate, and magnesium carbonate.

[0190] In some embodiments, the salt is selected from the group consisting of NaCl, KCl, NH 4 Cl, Na 3 PO 4 、K 3 PO 4 、Na 2 SO 4 、K 2 HPO 4 、KH 2 PO 4 、Na 2 HPO 4 、NaH 2 PO 4 、(NH 4 ) 3 PO 4 、(NH 4 ) 2 HPO4 、 NH 4 H 2 PO 4 、 potassium citrate, (NH 4 ) 2 SO 4 、 sodium citrate, sodium acetate, magnesium acetate, sodium oxalate, sodium borate, and ammonium acetate.

[0191] In some embodiments, the salt is selected from the group consisting of: (NH 4 ) 3 PO 4 、 sodium formate, ammonium formate, K 2 CO 3 、 KHCO 3 、 Na 2 CO 3 、 NaHCO 3 、 MgSO 4 、 MgCO 3 、 CaCO 3 、 CsOH, Cs 2 CO 3 、 Ba(OH) 2 and BaCO 3 .

[0192] In some embodiments, the salt is selected from the group consisting of: NH 4 Cl, NH 4 OH, tetramethylammonium chloride, tetrabutylammonium chloride, tetramethylammonium hydroxide, and tetrabutylammonium hydroxide.

[0193] In some embodiments, the surfactant is dissolved in an aqueous solution at a concentration of 0.05% - 10% (w / w).

[0194] In some embodiments, the surfactant is selected from the group consisting of: anionic surfactants, nonionic surfactants, cationic surfactants, and amphoteric surfactants; and wherein the anionic surfactant is carboxylate, sulfonate, petroleum sulfonate, alkylbenzene sulfonate, naphthalene sulfonate, olefin sulfonate, alkyl sulfate, sulfate, sulfated natural oil, sulfated natural fat, sulfated ester, sulfated alkanolamide, sulfated alkylphenol, ethoxylated alkylphenol, or sodium N-lauroylsarcosinate (NLS); the nonionic surfactant is ethoxylated fatty alcohol, polyoxyethylene surfactant, carboxylic acid ester, polyethylene glycol ester, sorbitan ester, fatty acid diol ester, carboxamide, monoalkanolamine condensate, or polyoxyethylene fatty acid amide; the cationic surfactant is quaternary ammonium salt, amine having an amide bond, polyoxyethylene alkylamine, polyoxyethylene alicyclic amine, n’n,n',n'-tetrasubstituted ethylenediamine, or 2-alkyl-1-hydroxyethyl-2-imidazoline; and the amphoteric surfactant is n-cocoyl 3-aminopropionic acid or its sodium salt, n-tallow 3-iminodipropionate or its disodium salt, n-carboxymethyl n-dimethyl n-9-octadecenyl ammonium hydroxide, or n-cocoylamidoethyl n-hydroxyethyl glycine or its sodium salt.

[0195] In some embodiments, the surfactant is Triton X-100, Triton X-114, Triton X-45, Tween 20, Igepal CA630, Brij 58, Brij O10, Brij L23, Pluronic L-61, Pluronic F-127, UCON, sodium dodecyl sulfate, sodium cholate, sodium deoxycholate, sodium N-lauroylsarcosinate (NLS), cetyltrimethylammonium bromide, or span 80.

[0196] In some embodiments, the binding buffer is a chaotropic agent, and the chaotropic agent includes anions selected from the group consisting of: thiocyanate, isothiocyanate, perchlorate, acetate, trichloroacetate, trifluoroacetate, chloride, and iodide.

[0197] In some embodiments, the binding buffer is a chaotropic agent, and the chaotropic agent is selected from the group consisting of: guanidine hydrochloride (GHCl), guanidine thiocyanate, guanidine isothiocyanate (GITC), sodium thiocyanate, sodium iodide, sodium perchlorate, sodium trichloroacetate, sodium trifluoroacetate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, thiourea, and urea.

[0198] In some embodiments, the binding buffer is a chaotropic agent, and the chaotropic agent is selected from the group consisting of: guanidine hydrochloride, magnesium chloride, and guanidine thiocyanate.

[0199] In some embodiments, the binding buffer includes a chaotropic agent at a concentration of 2-7 M.

[0200] In some embodiments, the first ATPS composition comprises the polymer at a concentration of 5%-80% (w / v), the salt at a concentration of 0.1%-80% (w / v), and the surfactant at a concentration of 0%-10% (w / v); the volume ratio of the first phase solution to the second phase solution is A:B, where A is 1, and B is 0.9 to 13.

[0201] In some embodiments, the second ATPS composition comprises the polymer at a concentration of 0.5%-30% (w / v), the salt at a concentration of 0.1%-10% (w / v), and the surfactant at a concentration of 0%-10% (w / v); the volume ratio of the third phase solution to the fourth phase solution is C:D; and where C is 1 and D is 1-24.

[0202] In some embodiments, the first ATPS composition comprises 5%-80% polymer (w / v) and 0.1%-80% salt (w / v); and the second ATPS composition comprises 0.5%-30% polymer (w / v) and 5%-60% salt (w / v).

[0203] In some embodiments, the first ATPS composition comprises 5%-60% polymer (w / v) and 0.5%-50% salt (w / v); and the second ATPS composition comprises 0.5%-30% polymer (w / v) and 5%-60% salt (w / v).

[0204] In some embodiments, the first ATPS composition comprises 12%-50% polymer (w / v) and 0.1%-20% salt (w / v); and the second ATPS composition comprises 0.5%-30% polymer (w / v) and 5%-60% salt (w / v).

[0205] In some embodiments, the first ATPS composition further comprises 0.5-2 mM ethylenediaminetetraacetic acid (EDTA), and 0.01%-10% surfactant; and the second ATPS composition further comprises 0.5-2 mM EDTA.

[0206] In some embodiments, the volume ratio between the first phase solution and the second phase solution of the first ATPS composition is A:B. Where A is 0.1 to 19 and B is 1.

[0207] In some embodiments, A is 0.9 to 13 and B is 1.

[0208] In some embodiments, A:B is 13:1, 6:1 or 0.9:1.

[0209] In some embodiments, the volume ratio between the third-phase solution and the fourth-phase solution of the second ATPS composition is C:D, where C is 1 and D is greater than or equal to 4.

[0210] In some embodiments, D is 4 - 100.

[0211] In some embodiments, D is 24.

[0212] In some embodiments, A is 5 - 15; B is 1; C is 1; and D is 20 - 100.

[0213] In some embodiments, an ATPS composition is provided that is selected from the group consisting of A1, A2, A3, A4, AA1, AA2, AA3, and AA4.

[0214] In some embodiments, a method 1 is provided that analyzes a final solution, and if the target analyte indicates that the patient has bladder cancer or is at risk of developing bladder cancer, the patient is treated with a cancer therapeutic agent.

[0215] In some embodiments, a kit is provided that includes a first ATPS composition selected from the group consisting of A1, A2, A3, and A4; a second ATPS composition selected from the group consisting of AA1, AA2, AA3, and AA4; and a binding buffer selected from the group consisting of B1, B2, and B3.

[0216] In some embodiments, the kit further includes an extraction column.

[0217] In some embodiments, the concentration of the polymer is 0.5% - 80% (w / v) of the first ATPS and / or the second ATPS. In some embodiments, the concentration of the polymer is 0.5% - 30% (w / v) of the first ATPS and / or the second ATPS. In some embodiments, the concentration of the polymer is 5% - 60% (w / v) of the first ATPS and / or the second ATPS. In some embodiments, the concentration of the polymer is 12% - 50% (w / v) of the first ATPS and / or the second ATPS.

[0218] In some embodiments, the concentration of the salt is 0.1%-80% (w / v) of the first ATPS and / or the second ATPS. In some embodiments, the concentration of the salt is 5%-60% (w / v) of the first ATPS and / or the second ATPS. In some embodiments, the concentration of the salt is 0.1%-50% (w / v) of the first ATPS and / or the second ATPS. In some embodiments, the concentration of the salt is 0.1%-20% (w / v) of the first ATPS and / or the second ATPS. In some embodiments, the concentration of the salt is 0.01%-30% (w / v). In some embodiments, the concentration of the salt is 0.01%-10% (w / v) of the first ATPS and / or the second ATPS.

[0219] In some embodiments, the concentration of the surfactant is 0.1%-50% (w / v) of the first ATPS and / or the second ATPS. In some embodiments, the concentration of the surfactant is 0.01%-10% (w / v) of the first ATPS and / or the second ATPS.

[0220] In some embodiments, the first ATPS composition is polymer-salt based and comprises at least one polymer at a concentration of 5%-80% (w / v) and at least one salt at a concentration of 0.1%-80% (w / v). In some embodiments, the first ATPS composition comprises at least one polymer at a concentration of 5%-60% (w / v) and at least one salt at a concentration of 0.5%-50% (w / v). In some embodiments, the first ATPS composition comprises at least one polymer at a concentration of 12%-50% (w / v) and at least one salt at a concentration of 0.1%-20% (w / v). In some embodiments, the first ATPS composition further comprises at least one surfactant at a concentration of 0.01%-10% (w / v).

[0221] In some embodiments, the second ATPS composition comprises at least one polymer at a concentration of 0.5%-30% (w / v) and at least one salt at a concentration of 5%-60% (w / v). In some embodiments, the second ATPS composition comprises at least one polymer at a concentration of 1%-6% (w / v) and at least one salt at a concentration of 10%-50% (w / v). In some embodiments, the second ATPS composition further comprises at least one surfactant at a concentration of 0.01%-10% (w / v).

[0222] In some embodiments, the first ATPS composition is polymer - salt based, which comprises at least one polymer at a concentration of 0.5% - 30% (w / v) and at least one salt at a concentration of 5% - 60% (w / v). In some embodiments, the first ATPS composition comprises at least one polymer at a concentration of 1% - 6% (w / v) and at least one salt at a concentration of 10% - 50% (w / v). In some embodiments, the first ATPS composition further comprises at least one surfactant at a concentration of 0.01% - 10% (w / v).

[0223] In some embodiments, the second ATPS composition comprises at least one polymer at a concentration of 5% - 80% (w / v) and at least one salt at a concentration of 0.1% - 80% (w / v). In some embodiments, the second ATPS composition comprises at least one polymer at a concentration of 5% - 60% (w / v) and at least one salt at a concentration of 0.5% - 50% (w / v). In some embodiments, the second ATPS composition comprises at least one polymer at a concentration of 12% - 50% (w / v) and at least one salt at a concentration of 0.1% - 20% (w / v). In some embodiments, the second ATPS composition further comprises at least one surfactant at a concentration of 0.01% - 10% (w / v).

[0224] In some embodiments, the first ATPS composition is polymer - polymer based, which comprises at least one polymer at a concentration of 0.2% - 50% (w / v). In some embodiments, the first ATPS composition further comprises at least one salt at a concentration of 0.01% - 10% (w / v). In some embodiments, the first ATPS composition further comprises at least one surfactant at a concentration of 0.01% - 10% (w / v).

[0225] In some embodiments, the first ATPS composition is surfactant based, which comprises at least one surfactant at a concentration of 0.1% - 50% (w / v). In some embodiments, the first ATPS composition further comprises at least one salt at a concentration of 0.01% - 30% (w / v).

[0226] Although the description refers to specific embodiments, the present disclosure should not be construed as limited to the embodiments set forth herein.

[0227] Examples

[0228] Examples are provided herein that describe in more detail certain embodiments of the present disclosure. The examples provided herein are for illustrative purposes only and are not meant to limit the scope of the invention in any way. All references given below and elsewhere in this application are hereby incorporated by reference herein.

[0229] The following equipment is used in the methods of Examples 1 and 2 below:

[0230] 1. A centrifuge (e.g., for 15 and 50 mL conical tubes).

[0231] 2. A benchtop microcentrifuge (e.g., for 1 and 2 mL tubes).

[0232] 3. Pipettes and pipette tips (e.g., 20 μL, 200 μL, and 1000 μL capacity pipettes).

[0233] 4. Pipette aids and serological pipette tips (e.g., 5 mL, 10 mL, and 50 mL).

[0234] 5. A water bath (e.g., set at 37 °C).

[0235] Example 1: Concentration and separation of the target analyte from a 40 mL sample

[0236] The following is an example method for concentrating and separating a target analyte from a biological sample having a volume of at least 40 mL. In this example, the sample is prepared as follows:

[0237] 1. Mix 40 mL of the biological sample with at least one lysis reagent (optional) using methods known to those skilled in the art to form one or more sample lysates.

[0238] 2. Transfer a portion (22.6 mL) of the sample lysate to a first tube containing a first ATPS composition (ATPS#1) to form an ATPS#1 solution. Pour the remaining sample lysate into a second tube also containing ATPS#1 to form an ATPS#1 solution.

[0239] 3. Vortex the two tubes containing the ATPS#1 solution thoroughly until homogeneous, and then centrifuge each at 2300 RCF for 6 minutes.

[0240] 4. Transfer the bottom phase (e.g., approximately 3.5 - 5 mL volume) (e.g., using a 10 mL serological pipette) from the two ATPS#1 solutions to a tube containing a second ATPS composition (ATPS#2) to form an ATPS#2 solution. Vortex the ATPS#2 solution thoroughly until homogeneous, and then centrifuge at 2300 RCF for 6 minutes.

[0241] 5. Transfer all of the top phase (approximately 400 - 600 μL) of the ATPS#2 solution to a 5 mL microcentrifuge tube. Add approximately 2 mL of binding buffer to the microcentrifuge tube containing the ATPS#2 top phase, and briefly vortex the tube.

[0242] 6. Transfer 800 uL of the ATPS#2 top phase to a centrifugal column and centrifuge at 12,000 rcf for 30 seconds. Discard the flow-through (also known as the "supernatant"). Repeat this centrifugal column step (Step 6) until all samples have passed through the centrifugal column.

[0243] 7. Add the wash buffer (500 uL) to the centrifugal column containing the mixture and centrifuge the centrifugal column containing the mixture at 12,000 rcf for 30 seconds. Discard the flow-through.

[0244] 8. Centrifuge the centrifugal column containing the mixture at 16,000 rcf for 2 minutes to remove any excess wash buffer.

[0245] 9. Place the centrifugal column containing the mixture in a new 1.5 mL collection tube. Pipette 1X TE buffer (20 - 100 uL) into the center of the centrifugal column membrane. Incubate the centrifugal column containing the mixture for 3 minutes and centrifuge at 12,000 rcf for 1 minute to elute the sample solution containing the concentrated target analyte. Store the sample solution in a refrigerator at -20 °C or lower for optional further processing.

[0246] The above example procedure is just an example, and alternative methods and conditions can also be used.

[0247] For example, in Step 2, the 40 mL sample after being subjected to the lysis agent is roughly divided into two parts. However, a large volume of fluid sample can be divided into many different arrangements.

[0248] Specific examples of ATPS#1, ATPS#2, and binding buffer that can be used in the above protocol are shown in Table 1a below.

[0249] Example 2: Concentration and separation of the target analyte from a 160 mL sample

[0250] In this example, a larger volume sample of approximately 160 mL is prepared according to the following steps:

[0251] 1. Divide the 160 mL sample into four separate 40 mL portions. For each 40 mL volume of sample input, perform Steps (1) to (7) in Example 1 above.

[0252] 2. Transfer the top phase of each ATPS#2 (about 400 uL - 600 uL) to a 15 mL microcentrifuge tube. This extraction step can be done with a pipette, for example, a P200 pipette set to 200 uL for the first extraction.

[0253] 3. For each 40 mL starting sample, add binding buffer (2 mL) to the tube containing the top phase of ATPS#2 (i.e., for 160 mL sample amplification, 4x ATPS#2 and 8 mL binding buffer are required). Briefly vortex the tube.

[0254] 4. Transfer 800 μL of the mixture (starting sample and binding buffer) to a centrifugal column.

[0255] 5. Centrifuge the mixture at 12,000 rcf for 30 seconds.

[0256] 6. Discard the flow-through (also known as the "supernatant"). Repeat steps 4 - 6 for the remaining sample until the entire mixture has passed through the centrifugal column. (For example, for an 800 μL centrifugal column capacity, a 160 mL starting sample input volume would require approximately 12 cycles)

[0257] 7. Add wash buffer to the centrifugal column containing the mixture (500 μL).

[0258] 8. Centrifuge the centrifugal column containing the mixture at 12,000 rcf for 30 seconds.

[0259] 9. Discard the flow-through.

[0260] 10. Centrifuge the centrifugal column containing the mixture at 16,000 rcf for 2 minutes to remove any excess wash buffer.

[0261] 11. Place the centrifugal column containing the mixture in a new 1.5 mL collection tube.

[0262] 12. Pipette 1X TE buffer (20 - 100 μL) into the center of the centrifugal column membrane.

[0263] 13. Incubate the centrifugal column containing the mixture for 3 minutes and centrifuge at 12,000 rcf for 1 minute to elute the sample solution containing the concentrated target analyte.

[0264] 14. Store the sample solution in a refrigerator at -20 °C or lower for optional further processing.

[0265] Example 3: Performance evaluation of the disclosed method

[0266] The performance of the methods and kits disclosed below can be evaluated according to the following steps:

[0267] 1. Prepare several large-volume extraction kit components by varying the following components:

[0268] a. ATPS#1

[0269] i. Polymer

[0270] ii. Salt

[0271] iii. Surfactant

[0272] b. ATPS#2

[0273] i. Polymer

[0274] ii. Salt

[0275] c. Extraction column

[0276] d. Binding buffer

[0277] i. Dispersing agent

[0278] 2. Prepare a sample solution to evaluate and spike a known amount of target DNA.

[0279] 3. Use a variant of the large - volume extraction kit prepared in step 1 above and an industry - standard extraction kit to perform extraction using their designated procedures.

[0280] 4. Quantify the target DNA using standard qPCR or ddPCR procedures

[0281] Table 1a. List of exemplary compositions

[0282] Specific examples of ATPS#1, ATPS#2, and binding buffer are shown below.

[0283] According to the method described in Example 2, use different combinations of the ATPS compositions and binding buffers shown in Table 1a to process sample solutions spiked with a known amount of DNA.

[0284] Results

[0285] It was found that the methods disclosed herein can effectively separate and concentrate target DNA from a large volume of fluid samples.

[0286] The above examples are for illustrative purposes only and are not intended to exhaustively list all possible embodiments of the invention. Other example embodiments are discussed herein.

[0287] Example 4a: Urine extraction using a centrifugal column with and without a prior ATPS step

[0288] In this example, the efficiency of recovering DNA from a large volume of urine using a spin column was compared under the following conditions: (i) using an ATPS system for pre - phase separation (also referred to as the "ATPS step") according to the methods disclosed herein; and (ii) without a pre - ATPS step.

[0289] Urine lysis

[0290] Urine samples were collected from 4 different donors. Samples from each donor were aliquoted into tubes of 40 mL each and divided into 3 groups, with each group containing 1 sample from each donor.

[0291] All 3 groups of urine samples were pretreated with 200 μL of 0.1 M EDTA per 10 mL of urine sample, vortexed thoroughly and centrifuged at 3000 rcf for 10 minutes. The supernatant was transferred to a new tube while the pellet was discarded.

[0292] Unwanted proteins and cells present in the pretreated urine samples were lysed by adding 5.2 mL of a suitable lysis buffer to the 40 mL samples from each donor. 100 fg of 145 bp double-stranded DNA (dsDNA) and 100 ng of 1 kb+ DNA ladder were spiked into the above samples. The samples were then vortexed thoroughly until homogeneous and then incubated in a preheated water bath at 37 °C for 15 minutes.

[0293] Two-phase system

[0294] Two different aqueous two-phase systems (ATPS) (also referred to as “dual ATPS system” or “sequential ATPS” in some embodiments) were prepared to extract cell-free DNA (cfDNA) from urine samples. The first ATPS (polymer, salt and / or surfactant) was used for the preliminary extraction of urine samples, where the expected cfDNA was significantly partitioned into the bottom salt-rich phase. The bottom phase of the first ATPS was then extracted and added to the second ATPS (polymer, salt and / or surfactant), which was used to concentrate the target cfDNA into a small volume (400 μL–600 μL) for user-friendly downstream processing.

[0295] In the case of 22600 μL of lysed urine samples, the first ATPS consisted of 31%–35% (w / v) polymer, 6%–9% (w / v) salt, 1.0–1.5 mM EDTA, 0.05%–0.35% (v / v) surfactant.

[0296] In the case where the bottom phase of the first ATPS was 3.5 mL–5 mL, the second ATPS consisted of 3%–11% (w / v) polymer, 18%–28% (w / v) salt, 1.0–1.5 mM EDTA.

[0297] For urine sample group 1, the pretreated urine sample of each donor was divided into two halves (22.6 mL), and then added to 2 first ATPS tubes for parallel phase separation (also known as "parallel ATPS"). The first ATPS was vortexed thoroughly and then centrifuged at 2300 rcf for 6 minutes. Then the salt-rich bottom phases from the two first ATPS (the same donor) were extracted, recombined and added to a second ATPS tube, which was vortexed thoroughly and centrifuged to allow phase separation. The polymer-rich top phase of the second ATPS system was extracted and placed into a new tube.

[0298] Urine sample group 2 was pretreated and processed, but not concentrated and purified through a dual ATPS system.

[0299] Purification of DNA

[0300] The target cfDNA in the urine sample was partitioned into the polymer-rich top phase in the second ATPS and concentrated to 400 μL - 600 μL. The top phase was separated for further processing.

[0301] A 3 - 7 M guanidine salt solution was used as the binding buffer. 2 mL of the binding buffer was added to the polymer-rich top phase (about 400 μL - 600 μL) of the second ATPS extracted from urine group 1 and vortexed thoroughly. The urine sample group 2 (40 mL) that was not purified and concentrated through ATPS was mixed with 2 mL of the binding buffer and vortexed thoroughly. Then each urine sample was added to an EconoSpin DNA column connected to a QIAvac24Plus vacuum manifold with appropriate extension tubes (3 mL and 20 mL). A pressure of 900 mbar was applied to the vacuum manifold, and the sample lysate was allowed to flow through the centrifugal column. The target cfDNA would bind to the centrifugal column and be retained, while the flow-through sample lysate was discarded through the vacuum manifold. After all possible sample lysates had flowed through the centrifugal column, the extension tubes were removed and discarded. The centrifugal column was removed from the manifold and inserted into a 2 mL waste tube. 500 μL of RPE wash buffer (80% v / v EtOH, 0.1 M sodium chloride, 0.01 M Tris-HCl) was added to the centrifugal column and centrifuged at 12000 rcf for 30 seconds. The flow-through was discarded, and the centrifugal column was centrifuged at 16000 rcf for a further 2 minutes to remove any excess RPE wash buffer. Then the centrifugal column was placed into a new 1.5 mL centrifuge tube, where 80 μL of elution buffer (0.01 M Tris-HCl, 1 mM EDTA) was directly transferred onto the silica membrane and incubated at room temperature for 3 minutes. The centrifugal column was centrifuged at 12000 rcf for 1 minute to elute the target cfDNA into the 1.5 mL centrifuge tube.

[0302] Detection of DNA

[0303] The recovery of spiked DNA (145bp and 2000bp DNA) in the extracted samples was quantitatively analyzed by qPCR using Quant Studio 5. The qPCR master mix for each reaction was prepared as follows: 5 uL TaqMan Fast Advanced Master Mix (Applied Biosystems, reference: 4444557), 0.5 uL 20x custom premixed custom oligonucleotide PSI-145FAM Dental, 0.4 uL universal Spike II primer (TATAA, DS25SII), 0.2 uL universal Spike II probe (TATAA, DSSII), 1.9 uL ultrapure water. The results were expressed as the average CT value. The lower the average CT value, the higher the amount of target DNA in the extracted sample, and the higher the CT value, the lower the amount of target DNA in the extracted sample.

[0304] Results

[0305] Column flow rate

[0306] Although the vacuum suction levels were the same, different groups of urine samples flowed through the silica membrane column at different rates. The amounts and times of the sample lysates of different groups of urine samples with different purification steps passing through the column are summarized in Table 2a below.

[0307] Table 2a. Summary of the results of different purification steps

[0308] Recovery of DNA

[0309] Now referring to Figure 1A - 1B and Table 2b, it shows the 145bp DNA ( Figure 1A ) and 2000bp DNA ( Figure 1B) average CT value. For 145bp DNA, Urine Sample Group 1 (Sample #1) had high recovery (average CT value of 25.36), but poor recovery was observed from Urine Sample #2 (average CT value of 38.15) (as shown in Table 2b). Similar results were observed for 200bp DNA recovery. Referring to Table 2a, in Urine Sample #1, all lysate samples flowed through the spin column within 1 minute. In Urine Sample #2, only 25% of the sample lysates passed through the spin column within the first 60 minutes. In the absence of parallel ATPS, the flow time needed to be increased (1 hour) to allow the untreated lysate to completely pass through the column. The results showed that concentrating urine lysates by parallel ATPS prior to spin column extraction improved the efficiency of downstream processing and significantly improved the recovery of 145bp and 2000bp DNA.

[0310] Table 2b: qPCR results of 145bp and 2000bp DNA oligonucleotide recovery in urine using spin columns with or without two-step ATPS.

[0311] In addition, compared to samples without ATPS treatment, adding a parallel ATPS step in large sample volume workflows such as urine extraction has the following advantages:

[0312] Significant reduction in reagent consumption

[0313] As shown in the examples herein, adding a parallel ATPS step significantly reduced the amount of binding buffer required for untreated lysates, for example, from 40 mL to only 2 mL. This is due to the smaller top polymer-rich phase generated by the second ATPS. When the sample input volume increases, the reduction in reagent consumption becomes more pronounced because a larger input volume would exponentially require more binding buffer, while the ATPS treatment can be modified to keep the top phase volume constant.

[0314] Significant reduction in column throughput time

[0315] As shown in the examples herein, adding a parallel ATPS step also significantly and surprisingly shortened the column throughput time from 1 hour to 1 minute.

[0316] Simplified laboratory setup

[0317] In addition, as can be seen from the examples herein, since the volume of the sample lysate is much smaller, there is no need for a custom large-volume extension tube when applying ATPS sample lysate concentration. No specific vacuum manifold is required, and a centrifuge can be used to pass the sample lysate through.

[0318] Further examples will be discussed below that demonstrate the unexpected efficiency of a system combined with one or more ATPS extraction workflows for extracting and purifying target analytes from large volume samples.

[0319] Example 4b: Urine extraction using magnetic beads with and without a prior ATPS step

[0320] Repeat the experiment discussed in Example 4a, except that the purification step is carried out using magnetic beads as the solid phase.

[0321] Prepare, pretreat and lyse two groups of urine samples according to the procedure discussed in Example 4a. The groups of urine samples in this example (Sample #3 and Sample #4) are summarized in Table 2c below.

[0322] Table 2c: Summary of test conditions for urine extraction using magnetic beads

[0323] Purification of DNA

[0324] The target cfDNA in the urine sample is partitioned into the polymer-rich top phase of the second ATPS and concentrated to 400 μL - 600 μL. The top phase is separated for further processing.

[0325] Use 3 - 7 M guanidine salt as the binding buffer. Add 2 mL of the binding buffer to the extracted top phase of Sample #1 and the lysed Sample #2 that has not undergone ATPS, respectively. Add 24 μL of magnetic beads to each tube. Then incubate the mixture on a rotator for 5 minutes to prevent bead precipitation. Then centrifuge the tubes briefly and place them on a magnetic rack for 2 minutes to immobilize the beads on the tube wall. Discard the supernatant without disturbing the beads. Add 2 mL of the binding buffer to each tube and rotate the tubes a total of 720° slowly on the magnetic rack. Pipette and discard the supernatant again. Add 800 μL of wash buffer (70% ethanol, 0.001 M EDTA, 0.01 M Tris-HCl) to the samples and rotate the tubes a total of 720° on the rack. Discard the supernatant. The washing step is carried out twice. To improve drying, briefly centrifuge the tubes using a benchtop microcentrifuge with the hinge facing outwards to collect any remaining wash buffer. Then dry the beads on the magnetic rack with the lid open for 7 minutes. Resuspend the bead complex in 80 μL of elution buffer (0.01 M Tris-HCl, 0.001 M EDTA) by mixing with a continuous pipette and then perform a gentle vortex. Then place the tube on the magnetic rack for 1 minute. Carefully collect the supernatant into a DNA low-binding tube (purchased from Eppendorf, catalog number 0030108035) without disturbing the magnetic beads for detection.

[0326] Detection of DNA

[0327] The steps for DNA detection of each sample are the same as or similar to those discussed above for Example 4a. For the sake of brevity and simplicity of this disclosure, the discussion of the detection steps is not repeated here. The results are expressed as average CT values.

[0328] Results

[0329] Recovery of DNA

[0330] Now refer to Figure 1C - 1D and Table 2d, which shows the average CT values of 145bp DNA ( Figure 1C ) and 2000bp DNA ( Figure 1D ) recovered from urine using magnetic beads with a previous ATPS step (Sample #3) and without a previous ATPS step (Sample #4). High recovery of 145bp DNA (average CT value of 27.47) and 2000bp DNA (average CT value of 27.09) was shown in urine sample #3, while no detectable target DNA recovery was observed in urine sample #4 (as shown in Table 2d). The results indicate that under the same conditions, by incorporating a parallel ATPS step prior to magnetic bead extraction, DNA recovery from large-volume samples such as urine can be significantly improved.

[0331] Table 2d: qPCR results of 145bp and 2000bp DNA oligonucleotide recovery from urine using magnetic beads with or without two-step ATPS.

[0332] Example 5: Urine extraction by dividing the sample matrix into 2x first ATPS

[0333] In this example, urine samples were divided into several first ATPS and / or second ATPS (i.e., parallel ATPS), and their DNA recovery was compared with urine samples processed by a single first and second ATPS.

[0334] Urine lysis

[0335] In this example, the urine sample was pretreated with 200 μL of 0.1 M EDTA per 10 mL of urine sample, vortexed thoroughly and centrifuged at 3000 rcf for 10 minutes. The supernatant was transferred to a new tube while discarding the pellet. To digest unwanted proteins and cells in the pretreated urine sample, 600 μL of proteinase K (28.57 mg / mL) and 2 mL of a suitable lysis buffer were added to 20 mL of the sample. 100 fg of 145 bp dsDNA and 100 ng of 1 kb+ DNA ladder were spiked into the above sample. The sample was then vortexed thoroughly until homogeneous and then incubated in a preheated water bath at 37 °C for 15 minutes.

[0336] Two-phase system

[0337] In this example, the extraction procedure involved two consecutive aqueous two-phase systems (ATPS) to separate, purify and concentrate DNA in the urine sample. In the first ATPS, DNA partitioned into the bottom phase and proteins partitioned into the top phase. The amount of the bottom phase was approximately 5 mL, which was carefully extracted and transferred to the second ATPS. In the second ATPS, DNA significantly partitioned into the top phase (an amount of approximately 500 μL), effectively concentrating the 20 mL sample matrix into 500 μL of a target-rich phase containing the target cfDNA.

[0338] Urine samples from 3 donors were divided into 3 independent groups (groups 1 - 3). For group 1, 22.6 mL of the urine sample was split in half and added to 2 separate first ATPS (2x first ATPS). The top phases of these first ATPS were added to 2 separate second ATPS (2x second ATPS). For group 2, similarly 22.6 mL of the urine sample was split in half and added to 2 separate first ATPS (2x first ATPS), however, the top phases of these first ATPS were combined and added to 1 single larger second ATPS (1x second ATPS). For group 3, 22.6 mL of the urine sample was directly added to 1 single large first ATPS (1x first ATPS), and then the top phase was extracted into 1 single large second ATPS (1x second ATPS). To ensure equal comparison, the first and second ATPS were scaled accordingly to ensure that the composition of salts and polymers was the same between conditions. The extraction conditions of the first and second ATPS used in this experiment are summarized in Table 3. Table 4 shows example polymer and salt combinations in the first and second ATPS compositions. The concentrations of polymers and salts and / or the volumes of the first and second ATPS compositions were adjusted accordingly based on the volume of the lysate used in Table 3 (11.3 mL, 11.3 mL, and 22.6 mL for groups 1 - 3 respectively).

[0339] Table 3. Overall summary of the workflow between different ATPS conditions.

[0340] Table 4. Concentrations of one or more polymers and one or more salts in the first and second ATPS, respectively. First ATPS Second ATPS 20% - 40% PAG, 0.1% - 20% phosphate 3% - 20% PAG, 0.1% - 20% phosphate

[0341] All concentrations are w / v ratios.

[0342] Purification of DNA

[0343] Purification of DNA was accomplished by spin column extraction. The top phase of the second ATPS was transferred to a tube containing 1 mL of binding buffer (3 - 7 M guanidine salt) and mixed well. 800 μL of the solution was transferred to a spin column (EconoSpin) and centrifuged at 12,000 rcf for 30 seconds. The flow-through was discarded. This process was repeated until all samples had passed through the spin column. 500 μL of wash buffer (80% ethanol v / v, 0.1 M NaCl, 0.01 M Tris-HCl) was added to the spin column and centrifuged at 12,000 rcf for 30 seconds. Then the spin column was centrifuged at 16,000 rcf for 2 minutes to dry. 40 μL of elution buffer (0.01 M Tris-HCl, 0.001 M EDTA) was added to the spin column membrane. The spin column was incubated at room temperature for 3 minutes. The eluate was collected in a collection tube by centrifuging at 12,000 rcf for 1 minute for detection.

[0344] Detection of DNA

[0345] The steps for DNA detection of each sample were the same or similar to those discussed above for Example 4a. For the sake of brevity and simplicity of this disclosure, the discussion of the detection steps is not repeated here. The results are presented as average Ct values.

[0346] Results

[0347] Figure 2A The Ct values obtained by qPCR of 145 bp dsDNA recovered under different extraction conditions (Groups 1 - 3) according to Table 3 are shown. The results are also summarized in Table 5 below.

[0348] Table 5. Average Ct values of qPCR of recovered 145 bp dsDNA.

[0349] Those skilled in the art would expect that as the number of ATPS increases, the recovery of the target cfDNA would be significantly reduced because there are more potential sources of cfDNA loss, such as losses due to manual handling and pipetting errors. However, now referring to Figure 2A and Table 5, the average Ct values of the 145bp dsDNA for all conditions (Groups 1 - 3) are within 0.2 Ct of each other, indicating that there is no significant loss of the target cfDNA despite the increase in the number of ATPS used to process the samples. The results show that splitting a large volume sample into multiple smaller parallel ATPS surprisingly preserves the cfDNA yield and does not affect performance when compared to a single large ATPS.

[0350] Example 6: Urine extraction by dividing the sample matrix into 4x first ATPS

[0351] In this example, the urine sample was divided into 4 separate first ATPS and further underwent either 2 separate second ATPS or a single second ATPS. Their DNA recoveries were compared.

[0352] Urine lysis

[0353] In this example, the urine sample was pretreated by the same procedure discussed in Example 5. To digest the unwanted proteins and cells in the pretreated urine sample, 2400 μL of proteinase K (28.57 mg / mL) and 8 mL of a suitable lysis buffer were added to 80 mL of the sample. 100 fg of 145bp dsDNA and 100 ng of 1kb+ DNA ladder were spiked into the above sample. Then the sample was vortexed thoroughly until homogeneous and then incubated in a preheated 37 °C water bath for 15 minutes.

[0354] Two-phase system

[0355] The extraction procedure is similar to the one discussed in Example 5. In the first ATPS, DNA is partitioned into the bottom phase and protein is partitioned into the top phase. The amount of the bottom phase is approximately 5 mL, which is carefully extracted and transferred to the second ATPS. In the second ATPS, DNA is significantly partitioned into the top phase (an amount of approximately 500 μL), effectively concentrating a 20 mL sample matrix into a 500 μL polymer-rich phase containing the target cfDNA. In this experiment, we demonstrated that using multiple smaller ATPSs in parallel does not affect performance compared to using a single large ATPS. Urine samples from 2 donors were divided into 2 separate groups (Group 4 and Group 5). For Group 4, a 90.4 mL urine sample was evenly divided into 4 separate first ATPSs (4x first ATPS). The top phases of 2 of the 4 first ATPSs were combined and added to 1 second ATPS, resulting in a total of 2 separate second ATPSs (2x second ATPS). For Group 5, similarly, a 90.4 mL urine sample was evenly divided into 4 separate first ATPSs, but all the top phases of these first ATPSs were combined and added to 1 single larger second ATPS (1x second ATPS). To ensure an equal comparison, the second ATPSs were scaled accordingly based on the conditions to ensure that the composition of salt and polymer was the same between the conditions. The extraction conditions for the first and second ATPSs used in this experiment are summarized in Table 6. The concentration of polymer and salt and / or the volume of the first and second ATPS compositions were adjusted accordingly based on the volume of the lysate used in Table 6 (22.6 mL in this example).

[0356] Table 6. Overall summary of the workflow between different ATPS conditions.

[0357] Table 7. Concentrations of polymer and salt in the first and second ATPSs respectively. First ATPS Second ATPS 20% - 40% PAG, 0.1% - 20% phosphate 3% - 20% PAG, 0.1% - 20% phosphate

[0358] All concentrations are w / v ratios.

[0359] Purification of DNA

[0360] The purification procedure used in this example is the same as the one discussed in Example 5, except that 4 mL of binding buffer (3 - 7 M guanidine salt) was used. For the sake of brevity and simplicity of this disclosure, the discussion of the purification steps is not repeated here.

[0361] Detection of DNA

[0362] The steps for DNA testing of each sample are the same as or similar to those discussed above for Example 4a. For the sake of brevity and simplicity of this disclosure, the discussion of the testing steps will not be repeated here. The results are presented as average Ct values.

[0363] Results

[0364] Figure 2B Shown are the Ct values obtained by qPCR of 145bp dsDNA recovered under different extraction conditions (Groups 4 - 5) according to Table 6. The results are also summarized in Table 8 below.

[0365] The Ct values of 145bp dsDNA under all conditions are within 0.1 Ct of each other, indicating that there is no significant loss of the target cfDNA with increasing amounts of ATPS.

[0366] Table 8. Average Ct values of qPCR of recovered 145bp dsDNA.

[0367] Now referring Figure 2B to Table 8, the average Ct values of 145bp dsDNA for all conditions (Groups 4 - 5) are within 0.1 Ct of each other, indicating that there is no significant loss of the target cfDNA with increasing amounts of the second ATPS. The results show that processing samples extracted from the first ATPS in multiple smaller second ATPSs in parallel surprisingly preserves the cfDNA yield and does not affect performance when compared to a single second ATPS.

[0368] Example 7: Urine extraction using ATPS with extreme volume ratios

[0369] This experiment tested the robustness of DNA recovery at a range of different volume ratios in the 1st ATPS and the 2nd ATPS. In this example, 0.25x PBS and urine samples were tested under different ATPS conditions that produced different phase volume ratios.

[0370] Urine lysis

[0371] In this example, a set of data was generated using 0.25x PBS as the sample matrix, and another set of data was obtained using urine samples from three separate donors. Similar to Examples 5 and 6, each 10 mL urine sample was pretreated with 200 μL of 0.1 M EDTA, vortexed thoroughly, and centrifuged at 3000 rcf for 10 minutes. The supernatant was transferred to a new tube while discarding the pellet. To digest unwanted proteins and cells in the pretreated urine samples, 2400 μL of Proteinase K (28.57 mg / mL) and 8 mL of a suitable lysis buffer were added to 80 mL of the sample. 100 fg of 145 bp dsDNA and 100 ng of 1 kb+ DNA ladder were spiked into the above samples. The samples were then vortexed well until homogeneous and then incubated in a preheated 37 °C water bath for 15 minutes.

[0372] Two-phase system

[0373] The extraction procedure involved two consecutive aqueous two-phase systems (ATPS) to separate, purify, and concentrate DNA from urine samples. 22.6 mL of the lysate was transferred to the first ATPS, where DNA partitioned into the bottom phase and proteins partitioned into the top phase. The bottom phase was carefully extracted and transferred to the second ATPS. In the second ATPS, DNA significantly partitioned into the top phase, effectively concentrating more than 20 mL of the sample matrix into a much smaller polymer-rich phase containing the target cfDNA.

[0374] The experiment was divided into two parts: varying the top:bottom volume ratio in the 1st ATPS while keeping the 2nd ATPS volume ratio constant, and the other part focused on keeping the 1st ATPS constant while varying the volume ratio of the 2nd ATPS. To test whether DNA recovery would be affected by changes in the 1st ATPS phase volume ratio, various formulations giving top:bottom phase volume ratios ranging from 0.9:1 to 13:1 were tested while keeping the 2nd ATPS volume ratio constant at 1:24. The formulation details are summarized in Table 9. The concentrations of the polymer and salt and the volumes of the first and second ATPS compositions were adjusted accordingly such that the 1st ATPS formed separate top:bottom volume ratios of 13:1, 6:1, and 0.9:1 after mixing with 22.6 mL of the lysate, and the 2nd ATPS formed a constant top:bottom volume ratio of 1:24 under all three conditions (Condition 1 = 2.6 mL, Condition 2 = 5 mL, Condition 3 = 20 mL) after mixing with different volumes of the bottom phase of the 1st ATPS.

[0375] Table 9. Test formulations to verify 1st ATPS volume ratio variability

[0376] *All 1st ATPS compositions contain PAG, phosphate, 1 mM EDTA, and 0.6% Triton X-114, and all 2nd ATPS compositions contain PAG, phosphate, and 0.7 mM EDTA.

[0377] To test whether DNA recovery is affected by different 2nd ATPS top:bottom phase volume ratios, the 1st ATPS formulation was kept constant, and 2nd ATPS formulations giving 1:1 and 1:24 (Table 10) were tested. The concentrations of the polymer and salt and the volumes of the 1st and 2nd ATPS compositions were adjusted accordingly such that the 1st ATPS formed a bottom phase of approximately 3.5 - 6 mL and a top:bottom volume ratio of 6:1 after mixing with 22.6 mL of lysate, and the 2nd ATPS formed top:bottom volume ratios of 1:24 and 1:1 respectively after mixing with the bottom phase of the 1st ATPS (approximately 5 mL in this example).

[0378] Table 10. Test formulations to verify 1ATPS volume ratio variability

[0379] *All 1st ATPS compositions contain PAG, phosphate, 1 mM EDTA, and 0.6% Triton X-114, and all 2nd ATPS compositions contain PAG, phosphate, and 0.7 mM EDTA.

[0380] Purification of DNA

[0381] Similar to the procedures discussed in Examples 5 and 6, DNA was purified from the extraction phase by spin column extraction. The top phase of the 2nd ATPS was transferred to a tube containing binding buffer (3 - 7 M guanidine salt), and the amount of buffer was scaled to the top phase volume at a binding buffer ratio of 1:4. The extracted top phase and binding buffer were mixed well, 800 μL of the solution was transferred to a spin column (EconoSpin), and centrifuged at 12,000 rcf for 30 seconds. The flow-through was discarded. This process was repeated until all samples had passed through the spin column. 500 μL of wash buffer (80% ethanol v / v, 0.1 M NaCl, 0.01 M Tris-HCl) was added to the spin column, and the spin column was centrifuged at 12,000 rcf for 30 seconds. Then the spin column was centrifuged at 16,000 rcf for 2 minutes to dry. 40 μL of elution buffer (0.01 M Tris-HCl, 0.001 M EDTA) was added to the spin column membrane. The spin column was incubated at room temperature for 3 minutes. The eluate was collected into a collection tube by centrifuging at 12,000 rcf for 1 minute for detection.

[0382] Detection of DNA

[0383] DNA testing was performed using the same method discussed in the previous example.

[0384] Results

[0385] Now referring to Figure 3A - 3D , which shows the DNA recovery obtained using different volume ratios according to the conditions in Tables 9 and 10 (the respective first ATPS volume ratios as described in Conditions 1 - 3, and the respective second ATPS volume ratios as described in Conditions 4 - 5). Figure 3A Shows the recovery of 145bp dsDNA spiked when using the 1st ATPS with different top:bottom phase volume ratios in the case of urine samples from three separate donors. The results are also summarized in Table 11. Figure 3B Shows the recovery of 145bp dsDNA spiked when using the 1st ATPS with different top:bottom phase volume ratios in 0.25x PBS as the sample matrix. The results are also summarized in Table 12.

[0386] Table 11. Average CT values of 145bp DNA in urine samples.

[0387] Table 12. Average CT values of 145bp DNA in 0.25x PBS.

[0388] Figure 3C Shows the recovery of 145bp dsDNA spiked when using the 2nd ATPS with different top:bottom phase volume ratios in the case of urine samples from three separate donors. The results are also summarized in Table 13. Figure 3D Shows the recovery of 145bp dsDNA spiked when using the 2nd ATPS with different top:bottom phase volume ratios in 0.25x PBS as the sample matrix. The results are also summarized in Table 14.

[0389] Table 13. Average CT values of 145bp DNA in urine samples.

[0390] Table 14. Average CT values of 145bp DNA in 0.25x PBS.

[0391] The results show that the ATPS system can function at different volume ratios and is particularly effective at certain volume ratios. This experiment demonstrates that by changing the 1st ATPS ( Figure 3A and3B ) and the top:bottom phase volume ratio in the 2nd ATPS Figure 3C and 3D ) The top:bottom phase volume ratio can still recover DNA. DNA recovery in all ATPS systems tested using spin columns is better than in systems without ATPS (see urine sample #2 in Example 4a).

[0392] The above examples demonstrate the robustness and stability of ATPS in various scenarios for processing large volume or large quantity of fluid samples. This highlights the advantages of the methods, kits, and embodiments described in this disclosure, which can be used or adjusted by those skilled in the art in different settings to achieve comparable DNA recovery from large volume or large quantity of fluid samples (such as urine), while minimizing errors and sample losses associated with sample handling and processing.

[0393] Example 8: Comparison of total DNA recovery using the disclosed method and a commercially available extraction kit

[0394] Example 8a

[0395] DNA was extracted from urine using the exemplary method and kit described in Example 4a (referred to herein as "this extraction method" or "phase") and compared with the Zymo Quick-DNA Urine Kit ("Zymo"), NextPrep-Mag Urine cfDNA Isolation Kit ("NextPrep Kit"), Norgen Urine DNA Isolation Kit - Spin Column ("Norgen"), and Wizard Plus Minipreps DNA Purification System ("Wizard"), all of which are commercially available. For each commercially available kit, the maximum urine sample input volume specified by the manufacturer was used, and extraction was performed according to the manufacturer's instructions. Cell-free urine was used to compare the commercially available kits (conditions A - D in Table 15) and this extraction method (condition E in Table 15). Additionally, for comparison, we performed a batch of 40 mL extractions of crude urine (unspun urine with cells) with the current extraction method (condition F in Table 15) to evaluate whether the current extraction method can perform equally well in the presence of cells. Urine samples were provided by 4 males and 4 females (n = 8 for each kit). The input and elution volumes for each kit were normalized to a 100:1 ratio, and extraction times were compared in Table 15.

[0396] Table 15. Comparison of Yield and Efficiency at Normalized Input:Elution Volume Ratio

[0397] Now refer to Figure 4A, which shows the recovery of 145bp DNA spike-in (copies / μL) using Conditions A - F of Table 15. The 145bp spike-in was detected by Droplet Digital PCR ddPCR. As Figure 4A shown, the recovery efficiency of 145bp spike-in DNA using this extraction method (Condition E) is comparable, if not higher, to that of the NextPrep (Condition A) and Wizard (Condition D) extraction kits, and significantly higher than that of the Zymo (Condition B) and Norgen (Condition C) extraction kits. Compared to the NextPrep (Condition A), Norgen (Condition C), and Wizard (Condition D) extraction kits, this extraction method can handle a larger input volume, but the extraction time is comparable if not shorter. Compared to Zymo (Condition B) with the same input volume, this extraction method has a shorter extraction time, significantly higher yield, and more consistent performance between samples. When using urine with cells (Condition F), even in the presence of cells, the average DNA recovery of this extraction method is significantly better than all commercially available kits, with satisfactory precision (476.1 ± 32.2 copies / μL). This indicates that this extraction method performs well in recovering the target DNA from crude urine as well as processed centrifuged urine. In summary, compared to commercially available extraction kits of industrial standards, the overall target DNA extraction performance (in terms of yield, input volume, and extraction time) using the method disclosed herein is surprisingly better.

[0398] Example 8b

[0399] A further comparison of DNA recovery from urine between this extraction method (also referred to herein as "Phase") and commercially available extraction kits (Zymo Quick-DNA Urine Kit ("Zymo"), Qiagen QIAamp Circulating Nucleic Acid Kit ("Qiagen" or "QCNA"), Norgen Urine DNA Isolation Kit - Spin Column ("Norgen"), and Wizard Plus Minipreps DNA Purification System ("Wizard")) was performed using this extraction method with a maximum urine sample input volume of 160 mL, while the commercially available extraction kits used the maximum sample input volume and optimal output volume recommended by the manufacturer and were extracted according to the manufacturer's instructions. Urine samples were provided by 4 males and 4 females (n = 8 for each kit). The conditions are summarized in Table 16.

[0400] Table 16. Comparison of Yield and Efficiency Using Recommended Input and Output Volumes

[0401] Now refer to Figure 4B, which shows the average concentration (copy number / μL) of DNA recovered using the kit and conditions according to Table 16. The 140 bp spike-in was detected by Droplet digital PCR ddPCR. The results show that due to the ability to handle high input volumes and concentrate them into low output volumes, this extraction method is significantly superior to all commercially available kits.

[0402] In summary, the experiments in Examples 8a and 8b show that due to the increased sample input volume allowed by this extraction method, the low output / elution volume, and the higher recovery efficiency of the target DNA, the total DNA recovery of this extraction method is significantly greater than that of all other commercially available kits.

[0403] The exemplary embodiments of the present invention have thus been fully described. Although the description refers to specific embodiments, those skilled in the art will appreciate that the present invention can be practiced with variations of these specific details. Therefore, the present invention should not be construed as limited to the embodiments set forth herein.

[0404] Numbered Example 1

[0405] Example 1. A method for concentrating and purifying one or more target analytes from a large volume of fluid sample, the method comprising the steps of: (a) preparing a first aqueous two-phase system (ATPS) composition, wherein the first ATPS composition comprises a polymer, a salt, a surfactant, or a combination thereof dissolved in an aqueous solution to form a first phase solution and a second phase solution; (b) adding a sample solution prepared from the large volume of fluid sample containing the one or more target analytes to the first ATPS composition such that the one or more target analytes partition into the first phase solution; (c) collecting the first phase solution and mixing the first phase solution with a second ATPS composition, wherein the second ATPS composition comprises a polymer, a salt, a surfactant, or a combination thereof dissolved in an aqueous solution to form a third phase solution and a fourth phase solution, such that the one or more target analytes partition into the third phase solution and are concentrated in the third phase solution; (d) collecting the third phase solution and mixing the third phase solution with a binding buffer to form a mixed solution, wherein the binding buffer comprises at least one dissociating agent; (e) loading the mixed solution onto an extraction column configured to selectively extract and purify the one or more target analytes; (f) eluting and collecting the one or more target analytes from the extraction column to obtain a final solution containing the concentrated and purified one or more target analytes.

[0406] Example 2. The method according to Example 1, wherein the sample solution is prepared by dividing the large volume of fluid sample containing the one or more target analytes into at least two aliquots of the sample solution, and the first ATPS composition is divided into at least two aliquots, wherein step (b) further comprises the steps of: (i) adding each aliquot of the sample solution prepared from the large volume of fluid sample containing the one or more target analytes to each aliquot of the first ATPS composition such that the one or more target analytes are partitioned into the first phase solution; (ii) collecting and combining the first phase solutions of the at least two aliquots of the first ATPS composition to form the first phase solution for step (c).

[0407] Example 3. The method according to any one of the preceding examples, wherein the extraction column is a centrifugal column, and wherein step (e) further comprises the steps of: (i) loading a portion of the mixed solution onto the extraction column; (ii) centrifuging the extraction column and discarding the flow-through or supernatant; and (iii) repeating steps (i) and (ii) above until all of the mixed solution has passed through the extraction column.

[0408] Example 4. The method according to any one of the preceding examples, the method further comprising the step of: (g) performing a diagnostic assay on the final solution to detect and quantify the one or more target analytes.

[0409] Example 5. A method for concentrating and purifying one or more target analytes from a large volume of fluid sample, the method comprising the steps of: (a) dividing the large volume of fluid sample containing the one or more target analytes into at least two aliquots of sample solution; (b) preparing at least two aliquots of a first aqueous two-phase system (ATPS) composition, wherein the first ATPS composition comprises a polymer, a salt, a surfactant, or a combination thereof dissolved in an aqueous solution to form a first-phase solution and a second-phase solution; (c) adding each aliquot of the sample solution containing the one or more target analytes to each aliquot of the first ATPS composition such that the one or more target analytes partition into the first-phase solution; (d) collecting the first-phase solution of the at least two aliquots of the first ATPS composition and mixing the first-phase solution with a second ATPS composition, wherein the second ATPS composition comprises a polymer, a salt, a surfactant, or a combination thereof dissolved in an aqueous solution to form a third-phase solution and a fourth-phase solution, such that the one or more target analytes partition into the third-phase solution and are concentrated in the third-phase solution; (e) collecting the third-phase solution and mixing the third-phase solution with a binding buffer to form a mixed solution, wherein the binding buffer comprises at least one dissociating agent; (f) loading the mixed solution onto an extraction column configured to selectively extract and purify the one or more target analytes; (g) eluting and collecting the one or more target analytes from the extraction column.

[0410] Example 6. The method according to any one of the preceding examples, wherein the large volume of fluid sample is selected from the group consisting of blood, plasma, serum, cerebrospinal fluid, urine, saliva, feces, tears, sputum, nasopharyngeal mucus, vaginal secretions, and penile secretions.

[0411] Example 7. The method according to any one of the preceding examples, wherein the large volume of fluid sample is urine.

[0412] Example 8. The method according to any one of the preceding examples, wherein the volume of the large volume of fluid sample is 40 mL or greater.

[0413] Example 9. The method according to any one of the preceding examples, wherein the volume of each aliquot of the sample solution is at most 40 ml.

[0414] Example 10. The method according to any one of the preceding examples, wherein the target analytes are selected from the group consisting of nucleic acids, proteins, antigens, biomolecules, sugar moieties, lipids, sterols, and combinations thereof.

[0415] Example 11. The method according to any one of the preceding examples, wherein the target analyte is DNA.

[0416] Example 12. The method according to any one of the preceding examples, wherein the target analyte is free DNA or circulating tumor DNA.

[0417] Example 13. The method according to any one of the preceding examples, wherein the polymer is dissolved in an aqueous solution at a concentration of 4% - 84% (w / w).

[0418] Example 14. The method according to any one of the preceding examples, wherein the polymer is selected from the group consisting of polyalkylene glycols, such as hydrophobically modified polyalkylene glycols, poly(alkylene oxide) polymers, poly(alkylene oxide) copolymers, such as hydrophobically modified poly(alkylene oxide) copolymers, polyvinylpyrrolidone, polyvinyl alcohol, polyvinylcaprolactam, polyvinyl methyl ether, alkoxylated surfactants, alkoxylated starches, alkoxylated celluloses, alkyl hydroxyalkyl celluloses, silicone-modified polyethers, and poly(N-isopropylacrylamide) and its copolymers. The method according to any one of the preceding examples, wherein the polymer is selected from the group consisting of: polyethers, polyimines, polyalkylene glycols, vinyl polymers, alkoxylated surfactants, polysaccharides, alkoxylated starches, alkoxylated celluloses, alkyl hydroxyalkyl celluloses, polyether-modified silicones, polyacrylamides, polyacrylic acids, and their copolymers. The method according to any one of the preceding examples, wherein the polymer is selected from the group consisting of: dipropylene glycol, tripropylene glycol, polyethylene glycol, polypropylene glycol, poly(ethylene glycol-propylene glycol), poly(ethylene glycol-random-propylene glycol), polyvinylpyrrolidone, polyvinyl alcohol, polyvinylcaprolactam, polyvinyl methyl ether, dextran, carboxymethyl dextran, dextran sulfate, hydroxypropyl dextran, starch, carboxymethyl cellulose, polyacrylic acid, hydroxypropyl cellulose, methyl cellulose, ethyl hydroxyethyl cellulose, maltodextrin, polyethyleneimine, poly(N-isopropylacrylamide), and its copolymers. The method according to any one of the preceding examples, wherein the polymer is selected from the group consisting of: dipropylene glycol, tripropylene glycol, polyethylene glycol, polypropylene glycol, poly(ethylene glycol-propylene glycol), poly(ethylene glycol-random-propylene glycol), polyvinylpyrrolidone, polyvinyl alcohol, polyvinylcaprolactam, polyvinyl methyl ether, and poly(N-isopropylacrylamide). The method according to any one of the preceding examples, wherein the polymer is selected from the group consisting of: polyacrylamides, polyacrylic acids, and their copolymers. The method according to any one of the preceding examples, wherein the polymer is selected from the group consisting of: dextran, carboxymethyl dextran, dextran sulfate, hydroxypropyl dextran, and starch. The method according to any one of the preceding examples, wherein the polymer has an average molecular weight in the range of 200 - 1,000 Da, 200 - 35,000 Da, 425 - 2,000 Da, 400 - 35,000 Da, 980 - 12,000 Da, or 3,400 - 5,000,000 Da. The method according to any one of the preceding examples, wherein the polymer comprises ethylene oxide and propylene oxide units, and the polymer has an EO:PO ratio of 90:10 to 10:90.

[0419] Example 15.

[0420] Example 16. The method according to any one of the preceding examples, wherein the salt is dissolved in an aqueous solution at a concentration of 1% - 55% (w / w).

[0421] Example 17. The method according to any one of the preceding examples, wherein the salt is dissolved in an aqueous solution at a concentration of 8% - 55% (w / w).

[0422] Example 18. The method according to any one of the preceding examples, wherein the salt is selected from the group consisting of lyophilic salts, discrete salts, inorganic salts containing cations such as linear or branched trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium, and tetrabutylammonium, and inorganic salts containing anions such as phosphate, sulfate, nitrate, chloride, and bicarbonate, NaCl, Na 3 PO 4 、K 3 PO 4 、Na 2 SO 4 、potassium citrate, (NH 4 ) 2 SO 4 、sodium citrate, sodium acetate, ammonium acetate, magnesium salts, lithium salts, sodium salts, potassium salts, cesium salts, zinc salts, aluminum salts, bromide salts, iodide salts, fluoride salts, carbonates, sulfates, citrates, carboxylates, borates, phosphates, potassium phosphate, and ammonium sulfate.

[0423] Example 19. The method according to any one of the preceding examples, wherein the surfactant is dissolved in an aqueous solution at a concentration of 0.05% - 10% (w / w).

[0424] Example 20. The method according to any one of the preceding examples, wherein the surfactant is dissolved in an aqueous solution at a concentration of 0.05% - 9.8% (w / w).

[0425] Example 21. The method according to any one of the foregoing examples, wherein the surfactant is selected from the group consisting of Triton-X, Triton-114, Igepal CA-630, and Nonidet P-40, anionic surfactants (such as carboxylates, sulfonates, petroleum sulfonates, alkylbenzene sulfonates, naphthalene sulfonates, olefin sulfonates, alkyl sulfates, sulfates, sulfated natural oils, sulfated natural fats, sulfated esters, sulfated alkanolamides, ethoxylated alkylphenols, and sulfated alkylphenols), nonionic surfactants (such as ethoxylated fatty alcohols, polyoxyethylene surfactants, carboxylic acid esters, polyethylene glycol esters, sorbitan esters, fatty acid ethylene glycol esters, carboxylic acid amides, monoalkanolamine concentrates, polyoxyethylene fatty acid amides), cationic surfactants (such as quaternary ammonium salts, amines having amide bonds, polyoxyethylene alkyl and cycloaliphatic amines, n,n',n',n'-tetrasubstituted ethylenediamine, 2-alkyl-1-hydroxyethyl-2-imidazoline), and amphoteric surfactants (such as n-cocoyl 3-aminopropionic acid / sodium salt, n-tallow 3-iminodipropionate, disodium salt, n-carboxymethyl-n-dimethyl-n-9-octadecenyl ammonium hydroxide, n-cocoylamidoethyl n-hydroxyethyl glycine and its sodium salt).

[0426] Example 22. An ATPS composition selected from the group consisting of A1, A2, A3, A4, AA1, AA2, AA3, and AA4.

[0427] Example 23. A kit comprising a first ATPS composition selected from the group consisting of A1, A2, A3, and A4; a second ATPS composition selected from the group consisting of AA1, AA2, AA3, and AA4; and a binding buffer selected from the group consisting of B1, B2, and B3.

[0428] Example 24. The kit according to Example 23, further comprising an extraction column.

[0429] Numbered Example 2

[0430] Example 1. A method for concentrating and purifying one or more target analytes from a large volume of fluid sample, the method comprising the steps of: (a) dividing the large volume of fluid sample containing the one or more target analytes into at least two aliquots of sample solution; (b) preparing at least two first aqueous two-phase system (ATPS) compositions, wherein each first ATPS composition comprises a polymer, a salt, a surfactant, or any combination thereof dissolved in an aqueous solution to form a first phase solution and a second phase solution; (c) adding each aliquot of the sample solution prepared from the large volume of fluid sample containing the one or more target analytes to each first ATPS composition such that the one or more target analytes partition into each first phase solution; (d) further processing each first phase solution to form a final phase solution; (e) mixing the final phase solution with at least one purification composition to form a mixed solution; (f) contacting the mixed solution with a downstream purification system configured to selectively separate the one or more target analytes; and (g) collecting the one or more target analytes from the downstream purification system to obtain a final solution containing the concentrated and purified one or more target analytes.

[0431] Example 2. The method according to any one of the preceding examples, wherein the further processing in step (d) comprises collecting and combining each first phase solution to form the final phase solution.

[0432] Example 3. The method according to any one of the preceding examples, wherein the further processing in step (d) comprises the steps of: (i) collecting each first phase solution; (ii) mixing each first phase solution with a second ATPS composition, wherein the second ATPS composition comprises a polymer, a salt, a surfactant, or a combination thereof dissolved in an aqueous solution to form a third phase solution and a fourth phase solution, such that the one or more target analytes partition into the third phase solution and are concentrated in the third phase solution; (iii) collecting and combining the third phase solutions to form the final phase solution.

[0433] Example 4. The method according to any one of the preceding examples, wherein the further processing in step (d) comprises the steps of: (i) collecting and combining each first phase solution to form a combined first phase solution; (ii) mixing each combined first phase solution with a second ATPS composition, wherein the second ATPS composition comprises a polymer, a salt, a surfactant, or a combination thereof dissolved in an aqueous solution to form a third phase solution and a fourth phase solution, such that the one or more target analytes partition into the third phase solution and are concentrated in the third phase solution; (iii) collecting the third phase solution to form the final phase solution.

[0434] Example 5. The method according to any one of the preceding examples, wherein the purification composition is a binding buffer comprising at least one dispersing agent; the downstream purification system comprises a solid-phase medium; and step (f) further comprises the steps of: (i) contacting a portion of the mixed solution with the solid-phase medium such that the one or more target analytes bind to the solid-phase medium to form a solid-phase extraction complex; (ii) disturbing the solid-phase extraction complex and discarding the flow-through or supernatant; and (iii) optionally repeating steps (i) and (ii).

[0435] Example 6. The method according to any one of the preceding examples, wherein the solid-phase medium is a solid-phase extraction column.

[0436] Example 7. The method according to any one of the preceding examples, wherein the solid-phase extraction column is a centrifugal column.

[0437] Example 8. The method according to any one of the preceding examples, wherein the solid-phase medium is a plurality of beads.

[0438] Example 9. The method according to any one of the preceding examples, wherein the plurality of beads are magnetic beads, silica-based beads, carboxyl beads, hydroxyl beads, amine-coated beads, or any combination thereof.

[0439] Example 10. The method according to any one of the preceding examples, the method further comprises the step of: (h) performing a diagnostic assay on the final solution to detect, quantify, characterize, or any combination thereof, the one or more target analytes.

[0440] Example 11. A method for concentrating and purifying one or more target analytes from a large volume of fluid sample, the method comprising the steps of: (a) dividing the large volume of fluid sample containing the one or more target analytes into at least two aliquots of sample solution; (b) preparing at least two aliquots of a first aqueous two-phase system (ATPS) composition, wherein the first ATPS composition comprises a polymer, a salt, a surfactant, or a combination thereof dissolved in an aqueous solution to form a first-phase solution and a second-phase solution; (c) adding each aliquot of the sample solution containing the one or more target analytes to each aliquot of the first ATPS composition such that the one or more target analytes are partitioned into the first-phase solution; (d) collecting the first-phase solution of the at least two aliquots of the first ATPS composition and mixing the first-phase solution with a second ATPS composition, wherein the second ATPS composition comprises a polymer, a salt, a surfactant, or a combination thereof dissolved in an aqueous solution to form a third-phase solution and a fourth-phase solution, such that the one or more target analytes are partitioned into the third-phase solution and concentrated in the third-phase solution; (e) collecting the third-phase solution and mixing the third-phase solution with a binding buffer to form a mixed solution, wherein the binding buffer comprises at least one dissociating agent; (f) loading the mixed solution onto an extraction column configured to selectively extract and purify the one or more target analytes; (g) eluting and collecting the one or more target analytes from the extraction column.

[0441] Example 12. The method according to any one of the preceding examples, wherein the large volume of fluid sample is selected from the group consisting of blood, plasma, serum, cerebrospinal fluid, urine, saliva, feces, tears, sputum, nasopharyngeal mucus, vaginal secretions, and penile secretions.

[0442] Example 13. The method according to any one of the preceding examples, wherein the large volume of fluid sample is urine.

[0443] Example 14. The method according to any one of the preceding examples, wherein the volume of the large volume of fluid sample is at least 10 mL.

[0444] Example 15. The method according to any one of the preceding examples, wherein the volume of the large volume of fluid sample is 40 mL or greater.

[0445] Example 16. The method according to any one of the preceding examples, wherein the volume of each aliquot of the sample solution is at most 40 ml.

[0446] Example 17. The method according to any one of the preceding examples, wherein the volume of each aliquot of the sample solution is 10 to 40 mL.

[0447] Example 18. The method according to any one of the preceding examples, wherein the one or more target analytes are selected from the group consisting of nucleic acids, proteins, antigens, biomolecules, sugar moieties, lipids, sterols, and any combination thereof.

[0448] Example 19. The method according to any one of the preceding examples, wherein the one or more target analytes are DNA.

[0449] Example 20. The method according to any one of the preceding examples, wherein the one or more target analytes are gDNA, cDNA, plasmid DNA, mitochondrial DNA, cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), circulating fetal DNA, microbial cell-free DNA, microRNA (miRNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), circular RNA, long non-coding RNA (lncRNA), or a combination thereof.

[0450] Example 21. The method according to any one of the preceding examples, wherein the one or more target analytes are cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA).

[0451] Example 22. The method according to any one of the preceding examples, wherein the polymer is dissolved in an aqueous solution at a concentration of 0.2% - 80% (w / v).

[0452] Example 23. The method according to any one of the preceding examples, wherein the polymer is selected from the group consisting of polyethers, polyimines, polyalkylene glycols, vinyl polymers, alkoxylated surfactants, polysaccharides, alkoxylated starches, alkoxylated celluloses, alkyl hydroxyalkyl celluloses, polyether-modified silicones, polyacrylamides, polyacrylic acids, and copolymers thereof.

[0453] Example 24. The method according to any one of the preceding examples, wherein the polymer is selected from the group consisting of dipropylene glycol, tripropylene glycol, polyethylene glycol, polypropylene glycol, poly(ethylene glycol - propylene glycol), poly(ethylene glycol - random - propylene glycol), polyvinylpyrrolidone, polyvinyl alcohol, polyvinylcaprolactam, polyvinyl methyl ether, dextran, carboxymethyl dextran, dextran sulfate, hydroxypropyl dextran, starch, carboxymethyl cellulose, polyacrylic acid, hydroxypropyl cellulose, methyl cellulose, ethyl hydroxyethyl cellulose, maltodextrin, polyethyleneimine, poly N - isopropylacrylamide, and copolymers thereof.

[0454] Example 25. The method according to any one of the preceding examples, wherein the polymer is selected from the group consisting of: dipropylene glycol, tripropylene glycol, polyethylene glycol, polypropylene glycol, poly(ethylene glycol - propylene glycol), poly(ethylene glycol - random - propylene glycol), polyvinylpyrrolidone, polyvinyl alcohol, polyvinylcaprolactam, polyvinyl methyl ether, and poly N - isopropylacrylamide.

[0455] Example 26. The method according to any one of the preceding examples, wherein the polymer is selected from the group consisting of: polyacrylamide, polyacrylic acid, and copolymers thereof.

[0456] Example 27. The method according to any one of the preceding examples, wherein the polymer is selected from the group consisting of: dextran, carboxymethyl dextran, dextran sulfate, hydroxypropyl dextran, and starch.

[0457] Example 28. The method according to any one of the preceding examples, wherein the polymer has an average molecular weight in the range of 200 - 1,000 Da, 200 - 35,000 Da, 425 - 2,000 Da, 400 - 35,000 Da, 980 - 12,000 Da, or 3,400 - 5,000,000 Da.

[0458] Example 29. The method according to any one of the preceding examples, wherein the polymer comprises ethylene oxide and propylene oxide units, and the polymer has an EO:PO ratio of 90:10 to 10:90.

[0459] Example 30. The method according to any one of the preceding examples, wherein the salt is dissolved in an aqueous solution at a concentration of 0.1% to 80% (w / v).

[0460] Example 31. The method according to any one of the preceding examples, wherein the salt comprises a cation selected from the group consisting of sodium, potassium, calcium, ammonium, lithium, magnesium, aluminum, cesium, barium, straight - chain or branched trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium, and tetrabutylammonium.

[0461] Example 32. The method according to any one of the preceding examples, wherein the salt comprises an anion selected from the group consisting of phosphate, hydrogen phosphate, dihydrogen phosphate, sulfate, sulfide, sulfite, bisulfate, carbonate, bicarbonate, acetate, nitrate, nitrite, sulfite, chloride, fluoride, chlorate, perchlorate, chlorite, hypochlorite, bromide, bromate, hypobromite, iodide, iodate, cyanate, thiocyanate, isothiocyanate, oxalate, formate, chromate, dichromate, permanganate, hydroxide, hydrogen ion, citrate, borate, and tris(hydroxymethyl)aminomethane.

[0462] Embodiment 33. The method according to any one of the preceding embodiments, wherein the salt is selected from the group consisting of aluminum chloride, aluminum phosphate, aluminum carbonate, magnesium chloride, magnesium phosphate and magnesium carbonate.

[0463] Embodiment 34. The method according to any one of the preceding embodiments, wherein the salt is selected from the group consisting of: NaCl, KCl, NH 4 Cl, Na 3 PO 4 , K 3 PO 4 、Na 2 SO 4 , K 2 HPO 4 , KH 2 PO 4 、Na 2 HPO 4 、NaH 2 PO 4 NH 4 ) 3 PO 4 NH 4 ) 2 HPO 4 NH 4 H 2 PO 4 , potassium citrate, (NH 4 ) 2 SO 4 , sodium citrate, sodium acetate, magnesium acetate, sodium oxalate, sodium borate and ammonium acetate.

[0464] Embodiment 35. The method according to any one of the preceding embodiments, wherein the salt is selected from the group consisting of: (NH4)3PO4, sodium formate, ammonium formate, K 2 CO 3 , KHCO 3 、Na 2 CO 3 、NaHCO 3 MgSO 4 MgCO 3 、CaCO 3 , CsOH, Cs 2 CO 3 、Ba(OH) 2 and BaCO 3 .

[0465] Embodiment 36. The method according to any one of the preceding embodiments, wherein the salt is selected from the group consisting of: NH 4 Cl, NH4 OH, tetramethylammonium chloride, tetrabutylammonium chloride, tetramethylammonium hydroxide, and tetrabutylammonium hydroxide.

[0466] Example 37. The method according to any one of the preceding examples, wherein the surfactant is dissolved in an aqueous solution at a concentration of 0.05% - 10% (w / v).

[0467] Example 38. The method according to any one of the preceding examples, wherein the surfactant is selected from the group consisting of: anionic surfactants, nonionic surfactants, cationic surfactants, and amphoteric surfactants; and wherein the anionic surfactant is carboxylate, sulfonate, petroleum sulfonate, alkylbenzene sulfonate, naphthalene sulfonate, olefin sulfonate, alkyl sulfate, sulfate, sulfated natural oil, sulfated natural fat, sulfated ester, sulfated alkanolamide, sulfated alkylphenol, ethoxylated alkylphenol, or sodium N-lauroylsarcosinate (NLS); the nonionic surfactant is ethoxylated fatty alcohol, polyoxyethylene surfactant, carboxylic acid ester, polyethylene glycol ester, sorbitan ester, fatty acid diol ester, carboxamide, single alkanolamine condensate, or polyoxyethylene fatty acid amide; the cationic surfactant is quaternary ammonium salt, amine with amide bond, polyoxyethylene alkylamine, polyoxyethylene alicyclic amine, n,n,n',n'-tetrasubstituted ethylenediamine, or 2-alkyl-1-hydroxyethyl-2-imidazoline; and the amphoteric surfactant is n-cocoyl-3-aminopropionic acid or its sodium salt, n-tallow-3-iminodipropionate or its disodium salt, n-carboxymethyl-n-dimethyl-n-9-octadecenyl ammonium hydroxide, or n-cocoylamidoethyl-n-hydroxyethylglycine or its sodium salt.

[0468] Example 40. The method according to any one of the preceding examples, wherein the binding buffer is a dispersant, and the dispersant contains anions selected from the group consisting of: thiocyanate, isothiocyanate, perchlorate, acetate, trichloroacetate, trifluoroacetate, chloride, and iodide.

[0469] Example 39. The method according to any one of the preceding examples, wherein the surfactant is Triton X-100, Triton X-114, Triton X-45, Tween 20, Igepal CA630, Brij 58, Brij O10, Brij L23, Pluronic L-61, Pluronic F-127, sodium dodecyl sulfate, sodium cholate, sodium deoxycholate, sodium N-lauroylsarcosinate (NLS), cetyltrimethylammonium bromide, or span 80.

[0470] Example 41. The method according to any one of the foregoing examples, wherein the binding buffer is a dissociating agent selected from the group consisting of guanidine hydrochloride (GHCl), guanidine thiocyanate, guanidine isothiocyanate (GITC), sodium thiocyanate, sodium iodide, sodium perchlorate, sodium trichloroacetate, sodium trifluoroacetate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, thiourea, and urea.

[0471] Example 42. The method according to any one of the foregoing examples, wherein the binding buffer is a dissociating agent selected from the group consisting of guanidine hydrochloride, magnesium chloride, and guanidine thiocyanate.

[0472] Example 43. The method according to any one of the foregoing examples, wherein the first ATPS composition comprises the polymer at a concentration of 5% - 80% (w / v), the salt at a concentration of 0.1% - 80% (w / v), and the surfactant at a concentration of 0% - 10% (w / v); the volume ratio of the first phase solution to the second phase solution is A:B, where A is 1 and B is 0.9 to 13.

[0473] Example 44. The method according to any one of the foregoing examples, wherein the second ATPS composition comprises the polymer at a concentration of 0.5% - 30% (w / v), the salt at a concentration of 0.1% - 10% (w / v), and the surfactant at a concentration of 0% - 10% (w / v); the volume ratio of the third phase solution to the fourth phase solution is C:D; and where C is 1 and D is 1 - 24.

[0474] Example 45. The method according to any one of the foregoing examples, wherein the first ATPS composition comprises 5% - 80% polymer (w / v) and 0.1% - 80% salt (w / v); and the second ATPS composition comprises 0.5% - 30% polymer (w / v) and 5% - 60% salt (w / v).

[0475] Example 46. The method according to any one of the foregoing examples, wherein the first ATPS composition comprises 5% - 60% polymer (w / v) and 0.5% - 50% salt (w / v); and the second ATPS composition comprises 0.5% - 30% polymer (w / v) and 5% - 60% salt (w / v).

[0476] Example 47. The method according to any one of the foregoing examples, wherein the first ATPS composition or the second ATPS composition is a polymer - polymer system comprising at least two polymers, and each polymer is dissolved in an aqueous solution at a concentration of 0.2% - 50% (w / v).

[0477] Example 48. The method according to any one of the preceding examples, wherein the first ATPS composition or the second ATPS composition is a micellar system comprising one or more surfactants, and each surfactant is dissolved in an aqueous solution at a concentration of 0.1% - 90% (w / v).

[0478] Example 49. The method according to any one of the preceding examples, wherein the first ATPS composition comprises 12% - 50% polymer (w / v) and 0.1% - 20% salt (w / v); and the second ATPS composition comprises 0.5% - 30% polymer (w / v) and 5% - 60% salt (w / v).

[0479] Example 50. The method according to any one of the preceding examples, wherein the first ATPS composition further comprises 0.5 - 2 mM ethylenediaminetetraacetic acid (EDTA) and 0.01% - 10% surfactant; and the second ATPS composition further comprises 0.5 - 2 mM EDTA.

[0480] Example 51. The method according to any one of the preceding examples, wherein the volume ratio between the first phase solution and the second phase solution of the first ATPS composition is A:B, where A is from 0.1 to 19 and B is 1.

[0481] Example 52. The method according to any one of the preceding examples, wherein A is from 0.9 to 13 and B is 1.

[0482] Example 53. The method according to any one of the preceding examples, wherein A:B is 13:1, 6:1 or 0.9:1.

[0483] Example 54. The method according to any one of the preceding examples, wherein the volume ratio between the third phase solution and the fourth phase solution of the second ATPS composition is C:D, where C is 1 and D is greater than or equal to 4.

[0484] Example 55. The method according to any one of the preceding examples, wherein D is from 4 to 100.

[0485] Example 56. The method according to any one of the preceding examples, wherein D is 24.

[0486] Example 57. The method according to any one of the preceding examples, wherein A is from 5 to 15; B is 1; C is 1; and D is from 20 to 100.

[0487] Example 58. An ATPS composition selected from the group consisting of A1, A2, A3, A4, AA1, AA2, AA3 and AA4.

[0488] Example 59. A method for treating bladder cancer in a patient in need thereof, the method comprising obtaining a urine sample from the patient, concentrating and purifying at least one target analyte from the urine sample according to the method of any one of the foregoing examples, analyzing the final solution, and treating the patient with a cancer therapeutic agent if the target analyte indicates that the patient has bladder cancer or is at risk of developing bladder cancer.

[0489] Example 60. A kit comprising a first ATPS composition selected from the group consisting of A1, A2, A3, and A4; a second ATPS composition selected from the group consisting of AA1, AA2, AA3, and AA4; and a binding buffer selected from the group consisting of B1, B2, and B3.

[0490] Example 61. The kit according to any one of the foregoing examples, further comprising an extraction column.

Claims

1. A method for concentrating and purifying one or more target analytes from a large volume of fluid sample, the method comprising the steps of: (a) dividing the large volume of fluid sample containing the one or more target analytes into at least two aliquots of sample solution; (b) preparing at least two first aqueous two-phase system (ATPS) compositions, wherein each first ATPS composition comprises a polymer, a salt, a surfactant, or any combination thereof dissolved in an aqueous solution to form a first-phase solution and a second-phase solution; (c) adding each aliquot of the sample solution prepared from the large volume of fluid sample containing the one or more target analytes to each first ATPS composition such that the one or more target analytes partition into each first-phase solution; (d) further processing each first-phase solution to form a final-phase solution; (e) mixing the final-phase solution with at least one purification composition to form a mixed solution; (f) contacting the mixed solution with a downstream purification system configured to selectively separate the one or more target analytes; and (g) collecting the one or more target analytes from the downstream purification system to obtain a final solution containing the concentrated and purified one or more target analytes.

2. The method according to claim 1, wherein the further processing in step (d) comprises collecting and combining each first-phase solution to form the final-phase solution.

3. The method according to claim 1, wherein the further processing in step (d) comprises the steps of (i) collecting each first-phase solution; (ii) mixing each first-phase solution with a second ATPS composition, wherein the second ATPS composition comprises a polymer, a salt, a surfactant, or a combination thereof dissolved in an aqueous solution to form a third-phase solution and a fourth-phase solution, such that the one or more target analytes partition into the third-phase solution and are concentrated in the third-phase solution; (iii) collecting and combining the third-phase solutions to form the final-phase solution.

4. The method according to claim 1, wherein the further processing in step (d) comprises the steps of (i) collecting and combining each first-phase solution to form a combined first-phase solution; (ii) mixing each combined first-phase solution with a second ATPS composition, wherein the second ATPS composition comprises a polymer, a salt, a surfactant, or a combination thereof dissolved in an aqueous solution to form a third-phase solution and a fourth-phase solution, such that the one or more target analytes partition into the third-phase solution and are concentrated in the third-phase solution; (iii) collecting the third-phase solution to form the final-phase solution.

5. The method according to any one of claims 1-4, wherein the purification composition is a binding buffer comprising at least one dissociating agent; the downstream purification system comprises a solid-phase medium; and step (f) further comprises the steps of: (i) contacting a portion of the mixed solution with the solid-phase medium such that the one or more target analytes bind to the solid-phase medium to form a solid-phase extraction complex; (ii) Agitate the solid-phase extraction complex and discard the flow-through or supernatant; and (iii) Optionally repeat steps (i) and (ii).

6. The method according to claim 5, wherein the solid-phase medium is a solid-phase extraction column.

7. The method according to claim 6, wherein the solid-phase extraction column is a centrifugal column.

8. The method according to claim 5, wherein the solid-phase medium is a plurality of beads.

9. The method according to claim 8, wherein the plurality of beads are magnetic beads, silica-based beads, carboxyl beads, hydroxyl beads, amine-coated beads, or any combination thereof.

10. The method according to any one of the preceding claims, further comprising the following steps: (h) Perform a diagnostic assay on the final solution to detect, quantify, characterize, or any combination thereof, the one or more target analytes.

11. A method for concentrating and purifying one or more target analytes from a large volume of fluid sample, the method comprising the following steps: (a) Divide the large volume of fluid sample containing the one or more target analytes into at least two aliquots of sample solution; (b) Prepare at least two aliquots of a first aqueous two-phase system (ATPS) composition, wherein the first ATPS composition comprises a polymer, salt, surfactant, or any combination thereof, dissolved in an aqueous solution to form a first-phase solution and a second-phase solution; (c) Add each aliquot of the sample solution containing the one or more target analytes to each aliquot of the first ATPS composition such that the one or more target analytes partition into the first-phase solution; (d) Collect the first-phase solution of the at least two aliquots of the first ATPS composition and mix the first-phase solution with a second ATPS composition, wherein the second ATPS composition comprises a polymer, salt, surfactant, or any combination thereof, dissolved in an aqueous solution to form a third-phase solution and a fourth-phase solution, such that the one or more target analytes partition into the third-phase solution and are concentrated in the third-phase solution; (e) Collect the third-phase solution and mix the third-phase solution with a binding buffer to form a mixed solution, wherein the binding buffer comprises at least one dissociating agent; (f) Load the mixed solution onto an extraction column configured to selectively extract and purify the one or more target analytes; (g) Elute and collect the one or more target analytes from the extraction column.

12. The method according to any one of the preceding claims, wherein the large volume of fluid sample is selected from the group consisting of: blood, plasma, serum, cerebrospinal fluid, urine, saliva, feces, tears, sputum, nasopharyngeal mucus, vaginal secretions, and penile secretions.

13. The method according to claim 12, wherein the large volume of fluid sample is urine.

14. The method according to any one of the preceding claims, wherein the large volume of fluid sample has a volume of at least 10 mL.

15. The method according to claim 14, wherein the large volume of fluid sample has a volume of 40 mL or greater.

16. The method according to claim 14, wherein each aliquot of the sample solution has a volume of up to 40 ml.

17. The method according to claim 16, wherein each aliquot of the sample solution has a volume of 10 to 40 mL.

18. The method according to any one of the preceding claims, wherein the one or more target analytes are selected from the group consisting of nucleic acids, proteins, antigens, biomolecules, sugar moieties, lipids, sterols, and any combination thereof.

19. The method according to claim 18, wherein the one or more target analytes are DNA.

20. The method according to claim 19, wherein the one or more target analytes are gDNA, cDNA, plasmid DNA, mitochondrial DNA, cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), circulating fetal DNA, microbial cell-free DNA, microRNA (miRNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), circular RNA, long non-coding RNA (lncRNA), or a combination thereof.

21. The method according to claim 20, wherein the one or more target analytes are cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA).

22. The method according to any one of the preceding claims, wherein the polymer is dissolved in an aqueous solution at a concentration of 0.2% - 80% (w / v).

23. The method according to any one of the preceding claims, wherein the polymer is selected from the group consisting of polyethers, polyimines, polyalkylene glycols, vinyl polymers, alkoxylated surfactants, polysaccharides, alkoxylated starches, alkoxylated celluloses, alkyl hydroxyalkyl celluloses, polyether-modified silicones, polyacrylamides, polyacrylic acids, and copolymers thereof.

24. The method according to any one of the preceding claims, wherein the polymer is selected from the group consisting of dipropylene glycol, tripropylene glycol, polyethylene glycol, polypropylene glycol, poly(ethylene glycol - propylene glycol), poly(ethylene glycol - random - propylene glycol), polyvinylpyrrolidone, polyvinyl alcohol, polyvinylcaprolactam, polyvinyl methyl ether, dextran, carboxymethyl dextran, dextran sulfate, hydroxypropyl dextran, starch, carboxymethyl cellulose, polyacrylic acid, hydroxypropyl cellulose, methyl cellulose, ethyl hydroxyethyl cellulose, maltodextrin, polyethyleneimine, poly(N - isopropylacrylamide), and copolymers thereof.

25. The method according to any one of the preceding claims, wherein the polymer is selected from the group consisting of dipropylene glycol, tripropylene glycol, polyethylene glycol, polypropylene glycol, poly(ethylene glycol - propylene glycol), poly(ethylene glycol - random - propylene glycol), polyvinylpyrrolidone, polyvinyl alcohol, polyvinylcaprolactam, polyvinyl methyl ether, and poly(N - isopropylacrylamide).

26. The method according to any one of the preceding claims, wherein the polymer is selected from the group consisting of polyacrylamides, polyacrylic acids, and copolymers thereof.

27. The method according to any one of the preceding claims, wherein the polymer is selected from the group consisting of: dextran, carboxymethyl dextran, dextran sulfate, hydroxypropyl dextran, and starch.

28. The method according to any one of the preceding claims, wherein the polymer has an average molecular weight in the range of 200 - 1,000 Da, 200 - 35,000 Da, 425 - 2,000 Da, 400 - 35,000 Da, 980 - 12,000 Da, or 3,400 - 5,000,000 Da.

29. The method according to any one of the preceding claims, wherein the polymer comprises ethylene oxide (EO) and propylene oxide (PO) units, and the polymer has an EO:PO ratio of 90:10 to 10:

90.

30. The method according to any one of the preceding claims, wherein the salt is dissolved in an aqueous solution at a concentration of 0.1% to 80% (w / v).

31. The method according to any one of the preceding claims, wherein the salt comprises a cation selected from the group consisting of: sodium, potassium, calcium, ammonium, lithium, magnesium, aluminum, cesium, barium, straight-chain or branched trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium, and tetrabutylammonium.

32. The method according to any one of the preceding claims, wherein the salt comprises an anion selected from the group consisting of: phosphate, hydrogen phosphate, dihydrogen phosphate, sulfate, sulfide, sulfite, bisulfate, carbonate, bicarbonate, acetate, nitrate, nitrite, sulfite, chloride, fluoride, chlorate, perchlorate, chlorite, hypochlorite, bromide, bromate, hypobromite, iodide, iodate, cyanate, thiocyanate, isothiocyanate, oxalate, formate, chromate, dichromate, permanganate, hydroxide, hydrogen ion, citrate, borate, and tris(hydroxymethyl)aminomethane.

33. The method according to any one of the preceding claims, wherein the salt is selected from the group consisting of: aluminum chloride, aluminum phosphate, aluminum carbonate, magnesium chloride, magnesium phosphate, and magnesium carbonate.

34. The method according to any one of the preceding claims, wherein the salt is selected from the group consisting of: NaCl, KCl, NH 4 Cl, Na 3 PO 4 、K 3 PO 4 、Na 2 SO 4 、K 2 HPO 4 、KH 2 PO 4 、Na 2 HPO 4 、NaH 2 PO 4 、(NH 4 ) 3 PO 4 、(NH 4 ) 2 HPO 4 、NH 4 H 2 PO 4 、 potassium citrate, (NH 4 ) 2 SO 4 、 sodium citrate, sodium acetate, magnesium acetate, sodium oxalate, sodium borate, and ammonium acetate.

35. The method according to any one of the preceding claims, wherein the salt is selected from the group consisting of: (NH 4 ) 3 PO 4 , sodium formate, ammonium formate, K 2 CO 3 , KHCO 3 , Na 2 CO 3 , NaHCO 3 , MgSO 4 , MgCO 3 , CaCO 3 , CsOH, Cs 2 CO 3 , Ba(OH) 2 and BaCO 3 .

36. The method according to any one of the preceding claims, wherein the salt is selected from the group consisting of: NH 4 Cl, NH 4 OH, tetramethylammonium chloride, tetrabutylammonium chloride, tetramethylammonium hydroxide, and tetrabutylammonium hydroxide.

37. The method according to any one of the preceding claims, wherein the surfactant is dissolved in an aqueous solution at a concentration of 0.05% - 10% (w / v).

38. The method according to any one of the preceding claims, wherein the surfactant is selected from the group consisting of: anionic surfactants, nonionic surfactants, cationic surfactants, and amphoteric surfactants; and wherein the anionic surfactant is carboxylate, sulfonate, petroleum sulfonate, alkylbenzene sulfonate, naphthalene sulfonate, olefin sulfonate, alkyl sulfate, sulfate, sulfated natural oil, sulfated natural fat, sulfated ester, sulfated alkanolamide, sulfated alkylphenol, ethoxylated alkylphenol, or sodium N-lauroylsarcosine (NLS); The non-ionic surfactant is ethoxylated fatty alcohol, polyoxyethylene surfactant, carboxylic acid ester, polyethylene glycol ester, sorbitan ester, fatty acid diol ester, carboxamide, single-chain alkanolamine condensate or polyoxyethylene fatty acid amide; The cationic surfactant is quaternary ammonium salt, amine with amide bond, polyoxyethylene alkylamine, polyoxyethylene alicyclic amine, N,N,N',N'-tetrasubstituted ethylenediamine or 2-alkyl-1-hydroxyethyl-2-imidazoline; and The zwitterionic surfactant is N-cocoyl-3-aminopropionic acid or its sodium salt, N-tallow-3-iminodipropionate or its disodium salt, N-carboxymethyl-N,N-dimethyl-N-9-octadecenyl ammonium hydroxide or N-cocoylamidoethyl-N-hydroxyethyl glycine or its sodium salt.

39. The method according to claim 38, wherein the surfactant is Triton X-100, Triton X-114, Triton X-45, Tween 20, Igepal CA630, Brij 58, Brij O10, Brij L23, Pluronic L-61, Pluronic F-127, sodium dodecyl sulfate, sodium cholate, sodium deoxycholate, sodium N-lauroylsarcosinate (NLS), cetyltrimethylammonium bromide or span 80.

40. The method according to claim 5, wherein the binding buffer is a chaotropic agent, and the chaotropic agent comprises anions selected from the group consisting of thiocyanate, isothiocyanate, perchlorate, acetate, trichloroacetate, trifluoroacetate, chloride and iodide.

41. The method according to claim 40, wherein the binding buffer is a chaotropic agent, and the chaotropic agent is selected from the group consisting of guanidine hydrochloride (GHCl), guanidine thiocyanate, guanidine isothiocyanate (GITC), sodium thiocyanate, sodium iodide, sodium perchlorate, sodium trichloroacetate, sodium trifluoroacetate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, thiourea and urea.

42. The method according to claim 40, wherein the binding buffer is a chaotropic agent, and the chaotropic agent is selected from the group consisting of guanidine hydrochloride, magnesium chloride and guanidine thiocyanate.

43. The method according to any one of the preceding claims, wherein the first ATPS composition comprises the polymer at a concentration of 5% - 80% (w / v), the salt at a concentration of 0.1% - 80% (w / v), and the surfactant at a concentration of 0% - 10% (w / v); the volume ratio of the first phase solution to the second phase solution is A:B, where A is 1, and B is 0.9 to 13.

44. The method according to claim 3, wherein the second ATPS composition comprises the polymer at a concentration of 0.5% - 30% (w / v), the salt at a concentration of 0.1% - 10% (w / v), and the surfactant at a concentration of 0% - 10% (w / v); the volume ratio of the third phase solution to the fourth phase solution is C:D; and wherein C is 1 and D is 1 - 24.

45. The method according to any one of the preceding claims, wherein the first ATPS composition comprises 5% - 80% polymer (w / v) and 0.1% - 80% salt (w / v); and the second ATPS composition comprises 0.5% - 30% polymer (w / v) and 5% - 60% salt (w / v).

46. The method according to any one of the preceding claims, wherein the first ATPS composition comprises 5% - 60% polymer (w / v) and 0.5% - 50% salt (w / v); and the second ATPS composition comprises 0.5% - 30% polymer (w / v) and 5% - 60% salt (w / v).

47. The method according to any one of the preceding claims, wherein the first ATPS composition or the second ATPS composition is a polymer - polymer system comprising at least two polymers, and each polymer is dissolved in an aqueous solution at a concentration of 0.2% - 50% (w / v).

48. The method according to any one of the preceding claims, wherein the first ATPS composition or the second ATPS composition is a micellar system comprising one or more surfactants, and each surfactant is dissolved in an aqueous solution at a concentration of 0.1% - 90% (w / v).

49. The method according to any one of the preceding claims, wherein the first ATPS composition comprises 12% - 50% polymer (w / v) and 0.1% - 20% salt (w / v); and the second ATPS composition comprises 0.5% - 30% polymer (w / v) and 5% - 60% salt (w / v).

50. The method according to any one of the preceding claims, wherein the first ATPS composition further comprises 0.5 - 2 mM ethylenediaminetetraacetic acid (EDTA) and 0.01% - 10% surfactant; and the second ATPS composition further comprises 0.5 - 2 mM EDTA.

51. The method according to any one of the preceding claims, wherein the volume ratio between the first phase solution and the second phase solution of the first ATPS composition is A:B, where A is 0.1 to 19 and B is 1.

52. The method according to claim 51, wherein A is 0.9 to 13 and B is 1.

53. The method according to claim 51, wherein A:B is 13:1, 6:1 or 0.9:

1.

54. The method according to any one of the preceding claims, wherein the volume ratio between the third phase solution and the fourth phase solution of the second ATPS composition is C:D, where C is 1 and D is greater than or equal to 4.

55. The method according to claim 54, wherein D is 4 - 100.

56. The method according to claim 54, wherein D is 24.

57. The method according to claim 54, wherein A is 5 - 15; B is 1; C is 1; and D is 20 - 100.

58. An ATPS composition selected from the group consisting of A1, A2, A3, A4, AA1, AA2, AA3, and AA4.

59. A method of treating bladder cancer in a patient in need thereof, the method comprising obtaining a urine sample from the patient, concentrating and purifying at least one target analyte from the urine sample according to the method of claim 1, analyzing the final solution, and treating the patient with a cancer therapeutic agent if the target analyte indicates that the patient has bladder cancer or is at risk of developing bladder cancer.

60. A kit comprising a first ATPS composition selected from the group consisting of A1, A2, A3, and A4; a second ATPS composition selected from the group consisting of AA1, AA2, AA3, and AA4; and a binding buffer selected from the group consisting of B1, B2, and B3.

61. The kit according to claim 60, further comprising an extraction column.

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