Methods for isolating target analytes from biological samples using ATPS and solid phase media

Abstract

The present disclosure relates to methods, compositions, and kits for enriching and purifying at least one target analyte from a clinical biological sample. In some embodiments, the methods involve one or more aqueous two-phase system (ATPS) compositions and at least one solid phase medium. Some embodiments provide kits including one or more ATPS compositions, a binding buffer, and a solid phase medium. Other embodiments provide methods for treating cancer or an infectious disease in a patient in need thereof.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 381,932, filed November 2, 2022, and U.S. Provisional Patent Application No. 63 / 381,933, filed November 2, 2022, the contents of all of the foregoing applications are incorporated herein by reference in their entirety for all purposes. [Technical Field]

[0002] This application relates to compositions, methods, and kits for concentrating and purifying one or more target analytes using an aqueous two-phase system (ATPS) and a solid phase medium. More particularly, this application relates to ATPS compositions, methods, and kits for concentrating and purifying at least one target analyte from a clinical biological sample. [Background technology]

[0003] Effectively concentrating and isolating target analytes (e.g., nucleic acids) from clinical biological samples for downstream applications (e.g., diagnostic tests) is a challenging task. For example, the concentrations of some target analytes in clinical biological samples are very low, and the yield of purified target analytes using conventional extraction methods is usually so low that subsequent analysis may not achieve sufficient diagnostic sensitivity and specificity. In some cases, the volume of binding buffer required for untreated sample digests is too large to be suitable for integration into conventional extraction workflows. Therefore, there is a need for improved methods for concentrating and purifying target analytes from clinical biological samples that are simple, stable, robust, and effective. Summary of the Invention

[0004] Disclosed herein are compositions, kits, methods and uses for isolating, concentrating and / or purifying target analytes (e.g., nucleic acids) using at least one aqueous two-phase system (ATPS) and a solid phase medium (e.g., beads or columns).

[0005] In some embodiments, a method is provided for concentrating and purifying at least one target analyte from a clinical biological sample, the method comprising: (a) combining the clinical biological sample with a first aqueous two-phase system (ATPS) composition comprising a polymer dissolved in an aqueous solution, a salt component comprising at least one salt, a surfactant, or any combination thereof, to form a target-rich phase solution and a target-poor phase solution; (b) collecting the target-rich phase; (c) optionally adding the target-rich phase to a second ATPS composition comprising a polymer, a salt component comprising at least one salt, a surfactant, or any combination thereof dissolved in an aqueous solution to form a second target-rich phase solution and a second target-poor phase solution, and collecting the second target-rich phase; (d) optionally mixing the target-rich phase from step (b) or the second target-rich phase from step (c) with a binding buffer to form a mixed solution; (e) contacting the target-rich phase from step (b), the second target-rich phase from step (c), or the mixed solution from step (d) with a solid phase medium configured to selectively bind the target analyte, causing the solid phase medium to bind the target analyte; and (f) eluting the target analyte from the solid phase medium using an elution solution and collecting, to obtain a final solution containing the concentrated and purified target analyte.

[0006] In some embodiments, there is provided a method of treating cancer or an infectious disease in a patient in need thereof, the method comprising: (i) obtaining a clinical biological sample from the patient; (ii) enriching and purifying at least one target analyte from the clinical biological sample according to the above embodiments; (iii) analyzing the final solution; and (iv) treating the patient if information obtained from the target analyte indicates that the patient has or is at risk for having cancer.

[0007] In some embodiments, a kit is provided, the kit comprising a first ATPS composition, a second ATPS composition, a binding buffer, and a solid phase medium.

[0008] advantage

[0009] Various embodiments of the present disclosure have many advantages.

[0010] In some embodiments, the disclosed methods, compositions, and kits provide a simple, inexpensive, and effective means for purifying target analytes from different clinical / biological samples of different volumes. The disclosed methods and kits involve an aqueous two-phase system (ATPS) in upstream processes, which effectively removes interfering salts and proteins in the sample matrix, which may prevent optimal binding of target analytes (e.g., DNA and free DNA (cfDNA)) to solid-phase media during the purification process. The disclosed methods and kits also surprisingly effectively minimize reagent usage without requiring specialized laboratory equipment.

[0011] In some examples, the methods described herein surprisingly effectively reduce the amount of binding buffer required for raw sample digests, thereby enabling these methods to be incorporated into conventional extraction workflows.

[0012] In some examples, surprisingly, although the upstream processes of the disclosed methods include additional ATPS steps, which are potential sources of target analyte loss (e.g., due to incomplete target partitioning in the ATPS), the recovery efficiency of target analytes using the disclosed methods is surprisingly high compared to the recovery efficiency of target analytes purified from clinical biological samples using methods without an ATPS.

[0013] One factor that influences target extraction efficiency is the composition of the ATPS, due to the different interactions between various components and the target. In some embodiments, the ATPS composition of the present disclosure is highly tunable depending on the target analyte being recovered, due to the different interactions between various components and the target analyte. The methods, compositions, and kits of the present disclosure can be suitable for a wide range of target analytes and clinical / biological samples.

[0014] By using the methods and kits disclosed in the present invention, target analytes (e.g., cfDNA) present in biological samples at very low concentrations can be effectively concentrated and purified, removing unwanted proteins and ions that may interfere with downstream detection.

[0015] In some embodiments, a large clinical / biological sample volume forms a large first ATPS bottom phase. In some embodiments, a second ATPS is used to concentrate the large first ATPS bottom phase into a smaller, more concentrated upper phase volume for more user-friendly downstream processing. In some embodiments, a two-step ATPS process is used on DNA to generate a more concentrated sample for detection.

[0016] In some embodiments, the methods and kits of the present invention are used in diagnostic tests for cancer (eg, bladder cancer) and infectious diseases (eg, human papillomavirus (HPV)).

[0017] These and other features and characteristics, and methods of use and functions of the associated components, will become more apparent from a consideration of the following detailed description and the appended claims in the accompanying drawings, all of which form a part hereof, wherein like reference numerals represent corresponding parts throughout the drawings. It is to be expressly understood, however, that the drawings are for the purpose of illustration and description only and are not intended as a definition of the limits of the claims. [Brief explanation of the drawings]

[0018] [Figure 1A]FIG. 1 shows an exemplary workflow for bead purification according to an exemplary embodiment described in Example 1a. [Figure 1B] FIG. 1 shows an exemplary workflow for column purification according to an exemplary embodiment described in Example 1b. [Figure 2A] FIG. 1 shows the mean CT values ​​of 145 bp DNA recovered from plasma using magnetic beads with and without a prior ATPS step according to the exemplary method described in Example 2a. [Figure 2B] FIG. 2a shows the mean CT values ​​of 2000 bp DNA recovered from plasma using magnetic beads with and without a prior ATPS step according to the exemplary method described in 2a. [Figure 2C] FIG. 1 shows the average CT values ​​of 145 bp DNA recovered from plasma using ATPS conditions according to Table 1.4 in Example 2b. [Figure 2D] FIG. 1 shows the average CT values ​​of 145 bp DNA recovered from plasma using ATPS conditions according to Table 1.5 in Example 2b. [Figure 2E] FIG. 1 shows the average CT values ​​of 145 bp DNA recovered from plasma using three different types of ATPS and magnetic beads according to Example 2c. [Figure 2F] FIG. 1 shows the average CT values ​​of 2000 bp DNA recovered from plasma using three different types of ATPS and magnetic beads according to Example 2c. [Figure 3A] FIG. 1 shows the mean CT values ​​of 145 bp DNA recovered from plasma using a spin column with and without a prior ATPS step according to the exemplary method described in Example 3a. [Figure 3B] FIG. 1 shows the mean CT values ​​of 2000 bp DNA recovered from plasma using a centrifugation column with and without a prior ATPS step according to the exemplary method described in Example 3a. [Figure 3C] FIG. 2 shows the average CT values ​​of 145 bp DNA recovered from plasma using ATPS conditions according to Table 2.3 in Example 3b. [Figure 3D] FIG. 2 shows the average CT values ​​of 145 bp DNA recovered from plasma using ATPS conditions according to Table 2.4 in Example 3b. [Figure 3E] FIG. 1 shows the mean CT values ​​of 145 bp DNA recovered from plasma using three different types of ATPS and centrifugal columns according to Example 3c. [Figure 3F] FIG. 1 shows the mean CT values ​​of 2000 bp DNA recovered from plasma using three different types of ATPS and centrifugal columns according to Example 3c. [Figure 4A] FIG. 1 shows the mean CT values ​​of 145 bp DNA recovered from urine using magnetic beads with and without a prior ATPS step according to the exemplary method described in Example 4a. [Figure 4B] FIG. 1 shows the mean CT values ​​of 2000 bp DNA recovered from urine using magnetic beads with and without a prior ATPS step according to the exemplary method described in Example 4a. [Figure 4C] FIG. 3 shows the average CT values ​​of 145 bp DNA recovered from urine using ATPS conditions according to Table 3.3 in Example 4b. [Figure 4D] FIG. 3 shows the average CT values ​​of 145 bp DNA recovered from urine using ATPS conditions according to Table 3.4 in Example 4b. [Figure 4E] FIG. 4 shows the average CT values ​​of 145 bp DNA recovered from urine using three different types of ATPS and magnetic beads according to Example 4c. [Figure 5A] FIG. 1 shows the mean CT values ​​of 145 bp DNA recovered from urine using a centrifugation column with and without a prior ATPS step according to the exemplary method described in Example 5a. [Figure 5B] FIG. 1 shows the mean CT values ​​of 2000 bp DNA recovered from urine using a centrifugation column with and without a prior ATPS step according to the exemplary method described in Example 5a. [Figure 5C]FIG. 5 shows the average CT values ​​of 145 bp DNA recovered from urine using ATPS conditions according to Table 4.3 in Example 5b. [Figure 5D] FIG. 5 shows the average CT values ​​of 145 bp DNA recovered from urine using ATPS conditions according to Table 4.4 in Example 5b. [Figure 5E] FIG. 5 shows the mean CT values ​​of 145 bp DNA recovered from urine using three different types of ATPS and centrifugation columns according to Example 5c. [Figure 6A] 10 is a combined data representation showing the mean CT values ​​of 145 bp DNA recovered from plasma and urine using magnetic beads according to Examples 2a-c and 4a-c. [Figure 6B] 10 is a combined data representation showing the mean CT values ​​of 145 bp DNA recovered from plasma and urine using centrifugal columns according to Examples 3a-c and 5a-c. [Figure 7A] FIG. 10 shows recovery using a 145 bp DNA spike-in (copies / uL) with conditions AF from Table 6. [Figure 7B] This figure shows the average concentration (copies / uL) of DNA recovered using the kits and conditions in Table 7. DETAILED DESCRIPTION OF THE INVENTION

[0019] Unless otherwise specified, the terms used in this specification (including technical and scientific terms) have the same meaning as commonly understood by those skilled in the art to which this invention belongs, and omit detailed descriptions of well-known functions and structures that may obscure the gist of the present invention.

[0020] As used in this specification and claims, "comprises" and "includes" mean including the following elements, but not excluding other elements.

[0021] As used in this specification and claims, the terms "comprises" (or any related form, e.g., "comprises" and "comprising"), "includes" (or any related form, e.g., "includes" or "including"), "containing" (or any related form, e.g., "contains" or "containing"), or "having" (or any related form, e.g., "has" or "having") mean the inclusion of the following elements, but do not exclude other elements. It is to be understood that for each embodiment of the term "comprises" (or any related form, e.g., "comprises" and "comprising"), "includes" (or any related form, e.g., "comprises" or "comprising"), or "containing" (or any related form, e.g., "contains" or "containing") used therein, the present disclosure / application further includes alternative embodiments in which the term "comprises", "comprises", "contains", or "having" therein is replaced with "consisting essentially of" or "consisting of". These alternative embodiments using "consisting of" or "consisting essentially of" are understood to be smaller scope embodiments of the "comprising," "including," or "containing" embodiments.

[0022] 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." The present disclosure / application includes the latter two embodiments even though they are not explicitly recited. It should be understood that the scope of the three embodiments listed above is different.

[0023] For clarity, "comprising," "including," and "containing," and any related forms, are open-ended terms, allowing for other elements or features than the required elements specified, whereas "consisting of..." is closed-ended terms, limited to the elements recited in the claim and does not include any element, step, or ingredient not specified in the claim.

[0024] "Consisting essentially of" limits the scope of a claim to certain materials, elements, or steps ("essential elements") that do not materially affect the essential characteristic(s) of the claimed invention. In some embodiments, the essential characteristic(s) are the basic and novel characteristic(s) of the claimed invention.

[0025] As used herein, the singular forms "a / an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0026] When a range is referred to in the specification, it is understood that the range includes at least each discrete point within the range. For example, in some embodiments, 1 to 7 refers to 1, 2, 3, 4, 5, 6, and 7. Unless otherwise specified, a range is intended to include all values ​​within that range, including integers, fractions, and portions thereof. For example, a range of 1 to 7 in the claims refers to a range that includes values ​​and subranges, e.g., 1, 1.5, 2 to 3, 6, and 7. Unless otherwise specified, a range is intended to include all values ​​within that range, including integers, fractions, and portions thereof.

[0027] As used herein, the term "about" is understood to be within normal tolerances in the art and not exceed ±10% of the stated value. By way of example only, about 50 means 45 to 55, including all values ​​therebetween. As used herein, the phrase "about" a particular value also includes that particular value, for example, about 50 includes 50.

[0028] As used herein, "aqueous" refers to the properties of a solvent / solute system in which the solvate has predominantly hydrophilic properties. Examples of aqueous solvent / solute systems include systems in which water or a water-containing composition is the primary solvent. The polymer and / or surfactant components (the use of which is described in the Examples) are "aqueous" because they form an aqueous phase when combined with a solvent (e.g., water). As will be understood by those skilled in the art, the term "liquid mixture" as used herein refers only to the combination of components as defined herein.

[0029] As used herein, aqueous two-phase system (ATPS) refers to a liquid-liquid separation system that can achieve analyte isolation or concentration through partitioning, where two phases, portions, regions, components, etc., interact differently with at least one analyte to which they are exposed or, optionally, dissolved. An ATPS forms when two immiscible phase-forming components with certain concentrations, such as a salt and a polymer, or two incompatible polymers (e.g., PEG and dextran), are mixed in an aqueous solution. ATPS methods are relatively inexpensive and scalable because they use two-phase partitioning to isolate analytes (e.g., nucleic acids) from contaminants.

[0030] As used herein, the term "isolated" refers to removing and thus altering an analyte from its original environment. For example, the provided isolated nucleic acid typically has fewer non-nucleic acid components (e.g., proteins, lipids) compared to the amount of components present in the original sample. A composition containing an isolated analyte (e.g., sample nucleic acid) can be substantially isolated (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% free of non-analyte components (e.g., non-nucleic acid components)).

[0031] As used herein, "concentrated" means that the mass ratio of the analyte of interest to the solution of suspended analyte is higher than the mass ratio of the analyte in its preconcentrated solution, e.g., it may be slightly higher, or more preferably at least 2-fold, 10-fold, or 100-fold higher.

[0032] As used herein, the term "polymer" refers to any polymer containing 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 made from two monomer types; similarly, a terpolymer refers to a polymer made from three monomer types. The two or three monomer types may be the same or different. Polymers also include various morphologies, including, but not limited to, linear polymers, branched polymers, random polymers, crosslinked polymers, and dendrimer systems. In some embodiments, the polymer further comprises other chemically modified equivalents, such as hydrophobically modified equivalents or silicone modified equivalents. For example, polyacrylamide polymer refers to any polymer containing at least one substituted or unsubstituted acrylamide unit, such as a homopolymer, copolymer, terpolymer, random copolymer, block copolymer, or terpolymer of polyacrylamide; the polyacrylamide may be a linear, branched, random, crosslinked polymer, or dendrimer of polyacrylamide; the polyacrylamide may be a hydrophobically modified polyacrylamide, or a silicone modified polyacrylamide.

[0033] In some embodiments, examples of polymers include, but are not limited to, polyethers, polyimides, polyacrylates, polyalkylene glycols, vinyl polymers, alkoxylated surfactants, polysaccharides, polyether-modified silicones, polyacrylamides, polyacrylic acids, and copolymers thereof. In some embodiments, the polymers are hydrophobically modified or silicone-modified. In some embodiments, the polymers are polyvinyl methyl ether, hydroxypropyl cellulose, or polyethyleneimine. In some embodiments, the polymers are PEG-PPG, polyvinylpyrrolidone, or methylcellulose. In some embodiments, the polymers are polyethylene glycol, polypropylene glycol, or dextran. In some embodiments, the polymers are alkylbenzene sulfonates, polyacrylamides, or isopropylacrylamide. In some embodiments, the polymers are acrylamides. In some embodiments, the polymers are dextran. In some embodiments, the polymers are polyalkylene glycols.

[0034] Examples of polyalkylene glycols (also referred to as "PAGs" or "poly(oxyalkylenes)" or "poly(alkylene oxides)") include, but are not limited to, hydrophobically modified polyalkylene glycols, poly(oxyalkylene) polymers, poly(oxyalkylene) copolymers, hydrophobically modified poly(oxyalkylene) copolymers, dipropylene glycol, tripropylene glycol, polyethylene glycol (also referred to as "PEGs"), and polypropylene glycol (also referred to as "PPGs"). In some embodiments, examples of PAG copolymers include, but are not limited to, poly(ethylene glycol-propylene glycol) (also referred to as "PEG-PPG" or "UCON") and poly(ethylene glycol-ran-propylene glycol) (also referred to as "PEG-ran-PPG"). In some embodiments, the PEG-PPG comprises a random copolymer, a block copolymer, or a combination thereof. In some embodiments, the PEG-PPG comprises a random copolymer and a block copolymer. In some embodiments, the PEG-PPG is PEG-ran-PPG.

[0035] As used herein, "vinyl polymer" refers to a group of polymers derived from substituted vinyl (HC=CHR) monomers. Examples of vinyl polymers include, but are not limited to, polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, and polyvinyl methyl ether.

[0036] 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 an alkoxylated starch, alkoxylated cellulose, or alkyl hydroxyalkyl cellulose.

[0037] Examples of polyacrylamides include, but are not limited to, poly N-isopropylacrylamide.

[0038] Examples of polyimides include, but are not limited to, polyethyleneimine.

[0039] 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 acid esters, polyethylene glycol esters, sorbitan esters, fatty acid glycol esters, carboxyamides, monoalkanolamine condensates, and polyoxyethylene fatty acid amides.

[0040] 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 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 a range between any two of the above molecular weights.

[0041] 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 a range between any two of the above PEG molecular weights. In some embodiments, the PPG has an average molecular weight within a range between any two PPG molecular weights listed above.

[0042] In some embodiments, the polymer comprises ethylene oxide (EO) and propylene oxide (PO) units, and the ethylene oxide:propylene oxide (EO:PO) ratio is from 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 a range between any two of the above ratios.

[0043] In some embodiments, the polymer is a PAG having an average molecular weight of about 980 to 12,000 Da and an EO:PO ratio of 50:50 to 75:25. Examples include, but are not limited to, PEG-PPG having average molecular weights 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 a range between any two of the above PEG-PPG molecular weights. In some embodiments, the PEG-PPG has an EO:PO ratio of 50:50 or 75:25. In some embodiments, the polymer is PEG-ran-PPG with an average molecular weight of about 2,500 or 12,000 Da and an EO:PO ratio of about 75:25.

[0044] In some embodiments, the polymer is a vinyl polymer having an average molecular weight of about 2,500 to 2,500,000 Da. Examples include, but are not limited to, polyvinylpyrrolidone having average molecular weights 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 a range between any two of the above molecular weights.

[0045] In some embodiments, the polymer is a polysaccharide and has an average molecular weight of about 6,000 to 5,000,000 Da, examples of which include, but are not limited to, dextrans having average molecular weights 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 a range between any two of the molecular weights above.

[0046] In some embodiments, the polymer is a polyether and has an average molecular weight of about 200 to 35,000 Da, examples of which include, but are not limited to, silicone-modified polyethers (or "polyether-modified silicones") having an average molecular weight of about 200 to 35,000 Da.

[0047] In some embodiments, the polymer is polyacrylamide and has an average molecular weight of 1,000 to 5,000,000 Da. Examples include, but are not limited to, polyacrylamides or poly(N-isopropylacrylamide) having average molecular weights of 1,000 Da, 2,000 Da, 5,000 Da, 10,000 Da, 40,000 Da, 85,000 Da, and 5,000,000 Da. In some embodiments, the polyacrylamide has an average molecular weight within a range between any two of the above molecular weights.

[0048] In some embodiments, the polymer is polyacrylic acid and has an average molecular weight of about 1,250 to 4,000,000 Da. Examples include polyacrylic acids having average molecular weights 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 polyacrylic acid has an average molecular weight within a range between any two of the above molecular weights.

[0049] 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 in which the cation is sodium, potassium, calcium, ammonium, lithium, magnesium, aluminum, cesium, barium, linear or branched chain trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium, and tetrabutylammonium, and / or salts in which the anion is phosphate, hydrogenphosphate, dihydrogenphosphate, sulfate, sulfide, sulfite, or the like. The salt may be selected from the group consisting of ammonium, ammonium ion ...

[0050] As used herein, "surfactant" includes, but is not limited to, anionic surfactants, nonionic surfactants, cationic surfactants, zwitterionic surfactants, or amphoteric surfactants.

[0051] Examples of anionic surfactants include, but are not limited to, carboxylates, sulfonates (also called "sulfonates"), petroleum sulfonates, alkyl benzene 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-lauroyl sarcosinate (NLS).

[0052] 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 glycol esters, carboxyamides, monoalkanolamine condensates, and polyoxyethylene fatty acid amides.

[0053] 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; Examples of amphoteric surfactants include, but are not limited to, n-cocoyl 3-aminopropionic acid or its sodium salt, n-tallow 3-iminodipropionic acid ester or its disodium salt, n-carboxymethyl n-dimethyl n-9 octadecenyl ammonium hydroxide, n-cocamidoethyl n-hydroxyethylglycine or its sodium salt, and N-lauroyl sarcosinate sodium (NLS).

[0054] In some embodiments, the surfactant comprises a polymer, such as PAG. In some embodiments, the surfactant comprises EO. x -PO y -EO x where EO refers to an ethylene oxide unit, PO refers to a propylene oxide unit, and x and y each represent the number of monomers. In some embodiments, x is 2 to 136. In some embodiments, y is 16 to 62. In some embodiments, an example surfactant is (C2H4O) 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), Brij 58, Brij O10, Brij L23, EO x -PO y -EOx , where x=2-136, y=16-62 (e.g., Pluronic L-61, Pluronic F-127), sodium dodecyl sulfate, sodium cholate, sodium deoxycholate, N-lauroyl sarcosine sodium salt (NLS), cetyltrimethylammonium bromide, or span 80.

[0055] As used herein, the terms "clinical biological sample," "clinical / biological sample," "clinical sample," or "biological sample" refer 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 biological samples include, but are not limited to, blood, plasma, urine, saliva, feces, cerebrospinal fluid (CSF), lymph, serum, sputum, peritoneal fluid, sweat, tears, nasal swabs, vaginal swabs, cervical swabs, semen, breast milk, and other bodily fluids. In some embodiments, the clinical biological sample is obtained from a biological sample treated with a degradation composition.

[0056] In some embodiments, the target analyte is a nucleic acid, a protein, an antigen, a biomolecule, a sugar moiety, a lipid, a sterol, an exosome, or any combination thereof. In some embodiments, examples of target analytes include, but are not limited to, genomic DNA (gDNA), cDNA, plasmid DNA, mitochondrial DNA, free DNA (cfDNA), circulating tumor DNA (ctDNA), circulating fetal DNA, microbial free DNA, microRNA (miRNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), circular RNA, long non-coding RNA (lncRNA), or a combination thereof.

[0057] As used herein, "free DNA" (cfDNA) is DNA that is present outside of cells, e.g., DNA that is present in a sample obtained from a subject (e.g., blood, plasma, serum, or urine).

[0058] When referring to a liquid sample in this disclosure, the terms "large volume," "large quantity," "high volume," or "bulk fluid" or "bulk 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 sample volume is 1-5 mL, 1-10 mL, 15-20 mL, 10-20 mL, 20-30 mL, or 30-40 mL. In some embodiments, the sample volume is at least 40 mL. In some embodiments, the sample volume ranges from 10 mL to 40 mL, 10 mL to 50 mL, 10 mL to 100 mL, 40 mL to 50 mL, 40 mL to 60 mL, 40 mL to 100 mL, 40 mL to 160 mL, 40 mL to 200 mL, 50 mL to 100 mL, 50 mL to 200 mL, or 50 mL to 300 mL. In some embodiments, the sample volume is 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.

[0059] As used herein, the term "chaotropic agent" refers to a substance that disrupts the hydrogen bond network between water molecules in a solution. In some embodiments, the chaotropic agent is a thiocyanate, isothiocyanate, perchlorate, acetate, trichloroacetate, trifluoroacetate, chloride, or iodide. Examples of chaotropic 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, and the like.

[0060] As used herein, the terms "Ct," "CT," "Ct value," or "CT value" refer to the cycle threshold and represent the cycle of a PCR amplification assay at which a signal (e.g., fluorescence) from a reporter, indicating amplicon production, first becomes detectable above background levels. In some embodiments, the CT value indirectly indicates the amount of target nucleic acid detected in a particular sample. Generally, the lower the CT value, the higher the amount of target nucleic acid in the sample, while the higher the CT value, the lower the amount of target nucleic acid in the sample.

[0061] As used herein, the term "solid-phase purification system" refers to a device, product, method, or process for purifying and / or selectively isolating a target analyte by chemical or physical means. In some embodiments, a solid-phase purification system comprises a solid-phase medium. In some embodiments, a solid-phase purification system refers to a magnetic bead workflow or a centrifugation column workflow.

[0062] As used herein, the term "solid phase medium" refers to a material that selectively isolates a target analyte by chemical or physical means. In some embodiments, the solid phase medium is provided in the form of a solid or a solid supported in a container. In some embodiments, the solid phase medium is a solid phase extraction column, e.g., a centrifugation column. In some embodiments, the solid phase medium is a plurality of beads, a silica resin, a silica membrane, silica gel, an alumina gel, a size exclusion resin, or an ion exchange resin.

[0063] As used herein, the terms "flow-through" and "supernatant" both refer to a liquid or solution isolated by or from a solid phase medium, which may be removed or isolated from the solid phase medium. In some examples, supernatant refers to the flow-through that passes through a column.

[0064] As used herein, the terms "disturbance" or "agitation" refer to the process of introducing physical forces and disturbances into a given system. In some embodiments, disturbance of a solid-phase extraction complex involves the introduction of centrifugal force, magnetic force, or a combination thereof, which causes isolation of one or more target analytes from or into the solid-phase medium or supernatant. In some embodiments, examples of disturbance or agitation include, but are not limited to, centrifugation, vacuuming, magnetization, vortexing, rotation, "spinning down," "spun down," swirling, rotating, shaking, stirring, rocking, and combinations thereof. In some embodiments, centrifugation or vortexing is achieved using a centrifuge or vortex. In some embodiments, vacuuming refers to contacting the solid-phase extraction complex with a vacuum manifold to generate a flow-through or supernatant. In some embodiments, agitation, e.g., magnetization, rotation, swirling, spinning, shaking, stirring, and shaking, is accomplished manually or by a suitable instrument (e.g., a benchtop microcentrifuge). In some embodiments, centrifugation and magnetization are performed simultaneously.

[0065] As used herein, the term "polymer-salt system" refers to any ATPS composition that consists essentially of a polymer and a salt component.

[0066] As used herein, the term "polymer-polymer system" refers to any ATPS composition that consists essentially of at least two polymers.

[0067] As used herein, the terms "micellar system" or "surfactant system" refer to any ATPS composition that consists essentially of at least two surfactants.

[0068] In some embodiments, the first ATPS composition or the second ATPS composition is a polymer-salt system comprising a polymer, a salt component, and a surfactant.

[0069] As used herein, the term "salt component" refers to a mixture having at least one salt. In some embodiments, the salt component comprises a salt and an acid.

[0070] Examples of the present invention

[0071] Example 1

[0072] Exemplary methods, compositions and kits for concentrating target analytes from bulk fluid samples

[0073] In one aspect, a method is provided for concentrating and purifying one or more target analytes from a bulk fluid sample, the method comprising: (a) preparing a first aqueous two-phase system (ATPS) composition, the first ATPS composition comprising a polymer, a salt, a surfactant, or a combination thereof, that dissolves in an aqueous solution to form a first phase solution and a second phase solution; (b) adding a sample solution prepared from the bulk fluid sample containing one or more target analytes to the first ATPS composition to apportion the one or more target analytes into the first phase solution; (c) collecting the first phase solution and mixing the first phase solution with a second ATPS composition, the second ATPS composition comprising a polymer, a salt, a surfactant, or a combination thereof, that dissolves in aqueous solution to form a third phase solution and a fourth phase solution, thereby allocating and concentrating the one or more target analytes 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, the binding buffer comprising at least one chaotropic 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 concentrated and purified target analytes.

[0074] In some embodiments, the sample solution is prepared by dividing the bulk fluid sample containing the one or more target analytes into at least two aliquots of sample solution, and dividing the first ATPS composition into at least two aliquots, wherein step (b) comprises: (i) adding aliquots of the sample solution prepared with the bulk fluid sample containing the one or more target analytes to aliquots of the first ATPS composition to apportion the one or more target analytes in the first phase solution; (ii) collecting and combining the first phase solution of at least two aliquots of said first ATPS composition to form the first phase solution for step (c).

[0075] In some embodiments, the extraction column is a centrifugation column, and wherein step (e) comprises: (i) loading a portion of the mixed solution onto an extraction column; (ii) centrifuging the extraction column and discarding the flow-through (also called "supernatant"); (iii) repeating steps (i) and (ii) above until all of the mixed solution has passed through the extraction column.

[0076] In some embodiments, the method further comprises: (g) subjecting said final solution to a diagnostic assay to detect and quantify said one or more target analytes.

[0077] In another aspect, a method is provided for concentrating and purifying one or more target analytes from a bulk fluid sample, the method comprising: (a) dividing a bulk fluid sample containing said 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, the first ATPS composition comprising a polymer, a salt, a surfactant, or a combination thereof, that dissolves in an aqueous solution to form a first phase solution and a second phase solution; (c) adding aliquots of the sample solution containing the one or more target analytes to aliquots of the first ATPS composition to apportion the one or more target analytes in the first phase solution; (d) collecting at least two aliquots of a first phase solution of the first ATPS composition, and mixing the first phase solution with a second ATPS composition, the second ATPS composition comprising a polymer, a salt, a surfactant, or a combination thereof, that dissolves in aqueous solution to form a third phase solution and a fourth phase solution, thereby allocating and concentrating the one or more target analytes 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, the binding buffer comprising at least one chaotropic 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 to obtain a final solution containing concentrated and purified target analytes.

[0078] In another aspect, a method is provided for concentrating and purifying one or more target analytes from a bulk fluid sample, the method comprising: (a) dividing a bulk fluid sample containing said 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, the first ATPS composition comprising a polymer, a salt, a surfactant, or a combination thereof, that dissolves in an aqueous solution to form a first phase solution and a second phase solution; (c) adding aliquots of the sample solution containing the one or more target analytes to aliquots of the first ATPS composition to apportion the one or more target analytes in the first phase solution; (d) collecting at least two aliquots of a first phase solution of the first ATPS composition, and mixing the first phase solution with a second ATPS composition, the second ATPS composition comprising a polymer, a salt, a surfactant, or a combination thereof, that dissolves in aqueous solution to form a third phase solution and a fourth phase solution, thereby allocating and concentrating the one or more target analytes 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, the binding buffer comprising at least one chaotropic agent; (f) loading a portion of the mixed solution onto an extraction column configured to selectively extract and purify the one or more target analytes; (g) centrifuging the extraction column and discarding the flow-through; (h) repeating steps (f) and (g) above until all of the mixed solution has passed through the extraction column; (i) eluting, collecting, and concentrating the one or more target analytes from the extraction column to obtain a final solution containing the one or more purified target analytes; (j) subjecting said final solution to a diagnostic assay to detect and quantify said one or more target analytes.

[0079] 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, for example, fecal material dissolved in a suitable volume (e.g., 500 mL) of water.

[0080] In some embodiments, the bulk fluid sample is urine.

[0081] In some embodiments, the volume of the bulk fluid sample is 40 mL or more, for example, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 200 mL, 300 mL, 400 mL, 500 mL or more.

[0082] 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.

[0083] In some embodiments, these target analytes are selected from the group consisting of nucleic acids, proteins, antigens, biomolecules, sugar moieties, lipids, sterols, and combinations thereof.

[0084] In some embodiments, the target analytes are DNA.

[0085] In some embodiments, these target analytes are free DNA or circulating tumor DNA.

[0086] In some embodiments, the polymer is dissolved in an aqueous solution at a concentration of 4% to 84% (w / w).

[0087] In some embodiments, the salt is dissolved in an aqueous solution at a concentration of 1% to 55% (w / w). In some embodiments, the salt is dissolved in an aqueous solution at a concentration of 8% to 55% (w / w).

[0088] In some embodiments, the surfactant is dissolved in the aqueous solution at a concentration of 0.05% to 10% (w / w). In some embodiments, the surfactant is dissolved in the aqueous solution at a concentration of 0.05% to 9.8% (w / w).

[0089] In another embodiment, an ATPS composition selected from the group consisting of A1, A2, A3, A4, A1, AA2, AA3, and AA4 in Table I is provided.

[0090] In another aspect, a kit is provided that includes a first ATPS composition selected from the group consisting of A1, A2, A3, and A4 in Table I, a second ATPS composition selected from the group consisting of A1, AA2, AA3, and AA4 in Table I, and a binding buffer selected from the group consisting of B1, B2, and B3 in Table I.

[0091] In some embodiments, the kit further comprises an extraction column.

[0092] Various ATPS systems that may be used 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.

[0093] In one embodiment, the first and / or second ATPS compositions comprise a polymer. In some embodiments, usable polymers include, but are not limited to, polyalkylene glycols (e.g., hydrophobically modified polyalkylene glycols), poly(oxyalkylene) polymers, poly(oxyalkylene) copolymers (e.g., hydrophobically modified poly(oxyalkylene) copolymers), polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, polyvinyl methyl ether, alkoxylated surfactants, alkoxylated starch, alkoxylated cellulose, alkyl hydroxyalkyl cellulose, silicone-modified polyethers, and poly-N-isopropylacrylamide and copolymers thereof. 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, polyimides, 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 selected from the group consisting of dipropylene glycol, tripropylene glycol, polyethylene glycol, polypropylene glycol, poly(ethylene glycol-propylene glycol), poly(ethylene glycol-ran-propylene glycol), polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, 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.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-ran-propylene glycol), polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, polyvinyl methyl ether, and poly(N-isopropylacrylamide). In some embodiments, the polymer is selected from the group consisting of polyacrylamide, polyacrylic acid, and copolymers thereof. 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 average molecular weight of the polymer is within the range of 200 to 1,000 Da, 200 to 35,000 Da, 425 to 2,000 Da, 400 to 35,000 Da, 980 to 12,000 Da, or 3,400 to 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.

[0094] In one embodiment, the polymer concentration of the first and / or second ATPS compositions is, by weight, in the range of about 4% to about 84% (w / w) of the total weight of the aqueous solution. In each example, the polymer solution may be 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, approximately 55% w / w, approximately 55.5% w / w, approximately 56% w / w, approximately 56.5% w / w, approximately 57% w / w, approximately 57.5% w / w, approximately 58% w / w, approximately 58.5% w / w, approximately 59% w / w, approximately 59.5% w / w, approximately 60% w / w, approximately 60.5% w / w, approximately 61% w / w, approximately 61.5% w / w, approximately 62% w / w, approximately 62.5% w / w, approximately 63% w / w, approximately 63.5% w / w, approximately 64% w / w, approximately 64.5% w / w, approximately 65% ​​w / w, approximately 65.5% w / w, approximately 66% w / w, approximately 66.5% w / w, approximately 67% w / w, approximately 67.5% w / w, approximately 68% w / w, approximately 68.5% w / w, approximately 69% w / w, approximately 69.5% w / w, approximately 70% w / w, approximately 70.5% w / w, approximately 71% w / w, approximately 71.5% w / w, approximately 72% w / w, approximately 72.5% w / w, approximately 73% The polymer solution may be selected from 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.

[0095] In one embodiment, the first and / or second ATPS compositions include salts, thereby forming salt solutions. In some embodiments, the salts include, but are not limited to, kosmotropic salts, chaotropic salts, cation-containing inorganic salts, such as linear or branched trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium, and tetrabutylammonium, and anion-containing inorganic salts, such as phosphates, sulfates, nitrates, chlorides, and bicarbonates. In another embodiment, the salts include NaCl, Na3PO4, K3PO4, Na2SO4, potassium citrate, (NH4)2SO4, sodium citrate, sodium acetate, or combinations thereof. Other salts, such as ammonium acetate, may also be used. In another embodiment, the salts may be selected from magnesium salts, lithium salts, sodium salts, potassium salts, cesium salts, zinc salts, and aluminum salts. In some embodiments, the salt may be selected from bromides, iodides, fluorides, carbonates, sulfates, citrates, carboxylates, borates, and phosphates. In some embodiments, the salt includes potassium phosphate. In some embodiments, the salt includes ammonium sulfate.

[0096] In one embodiment, the total salt concentration ranges from about 0.01% to about 90%. One skilled in the art will appreciate that the amount of salt required to form an aqueous two-phase system is affected by the molecular weight, concentration, and physical state of the polymer.

[0097] In each example, the salt concentration is about 1% to 55% w / w. In each example, 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, approximately 39% w / w, approximately 39.5% w / w, approximately 40% w / w, approximately 40.5% w / w, approximately 41% w / w, approximately 41.5% w / w, approximately 42% w / w, approximately 42.5% w / w, approximately 43% w / w, approximately 43.5% w / w, approximately 44% w / w, approximately 44.5% w / w, approximately 45% w / w, approximately 45.5% w / w, approximately 46% w / w, approximately 46.5% w / w, approximately 47% w / w, approximately 47.5% w / w, approximately 48% w / w, approximately 48.5% w / w, approximately 49% w / w, approximately 49.5% w / w, approximately 50% w / w, approximately 50.5% w / w, approximately 51% w / w, approximately 51.5% w / w, approximately 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, approximately 60% w / w, approximately 60.5% w / w, approximately 61% w / w, approximately 61.5% w / w, approximately 62% w / w, approximately 62.5% w / w, approximately 63% w / w, approximately 63.5% w / w, approximately 64% w / w, approximately 64.5% w / w, approximately 65% ​​w / w, approximately 65.5% w / w, approximately 66% w / w, approximately 66.5% w / w, approximately 67% w / w, approximately 67.5% w / w, approximately 68% w / w, approximately 68.5% w / w, approximately 69% w / w, approximately 69.5% w / w, approximately 70% w / w, approximately 70.5% w / w, approximately 71% w / w, approximately 71.5% w / w, approximately 72% w / w, approximately 72.5% w / w, approximately 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.

[0098] In one embodiment, the first and / or second ATPS compositions include a surfactant. In some embodiments, usable surfactants include Triton-X, Triton-114, Igepal CA-630, and Nonidet P-40, anionic surfactants (e.g., 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, and ethoxylated alkylphenols), nonionic surfactants (e.g., ethoxylated fatty alcohols, polyoxyethylene surfactants, carboxylic acid esters, polyethylene glycol esters, sorbitan esters, fatty acid ethylene glycol esters, carboxylic acid amides, and monoalkanolamine condensates), and the like. and polyoxyethylene fatty acid amides), cationic surfactants (e.g., quaternary ammonium salts, amines with an amide bond, polyoxyethylene alkyl and alicyclic amines, n,n,n',n' tetrasubstituted ethylenediamines, 2-alkyl 1-hydroxyethyl 2-imidazolines), and amphoteric surfactants (e.g., n-cocoyl 3-aminopropionic acid and its sodium salt, n-tallow 3-iminodipropionate and its disodium salt, n-carboxymethyl n-dimethyl n-9 octadecenyl ammonium hydroxide, n-cocamidoethyl n-hydroxyethylglycine and its sodium salt).

[0099] In one embodiment, the surfactant concentration of the first ATPS composition is in the range of about 0.05% w / w to about 10% w / w. In each example, 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, approx. 2.7% w / w, approx. 2.8% w / w, approx. 2.9% w / w, approx. 3% w / w, 3.1% w / w, approx. 3.2% w / w, approx. 3.3% w / w, approx. 3.4% w / w, approx. 3.5% w / w, approx. 3.6% w / w, approx. 3.7% w / w, approx. 3.8% w / w, approx. 3.9% w / w, approx. 4% w / w, approx. 4.1% w / w, approx. 4.2% w / w, approx. 4.3% w / w, approx. 4.4% w / w, approx. 4.5% w / w, approx. 4.6% w / w, approx. 4.7% w / w, approx. 4.8% w / w, approx. 4.9% w / w, approx. 5% w / w, approx. 5.1% w / w, approx. 5.2% w / w, approx. 5.3% w / w, approx. 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, approx. 8.3% w / w, approx. 8.4% w / w, approx. 8.5% w / w, approx. 8.6% w / w, approx. 8.7% w / w, approx. 8.8% w / w, approx. 8.9% w / w, approx. 9% w / w, 9.1% w / w, approx. 9.2% w / w, approx. 9.3% w / w, approx. 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.

[0100] 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.

[0101] In one embodiment, the concentration of the chaotropic agent in the binding buffer is in the range of about 0.1M to 8M. In each example, 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 5 M, about 6 M, about 7 M, about 8 M, about 9 M, about 10 M, about 11 M, about 12 M, about 13 M, about 14 M, about 15 M, about 16 M, about 17 M, about 18 M, about 19 M, about 20 M, about 21 M, about 22 M, about 23 M, about 24 M, about 25 M, about 26 M, about 27 M, about 28 M, about 29 M, about 30 M, about 31 M, about 32 M, about 33 M, about 34 M, about 35 M, about 36 M, about 37 M, about 38 M, about 39 M, about 40 M, about 41 M, about 42 M, about 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.

[0102] In one example, extraction columns that can be used include, but are not limited to, Epoch life science-EconoSpin Silica Membrane Mini Centrifugal Column-1920-250, HiBinds RNA mini-RNACOL-02, Corbition Silica Centrifugal Column-PC0054, PuroSpin Micro Silica Centrifugal Column-Luna Nano USP003, Purospin Nano Silica Centrifugal Column-Lunonano USP002, Qiagen RNEasy minElute, Qiagen minElute-700384 Qiagen GMBH and Qiagen mini.

[0103] Exemplary regimen for processing a 40 mL sample

[0104] The following equipment was used in the methods of Examples 1 and 2 below: 1. Centrifuge (e.g., for 15 and 50 mL conical tubes).

[0105] 2. Tabletop microcentrifuge (e.g., for 1 and 2 mL tubes).

[0106] 3. Pipettes and pipette tips (e.g., 20 μL, 200 μL, and 1000 μL capacity pipettes).

[0107] 4. Pipette aids and serological pipette tips (e.g., 5 mL, 10 mL, and 50 mL).

[0108] 5. Water bath (e.g., set to 37°C).

[0109] Specific examples of ATPS #1, ATPS #2 and binding buffers that can be used in the above regimen are shown in Table I below.

[0110] The following is an exemplary method of how to concentrate and isolate a target analyte from a biological sample having a volume of at least 40 mL. In this example, the sample is prepared according to the following steps: 40 mL of the biological sample was mixed with at least one lysis reagent (optional) using methods known to those skilled in the art to form one or more sample lysates.

[0111] A portion of the sample digest (22.6 mL) was transferred to a first tube containing a first ATPS composition (ATPS #1) to form ATPS #1 solution, and the remaining sample digest was poured into a second tube containing ATPS #1 to form ATPS #1 solution.

[0112] The two tubes containing ATPS #1 solution were vortexed thoroughly until homogenous and then centrifuged at 2300 RCF for 6 minutes each.

[0113] The bottom phase (e.g., approximately 3.5-5 mL volume) from each of the two ATPS #1 solutions was transferred (e.g., using a 10 mL serological pipette) to a tube containing the second ATPS composition (ATPS #2) to form ATPS #2 solution. The ATPS #2 solution was vortexed thoroughly until homogeneous and centrifuged at 2300 RCF for 6 minutes.

[0114] The entire upper phase of the ATPS #2 solution (approximately 400-600 μL) was transferred to a 5 mL microcentrifuge tube. Approximately 2 mL of binding buffer was added to the microcentrifuge tube containing the ATPS #2 upper phase, and the tube was vortexed briefly.

[0115] 800 μL of the ATPS #2 upper phase was transferred to a centrifuge column and centrifuged at 12,000 rcf for 30 seconds. The flow-through was discarded. This centrifuge column step (Step 6) was repeated until the entire sample had passed through the centrifuge column.

[0116] Wash buffer (500 uL) was added to the centrifuge column containing the mixture, and the centrifuge column containing the mixture was centrifuged at 12,000 rcf for 30 seconds. The flow-through was discarded.

[0117] The centrifuge column containing the mixture was centrifuged at 16,000 rcf for 2 minutes to remove any excess wash buffer.

[0118] The centrifugation column containing the mixture was placed into a new 1.5 mL collection tube. 1X TE buffer (20-100 uL) was pipetted onto the center of the centrifugation column membrane. The centrifugation column containing the mixture was incubated for 3 minutes and centrifuged at 12,000 rcf for 1 minute to elute the sample solution containing the concentrated target analytes. The sample solution was stored in a refrigerator at -20°C or lower for optional further processing.

[0119] The exemplary procedures above are merely examples and alternative methods and conditions may be used.

[0120] For example, in step 2, the 40 mL sample that was subjected to the disintegrant was roughly divided into two portions. However, the bulk fluid sample may be divided into many different arrangements.

[0121] Specific examples of ATPS #1, ATPS #2 and binding buffers that can be used in the above regimen are shown in Table I below.

[0122] Exemplary regimen amplified to 160 mL sample input

[0123] In this example, a large volume sample of approximately 160 mL was prepared by following these steps: 1. The 160 mL sample was divided into four individual 40 mL portions. Steps (1) through (7) in Example 1 above were performed for each 40 mL sample input.

[0124] 2. The upper phase of each ATPS #2 (approximately 400-600 uL) was transferred to a 15 mL microcentrifuge tube. The extraction step may be completed with a pipette, e.g., a P200 pipette set to 200 uL for the first extraction.

[0125] 3. For each 40 mL starting sample, Binding Buffer (2 mL) was added to the tube containing the ATPS #2 upper phase (i.e., amplification of a 160 mL sample requires 4x ATPS #2 and 8 mL of Binding Buffer). The tube was vortexed briefly.

[0126] 4. 800 uL of the mixture (starting sample and binding buffer) was transferred to a centrifuge column.

[0127] 5. The mixture was centrifuged at 12,000 rcf for 30 seconds.

[0128] 6. Discard the flow-through. Repeat steps 4-6 for the remaining sample until the entire mixture has passed through the centrifuge column. (For example, for an 800 uL centrifuge column volume, a starting sample load volume of 160 mL will require approximately 12 cycles.) 7. Wash buffer was added to the centrifuge column containing the mixture (500 uL).

[0129] 8. The centrifuge column containing the mixture was centrifuged at 12,000 rcf for 30 seconds.

[0130] 9. The flow-through was discarded.

[0131] 10. The centrifuge column containing the mixture was centrifuged at 16,000 rcf for 2 minutes to remove any excess wash buffer.

[0132] 11. The centrifuge column containing the mixture was placed into a new 1.5 mL collection tube.

[0133] 12. Pipet 1X TE buffer (20-100 uL) onto the center of the centrifuge column membrane.

[0134] 13. The centrifuge column containing the mixture was incubated for 3 minutes and centrifuged at 12,000 rcf for 1 minute to elute the sample solution containing the concentrated target analytes.

[0135] 14. The sample solution was stored in a refrigerator at -20°C or colder for optional further processing.

[0136] Performance Evaluation of Exemplary Extraction Kits

[0137] The performance of the methods and kits disclosed below may be evaluated by the following steps: 1. Prepare several large volume extraction kit components by modifying the following components: a. ATPS #1 i. Polymer ii. Salt iii. Surfactants b. ATPS #2 i. Polymer ii. Salt c. Extraction column d. Binding buffer i. Chaotropic Agents 2. Sample solutions were prepared to assess and spike in known amounts of target DNA.

[0138] 3. Extractions were performed using variations of the large-capacity extraction kit prepared in step 1 above and industry-standard extraction kits according to their designated procedures.

[0139] 4. Target DNA was quantified using standard qPCR or ddPCR procedures.

[0140] It was discovered that samples extracted using the disclosed method contained higher concentrations of target DNA compared to industry standard extraction kits.

[0141] Specific examples of ATPS #1, ATPS #2, and binding buffer are shown below.

[0142] Table I: Exemplary ATPS #1, ATPS #2 and binding buffer. [Table I]

[0143] Example 2

[0144] Exemplary Methods for Using ATPS with Magnetic Beads

[0145] In another aspect, a method is provided for concentrating and purifying one or more target analytes from a sample solution, the method comprising: (a) adding a sample solution containing the one or more target analytes to a first aqueous two-phase system (ATPS) to form a mixture that separates into a first phase and a second phase, and the one or more target analytes are concentrated in the first phase; (b) isolating the first phase containing the enriched one or more target analytes to obtain an enriched solution; (c) applying magnetic beads to the concentrated solution and allowing the magnetic beads to bind to the one or more target analytes to form bead-analyte complexes; (d) recovering the one or more target analytes from the bead-analyte complexes to obtain a final solution containing concentrated and purified one or more target analytes.

[0146] In some embodiments, step (b) comprises: (i) adding the isolated first phase containing the enriched target analyte(s) to a second ATPS to form a second mixture that separates into a third phase and a fourth phase, wherein the one or more target analytes are enriched in the third phase; (ii) isolating a third phase containing the enriched target analyte(s) in step (b) to form a concentrated solution for step (c).

[0147] In some embodiments, the concentrated solution for step (c) is obtained by mixing the concentrated solution of step (b) with a binding buffer comprising at least one chaotropic agent selected from n-butanol, ethanol, guanidine chloride, guanidine thiocyanate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea.

[0148] In some embodiments, step (d) comprises: a. mixing the bead-analyte complex with a fractionation buffer comprising a polymer, a salt, a surfactant, a chaotropic agent, or a combination thereof to form a fractionation solution, and releasing one or more target analytes smaller than a target size from the bead-analyte complex into the fractionation solution; b. immobilizing the bead-analyte complex using a magnetic support; c. isolating from the immobilized bead-analyte complexes the one or more target analytes smaller than the target size in a fractionation solution.

[0149] In some embodiments, step (d) comprises: (iv) adding the isolated one or more target analytes smaller than the target size to a second binding buffer containing at least one chaotropic agent selected from n-butanol, ethanol, guanidine chloride, guanidine thiocyanate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea; (v) applying magnetic beads to the mixture of the isolated one or more target analytes smaller than the target size and the second binding buffer, so that the magnetic beads bind to the one or more target analytes smaller than the target size to form second bead-analyte complexes; (vi) recovering the one or more target analytes from the second bead-analyte complex.

[0150] In some embodiments, the method further comprises: (e) subjecting said final solution to a diagnostic assay to detect and quantify said one or more target analytes.

[0151] 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 combinations thereof.

[0152] In some embodiments, the one or more target analytes is DNA.

[0153] In some embodiments, the one or more target analytes is free DNA or circulating tumor DNA.

[0154] In some embodiments, the first ATPS comprises a first ATPS component capable of forming a first phase and a second phase when the first ATPS component is dissolved in an aqueous solution, wherein the first ATPS component is selected from the group consisting of a polymer, a salt, a surfactant, and combinations thereof.

[0155] In some embodiments, the second ATPS comprises a second ATPS component capable of forming a third phase and a fourth phase when the second ATPS component dissolves in an aqueous solution, wherein the second ATPS component is selected from the group consisting of a polymer, a salt, a surfactant, and combinations thereof.

[0156] In some embodiments, the polymer is dissolved in an aqueous solution at a concentration of 4% to 84% (w / w).

[0157] In some embodiments, the salt is dissolved in an aqueous solution at a concentration of 1% to 80% (w / w). In some embodiments, the salt is dissolved in an aqueous solution at a concentration of 8% to 80% (w / w).

[0158] In some embodiments, the surfactant is dissolved in the aqueous solution at a concentration of 0.05% to 10% (w / w). In some embodiments, the surfactant is dissolved in the aqueous solution at a concentration of 0.05% to 9.8% (w / w).

[0159] In some embodiments, step (a) comprises: (i) embedding a porous material in a composition capable of forming a first ATPS; (ii) contacting the sample solution with a porous material embedded with components, the components forming a first phase and a second phase as the sample solution passes through the porous material.

[0160] In one aspect, a method is provided for concentrating and purifying one or more target analytes from a sample solution, the method comprising: (a) adding a sample solution containing the one or more target analytes to a first aqueous two-phase system (ATPS) to form a mixture that separates into a first phase and a second phase, and the one or more target analytes are concentrated in the first phase; (b) isolating the first phase containing the enriched target analyte(s); (c) adding the isolated first phase containing the enriched target analytes to a second ATPS to form a second mixture that separates into a third phase and a fourth phase, wherein the one or more target analytes are enriched in the third phase; (d) obtaining a concentrated solution by isolating a third phase containing the concentrated target analyte(s); (e) mixing the concentrated solution with a binding buffer containing at least one chaotropic agent selected from n-butanol, ethanol, guanidine chloride, guanidine thiocyanate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea; (f) applying magnetic beads to the mixture of the concentrated solution and binding buffer, and allowing the magnetic beads to bind to the one or more target analytes to form bead-analyte complexes; (g) combining the bead-analyte complex with a fractionation buffer comprising a polymer, a salt, a surfactant, a chaotropic agent, or a combination thereof to form a fractionation solution, and releasing one or more target analytes smaller than a target size from the bead-analyte complex into the fractionation solution; (h) immobilizing the bead-analyte complex using a magnetic support; (i) isolating one or more target analytes from the immobilized bead-analyte complexes that are smaller than the target size in a fractionation solution; (j) adding the isolated target analyte(s) smaller than the target size to a second binding buffer containing at least one chaotropic agent selected from n-butanol, ethanol, guanidine chloride, guanidine thiocyanate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea; (k) applying magnetic beads to a mixture of isolated target analytes or target analytes smaller than the target size and a second binding buffer, so that the magnetic beads bind to the target analytes or target analytes smaller than the target size to form second bead-analyte complexes; (l) recovering one or more target analytes smaller than the target size from the second bead-analyte complexes to obtain a final solution containing one or more concentrated and purified target analytes smaller than the target size; (m) subjecting said final solution to a diagnostic assay to detect and quantify said one or more target analytes smaller than the target size.

[0161] Various ATPS systems that may be used 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. Examples of polymers, salts, and surfactants and their concentrations that may be used in the ATPS systems (first and / or second ATPS) include, but are not limited to, those described in Example 1 above.

[0162] In some embodiments, the fractionation buffer comprises a polymer, a salt, a detergent, a chaotropic agent, or a combination thereof. In some embodiments, polymers, salts, detergents, and chaotropic agents that can be used include, but are not limited to, those described in Example 1 above.

[0163] In some embodiments, magnetic beads that can be used include, but are not limited to, those listed in Table II below.

[0164] Table II: Examples of magnetic beads [Table II]

[0165] Exemplary Regimen Without Fractionation Buffer

[0166] The following is an exemplary method of how to concentrate and purify a target analyte according to the present disclosure. In this example, the target analyte is DNA.

[0167] The regimen steps are performed as follows: 1. A desired volume of processed biological sample (e.g., plasma) (e.g., 2-3 mL) was added to the first ATPS (Solution B) to form Solution B'. Methods of processing biological samples include, but are not limited to, digestion to form sample digests.

[0168] 2. Solution B' was vortexed thoroughly (e.g., for about 10 seconds) until homogeneous, and then centrifuged at 2,300 xg for 6 minutes.

[0169] 3. The bottom phase of solution B' was transferred to a second ATPS (solution C) to form solution C'.

[0170] 4. Solution C' was vortexed thoroughly for 10 seconds until homogeneous, and then centrifuged at 7,000 xg for 1 minute.

[0171] 5. 800 uL of Binding Buffer (e.g., Binding Buffer BB1, BB2, or BB3) was added to a new 2 mL microcentrifuge tube.

[0172] 6. The upper phase of Solution C' containing the enriched target analytes was transferred to a microcentrifuge tube from step 5.

[0173] 7. The provided magnetic beads (e.g., magnetic beads selected from Table II, 12 μL) were vortexed before use and added to the microcentrifuge tube in step 6, allowing the magnetic beads to bind with the target analyte to form a bead-analyte complex.

[0174] 8. The microcentrifuge tube was tilted, rotated, incubated for 5 minutes, and then placed on a magnetic support for 2 minutes to immobilize the bead-analyte complexes to the tube wall.

[0175] 9. The supernatant in the microcentrifuge tube was pipetted and discarded, without disturbing the bead-analyte complex, and the microcentrifuge tube was removed from the magnetic support.

[0176] 10. 800 uL of binding buffer (e.g., Binding Buffer BB1, BB2, or BB3) was added to the microcentrifuge tube. The microcentrifuge tube was vortexed for 20 seconds and placed on a magnetic support for 2 minutes to immobilize the bead-analyte complexes to the tube wall.

[0177] 11. The supernatant in the microcentrifuge tube was pipetted and discarded, providing the bead-analyte complex was not disturbed.

[0178] 12. An appropriate wash buffer (e.g., 800 μL) known to those skilled in the art was added to the microcentrifuge tube, which was then spun 120 degrees per rotation on the magnetic support for a total of 720 degrees. After spinning, the supernatant in the microcentrifuge tube was pipetted and discarded, provided that the bead-analyte complex was not disturbed.

[0179] 13. Step 12 was repeated at least once.

[0180] 14. The microcentrifuge tube was then briefly spun with the hinge facing outwards to collect any wash buffer remaining in the tube.

[0181] 15. The microcentrifuge tube was placed back on the magnetic support and left for 1 minute to immobilize the bead-analyte complexes to the tube wall.

[0182] 16. Carefully discard all supernatant (e.g., using a 10 μl pipette tip) without disturbing the bead-analyte complex.

[0183] 17. The tube was opened and the bead-analyte complex was dried on a magnetic rack for 7 minutes.

[0184] 18. After drying, the microcentrifuge tube was then removed from the magnetic support.

[0185] 19. An appropriate elution buffer known to one of skill in the art (eg, 40 μL) was added directly to the bead-analyte complex (in the microcentrifuge tube).

[0186] 20. Using a pipette, resuspend the bead-analyte complexes by continuously stirring and pipetting up and down 5 times.

[0187] 21. Vortex the microcentrifuge tube briefly (e.g., for 15 seconds).

[0188] 22. The microcentrifuge tube was incubated at room temperature for 3 minutes.

[0189] 23. The microcentrifuge tube was placed on the magnetic support for 1 minute.

[0190] 24. The supernatant containing the purified target analyte was carefully collected into a clean max collection tube, provided that the magnetic beads were not disturbed.

[0191] 25. The purified target analyte was used immediately or stored at -20°C or below.

[0192] Exemplary Regimen Using Fractionation Buffer

[0193] The following is another exemplary method of how to enrich and purify a target analyte according to the present disclosure. In this example, the target analyte is DNA.

[0194] The regimen steps are performed as follows: 1. A desired volume of processed biological sample (e.g., plasma) (e.g., 2-3 mL) was added to the first ATPS (Solution B) to form Solution B'. Methods of processing biological samples include, but are not limited to, digestion to form sample digests.

[0195] 2. Solution B' was vortexed thoroughly for 10 seconds until homogeneous, and then centrifuged at 2,300 xg for 6 minutes.

[0196] 3. The bottom phase of solution B' was transferred to a second ATPS (solution C) to form solution C'.

[0197] 4. Solution C' was vortexed thoroughly for 10 seconds until homogeneous and centrifuged at 7,000 xg for 1 minute.

[0198] 5. 800 uL of Binding Buffer (e.g., Binding Buffer BB1, BB2, or BB3) was added to a new 2 mL microcentrifuge tube.

[0199] 6. The upper phase of Solution C' containing the concentrated target analyte was transferred to the tube containing Binding Buffer from step 5.

[0200] 7. The provided magnetic beads (e.g., magnetic beads selected from Table 1, 12 μL) were vortexed before use and added to the microcentrifuge tube in step 6, allowing the magnetic beads to bind with the target analyte to form a bead-analyte complex.

[0201] 8. The microcentrifuge tube was tilted, rotated, incubated for 5 minutes, and then placed on a magnetic support for 2 minutes to immobilize the bead-analyte complexes to the tube wall.

[0202] 9. The supernatant in the microcentrifuge tube was pipetted and discarded, without disturbing the bead-analyte complex, and the microcentrifuge tube was removed from the magnetic support.

[0203] 10. 300 uL of fractionation buffer (e.g., fractionation buffer F1, F2, or F3) was added to the microcentrifuge tube. The microcentrifuge tube was vortexed for 20 seconds, tilted and rotated, incubated for 5 minutes, and placed on a magnetic support for 2 minutes to immobilize the bead-analyte complexes to the tube wall and release target analytes smaller than the target size from the bead-analyte complexes into the supernatant.

[0204] 11. Add 600 uL of the second binding buffer (e.g., binding buffer BB1, BB2, or BB3) to a new 2 mL microcentrifuge tube.

[0205] 12. The supernatant from step 10 was transferred to the tube containing the second binding buffer from step 11.

[0206] 13. Pipette the mixture up and down to ensure all of the supernatant was transferred and mixed thoroughly with the second binding buffer.

[0207] 14. The provided magnetic beads (e.g., magnetic beads selected from Table 1, 6 μL) were vortexed before use and added to the microcentrifuge tube in step 13, allowing the magnetic beads to bind target analytes smaller than the target size to form second bead-analyte complexes.

[0208] 15. The microcentrifuge tube was tilted, rotated, incubated for 5 minutes, and then placed on a magnetic support for 4 minutes to immobilize the second bead-analyte complex to the tube wall.

[0209] 16. The supernatant in the microcentrifuge tube was pipetted and discarded, provided that the second bead-analyte complex was not disturbed.

[0210] 17. An appropriate wash buffer (e.g., 800 uL) known to those skilled in the art was added to the microcentrifuge tube, and the tube was spun 120 degrees at a time on the magnetic support for a total of 720 degrees. After spinning, the supernatant in the microcentrifuge tube was pipetted and discarded, provided that the second bead-analyte complex was not disturbed.

[0211] 18. Step 17 was repeated.

[0212] 19. The tube was opened and the beads were dried on a magnetic rack for 15 minutes.

[0213] 20. After drying, the tube was removed from the magnetic support.

[0214] 21. An appropriate elution buffer known to one of skill in the art (eg, 40 μL) was added directly to the second bead-analyte complex (in the microcentrifuge tube).

[0215] 22. Using a pipette, resuspend the second bead-analyte complex by continuously stirring and pipetting up and down five times.

[0216] 23. The microcentrifuge tube was briefly vortexed for 10 seconds.

[0217] 24. The microcentrifuge tube was incubated at room temperature for 3 minutes.

[0218] 25. The microcentrifuge tube was placed on the magnetic support for 1 minute.

[0219] 26. Supernatant containing purified target analytes smaller than the target size was carefully collected into a clean max collection tube, provided that the magnetic beads were not disturbed.

[0220] 27. Purified target analytes smaller than the target size were used immediately or stored at -20°C or below.

[0221] Performance evaluation of exemplary methods

[0222] The performance of the methods and kits disclosed below may be evaluated by the following steps: Prepare several magnetic bead extraction kit components by modifying the following components: Solution B (first ATPS) Polymer Salt surfactants Solution C (second ATPS) Polymer Salt Magnetic beads · Binding buffer Chaotropic agents Polymer Fractionation buffer Chaotropic agents Polymer Sample solutions were prepared to assess and spike in known amounts of target DNA.

[0223] Extraction was performed using a variation of the magnetic bead extraction kit prepared in step 1 above and an industry-standard extraction kit according to their designated procedures.

[0224] Target DNA was quantified using standard qPCR or ddPCR procedures.

[0225] Table III: Exemplary ATPS #1, ATPS #2 and binding buffers.

[0226] In each exemplary embodiment, Solution B, Solution C, Binding Buffer and Fractionation Buffer are selected from the embodiments shown below in various different combinations.

[0227] [Table III]

[0228] Example 3

[0229] In some embodiments, a method is provided for concentrating and purifying at least one target analyte from a clinical biological sample, the method comprising: (a) combining the clinical biological sample with a first aqueous two-phase system (ATPS) composition comprising a polymer dissolved in an aqueous solution, a salt component comprising at least one salt, a surfactant, or any combination thereof, to form a target-rich phase solution and a target-poor phase solution; (b) collecting the target-rich phase; and (c) optionally combining the target-rich phase with a second ATPS composition comprising a polymer dissolved in an aqueous solution, a salt component comprising at least one salt, a surfactant, or any combination thereof, to form a second target-rich phase solution and a second target-poor phase solution. (d) optionally mixing the target-rich phase from step (b) or the second target-rich phase from step (c) with a binding buffer to form a mixed solution; (e) contacting the target-rich phase from step (b), the second target-rich phase from step (c), or the mixed solution from step (d) with a solid phase medium configured to selectively bind the target analyte, causing the solid phase medium to bind the target analyte; and (f) eluting the target analyte from the solid phase medium using an elution solution and collecting, to obtain a final solution containing the concentrated and purified target analyte.

[0230] In some embodiments, the method further comprises washing the solid phase medium with one or more suitable solvents to remove impurities after step (e) and before step (f).

[0231] In some embodiments, the suitable solvent is a binding buffer.

[0232] In some embodiments, the method further comprises, prior to step (a), treating the clinical biological sample with a degradation composition.

[0233] In some embodiments, the binding buffer comprises a chaotropic agent comprising an anion selected from the group consisting of thiocyanate, isothiocyanate, peroxide, acetate, trichloroacetate, trifluoroacetate, chloride, or iodide.

[0234] In some embodiments, the binding buffer comprises a chaotropic 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.

[0235] In some embodiments, the binding buffer contains a chaotropic agent at a concentration of 2M to 7M.

[0236] In some embodiments, the binding buffer further comprises a polymer at a concentration of 5-20% (w / v).

[0237] In some embodiments, the clinical biological sample is blood, plasma, urine, saliva, feces, cerebrospinal fluid (CSF), lymph, serum, sputum, peritoneal fluid, sweat, tears, nasal swab, vaginal swab, cervical swab, semen, or breast milk.

[0238] In some embodiments, the clinical biological sample is <500 mL.

[0239] In some embodiments, the target analyte is selected from the group consisting of a nucleic acid, a protein, an antigen, a biomolecule, a sugar moiety, a lipid, a sterol, an exosome, and any combination thereof.

[0240] In some embodiments, the target analyte is a nucleic acid, and the nucleic acid is gDNA, cDNA, plasmid DNA, mitochondrial DNA, free DNA (cfDNA), circulating tumor DNA (ctDNA), circulating fetal DNA, microbial free DNA, microRNA (miRNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), circular RNA, long non-coding RNA (lncRNA), or a combination thereof.

[0241] In some embodiments, the nucleic acid is cfDNA, fetal free DNA, mitochondrial DNA, microbial free DNA, or ctDNA.

[0242] In some embodiments, step (e) includes (i) contacting a portion of the mixed solution with the solid phase medium to bind the target analyte to the solid phase medium and form a solid phase extraction complex; (ii) perturbing the solid phase extraction complex and discarding the flow-through (also referred to as "supernatant"); and (iii) optionally repeating steps (i) and (ii).

[0243] In some embodiments, a method or method according to claim 16 is provided, wherein the solid phase medium is a plurality of beads.

[0244] In some embodiments, the beads are selected from the group consisting of magnetic beads, silica-based beads, carboxy beads, hydroxy beads, and amine-coated beads.

[0245] In some embodiments, the solid phase extraction complex is a bead-analyte complex, the agitation is rotation, and the flow-through is a supernatant.

[0246] In some embodiments, the target analytes are nucleic acids smaller than a target size, the plurality of beads bind the target analytes and other nucleic acids, and the elution solution is a fractionation buffer that, upon contact with the beads during elution step (f), releases the target analytes while not releasing the other nucleic acids, thereby obtaining a final solution containing concentrated and purified one or more target analytes.

[0247] In some embodiments, the fractionation buffer comprises a polymer, a chaotropic agent, or any combination thereof.

[0248] In some embodiments, the solid phase medium is an extraction column.

[0249] In some embodiments, the extraction column is a centrifugation column.

[0250] In some embodiments, step (e) further comprises: (i) loading a portion of the mixed solution from step (d) onto the extraction column; (ii) centrifuging the extraction column and discarding the flow-through; and (iii) optionally repeating steps (i) and (ii) one or more times until all of the mixed solution has passed through the extraction column.

[0251] In some embodiments, the salt comprises a cation selected from the group consisting of sodium, potassium, calcium, ammonium, lithium, magnesium, aluminum, cesium, barium, straight or branched chain trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium, and tetrabutylammonium.

[0252] In some embodiments, the salt comprises an anion selected from the group consisting of phosphate, hydrogen phosphate, dihydrogen phosphate, sulfate, sulfide, sulfite, hydrogen sulfate, 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, polyacrylate, hydroxide, hydride, citrate, borate, and tris(hydroxymethyl)aminomethane.

[0253] In some embodiments, the polymer is selected from the group consisting of polyethers, polyimides, polyacrylates, polyalkylene glycols, vinyl polymers, alkoxylated surfactants, polysaccharides, alkoxylated starches, alkoxylated celluloses, alkylhydroxyalkyl celluloses, polyether-modified silicones, polyacrylamides, polyacrylic acids, and copolymers thereof, hi some embodiments, the polymer is hydrophobically or silicone-modified.

[0254] 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-ran-propylene glycol), polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, 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.

[0255] In some embodiments, the polymer is dipropylene glycol, tripropylene glycol, polyethylene glycol, polypropylene glycol, poly(ethylene glycol-propylene glycol), poly(ethylene glycol-ran-propylene glycol), polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, polyvinyl methyl ether, and poly N-isopropylacrylamide.

[0256] In some embodiments, the polymer is polyacrylamide, polyacrylic acid, or a copolymer thereof. In some embodiments, the polymer is dextran, carboxymethyl dextran, dextran sulfate, hydroxypropyl dextran, or starch.

[0257] In some embodiments, the average molecular weight of the polymer is in the range of 200 to 1,000 Da, 200 to 35,000 Da, 425 to 2,000 Da, 400 to 35,000 Da, 980 to 12,000 Da, or 3,400 to 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.

[0258] In some embodiments, the surfactant is selected from the group consisting of anionic surfactants, nonionic surfactants, cationic surfactants, and amphoteric surfactants, wherein the anionic surfactant is a carboxylate, a sulfonate, a petroleum sulfonate, an alkylbenzene sulfonate, a naphthalene sulfonate, an olefin sulfonate, an alkyl sulfate, a sulfate, a sulfated natural oil, a sulfated natural fat, a sulfated ester, a sulfated alkanolamide, a sulfated alkylphenol, an ethoxylated alkylphenol, or sodium N-lauroyl sarcosinate (NLS), and the nonionic surfactant is an ethoxylated fatty alcohol, a polyoxyethylene surfactant, a carboxylic acid ester, a polyethylene glycol ester, a sorbitan ester, or a sorbitan ester. The cationic surfactant is a quaternary ammonium salt, an amine having an amide bond, a polyoxyethylene alkylamine, a polyoxyethylene alicyclic amine, an n,n,n',n'-tetrasubstituted ethylenediamine, or a 2-alkyl 1-hydroxyethyl 2-imidazoline; and the amphoteric surfactant is n-cocoyl 3-aminopropionic acid or its sodium salt, n-tallow 3-iminodipropionic acid ester or its disodium salt, n-carboxymethyl n-dimethyl n-9 octadecenyl ammonium hydroxide, or n-cocamidoethyl n-hydroxyethylglycine or its sodium salt.

[0259] 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, sodium dodecyl sulfate, sodium cholate, sodium deoxycholate, sodium N-lauroylsarcosinate (NLS), cetyltrimethylammonium bromide, or span 80.

[0260] 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.

[0261] In some embodiments, the salt component comprises at least two salts, wherein at least one salt is selected from the group consisting of NaCl, KCl, NH4Cl, Na3PO4, K3PO4, Na2SO4, K2HPO4, KH2PO4, Na2HPO4, NaH2PO4, (NH4)3PO4, (NH4)2HPO4, NH4H2PO4, potassium citrate, (NH4)2SO4, sodium citrate, sodium acetate, magnesium acetate, sodium oxalate, sodium borate, ammonium acetate, (NH4)3PO4, sodium formate, ammonium formate, sodium polyacrylate, K2CO3, KHCO3, Na2CO3, NaHCO3, MgSO4, MgCO3, CaCO3, CsOH, Cs2CO3, Ba(OH)2, BaCO3, NH4Cl, NH4OH, tetramethyl ammonium chloride, tetrabutyl ammonium chloride, tetramethyl ammonium hydroxide, and tetrabutyl ammonium hydroxide.

[0262] In some embodiments, the polymer of the first ATPS composition or the second ATPS composition is soluble in an aqueous solution at a concentration of 0.5 to 80% (w / v).

[0263] In some embodiments, the polymer of the first ATPS composition or the second ATPS composition is soluble in an aqueous solution at a concentration of 0.2 to 50% (w / v).

[0264] In some embodiments, the salt component of the first ATPS composition or the second ATPS composition is soluble in an aqueous solution at a concentration of 0.1% to 80% (w / v).

[0265] In some embodiments, the surfactant of the first ATPS composition or the second ATPS composition is soluble in aqueous solution at a concentration of 0.1% to 50% (w / v).

[0266] In some embodiments, the first ATPS composition or the second ATPS composition is a polymer-salt system, a polymer-polymer system, or a micellar system.

[0267] In some embodiments, the first ATPS composition or the second ATPS composition is a polymer-salt system, wherein the polymer is soluble in aqueous solution at a concentration of 0.5% to 80% (w / v) and the salt component is soluble in aqueous solution at a concentration of 0.1% to 80% (w / v).

[0268] In some embodiments, the polymer of the first ATPS composition is soluble in aqueous solution at a concentration of 5% to 80% (w / v), and the salt component of the first ATPS composition is soluble in aqueous solution at a concentration of 0.1% to 80% (w / v), and the polymer of the second ATPS composition is soluble in aqueous solution at a concentration of 0.5% to 30% (w / v), and the salt component of the second ATPS composition is soluble in aqueous solution at a concentration of 5% to 60% (w / v).

[0269] In some embodiments, the first ATPS composition or the second ATPS composition further comprises at least one salt at a concentration of 0.01% to 10% (w / v) or at least one surfactant at a concentration of 0.01% to 10% (w / v).

[0270] In some embodiments, the polymer of the first ATPS composition is a polyether, a polyimide, a polyacrylate, a polyalkylene glycol, a vinyl polymer, an alkoxylated surfactant, a polysaccharide, an alkoxylated starch, an alkoxylated cellulose, an alkylhydroxyalkyl cellulose, a polyether-modified silicone, a polyacrylamide, or a polyacrylic acid, and the salt of the first ATPS composition is NaCl, KCl, NH4Cl, Na3PO4, K3PO4, Na2SO4, K2HPO4, KH2PO4, Na2HPO4 , NaH2PO4, (NH4)3PO4, (NH4)2HPO4, NH4H2PO4, potassium citrate, (NH4)2SO4, sodium citrate, sodium acetate, magnesium acetate, sodium oxalate, sodium borate, ammonium acetate, (NH4)3PO4, sodium formate, ammonium formate, sodium polyacrylate, K2CO3, KHCO3, Na2CO3, NaHCO3, MgSO4, MgCO3, CaCO3, CsOH, Cs2CO3, Ba(OH)2, BaCO3, NH4OH, tetramethylammonium chloride, tetrabutylammonium chloride the polymer of the second ATPS composition is a polyether, a polyimide, a polyacrylic acid ester, a polyalkylene glycol, a vinyl polymer, an alkoxylated surfactant, a polysaccharide, an alkoxylated starch, an alkoxylated cellulose, an alkylhydroxyalkyl cellulose, a polyether-modified silicone, a polyacrylamide, or a polyacrylic acid; and the salt of the second ATPS composition is NaCl, KCl, NH4Cl, Na3PO4, K3PO4, Na2SO4, K2HPO4, KH2PO4, Na2HPO4, NaH2PO4, (NH4)3PO4, (NH4)2HPO4, NH4H2PO4, potassium citrate, (NH4)2SO4, sodium citrate, sodium acetate, magnesium acetate, sodium oxalate, sodium borate, ammonium acetate, (NH4)3PO4, sodium formate, ammonium formate, sodium polyacrylate, K2CO3, KHCO3, Na2CO3, NaHCO3, MgSO4, MgCO3, CaCO3, CsOH, Cs2CO3, Ba(OH)2,The polymer may be BaCO3, NH4OH, tetramethyl ammonium chloride, tetrabutyl ammonium chloride, tetramethyl ammonium hydroxide, or tetrabutyl ammonium hydroxide. In some embodiments, the first ATPS composition or the second ATPS composition is a polymer-polymer system comprising at least two polymers, and each polymer is soluble in aqueous solution at a concentration of 0.2 to 50% (w / v).

[0271] In some embodiments, the first ATPS composition or the second ATPS composition further comprises at least one salt at a concentration of 0.01% to 10% (w / v) or at least one surfactant at a concentration of 0.01% to 10% (w / v).

[0272] In some embodiments, the at least one salt comprises a cation selected from the group consisting of sodium, potassium, calcium, ammonium, lithium, magnesium, aluminum, cesium, barium, linear or branched trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium, and tetrabutylammonium, and at least one surfactant selected from the group consisting of anionic surfactants, nonionic surfactants, cationic surfactants, and amphoteric surfactants, wherein the anionic surfactant is selected from the group consisting of 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, or is N-lauroyl sarcosinate sodium (NLS), the nonionic surfactant is an ethoxylated fatty alcohol, a polyoxyethylene surfactant, a carboxylic acid ester, a polyethylene glycol ester, a sorbitan ester, a fatty acid glycol ester, a carboxyamide, a monoalkanolamine condensate, or a polyoxyethylene fatty acid amide, the cationic surfactant is a quaternary ammonium salt, an amine having an amide bond, a polyoxyethylene alkylamine, a polyoxyethylene alicyclic amine, an n,n,n',n'-tetrasubstituted ethylenediamine, or a 2-alkyl 1-hydroxyethyl 2-imidazoline, and the amphoteric surfactant is n-cocoyl 3-aminopropionic acid or its sodium salt, n-tallow 3-iminodipropionic acid ester or its disodium salt, n-carboxymethyl n-dimethyl n-9 octadecenyl ammonium hydroxide, or n-cocamidoethyl n-hydroxyethylglycine or its sodium salt.

[0273] In some embodiments, the first ATPS composition or the second ATPS composition is a micellar system comprising at least two surfactants, and each surfactant is soluble in aqueous solution at a concentration of 0.1% to 50% (w / v).

[0274] In some embodiments, the first ATPS composition or the second ATPS composition further comprises at least one salt at a concentration of 0.01% to 30% (w / v).

[0275] In some embodiments, the at least one salt comprises a cation selected from the group consisting of sodium, potassium, calcium, ammonium, lithium, magnesium, aluminum, cesium, barium, straight or branched chain trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium, and tetrabutylammonium.

[0276] In some embodiments, the polymer of the first ATPS composition is 6% to 40% PEG 600 (w / v), and the salt components of the first ATPS composition are 0.1% to 12% KHPO (w / v) and 0.1% to 20% KHPO (w / v).

[0277] In some embodiments, the polymer of the second ATPS composition is 2% to 20% PEG (w / v), and the salt components of the second ATPS composition are 1% to 50% KHPO (w / v) and 0.1% to 40% KHPO (w / v).

[0278] In some embodiments, the polymer of the first ATPS composition is 6% to 36% PEG 600 (w / v), and the salt components of the first ATPS composition are 2% to 12% Na2SO4 (w / v), 0.01 to 0.1 M Na2HPO4, and 0.01 to 0.1 M NaH2PO4.

[0279] In some embodiments, the polymer of the second ATPS composition is 2% to 12% PEG (w / v), and the salt components of the second ATPS composition are 5% to 20% NaSO (w / v), 0.01 to 0.1 M NaHPO, and 0.01 to 0.1 M NaHPO.

[0280] In some embodiments, the polymer of the first ATPS composition is 20% to 40% PEG 1000 (w / v), and the salt components of the first ATPS composition are 0.1% to 8% KHPO (w / v) and 0.1% to 12% KHPO (w / v).

[0281] In some embodiments, the polymer of the second ATPS composition is 3% to 13% PEG (w / v), and the salt components of the second ATPS composition are 1% to 5% KHPO (w / v) and 0.1% to 20% KHPO (w / v).

[0282] In some embodiments, the polymer of the first ATPS composition is 10% to 35% PEG 3000 (w / v), and the salt components of the first ATPS composition are 0.1% to 6% KHPO (w / v) and 0.1% to 8% KHPO (w / v).

[0283] In some embodiments, the polymer of the second ATPS composition is 5% to 17% PEG (w / v), and the salt components of the second ATPS composition are 0.1% to 12% KHPO (w / v) and 0.1% to 16% KHPO (w / v).

[0284] In some embodiments, the polymer of the first ATPS composition is 10% to 32% PEG 8000 (w / v), and the salt components of the first ATPS composition are 0.1% to 6% KHPO (w / v) and 0.1% to 9% KHPO (w / v).

[0285] In some embodiments, the polymer of the second ATPS composition is 2% to 9% PEG (w / v), and the salt components of the second ATPS composition are 1% to 30% KHPO (w / v) and 1% to 40% KHPO (w / v).

[0286] In some embodiments, the polymer of the first ATPS composition is 40% to 60% PPG 425 (w / v), and the salt components of the first ATPS composition are 0.1% to 5% KHPO (w / v) and 0.1% to 5% KHPO (w / v).

[0287] In some embodiments, the polymer of the second ATPS composition is 5% to 15% PEG (w / v), and the salt components of the second ATPS composition are 0.1% to 20% KHPO (w / v) and 0.1% to 20% KHPO (w / v).

[0288] In some embodiments, the polymer of the first ATPS composition is 10%-50% PEG-ran-PPG, having an average molecular weight of about 12,000 Da, and an EO:PO ratio of about 75:25 (w / v), and the salt components of the first ATPS composition are 0.1%-5% KHPO (w / v) and 0.1%-12% KHPO (w / v).

[0289] In some embodiments, the polymer of the second ATPS composition is 2% to 12% PEG (w / v), and the salt components of the second ATPS composition are 0.1% to 30% KHPO (w / v) and 1% to 40% KHPO (w / v).

[0290] In some embodiments, the polymer of the first ATPS composition is 10% to 32% PEG-ran-PPG, having an average molecular weight of about 2,500 Da and an EO:PO ratio of about 75:25 (w / v), and the salt component of the first ATPS composition is 0.1% to 5% sodium citrate (w / v) and 0.1% to 12% citric acid (w / v).

[0291] In some embodiments, the polymer of the second ATPS composition is 3% to 13% PEG (w / v), and the salt components of the second ATPS composition are 0.1% to 20% KHPO (w / v) and 1% to 36% KHPO (w / v).

[0292] In some embodiments, the first ATPS composition comprises 8%-18% PEG 600 (w / v), 7%-20% KHPO (w / v), and 0.1%-5% KHPO, and the second ATPS composition comprises 8%-25% PEG 200 (w / v), 40%-50% KHPO (w / v), and 4%-16% KHPO.

[0293] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition comprising 10%-25% polyvinylpyrrolidone 3,500 Da (w / v) and 15%-20% potassium phosphate (w / v).

[0294] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition comprising 25%-35% polyvinylpyrrolidone 3,500 Da (w / v) and 10%-15% potassium phosphate (w / v).

[0295] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition comprising 35%-50% polyvinylpyrrolidone 3,500 Da (w / v) and 5%-10% potassium phosphate (w / v).

[0296] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition, comprising 2%-12% PEG 600 (w / v) and 35%-50% sodium phosphate (w / v).

[0297] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition, comprising 12%-18% PEG 600 (w / v) and 20%-35% sodium phosphate (w / v).

[0298] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition, comprising 18%-25% PEG 600 (w / v) and 10%-20% sodium phosphate (w / v).

[0299] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition comprising 5%-20% polyvinylpyrrolidone 10,000 Da (w / v) and 30%-40% sodium citrate (w / v).

[0300] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition comprising 20%-35% polyvinylpyrrolidone 10,000 Da (w / v) and 20%-30% sodium citrate (w / v).

[0301] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition comprising 35%-50% polyvinylpyrrolidone 10,000 Da (w / v) and 10%-20% sodium citrate (w / v).

[0302] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition, comprising 2%-10% PEG 300 (w / v) and 49%-60% sodium citrate (w / v).

[0303] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition and comprises 10%-18% PEG 300 (w / v) and 37%-49% sodium citrate (w / v).

[0304] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition and comprises 18%-25% PEG 300 (w / v) and 20%-37% sodium citrate (w / v).

[0305] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition comprising 10%-35% PPG 425 (w / v) and 14%-20% sodium sulfate (w / v).

[0306] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition comprising 35%-50% PPG 425 (w / v) and 8%-14% sodium sulfate (w / v).

[0307] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition comprising 55%-70% PPG 425 (w / v) and 3%-8% sodium sulfate (w / v).

[0308] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition comprising 5%-16% PPG 425 (w / v) and 11%-15% sodium polyacrylate 240,000 (w / v).

[0309] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition and includes 16%-24% PPG 425 (w / v) and 5%-11% sodium polyacrylate 240,000 (w / v).

[0310] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition comprising 24%-40% PPG 425 (w / v) and 0.5%-5% sodium polyacrylate 240,000 (w / v).

[0311] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition, comprising 4%-12% PEG 8000 (w / v) and 21%-30% sodium carbonate (w / v).

[0312] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition and comprises 12%-21% PEG 8000 (w / v) and 11%-21% sodium carbonate (w / v).

[0313] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition and comprises 21%-30% PEG 8000 (w / v) and 3%-11% sodium carbonate (w / v).

[0314] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition, comprising 1%-5% PEG 8000 (w / v) and 14%-20% sodium polyacrylate 16,000 (w / v).

[0315] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition and includes 6%-11% PEG 8000 (w / v) and 7%-14% sodium polyacrylate 16,000 (w / v).

[0316] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition and includes 11%-15% PEG 8000 (w / v) and 2%-7% sodium polyacrylate 16,000 (w / v).

[0317] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition, comprising 10%-25% polyvinyl alcohol 3,500 Da (w / v) and 7%-10% potassium phosphate (w / v).

[0318] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition, comprising 25%-35% polyvinyl alcohol 3,500 Da (w / v) and 4%-7% potassium phosphate (w / v).

[0319] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition, comprising 35%-50% polyvinyl alcohol 3,500 Da (w / v) and 2%-4% potassium phosphate (w / v).

[0320] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition, comprising 2%-5% PEG 400 (w / v) and 34%-50% potassium phosphate (w / v).

[0321] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition, comprising 6%-11% PEG 400 (w / v) and 22%-34% potassium phosphate (w / v).

[0322] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition, comprising 11%-15% PEG 400 (w / v) and 10%-22% potassium phosphate (w / v).

[0323] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition, comprising 4%-12% polyvinyl alcohol 8,000 Da (w / v) and 21%-30% sodium citrate (w / v).

[0324] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition, comprising 12%-21% polyvinyl alcohol 8,000 Da (w / v) and 11-21% sodium citrate (w / v).

[0325] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition, comprising 21%-30% polyvinyl alcohol 8,000 Da (w / v) and 3%-11% sodium citrate (w / v).

[0326] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition and comprises 1%-5% PPG 425 (w / v) and 20%-30% sodium citrate (w / v).

[0327] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition and comprises 6%-11% PPG 425 (w / v) and 9%-20% sodium citrate (w / v).

[0328] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition and comprises 11% to 15% PPG 425 (w / v) and 0.5% to 9% sodium citrate (w / v).

[0329] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition comprising 2%-12% polyvinyl alcohol 20,000 Da (w / v) and 21%-30% sodium citrate (w / v).

[0330] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition comprising 12%-21% polyvinyl alcohol 20,000 Da (w / v) and 11%-21% sodium citrate (w / v).

[0331] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition, comprising 21%-30% polyvinyl alcohol 20,000 Da (w / v) and 3%-11% sodium citrate (w / v).

[0332] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition, comprising 0.5%-18% PEG-ran-PPG 12,000 Da (w / v) and 14%-20% sodium sulfate (w / v).

[0333] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition and comprises 18%-35% PEG-ran-PPG 12,000 Da (w / v) and 7%-14% sodium sulfate (w / v).

[0334] In some embodiments, the first or second ATPS composition is a polymer-salt-based ATPS composition comprising 35%-50% PEG-ran-PPG 12,000 Da (w / v) and 2%-7% sodium sulfate (w / v).

[0335] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 3%-18% PEG 6000 (w / v), 1%-6% dextran 450-650 kDa (w / v), and 5-50 mM NaHPO / NaHPO.

[0336] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 1%-9% PEG 6000 (w / v) and 17%-25% dextran 2,200,000 Da (w / v).

[0337] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 9%-18% PEG 6000 (w / v) and 9%-17% dextran 2,200,000 Da (w / v).

[0338] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 18%-25% PEG 6000 (w / v) and 0.5%-9% dextran 2,200,000 Da (w / v).

[0339] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 1%-9% PEG 6000 (w / v) and 20%-30% dextran 460,000 Da (w / v).

[0340] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 9%-18% PEG 6000 (w / v) and 9%-20% dextran 460,000 Da (w / v).

[0341] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 18%-25% PEG 6000 (w / v) and 0.5%-9% dextran 460,000 Da (w / v).

[0342] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 1%-9% PEG 6000 (w / v) and 17%-25% dextran 179,000 Da (w / v).

[0343] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 9%-18% PEG 6000 (w / v) and 9%-17% dextran 179,000 Da (w / v).

[0344] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 18%-25% PEG 6000 (w / v) and 0.5%-9% dextran 179,000 Da (w / v).

[0345] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 1%-9% PEG 8000 (w / v) and 17%-25% dextran 70,000 Da (w / v).

[0346] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 9%-18% PEG 8000 (w / v) and 9%-17% dextran 70,000 Da (w / v).

[0347] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 18%-25% PEG 8000 (w / v) and 0.5%-9% dextran 70,000 Da (w / v).

[0348] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 1%-16% PEG 6000 (w / v) and 33%-50% dextran 3,400 Da (w / v).

[0349] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition and comprises 16%-33% PEG 6000 (w / v) and 16%-33% dextran 3,400 Da (w / v).

[0350] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 33%-50% PEG 6000 (w / v) and 1%-16% dextran 3,400 Da (w / v).

[0351] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 1%-16% PEG 4000 (w / v) and 33%-50% dextran 3,400 Da (w / v).

[0352] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 16%-33% PEG 4000 (w / v) and 16%-33% dextran 3,400 Da (w / v).

[0353] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 33%-50% PEG 4000 (w / v) and 1%-16% dextran 3,400 Da (w / v).

[0354] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 1%-5% PEG 20000 (w / v) and 20%-30% dextran 500,000 Da (w / v).

[0355] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 6%-11% PEG 20000 (w / v) and 9%-20% dextran 500,000 Da (w / v).

[0356] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 11%-15% PEG 20000 (w / v) and 0.5%-9% dextran 500,000 Da (w / v).

[0357] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 1%-7% PEG 8000 (w / v) and 20%-30% dextran 500,000 Da (w / v).

[0358] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 7%-14% PEG 8000 (w / v) and 9%-20% dextran 500,000 Da (w / v).

[0359] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 14%-20% PEG 8000 (w / v) and 0.5%-9% dextran 500,000 Da (w / v).

[0360] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 2%-16% PEG 3400 (w / v) and 20%-30% dextran 500,000 Da (w / v).

[0361] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 16%-33% PEG 3400 (w / v) and 9%-20% dextran 500,000 Da (w / v).

[0362] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 33%-50% PEG 3400 (w / v) and 0.5%-9% dextran 500,000 Da (w / v).

[0363] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 0.1% to 7% methylcellulose (w / v) and 14% to 20% dextran 2,200,000 Da (w / v).

[0364] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 7% to 14% methylcellulose (w / v) and 7% to 14% dextran 2,200,000 Da (w / v).

[0365] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition, comprising 14% to 20% methylcellulose (w / v) and 0.1% to 7% dextran 2,200,000 Da (w / v).

[0366] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 0.5%-16% PolyViol 28 / 20 (w / v) and 33%-50% dextran 2,200,000 Da (w / v).

[0367] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 16%-33% PolyViol 28 / 20 (w / v) and 16%-33% dextran 2,200,000 Da (w / v).

[0368] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 33%-50% PolyViol 28 / 20 (w / v) and 0.5%-16% dextran 2,200,000 Da (w / v).

[0369] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition and comprises 4%-21% PEG 600 (w / v) and 58%-70% PPG 400 (w / v).

[0370] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition and comprises 21%-42% PEG 600 (w / v) and 36%-58% PPG 400 (w / v).

[0371] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition and comprises 42%-60% PEG 600 (w / v) and 4%-36% PPG 400 (w / v).

[0372] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 0.5%-7% hydroxypropyl dextran 70 (w / v) and 14%-20% dextran 2,200,000 Da (w / v).

[0373] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 7%-14% hydroxypropyl dextran 70 (w / v) and 7%-14% dextran 2,200,000 Da (w / v).

[0374] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 14%-20% hydroxypropyl dextran 70 (w / v) and 0.5%-7% dextran 2,200,000 Da (w / v).

[0375] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 0.5%-7% PEG 8000 (w / v) and 20%-30% hydroxypropyl starch 60-70 kDa (w / v).

[0376] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 7%-14% PEG 8000 (w / v) and 10%-20% hydroxypropyl starch 60-70 kDa (w / v).

[0377] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 14%-20% PEG 8000 (w / v) and 1%-10% hydroxypropyl starch 60-70 kDa (w / v).

[0378] In some embodiments, the first or second ATPS composition is a micelle-based ATPS composition, comprising 5% to 17% (C2H4O) (4.5) C 14 H 22 O (Triton X-45) and 60% to 90% (C2H4O) (9-10) C 14 H 22 Contains O (Triton X-114).

[0379] In some embodiments, the first or second ATPS composition is a micelle-based ATPS composition and includes 10% to 20% Triton X-100 (w / v).

[0380] In some embodiments, the first or second ATPS composition is a micelle-based ATPS composition and includes 20% to 30% Triton X-100 (w / v).

[0381] In some embodiments, the first or second ATPS composition is a micelle-based ATPS composition and comprises 30% to 40% Triton X-100 (w / v).

[0382] In some embodiments, the first or second ATPS composition is a micelle-based ATPS composition and comprises 5% to 18% Triton X-114 (w / v).

[0383] In some embodiments, the first or second ATPS composition is a micelle-based ATPS composition and comprises 18% to 29% Triton X-114 (w / v).

[0384] In some embodiments, the first or second ATPS composition is a micelle-based ATPS composition and comprises 29% to 40% Triton X-114 (w / v).

[0385] In some embodiments, the first or second ATPS composition is a micelle-based ATPS composition and comprises 3% to 19% decyl tetraethylene glycol ether (C10E4) (w / v).

[0386] In some embodiments, the first or second ATPS composition is a micelle-based ATPS composition and comprises 19% to 34% decyl tetraethylene glycol ether (C10E4) (w / v).

[0387] In some embodiments, the first or second ATPS composition is a micelle-based ATPS composition and comprises 34% to 50% decyl tetraethylene glycol ether (C10E4) (w / v).

[0388] In some embodiments, the first or second ATPS composition is a micelle-based ATPS composition, comprising 1% to 10% Pluronic F68 (MW 8,400 Da, EO 82 -PO 31 -EO 82 ) (w / v) and 14% to 20% potassium phosphate (w / v).

[0389] In some embodiments, the first or second ATPS composition is a micelle-based ATPS composition and comprises 10%-20% Pluronic F68 (w / v) and 7%-14% potassium phosphate (w / v).

[0390] In some embodiments, the first or second ATPS composition is a micelle-based ATPS composition and comprises 20%-30% Pluronic F68 (w / v) and 1%-7% potassium phosphate (w / v).

[0391] In some embodiments, the first or second ATPS composition is a micelle-based ATPS composition, comprising 5% to 21% Pluronic 17R4 (MW 2,650 Da, PO 14 -EO 24 -PO 14 ) (w / v) and 7% to 10% potassium phosphate (w / v).

[0392] In some embodiments, the first or second ATPS composition is a micelle-based ATPS composition and comprises 21%-42% Pluronic 17R4 (w / v) and 3%-7% potassium phosphate (w / v).

[0393] In some embodiments, the first or second ATPS composition is a micelle-based ATPS composition and comprises 42%-60% Pluronic 17R4 (w / v) and 1%-3% potassium phosphate (w / v).

[0394] In some embodiments, the first or second ATPS composition is a micelle-based ATPS composition, comprising 2% to 21% Pluronic L-35 (MW 1,900 Da, EO 10 -PO 16 -EO 10 ) (w / v) and 14% to 20% sodium sulfate (w / v).

[0395] In some embodiments, the first or second ATPS composition is a micelle-based ATPS composition and comprises 21%-42% Pluronic L-35 (w / v) and 7%-14% sodium sulfate (w / v).

[0396] In some embodiments, the first or second ATPS composition is a micelle-based ATPS composition and comprises 42%-60% Pluronic L-35 (w / v) and 1%-7% sodium sulfate (w / v).

[0397] In some embodiments, the first ATPS composition and / or the second ATPS composition is selected from Tables 1.0a-1.0d.

[0398] In some embodiments, the first ATPS composition or the second ATPS composition further comprises 0.5 to 2 mM ethylenediaminetetraacetic acid (EDTA).

[0399] In some embodiments, the method further comprises analyzing the final solution from step (f) using a method selected from the group consisting of qPCR, ddPCR, qubit, ELISA, NGS sequencer, bisulfite, RT-PCR, Sanger sequencing, nanodroplet, nanopore sequencing, nucleic acid sequencing, and a PCR-based assay.

[0400] In some embodiments, the target analyte is a biomarker that indicates the presence or risk of a medical disorder or disease in a patient, wherein the medical disorder or disease is an infectious disease, cancer, or a genetic disease.

[0401] In some embodiments, the clinical biological sample is a bulk fluid sample having a volume of >10 mL, and prior to step (a), the method further comprises dividing the bulk fluid sample into at least two aliquots of sample solution, wherein in step (a), each aliquot is individually combined with the first ATPS, and optionally, in step (c), the collected target-rich phase from step (b) is individually combined with the second ATPS to form a target-rich phase for each aliquot.

[0402] In some embodiments, the target-rich phase of each aliquot from step (b) or the second target-rich phase of each aliquot from step (c) are combined to form the final target-rich phase used in step (d).

[0403] In some embodiments, the bulk fluid sample is urine.

[0404] In some embodiments, there is provided a method of treating cancer or an infectious disease in a patient in need thereof, the method comprising: (i) obtaining a clinical biological sample from the patient; (ii) enriching and purifying at least one target analyte from the clinical biological sample according to the above embodiments; (iii) analyzing the final solution; and (iv) treating the patient if information obtained from the target analyte indicates that the patient has or is at risk for having cancer.

[0405] In some embodiments, the cancer is CNS cancer, breast cancer, bladder cancer, pancreatic cancer, lung cancer, melanoma, colon cancer, hematopoietic cancer, or ovarian cancer, and wherein the infection is HPV.

[0406] In some embodiments, the target analyte is kidney cFDNA or ctDNA, and wherein the cancer is a systemic cancer.

[0407] In some embodiments, the target analyte is urogenital cFDNA or ctDNA, and wherein the cancer is a urogenital cancer.

[0408] In some embodiments, the target analyte is bladder cancer DNA and the medical disorder or disease is bladder cancer.

[0409] In some embodiments, the target analyte is HPV viral RNA or HPV viral DNA and the medical disorder or disease is HPV.

[0410] In some embodiments, a kit is provided, the kit comprising a polymer from any one of the above embodiments, a salt component comprising at least one salt, an ATPS composition comprising a binding buffer and a solid phase medium.

[0411] In some embodiments, a kit is provided, the kit comprising a first ATPS composition according to any one of the above embodiments, a second ATPS composition, a binding buffer, and a solid phase medium.

[0412] In some embodiments, the polymer is soluble in aqueous solution at a concentration of 0.5% to 80% (w / v), and the salt is soluble in aqueous solution at a concentration of 0.1% to 80% (w / v).

[0413] In some embodiments, the first ATPS composition comprises the polymer soluble in aqueous solution at a concentration of 5% to 80% (w / v) and the salt soluble in aqueous solution at a concentration of 0.1% to 80% (w / v), and the second ATPS composition comprises the polymer soluble in aqueous solution at a concentration of 0.5% to 30% (w / v) and the salt soluble in aqueous solution at a concentration of 5% to 60% (w / v).

[0414] In some embodiments, the concentration of the polymer is 0.5% to 80% (w / v) of the first ATPS and / or the second ATPS. In some embodiments, the concentration of the polymer is 0.5% to 30% (w / v) of the first ATPS and / or the second ATPS. In some embodiments, the concentration of the polymer is 5% to 60% (w / v) of the first ATPS and / or the second ATPS. In some embodiments, the concentration of the polymer is 12% to 50% (w / v) of the first ATPS and / or the second ATPS.

[0415] In some embodiments, the salt concentration is 0.1% to 80% (w / v) of the first ATPS and / or the second ATPS. In some embodiments, the salt concentration is 5% to 60% (w / v) of the first ATPS and / or the second ATPS. In some embodiments, the salt concentration is 0.1% to 50% (w / v) of the first ATPS and / or the second ATPS. In some embodiments, the salt concentration is 0.1% to 20% (w / v) of the first ATPS and / or the second ATPS. In some embodiments, the salt concentration is 0.01% to 30% (w / v). In some embodiments, the salt concentration is 0.01% to 10% (w / v) of the first ATPS and / or the second ATPS.

[0416] In some embodiments, the surfactant concentration is 0.1% to 90% (w / v) or 0.1% to 50% (w / v) of the first ATPS and / or the second ATPS, hi some embodiments, the surfactant concentration is 0.01% to 10% (w / v) of the first ATPS and / or the second ATPS.

[0417] In some embodiments, the first ATPS composition is a polymer-salt-based system comprising 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).

[0418] In some embodiments, the second ATPS composition comprises at least one polymer at a concentration of 0.5% to 30% (w / v) and at least one salt at a concentration of 5% to 60% (w / v). In some embodiments, the second ATPS composition comprises at least one polymer at a concentration of 1% to 6% (w / v) and at least one salt at a concentration of 10% to 50% (w / v). In some embodiments, the second ATPS composition further comprises at least one surfactant at a concentration of 0.01% to 10% (w / v).

[0419] In some embodiments, the first ATPS composition is a polymer-salt system that includes at least one polymer at a concentration of 0.5% to 30% (w / v) and at least one salt at a concentration of 5% to 60% (w / v). In some embodiments, the first ATPS composition includes at least one polymer at a concentration of 1% to 6% (w / v) and at least one salt at a concentration of 10% to 50% (w / v). In some embodiments, the first ATPS composition further includes at least one surfactant at a concentration of 0.01% to 10% (w / v).

[0420] In some embodiments, the second ATPS composition comprises at least one polymer at a concentration of 5% to 80% (w / v) and at least one salt at a concentration of 0.1% to 80% (w / v). In some embodiments, the second ATPS composition comprises at least one polymer at a concentration of 5% to 60% (w / v) and at least one salt at a concentration of 0.5% to 50% (w / v). In some embodiments, the second ATPS composition comprises at least one polymer at a concentration of 12% to 50% (w / v) and at least one salt at a concentration of 0.1% to 20% (w / v). In some embodiments, the second ATPS composition further comprises at least one surfactant at a concentration of 0.01% to 10% (w / v).

[0421] In some embodiments, the first ATPS composition is a polymer-polymer system comprising at least two polymers, each polymer soluble in aqueous solution at a concentration of 0.2% to 50% (w / v). In some embodiments, the first ATPS composition further comprises at least one salt at a concentration of 0.01% to 10% (w / v). In some embodiments, the first ATPS composition further comprises at least one surfactant at a concentration of 0.01% to 10% (w / v).

[0422] In some embodiments, the first ATPS composition is a micellar system that includes at least two surfactants, each of which is dissolved in aqueous solution at a concentration of 0.1% to 90% (w / v). In some embodiments, the first ATPS composition further includes at least one salt at a concentration of 0.01% to 30% (w / v).

[0423] In some embodiments, the polymer of the first ATPS composition or the second ATPS composition is soluble in aqueous solution at a concentration of 0.5% to 80% (w / v), 0.5% to 60% (w / v), 5% to 80% (w / v), 5% to 60% (w / v), 12% to 50% (w / v), or 0.5% to 30% (w / v).

[0424] In some embodiments, the salt component of the first ATPS composition or the second ATPS composition is soluble in aqueous solution at a concentration of 0.1% to 80% (w / v), 0.5% to 50% (w / v), 0.1% to 20% (w / v), 5% to 60% (w / v), or 10% to 50% (w / v).

[0425] In some embodiments, the binding buffer comprises a guanidine salt solution of 2 M to 7 M. In some embodiments, the binding buffer further comprises a polymer at a concentration of 5% to 20% (w / v).

[0426] In some embodiments, the kit provided includes an ATPS composition, which includes a polymer from any one of the above embodiments, a salt component including at least one salt, a binding buffer, and a solid phase medium.

[0427] Although specific examples have been mentioned, the present disclosure should not be construed as being limited to the examples described herein.

[0428] Other illustrative examples are also discussed herein.

[0429] example

[0430] This specification provides several example embodiments that more fully describe the contents of the present disclosure. The examples provided herein are for illustrative purposes only and are not intended to limit the scope of the present invention in any way. All references cited below and elsewhere in this application are incorporated herein by reference.

[0431] Exemplary Workflow for Enrichment and Purification of One or More Target Analytes from Clinical Biological Samples

[0432] This specification provides exemplary workflows for the enrichment and purification of one or more target analytes (e.g., target nucleic acids) from clinical biological samples using aqueous two-phase systems (ATPS) and solid phase media. Tables 1.0a-1.0d summarize exemplary ATPS compositions for use in the first and / or second ATPS steps. These examples are for illustrative purposes only and are not intended to be a comprehensive list of all possible embodiments of the present invention.

[0433] Tables 1.0a-1.0d: Exemplary ATPS Compositions. Unless otherwise noted, all concentrations are given as w / v ratios. [Table 1.0a] [Table 1.0b] [Table 1.0c] [Table 1.0d]

[0434] Example 1a Bead purification

[0435] Referring now to FIG. 1A, which illustrates an exemplary workflow for bead purification (also referred to in some embodiments as "bead purification," "magnetic bead workflow," or "magnetic bead purification") integrated with a previous ATPS step. In this example, in a first, optional digestion step 111, an appropriate digestion buffer is added to a sample (e.g., blood, plasma, serum, cerebrospinal fluid, urine, saliva, feces, tears, sputum, nasopharyngeal mucus, vaginal secretions, and penile secretions) to digest cells in the sample and release biomolecules into the digestion buffer solution. The digested sample is then passed through two sequential aqueous two-phase systems (ATPS) to isolate and concentrate DNA.

[0436] In step 112, the digested sample was mixed with a first ATPS. The mixture was vigorously vortexed and centrifuged to separate it into a top phase and a bottom phase. In some embodiments, the top phase is a target-rich phase (also referred to as a "first target-rich phase"). In some embodiments, the bottom phase is a target-rich phase containing the target analyte.

[0437] In step 113, all target-rich phases assigned to the DNA from step 112 are subjected to a second ATPS (2 nd The mixture was transferred to a centrifuge (ATPS) and mixed thoroughly. The mixture was vortexed and centrifuged to separate it into an upper and lower phase. In some embodiments, the upper phase is a second target-rich phase containing the target analyte. In some embodiments, the lower phase is a second target-rich phase containing the target analyte.

[0438] In step 114, the second target-rich phase, assigned to the target DNA from step 113, was extracted and placed in an empty microcentrifuge tube. A binding buffer (e.g., as described in Examples 2a-5c below) was added to the tube and thoroughly mixed with the upper phase from step 113 and a plurality of beads (e.g., as described in Table 1.1), which were also added to the tube to form solid-phase extraction complexes (i.e., bead-analyte complexes in this example). The mixture containing the bead-analyte complexes was incubated for a period of time and then centrifuged to immobilize the bead-analyte complexes. The supernatant was discarded, provided that the bead-analyte complexes were not disturbed. In some examples, the bead-analyte complexes were immobilized to the tube wall using a magnetic rack.

[0439] In step 115, the bead-analyte complexes are further purified by washing with a suitable solvent and discarding the supernatant containing impurities. In some embodiments, the suitable solvent is a binding buffer or a wash buffer. Optionally, step 115 is repeated.

[0440] In step 116, the bead-analyte complexes were then dried with the lid open, and the bead-analyte complexes were resuspended in an appropriate elution solution (e.g., elution buffer) and mixed thoroughly to release the beads from the bead-analyte complexes. The beads were then immobilized, and the final solution containing the purified target DNA was collected for further use.

[0441] Table 1.1: Examples of magnetic beads. [Table 1.1]

[0442] Example 1b Column purification

[0443] Referring now to FIG. 1B, which illustrates an exemplary workflow of column purification integrated with a previous ATPS step (also referred to in some embodiments as "centrifugation column purification" or "centrifugation column workflow"). In this example, in a first optional digestion step 121, an appropriate digestion buffer is added to a sample (e.g., blood, plasma, serum, cerebrospinal fluid, urine, saliva, feces, tears, sputum, nasopharyngeal mucus, vaginal secretions, and penile secretions) to digest cells in the sample and release biomolecules into the digestion buffer solution. The digested sample is then passed through two sequential aqueous two-phase systems (ATPS) to isolate and concentrate DNA.

[0444] In step 122, the digested sample is mixed with a first ATPS. The mixture is vigorously vortexed and centrifuged to separate it into an upper and a lower phase. In some embodiments, the upper phase is a target-rich phase (also referred to as a "first target-rich phase"). In some embodiments, the lower phase is a target-rich phase containing the target analyte.

[0445] In step 123, all target-rich phases assigned to DNA from step 122 are subjected to a second ATPS (2 nd The mixture was transferred to a centrifuge (ATPS) and mixed thoroughly. The mixture was vortexed and centrifuged to separate it into an upper and lower phase. In some embodiments, the upper phase is a second target-rich phase containing the target analyte. In some embodiments, the lower phase is a second target-rich phase containing the target analyte.

[0446] In step 124, the second target-rich phase (also referred to as the "second target-rich phase") assigned to the target DNA from step 123 was extracted and placed into an empty microcentrifuge tube. Binding buffer (e.g., as described in Examples 2a-5c below) was added to the tube and thoroughly mixed with the upper phase from step 123 to form a mixed solution. The mixed solution was transferred to a centrifugation column to form the solid-phase extraction complex, which was then centrifuged. The flow-through (also referred to as the "supernatant") from the centrifugation column was discarded. Step 124 was repeated until all of the sample had passed through the centrifugation column.

[0447] In step 125, the solid phase extraction complex is further purified by washing with a suitable solvent and discarding the supernatant containing impurities. In some embodiments, the suitable solvent is a binding buffer or a wash buffer. Optionally, step 125 is repeated.

[0448] In step 126, the complex was then extracted by centrifuging and drying the solid phase. An appropriate elution buffer was added to the solid phase extraction complex, which was then incubated for a period of time to form a final solution. The final solution containing the purified target DNA was collected by centrifugation into a collection tube for further use.

[0449] Example 2a: Comparison of the efficiency of DNA recovery from plasma with magnetic beads with and without a prior aqueous two-phase system (ATPS) step

[0450] In this example, the efficiency of DNA recovery from plasma using magnetic beads (also referred to as "magnetic beads") was compared (i) using phase separation prior to the ATPS system (also referred to as "ATPS step," "ATPS extraction," or "ATPS workflow") according to the methods of the present disclosure, and (ii) without using a prior ATPS step.

[0451] material

[0452] In this example, citrated mixed-gender pooled plasma (catalog number PR-100) was purchased from TCS Biosciences Ltd. The aqueous two-phase system (ATPS) contained polymer-salt-based ATPS components (also referred to as the "polymer-salt system"). The binding buffer consisted of 1-5 M guanidine salt, Tris-HCl, and EDTA. Unless otherwise noted, 145 bp DNA oligonucleotides and / or 2000 bp DNA oligonucleotides were spiked in for DNA recovery testing. All other chemicals were of analytical grade.

[0453] TaqMan used in PCRTM Fast Advance Master Mix (2X) and Custom TaqMan TM Assays are provided by Thermofisher Scientific. Custom TaqMan TM The assay consists of a forward primer, a reverse primer, and a FAM dye-labeled TaqMan MGB probe.

[0454] Degradation of plasma cells

[0455] Pooled human plasma was spiked with 100 fg 145 bp DNA per 2 mL plus 1.5 μL of 2000 bp DNA (TATAA) stock solution. 160 μL of the appropriate digestion buffer and 60 μL of proteinase K (28.5 mg / mL) were added per 2 mL of spiked-in plasma. The mixture was vortexed thoroughly and digested in a preheated 60°C heating block for 15 minutes.

[0456] Extraction Process

[0457] One group of degraded plasma samples (sample #1) was treated with two consecutive ATPS steps (also referred to in some examples as "double ATPS," "two-step ATPS," or "first and second ATPS") to enrich and separate DNA prior to magnetic bead purification, while the other groups of degraded plasma (samples #2 and #3) underwent direct magnetic bead purification without a prior ATPS step.

[0458] For sample #1, ATPS 67, listed in Table 1.0b, was used as the first ATPS, and ATPS 94, listed in Table 1.0b, was used as the second ATPS. 2.22 mL of digested sample #1 was transferred to 1.170 mL of the first ATPS and vortexed to mix. The mixture was centrifuged at 2,300 rcf for 6 minutes. The entire salt-rich bottom phase (approximately 1 mL) (also referred to as the "target-rich phase" in some examples) was transferred to 265 μL of the second ATPS and mixed thoroughly. The mixture was then centrifuged at 7,000 rcf for 1 minute. The polymer-rich upper phase (approximately 150 μL) (also referred to as the "second target-rich phase" in some examples) was carefully extracted and placed in an empty microcentrifuge tube for further purification.

[0459] DNA purification

[0460] 800 μL of binding buffer was added to the extracted upper phase of sample #1 and to the digested sample #2 that had not been subjected to ATPS. For sample #3, 11.43 mL of binding buffer was added to digested sample #3, so that the ratio of binding buffer to digested sample was the same as the ratio of binding buffer to upper phase in sample #1. Samples #1, #2, and #3 are summarized in Table 1.2 below. 12 μL of magnetic beads was added to each tube. The mixture was then incubated on a rotator for 5 minutes to prevent bead settling. The tubes were then briefly centrifuged to sediment and placed on a magnetic rack for 2 minutes to fix the beads to the tube wall. The supernatant was discarded without disturbing the beads. 800 μL of binding buffer was added to each tube, and the tubes were gently rotated on the magnetic support for a total of 720°. Pipetting was repeated, and the supernatant was discarded. To the sample, 800 μL of wash buffer (70% ethanol, 0.001 M EDTA, 0.01 M Tris-HCl) was added, and the tube was rotated a total of 720° on the support. The supernatant was discarded. This wash step was performed twice. To improve drying efficiency, the tube was briefly centrifuged in a tabletop microcentrifuge with the hinge facing outward to collect any remaining wash buffer. The beads were then dried on the magnetic support with the lid open for 7 minutes. The bead complex was resuspended in 40 μL of elution buffer (0.01 M Tris-HCl, 0.001 M EDTA) by repeated pipette mixing and briefly vortexed. The tube was then placed on a magnetic rack for 1 minute. The supernatant was carefully collected into a DNA low-binding tube (purchased from Eppendorf, catalog number 0030108035) without disturbing the magnetic beads for detection.

[0461] Table 1.2: Summary of test conditions for plasma extraction and purification. [Table 1.2]

[0462] DNA detection

[0463] All recoveries of DNA oligonucleotides discussed in this disclosure were quantified by quantitative real-time polymerase chain reaction (qPCR) using Quant Studio 5. The qPCR reaction mixture (each reaction) consisted of 5 μL of TaqMan Fast Advance Master Mix (Applied Biosystems, see 4444557), 0.5 μL of 20x custom premixed oligonucleotides PSI-145 FAM Dental, 0.4 μL of universal Spike II primers (TATAA, DS25SII), 0.2 μL of universal Spike II probes (TATAA, DSSII), and 1.9 μL of ultrapure water.

[0464] 8 μL PCR reaction mixture was allocated to each PCR well, and 2 μL of purified DNA sample was added. qPCR was performed using the QuantStudio TM The PCR was performed using a QuantStudio 3 real-time PCR system (Thermofisher Scientific). The heat cycle consisted of 40 cycles consisting of 50°C for 2 minutes, 95°C for 2 minutes, and finally 95°C for 1 second and 60°C for 20 seconds. TM Design and analysis software was used to determine the cycle threshold (CT), where the fluorescent signal of the amplification reaction was higher than background fluorescence. Data analysis was performed in Excel and GraphPad Prism against the original CT.

[0465] All results were expressed as mean CT values. The lower the mean 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.

[0466] result

[0467] DNA recovery

[0468] Referring now to Figures 2A-B, the average CT values ​​of 145 bp and 2000 bp DNA oligonucleotides recovered from magnetic bead-purified plasma (Sample #1: using a double ATPS step, Sample #2: not using an ATPS step and unscaled binding buffer volume, and Sample #3: not using an ATPS step and scaled binding buffer volume, per Table 1.2) are shown. The results are summarized in Table 1.3. For 145 bp DNA, plasma sample #1 had a high recovery (average CT value of 27.21), while plasma sample #2 had a low recovery (average CT value of 34.83). For sample #3, while the volume of binding buffer was increased proportionally, the recovery rate (average CT value of 32.07) improved slightly compared to sample #2, but was still significantly lower than that of sample #1. Similar results were also observed for 2000 bp DNA recovery. The results showed that when ATPS was added to the extraction process before the magnetic bead binding step, the recovery of 145 bp and 2000 bp DNA oligonucleotides was significantly improved, and surprisingly, even when the volume of binding buffer was increased proportionally compared to samples without the ATPS step, the DNA recovery was still significantly improved.

[0469] Table 1.3: qPCR results for recovery of 145 bp and 2000 bp DNA oligonucleotides from plasma with or without a two-step ATPS using magnetic beads. [Table 1.3]

[0470] Overall, the recovery results surprisingly demonstrate that incorporating an ATPS into the plasma extraction workflow prior to magnetic bead purification can achieve higher DNA recovery, even with additional steps for ATPS concentration and separation (which are potential sources of target analyte loss during sample processing). Using an ATPS prior to magnetic bead purification significantly improves DNA recovery, requiring fewer magnetic beads to alter the binding balance and obtain better yields, thereby reducing the amount of magnetic beads (which are expensive) and hazardous binding buffers required to achieve effective purification.

[0471] Example 2b: Plasma extraction performance with different polymers and salts in ATPS compositions

[0472] In this example, ATPSs with different polymer molecular weights and component concentrations were used to test their ability to bind to magnetic beads and recover DNA from plasma samples, and ATPSs with different polymer and salt chemistries were used to test their ability to bind to magnetic beads and recover DNA from plasma samples.

[0473] The reagents used in this example are similar or the same as those discussed in the previous example.

[0474] Degradation of plasma cells

[0475] To digest unwanted proteins and cells in plasma, 160 μL of the appropriate digestion buffer and 60 μL of proteinase K (28.57 mg / mL) were added to a 2 mL aliquot of citrate (TCS) plasma. 100 fg 145 bp DNA and 100 ng GR 1 kb+ standard were spiked into the sample. After vortexing for 15 seconds, the sample was incubated at 60°C for 15 minutes. The digest was used for further extraction.

[0476] Two-phase system

[0477] The extraction procedure involves two sequential aqueous two-phase systems (ATPS), including a first ATPS and a second ATPS composition for isolating, purifying, and concentrating DNA from plasma samples. In the first ATPS, DNA was allocated to the bottom phase (also called the "target-rich phase"), and protein was allocated to the top phase (also called the "target-poor phase"). Approximately 1 mL of the bottom phase was carefully extracted and transferred to the second ATPS. DNA was allocated to the top phase (also called the "second target-rich phase") of the second ATPS, and the top phase was carefully extracted for further purification and detection.

[0478] In this example, the second ATPS composition was varied according to the corresponding first ATPS composition to achieve a small upper phase volume of approximately 150 μL.

[0479] Extraction Process

[0480] This example describes an ATPS-based method for cfDNA isolation and purification. 2.22 mL of digest was added to the first ATPS. A dye was added to visualize the two phases, creating a colored upper phase. In this example, most of the DNA was located in the bottom phase, while the protein was located in the upper phase. The solution was made turbid by vigorous vortexing for 15 seconds. Phase separation was then facilitated by centrifugation at 2300 rcf for 6 minutes. After phase separation, the bottom phase volume was approximately 1 mL. The entire bottom phase was carefully extracted and transferred to a second ATPS. The second ATPS mixture was vortexed for 15 seconds to create a turbid solution, and then centrifuged at 7000 rcf for 1 minute to achieve phase separation. The resulting upper phase volume was approximately 150 μL and carefully extracted for further DNA purification and detection.

[0481] DNA purification

[0482] DNA purification was completed by magnetic bead extraction. The upper phase from the second ATPS was transferred to a tube containing 800 μL of binding buffer (1-5 M guanidine salt). 12 μL of MagQu magnetic beads were added to the tube, and the sample was incubated for 5 minutes with tilt and rotation. The tube was then briefly centrifuged to sediment and placed on a magnetic support for 2 minutes to immobilize the beads to the tube wall. The supernatant was discarded after pipetting without disturbing the magnetic beads. 800 μL of binding buffer was added to the sample. The tube was rotated a total of 720° on the support. The supernatant was discarded after pipetting. 800 μL of wash buffer (70% ethanol, 0.001 M EDTA, 0.01 M Tris-HCl) was added to the sample, and the tube was rotated a total of 720° on the support. The supernatant was discarded after pipetting. After washing twice, the beads were dried by placing the tube on a support with the lid open. 40 μL of elution buffer (0.01 M Tris-HCl, 0.001 M EDTA) was added to the sample. The bead complex was resuspended by repeated pipetting and briefly vortexed. The sample was incubated at room temperature for 3 minutes, briefly centrifuged, and then sedimented. The tube was then placed on a magnetic rack for 1 minute. The supernatant was carefully collected without disturbing the magnetic beads for detection.

[0483] Extraction efficiency

[0484] The DNA can be further concentrated for detection by using a second ATPS after the first, both of which contain a mixture of polymer and salt, where the resulting top phase is rich in polymer and the bottom phase is rich in salt.

[0485] In this example, we investigated the DNA extraction efficiency of different ATPS compositions while maintaining a constant volume ratio of the target-rich phase to the target-poor phase in the first and second ATPSs. To evaluate DNA recovery performance, a 2 mL sample containing 100 ng of DNA standard in human plasma was prepared. Assuming a 100% recovery rate, this would yield 2.5 ng per μL of eluate.

[0486] DNA detection

[0487] The steps for performing DNA detection for each sample were the same or similar to those discussed above for Example 1a. For the sake of brevity and simplicity of this disclosure, the discussion of the detection steps will not be repeated here. Results were expressed as mean CT values.

[0488] result

[0489] Next, referring to Figure 2C, we compared the average C values ​​for 145 bp DNA recovery using the first and second ATPS compositions under conditions 5, 7, 6, and 8 in Table 1.4. Conditions 5, 7, 6, and 8 correspond to the same polymer and salt combinations with different polymer molecular weights, and the respective 145 bp DNA recovery results are shown in Table 1.4. The results show that when coupled with the magnetic bead workflow, all systems with different polymer molecular weights in the first ATPS can recover detectable levels of DNA from plasma.

[0490] Table 1.4: Recovery of DNA from plasma using ATPS and magnetic beads with different polymer molecular weights. [Table 1.4]

[0491] Referring now to Figure 2D, the average C values ​​for 145 bp DNA recovery using the first and second ATPS compositions under conditions 9, 10, and 11 in Table 1.5 were compared. Conditions 9, 10, and 11 correspond to three different combinations of polymer and salt, and Table 1.5 shows the respective 145 bp DNA recovery results. The results show that all systems with various combinations of polymer and salt in either the first or second ATPS recovered satisfactory levels of DNA (e.g., levels sufficient for detection purposes in downstream applications, such as diagnostic applications).

[0492] Table 1.5: DNA recovery from plasma using ATPS and magnetic beads with different combinations of polymer and salt. [Table 1.5]

[0493] In summary, for systems with the same type of polymer and salt but different polymer sizes (conditions 5, 7, 6, and 8, Table 1.4), satisfactory amounts of DNA were recovered for various polymer sizes in the first ATPS. For systems using different salts (e.g., various cations and anions) and polymers of different properties (conditions 9, 10, and 11, Table 1.5), DNA recovery was also good.

[0494] Example 2c: Plasma extraction performance using various ATPS systems and magnetic beads

[0495] In this example, we demonstrate the performance of different classes of ATPS systems (polymer-salt, polymer-polymer, micelles) in combination with magnetic beads to extract DNA from plasma. The materials used in this example are the same as or similar to those discussed in previous examples. For the sake of brevity and simplicity of this disclosure, we will not repeat the discussion of the materials here.

[0496] Degradation of plasma

[0497] In this example, K2 EDTA plasma (TCS Biosciences, Lot No. 23014500) was digested according to the method for optimizing the respective downstream ATPS system. Samples were spiked with 100 fg of 145 bp DNA and 1.5 uL of stock 2000 bp DNA.

[0498] For polymer-polymer-based ATPS systems, 500 uL of the appropriate digestion buffer and 30 uL of protease K (28.57 mg / mL) were added to 1 mL of K2 EDTA plasma, vortexed thoroughly, and incubated in a preheated 60°C water bath for 15 min.

[0499] For the micelle-based ATPS system, 300 μL of the appropriate lysis buffer was added to 1 mL of K2EDTA plasma, vortexed thoroughly, and incubated at room temperature for 3 minutes. Then, 112.5 μL of protein precipitation buffer A (PPt A) (20% ZnCl2 w / v, 1% acetic acid w / v) was added, vortexed thoroughly until homogenous, and centrifuged at 12,000 rcf for 3 minutes. Other appropriate protein precipitation buffers may be used in place of protein precipitation buffer A in the above method. The supernatant was extracted for further downstream processing, and the pellet was discarded.

[0500] For the polymer-salt-based ATPS system, 80 uL of the appropriate digestion buffer and 30 uL of protease K (28.57 mg / mL) were added to 1 mL of K2 EDTA plasma, vortexed thoroughly, and incubated in a preheated 60°C water bath for 15 min.

[0501] Extraction Process

[0502] To effectively extract cfDNA from plasma samples, different types of aqueous two-phase systems (ATPS) were prepared. Table 1.6 summarizes the three different types of ATPS and the subsequent binding steps.

[0503] Table 1.6: Summary of experimental conditions with alternative ATPS classes. [Table 1.6]

[0504] The sample digests were added to each ATPS system: 1530 μL of sample digest was added to 500 μL of the polymer-polymer-based system, and 1110 μL of sample digest was added to 1.17 mL of the polymer-salt-based first system. For the micellar system, 1200 μL of the extracted supernatant after protein precipitation was added to 500 mg of the micellar system. In some examples, the first ATPS system was a single ATPS system without a second ATPS system following it.

[0505] After adding the sample digest to all ATPS tubes, vortex thoroughly and vortex thoroughly.

[0506] The polymer-polymer ATPS system was centrifuged at 2300 rcf for 6 minutes. The target cfDNA was concentrated in the polymer-rich bottom phase. The entire bottom phase (approximately 300 μL) was extracted and transferred to a new 2 mL centrifuge tube for further processing.

[0507] The micellar ATPS system was centrifuged at 12,000 rcf for 5 minutes. The target cfDNA was concentrated in the detergent-rich upper phase. The entire upper phase (approximately 280 μL) was extracted and transferred to a new 2 mL centrifuge tube for further processing.

[0508] For the polymer-salt ATPS system, the extraction process was the same or similar to the method discussed in the previous example. The first polymer-salt ATPS system was centrifuged at 2300 rcf for 6 minutes. The target cfDNA was concentrated in the salt-rich bottom phase. The entire bottom phase (approximately 1000 μL) was extracted and transferred to a second ATPS tube containing 265 μL of the second ATPS composition, which was vortexed thoroughly and centrifuged at 7000 rcf for 1 minute. The target cfDNA was concentrated in the polymer-rich upper phase (approximately 120 μL), which was then extracted and transferred to a new tube for further processing.

[0509] DNA purification

[0510] After extraction of the target cfDNA-containing phase from each ATPS system, downstream processing involves adding a solid phase medium and binding buffer to isolate the nucleic acid from the liquid medium, and washing to remove unwanted ions and molecules before the target nucleic acid is eluted with an elution buffer.

[0511] 1500 μL of binding buffer (3–7 M guanidine salt, 50 mM pH 7 NaHPO / NaHPO) was added to the extracted polymer-rich bottom phase of the polymer-polymer system. 500 μL of binding buffer (3–7 M guanidine salt) was added to the extracted surfactant-rich top phase of the micellar system. 800 μL of binding buffer (3–7 M guanidine salt) was added to the extracted polymer-rich top phase of the second polymer-salt ATPS system. 6 μL of magnetic beads (MagQu) were then added to all centrifuge tubes containing the mixture of extraction phase and binding buffer, placed on a rotator, and incubated for 5 minutes. During incubation, the target cfDNA bound to the magnetic beads. After incubation, all tubes were briefly centrifuged and placed on a magnetic rack, which attracted the cfDNA-bound magnetic beads. The supernatant was then removed and discarded, allowing the magnetic beads to bind to the inner wall of the centrifuge tube. A second binding buffer wash step was performed on the polymer-polymer and polymer-salt systems. 800 μL of binding buffer (3-7 M guanidine salt, 50 mM pH 7 NaHPO / NaHPO) was added to the magnetic beads from the polymer-polymer system, and 800 μL of binding buffer (3-7 M guanidine salt) was added to the magnetic beads from the polymer-salt system. The tubes were then vortexed thoroughly, centrifuged briefly, and returned to the magnetic rack. Once all the magnetic beads had bound to the inside of the centrifuge wall, the supernatant was discarded.

[0512] 800 μL of the original wash buffer (70% v / v EtOH, 1 mM EDTA, 10 mM Tris-HCl) was added to the magnetic beads from the polymer-polymer and polymer-salt systems, and 500 μL of RPE wash buffer (80% v / v EtOH, 0.1 M NaCl, 10 mM Tris-HCl) was added to the magnetic beads from the micelle system. Before discarding the wash buffer, the centrifuge tube was rotated 720° on the magnetic rack. The above procedure was repeated for a second wash. The centrifuge tube was then briefly centrifuged before returning it to the magnetic rack, and any excess wash buffer remaining in the centrifuge tube was discarded. For the polymer-polymer and polymer-salt systems, the magnetic beads were allowed to dry at room temperature for 7 minutes, and for the micelle system, for 10 minutes.

[0513] After the magnetic beads dried, they were resuspended in 20 μL of elution buffer (10 mM Tris-HCl, 1 mM EDTA) and incubated at room temperature for 3 minutes. The target cfDNA was eluted from the magnetic beads into the elution buffer. The tube was then returned to the magnetic rack. Once all of the magnetic beads were bound, the elution buffer was extracted.

[0514] DNA detection

[0515] The steps for performing DNA detection for each sample were the same or similar to those discussed above for Example 2a. For the sake of brevity and simplicity of this disclosure, the discussion of the detection steps will not be repeated here. Results were expressed as mean CT values.

[0516] result

[0517] allocation

[0518] The first ATPSs from the polymer-polymer (PP) and polymer-salt (PS)-based classes effectively allocated target-free DNA (cfDNA) from digested samples to the bottom phase of the first ATPS, while excess protein allocated to the top phase of the ATPS system. The opposite trend was observed for micellar ATPS systems, where cfDNA allocated to the top phase. The PS second ATPS is used to concentrate the large bottom phase of the first ATPS into a more concentrated, smaller top-phase volume for more user-friendly downstream processing.

[0519] DNA recovery

[0520] Next, referring to Figures 2E-2F, the average CT values ​​for recovering 145 bp DNA and 2000 bp DNA from plasma using three different types of ATPS systems (i.e., polymer-polymer, micelle, and polymer-salt systems) are shown. For 145 bp DNA recovery, the three systems were comparable in efficiency, as the differences in average CT values ​​between the polymer-polymer system (average CT value = 24.81), micelle system (average CT value = 24.93), and polymer-salt system (average CT value = 24.73) were all within 1 CT value (shown in Table 1.7). Similar results were observed for the recovery of 2000 bp DNA. The results show that the recoveries of 145 bp and 2000 bp DNA for the different ATPS classes are comparable.

[0521] Table 1.7: qPCR results for recovery of 145 bp and 2000 bp DNA oligonucleotides from plasma by different ATPS classes using magnetic beads. [Table 1.7]

[0522] Example 3a: Comparison of the efficiency of DNA recovery from plasma by centrifugal columns with and without a prior aqueous two-phase system (ATPS) step

[0523] In this example, the efficiency of DNA recovery from plasma using a centrifugal column was compared (i) using phase separation prior to the ATPS system according to the methods of the present disclosure, and (ii) without using a prior ATPS step. The materials used in this example are the same as or similar to those discussed in the previous examples. For the sake of brevity and simplicity of this disclosure, the discussion of the materials will not be repeated here.

[0524] material

[0525] The materials used in this example are the same or similar to those discussed in the previous examples, with the difference being the use of centrifugation columns (EconoSpin) instead of magnetic beads for DNA purification. For the sake of brevity and simplicity of this disclosure, the discussion of the materials will not be repeated here.

[0526] EconoSpin DNA / RNA mini centrifugation columns were purchased from Epoch Life Sciences, universal DNA spike II template was purchased from TATAA Biocenter, and GeneRuler 1 kb+ DNA standard was purchased from ThermoFisher.

[0527] Degradation of plasma cells

[0528] Each 2 mL of human pooled plasma was spiked with 100 fg of a 145 bp double-stranded DNA (dsDNA) oligonucleotide, 1.5 μL of a stock 2000 bp dsDNA oligonucleotide, and 100 ng of a 1 kb+ standard. Each 2 mL of spiked-in plasma was spiked with 160 μL of the appropriate digestion buffer and 60 μL of proteinase K (28.5 mg / mL). The mixture was vortexed thoroughly and digested in a preheated 60°C heating block for 15 minutes.

[0529] Extraction Process

[0530] One group of digested plasma samples (sample #4) was treated with two successive ATPS steps to isolate and enrich DNA prior to centrifugal column purification, while the other groups of digested plasma samples (samples #5 and #6) were directly subjected to centrifugal column purification without the previous ATPS step.

[0531] In this example, the steps for preparing Samples #4-6 are the same or similar to the materials discussed for Example 2a. For the sake of brevity and simplicity of this disclosure, the discussion of the two-phase steps will not be repeated here.

[0532] DNA purification

[0533] In this example, a binding buffer containing 1-5 M guanidine salt was used.

[0534] 800 μL of binding buffer was added to the extracted upper phase of sample #4 and to the digested sample #5, which had not been subjected to ATPS. For sample #6, 11.43 mL of binding buffer was added to digested sample #6, making the ratio of digested sample to binding buffer the same as the ratio of upper phase to binding buffer in sample #4. Samples #4-6 are summarized in Table 2.0 below. All samples were vortexed thoroughly before application to the EconoSpin column, and centrifuged at 12,000 rcf for 30 seconds in 800 μL until all sample digests had passed through the column. The flow-through (also referred to as "supernatant") was then discarded. 800 μL of wash buffer (70% ethanol, 0.001 M EDTA, 0.01 M Tris-HCl) was added to the centrifuge column, which was then centrifuged at 12,000 rcf for 30 seconds. The flow-through was discarded, and the column was centrifuged at 16,000 rcf for 2 minutes to remove excess wash buffer. 40 μL of elution buffer (0.01 M Tris-HCl, 0.001 M EDTA) was added to the column, which was then incubated for 3 minutes. The column was centrifuged at 12,000 rcf for 1 minute to elute the target DNA-containing buffer into a DNAase-free tube.

[0535] Table 2.0: Summary of plasma extraction and purification test conditions. [Table 2.0]

[0536] DNA detection

[0537] The steps for performing DNA detection for each sample were the same or similar to those discussed above for Example 1a. For the sake of brevity and simplicity of this disclosure, the discussion of the detection steps will not be repeated here. Results were expressed as mean CT values.

[0538] result

[0539] DNA recovery

[0540] Referring now to Figures 3A-3B, the average CT values ​​of 145 bp and 2000 bp DNA oligonucleotides recovered from plasma purified with a centrifugal column (Sample #4: with ATPS step, Sample #5: without ATPS step and unscaled binding buffer volume, and Sample #6: without ATPS step and scaled binding buffer volume, per Table 2.0) are shown. For 145 bp DNA, plasma sample #4 had a high recovery rate (average CT value of 27.15), while plasma sample #5 had a low recovery rate (average CT value of 37.49). For sample #6, proportionally increasing the binding buffer volume resulted in some improvement in recovery rate compared to sample #5 (average CT value of 31.71), but it was still significantly lower than the recovery rate of sample #4. Similar results could be observed from the recovery of 2000 bp DNA. The data show that concentrating and purifying plasma samples with a two-step ATPS step prior to the column binding step improved DNA recovery, and surprisingly, even with a proportional increase in the volume of binding buffer, DNA recovery was still significantly improved compared to samples without the ATPS step.

[0541] Table 2.1: qPCR results for recovery of 145 bp and 2000 bp DNA oligonucleotides from plasma with or without a two-step ATPS using a centrifugal column. [Table 2.1]

[0542] Overall, the recovery results surprisingly show that higher DNA recoveries can be achieved when ATPS is incorporated into the plasma extraction workflow prior to purification on a centrifugation column, even with the additional steps of ATPS concentration and separation (which are potential sources of target analyte loss during sample processing).

[0543] Example 3b: Plasma extraction performance with different polymers and salts in ATPS compositions

[0544] In this example, we demonstrate the ability to bind ATPSs with different polymer molecular weights and component concentrations to a centrifugal column to recover DNA from a plasma sample, and the ability to bind ATPSs with different polymer and salt chemistries to a centrifugal column to recover DNA from a plasma sample. The materials used in this example are the same as or similar to those discussed in the previous examples. For the sake of brevity and simplicity of this disclosure, we will not repeat the discussion of the materials here.

[0545] Degradation of plasma cells

[0546] In this example, 160 μL of the appropriate digestion buffer and 60 μL of protease K (28.57 mg / mL) were added to a 2 mL aliquot of EDTA (K2 TCS) plasma. 100 fg 145 bp DNA and 100 ng GR 1 kb+ standard were spiked into the sample. After vortexing for 15 seconds, the sample was incubated at 60°C for 15 minutes. The digest was used for further extraction.

[0547] Two-phase system

[0548] The extraction procedure involved two aqueous two-phase systems (ATPS) to isolate, purify, and concentrate DNA from plasma samples. The steps for performing the ATPS extraction in this example are the same as or similar to those discussed in Example 2b.

[0549] Extraction Process

[0550] This example describes an ATPS-based method for cfDNA isolation and purification. 2.22 mL of digest was added to the first ATPS. A dye was added to visualize the two phases, creating a colored upper phase. In this example, most of the DNA was located in the bottom phase, while the protein was located in the upper phase. The solution was made turbid by vigorous vortexing for 15 seconds. Phase separation was then facilitated by centrifugation at 2300 rcf for 6 minutes. After phase separation, the bottom phase volume was approximately 1 mL. The entire bottom phase was carefully extracted and transferred to a second ATPS. The second ATPS mixture was vortexed for 15 seconds to create a turbid solution, and then centrifuged at 7000 rcf for 1 minute to achieve phase separation. The resulting upper phase volume was approximately 150 μL and carefully extracted for further DNA purification and detection.

[0551] DNA purification

[0552] DNA purification was completed by centrifugal column extraction. The upper phase from the second ATPS was transferred to a test tube containing 800 μL of binding buffer (1-5 M guanidine salt). After thorough mixing, 800 μL of the solution was transferred to a centrifugal column (EconoSpin) and centrifuged at 12,000 rcf for 30 seconds. The flow-through was discarded. The process was repeated until the entire sample had passed through the centrifugal column. 800 μL of wash buffer (70% ethanol, 0.001 M EDTA, 0.01 M Tris-HCl) was added to the centrifugal column and centrifuged at 12,000 rcf for 30 seconds. After two washes, the tube was centrifuged at 16,000 rcf for 2 minutes to dry it. 40 μL of elution buffer (0.01 M Tris-HCl, 0.001 M EDTA) was added to the sample. The sample was incubated at room temperature for 3 minutes. The eluate was collected in a collection tube for detection by centrifugation at 12,000 rcf for 1 minute.

[0553] Extraction efficiency

[0554] The DNA extraction efficiency was investigated for different ATPS compositions while maintaining a constant volume ratio between the first and second ATPSs. To evaluate DNA recovery performance, a 2 mL sample containing 100 ng of DNA standard in human plasma was prepared. A 100% recovery yield would be 2.5 ng per μL of eluate.

[0555] DNA detection

[0556] The steps for performing DNA detection for each sample were the same or similar to those discussed above for Example 1a. For the sake of brevity and simplicity of this disclosure, the discussion of the detection steps will not be repeated here. Results were expressed as mean CT values.

[0557] result

[0558] Referring now to Figure 3C, the average C values ​​for 145 bp DNA recovery from plasma using the first and second ATPS compositions under conditions 5, 7, and 8 in Table 2.3 were compared. Conditions 5, 7, and 8 correspond to the same polymer and salt combinations with different polymer molecular weights, and the respective 145 bp DNA recovery results are shown in Table 2.3. The results show that when coupled with a centrifugal column workflow, all systems with alternative polymer molecular weights are able to recover detectable levels of DNA.

[0559] Table 2.3: Recovery of DNA from plasma using ATPS with different polymer molecular weights and centrifugal columns. [Table 2.3]

[0560] Referring now to Figure 3D, the average C values ​​for 145 bp DNA recovery from plasma using the first and second ATPS compositions under conditions 9, 22, 10, and 11 in Table 2.4 were compared. Conditions 9, 10, and 11 correspond to three different combinations of polymer and salt, and Table 2.4 shows the respective 145 bp DNA recovery results. The results show that when coupled with a centrifugation column workflow, all systems with alternative polymers and salts are able to recover detectable DNA.

[0561] Table 2.4: DNA recovery from plasma using ATPS and centrifugal columns with different polymers and salts. [Table 2.4]

[0562] Example 3c: Plasma extraction performance with different ATPS systems using a centrifugal column

[0563] In this example, the performance of different classes of ATPS systems (polymer-salt, polymer-polymer, micellar) used in conjunction with a centrifugal column was demonstrated. The materials used in this example are the same as or similar to those discussed in the previous examples. For the sake of brevity and simplicity of this disclosure, the discussion of the materials will not be repeated here.

[0564] Degradation of plasma

[0565] In this example, K2 EDTA plasma (TCS Biosciences, Lot No. 23014500) was digested in three different ways, thereby optimizing for each downstream ATPS system. The digestion samples used in this example are the same as or similar to those discussed in Example 2c. For the sake of brevity and simplicity of this disclosure, the discussion of the digestion samples will not be repeated here.

[0566] Extraction Process

[0567] The extraction process and three different classes of ATPS systems prepared were the same as those discussed in Example 2c and Table 1.6: 1. Polymer-based-polymer (PP), 2. Micellar, and 3. Double ATPS-polymer-based-salt (PS). For the sake of brevity and simplicity of this disclosure, the discussion of the preparation and extraction process of the ATPS compositions above will not be repeated here. Table 2.5 summarizes the three different types of ATPS and the subsequent conjugation steps.

[0568] Table 2.5: Summary of experimental conditions with alternative ATPS classes. [Table 2.5]

[0569] DNA purification

[0570] After extraction of the target cfDNA-containing phase from each ATPS system, downstream processing involves adding a binding buffer to a centrifugation column containing a porous membrane to isolate the nucleic acid from the liquid medium, followed by washing to remove unwanted ions and molecules before the target nucleic acid is eluted with an elution buffer. Table 2.5 summarizes the respective binding buffers used for each sample.

[0571] 1500 μL of binding buffer (3–7 M guanidine salt, 50 mM pH 7 NaHPO / NaHPO) was added to the extracted polymer-rich bottom phase of the polymer-polymer system. 500 μL of binding buffer (3–7 M guanidine salt) was added to the extracted surfactant-rich top phase of the micellar system. 800 μL of binding buffer (1–5 M guanidine salt) was added to the extracted polymer-rich top phase of the second polymer-salt ATPS system. 800 μL of each sample was then added to a separate EconoSpin column to obtain DNA, which was centrifuged at 12,000 rcf for 30 seconds. The target cfDNA was bound and retained on the centrifugation column, while the flow-through sample digest passed through a vacuum manifold and was discarded. The flow-through was discarded and the process was repeated until all samples had flowed through the centrifugation column. 800 μL of wash buffer (70% EtOH v / v, 1 mM EDTA, 10 mM Tris-HCl) was added to the polymer-polymer and polymer-salt centrifuge columns, and 400 μL of RPE wash buffer (80% EtOH, 100 mM NaCl, 10 mM Tris-HCl) was added to the micelle-based centrifuge column. All centrifuge columns were then centrifuged at 12,000 rcf for 30 seconds. This wash step removed impurities (e.g., proteins and other ions) bound to the silica centrifuge column membrane. The flow-through was discarded, and the centrifuge columns were centrifuged at 16,000 rcf for an additional 2 minutes to remove any excess RPE wash buffer. The centrifuge columns were then placed in new 1.5 mL centrifuge tubes, and 80 μL of elution buffer (0.01 M Tris-HCl, 1 mM EDTA) was pipetted directly onto the silica membrane and incubated at room temperature for 3 minutes. The centrifuge column was centrifuged at 12,000 rcf for 1 minute to elute the target cfDNA into a 1.5 mL centrifuge tube.

[0572] DNA detection

[0573] The steps for performing DNA detection for each sample were the same or similar to those discussed above for Example 1a. For the sake of brevity and simplicity of this disclosure, the discussion of the detection steps will not be repeated here. Results were expressed as mean CT values.

[0574] DNA recovery

[0575] Next, referring to Figures 3E-3F, the average CT values ​​of 145 bp and 2000 bp DNA recovered from plasma were comparable among the three different ATPS systems, and acceptable recoveries were all within a difference of approximately 1 CT value, as shown in Table 2.6. This indicated that the different ATPS classes were able to recover DNA to a similar extent during optimization.

[0576] Table 2.6: qPCR results for recovery of 145 bp and 2000 bp DNA oligonucleotides from plasma by different ATPS classes using centrifugation columns. [Table 2.6]

[0577] Example 4a: Comparison of the efficiency of DNA recovery from urine by magnetic beads with and without a prior aqueous two-phase system (ATPS) step

[0578] In this example, the efficiency of DNA recovery from urine using magnetic beads was compared (i) using phase separation prior to the ATPS system (also referred to as "ATPS step," "ATPS extraction," or "ATPS workflow") according to the methods of the present disclosure, and (ii) without using a prior ATPS step. The materials used in this example are the same as or similar to those discussed in the previous examples. For the sake of brevity and simplicity of this disclosure, the discussion of the materials will not be repeated here.

[0579] Breakdown of urine

[0580] Urine samples were collected from three different donors. The samples from each donor were divided equally into 40 mL tubes and separated into two groups, each containing one sample from each donor.

[0581] All urine samples from both groups 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 and the pellet was discarded.

[0582] Unwanted proteins and cells present in the pretreated urine samples were digested by adding 1200 μL (28.57 mg / mL) proteinase K and 4 mL of the appropriate digestion buffer to each 40 mL sample from each donor. The samples were then thoroughly lubricated to homogeneity and incubated in a preheated 37°C water bath for 15 minutes.

[0583] Extraction Process

[0584] To effectively extract cfDNA from urine samples, two different aqueous two-phase systems (ATPS) were prepared. The first ATPS was used for the initial extraction of urine samples, where the desired cfDNA was concentrated in the salt-rich bottom phase. To enable user-friendly downstream processing, the bottom phase from the first ATPS was extracted and added to the second ATPS, concentrating the target cfDNA in a small volume (400 µL–600 µL).

[0585] In this example, the first ATPS consists of ATPS 38 composition according to Table 1.0a, containing 22600 uL of digested urine sample.

[0586] The second ATPS consists of ATPS 71 composition according to Table 1.0a, where it contains 3.5 mL to 5 mL of the first ATPS bottom phase.

[0587] For urine sample group 1 (sample #1), the urine sample from each donor was split in half and added to two first ATPS tubes. The first ATPS was vortexed thoroughly and centrifuged at 2300 rcf for 6 minutes. The salt-rich bottom phase from the two first ATPSs (from the same donor) was then extracted, reconstituted, and added to a second ATPS tube, which was vortexed thoroughly and centrifuged to allow phase separation. The polymer-rich upper phase of the second ATPS system was extracted and placed in a new tube.

[0588] Urine sample group 2 (sample #2) was pretreated and processed but was concentrated and not subjected to the double ATPS system for purification. The urine sample groups are summarized in Table 3.0 below.

[0589] Table 3.0: Summary of test conditions for urine extraction and purification. [Table 3.0]

[0590] DNA purification

[0591] The target cfDNA in the urine sample was concentrated to 400-600 μL in the polymer-rich upper phase of the second ATPS. A binding buffer containing 3-7 M guanidine salt was used.

[0592] Two milliliters of binding buffer were added to the extracted upper phase of sample #1 and to the digested sample #2 that had not been subjected to ATPS. 24 μL of magnetic beads were added to each tube. The mixture was then incubated on a rotator for 5 minutes to prevent bead sedimentation. The tubes were then briefly centrifuged to sediment and placed on a magnetic rack for 2 minutes to immobilize the beads to the tube wall. The supernatant was discarded without disturbing the beads. 2 mL of binding buffer was added to each tube and gently spun on the magnetic support for a total of 720°. Pipetting was repeated, and the supernatant was discarded. 800 μL of wash buffer (70% ethanol, 0.001 M EDTA, 0.01 M Tris-HCl) was added to the samples, and the tubes were rotated on the support for a total of 720°. The supernatant was discarded. This washing step was performed twice. To improve drying, the tubes were briefly centrifuged and spun in a tabletop microcentrifuge with the hinge facing outward to collect any remaining wash buffer. The beads were then dried on the magnetic support with the lid open for 7 minutes. The bead-analyte complexes were resuspended in 80 μL of elution buffer (0.01 M Tris-HCl, 0.001 M EDTA) by repeated pipetting and vortexing briefly. The tubes were then placed on a magnetic rack for 1 minute. The supernatant was carefully collected into a low-DNA binding tube without disturbing the magnetic beads for detection.

[0593] DNA detection

[0594] The steps for performing DNA detection for each sample were the same or similar to those discussed above for Example 2a. For the sake of brevity and simplicity of this disclosure, the discussion of the detection steps will not be repeated here. Results were expressed as mean CT values.

[0595] result

[0596] DNA recovery

[0597] Next, referring to Figures 4A-4B, we show the average CT values ​​of 145 bp DNA (Figure 3A) and 2000 bp DNA (Figure 4B) recovered from urine using magnetic beads with and without a prior ATPS step (sample #1 and sample #2). While we demonstrated high recovery rates of 145 bp DNA (average CT value of 27.47) and 2000 bp DNA (average CT value of 27.09) from urine sample #1, we did not observe detectable target DNA recovery from urine sample #2 (shown in Table 3.1). The results demonstrate that, under the same conditions, increasing the number of parallel ATPS steps prior to magnetic bead purification significantly improves DNA recovery from large-volume samples such as urine.

[0598] Table 3.1: qPCR results for recovery of 145 bp and 2000 bp DNA oligonucleotides from urine using magnetic beads with or without a two-step ATPS. [Table 3.1]

[0599] Overall, the recovery results surprisingly show that higher DNA recoveries can be achieved when ATPS is incorporated into the plasma extraction workflow prior to purification on a centrifugation column, even with the additional steps of ATPS concentration and separation (which are potential sources of target analyte loss during sample processing).

[0600] Example 4b: Urine extraction performance with different polymers and salts in ATPS compositions

[0601] In this example, ATPSs with different polymer molecular weights and component concentrations were used to demonstrate their ability to bind to magnetic beads and recover DNA from urine samples. The materials used in this example are the same as or similar to those discussed in the previous examples. For the sake of brevity and simplicity of this disclosure, the discussion of the materials will not be repeated here.

[0602] Breakdown of urine

[0603] 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, and the pellet was discarded. To digest unwanted proteins and cells, 60 μL of proteinase K (28.57 mg / mL) and 200 μL of the appropriate digestion buffer were added to 2 mL of sample. 100 fg of 145 bp oligonucleotide and 100 ng of 1 kb+ DNA standard were spiked into the sample. The sample was then thoroughly lutixed to homogeneity and incubated in a preheated 37°C water bath for 15 minutes.

[0604] Two-phase system

[0605] The extraction procedure involved two aqueous two-phase systems (ATPSs) by which DNA was isolated, purified, and concentrated from urine samples. To evaluate the suitability of such systems, the phase separation characteristics and DNA extraction efficiency of various polymers and salts in the first ATPS were investigated. In the first ATPS, DNA was assigned to the bottom phase, and proteins to the upper phase. Approximately 1 mL of the bottom phase was carefully extracted and transferred to the second ATPS. Because the composition of the ATPS bottom phase strongly depends on the ATPS components, the composition of the bottom phase of the first ATPS differs from each other depending on the various polymers and salts used. Therefore, the composition of the second ATPS was changed accordingly to achieve a small upper phase volume of approximately 150 μL. DNA was assigned to the upper phase of the second ATPS, and the upper phase was carefully extracted for further purification and detection.

[0606] Extraction Process

[0607] 2.26 mL of digest was added to the first ATPS. A dye was added to visualize the two phases, creating a colored upper phase. To optimize and combine interactions with the ATPS components, most of the DNA was assigned to the bottom phase, while the proteins were assigned to the upper phase. The solution was turbid by vigorous vortexing for 15 seconds. Phase separation was then facilitated by centrifugation at 2300 rcf for 6 minutes. After phase separation, the bottom phase volume was approximately 1 mL. The entire bottom phase was carefully extracted and transferred to a second ATPS. The second ATPS was configured to assign DNA to the upper phase. The mixture was vortexed for 15 seconds to create a turbid solution, and then centrifuged at 7000 rcf for 1 minute to achieve phase separation. The volume of the upper phase in the resulting solution was approximately 150 μL. The small volume ratio between the upper and bottom phases favors the concentration of DNA in the upper phase. The upper phase was then carefully extracted for further DNA purification and detection.

[0608] DNA purification

[0609] DNA purification was completed by magnetic bead extraction (also known as the "magnetic bead workflow"). The upper phase from the second ATPS was transferred to a tube containing 600 μL of binding buffer (3-7 M guanidine salt). 6 μL of magnetic beads were added to the tube, and the sample was incubated with tilt and rotation for 5 minutes. The tube was then briefly centrifuged to sediment and placed on a magnetic support for 2 minutes to immobilize the beads to the tube wall. The supernatant was discarded after pipetting without disturbing the magnetic beads. 800 μL of binding buffer was added to the sample. The tube was rotated a total of 720° on the support. The supernatant was discarded after pipetting. 800 μL of wash buffer (70% ethanol, 0.001 M EDTA, 0.01 M Tris-HCl) was added to the sample, and the tube was rotated a total of 720° on the support. The supernatant was discarded after pipetting. Two washing steps were performed, and the tubes were placed on a support with the lids open to allow the beads to dry. 40 μL of elution buffer (0.01 M Tris-HCl, 0.001 M EDTA) was added to the samples. The bead complexes were resuspended by repeated pipetting and briefly vortexed. The samples were incubated at room temperature for 3 minutes and briefly centrifuged. The tubes were then placed on a magnetic rack for 1 minute. The supernatant was carefully collected without disturbing the magnetic beads for detection.

[0610] Extraction efficiency

[0611] Both the first and second ATPSs contained a mixture of polymer and salt, resulting in a polymer-rich upper phase and a salt-rich lower phase. To investigate the compatibility of the current system with different reagents, we investigated the DNA extraction efficiency of different ATPS compositions while maintaining a constant volume ratio of the first and second ATPSs. To evaluate DNA recovery performance, a sample containing 100 fg of 145 bp dsDNA was prepared, which, assuming a 100% recovery, would yield 2.5 ng per μL of eluate.

[0612] DNA detection

[0613] The steps for performing DNA detection for each sample were the same or similar to those discussed above for Example 1a. For the sake of brevity and simplicity of this disclosure, the discussion of the detection steps will not be repeated here. Recovery was quantified by qPCR, and the results are summarized in Figures 3C-3D. Results were expressed as mean C values.

[0614] result

[0615] Referring now to Figure 4C, the average C values ​​for 145 bp DNA recovery using the first and second ATPS compositions under conditions 33, 41, 34, and 35 in Table 3.3 were compared. Conditions 33, 41, 34, and 35 correspond to the same polymer and salt combinations with different polymer molecular weights, and the respective 145 bp DNA recovery results are shown in Table 3.3. The results show that when coupled with a magnetic bead workflow, all systems with alternative polymer molecular weights are able to recover detectable levels of DNA from urine.

[0616] Table 3.3: Recovery of DNA from urine using ATPS and magnetic beads with different polymer molecular weights. [Table 3.3]

[0617] Referring next to Figure 4D, the average C values ​​for 145 bp DNA recovery from urine using the first and second ATPS compositions under conditions 36, 6, 38, and 39 in Table 3.4 were compared. Conditions 36, 6, 38, and 39 correspond to four different combinations of polymer and salt, and Table 3.4 shows the respective 145 bp DNA recovery results. The results show that all systems with alternative polymers and salts recovered detectable DNA from urine using magnetic beads. All samples showed acceptable DNA recovery, demonstrating the successful extraction of DNA using multiple reagents.

[0618] Table 3.4: Recovery of DNA from urine using ATPS and magnetic beads with different ranges of polymers and salts. [Table 3.4]

[0619] Example 4c: Urine extraction performance with various ATPS systems using magnetic beads

[0620] In this example, we demonstrate the ability to recover DNA from urine samples using different classes of ATPS systems (polymer-salt, polymer-polymer, micelles) in conjunction with magnetic beads. The materials used in this example are the same as or similar to those discussed in previous examples. For the sake of brevity and simplicity of this disclosure, we will not repeat the discussion of the materials here.

[0621] Breakdown of urine

[0622] Urine samples extracted with the polymer-polymer and polymer-salt ATPS systems were pretreated with 200 μL of 0.1 M EDTA per 10 mL of urine sample, thoroughly vortexed, and centrifuged at 3000 rcf for 10 minutes. The supernatant was transferred to a new tube, and the pellet was discarded. Unwanted proteins and cells present in the pretreated urine sample were digested by adding 60 μL of proteinase K (28.57 mg / mL) and 200 μL of the appropriate digestion buffer to a 2 mL sample. The sample was then thoroughly lutixed until homogenous and incubated in a preheated 37°C water bath for 15 minutes.

[0623] For samples extracted with the micelle-based ATPS system, urine samples were pretreated with 200 μL of 0.1 M EDTA per 10 mL of urine sample, centrifuged at 3,000 rcf for 10 minutes, and the supernatant was collected. 300 μL of the appropriate lysis buffer was added to 1 mL of pretreated urine sample, vortexed thoroughly, and incubated at room temperature for 3 minutes. 60 μL of protein precipitation buffer A (PPt A) (20% ZnCl w / v, 1% acetic acid w / v) was then added to the sample, vortexed thoroughly until homogenous, and centrifuged at 12,000 rcf for 3 minutes. Other appropriate protein precipitation buffers may be used in place of protein precipitation buffer A in the above method. The supernatant was extracted for further downstream processing, and the pellet was discarded.

[0624] Extraction Process

[0625] To effectively extract cfDNA from urine samples, different types of aqueous two-phase systems (ATPS) similar to those discussed in Example 2c were prepared. Table 3.5 summarizes the three different types of ATPS systems that were prepared. In the polymer-polymer and polymer-salt based ATPS systems, target cfDNA was allocated to the bottom phase from the degraded sample without apportioning excess protein to the top phase. The opposite trend was observed in the micelle-based ATPS system.

[0626] Table 3.5: Summary of experimental conditions with alternative ATPS classes. [Table 3.5]

[0627] The sample digest was added to each ATPS system, with 1130 uL of sample digest added to the polymer-polymer-based system and 1360 uL of extracted supernatant added to the micelle-based system. After adding the sample digest to all ATPS tubes, they were vortexed thoroughly.

[0628] The polymer-polymer ATPS system was centrifuged at 2300 rcf for 6 minutes. The target cfDNA was concentrated in the polymer-rich bottom phase. The entire bottom phase (approximately 300 μL) was extracted and transferred to a new 2 mL centrifuge tube for further processing.

[0629] The micellar ATPS system was centrifuged at 12,000 rcf for 5 minutes. The target cfDNA was concentrated in the detergent-rich upper phase. The entire upper phase (approximately 280 μL) was extracted and transferred to a new 2 mL centrifuge tube for further processing.

[0630] The first polymer-salt ATPS system was centrifuged at 2300 rcf for 6 minutes. The target cfDNA was concentrated in the salt-rich bottom phase. The entire bottom phase (approximately 1000 μL) was extracted and transferred to the second ATPS, which was thoroughly vortexed and centrifuged at 7000 rcf for 1 minute. The target cfDNA was concentrated in the polymer-rich upper phase (approximately 120 μL), which was then extracted and transferred to a new tube for further processing.

[0631] DNA purification

[0632] After extraction of the target cfDNA-containing phase from each ATPS system, downstream processing involves adding a solid phase medium and binding buffer to isolate the nucleic acid from the liquid medium, and washing to remove unwanted ions and molecules before the target nucleic acid is eluted with an elution buffer.

[0633] 1500 μL of binding buffer (3–7 M guanidine salt, 50 mM pH 7 NaHPO / NaHPO) was added to the extracted polymer-rich bottom phase of the polymer-polymer system. 500 μL of binding buffer (3–7 M guanidine salt) was added to the extracted surfactant-rich top phase of the micellar system. 800 μL of binding buffer (1–5 M guanidine salt) was added to the extracted polymer-rich top phase of the second polymer-salt ATPS system. 6 μL of magnetic beads were then added to all centrifuge tubes containing the mixture of extraction phase and binding buffer, placed on a rotator, and incubated for 5 minutes. The target cfDNA bound to the magnetic beads, forming bead-analyte complexes. After incubation, all tubes were briefly centrifuged and placed on a magnetic rack, which applied a magnetic field to attract the cfDNA-bound magnetic beads. The supernatant was then removed and discarded, and the magnetic beads were allowed to bind to the inner wall of the centrifuge tube. A second binding buffer wash step was performed on the polymer-polymer and polymer-salt systems. 800 μL of binding buffer (3-7 M guanidine salt, 50 mM pH 7 NaHPO / NaHPO) was added to the magnetic beads from the polymer-polymer system, and 800 μL of binding buffer (1-5 M guanidine salt) was added to the magnetic beads from the polymer-salt system. The tubes were then vortexed thoroughly, centrifuged briefly, and returned to the magnetic rack. Once all the magnetic beads had bound to the inside of the centrifuge wall, the supernatant was discarded.

[0634] 800 μL of the original wash buffer (70% v / v EtOH, 1 mM EDTA, 10 mM Tris-HCl) was added to the magnetic beads from the polymer-polymer and polymer-salt systems, and 500 μL of RPE wash buffer (80% v / v EtOH, 0.1 M NaCl, 10 mM Tris-HCl) was added to the magnetic beads from the micelle system. Before discarding the wash buffer, the centrifuge tube was rotated 720° on the magnetic rack. The above procedure was repeated for a second wash. The centrifuge tube was then briefly centrifuged before returning it to the magnetic rack, and any excess wash buffer remaining in the centrifuge tube was discarded. For the polymer-polymer and polymer-salt systems, the magnetic beads were allowed to dry at room temperature for 7 minutes, and for the micelle system, for 10 minutes.

[0635] After the magnetic beads dried, they were resuspended in 20 μL of elution buffer (10 mM Tris-HCl, 1 mM EDTA) and incubated at room temperature for 3 minutes. The target cfDNA was eluted from the magnetic beads into the elution buffer. The tube was then returned to the magnetic rack. Once all of the magnetic beads were bound, the elution buffer was extracted.

[0636] DNA detection

[0637] The steps for performing DNA detection for each sample were the same or similar to those discussed above for Example 1a. For the sake of brevity and simplicity of this disclosure, the discussion of the detection steps will not be repeated here. Results were expressed as mean CT values.

[0638] result

[0639] Referring now to Figure 4E, Table 3.6 shows the average CT values ​​for 145 bp DNA recovery from urine when extracted using different types of ATPS systems. The results show that the three systems have comparable DNA recovery efficiencies, with the average CT values ​​for the polymer-polymer system (average CT value = 28.73), micellar system (average CT value = 27.99), and polymer-salt system (average CT value = 27.85) all differing within 1 CT value. This indicates that different ATPS classes can recover DNA to a similar extent when optimized.

[0640] Table 3.6: qPCR results of 145 bp DNA recovery from urine by different ATPS classes using magnetic beads. [Table 3.6]

[0641] Example 5a: Comparison of the efficiency of DNA recovery from urine by centrifugal columns with and without a prior aqueous two-phase system (ATPS) step

[0642] In this example, the efficiency of DNA recovery from urine using a centrifugal column was compared (i) using a prior phase separation of the ATPS system according to the methods of the present disclosure, and (ii) without using a prior ATPS step. The materials used in this example are the same as or similar to those discussed in the previous example. For the sake of brevity and simplicity of this disclosure, the discussion of the materials will not be repeated here.

[0643] Breakdown of urine

[0644] Urine samples were collected from four different donors. The samples from each donor were divided equally into 40 mL tubes and separated into two groups, each containing one sample from each donor.

[0645] All urine samples from both groups were treated 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 and the pellet was discarded.

[0646] Unwanted proteins and cells present in the pretreated urine samples were digested by adding 1200 μL (28.57 mg / mL) proteinase K and 4 mL of the appropriate digestion buffer to each 40 mL sample from each donor. The samples were then thoroughly lubricated to homogeneity and incubated in a preheated 37°C water bath for 15 minutes.

[0647] Extraction Process

[0648] To effectively extract cfDNA from urine samples, two different aqueous two-phase systems (ATPS) were prepared. In this example, the steps to perform the ATPS extraction to prepare urine sample #3 and urine sample #4 were the same or similar to those discussed for Example 4a. For the sake of brevity and simplicity of this disclosure, the discussion of the ATPS steps will not be repeated here. The urine sample groups are summarized in Table 4.0 below.

[0649] Table 4.0. Summary of test conditions for urine extraction and purification. [Table 4.0]

[0650] DNA purification

[0651] The target cfDNA in the urine sample was concentrated in the polymer-rich upper phase of the second ATPS, which was then isolated for further processing.

[0652] A 3-7 M guanidine salt solution was used as the binding buffer. 2 mL of 3-7 M guanidine salt (approximately 400 μL to 600 μL) was added to the polymer-rich upper phase of the second ATPS extracted from urine group 3 (sample #3) and vortexed thoroughly. Urine sample group 4 (sample #4), which had not undergone ATPS purification or concentration, was mixed with 2 mL of 3-7 M guanidine salt and vortexed thoroughly. Each urine sample was then added to an EconoSpin column, and the ligated DNA was obtained by loading the appropriate bulking agent (3 mL and 20 mL) onto a QIAvac 24 Plus vacuum manifold. A pressure of 900 mbar was applied to the vacuum manifold, allowing the sample lysate to flow through the centrifugation column. The target cfDNA was bound and retained on the centrifugation column, while the flow-through sample lysate (also referred to as the "supernatant") passed through the vacuum manifold and was discarded. After all possible sample lysate had flowed through the centrifugation column, the bulking agent was removed and discarded. The centrifugal column was removed from the manifold and a 2 mL waste tube was inserted. 500 μL of RPE wash buffer (80% v / v EtOH, 0.1 M NaCl, 0.01 M Tris-HCl) was added to the centrifugal column and centrifuged at 12,000 rcf for 30 seconds. The flow-through was discarded, and the centrifugal column was centrifuged at 16,000 rcf for an additional 2 minutes to remove any excess RPE wash buffer. The centrifugal column was then placed in a new 1.5 mL centrifuge tube, and 80 μL of elution buffer (0.01 M Tris-HCl, 1 mM EDTA) was pipetted directly onto the silica membrane and incubated at room temperature for 3 minutes. The centrifugal column was centrifuged at 12,000 rcf for 1 minute to elute the target cfDNA into the 1.5 mL centrifuge tube.

[0653] DNA detection

[0654] The steps for performing DNA detection for each sample were the same or similar to those discussed above for Example 1a. For the sake of brevity and simplicity of this disclosure, the discussion of the detection steps will not be repeated here. Results were expressed as mean CT values.

[0655] result

[0656] DNA recovery

[0657] In urine sample #3, the entire digest flowed through the centrifuge column within 1 minute. In urine sample #4, only 25% of the sample digest passed through the centrifuge column within the first 60 minutes. In the case of parallel ATPS, the flow time (1 hour) needed to be increased to allow the unprocessed digest to pass completely through the column.

[0658] Next, referring to Figures 5A-5B, we show the average CT values ​​of 145 bp and 2000 bp DNA recovered from urine using a centrifugal column with and without a prior ATPS step (sample #3 and sample #4). According to Table 4.1, for 145 bp DNA, urine sample #3 had a high recovery rate (average CT value of 25.36 ± 0.08), while urine sample #4 had a low recovery rate (average CT value of 38.15 ± 2.10). Similar results were observed for 2000 bp DNA recovery. As shown in the results, concentrating the urine digest by parallel ATPS prior to centrifugal column extraction significantly improved the recovery of 145 bp and 2000 bp DNA, improving the efficiency of downstream processing.

[0659] Table 4.1: qPCR results for recovery of 145 bp and 2000 bp DNA oligonucleotides from urine using centrifugal columns with or without a two-step ATPS. [Table 4.1]

[0660] Overall, the recovery results surprisingly show that higher DNA recoveries can be achieved when ATPS is incorporated into the plasma extraction workflow prior to purification on a centrifugation column, even with the additional steps of ATPS concentration and separation (which are potential sources of target analyte loss during sample processing).

[0661] Example 5b: Urine extraction performance with different polymers and salts in ATPS compositions

[0662] In this example, ATPSs with different polymer MWs and component concentrations were used to demonstrate the ability to bind to a centrifugal column to recover DNA from a urine sample. The materials used in this example are the same as or similar to those discussed in the previous examples. For the sake of brevity and simplicity of this disclosure, the discussion of the materials will not be repeated here.

[0663] Breakdown of urine

[0664] In this example, urine samples were pretreated with 200 μL of 0.1 M EDTA per 10 mL of urine sample, thoroughly vortexed, and centrifuged at 3000 rcf for 10 minutes. The supernatant was transferred to a new tube, and the pellet was discarded. To digest unwanted proteins and cells present in the pretreated urine sample, 1200 μL of proteinase K (28.57 mg / mL) and 4 mL of the appropriate digestion buffer were added to the 40 mL sample. 100 μg of 145 bp DNA and 100 ng of GR 1 kb+ standard were spiked into the sample. The sample was then thoroughly lutixed to homogeneity and incubated in a preheated 37°C water bath for 15 minutes.

[0665] Two-phase system

[0666] The extraction procedure, involving two aqueous two-phase systems (ATPS), used to isolate, purify, and concentrate DNA from urine samples was similar to that described in the previous example. In the first ATPS, DNA was assigned to the bottom phase, and proteins to the upper phase. Approximately 1 mL of the bottom phase was carefully extracted and transferred to the second ATPS. Because the composition of the ATPS bottom phase strongly depends on the ATPS components, the composition of the bottom phase of the first ATPS differs from each other due to the various polymers and salts used. Therefore, the composition of the second ATPS was changed accordingly to achieve a small upper phase volume of approximately 150 μL. DNA was assigned to the upper phase of the second ATPS, and the upper phase was carefully extracted for further purification and detection.

[0667] Extraction Process

[0668] 2.22 mL of digest was added to the first ATPS. A dye was added to visualize the two phases, creating a colored upper phase. In this example, most of the DNA was located in the bottom phase, while the protein was located in the upper phase. The solution was made cloudy by vigorously vortexing for 15 seconds. Phase separation was then facilitated by centrifugation at 2300 rcf for 6 minutes. After phase separation, the bottom phase volume was approximately 1 mL. The entire bottom phase was carefully extracted and transferred to the second ATPS. The second ATPS mixture was vortexed for 15 seconds to create a cloudy solution, and then centrifuged at 7000 rcf for 1 minute to achieve phase separation. The resulting upper phase volume was approximately 150 μL and carefully extracted for further DNA purification and detection.

[0669] DNA purification

[0670] DNA purification was performed by centrifugal column extraction, similar to the steps discussed in Example 5a. The upper phase from the second ATPS was transferred to a test tube containing 800 μL of binding buffer (1-5 M guanidine salt). After thorough mixing, 800 μL of the solution was transferred to a centrifugal column (EconoSpin) and centrifuged at 12,000 rcf for 30 seconds. The flow-through was discarded. The above process was repeated until the entire sample had passed through the centrifugal column. 800 μL of wash buffer (70% ethanol, 0.001 M EDTA, 0.01 M Tris-HCl) was added to the centrifugal column and centrifuged at 12,000 rcf for 30 seconds. After two washes, the tube was centrifuged at 16,000 rcf for 2 minutes and dried. 40 μL of elution buffer (0.01 M Tris-HCl, 0.001 M EDTA) was added to the sample. The sample was incubated at room temperature for 3 minutes. The eluate was collected in a collection tube for detection by centrifugation at 12,000 rcf for 1 minute.

[0671] Extraction efficiency

[0672] Both the first and second ATPSs contained a mixture of polymer and salt, resulting in a polymer-rich upper phase and a salt-rich lower phase. In this example, the DNA extraction efficiency of different ATPS compositions was investigated while maintaining a constant volume ratio between the first and second ATPSs. To evaluate DNA recovery performance, a 2 mL sample containing 100 ng of DNA standard in urine was prepared. A 100% recovery yield would be 2.5 ng per μL of eluate.

[0673] DNA detection

[0674] The steps for performing DNA detection for each sample were the same or similar to those discussed above for Example 1a. For the sake of brevity and simplicity of this disclosure, the discussion of the detection steps will not be repeated here. Results were expressed as mean CT values.

[0675] result

[0676] Referring now to Figure 5C, the average C values ​​for 145 bp DNA recovery from urine using the first and second ATPS compositions under conditions 33, 34, and 35 in Table 4.3 were compared. Conditions 33, 34, and 35 correspond to the same polymer and salt combinations with three different polymer molecular weights, and the respective 145 bp DNA recovery results are shown in Table 4.3. The results show that when coupled with a centrifugation column workflow, all systems with alternative polymer molecular weights are able to recover detectable DNA from urine.

[0677] Table 4.3: Recovery of DNA from urine using ATPS with different polymer molecular weights and centrifugal columns. [Table 4.3]

[0678] Referring now to Figure 5D, the average C values ​​for 145 bp DNA recovery from urine using the first and second ATPS compositions under conditions 36-39 in Table 4.4 were compared. Conditions 36, 37, 38, and 39 correspond to four different combinations of polymer and salt, and the 145 bp DNA recovery results for each are shown in Table 4.4. The results show that all systems with alternate polymers and salts recovered detectable DNA from urine using a centrifugation column.

[0679] Table 4.4: DNA recovery from urine using ATPS and centrifugation columns with different polymers and salts. [Table 4.4]

[0680] Example 5c: Urine extraction performance with different ATPS systems using a centrifugal column

[0681] In this example, we demonstrate the ability to recover DNA from urine samples using different classes of ATPS systems (polymer-salt, polymer-polymer, micelles) in combination with a centrifugal column. The materials used in this example are the same as or similar to those discussed in the previous examples. For the sake of brevity and simplicity of this disclosure, the discussion of the materials will not be repeated here.

[0682] Breakdown of urine

[0683] In this example, urine samples extracted with the polymer-polymer and polymer-salt ATPS systems were pretreated with 200 μL of 0.1 M EDTA per 10 mL of urine sample, thoroughly vortexed, and centrifuged at 3000 rcf for 10 minutes. The supernatant was transferred to a new tube, and the pellet was discarded. Unwanted proteins and cells present in the pretreated urine sample were digested by adding 1200 μL of proteinase K (28.57 mg / mL) and 4 mL of the appropriate digestion buffer to the 40 mL sample. The sample was then thoroughly lutixed until homogenous and incubated in a preheated 37°C water bath for 15 minutes.

[0684] For samples extracted with the micelle-based ATPS system, urine samples were pretreated with 200 μL of 0.1 M EDTA per 10 mL of urine sample, centrifuged at 3,000 rcf for 10 minutes, and the supernatant was collected. 300 μL of the appropriate digest was added to 1 mL of pretreated urine sample, vortexed thoroughly, and incubated at room temperature for 3 minutes. 112.5 μL of protein precipitation buffer A (PPt A) (20% ZnCl w / v, 1% acetic acid w / v) was then added to the sample, vortexed thoroughly until homogenous, and centrifuged at 12,000 rcf for 3 minutes. Other appropriate protein precipitation buffers may be used in place of protein precipitation buffer A in the above method. The supernatant was extracted for further downstream processing, and the pellet was discarded.

[0685] Extraction Process

[0686] The extraction process and the three different classes of ATPS systems prepared were all the same as discussed in Example 4c: 1. Polymer-based-polymer (PP), 2. Micellar, and 3. Double ATPS-polymer-based-salt (PS). For the sake of brevity and simplicity of this disclosure, the discussion of the preparation and extraction process of the ATPS compositions described above will not be repeated here.

[0687] Table 4.5 summarizes the three different types of ATPS and the subsequent binding steps used in this example.

[0688] Table 4.5: Summary of experimental conditions with alternative ATPS classes. [Table 4.5]

[0689] The sample digest was added to each ATPS system, 1130 uL of sample digest was added to the polymer-polymer based system, and 1360 uL of extracted supernatant was added to the micellar system. After adding the sample digest to all ATPS tubes, they were vortexed thoroughly.

[0690] The polymer-polymer ATPS system was centrifuged at 2300 rcf for 6 minutes. The target cfDNA was concentrated in the polymer-rich bottom phase. The entire bottom phase (approximately 300 μL) was extracted and transferred to a new 2 mL centrifuge tube for further processing.

[0691] The micellar ATPS system was centrifuged at 12,000 rcf for 5 minutes. The target cfDNA was concentrated in the detergent-rich upper phase. The entire upper phase (approximately 280 μL) was extracted and transferred to a new 2 mL centrifuge tube for further processing.

[0692] The first polymer-salt ATPS system was centrifuged at 2300 rcf for 6 minutes. The target cfDNA was concentrated in the salt-rich bottom phase. The entire bottom phase (approximately 1000 μL) was extracted and transferred to the second ATPS, which was thoroughly vortexed and centrifuged at 7000 rcf for 1 minute. The target cfDNA was partitioned and concentrated in the polymer-rich upper phase (approximately 120 μL), which was then extracted and transferred to a new tube for further processing.

[0693] DNA purification

[0694] After extraction of the target cfDNA-containing phase from each ATPS system, downstream processing involves adding a solid phase medium and binding buffer to isolate the nucleic acid from the liquid medium, and washing to remove unwanted ions and molecules before the target nucleic acid is eluted with an elution buffer.

[0695] 1500 μL of binding buffer (3–7 M guanidine salt, 50 mM pH 7 NaHPO / NaHPO) was added to the extracted polymer-rich bottom phase of the polymer-polymer system. 500 μL of binding buffer (3–7 M guanidine salt) was added to the extracted surfactant-rich top phase of the micellar system. 800 μL of each sample was then added to individual EconoSpin columns to obtain DNA and centrifuged at 12,000 rcf for 30 seconds. The target cfDNA was bound and retained on the centrifugal column, while the flow-through sample digest passed through a vacuum manifold and was discarded. The flow-through was discarded and the process repeated until the entire sample had flowed through the centrifugal column. 500 μL of RPE wash buffer (80% v / v EtOH, 0.1 M NaCl, 0.01 M Tris-HCl) was added to the centrifugal column and centrifuged at 12,000 rcf for 30 seconds. The flow-through was discarded, and the column was centrifuged at 16,000 rcf for an additional 2 minutes to remove any excess RPE wash buffer. The column was then placed in a new 1.5 mL centrifuge tube, and 80 μL of elution buffer (0.01 M Tris-HCl, 1 mM EDTA) was pipetted directly onto the silica membrane and incubated at room temperature for 3 minutes. The column was centrifuged at 12,000 rcf for 1 minute to elute the target cfDNA into the 1.5 mL tube.

[0696] DNA detection

[0697] The steps for performing DNA detection for each sample were the same or similar to those discussed above for Example 2a. For the sake of brevity and simplicity of this disclosure, the discussion of the detection steps will not be repeated here. Results were expressed as mean CT values.

[0698] DNA recovery

[0699] result

[0700] Referring now to Figure 5E, Table 4.6 shows the average C values ​​for 145 bp DNA recovery from urine using different types of ATPS systems (polymer-polymer, micelle, polymer-salt). The results show that all three systems can effectively provide satisfactory DNA recovery from urine.

[0701] Table 4.6: qPCR results of 145 bp DNA recovery from urine by different ATPS classes using centrifugation columns. [Table 4.6]

[0702] Example 6: Matching results

[0703] Magnetic beads were used as the solid phase.

[0704] Referring now to Figure 6A, we present a combined data representation of the Ct values ​​of 145 bp DNA recovered from plasma and urine using magnetic beads (associated with Examples 2a-c and 4a-c). As shown in Figure 6A, compared to the conditions without a preceding ATPS step, all conditions with a preceding ATPS step achieved higher DNA recovery rates, i.e., plasma sample #2 and urine sample #2 in Figure 6A both yielded Ct values ​​close to 40 (see sample #2 in Example 2a and sample #2 in Example 4a). This highlights the importance of incorporating an ATPS step into urine extraction prior to magnetic bead purification to achieve satisfactory DNA recovery.

[0705] A centrifugation column was used as the solid phase.

[0706] Referring now to Figure 6B, a combined data representation of Ct values ​​for 145 bp DNA recovered from plasma and urine using a centrifugal column is shown (associated with Examples 3a-c and 5a-c). Similarly, as shown in Figure 6B, compared to conditions without a preceding ATPS step, all conditions with a preceding ATPS step achieved higher DNA recovery, i.e., plasma sample #5 and urine sample #4 in Figure 6B both yielded Ct values ​​close to 40 (see sample #5 in Example 3a and sample #4 in Example 5a). This highlights the importance of incorporating an ATPS step into plasma extraction prior to centrifugal column purification to achieve satisfactory DNA recovery.

[0707] In summary, compared to samples not treated with ATPS, adding an ATPS step prior to the solid phase extraction workflow offers the following advantages: Significant reduction in reagent consumption

[0708] As can be seen from the examples presented herein, in some cases, the addition of one or more ATPS steps significantly reduced the amount of binding buffer required for unprocessed digests. For example, the addition of a two-step ATPS system prior to solid-phase extraction reduced the binding buffer required for a 40 mL bulk fluid sample to only 2 mL. This is due to the small target-rich phase created by the first or second ATPS. The extent of the reduction in reagent consumption becomes more pronounced when sample input volumes are increased; while larger input volume indices require more binding buffer, the ATPS process can be modified to maintain a constant upper phase volume.

[0709] In some instances, high DNA recovery was achieved without the need to alter the binding balance with more magnetic beads to obtain better yields, thereby reducing the use of expensive magnetic beads and hazardous binding buffers.

[0710] Significantly reduced column flow time

[0711] In some instances, the addition of a parallel ATPS step significantly and surprisingly reduced the column flow-through time from 1 hour to 1 minute.

[0712] Simplified laboratory setup

[0713] In some instances, large volumes of custom bulking agents are not required when applying ATPS sample digest concentrations because the sample digest volume is much smaller, specific vacuum manifolds are not required, and a centrifuge may be used to pass the sample digest.

[0714] In summary, the results demonstrate the surprising efficiency of systems combining one or more ATPS extraction workflows with different solid phase media for the purification of extracted analytes from different clinical biological samples.

[0715] Example 7: Comparison of total DNA recovery using the disclosed method and a commercially available extraction kit

[0716] Example 7a

[0717] DNA extraction from urine using the exemplary method and kit described in Example 5a (referred to herein as the "extraction method" or "phase") was compared with the Zymo Rapid DNA Urine Kit ("Zymo"), the NextPrep-Mag Urine cfDNA Isolation Kit ("NextPrep Kit"), the Norgen Urine DNA Isolation Kit-Centrifugation Column ("Norgen"), and the Wizard Plus Mini Preparative DNA Purification System ("Wizard"), all of which are commercially available. The competitor kits were extracted according to the manufacturer's instructions, using the maximum urine sample input volume specified by the manufacturer. Cell-free urine was used to compare the commercial kit (condition AD in Table 6) and the extraction method (condition E in Table 6). As a comparison, the inventors extracted a 40 mL batch using crude urine (unspun urine containing cells) and the current extraction method (condition F in Table 6) to evaluate whether the current extraction method would perform equally well in the presence of cells. Urine samples were provided by four males and four females (n = 8 for each kit). The input and elution volumes for each kit were normalized to a ratio of 100:1 and the extraction times were compared in Table 6.

[0718] Table 6: Comparison of yield and efficiency at normalized input:elution volume ratios. [Table 6]

[0719] Referring now to Figure 7A, which shows the recovery using 145-bp DNA spike-in (copies / µL) using conditions A and B in Table 6. The 145-bp spike-in was detected by droplet digital PCR (ddPCR). As shown in Figure 7A, the recovery efficiency of 140-bp spike-in DNA using this extraction method (condition E) was comparable, if not higher, than 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 with the NextPrep (condition A), Norgen (condition C), and Wizard (condition D) extraction kits, this extraction method can process a larger input volume but requires comparable, if not shorter, extraction times. Compared with Zymo (condition B) with the same input volume, this extraction method has a shorter extraction time, significantly higher yields, and more consistent inter-sample performance. When using urine containing cells (condition F), the average DNA recovery rate of the present extraction method was significantly superior to all competitors, even in the presence of cells, with satisfactory precision (476.1 ± 32.2 copies / uL). This demonstrated that the present extraction method is superior for recovering target DNA using crude urine and processed, centrifuged urine. In summary, the overall target DNA extraction performance (in terms of yield, input volume, and extraction time) using the disclosed method is surprisingly superior compared to industry-standard commercially available extraction kits.

[0720] Example 7b

[0721] We further compared DNA recovery from urine between this extraction method (also referred to herein as "phase") and commercially available extraction kits (Zymo Rapid DNA Urine Kit ("Zymo"), Qiagen QIAamp Circulating Nucleic Acid Kit ("Qiagen" or "QCNA"), Norgen Urine DNA Isolation Kit-Centrifugation Column ("Norgen"), and Wizard Plus Mini Preparative DNA Purification System ("Wizard")) by using a maximum urine sample input volume of 160 mL, while the commercial extraction kits used the manufacturer's recommended maximum sample input volume and optimal export volume and were extracted according to the manufacturer's instructions. Urine samples were provided by four males and four females (n = 8 for each kit). Conditions are summarized in Table 7.

[0722] Table 7: Comparison of yield and efficiency using recommended input and export volumes. [Table 7]

[0723] Referring now to Figure 7B, which shows the average concentration (copies / uL) of DNA recovered using the kits and conditions from Table 7. The 140 bp spike-in was detected by Droplet Digital PCR ddPCR. The results show that the ability to process high input volumes and concentrate into low export volumes allows this extraction method to significantly outperform all competing kits.

[0724] In summary, the experiments in Examples 7a and 7b show that the total DNA recovery rate of this extraction method is significantly higher than that of all other commercially available kits because the extraction method allows for increased sample input volume, has lower export / elution volumes, and has higher target DNA recovery efficiency.

[0725] Illustrative embodiments of the present invention have now been fully described. While the description has referred to particular embodiments, it will be apparent to those skilled in the art that the present invention may be practiced with variations of these specific details. Therefore, the present invention should not be construed as being limited to the embodiments set forth herein.

[0726] Numbered Example 1

[0727] Example 1. A method for concentrating and purifying one or more target analytes from a bulk fluid sample, the method comprising: (a) preparing a first aqueous two-phase system (ATPS) composition, the first ATPS composition comprising a polymer, a salt, a surfactant, or a combination thereof, that dissolves in an aqueous solution to form a first phase solution and a second phase solution; (b) adding a sample solution prepared from the bulk fluid sample containing the one or more target analytes to the first ATPS composition, thereby allocating the one or more target analytes in the first phase solution; and (c) collecting the first phase solution and mixing the first phase solution with a second ATPS composition, the second ATPS composition dissolving in an aqueous solution to form a third phase solution and a fourth phase solution. (d) collecting the third phase solution and mixing the third phase solution with a binding buffer to form a mixed solution, the binding buffer comprising at least one chaotropic agent; (e) loading the mixed solution onto an extraction column configured to selectively extract and purify the one or more target analytes; and (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.

[0728] Example 2. The method of any one of the preceding examples, wherein the sample solution is prepared by dividing the bulk fluid sample containing the one or more target analytes into at least two aliquots of sample solution, and the first ATPS composition is divided into at least two aliquots, and wherein step (b) further comprises: (i) adding each aliquot of the sample solution prepared with the bulk fluid sample containing the one or more target analytes to each aliquot of the first ATPS composition to allocate the one or more target analytes in the first phase solution; and (ii) collecting and combining the first phase solutions of the at least two aliquots of the first ATPS composition to form a first phase solution for step (c).

[0729] Example 3. The method of any one of the preceding Examples, wherein the extraction column is a centrifugation column, and wherein step (e) further comprises: (i) loading a portion of the mixed solution onto the extraction column; (ii) centrifuging the extraction column and discarding the flow-through; and (iii) repeating steps (i) and (ii) until all of the mixed solution has passed through the extraction column.

[0730] Example 4. The method of any one of the preceding Examples, wherein the method further comprises (g) subjecting the final solution to a diagnostic assay to detect and quantify the one or more target analytes.

[0731] Example 5. A method for concentrating and purifying one or more target analytes from a bulk fluid sample, the method comprising: (a) dividing the bulk 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, the first ATPS composition comprising a polymer, a salt, a surfactant, or a combination thereof, that dissolves 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, thereby allocating the one or more target analytes in the first phase solution; and (d) dissolving each aliquot of the at least two aliquots of the first ATPS composition. (e) collecting the first phase solution and mixing the first phase solution with a second ATPS composition, the second ATPS composition comprising a polymer, a salt, a surfactant, or a combination thereof, that dissolves in aqueous solution to form a third phase solution and a fourth phase solution, thereby assigning and concentrating the one or more target analytes 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, the binding buffer comprising at least one chaotropic agent; (f) loading the mixed solution onto an extraction column configured to selectively extract and purify the one or more target analytes; and (g) eluting and collecting the one or more target analytes from the extraction column.

[0732] Example 6. The method of any one of the preceding Examples, wherein 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.

[0733] Example 7. The method of any one of the preceding examples, wherein the bulk fluid sample is urine.

[0734] Example 8. The method of any one of the preceding examples, wherein the volume of the bulk fluid sample is 40 mL or greater.

[0735] Example 9. The method of any one of the preceding examples, wherein the volume of each aliquot of the sample solution is at most 40 ml.

[0736] Example 10. The method of any one of the preceding examples, wherein the target analyte is selected from the group consisting of a nucleic acid, a protein, an antigen, a biomolecule, a sugar moiety, a lipid, a sterol, and combinations thereof.

[0737] Example 11. The method of any one of the preceding examples, wherein the target analyte is DNA.

[0738] Example 12. The method of any one of the preceding examples, wherein the target analyte is free DNA or circulating tumor DNA.

[0739] Example 13. The method of any one of the preceding examples, wherein the polymer is soluble in aqueous solution at a concentration of 4% to 84% (w / w).

[0740] Example 14. The method of any one of the preceding examples, wherein the polymer is selected from the group consisting of polyalkylene glycols, e.g., hydrophobically modified polyalkylene glycols, poly(oxyalkylene) polymers, poly(oxyalkylene) copolymers, e.g., hydrophobically modified poly(oxyalkylene) copolymers, polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, polyvinyl methyl ether, alkoxylated surfactants, alkoxylated starch, alkoxylated cellulose, alkyl hydroxyalkyl cellulose, silicone-modified polyethers, and poly-N-isopropylacrylamide and copolymers thereof. The method of any one of the preceding examples, wherein the polymer is selected from the group consisting of polyethers, polyimides, polyalkylene glycols, vinyl polymers, alkoxylated surfactants, polysaccharides, alkoxylated starch, alkoxylated cellulose, alkyl hydroxyalkyl cellulose, polyether-modified silicones, polyacrylamides, polyacrylic acids, and copolymers thereof. 3. The method of 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-ran-propylene glycol), polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, 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.The method of 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-ran-propylene glycol), polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, polyvinyl methyl ether, and poly N-isopropylacrylamide. The method of any one of the preceding Examples, wherein the polymer is selected from the group consisting of polyacrylamide, polyacrylic acid, and copolymers thereof. The method of 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 of any one of the preceding Examples, wherein the average molecular weight of the polymer is in the range of 200 to 1,000 Da, 200 to 35,000 Da, 425 to 2,000 Da, 400 to 35,000 Da, 980 to 12,000 Da, or 3,400 to 5,000,000 Da. The method of any one of the preceding Examples, wherein the polymer comprises ethylene oxide and propylene oxide units and has an EO:PO ratio of 90:10 to 10:90.

[0741] Example 15.

[0742] Example 16. The method of any one of the preceding examples, wherein the salt is dissolved in aqueous solution at a concentration of 1% to 55% (w / w).

[0743] Example 17. The method of any one of the preceding examples, wherein the salt is dissolved in aqueous solution at a concentration of 8% to 55% (w / w).

[0744] Example 18. The method of any one of the preceding examples, wherein the salt is selected from the group consisting of kosmotropic salts, chaotropic salts, cation-containing inorganic salts, such as linear or branched trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium, and tetrabutylammonium, and anion-containing inorganic salts, such as phosphates, sulfates, nitrates, chlorides, and bicarbonates, NaCl, Na3PO4, K3PO4, Na2SO4, potassium citrate, (NH4)2SO4, 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.

[0745] Example 19. The method of any one of the preceding examples, wherein the surfactant is dissolved in aqueous solution at a concentration of 0.05% to 10% (w / w).

[0746] Example 20. The method of any one of the preceding examples, wherein the surfactant is dissolved in aqueous solution at a concentration of 0.05% to 9.8% (w / w).

[0747] Example 21. The surfactants are Triton-X, Triton-114, Igepal CA-630 and Nonidet P-40, anionic surfactants (e.g., 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 (e.g., ethoxylated fatty alcohols, polyoxyethylene surfactants, carboxylic acid esters, polyethylene glycol esters, sorbitan esters, fatty acid ethylene glycol esters, carboxylic acid amides, monoalkanolamine condensates, polyoxyethylene The method of any one of the preceding Examples, wherein the surfactant is selected from the group consisting of n-cocoyl 3-aminopropionic acid / sodium salt, n-tallow 3-iminodipropionate, disodium salt, n-carboxymethyl n-dimethyl n-9 octadecenyl ammonium hydroxide, n-cocamidoethyl n-hydroxyethylglycine, and sodium salts thereof.

[0748] Example 22. An ATPS composition selected from the group consisting of A1, A2, A3, A4, AAl, AA2, AA3 and AA4.

[0749] 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 A1, AA2, AA3 and AA4, and a binding buffer selected from the group consisting of B1, B2 and B3.

[0750] Example 24 The kit of any one of the preceding Examples, wherein the kit further comprises an extraction column.

[0751] Numbered Example 2

[0752] Example 1. A method for concentrating and purifying one or more target analytes from a sample solution, the method comprising: (a) adding a sample solution containing one or more target analytes to a first aqueous two-phase system (ATPS) to form a mixture that separates into a first phase and a second phase, and the one or more target analytes are concentrated in the first phase; (b) isolating the first phase containing the concentrated one or more target analytes to obtain an enriched solution; (c) applying magnetic beads to the enriched solution, causing the magnetic beads to bind to the one or more target analytes to form bead-analyte complexes; and (d) recovering the one or more target analytes from the bead-analyte complexes to obtain a final solution containing the concentrated and purified one or more target analytes.

[0753] Example 2. The method of any one of the preceding Examples, wherein step (b) further comprises: (i) adding the isolated first phase containing the concentrated one or more target analytes to a second ATPS to form a second mixture that separates into a third and a fourth phase, wherein the one or more target analytes are concentrated in the third phase; and (ii) isolating the third phase containing the concentrated one or more target analytes in step (b) to form a concentrated solution for step (c).

[0754] Example 3. The method of any one of the preceding Examples, wherein the concentrated solution for step (c) is obtained by combining the concentrated solution of step (b) with a binding buffer comprising at least one chaotropic agent selected from n-butanol, ethanol, guanidine chloride, guanidine thiocyanate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea.

[0755] Example 4. The method of any one of the preceding Examples, wherein step (d) further comprises: (i) mixing the bead-analyte complexes with a fractionation buffer comprising a polymer, a salt, a surfactant, a chaotropic agent, or a combination thereof, to form a fractionation solution, and releasing the one or more target analytes smaller than a target size from the bead-analyte complexes into the fractionation solution; (ii) immobilizing the bead-analyte complexes using a magnetic support; and (iii) isolating the one or more target analytes smaller than a target size in the fractionation solution from the immobilized bead-analyte complexes.

[0756] Example 5. The method of any one of the preceding Examples, wherein step (d) further comprises: (iv) adding the one or more isolated target analytes that are smaller than the target size to a second binding buffer containing at least one chaotropic agent selected from n-butanol, ethanol, guanidine chloride, guanidine thiocyanate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea; (v) applying magnetic beads to the mixture of the one or more isolated target analytes that are smaller than the target size and the second binding buffer, wherein the magnetic beads bind to the one or more target analytes that are smaller than the target size to form second bead-analyte complexes; and (vi) recovering the one or more target analytes from the second bead-analyte complexes.

[0757] Example 6. The method of any one of the preceding Examples, wherein the method further comprises (e) subjecting the final solution to a diagnostic assay to detect and quantify the one or more target analytes.

[0758] Example 7. The method of 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 combinations thereof.

[0759] Example 8. The method of any one of the preceding Examples, wherein the one or more target analytes is DNA.

[0760] Example 9. The method of any one of the preceding Examples, wherein the one or more target analytes is free DNA or circulating tumor DNA.

[0761] Example 10. The method of any one of the preceding examples, wherein the first ATPS comprises a first ATPS component capable of forming a first phase and a second phase when the first ATPS component dissolves in an aqueous solution, wherein the first ATPS component is selected from the group consisting of a polymer, a salt, a surfactant, and combinations thereof.

[0762] Example 11. The method of any one of Examples 2-10, wherein the second ATPS comprises a second ATPS component capable of forming a third phase and a fourth phase when the second ATPS component dissolves in an aqueous solution, wherein the second ATPS component is selected from the group consisting of a polymer, a salt, a surfactant, and combinations thereof.

[0763] Example 12. The method of example 10 or 11, wherein the polymer is soluble in aqueous solution at a concentration of 4% to 84% (w / w).

[0764] Example 13. The method of any one of Examples 10-12, wherein the polymer is selected from the group consisting of polyalkylene glycols, e.g., hydrophobically modified polyalkylene glycols, poly(oxyalkylene) polymers, poly(oxyalkylene) copolymers, e.g., hydrophobically modified poly(oxyalkylene) copolymers, polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, polyvinyl methyl ether, alkoxylated surfactants, alkoxylated starch, alkoxylated cellulose, alkyl hydroxyalkyl cellulose, silicone-modified polyethers, and poly-N-isopropylacrylamide and copolymers thereof. The method of any one of the preceding Examples, wherein the polymer is selected from the group consisting of polyethers, polyimides, polyalkylene glycols, vinyl polymers, alkoxylated surfactants, polysaccharides, alkoxylated starch, alkoxylated cellulose, alkyl hydroxyalkyl cellulose, polyether-modified silicones, polyacrylamides, polyacrylic acids, and copolymers thereof. 3. The method of 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-ran-propylene glycol), polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, 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.The method of 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-ran-propylene glycol), polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, polyvinyl methyl ether, and poly N-isopropylacrylamide. The method of any one of the preceding Examples, wherein the polymer is selected from the group consisting of polyacrylamide, polyacrylic acid, and copolymers thereof. The method of 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 of any one of the preceding Examples, wherein the average molecular weight of the polymer is in the range of 200 to 1,000 Da, 200 to 35,000 Da, 425 to 2,000 Da, 400 to 35,000 Da, 980 to 12,000 Da, or 3,400 to 5,000,000 Da. The method of any one of the preceding Examples, wherein the polymer comprises ethylene oxide and propylene oxide units and has an EO:PO ratio of 90:10 to 10:90.

[0765] Example 14.

[0766] Example 15. The method of any one of Examples 10 to 14, wherein the salt is dissolved in aqueous solution at a concentration of 1% to 80% (w / w).

[0767] Example 16. The method of any one of Examples 10 to 15, wherein the salt is dissolved in aqueous solution at a concentration of 8% to 80% (w / w).

[0768] Example 17. The method of any one of Examples 10-16, wherein the salt is selected from the group consisting of kosmotropic salts, chaotropic salts, cation-containing inorganic salts, such as linear or branched trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium, and tetrabutylammonium, and anion-containing inorganic salts, such as phosphates, sulfates, nitrates, chlorides, and bicarbonates, NaCl, Na3PO4, K3PO4, Na2SO4, potassium citrate, (NH4)2SO4, 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.

[0769] Example 18. The method of any one of Examples 10 to 17, wherein the surfactant is dissolved in aqueous solution at a concentration of 0.05% to 10% (w / w).

[0770] Example 19. The method of any one of Examples 10 to 18, wherein the surfactant is dissolved in aqueous solution at a concentration of 0.05% to 9.8% (w / w).

[0771] Example 20. The surfactants are Triton-X, Triton-114, Igepal CA-630 and Nonidet P-40, anionic surfactants (e.g., 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 (e.g., ethoxylated fatty alcohols, polyoxyethylene surfactants, carboxylic acid esters, polyethylene glycol esters, sorbitan esters, fatty acid ethylene glycol esters, carboxylic acid amides, monoalkanolamine condensates, polyoxyethylene The method according to any one of Examples 10 to 19, wherein the surfactant is selected from the group consisting of: fatty acid amides), cationic surfactants (e.g., quaternary ammonium salts, amines having an amide bond, polyoxyethylene alkyl and alicyclic amines, n,n,n',n'-tetrasubstituted ethylenediamines, 2-alkyl 1-hydroxyethyl 2-imidazolines), and amphoteric surfactants (e.g., n-cocoyl 3-aminopropionic acid / sodium salt, n-tallow 3-iminodipropionate disodium salt, n-carboxymethyl n-dimethyl n-9 octadecenyl ammonium hydroxide, n-cocamidoethyl n-hydroxyethylglycine and sodium salts thereof).

[0772] Example 21. The method of any one of the preceding examples, wherein step (a) further comprises: (i) embedding a porous material in components capable of forming a first ATPS; and (ii) contacting a sample solution with the porous material embedded in the components, wherein the components form a first phase and a second phase as the sample solution passes through the porous material.

[0773] Numbered Example 3

[0774] Example 1. A method for concentrating and purifying at least one target analyte from a clinical biological sample, the method comprising: (a) combining the clinical biological sample with a first aqueous two-phase system (ATPS) composition comprising a polymer dissolved in an aqueous solution, a salt component comprising at least one salt, a surfactant, or any combination thereof, to form a target-rich phase solution and a target-poor phase solution; (b) collecting the target-rich phase; and (c) optionally adding the target-rich phase to a second ATPS composition comprising a polymer dissolved in an aqueous solution, a salt component comprising at least one salt, a surfactant, or any combination thereof, to form a second target-rich phase solution and a second target-poor phase solution. and collecting the second target-rich phase; (d) optionally mixing the target-rich phase from step (b) or the second target-rich phase from step (c) with a binding buffer to form a mixed solution; (e) contacting the target-rich phase from step (b), the second target-rich phase from step (c), or the mixed solution from step (d) with a solid phase medium configured to selectively bind the target analyte, causing the solid phase medium to bind the target analyte; and (f) eluting the target analyte from the solid phase medium with an elution solution and collecting, to obtain a final solution containing concentrated and purified target analyte.

[0775] Example 2. The method of any one of the preceding Examples, wherein the method further comprises washing the solid phase medium with one or more suitable solvents after step (e) and before step (f) to remove impurities.

[0776] Example 3. The method of any one of the preceding examples, wherein the suitable solvent is a binding buffer.

[0777] Example 4. The method of any one of the preceding Examples, wherein said method further comprises, prior to step (a), treating said clinical biological sample with a degradation composition.

[0778] Example 5. The method of any one of the preceding Examples, wherein the binding buffer comprises a chaotropic agent comprising an anion selected from the group consisting of thiocyanate, isothiocyanate, perchlorate, acetate, trichloroacetate, trifluoroacetate, chloride, and iodide.

[0779] Example 6. The method of any one of the preceding Examples, wherein the binding buffer comprises a chaotropic 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.

[0780] Example 7. The method of any one of the preceding Examples, wherein the binding buffer comprises a 2 M to 7 M chaotropic agent solution.

[0781] Example 8. The method of any one of the preceding Examples, wherein the binding buffer further comprises a polymer at a concentration of 5-20% (w / v).

[0782] Example 9. The method of any one of the preceding examples, wherein said clinical biological sample is blood, plasma, urine, saliva, feces, cerebrospinal fluid (CSF), lymph, serum, sputum, peritoneal fluid, sweat, tears, nasal swab, vaginal swab, cervical swab, semen, or breast milk.

[0783] Example 10 The method of any one of the preceding examples, wherein said clinical biological sample is <500 mL.

[0784] Example 11. The method of any one of the preceding examples, wherein the target analyte is selected from the group consisting of a nucleic acid, a protein, an antigen, a biomolecule, a sugar moiety, a lipid, a sterol, an exosome, and combinations thereof.

[0785] Example 12. The method of any one of the preceding examples, wherein the target analyte is a nucleic acid, and the nucleic acid is gDNA, cDNA, plasmid DNA, mitochondrial DNA, free DNA (cfDNA), circulating tumor DNA (ctDNA), circulating fetal DNA, microbial free DNA, microRNA (miRNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), circular RNA, long non-coding RNA (lncRNA), or a combination thereof.

[0786] Example 13. The method of any one of the preceding examples, wherein the nucleic acid is cfDNA, fetal free DNA, mitochondrial DNA, microbial free DNA, or ctDNA.

[0787] Example 14. The method of any one of the preceding examples, wherein step (e) comprises: (i) contacting a portion of the mixed solution with the solid phase medium and binding the target analyte to the solid phase medium to form a solid phase extraction complex; (ii) perturbing the solid phase extraction complex and discarding the flow-through; and (iii) optionally repeating steps (i) and (ii).

[0788] Example 15. The method of any one of the preceding examples, wherein the solid phase medium is a plurality of beads.

[0789] Example 16. The method of any one of the preceding examples, wherein the beads are selected from the group consisting of magnetic beads, silica-based beads, carboxy beads, hydroxy beads and amine-coated beads.

[0790] Example 17. The method of any one of the preceding examples, wherein the solid phase extraction complex is a bead-analyte complex, the agitation is rotation, and the flow-through is a supernatant.

[0791] Example 18. The method of any one of the preceding examples, wherein the target analytes are nucleic acids smaller than a target size, the plurality of beads bind the target analytes and other nucleic acids, and the elution solution is a fractionation buffer that, upon contact with the beads during elution step (f), releases the target analytes while not releasing the other nucleic acids, thereby obtaining a final solution containing concentrated and purified one or more target analytes.

[0792] Example 19. The method of any one of the preceding examples, wherein the fractionation buffer further comprises a polymer, a chaotropic agent, or any combination thereof.

[0793] Example 20. The method of any one of the preceding examples, wherein the solid phase medium is an extraction column.

[0794] Example 21 The method of any one of the preceding examples, wherein the extraction column is a centrifugation column.

[0795] Example 22. The method of any one of the preceding Examples, wherein step (e) further comprises: (i) loading a portion of the mixed solution from step (d) onto the extraction column; (ii) centrifuging the extraction column and discarding the flow-through; and (iii) optionally repeating steps (i) and (ii) one or more times until all of the mixed solution has passed through the extraction column.

[0796] Example 23. The method of 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 or branched chain trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium, and tetrabutylammonium.

[0797] Example 24. The method of 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, hydrogen sulfate, 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, polyacrylate, hydroxide, hydride, citrate, borate, and tris(hydroxymethyl)aminomethane.

[0798] Example 25. The method of any one of the preceding examples, wherein the polymer is selected from the group consisting of polyethers, polyimides, polyacrylates, polyalkylene glycols, vinyl polymers, alkoxylated surfactants, polysaccharides, alkoxylated starches, alkoxylated celluloses, alkylhydroxyalkylcelluloses, polyether-modified silicones, polyacrylamides, polyacrylic acids, and copolymers of one or more thereof.

[0799] Example 26 The method of 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-ran-propylene glycol), polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, 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.

[0800] Example 27. The surfactant is selected from the group consisting of anionic surfactants, nonionic surfactants, cationic surfactants, and amphoteric surfactants, wherein the anionic surfactant is a carboxylate, a sulfonate, a petroleum sulfonate, an alkylbenzene sulfonate, a naphthalene sulfonate, an olefin sulfonate, an alkyl sulfate, a sulfate, a sulfated natural oil, a sulfated natural fat, a sulfated ester, a sulfated alkanolamide, a sulfated alkylphenol, an ethoxylated alkylphenol, or sodium N-lauroyl sarcosinate (NLS), and the nonionic surfactant is an ethoxylated fatty alcohol, a polyoxyethylene surfactant, a carboxylic acid ester, a polyethylene glycol ester, a sorbitan ester, a fatty acid glycol ester, or a hydroxypropyl ester. the cationic surfactant is a quaternary ammonium salt, an amine having an amide bond, a polyoxyethylene alkylamine, a polyoxyethylene alicyclic amine, an n,n,n',n'-tetrasubstituted ethylenediamine, or a 2-alkyl 1-hydroxyethyl 2-imidazoline; and the amphoteric surfactant is n-cocoyl 3-aminopropionic acid or its sodium salt, n-tallow 3-iminodipropionic acid ester or its disodium salt, n-carboxymethyl n-dimethyl n-9 octadecenyl ammonium hydroxide, or n-cocamidoethyl n-hydroxyethylglycine or its sodium salt.

[0801] Example 28. The method of 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.

[0802] Example 29. The method of any one of the preceding examples, wherein the average molecular weight of the polymer is in the range of 200 to 1,000 Da, 200 to 35,000 Da, 300 to 35,000 Da, 400 to 2,000 Da, 400 to 35,000 Da, 2,500 to 2,500,000 Da, 1,250 to 4,000,000 Da, or 6,000 to 5,000,000 Da.

[0803] Example 30. The method of any one of the preceding examples, wherein the polymer of the first ATPS composition or the second ATPS composition is soluble in aqueous solution at a concentration of 0.5% to 80% (w / v).

[0804] Example 31. The method of any one of the preceding examples, wherein the polymer of the first ATPS composition or the second ATPS composition is soluble in aqueous solution at a concentration of 0.2% to 50% (w / v).

[0805] Example 32. The method of any one of the preceding examples, wherein the salt component of the first ATPS composition or the second ATPS composition is dissolved in aqueous solution at a concentration of 0.1% to 80% (w / v).

[0806] Example 33. The method of any one of the preceding examples, wherein the surfactant of the first ATPS composition or the second ATPS composition is dissolved in aqueous solution at a concentration of 0.1% to 90% (w / v).

[0807] Example 34. The method of any one of the preceding examples, wherein the first ATPS composition or the second ATPS composition is a polymer-salt system, a polymer-polymer system, or a micelle system.

[0808] Example 35. The method of any one of the preceding examples, wherein the first ATPS composition or the second ATPS composition is a polymer-salt system, the polymer dissolves in aqueous solution at a concentration of 0.5% to 80% (w / v), and the salt component dissolves in aqueous solution at a concentration of 0.1% to 80% (w / v).

[0809] Example 36. A method according to any one of the preceding examples, wherein the polymer of the first ATPS composition is soluble in aqueous solution at a concentration of 5% to 80% (w / v), and the salt component of the first ATPS composition is soluble in aqueous solution at a concentration of 0.1% to 80% (w / v), and the polymer of the second ATPS composition is soluble in aqueous solution at a concentration of 0.5% to 30% (w / v), and the salt component of the second ATPS composition is soluble in aqueous solution at a concentration of 5% to 60% (w / v).

[0810] Example 37. The method of any one of the preceding examples, wherein the first ATPS composition or the second ATPS composition further comprises at least one salt at a concentration of 0.01% to 10% (w / v) or at least one surfactant at a concentration of 0.01% to 10% (w / v).

[0811] Example 38. The method of any one of the preceding 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 soluble in aqueous solution at a concentration of 0.2% to 50% (w / v).

[0812] Example 39. The method of any one of the preceding examples, wherein the first ATPS composition or the second ATPS composition further comprises at least one salt at a concentration of 0.01% to 10% (w / v) or at least one surfactant at a concentration of 0.01% to 10% (w / v).

[0813] Example 40. The at least one salt comprises a cation selected from the group consisting of sodium, potassium, calcium, ammonium, lithium, magnesium, aluminum, cesium, barium, linear or branched trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium, and tetrabutylammonium, and at least one surfactant selected from the group consisting of anionic surfactants, nonionic surfactants, cationic surfactants, and amphoteric surfactants, wherein the anionic surfactant is a 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 N-lauroyl sarcosine. sodium (NLS), the nonionic surfactant is an ethoxylated fatty alcohol, a polyoxyethylene surfactant, a carboxylic acid ester, a polyethylene glycol ester, a sorbitan ester, a fatty acid glycol ester, a carboxyamide, a monoalkanolamine condensate, or a polyoxyethylene fatty acid amide, the cationic surfactant is a quaternary ammonium salt, an amine having an amide bond, a polyoxyethylene alkylamine, a polyoxyethylene alicyclic amine, an n,n,n',n'-tetrasubstituted ethylenediamine, or a 2-alkyl 1-hydroxyethyl 2-imidazoline, and the amphoteric surfactant is n-cocoyl 3-aminopropionic acid or its sodium salt, n-tallow 3-iminodipropionic acid ester or its disodium salt, n-carboxymethyl n-dimethyl n-9 octadecenyl ammonium hydroxide, or n-cocamidoethyl n-hydroxyethylglycine or its sodium salt.

[0814] Example 41. The method of any one of the preceding examples, wherein the first ATPS composition or the second ATPS composition is a micellar system containing at least one surfactant, and each surfactant is dissolved in aqueous solution at a concentration of 0.1% to 90% (w / v).

[0815] Example 42. The method of any one of the preceding examples, wherein the first ATPS composition or the second ATPS composition further comprises at least one salt at a concentration of 0.01% to 30% (w / v).

[0816] Example 43. The method of any one of the preceding examples, wherein the at least one salt comprises a cation selected from the group consisting of sodium, potassium, calcium, ammonium, lithium, magnesium, aluminum, cesium, barium, straight or branched chain trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium, and tetrabutylammonium.

[0817] Example 44. The method of any one of the preceding examples, wherein the first ATPS composition or second ATPS composition is selected from Tables 1.0a-1.0d.

[0818] Example 45. The method of any one of the preceding examples, wherein the first ATPS composition or the second ATPS composition further comprises 0.5 to 2 mM ethylenediaminetetraacetic acid (EDTA).

[0819] Example 46. The method of any one of the preceding examples, wherein the method further comprises analyzing the final solution from step (f) by a method selected from the group consisting of qPCR, ddPCR, qubit, ELISA, NGS sequencer, bisulfite, RT-PCR, Sanger sequencing, nanodroplet, nanopore sequencing, nucleic acid sequencing, and a PCR-based assay.

[0820] Example 47. The method of any one of the preceding examples, wherein the target analyte is a biomarker indicative of the presence or risk of a medical disorder or disease in a patient, wherein the medical disorder or disease is an infectious disease, cancer, or a genetic disease.

[0821] Example 48. The method of any one of the preceding examples, wherein the clinical biological sample is a bulk fluid sample having a volume >10 mL, and further comprising, prior to step (a), dividing the bulk fluid sample into at least two aliquots of sample solution, wherein in step (a) each aliquot is individually combined with the first ATPS, and optionally, in step (c) the collected target-rich phase from step (b) is individually combined with the second ATPS to form a target-rich phase for each aliquot.

[0822] Example 49. The method of any one of the preceding examples, wherein the target-rich phase of each aliquot from step (b) or the second target-rich phase of each aliquot from step (c) is combined to form the final target-rich phase used in step (d).

[0823] Example 50 The method of any one of the preceding examples, wherein the bulk fluid sample is urine.

[0824] Example 51. A method of treating cancer or an infectious disease in a patient in need thereof, the method comprising: (i) obtaining a clinical biological sample from the patient; (ii) enriching and purifying at least one target analyte from the clinical biological sample according to the method of any one of the preceding Examples; (iii) analyzing the final solution; and (iv) treating the patient if information obtained from the target analyte indicates that the patient has or is at risk of having cancer.

[0825] Example 52 The method of any one of the preceding examples, wherein the cancer is CNS cancer, breast cancer, bladder cancer, pancreatic cancer, lung cancer, melanoma, colon cancer, hematopoietic cancer, or ovarian cancer, and wherein the infection is HPV.

[0826] Example 53 The method of any one of the preceding Examples, wherein the target analyte is kidney cFDNA or ctDNA, and wherein the cancer is a systemic cancer.

[0827] Example 54 The method of any one of the preceding Examples, wherein the target analyte is urogenital cFDNA or ctDNA, and wherein the cancer is a urogenital cancer.

[0828] Example 55 The method of any one of the preceding examples, wherein the target analyte is bladder cancer DNA and the medical disorder or disease is bladder cancer.

[0829] Example 56 The method of any one of the preceding examples, wherein the target analyte is HPV viral RNA or HPV viral DNA and the medical disorder or disease is HPV.

[0830] Example 57. A kit comprising a first ATPS composition, a second ATPS composition, a binding buffer, and a solid phase medium, wherein the first ATPS composition, the second ATPS composition, the binding buffer, and the solid phase medium are selected from those described in any one of the preceding examples.

Claims

1. 1. A method for enriching and purifying at least one target analyte from a clinical biological sample, the method comprising: (a) combining the clinical biological sample with a first aqueous two-phase system (ATPS) composition comprising a polymer dissolved in an aqueous solution, a salt component comprising at least one salt, a surfactant, or any combination thereof, to form a target-rich phase solution and a target-poor phase solution; (b) collecting the target-rich phase; (c) optionally, adding the target-rich phase to a second ATPS composition comprising a polymer dissolved in an aqueous solution, a salt component comprising at least one salt, a surfactant, or any combination thereof to form a second target-rich phase solution and a second target-poor phase solution, and collecting the second target-rich phase; (d) optionally mixing the target-rich phase from step (b) or the second target-rich phase from step (c) with a binding buffer to form a mixed solution; (e) contacting the target-rich phase from step (b), the second target-rich phase from step (c), or the mixed solution from step (d) with a solid phase medium configured to selectively bind the target analyte, thereby binding the solid phase medium to the target analyte; (f) eluting and collecting the target analyte from the solid phase medium with an elution solution to obtain a final solution containing the concentrated and purified target analyte.

2. 10. The method of claim 1, wherein the method further comprises washing the solid phase medium with one or more suitable solvents after step (e) and before step (f) to remove impurities.

3. The method of claim 2 , wherein the suitable solvent is a binding buffer.

4. 10. The method of claim 1, wherein the method further comprises treating the clinical biological sample with a degradation composition prior to step (a).

5. 2. The method of claim 1, wherein the binding buffer comprises a chaotropic agent comprising an anion selected from the group consisting of thiocyanate, isothiocyanate, perchlorate, acetate, trichloroacetate, trifluoroacetate, chloride, and iodide.

6. 2. The method of claim 1, wherein the binding buffer comprises a chaotropic 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.

7. The method of claim 6, wherein the binding buffer comprises a 2 M to 7 M chaotropic agent solution.

8. The method according to any one of claims 5 to 7, wherein the binding buffer further comprises a polymer at a concentration of 5 to 20% (w / v).

9. 2. The method of claim 1, wherein the clinical biological sample is blood, plasma, urine, saliva, feces, cerebrospinal fluid (CSF), lymph, serum, sputum, peritoneal fluid, sweat, tears, nasal swab, vaginal swab, cervical swab, semen, or breast milk.

10. 10. The method of claim 9, wherein the clinical biological sample is <500 mL.

11. 10. The method of claim 1, wherein the target analyte is selected from the group consisting of a nucleic acid, a protein, an antigen, a biomolecule, a sugar moiety, a lipid, a sterol, an exosome, and any combination thereof.

12. 12. The method of claim 11, wherein the target analyte is a nucleic acid, and the nucleic acid is gDNA, cDNA, plasmid DNA, mitochondrial DNA, free DNA (cfDNA), circulating tumor DNA (ctDNA), circulating fetal DNA, microbial free DNA, microRNA (miRNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), circular RNA, long non-coding RNA (lncRNA), or a combination thereof.

13. 13. The method of claim 12, wherein the nucleic acid is cfDNA, fetal free DNA, mitochondrial DNA, microbial free DNA, or ctDNA.

14. Step (e) (i) contacting a portion of the mixed solution with the solid phase medium to bind the target analyte to the solid phase medium to form a solid phase extraction complex; (ii) perturbing the solid phase extraction complex and discarding the flow-through; 10. The method of claim 1, further comprising: (iii) optionally repeating steps (i) and (ii).

15. 15. The method of claim 1 or 14, wherein the solid phase medium is a plurality of beads.

16. 16. The method of claim 15, wherein the beads are selected from the group consisting of magnetic beads, silica-based beads, carboxy beads, hydroxy beads, and amine-coated beads.

17. 16. The method of claim 15, wherein the solid phase extraction complex is a bead-analyte complex, the agitation is rotation, and the flow-through is a supernatant.

18. the target analyte is a nucleic acid smaller than a target size; the plurality of beads bind to the target analyte and other nucleic acids; 16. The method of claim 15, wherein the elution solution is a fractionation buffer that, upon contact with the beads during elution step (f), releases the target analytes while not releasing the other nucleic acids, resulting in a final solution containing concentrated and purified target analytes or analytes.

19. 20. The method of claim 18, wherein the fractionation buffer comprises a polymer, a chaotropic agent, or any combination thereof.

20. The method of claim 1 , wherein the solid phase medium is an extraction column.

21. 21. The method of claim 20, wherein the extraction column is a centrifugation column.

22. Step (e) (i) loading a portion of the mixed solution from step (d) onto the extraction column; (ii) centrifuging the extraction column and discarding the flow-through; (iii) optionally repeating steps (i) and (ii) one or more times until all of the mixed solution has passed through the extraction column.

23. 10. The method of claim 1, wherein the salt comprises a cation selected from the group consisting of sodium, potassium, calcium, ammonium, lithium, magnesium, aluminum, cesium, barium, straight or branched chain trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium, and tetrabutylammonium.

24. 2. The method of claim 1, wherein the salt comprises an anion selected from the group consisting of phosphate, hydrogen phosphate, dihydrogen phosphate, sulfate, sulfide, sulfite, hydrogen sulfate, 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, polyacrylate, hydroxide, hydride, citrate, borate, and tris(hydroxymethyl)aminomethane.

25. 10. The method of claim 1, wherein the polymer is selected from the group consisting of polyethers, polyimides, polyacrylates, polyalkylene glycols, vinyl polymers, alkoxylated surfactants, polysaccharides, alkoxylated starches, alkoxylated celluloses, alkylhydroxyalkylcelluloses, polyether-modified silicones, polyacrylamides, polyacrylic acids, and copolymers of one or more thereof.

26. 2. The method of claim 1, 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-ran-propylene glycol), polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, 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.

27. the surfactant is selected from the group consisting of anionic surfactants, nonionic surfactants, cationic surfactants, and amphoteric surfactants; where: The anionic surfactant is a carboxylate, a sulfonate, a petroleum sulfonate, an alkylbenzene sulfonate, a naphthalene sulfonate, an olefin sulfonate, an alkyl sulfate, a sulfate, a sulfated natural oil, a sulfated natural fat, a sulfated ester, a sulfated alkanolamide, a sulfated alkylphenol, an ethoxylated alkylphenol, or sodium N-lauroyl sarcosinate (NLS); the nonionic surfactant is an ethoxylated fatty alcohol, a polyoxyethylene surfactant, a carboxylic acid ester, a polyethylene glycol ester, a sorbitan ester, a fatty acid glycol ester, a carboxyamide, a monoalkanolamine condensate, or a polyoxyethylene fatty acid amide; The cationic surfactant is a quaternary ammonium salt, an amine having an amide bond, a polyoxyethylene alkylamine, a polyoxyethylene alicyclic amine, an n,n,n',n' tetrasubstituted ethylenediamine, and a 2-alkyl 1-hydroxyethyl 2-imidazoline; and 2. The method of claim 1, wherein the amphoteric surfactant is n-cocoyl 3-aminopropionic acid or its sodium salt, n-tallow 3-iminodipropionic acid ester or its disodium salt, n-carboxymethyl n-dimethyl n-9 octadecenyl ammonium hydroxide, or n-cocamidoethyl n-hydroxyethylglycine or its sodium salt.

28. 28. The method of claim 27, 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.

29. 10. The method of claim 1, wherein the average molecular weight of the polymer is in the range of 200 to 1,000 Da, 200 to 35,000 Da, 300 to 35,000 Da, 400 to 2,000 Da, 400 to 35,000 Da, 2,500 to 2,500,000 Da, 1,250 to 4,000,000 Da, or 6,000 to 5,000,000 Da.

30. 10. The method of claim 1, wherein the polymer of the first ATPS composition or the second ATPS composition is soluble in an aqueous solution at a concentration of 0.5 to 80% (w / v).

31. 31. The method of claim 30, wherein the polymer of the first ATPS composition or the second ATPS composition is soluble in an aqueous solution at a concentration of 0.2 to 50% (w / v).

32. 25. The method of claim 1, 23, or 24, wherein the salt component of the first ATPS composition or the second ATPS composition is soluble in aqueous solution at a concentration of 0.1% to 80% (w / v).

33. 27. The method of claim 1, 25, or 26, wherein the surfactant of the first ATPS composition or the second ATPS composition is soluble in aqueous solution at a concentration of 0.1% to 90% (w / v).

34. The method of claim 1 , wherein the first ATPS composition or the second ATPS composition is a polymer-salt system, a polymer-polymer system, or a micelle system.

35. the first ATPS composition or the second ATPS composition is a polymer-salt system; The polymer is soluble in aqueous solution at a concentration of 0.5% to 80% (w / v), and 2. The method of claim 1, wherein the salt component is soluble in the aqueous solution at a concentration of 0.1% to 80% (w / v).

36. the polymer of the first ATPS composition is soluble in aqueous solution at a concentration of 5% to 80% (w / v); and the salt component of the first ATPS composition is dissolved in an aqueous solution at a concentration of 0.1% to 80% (w / v); and the polymer of the second ATPS composition is soluble in aqueous solution at a concentration of 0.5% to 30% (w / v); and 36. The method of claim 35, wherein the salt component of the second ATPS composition is soluble in aqueous solution at a concentration of 5% to 60% (w / v).

37. 37. The method of claim 36, wherein the first ATPS composition or the second ATPS composition further comprises at least one salt at a concentration of 0.01% to 10% (w / v) or at least one surfactant at a concentration of 0.01% to 10% (w / v).

38. 2. The method of claim 1, wherein the first ATPS composition or the second ATPS composition is a polymer-polymer system comprising at least two polymers, and each polymer is soluble in aqueous solution at a concentration of 0.2% to 50% (w / v).

39. 39. The method of claim 38, wherein the first ATPS composition or the second ATPS composition further comprises at least one salt at a concentration of 0.01% to 10% (w / v) or at least one surfactant at a concentration of 0.01% to 10% (w / v).

40. The at least one salt comprises a cation selected from the group consisting of sodium, potassium, calcium, ammonium, lithium, magnesium, aluminum, cesium, barium, linear or branched trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium, and tetrabutylammonium, and at least one surfactant selected from the group consisting of anionic surfactants, nonionic surfactants, cationic surfactants, and amphoteric surfactants, wherein The anionic surfactant is a carboxylate, a sulfonate, a petroleum sulfonate, an alkylbenzene sulfonate, a naphthalene sulfonate, an olefin sulfonate, an alkyl sulfate, a sulfate, a sulfated natural oil, a sulfated natural fat, a sulfated ester, a sulfated alkanolamide, a sulfated alkylphenol, an ethoxylated alkylphenol, or sodium N-lauroyl sarcosinate (NLS); the nonionic surfactant is an ethoxylated fatty alcohol, a polyoxyethylene surfactant, a carboxylic acid ester, a polyethylene glycol ester, a sorbitan ester, a fatty acid glycol ester, a carboxyamide, a monoalkanolamine condensate, or a polyoxyethylene fatty acid amide; The cationic surfactant is a quaternary ammonium salt, an amine having an amide bond, a polyoxyethylene alkylamine, a polyoxyethylene alicyclic amine, an n,n,n',n' tetrasubstituted ethylenediamine, and a 2-alkyl 1-hydroxyethyl 2-imidazoline; and 40. The method of claim 39, wherein the amphoteric surfactant is n-cocoyl 3-aminopropionic acid or its sodium salt, n-tallow 3-iminodipropionic acid ester or its disodium salt, n-carboxymethyl n-dimethyl n-9 octadecenyl ammonium hydroxide, or n-cocamidoethyl n-hydroxyethylglycine or its sodium salt.

41. 2. The method of claim 1, wherein the first ATPS composition or the second ATPS composition is a micellar system comprising at least two surfactants, and each surfactant is soluble in aqueous solution at a concentration of 0.1% to 90% (w / v).

42. 42. The method of claim 41, wherein the first ATPS composition or the second ATPS composition further comprises at least one salt at a concentration of 0.01% to 30% (w / v).

43. 43. The method of claim 42, wherein the at least one salt comprises a cation selected from the group consisting of sodium, potassium, calcium, ammonium, lithium, magnesium, aluminum, cesium, barium, straight or branched chain trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium, and tetrabutylammonium.

44. 10. The method of claim 1, wherein the first ATPS composition and / or the second ATPS composition is selected from Tables 1.0a to 1.0d.

45. 10. The method of claim 1, wherein the first ATPS composition or the second ATPS composition further comprises 0.5 to 2 mM ethylenediaminetetraacetic acid (EDTA).

46. 10. The method of claim 1, further comprising analyzing the final solution from step (f) by a method selected from the group consisting of qPCR, ddPCR, qubit, ELISA, NGS sequencer, bisulfite, RT-PCR, Sanger sequencing, nanodroplet, nanopore sequencing, nucleic acid sequencing, and a PCR-based assay.

47. 10. The method of claim 1, wherein the target analyte is a biomarker that indicates the presence or risk of a medical disorder or disease in a patient, wherein the medical disorder or disease is an infectious disease, cancer, or a genetic disease.

48. 2. The method of claim 1, wherein the clinical biological sample is a bulk fluid sample of >10 mL volume, and further comprising, prior to step (a), dividing the bulk fluid sample into at least two aliquots of sample solution, wherein in step (a), each aliquot is individually combined with the first ATPS, and optionally, in step (c), the collected target-rich phase from step (b) is individually combined with the second ATPS to form a target-rich phase for each aliquot.

49. 49. The method of claim 48, wherein the target-rich phase of each aliquot from step (b) or the second target-rich phase of each aliquot from step (c) is combined to form the final target-rich phase used in step (d).

50. 50. The method of claim 48 or 49, wherein the bulk fluid sample is urine.

51. 1. A method of treating cancer or an infectious disease in a patient in need thereof, said method comprising: (i) obtaining a clinical biological sample from said patient; (ii) enriching and purifying at least one target analyte from the clinical biological sample according to the method of claim 1; (iii) analyzing the final solution; (iv) treating the patient if the information obtained from the target analyte indicates that the patient has or is at risk for having cancer.

52. 52. The method of claim 51, wherein the cancer is CNS cancer, breast cancer, bladder cancer, pancreatic cancer, lung cancer, melanoma, colon cancer, hematopoietic cancer, or ovarian cancer, and wherein the infectious disease is HPV.

53. 52. The method of claim 51, wherein the target analyte is kidney cFDNA or ctDNA and wherein the cancer is a systemic cancer.

54. 52. The method of claim 51, wherein the target analyte is urogenital cFDNA or ctDNA and wherein the cancer is a urogenital cancer.

55. 52. The method of claim 51, wherein the target analyte is bladder cancer DNA and the medical disorder or disease is bladder cancer.

56. 52. The method of claim 51, wherein the target analyte is HPV viral RNA or HPV viral DNA and the medical disorder or disease is HPV.

57. A kit, the kit comprising: a first ATPS composition; and a second ATPS composition; and Binding buffer; and a solid phase medium, wherein the first ATPS composition, the second ATPS composition, the binding buffer, and the solid phase medium are selected from those described in any one of the preceding claims.

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