Method for separating target analyte from biological sample using ATPS and solid phase medium

Abstract

The present disclosure relates to methods, compositions and kits for concentrating 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 a kit comprising one or more ATPS compositions, a binding buffer; and a solid phase medium. Other embodiments provide methods of treating cancer or 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 Application Serial No. 63 / 381,932, filed on November 2, 2022, and U.S. Provisional Application Serial No. 63 / 381,933, filed on November 2, 2022. The contents of the foregoing applications are hereby incorporated by reference in their entirety for all purposes. Technical Field

[0002] The present 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, the present application relates to ATPS compositions, methods and kits for concentrating and purifying at least one target analyte from a clinical biological sample. Background Art

[0003] Effectively concentrating and isolating target analytes (e.g., nucleic acids) from clinical biological samples for downstream applications (e.g., for diagnostic testing) is a challenging task. For example, some target analytes in clinical biological samples are at extremely low concentrations, and the yield of target analytes purified using traditional extraction methods is typically very low, so that subsequent analysis may not achieve sufficient diagnostic sensitivity and specificity. In some cases, the amount of binding buffer required for untreated sample lysates would be very large and unsuitable for inclusion in traditional extraction workflows. Therefore, there is a need for simple, stable, robust, and effective improved methods for concentrating and purifying target analytes from clinical biological samples. Summary of the invention

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

[0005] In some embodiments, a method for concentrating and purifying at least one target analyte from a clinical biological sample is provided, the method comprising the following steps: (a) combining the clinical biological sample with a first aqueous two-phase system (ATPS) composition to form a target-rich phase solution and a target-poor phase solution, the 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; (b) collecting the target-rich phase; (c) optionally adding the target-rich phase to a second ATPS composition to form a second target-rich phase solution and a second target-poor phase solution, and collecting the second target-rich phase, the 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; (d) optionally adding the target-rich phase to a second ATPS composition to form a second target-rich phase solution and a second target-poor phase solution; The target enriched phase of step (b) or the second target enriched phase from step (c) is mixed with a binding buffer to form a mixed solution; (e) the target enriched phase from step (b), the second target enriched phase from step (c) or the mixed solution from step (d) is contacted with a solid phase medium, which is configured to selectively bind the target analyte, so that the solid phase medium binds the target analyte; and (f) the target analyte is eluted from the solid phase medium with an elution solution and collected to obtain a final solution containing a concentrated and purified target analyte.

[0006] In some embodiments, a method for treating cancer or an infectious disease in a patient in need thereof is provided, the method comprising the following steps: (i) obtaining a clinical biological sample from the patient; (ii) concentrating 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 the information obtained from the target analyte indicates that the patient has cancer or is at risk of developing 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] There are many advantages to the various embodiments of the present disclosure.

[0010] In some embodiments, the methods, compositions and kits disclosed herein provide simple, relatively cheap, and effective means to purify target analytes from different clinical / biological samples of different volumes. The methods and kits disclosed herein relate to an aqueous two-phase system (ATPS) in an upstream process that effectively removes interfering salts and proteins in the sample matrix that may prevent optimal binding of target analytes (such as DNA and free DNA (cfDNA)) to a solid phase medium during purification. The methods and kits disclosed herein also surprisingly effectively minimize the use of reagents without the need for specialized laboratory equipment.

[0011] In some embodiments, the methods described herein are surprisingly effective in reducing the amount of binding buffer required for unprocessed sample lysates, thereby allowing the methods to be incorporated into traditional extraction workflows.

[0012] In some embodiments, it was surprisingly found that although the disclosed methods include additional ATPS steps in the upstream process, which are potential sources of target analyte loss (e.g., due to imperfect target partitioning in ATPS), the recovery efficiency of the target analyte using the disclosed methods is surprisingly higher than the recovery efficiency of purifying the target analyte from a clinical biological sample using a method without ATPS.

[0013] One of the factors that affects the efficiency of target extraction is the composition of the ATPS, as various components interact differently with the target. In some embodiments, the composition of the ATPS in the present disclosure is highly tunable, depending on the target analyte to be recovered, as various components interact differently with the target analyte. The methods, compositions, and kits of the present disclosure can accommodate a wide range of target analytes and clinical / biological samples.

[0014] Using the methods and kits disclosed in the present invention, target analytes (such as cell-free DNA (cfDNA)) present in very small concentrations in biological samples can be effectively concentrated and purified to remove unwanted proteins and ions that may interfere with downstream detection.

[0015] In some embodiments, large clinical / biological sample volumes produce a large first ATPS bottom phase. In some embodiments, a second ATPS is used to concentrate the large first ATPS bottom phase into a more concentrated small top phase volume for more user-friendly downstream processing. In some embodiments, a two-step ATPS process is used for DNA, producing a further concentrated sample for testing.

[0016] In some embodiments, the methods and kits of the 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, as well as methods of use and functions of related components, will become more apparent upon consideration of the following detailed description and appended claims with reference to the accompanying drawings, all of which form a part of this specification, wherein like reference numerals represent corresponding parts in the various figures. It should be expressly understood, however, that the drawings are for illustration and description purposes only and are not intended as a definition of the limitations of the claims. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A An example workflow for bead purification according to the example embodiment described in Example 1a is shown. Figure 1B An example workflow for column purification according to the example embodiment described in Example 1b is shown. Figure 2A is a graph showing average CT values ​​for 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 is a graph showing average CT values ​​for 2000 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 2C is a graph showing the average CT values ​​of 145 bp DNA recovered from plasma using ATPS conditions according to Table 1.4 in Example 2b. Figure 2D is a graph showing the average CT values ​​of 145 bp DNA recovered from plasma using ATPS conditions according to Table 1.5 in Example 2b. Figure 2E is a graph showing the average CT values ​​of 145 bp DNA recovered from plasma using 3 different types of ATPS and magnetic beads according to Example 2c. Figure 2F is a graph showing the average CT values ​​of 2000 bp DNA recovered from plasma using 3 different types of ATPS and magnetic beads according to Example 2c. Figure 3A is a graph showing average CT values ​​for 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 is a graph showing average CT values ​​for 2000 bp DNA recovered from plasma using spin columns with and without a prior ATPS step according to the exemplary method described in Example 3a. Figure 3Cis a graph showing the average CT values ​​of 145 bp DNA recovered from plasma using ATPS conditions according to Table 2.3 in Example 3b. Figure 3D is a graph showing the average CT values ​​of 145 bp DNA recovered from plasma using ATPS conditions according to Table 2.4 in Example 3b. Figure 3E is a graph showing the average CT values ​​of 145 bp DNA recovered from plasma using 3 different types of ATPS and spin columns according to Example 3c. Figure 3F is a graph showing the average CT values ​​of 2000 bp DNA recovered from plasma using 3 different types of ATPS and spin columns according to Example 3c. Figure 4A is a graph showing average CT values ​​for 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 is a graph showing average CT values ​​for 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 is a graph showing the average CT values ​​for 145 bp DNA recovered from urine using ATPS conditions according to Table 3.3 in Example 4b. Figure 4D is a graph showing the average CT values ​​for 145 bp DNA recovered from urine using ATPS conditions according to Table 3.4 in Example 4b. Figure 4E is a graph showing the average CT values ​​of 145 bp DNA recovered from urine using 3 different types of ATPS and magnetic beads according to Example 4c. Figure 5A is a graph showing average CT values ​​for 145 bp DNA recovered from urine using spin columns with and without a prior ATPS step according to the exemplary method described in Example 5a. Figure 5B is a graph showing average CT values ​​for 2000 bp DNA recovered from urine using spin columns with and without a prior ATPS step according to the exemplary method described in Example 5a. Figure 5C is a graph showing the average CT values ​​for 145 bp DNA recovered from urine using ATPS conditions according to Table 4.3 in Example 5b. Figure 5Dis a graph showing the average CT values ​​for 145 bp DNA recovered from urine using ATPS conditions according to Table 4.4 in Example 5b. Figure 5E is a graph showing the average CT values ​​of 145 bp DNA recovered from urine using 3 different types of ATPS and spin columns according to Example 5c. Fig. 6A is a combined data representation showing the average CT values ​​for 145 bp DNA recovered from plasma and urine using magnetic beads according to Examples 2a-c and 4a-c. Figure 6B is a combined data representation showing the average CT values ​​for 145 bp DNA recovered from plasma and urine using spin columns according to Examples 3a-c and Examples 5a-c. Fig. 7A Graphs showing the recovery of 145 bp DNA spike-in (copies / uL) using conditions AF according to Table 6. Figure 7B is a graph showing the average concentration (copy number / uL) of DNA recovered using the kit and conditions according to Table 7. DETAILED DESCRIPTION

[0018] Unless otherwise explained, terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs, and detailed description of well-known functions and constructions that may obscure the gist of the present invention is omitted.

[0019] As used herein and in the claims, "comprising" and "including" mean including the following elements but not excluding other elements.

[0020] As used herein and in the claims, the terms "comprising" (or any related forms, such as "comprise" and "comprises"), "including" (or any related forms, such as "include" or "includes"), "containing" (or any related forms, such as "contain" or "contains"), or "having" (or any related forms, such as "have" or "has") mean the inclusion of the following elements but not the exclusion of other elements. It should be understood that, for each embodiment in which the terms "comprising" (or any related forms, such as "comprise" and "comprises"), "including" (or any related forms, such as "include" or "includes"), or "containing" (or any related forms, such as "contain" or "contains") are used, the present disclosure / application also includes alternative embodiments in which the terms "comprising", "including", "containing", or "having" are replaced with "consisting essentially of" or "consisting of. These alternative embodiments using "consisting of" or "consisting essentially of are understood to be narrower embodiments of the "comprising", "including", or "containing" embodiments.

[0021] For example, alternative embodiments of "a solution comprising A, B, and C" would be "a solution consisting of A, B, and C" and "a solution consisting essentially of A, B, and C." Even though the latter two embodiments are not explicitly written out, the present disclosure / application includes those embodiments. Furthermore, it should be understood that the scope of the three embodiments listed above is different.

[0022] For clarity, "comprising," "including," and "containing" and any related forms are open terms that allow additional elements or features other than the specified essential elements, whereas "consisting of" is a closed term that is limited to the elements recited in the claim and does not include any element, step, or ingredient not specified in a claim.

[0023] "Consisting essentially of" limits the scope of a claim to certain materials, components, or steps ("essential elements") that do not materially affect the basic feature(s) of the claimed invention. In some embodiments, the essential features are the basic and novel feature(s) of the claimed invention.

[0024] 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.

[0025] When a range is mentioned in the specification, the range is understood to include at least each discrete point within the range. For example, in some embodiments, 1-7 refers to 1, 2, 3, 4, 5, 6, and 7. Unless otherwise specified, a range is intended to include all values ​​belonging to the range, including integers, fractions, parts, etc. For example, a range of 1-7 described in the claims refers to a range including values ​​and sub-ranges, such as 1, 1.5, 2-3, 6, and 7. Unless otherwise specified, a range is intended to include all values ​​belonging to the range, including integers, fractions, parts, etc.

[0026] As used herein, the term "about" is understood to be within the normal tolerance range of the art and not more than ±10% of the specified value. By way of example only, about 50 means from 45 to 55, including all values ​​in between. As used herein, the phrase "about" a particular value also includes the particular value, for example, about 50 includes 50.

[0027] As used herein, "aqueous" refers to the characteristic properties of a solvent / solute system in which the solvated substance has a predominantly hydrophilic character. Examples of aqueous solvent / solute systems include those in which water or an aqueous composition is the primary solvent. The polymer and / or surfactant components (the uses of which are described in the examples) are "aqueous" because they form an aqueous phase when combined with a solvent such as water. In addition, as will be appreciated by the skilled artisan, in this article, the term liquid "mixture" refers only to the combination of components defined herein.

[0028] As used herein, an aqueous two-phase system (ATPS) means a liquid-liquid separation system that can achieve separation or concentration of analytes by partitioning, wherein two phases, parts, regions, components, etc. interact in different ways with at least one analyte to which they are exposed and optionally dissolved. ATPS is formed when two immiscible phase-forming components, such as salts and polymers, or two incompatible polymers (e.g., PEG and dextran) with a certain concentration are mixed in an aqueous solution. ATPS methods are relatively inexpensive and scalable because they use two-phase partitioning to separate analytes (e.g., nucleic acids) from contaminants.

[0029] As used herein, the term "separated" refers to an analyte being removed from its original environment and therefore changed from its original environment. For example, the isolated nucleic acid provided generally has less non-nucleic acid components (e.g., protein, lipid) compared to the amount of components present in the source sample. The composition comprising the 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 greater than 99% free of non-analyte components (e.g., non-nucleic acid components)).

[0030] As used herein, "concentrated" means that the mass ratio of the analyte in question to the solution in which the analyte is suspended is higher than the mass ratio of the analyte in its pre-concentrated solution. For example, it can be slightly higher, or more preferably at least two, ten or one hundred times higher.

[0031] As used herein, the term "polymer" refers to any polymer comprising at least one substituted or unsubstituted monomer. Examples of polymers include, but are not limited to, homopolymers, copolymers, terpolymers, random copolymers, and block copolymers. Block copolymers include, but are not limited to, block, graft, dendritic polymers, and star polymers. As used herein, copolymers refer to polymers derived from two monomer species; similarly, terpolymers refer to polymers derived from three monomer species. The two or three monomer species may be of the same or different species. Polymers also include various morphologies, including, but not limited to, linear polymers, branched polymers, random polymers, crosslinked polymers, and dendritic polymer systems. In some embodiments, polymers also include chemically modified equivalents thereof, such as hydrophobically modified or silicone modified. For example, a polyacrylamide polymer refers to any polymer comprising at least one substituted or unsubstituted acrylamide unit, such as a homopolymer, copolymer, terpolymer, random copolymer, block copolymer, or terpolymer of polyacrylamide; polyacrylamide may be a linear polymer, a branched polymer, a random polymer, a crosslinked polymer, or a dendritic polymer of polyacrylamide; polyacrylamide may be a hydrophobically modified polyacrylamide, or a silicone modified polyacrylamide.

[0032] In some embodiments, examples of polymers include, but are not limited to, polyethers, polyimines, polyacrylates, polyalkylene glycols, vinyl polymers, alkoxylated surfactants, polysaccharides, polyether-modified silicones, polyacrylamides, polyacrylic acids, and copolymers thereof. In some embodiments, the polymer is hydrophobically modified or silicone-modified. In some embodiments, the polymer is polyvinyl methyl ether, hydroxypropyl cellulose, or polyethyleneimine. In some embodiments, the polymer is PEG-PPG, polyvinyl pyrrolidone, or methylcellulose. In some embodiments, the polymer is polyethylene glycol, polypropylene glycol, or dextran. In some embodiments, the polymer is alkylbenzene sulfonate, polyacrylamide, or isopropylacrylamide. In some embodiments, the polymer is acrylamide. In some embodiments, the polymer is dextran. In some embodiments, the polymer is polyalkylene glycol.

[0033] Examples of polyalkylene glycols (also referred to as "PAGs" or "poly(oxyalkylene)s" 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 "PEG"), polypropylene glycol (also referred to as "PPG"). In some embodiments, examples of copolymers of PAGs 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, PEG-PPG comprises a random copolymer, a block copolymer, or a combination thereof. In some embodiments, PEG-PPG comprises a random copolymer and a block copolymer. In some embodiments, PEG-PPG is PEG-ran-PPG.

[0034] As used herein, "vinyl polymer" refers to a group of polymers derived from substituted vinyl (H2C=CHR) monomers. Examples of vinyl polymers include, but are not limited to, polyvinyl pyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, and polyvinyl methyl ether.

[0035] 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, an alkoxylated cellulose, or an alkyl hydroxyalkyl cellulose.

[0036] Examples of polyacrylamides include, but are not limited to, poly-N-isopropylacrylamide.

[0037] Examples of polyimines include, but are not limited to, polyethyleneimine.

[0038] 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, carboxamides, monoalkanolamine condensates, and polyoxyethylene fatty acid amides.

[0039] In some embodiments, polymer has an average molecular weight of about 200-1,000Da, 200-35,000Da, 300-35,000Da, 400-2,000Da or 400-35,000Da. Examples include, but are not limited to, polyalkylene glycols (PAGs) having an average molecular weight of about 400Da, 500Da, 600Da, 700Da, 800Da, 900Da, 1,000Da, 2,000Da, 3,000Da, 4,000Da, 5,000Da, 6,000Da, 7,000Da, 8,000Da, 9,000Da, 10,000Da, 15,000Da, 20,000Da, 25,000Da, 30000Da and 35000Da. In some embodiments, PAG has an average molecular weight in the range between any one of the above two molecular weights.

[0040] Examples of PAG 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, PEG has an average molecular weight within the range between any of the two PEG molecular weights described above. In some embodiments, PPG has an average molecular weight within the range between any of the two PPG molecular weights described above.

[0041] 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 in a range between any of the above two ratios.

[0042] In some embodiments, the polymer is a PAG with an average molecular weight of about 980-12,000Da and an EO:PO ratio of 50:50 to 75:25. Examples thereof include, but are not limited to, PEG-PPG with an average molecular weight of about 980Da, 1,230Da, 1,590Da, 2,470Da, 2,660Da, 3,380Da, 3,930Da, 6,950Da, and 12,000Da. In some embodiments, the PEG-PPG has an average molecular weight in the range between any one of the above-mentioned two PEG-PPG molecular weights. In some embodiments, the PEG-PPG comprises an EO:PO ratio of 50:50 or 75:25. In some embodiments, the polymer is a PEG-ran-PPG with an average molecular weight of about 2,500 or 12,000Da and an EO:PO ratio of about 75:25.

[0043] In some embodiments, the polymer is a vinyl polymer having an average molecular weight of about 2,500-2,500,000 Da. Examples include, but are not limited to, polyvinylpyrrolidone having an average molecular weight of about 2,500 Da, 10,000 Da, 40,000 Da, 100,000 Da, and 2,500,000 Da. In some embodiments, the vinyl polymer has an average molecular weight within a range between any of the above two molecular weights.

[0044] In certain embodiments, the polymer is a polysaccharide and the average molecular weight is about 6,000-5,000,000Da. Examples include, but are not limited to, dextran having an average molecular weight of about 6,000Da, 12,000Da, 25,000Da, 60,000Da, 70,000Da, 80,000Da, 150,000Da, 270,000Da, 410,000Da, 450,000Da, 550,000Da, 650,000Da, 670,000Da, 1,500,000Da, 2,000,000Da, 2,800,000Da, 4,000,000Da and 5,000,000Da. In certain embodiments, the dextran has an average molecular weight in the range between any one of the above two molecular weights.

[0045] In some embodiments, the polymer is a polyether and has an average molecular weight of about 200-35,000 Da. Examples include, but are not limited to, silicon-modified polyethers (or "polyether-modified silicones") having an average molecular weight of about 200 Da-35,000 Da.

[0046] In some embodiments, the polymer is polyacrylamide and has an average molecular weight of 1,000-5,000,000 Da. Examples include, but are not limited to, polyacrylamide or poly(N-isopropylacrylamide) having an average molecular weight of 1,000, 2,000, 5,000, 10,000, 40,000, 85,000, 5,000,000 Da. In some embodiments, the polyacrylamide has an average molecular weight in the range between any of the above two molecular weights.

[0047] In some embodiments, the polymer is polyacrylic acid and has an average molecular weight of about 1,250-4,000,000 Da. Examples include, but are not limited to, polyacrylic acid having an average molecular weight of 1,200 Da, 2,100 Da, 5,100 Da, 8,000 Da, 8,600 Da, 8,700 Da, 16,000 Da, and 83,000 Da. In some embodiments, the polyacrylic acid has an average molecular weight in the range between any of the above two molecular weights.

[0048] As used herein, the term "salt" refers to a substance having at least one cation and at least one anion. Examples of salts include, but are not limited to, salts wherein the cation is sodium, potassium, calcium, ammonium, lithium, magnesium, aluminum, cesium, barium, linear or branched trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium or tetrabutylammonium, and / or wherein the anion is phosphate, hydrogenphosphate, dihydrogenphosphate, sulfate, sulfide, sulfite, hydrogensulfate, 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 or tris(hydroxymethyl)aminomethane. In some embodiments, the salt is a kosmotropic salt, a discrete salt, or an inorganic salt.

[0049] As used herein, "surfactant" includes, but is not limited to, anionic surfactants, nonionic surfactants, cationic surfactants, zwitterionic surfactants, or amphoteric surfactants.

[0050] Examples of anionic surfactants include, but are not limited to, carboxylates, sulfonates (also referred to as "sulfonates"), petroleum sulfonates, alkylbenzene sulfonates, naphthalene sulfonates, olefin sulfonates, alkyl sulfates, sulfates, sulfated natural oils, sulfated natural fats, sulfated esters, sulfated alkanolamides, sulfated alkylphenols, ethoxylated alkylphenols, and sodium N-lauroyl sarcosinate (NLS).

[0051] 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, carboxamides, monoalkanolamine condensates, and polyoxyethylene fatty acid amides.

[0052] Examples of cationic surfactants include, but are not limited to, quaternary ammonium salts, amines having an amide bond, polyoxyethylene alkylamines, polyoxyethylene alicyclic amines, n,n,n',n'tetrasubstituted ethylenediamines, and 2-alkyl 1-hydroxyethyl 2-imidazolines.

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

[0054] In some embodiments, the surfactant comprises a polymer such as PAG. In some embodiments, the surfactant has EO x-PO y -EO x The structure of wherein EO refers to ethylene oxide units, PO refers to propylene oxide units, and x and y are the monomer numbers, respectively. In some embodiments, x=2-136. In some embodiments, y=16-62. In some embodiments, examples of surfactants include, but are not limited to (C2H4O) n C 14 H 22 O, wherein n = 4-10 (e.g. Triton X-100, Triton X-114, Triton X-45, Tween 20, Igepal CA630), Brij58, Brij O10, Brij L23, EO x -PO y -EO x , wherein x=2-136 and y=16-62 (e.g., Pluronic L-61, Pluronic F-127), sodium lauryl 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 being). 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, endocervical swabs, semen, breast milk, and other body fluids. In some embodiments, the clinical biological sample is obtained from a biological sample treated with a lysis 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, cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), circulating fetal DNA, microbial cell-free DNA, microRNA (miRNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), circular RNA, long non-coding RNA (lncRNA), or a combination thereof.

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

[0058] When referring to a liquid sample in this disclosure, the terms "large volume", "large amount", "high volume" or "large amount of fluid", "large amount of fluid sample" refer to a biological sample with 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, 500 mL. In some embodiments, the volume of the sample is 1-5 mL, 1-10 mL, 15-20 mL, 10-20 mL, 20-30 mL or 30-40 mL. In some embodiments, the volume of the sample is at least 40 mL. In some embodiments, the volume of the sample ranges from 10mL-40mL, 10mL-50mL, 10mL-100mL, 40mL-50mL, 40mL-60mL, 40mL-100mL, 40mL-160mL, 40mL-200mL, 50mL-100mL, 50mL-200mL, or 50mL-300mL. In some embodiments, the volume of the sample is at least 10mL, 20mL, 30mL, 40mL, 50mL, 60mL, 70mL, 80mL, 90mL, or 100mL; at most 100mL, 200mL, 300mL, 400mL, or 500mL.

[0059] As used herein, the term "dispersant" refers to a substance that destroys the hydrogen bond network between water molecules in a solution. In certain embodiments, the dispersant is a thiocyanate, an isothiocyanate, a perchlorate, an acetate, a trichloroacetate, a trifluoroacetate, a chloride or an iodide. Examples of dispersants include, but are not limited to, guanidine hydrochloride (GHCl), guanidine thiocyanate, guanidine isothiocyanate (GITC), sodium thiocyanate, sodium iodide, sodium perchlorate, sodium trichloroacetate, sodium trifluoroacetate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, thiourea, urea, etc.

[0060] As used herein, the term "Ct", "CT", "Ct value" or "CT value" refers to a cycle threshold and represents a cycle of a PCR amplification assay in which a signal (e.g., fluorescence) generated by an indicator amplicon from a reporter first becomes detectable above background levels. In some embodiments, the CT value is an indirect indication of the amount of target nucleic acid detected in a particular sample. In general, a lower CT value indicates a higher amount of target nucleic acid in a sample, while a higher CT value indicates a lower amount of target nucleic acid in a 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, the solid phase purification system comprises a solid phase medium. In some embodiments, the solid phase purification system refers to a magnetic bead workflow or a spin column workflow.

[0062] As used herein, the term "solid phase medium" refers to a material that selectively separates target analytes 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, such as a centrifugal column. In some embodiments, the solid phase medium is a plurality of beads, a silica resin, a silica membrane, a silica gel, an alumina gel, a size exclusion resin, or an ion exchange resin.

[0063] As used herein, the terms "flow-through" and "supernatant" all refer to a liquid or solution that passes through or is separated from a solid medium, which can be removed or separated from the solid medium. In some embodiments, the supernatant refers to the flow-through of the column.

[0064] As used herein, the term "disturbance" or "perturbation" refers to the process of introducing physical forces and disturbances into the provided system. In some embodiments, perturbing the solid phase extraction complex introduces centrifugal force, magnetic force or a combination thereof, which causes one or more target analytes to be separated from or separated into the solid phase medium or supernatant. In some embodiments, examples of disturbance or disturbance are but are not limited to centrifugation, vacuuming, magnetization, vortexing, rotation, "spinned down", "spun down", swirl, rotation, vibration, stirring, swinging and combinations thereof. In some embodiments, centrifugation or vortexing is achieved by using a centrifuge (centrifuge) or vortex. In some embodiments, vacuuming refers to contacting the solid phase extraction complex with a vacuum manifold to produce a flow-through liquid or supernatant. In some embodiments, disturbances such as magnetization, rotation, swirl, rotation, vibration, stirring and swinging are achieved manually or by appropriate instruments (e.g., desktop microcentrifuges). In some embodiments, centrifugation and magnetization are performed simultaneously.

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

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

[0067] As used herein, the term "micellar system" or "surfactant system" refers to any ATPS composition consisting essentially of at least two surfactants.

[0068] In some embodiments, the first ATPS composition or the second ATPS composition is a polymer-salt system consisting of 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 consists of a salt and an acid. Embodiments of the present invention Example 1 Exemplary methods, compositions, and kits for concentrating target analytes from bulk fluid samples

[0070] In one aspect, a method for concentrating and purifying one or more target analytes from a bulk fluid sample is provided, the method comprising the steps of: (a) preparing a first aqueous two-phase system (ATPS) composition, wherein the first ATPS composition comprises a polymer, a salt, a surfactant, or a combination thereof dissolved in an aqueous solution to form a first phase solution and a second phase solution; (b) adding a sample solution prepared from the plurality of fluid samples containing one or more target analytes to the first ATPS composition, such that the one or more target analytes are partitioned into the first phase solution; (c) collecting the first phase solution and mixing the first phase solution with a second ATPS composition, wherein the second ATPS composition comprises a polymer, a salt, a surfactant, or a combination thereof dissolved in an aqueous solution to form a third phase solution and a fourth phase solution, such that the one or more target analytes are partitioned into and concentrated in the third phase solution; (d) collecting the third phase solution and mixing the third phase solution with a binding buffer to form a mixed solution, wherein the binding buffer comprises at least one discrete 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 one or more target analytes.

[0071] In some embodiments, the sample solution is prepared by dividing a bulk fluid sample containing the one or more target analytes into at least two aliquots of the sample solution, and the first ATPS composition is divided into at least two aliquots, wherein step (b) further comprises the following steps: (i) adding each aliquot of the sample solution prepared from the plurality of fluid samples containing the one or more target analytes to each aliquot of the first ATPS composition such that the one or more target analytes partition into the first phase solution; (ii) collecting and combining the first phase solutions of at least two samples of the first ATPS composition to form a first phase solution for step (c).

[0072] In some embodiments, the extraction column is a spin column, and wherein step (e) further comprises the following steps: (i) loading a portion of the mixed solution onto an extraction column; (ii) centrifuging the extraction column and discarding the flow-through (also referred to as 'supernatant'); and (iii) repeating the above steps (i) and (ii) until all of the mixed solution passes through the extraction column.

[0073] In some embodiments, the method further comprises the following steps: (g) subjecting the final solution to a diagnostic assay to detect and quantify the one or more target analytes.

[0074] In another aspect, a method is provided for concentrating and purifying one or more target analytes from a bulk fluid sample, the method comprising the steps of: (a) dividing a bulk fluid sample containing the one or more target analytes into at least two aliquots of sample solution; (b) preparing at least two samples of a first aqueous two-phase system (ATPS) composition, wherein the first ATPS composition comprises a polymer, a salt, a surfactant, or a combination thereof dissolved in an aqueous solution to form a first phase solution and a second phase solution; (c) adding each aliquot of the sample solution containing the one or more target analytes to each aliquot of the first ATPS composition such that the one or more target analytes partition into the first phase solution; (d) collecting first phase solutions of at least two samples of the first ATPS composition and mixing the first phase solutions with a second ATPS composition, wherein the second ATPS composition comprises a polymer, a salt, a surfactant, or a combination thereof dissolved in an aqueous solution to form a third phase solution and a fourth phase solution, so that the one or more target analytes are partitioned into and concentrated in the third phase solution; (e) collecting the third phase solution and mixing the third phase solution with a binding buffer to form a mixed solution, wherein the binding buffer comprises at least one discrete 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 one or more target analytes.

[0075] In another aspect, a method is provided for concentrating and purifying one or more target analytes from a bulk fluid sample, the method comprising the steps of: (a) dividing a bulk fluid sample containing the one or more target analytes into at least two aliquots of sample solution; (b) preparing at least two samples of a first aqueous two-phase system (ATPS) composition, wherein the first ATPS composition comprises a polymer, a salt, a surfactant, or a combination thereof dissolved in an aqueous solution to form a first phase solution and a second phase solution; (c) adding each aliquot of the sample solution containing the one or more target analytes to each aliquot of the first ATPS composition such that the one or more target analytes partition into the first phase solution; (d) collecting first phase solutions of at least two samples of the first ATPS composition and mixing the first phase solutions with a second ATPS composition, wherein the second ATPS composition comprises a polymer, a salt, a surfactant, or a combination thereof dissolved in an aqueous solution to form a third phase solution and a fourth phase solution, so that the one or more target analytes are partitioned into and concentrated in the third phase solution; (e) collecting the third phase solution and mixing the third phase solution with a binding buffer to form a mixed solution, wherein the binding buffer comprises at least one discrete 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 the above steps (f) and (g) until all of the mixed solution passes through the extraction column; (i) eluting and collecting the one or more target analytes from the extraction column; obtaining a final solution containing concentrated and purified one or more target analytes; and (j) subjecting the final solution to a diagnostic assay to detect and quantify the one or more target analytes.

[0076] 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 an appropriate volume (e.g., 500 mL) of water.

[0077] In some embodiments, the bulk fluid sample is urine.

[0078] In some embodiments, the volume of the plurality of fluid samples is 40 mL or greater, such as 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 200 mL, 300 mL, 400 mL, 500 mL or greater.

[0079] In some embodiments, the volume of each specimen 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.

[0080] In some embodiments, the target analytes are selected from the group consisting of nucleic acids, proteins, antigens, biomolecules, sugar moieties, lipids, sterols, and combinations thereof.

[0081] In some embodiments, the target analytes are DNA.

[0082] In some embodiments, the target analytes are cell-free DNA or circulating tumor DNA.

[0083] In some embodiments, the polymer is dissolved in the aqueous solution at a concentration of 4%-84% (w / w).

[0084] In some embodiments, the salt is dissolved in the aqueous solution at a concentration of 1%-55% (w / w). In some embodiments, the salt is dissolved in the aqueous solution at a concentration of 8%-55% (w / w).

[0085] In some embodiments, the surfactant is dissolved in the aqueous solution at a concentration of 0.05%-10% (w / w). In some embodiments, the surfactant is dissolved in the aqueous solution at a concentration of 0.05%-9.8% (w / w).

[0086] In another aspect, an ATPS composition is provided that is selected from the group consisting of A1, A2, A3, A4, AA1, AA2, AA3 and AA4 in Table I.

[0087] On the other hand, a kit is provided, which comprises a first ATPS composition selected from the group consisting of A1, A2, A3 and A4 in Table I; a second ATPS composition selected from the group consisting of AA1, AA2, AA3 and AA4 in Table I; and a binding buffer selected from the group consisting of B1, B2 and B3 in Table I.

[0088] In some embodiments, the kit further comprises an extraction column.

[0089] Various ATPS systems that can 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.

[0090] In one embodiment, the first and / or second ATPS composition comprises a polymer. In some embodiments, the polymer that can be used includes, but is not limited to, polyalkylene glycols, such as hydrophobically modified polyalkylene glycols, poly(oxyalkylene) polymers, poly(oxyalkylene) copolymers, such as hydrophobically modified poly(oxyalkylene) copolymers, polyvinyl pyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, polyvinyl methyl ether, alkoxylated surfactants, alkoxylated starch, alkoxylated cellulose, alkyl hydroxyalkyl cellulose, silicon-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, polyimines, polyalkylene glycols, vinyl polymers, alkoxylated surfactants, polysaccharides, alkoxylated starch, alkoxylated cellulose, alkyl hydroxyalkyl cellulose, 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), polyvinyl pyrrolidone, 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, polyethylene imine, poly-N-isopropyl acrylamide 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), polyvinyl pyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, polyvinyl methyl ether and poly-N-isopropyl acrylamide. 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 in the range of 200-1,000Da, 200-35,000Da, 425-2,000Da, 400-35,000Da, 980-12,000Da or 3,400-5,000,000Da. In some embodiments, the polymer includes ethylene oxide and propylene oxide units, and the polymer has an EO:PO ratio of 90:10 to 10:90.

[0091] In one embodiment, the polymer concentration of the first and / or second ATPS composition is in the range of about 4% to about 84% by weight (w / w) based on the total weight of the aqueous solution. In various embodiments, the polymer solution is selected from about 4% w / w, about 4.5% w / w, about 5% w / w, about 5.5% w / w, about 6% w / w, about 6.5% w / w, about 7% w / w, about 7.5% w / w, about 8% w / w, about 8.5% w / w, about 9% w / w, about 9.5% w / w, about 10% w / w, about 10.5% w / w, about 11% w / w, about 11.5% w / w, about 12% w / w, about 12.5% ​​w / w, about 13% w / w, about 13.5% w / w, about 14% w / w, about 14.5% w / w, about 15% w / w, about 15.5% w / w, about 16% w / w, about 16.5% w / w. 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 % w / w, about 40% w / w, about 40.5% w / w, about 41% w / w, about 41.5% w / w, about 42% w / w, about 42.5% w / w, about 43% w / w, about 43.5% w / w, about 44% w / w, about 44.5% w / w, about 45% w / w, about 45.5% w / w, about 46% w / w, about 46.5% w / w, about 47% w / w, about 47.5% w / w, about 48% w / w, about 48.5% w / w, about 49% w / w, about 49.5% w / w, about 50% w / w, about 50.5% w / w, about 51% w / w, about 51.5% w / w, about 52% w / w, about 52. 5% w / w, about 53% w / w, about 53.5% w / w, about 54% w / w, about 54.5% w / w, about 55% w / w, about 55.5% w / w, about 56% w / w, about 56.5% w / w, about 57% w / w, about 57.5% w / w, about 58% w / w, about 58.5% w / w, about 59% w / w, about 59.5% w / w, about 60% w / w, about 60.5% w / w, about 61% w / w, about 61.5% w / w, about 62% w / w, about 62.5% w / w, about 63% w / w, about 63.5% w / w, about 64% w / w, about 64.5% w / w, about 65% w / w, about 65.5% w / w, about 66% w / w, about 66.5% w / w, about 67% w / w, about 67.5% w / w, about 68% w / w, about 68.5% w / w, about 69% w / w, about 69.5% w / w, about 70% w / w, about 70.5% w / w, about 71% w / w, about 71.5% w / w, about 72% w / w, about 72.5% w / w, about 73% w / w, about 73.5% w / w, about 74% w / w, about 74.5% w / w, about 75% w / w, about 75.5% w / w, about 76% w / w, about 76.5% w / w, about 77% w / w, about 77.5% w / w, about 78% w / w, about 78.5% w / w, about 79% w / w, about 79.5% w / w, 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 polymer solutions.

[0092] In one embodiment, the first and / or second ATPS composition comprises a salt and thereby forms a salt solution. In some embodiments, the salt includes, but is not limited to, a kosmotropic salt, a discrete salt, an inorganic salt containing a cation, such as linear or branched trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium and tetrabutylammonium, and an inorganic salt containing an anion, such as a phosphate, a sulfate, a nitrate, a chloride and a bicarbonate. In another embodiment, the salt comprises NaCl, Na3PO4, K3PO4, Na2SO4, potassium citrate, (NH4)2SO4, sodium citrate, sodium acetate or a combination thereof. Other salts, such as ammonium acetate, may also be used. In another embodiment, the salt 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 bromide salts, iodide salts, fluoride salts, carbonates, sulfates, citrates, carboxylates, borates and phosphates. In some embodiments, the salt comprises potassium phosphate. In some embodiments, the salt comprises ammonium sulfate.

[0093] In one embodiment, the total salt concentration is in the range of about 0.01% to about 90%.One skilled in the art will appreciate that the amount of salt required to form a two-phase aqueous system will be affected by the molecular weight, concentration, and physical state of the polymer.

[0094] In various embodiments, the salt concentration is about 1%-55% w / w. In various embodiments, the salt concentration is about 1% w / w, about 1.5% w / w, about 2% w / w, about 2.5% w / w, about 3% w / w, about 3.5% w / w, about 4% w / w, about 4.5% w / w, about 5% w / w, about 5.5% w / w, about 6% w / w, about 6.5% w / w, about 7% w / w, about 7.5% w / w, about 8% w / w, about 8.5% w / w, about 9% w / w, about 9.5% w / w, about 10% w / w, about 10.5% w / w, about 11% w / w, about 11.5% w / w, about 12% w / w, about 12.5% ​​w / w, about 13% w / w, about 13.5% w / w, about 14% w / w, about 14.5% w / w. 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 w / w, about 38.5% w / w, about 39% w / w, about 39.5% w / w, about 40% w / w, about 40.5% w / w, about 41% w / w, about 41.5% w / w, about 42% w / w, about 42.5% w / w, about 43% w / w, about 43.5% w / w, about 44% w / w, about 44.5% w / w, about 45% w / w, about 45.5% w / w, about 46% w / w, about 46.5% w / w, about 47% w / w, about 47.5% w / w, about 48% w / w, about 48.5% w / w, about 49% w / w, about 49.5% w / w, about 50% w / w, about 50.5% w / w, about 51% w / w, about 51.5% w / w, about 52% w / w, about 52.5% w / w, about 53% w / w, about 53.5% w / w, about 54% w / w, about 54.5% w / w, about 55% w / w, about 55.5% w / w, about 56% w / w, about 56.5% w / w, about 57% w / w, about 57.5% w / w, about 58% w / w, about 58.5% w / w, about 59% w / w, about 59.5% w / w, about 60% w / w, about 60.5% w / w, about 61% w / w, about 61.5% w / w, about 62% w / w, about 62.5% w / w, about 63% w / w, about 63.5% w / w, about 64% w / w, about 64.5% w / w, about 65% w / w, about 65.5% w / w, about 66% w / w, about 66.5% w / w, about 67% w / w, about 67.5% w / w, about 68% w / w, about 68.5% w / w, about 69% w / w, about 69.5% w / w, about 70% w / w, about 70.5% w / w, about 71% w / w, about 71.5% w / w, about 72% w / w, about 72.5% w / w, about 73% w / w, about 73.5% w / w, about 74% w / w, about 74.5% w / w, about 75% w / w, about 75.5% w / w, about 76% w / w, about 76.5% w / w, about 77% w / w, about 77.5% w / w, about 78% w / w, about 78.5% w / w, about 79% w / w, about 79.5% w / w or about 80% w / w. .

[0095] In one embodiment, the first and / or second ATPS composition comprises a surfactant. In some embodiments, possible surfactants that can be used include, but are not limited to, Triton-X, Triton-114, Igepal CA-630, and Nonidet P-40, anionic surfactants (such as carboxylates, sulfonates, petroleum sulfonates, alkylbenzene sulfonates, naphthalene sulfonates, olefin sulfonates, alkyl sulfates, sulfates, sulfated natural oils, sulfated natural fats, sulfated esters, sulfated alkanolamides, sulfated alkylphenols, ethoxylated alkylphenols), nonionic surfactants (such as ethoxylated fatty alcohols, polyoxyethylene surfactants, carboxylates, polyethylene glycol esters, sorbitan esters, fatty acid glycol esters, carboxylic acid amides, monoalkanolamine concentrates, polyoxyethylene fatty acid amides), cationic surfactants (such as quaternary ammonium salts, amines with amide bonds, polyoxyethylene alkyl and alicyclic amines, n,n,n',n'tetrasubstituted ethylenediamines, 2-alkyl 1-hydroxyethyl 2-imidazolines), and amphoteric surfactants (such as n-coco 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-cocoamidoethyl n-hydroxyethylglycine and its sodium salt).

[0096] 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 various embodiments, the surfactant concentration is about 0.05% w / w, 0.1% w / w, about 0.2% w / w, about 0.3% w / w, about 0.4% w / w, about 0.5% w / w, about 0.6% w / w, about 0.7% w / w, about 0.8% w / w, about 0.9% w / w, about 1% w / w, 1.1% w / w, about 1.2% w / w, about 1.3% w / w, about 1.4% w / w, about 1.5% w / w, about 1.6% w / w, about 1.7% w / w, about 1.8% w / w, about 1.9% w / w, about 2% w / w, about 2.1% w / w, about 2.2% w / w, about 2.3% w / w w / w, about 2.4% w / w, about 2.5% w / w, about 2.6% w / w, about 2.7% w / w, about 2.8% w / w, about 2.9% w / w, about 3% w / w, 3.1% w / w, about 3.2% w / w, about 3.3% w / w, about 3.4% w / w, about 3.5% w / w, about 3.6% w / w, about 3.7% w / w, about 3.8% w / w, about 3.9% w / w, about 4% w / w, about 4.1% w / w, about 4.2% w / w, about 4.3% w / w, about 4.4% w / w, about 4.5% w / w, about 4.6% w / w, about 4.7% w / w, about 4.8% w / w, about 4.9% w / w, about 5.1% w / w, about 5.2% w / w, about 5.3% w / w, about 5.4% w / w, about 5.5% w / w, about 5.6% w / w, about 5.7% w / w, about 5.8% w / w, about 5.9% w / w % w / w, about 5% w / w, about 5.1% w / w, about 5.2% w / w, about 5.3% w / w, about 5.4% w / w, about 5.5% w / w, about 5.6% w / w, about 5.7% w / w, about 5.8% w / w, about 5.9% w / w, about 6% w / w, 6.1% w / w, about 6.2% w / w, about 6.3% w / w, about 6.4% w / w, about 6.5% w / w, about 6.6% w / w, about 6.7% w / w, about 6.8% w / w, about 6.9% w / w, about 7% w / w, about 7.1% w / w, about 7.2% w / w, about 7.3% w / w, about 7.4% w / w, about 7 In some embodiments, the present invention relates to a method for preparing the present invention in an amount of about 1% w / w to about 5% w / w of the present invention. In some embodiments, the present invention relates to a method for preparing the present invention in an amount of about 1% w / w to about 5% w / w of the present invention. In some embodiments, the present invention relates to a method for preparing the present invention in an amount of about 1% w / w to about 5% w / w of the present invention. In some embodiments, the present invention relates to a method for preparing the present invention in an amount of about 1% w / w to about 5% w / w of the present invention.

[0097] In one embodiment, the binding buffer comprises a dispersant. In some embodiments, possible dispersants 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.

[0098] In one embodiment, the concentration of the dispersant in the binding buffer is in the range of about 0.1 M to 8 M. In various embodiments, the concentration of the dispersant 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 4.1 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. .9M, about 4M, about 4.1M, about 4.2M, about 4.3M, about 4.4M, about 4.5M, about 4.6M, about 4.7M, about 4.8M, about 4.9M, about 5M, about 5.1M, about 5.2M, about 5.3M, about 5.4M, about 5.5M, about 5.6M, about 5.7M, about 5.8M, about 5.9M, about 6M, about 6.1M, about 6.2M, about 6.3M, about 6.4M, about 6.5M, about 6.6M, about 6.7M, about 6.8M, about 6.9M, about 7M, about 7.1M, about 7.2M, about 7.3M, about 7.4M, about 7.5M, about 7.6M, about 7.7M, about 7.8M, about 7.9M or about 8M.

[0099] In one embodiment, possible extraction columns that can be used include, but are not limited to, Epoch life science-EconoSpin silica membrane mini spin column-1920-250, HiBinds RNA mini-RNACOL-02, Corbition silica spin column-PC0054, PuroSpin micro silica spin column-Luna Nano USP003, Purospin nano silica spin column-Lunonano USP002, Qiagen RNEasy minElute, Qiagen minElute-700384 Qiagen GMBH and Qiagen mini. Example protocol for processing 40 mL of sample

[0100] 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). 2. Benchtop microcentrifuge (eg, for 1 and 2 mL tubes). 3. Pipettes and pipette tips (eg, 20 μL, 200 μL, and 1000 μL capacity pipettes). 4. Pipette aids and serological pipette tips (eg, 5 mL, 10 mL, and 50 mL). 5. Water bath (eg, set at 37°C).

[0101] Specific examples of ATPS#1, ATPS#2, and binding buffers that can be used in the above protocol are shown in Table I below.

[0102] The following is an example method for concentrating and isolating target analytes from a biological sample volume of at least 40 mL. In this example, the sample was prepared as follows:

[0103] 40 mL of the biological sample is mixed with at least one lysing reagent (optional) using methods known to those skilled in the art to form one or more sample lysates.

[0104] A portion of the sample lysate (22.6 mL) was transferred to a first tube containing a first ATPS composition (ATPS#1) to form an ATPS#1 solution. The remaining sample lysate was poured into a second tube also containing ATPS#1 to form an ATPS#1 solution.

[0105] The two tubes containing ATPS#1 solution were vortexed thoroughly until homogenous and then centrifuged separately at 2300 RCF for 6 minutes.

[0106] The bottom phase (e.g., about 3.5-5 mL volume) from the two ATPS#1 solutions was transferred (e.g., using a 10 mL serological pipette) to a tube containing a second ATPS composition (ATPS#2) to form an ATPS#2 solution. The ATPS#2 solution was vortexed thoroughly until homogenous and then centrifuged at 2300 RCF for 6 minutes.

[0107] Transfer all of the top phase of the ATPS#2 solution (approximately 400-600 uL) to a 5 mL microcentrifuge tube. Add approximately 2 mL of binding buffer to the microcentrifuge tube containing the top phase of ATPS#2 and vortex the tube briefly.

[0108] Transfer 800uL of ATPS#2 top phase to the spin column and centrifuge at 12,000rcf for 30 seconds. Discard the flow-through. Repeat this spin column step (step 6) until all sample has passed through the spin column.

[0109] Wash buffer (500 uL) was added to the spin column containing the mixture, and the spin column containing the mixture was centrifuged at 12,000 rcf for 30 seconds. The flow-through was discarded.

[0110] The spin column containing the mixture was centrifuged at 16,000 rcf for 2 minutes to remove any excess wash buffer.

[0111] Place the centrifuge column containing the mixture in a new 1.5 mL collection tube. Pipette 1X TE buffer (20-100 uL) onto the center of the centrifuge column membrane. Incubate the centrifuge column containing the mixture for 3 minutes and centrifuge at 12,000 rcf for 1 minute to elute the sample solution containing the concentrated target analyte. Store the sample solution in a refrigerator at -20°C or lower for optional further processing.

[0112] The sample program above is just an example and alternative methods and conditions can be used.

[0113] For example, a 40 mL sample after being subjected to a lysing agent is roughly divided into two portions in step 2. However, a large fluid sample can be divided into many different arrangements.

[0114] Specific examples of ATPS#1, ATPS#2, and binding buffers that can be used in the above protocol are shown in Table I below. Example protocol for scale-up to 160 mL sample input

[0115] In this example, a larger sample volume of approximately 160 mL was prepared as follows: 1. Divide the 160 mL sample into four separate 40 mL portions. For each 40 mL volume of sample input, perform steps (1) to (7) in Example 1 above. 2. Transfer the top phase (approximately 400uL-600uL) of each ATPS#2 to a 15mL microcentrifuge tube. This extraction step can be done with a pipette, such as a P200 pipette set to 200uL for the first extraction. 3. For each 40 mL starting sample, add Binding Buffer (2 mL) to the tube containing the top phase of ATPS#2 (ie, 160 mL sample scale-up requires 4x ATPS#2 and 8 mL Binding Buffer). Vortex the tube briefly. 4. Transfer 800uL of the mixture (starting sample and binding buffer) to the spin column. 5. Centrifuge the mixture at 12,000 rcf for 30 seconds. 6. Discard the flow-through. Repeat steps 4-6 for the remaining sample until the entire mixture has passed through the spin column. (For example, for an 800uL spin column capacity, 160mL of starting sample input volume requires approximately 12 cycles) 7. Add wash buffer to the spin column containing the mixture (500 uL). 8. Centrifuge the spin column containing the mixture at 12,000 rcf for 30 seconds. 9. Discard the flow-through. 10. Centrifuge the spin column containing the mixture at 16,000 rcf for 2 minutes to remove any excess wash buffer. 11. Place the spin column containing the mixture into a new 1.5 mL collection tube. 12. Pipette 1X TE buffer (20-100uL) onto the center of the spin column membrane. 13. Incubate the spin column containing the mixture for 3 minutes and centrifuge at 12,000 rcf for 1 minute to elute the sample solution containing the concentrated target analyte. 14. Store the sample solution in a freezer at -20°C or below for optional further processing. Performance Evaluation of Example Extraction Kits

[0116] The performance of the methods and kits disclosed below can be evaluated by following the following steps: 1. Prepare several large-volume extraction kit components by varying 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. Dispersant 2. Prepare sample solutions to evaluate and spike with known amounts of target DNA. 3. Perform extraction using a variation of the large volume extraction kit prepared in step 1 above as well as an industry standard extraction kit using their designated procedures. 4. Quantify target DNA using standard qPCR or ddPCR procedures

[0117] Compared to the industry standard kit, samples extracted from the disclosed method were found to contain higher concentrations of target DNA.

[0118] Specific examples of ATPS#1, ATPS#2, and binding buffer are shown below. Table I: Example ATPS#1, ATPS#2 and binding buffer. Example 2 Example method using ATPS with magnetic beads

[0119] Another aspect provides a method for concentrating and purifying one or more target analytes from a sample solution, the method comprising the steps of: (a) adding a sample solution containing the one or more target analytes to a first aqueous two-phase system (ATPS) to form a mixture separated into a first phase and a second phase, wherein the one or more target analytes are concentrated in the first phase; (b) separating the first phase containing the concentrated one or more target analytes to obtain a concentrated solution; (c) applying magnetic beads to the concentrated solution such that the magnetic beads 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 complex, thereby obtaining a final solution comprising concentrated and purified one or more target analytes.

[0120] In some embodiments, step (b) further comprises the following steps: (i) adding the separated first phase containing the concentrated one or more target analytes to a second ATPS to form a second mixture separated into a third phase and a fourth phase, wherein the one or more target analytes are concentrated in the third phase; and (ii) separating the third phase containing the concentrated one or more target analytes to form a concentrated solution in step (b) for use in step (c).

[0121] In some embodiments, the concentrated solution of step (b) is mixed with a binding buffer, wherein the binding buffer comprises at least one dispersant selected from the group consisting of n-butanol, ethanol, guanidine chloride, guanidine thiocyanate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea and urea, thereby obtaining a concentrated solution for step (c).

[0122] In some embodiments, step (d) further comprises the following steps: a. mixing the bead-analyte complex with a fractionation buffer comprising a polymer, a salt, a surfactant, a dispersant, or a combination thereof to form a fractionation solution, such that one or more target analytes below a target size are released from the bead-analyte complex into the fractionation solution; b. immobilizing the bead-analyte complex using a magnetic support; and c. separating the one or more target analytes below the target size in the fractionation solution from the immobilized bead-analyte complexes.

[0123] In some embodiments, step (d) further comprises the following steps: (iv) adding the separated one or more target analytes below the target size to a second binding buffer, wherein the second binding buffer comprises at least one dispersant selected from the group consisting of 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 separated one or more target analytes below the target size and the second binding buffer, wherein the magnetic beads bind to the one or more target analytes below the target size to form a second bead-analyte complex; and (vi) recovering the one or more target analytes from the second bead-analyte complex.

[0124] In some embodiments, the method further comprises the following steps: (e) subjecting the final solution to a diagnostic assay to detect and quantify the one or more target analytes.

[0125] 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.

[0126] In some embodiments, the one or more target analytes are DNA.

[0127] In some embodiments, the one or more target analytes are cell-free DNA or circulating tumor DNA.

[0128] 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.

[0129] 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 is dissolved 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.

[0130] In some embodiments, the polymer is dissolved in the aqueous solution at a concentration of 4%-84% (w / w).

[0131] In some embodiments, the salt is dissolved in the aqueous solution at a concentration of 1%-80% (w / w). In some embodiments, the salt is dissolved in the aqueous solution at a concentration of 8%-80% (w / w).

[0132] In some embodiments, the surfactant is dissolved in the aqueous solution at a concentration of 0.05%-10% (w / w). In some embodiments, the surfactant is dissolved in the aqueous solution at a concentration of 0.05%-9.8% (w / w).

[0133] In some embodiments, step (a) further comprises the following steps: (i) embedding a component capable of forming a first ATPS in a porous material; and (ii) contacting the sample solution with a porous material in which components are embedded, wherein the components form a first phase and a second phase when the sample solution passes through the porous material.

[0134] In one aspect, a method for concentrating and purifying one or more target analytes from a sample solution is provided, the method comprising the steps of: (a) adding a sample solution containing the one or more target analytes to a first aqueous two-phase system (ATPS) to form a mixture separated into a first phase and a second phase, wherein the one or more target analytes are concentrated in the first phase; (b) separating the first phase containing the concentrated one or more target analytes; (c) adding the separated first phase containing the concentrated one or more target analytes to a second ATPS to form a second mixture separated into a third phase and a fourth phase, wherein the one or more target analytes are concentrated in the third phase; (d) separating the third phase containing the concentrated one or more target analytes to obtain a concentrated solution; (e) mixing the concentrated solution with a binding buffer, wherein the binding buffer comprises at least one dispersant selected from the group consisting of 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 the binding buffer, such that the magnetic beads bind to the one or more target analytes to form bead-analyte complexes; and (g) mixing the bead-analyte complex with a fractionation buffer comprising a polymer, a salt, a surfactant, a dispersant, or a combination thereof to form a fractionation solution, such that one or more target analytes below a target size are released from the bead-analyte complex into the fractionation solution; (h) immobilizing the bead-analyte complex using a magnetic support; (i) separating one or more target analytes below a target size in the fractionation solution from the immobilized bead-analyte complex; (j) adding the separated one or more target analytes below the target size to a second binding buffer, wherein the second binding buffer comprises at least one dispersant selected from the group consisting of 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 the separated one or more target analytes below the target size and a second binding buffer, wherein the magnetic beads bind to the one or more target analytes below the target size to form a second bead-analyte complex; (l) recovering one or more target analytes below a target size from the second bead-analyte complex, thereby obtaining a final solution containing concentrated and purified one or more target analytes below a target size; and (m) subjecting the final solution to a diagnostic assay to detect and quantify the one or more target analytes below a target size.

[0135] Various ATPS systems that can 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 that can be used in the ATPS system (first and / or second ATPS) and their concentrations include, but are not limited to, those described in Example 1 above.

[0136] In some embodiments, the fractionation buffer comprises a polymer, a salt, a surfactant, a dispersant, or a combination thereof. In some embodiments, possible polymers, salts, surfactants, and dispersants that can be used include, but are not limited to, those described in Example 1 above.

[0137] In some embodiments, possible magnetic beads that may be used include, but are not limited to, those listed in Table II below. Table II: Magnetic Bead Examples Example protocol without fractionation buffer

[0138] The following is an example method of how to concentrate and separate a target analyte according to the present disclosure. In this example, the target analyte is DNA.

[0139] The program steps are as follows: 1. Add a desired volume of processed biological sample (eg, plasma) (eg, 2-3 mL) to the first ATPS (Solution B) to form Solution B'. Processing methods of biological samples include, but are not limited to, lysis to form a sample lysate. 2. Solution B' was vortexed thoroughly (eg, about 10 seconds) until homogenous, and then centrifuged at 2,300 xg for 6 minutes. 3. The bottom phase of solution B' was transferred to the second ATPS (solution C) to form solution C'. 4. Vortex Solution C' thoroughly for 10 seconds until homogenous, then centrifuge at 7,000 x g for 1 minute. 5. Add 800 uL of binding buffer (eg, binding buffer BB1, BB2, or BB3) to a new 2 mL microcentrifuge tube. 6. Transfer the top phase of solution C' containing the concentrated target analyte from step 5 to a microcentrifuge tube. 7. Vortex the provided magnetic beads (e.g., magnetic beads selected from Table II, 12 μL) before use, and then add them to the microcentrifuge tube from step 6 to allow the magnetic beads to bind to the target analyte to form a bead-analyte complex. 8. Incubate the microcentrifuge tube with tilting rotation for 5 minutes and then place on a magnetic stand for 2 minutes to fix the bead-analyte complex to the tube wall. 9. Without disturbing the bead-analyte complex, pipette and discard the supernatant in the microcentrifuge tube. Then remove the microcentrifuge tube from the magnetic stand. 10. Add 800 uL of binding buffer (e.g., binding buffer BB1, BB2, or BB3) to the microcentrifuge tube. Vortex the microcentrifuge tube for 20 seconds and then place it on a magnetic stand for 2 minutes to fix the bead-analyte complex to the tube wall. 11. Without disturbing the bead-analyte complex, pipette and discard the supernatant in the microcentrifuge tube. 12. Add a suitable wash buffer known to those skilled in the art (e.g., 800 uL) to the microcentrifuge tube and then rotate on a magnetic stand, 120 degrees each time, for a total of 720 degrees. After the rotation, pipette and discard the supernatant in the microcentrifuge tube without disturbing the bead-analyte complex. 13. Repeat step 12 at least once. 14. Then briefly spin the microcentrifuge tube with the hinge facing outward to collect any remaining wash buffer in the tube. 15. Place the microcentrifuge tube back on the magnetic stand for 1 minute to fix the bead-analyte complex to the tube wall. 16. Carefully discard all supernatant without disturbing the bead-analyte complex (eg, using a 10 μl pipette tip). 17. Open the tube cap and allow the bead-analyte complex to dry on the magnetic stand for 7 minutes. 18. Then remove the microcentrifuge tube from the magnetic stand after drying. 19. Add a suitable elution buffer (eg, 40 μL) known to those skilled in the art directly to the bead-analyte complex (in a microcentrifuge tube). 20. Use a pipette to resuspend the bead-analyte complex by continuous stirring and then pipetting up and down 5 times. 21. Vortex the microcentrifuge tube briefly (e.g., 15 seconds). 22. Incubate the microcentrifuge tube at room temperature for 3 minutes. 23. Place the microcentrifuge tube on the magnetic stand for 1 minute. 24. Carefully collect the supernatant containing the purified target analyte into a clean maximum recovery tube without disturbing the magnetic beads. 25. The purified target analyte is ready for immediate use or long-term storage at -20°C or below. Example protocol using fractionation buffers

[0140] The following is another example method of how to concentrate and separate a target analyte according to the present disclosure. In this example, the target analyte is DNA.

[0141] The program steps are as follows: 1. Add a desired volume of processed biological sample (eg, plasma) (eg, 2-3 mL) to the first ATPS (Solution B) to form Solution B'. Processing methods of biological samples include, but are not limited to, lysis to form a sample lysate. 2. Vortex Solution B' thoroughly for 10 seconds until homogenous, then centrifuge at 2,300 x g for 6 minutes. 3. The bottom phase of solution B' was transferred to the second ATPS (solution C) to form solution C'. 4. Vortex Solution C' thoroughly for 10 seconds until homogenous, then centrifuge at 7,000 x g for 1 minute. 5. Add 800 uL of binding buffer (eg, binding buffer BB1, BB2, or BB3) to a new 2 mL microcentrifuge tube. 6. Transfer the top phase of solution C' containing the concentrated target analyte from step 5 to the tube containing binding buffer. 7. Vortex the provided magnetic beads (e.g., magnetic beads selected from Table 1, 12 μL) before use, and then add them to the microcentrifuge tube from step 6 to allow the magnetic beads to bind to the target analyte to form a bead-analyte complex. 8. Incubate the microcentrifuge tube with tilting rotation for 5 minutes and then place on a magnetic stand for 2 minutes to fix the bead-analyte complex to the tube wall. 9. Without disturbing the bead-analyte complex, pipette and discard the supernatant in the microcentrifuge tube. Then remove the microcentrifuge tube from the magnetic stand. 10. Add 300uL of fractionation buffer (e.g., fractionation buffer F1, F2, or F3) to the microcentrifuge tube. Vortex the microcentrifuge tube for 20 seconds, incubate for 5 minutes with tilt rotation, and then place on a magnetic stand for 2 minutes to fix the bead-analyte complex to the tube wall, so that the target analyte below the target size is released from the bead-analyte complex into the supernatant. 11. Add 600 uL of second binding buffer (eg, binding buffer BB1, BB2, or BB3) to a new 2 mL microcentrifuge tube. 12. Transfer the supernatant from step 10 to the tube containing the second binding buffer from step 11. 13. Pipette the mixture up and down to ensure that all the supernatant is transferred and thoroughly mixed with the second binding buffer. 14. Vortex the provided magnetic beads (e.g., magnetic beads selected from Table 1, 6 μL) before use, and then add them to the microcentrifuge tube from step 13, so that the magnetic beads bind to the target analyte below the target size to form a second bead-analyte complex. 15. Incubate the microcentrifuge tube with tilt rotation for 5 minutes and then place on a magnetic stand for 4 minutes to fix the second bead-analyte complex to the tube wall. 16. Without disturbing the second bead-analyte complex, pipette and discard the supernatant in the microcentrifuge tube. 17. Add a suitable wash buffer known to those skilled in the art (e.g., 800 uL) to the microcentrifuge tube and then rotate on a magnetic stand, 120 degrees each time, for a total of 720 degrees. After the rotation, pipette and discard the supernatant in the microcentrifuge tube without disturbing the second bead-analyte complex. 18. Repeat step 17. 19. Open the tube cap and allow the beads to dry on the magnetic rack for 15 minutes. 20. Remove the tube from the magnetic stand after drying. 21. Add a suitable elution buffer (eg, 40 μL) known to those skilled in the art directly to the second bead-analyte complex (in a microcentrifuge tube). 22. Use a pipette to resuspend the second bead-analyte complex by continuous stirring and then pipetting up and down 5 times. 23. Gently vortex the microcentrifuge tube for 10 seconds. 24. Incubate the microcentrifuge tube at room temperature for 3 minutes. 25. Place the microcentrifuge tube on the magnetic stand for 1 minute. 26. Carefully collect the supernatant containing the purified target analyte below the target size into a clean Maxi-Recovery tube without disturbing the beads. 27. Purified target analytes below target size are ready for immediate use or long term storage at -20°C or below. Performance Evaluation of Example Methods

[0142] The performance of the methods and kits disclosed below can be evaluated by following the following steps:

[0143] Several magnetic bead extraction kit compositions were prepared by varying the following components: Solution B (first ATPS) Polymer Salt Surfactants Solution C (Second ATPS) Polymer Salt Magnetic beads Binding buffer o Dispersants Polymer ·Grading buffer o Dispersants Polymer

[0144] Prepare sample solutions for evaluation and spike with known amounts of target DNA.

[0145] Extractions were performed using a variation of the magnetic bead extraction kit prepared in step 1 above as well as an industry standard extraction kit using their designated protocols.

[0146] Quantify target DNA using standard qPCR or ddPCR procedures. Table III: Example ATPS#1, ATPS#2 and binding buffer.

[0147] In various exemplary embodiments, solution B, solution C, binding buffer, and fractionation buffer are selected from the embodiments shown below in various combinations. Example 3

[0148] In some embodiments, a method for concentrating and purifying at least one target analyte from a clinical biological sample is provided, the method comprising the following steps: (a) combining the clinical biological sample with a first aqueous two-phase system (ATPS) composition to form a target enriched phase solution and a target lean phase solution, the 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; (b) collecting the target enriched phase; (c) optionally adding the target enriched phase to a second ATPS composition to form a second target enriched phase solution and a second target lean phase solution, and collecting the second target enriched phase, the second ATPS composition comprising a solution (d) optionally mixing the target enriched phase from step (b) or the second target enriched phase from step (c) with a binding buffer to form a mixed solution; (e) contacting the target enriched phase from step (b), the second target enriched phase from step (c) or the mixed solution from step (d) with a solid phase medium, the solid phase medium being configured to selectively bind the target analyte, so that the solid phase medium binds the target analyte; and (f) eluting and collecting the target analyte from the solid phase medium with an elution solution to obtain a final solution containing a concentrated and purified target analyte.

[0149] In some embodiments, the method further comprises the step of washing the solid phase medium with one or more appropriate solvents to remove impurities after step (e) and before step (f).

[0150] In some embodiments, the suitable solvent is a binding buffer.

[0151] In some embodiments, the method further comprises the step of treating the clinical biological sample with a lysis composition prior to step (a).

[0152] In some embodiments, the binding buffer comprises a dispersant comprising an anion selected from the group consisting of thiocyanate, isothiocyanate, perchlorate, acetate, trichloroacetate, trifluoroacetate, chloride, or iodide.

[0153] In some embodiments, the binding buffer comprises a discrete 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.

[0154] In some embodiments, the binding buffer comprises the dispersant at a concentration of 2M to 7M.

[0155] In some embodiments, the binding buffer further comprises a polymer at a concentration of 5-20% (w / v).

[0156] In some embodiments, the clinical biological sample is blood, plasma, urine, saliva, feces, cerebrospinal fluid (CSF), lymph fluid, serum, sputum, peritoneal fluid, sweat, tears, nasal swab, vaginal swab, endocervical swab, semen, or breast milk.

[0157] In some embodiments, the clinical biological sample is <500 mL.

[0158] In some embodiments, the target analyte is selected from the group consisting of nucleic acids, proteins, antigens, biomolecules, sugar moieties, lipids, sterols, exosomes, and any combination thereof.

[0159] In some embodiments, the target analyte is a nucleic acid, and the nucleic acid is gDNA, cDNA, plasmid DNA, mitochondrial DNA, cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), circulating fetal DNA, microbial cell-free DNA, microRNA (miRNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), circular RNA, long noncoding RNA (lncRNA), or a combination thereof.

[0160] In some embodiments, the nucleic acid is cfDNA, fetal free DNA, mitochondrial DNA, microbial free DNA, or ctDNA.

[0161] In some embodiments, step (e) comprises the following steps: (i) contacting a portion of the mixed solution with the solid phase medium so that the target analyte binds to the solid phase medium to form a solid phase extraction complex; (ii) disturbing the solid phase extraction complex and discarding the flow-through liquid (also called "supernatant"); and (iii) optionally repeating steps (i) and (ii).

[0162] In some embodiments, a method or claim 16 is provided wherein the solid medium is a plurality of beads.

[0163] In some embodiments, the beads are selected from the group consisting of magnetic beads, silica-based beads, carboxyl beads, hydroxyl beads, and amine-coated beads.

[0164] In some embodiments, the solid phase extraction complex is a bead-analyte complex; the perturbation is rotation; and the flow-through is supernatant.

[0165] In some embodiments, 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; and the elution solution is a fractionation buffer which, when in contact with the beads during the elution step (f), causes the target analyte to be released without releasing the other nucleic acids, thereby obtaining a final solution containing one or more concentrated and purified target analytes.

[0166] In some embodiments, the grading buffer comprises a polymer, a dispersion agent, or any combination thereof.

[0167] In some embodiments, the solid phase medium is an extraction column.

[0168] In some embodiments, the extraction column is a spin column.

[0169] In some embodiments, step (e) comprises the following steps: (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 the above steps (i) and (ii) one or more times until all of the mixed solution passes through the extraction column.

[0170] In some embodiments, the salt includes 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.

[0171] In some embodiments, the salt includes 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.

[0172] In some embodiments, the polymer is selected from the group consisting of polyethers, polyimines, polyacrylates, polyalkylene glycols, vinyl polymers, alkoxylated surfactants, polysaccharides, alkoxylated starches, alkoxylated celluloses, alkyl hydroxyalkyl celluloses, polyether-modified silicones, polyacrylamides, polyacrylic acids, and copolymers thereof. In some embodiments, the polymer is hydrophobically modified or silicone-modified.

[0173] 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), polyvinyl pyrrolidone, 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.

[0174] 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), polyvinyl pyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, polyvinyl methyl ether, and poly-N-isopropylacrylamide.

[0175] 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.

[0176] In some embodiments, the polymer has an average molecular weight of 200-1,000Da, 200-35,000Da, 425-2,000Da, 400-35,000Da, 980-12,000Da, or 3,400-5,000,000Da. 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.

[0177] 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, sulfonate, petroleum sulfonate, alkylbenzene sulfonate, naphthalene sulfonate, olefin sulfonate, alkyl sulfate, sulfate, sulfated natural oil, sulfated natural fat, sulfated ester, sulfated alkanolamide, sulfated alkylphenol, ethoxylated alkylphenol or sodium N-lauroyl sarcosinate (NLS); the nonionic surfactant is an ethoxylated fatty alcohol, a polyoxyethylene surfactant, a carboxylate, Polyethylene glycol ester, sorbitan ester, fatty acid glycol ester, carboxamide, monoalkanolamine condensate or polyoxyethylene fatty acid amide; the cationic surfactant is a quaternary ammonium salt, an amine with an amide bond, a polyoxyethylene alkylamine, a polyoxyethylene alicyclic amine, n,n,n',n'tetrasubstituted ethylenediamine or 2-alkyl 1-hydroxyethyl 2-imidazoline; and the amphoteric surfactant is n-coconut 3-aminopropionic acid or its sodium salt, n-tallow 3-iminodipropionic acid or its disodium salt, n-carboxymethyl n-dimethyl n-9 octadecenyl ammonium hydroxide or n-coconut amide ethyl n-hydroxyethyl glycine or its sodium salt.

[0178] 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-lauroyl sarcosinate (NLS), hexadecyltrimethylammonium bromide or span 80.

[0179] 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.

[0180] In some embodiments, the salt component includes 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, tetramethylammonium chloride, tetrabutylammonium chloride, tetramethylammonium hydroxide and tetrabutylammonium hydroxide.

[0181] In some embodiments, the polymer of the first ATPS composition or the second ATPS composition is dissolved in an aqueous solution at a concentration of 0.5-80% (w / v).

[0182] In some embodiments, the polymer of the first ATPS composition or the second ATPS composition is dissolved in an aqueous solution at a concentration of 0.2-50% (w / v).

[0183] In some embodiments, the salt component of the first ATPS composition or the second ATPS composition is dissolved in an aqueous solution at a concentration of 0.1% to 80% (w / v).

[0184] In some embodiments, the surfactant of the first ATPS composition or the second ATPS composition is dissolved in an aqueous solution at a concentration of 0.1%-50% (w / v).

[0185] 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.

[0186] In some embodiments, the first ATPS composition or the second ATPS composition is a polymer-salt system; the polymer is dissolved in an aqueous solution at a concentration of 0.5%-80% (w / v); and the salt component is dissolved in an aqueous solution at a concentration of 0.1%-80% (w / v).

[0187] In some embodiments, the polymer of the first ATPS composition is dissolved in an aqueous solution at a concentration of 5%-80% (w / v), and the salt component of the first ATPS composition is dissolved in an aqueous solution at a concentration of 0.1%-80% (w / v); and the polymer of the second ATPS composition is dissolved in an aqueous solution at a concentration of 0.5%-30% (w / v), and the salt component of the second ATPS composition is dissolved in an aqueous solution at a concentration of 5%-60% (w / w).

[0188] In some embodiments, the first ATPS composition or the second ATPS composition further comprises at least one salt at a concentration of 0.01%-10% (w / v), or at least one surfactant at a concentration of 0.01%-10% (w / v).

[0189] In some embodiments, the polymer of the first ATPS composition is a polyether, a polyimine, a polyacrylate, a polyalkylene glycol, a vinyl polymer, an alkoxylated surfactant, a polysaccharide, an alkoxylated starch, an alkoxylated cellulose, an alkyl hydroxyalkyl 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, (N H4)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, tetramethylammonium hydroxide or tetrabutylammonium hydroxide ammonium hydroxide; the polymer of the second ATPS composition is a polyether, a polyimine, a polyacrylate, a polyalkylene glycol, a vinyl polymer, an alkoxylated surfactant, a polysaccharide, an alkoxylated starch, an alkoxylated cellulose, an alkyl hydroxyalkyl 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, BaCO3, NH4OH, tetramethylammonium chloride, tetrabutylammonium chloride, tetramethylammonium hydroxide or tetrabutylammonium 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 dissolved in the aqueous solution at a concentration of 0.2-50% (w / v).

[0190] In some embodiments, the first ATPS composition or the second ATPS composition further comprises at least one salt at a concentration of 0.01%-10% (w / v), or at least one surfactant at a concentration of 0.01%-10% (w / v).

[0191] 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 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 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 carboxamide, a monoalkanolamine condensate or a polyoxyethylene fatty acid amide; the cationic surfactant is a quaternary ammonium salt, an amine with 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-coconut 3-aminopropionic acid or its sodium salt, n-tallow 3-iminodipropionic acid or its disodium salt, n-carboxymethyl n-dimethyl n-9 octadecenyl ammonium hydroxide or n-coconut amide ethyl n-hydroxyethyl glycine or its sodium salt.

[0192] 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 dissolved in an aqueous solution at a concentration of 0.1%-50% (w / v).

[0193] In some embodiments, the first ATPS composition or the second ATPS composition further comprises at least one salt at a concentration of 0.01%-30% (w / v).

[0194] 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.

[0195] In some embodiments, the polymer of the first ATPS composition is 6%-40% PEG 600 (w / v); and the salt component of the first ATPS composition is 0.1%-12% K2HPO4 (w / v) and 0.1%-20% KH2PO4 (w / v).

[0196] In some embodiments, the polymer of the second ATPS composition is 2%-20% PEG (w / v), and the salt component of the second ATPS composition is 1%-50% K2HPO4 (w / v) and 0.1%-40% KH2PO4 (w / v).

[0197] In some embodiments, the polymer of the first ATPS composition is 6%-36% PEG 600 (w / v), and the salt component of the first ATPS composition is 2%-12% Na2SO4 (w / v), 0.01-0.1M Na2HPO4, and 0.01-0.1M NaH2PO4.

[0198] In some embodiments, the polymer of the second ATPS composition is 2%-12% PEG (w / v), and the salt component of the second ATPS composition is 5%-20% Na2SO4 (w / v), 0.01-0.1M Na2HPO4, and 0.01-0.1M NaH2PO4.

[0199] In some embodiments, the polymer of the first ATPS composition is 20%-40% PEG1000 (w / v), and the salt component of the first ATPS composition is 0.1%-8% K2HPO4 (w / v) and 0.1%-12% KH2PO4 (w / v).

[0200] In some embodiments, the polymer of the second ATPS composition is 3%-13% PEG (w / v), and the salt component of the second ATPS composition is 1%-5% K2HPO4 (w / v) and 0.1%-20% KH2PO4 (w / v).

[0201] In some embodiments, the polymer of the first ATPS composition is 10%-35% PEG 3000 (w / v), and the salt components of the first ATPS composition are 0.1%-6% K2HPO4 (w / v) and 0.1%-8% KH2PO4 (w / v).

[0202] In some embodiments, the polymer of the second ATPS composition is 5%-17% PEG (w / v), and the salt component of the second ATPS composition is 0.1%-12% K2HPO4 (w / v) and 0.1%-16% KH2PO4 (w / v).

[0203] In some embodiments, the polymer of the first ATPS composition is 10%-32% PEG 8000 (w / v), and the salt components of the first ATPS composition are 0.1%-6% K2HPO4 (w / v) and 0.1%-9% KH2PO4 (w / v).

[0204] In some embodiments, the polymer of the second ATPS composition is 2%-9% PEG (w / v), and the salt component of the second ATPS composition is 1%-30% K2HPO4 (w / v) and 1%-40% KH2PO4 (w / v).

[0205] In some embodiments, the polymer of the first ATPS composition is 40-60% PPG 425 (w / v), and the salt components of the first ATPS composition are 0.1%-5% K2HPO4 (w / v) and 0.1%-5% KH2PO4 (w / v).

[0206] In some embodiments, the polymer of the second ATPS composition is 5%-15% PEG (w / v), and the salt component of the second ATPS composition is 0.1%-20% K2HPO4 (w / v) and 0.1%-20% KH2PO4 (w / v).

[0207] In some embodiments, the polymer of the first ATPS composition is 10%-50% PEG-ran-PPG, which has 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% K2HPO4 (w / v) and 0.1%-12% KH2PO4 (w / v).

[0208] In some embodiments, the polymer of the second ATPS composition is 2%-12% PEG (w / v), and the salt component of the second ATPS composition is 0.1%-30% K2HPO4 (w / v) and 1%-40% KH2PO4 (w / v).

[0209] In some embodiments, the polymer of the first ATPS composition is 10%-32% PEG-ran-PPG, which has 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%-5% sodium citrate (w / v) and 0.1%-12% citric acid (w / v).

[0210] In some embodiments, the polymer of the second ATPS composition is 3%-13% PEG (w / v), and the salt component of the second ATPS composition is 0.1%-20% K2HPO4 (w / v) and 1%-36% KH2PO4 (w / v).

[0211] In some embodiments, the first ATPS composition comprises 8%-18% PEG 600 (w / v), 7%-20% K2HPO4 (w / v), and 0.1%-5% KH2PO4; and the second ATPS composition comprises 8%-25% PEG 200 (w / v), 40%-50% K2HPO4 (w / v), and 4%-16% KH2PO4.

[0212] 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).

[0213] 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).

[0214] 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).

[0215] 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).

[0216] 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).

[0217] 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).

[0218] 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).

[0219] 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).

[0220] 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).

[0221] 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).

[0222] In some embodiments, the first or second ATPS composition is a polymer-salt based ATPS composition comprising 10%-18% PEG 300 (w / v) and 37%-49% sodium citrate (w / v).

[0223] In some embodiments, the first or second ATPS composition is a polymer-salt based ATPS composition comprising 18%-25% PEG 300 (w / v) and 20%-37% sodium citrate (w / v).

[0224] 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).

[0225] 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).

[0226] 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).

[0227] 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).

[0228] In some embodiments, the first or second ATPS composition is a polymer-salt based ATPS composition comprising 16%-24% PPG 425 (w / v) and 5%-11% sodium polyacrylate 240,000 (w / v).

[0229] 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).

[0230] 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).

[0231] In some embodiments, the first or second ATPS composition is a polymer-salt based ATPS composition comprising 12%-21% PEG 8000 (w / v) and 11%-21% sodium carbonate (w / v).

[0232] In some embodiments, the first or second ATPS composition is a polymer-salt based ATPS composition comprising 21%-30% PEG 8000 (w / v) and 3%-11% sodium carbonate (w / v).

[0233] 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).

[0234] In some embodiments, the first or second ATPS composition is a polymer-salt based ATPS composition comprising 6%-11% PEG 8000 (w / v) and 7%-14% sodium polyacrylate 16,000 (w / v).

[0235] In some embodiments, the first or second ATPS composition is a polymer-salt based ATPS composition comprising 11%-15% PEG 8000 (w / v) and 2%-7% sodium polyacrylate 16,000 (w / v).

[0236] 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).

[0237] 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).

[0238] 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).

[0239] 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).

[0240] 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).

[0241] 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).

[0242] 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).

[0243] 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).

[0244] 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).

[0245] In some embodiments, the first or second ATPS composition is a polymer-salt based ATPS composition comprising 1%-5% PPG 425 (w / v) and 20%-30% sodium citrate (w / v).

[0246] In some embodiments, the first or second ATPS composition is a polymer-salt based ATPS composition comprising 6%-11% PPG 425 (w / v) and 9%-20% sodium citrate (w / v).

[0247] In some embodiments, the first or second ATPS composition is a polymer-salt based ATPS composition comprising 11%-15% PPG 425 (w / v) and 0.5%-9% sodium citrate (w / v).

[0248] 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).

[0249] 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).

[0250] 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).

[0251] 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).

[0252] In some embodiments, the first or second ATPS composition is a polymer-salt based ATPS composition comprising 18%-35% PEG-ran-PPG 12,000 Da (w / v) and 7%-14% sodium sulfate (w / v).

[0253] 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).

[0254] 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 Na2HPO4 / NaH2PO4.

[0255] 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).

[0256] 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).

[0257] 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).

[0258] 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).

[0259] 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).

[0260] 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).

[0261] 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).

[0262] 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).

[0263] 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).

[0264] 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).

[0265] 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).

[0266] 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).

[0267] 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).

[0268] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 16%-33% PEG 6000 (w / v) and 16%-33% dextran 3,400 Da (w / v).

[0269] 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).

[0270] 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).

[0271] 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).

[0272] 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).

[0273] 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).

[0274] 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).

[0275] 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).

[0276] 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).

[0277] 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).

[0278] 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).

[0279] 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).

[0280] 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).

[0281] 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).

[0282] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 0.1%-7% methylcellulose (w / v) and 14%-20% dextran 2,200,000 Da (w / v).

[0283] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 7%-14% methylcellulose (w / v) and 7%-14% dextran 2,200,000 Da (w / v).

[0284] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 14%-20% methylcellulose (w / v) and 0.1%-7% dextran 2,200,000 Da (w / v).

[0285] 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).

[0286] 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).

[0287] 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).

[0288] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 4%-21% PEG 600 (w / v) and 58%-70% PPG 400 (w / v).

[0289] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 21%-42% PEG 600 (w / v) and 36%-58% PPG 400 (w / v).

[0290] In some embodiments, the first or second ATPS composition is a polymer-polymer based ATPS composition comprising 42%-60% PEG 600 (w / v) and 4%-36% PPG 400 (w / v).

[0291] 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).

[0292] 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).

[0293] 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).

[0294] 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).

[0295] 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).

[0296] 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).

[0297] In some embodiments, the first or second ATPS composition is a micellar-based ATPS composition comprising 5%-17% (C2H4O) (4.5) C 14 H 22 O (Triton X-45) and 60%-90% (C2H4O) (9-10) C 14 H 22 O(Triton X-114).

[0298] In some embodiments, the first or second ATPS composition is a micellar-based ATPS composition comprising 10%-20% Triton X-100 (w / v).

[0299] In some embodiments, the first or second ATPS composition is a micellar-based ATPS composition comprising 20%-30% Triton X-100 (w / v).

[0300] In some embodiments, the first or second ATPS composition is a micellar-based ATPS composition comprising 30%-40% Triton X-100 (w / v).

[0301] In some embodiments, the first or second ATPS composition is a micellar-based ATPS composition comprising 5%-18% Triton X-114 (w / v).

[0302] In some embodiments, the first or second ATPS composition is a micellar-based ATPS composition comprising 18%-29% Triton X-114 (w / v).

[0303] In some embodiments, the first or second ATPS composition is a micellar-based ATPS composition comprising 29%-40% Triton X-114 (w / v).

[0304] In some embodiments, the first or second ATPS composition is a micellar-based ATPS composition comprising 3%-19% decyltetraethylene glycol ether (C10E4) (w / v).

[0305] In some embodiments, the first or second ATPS composition is a micellar-based ATPS composition comprising 19%-34% decyltetraethylene glycol ether (C10E4) (w / v).

[0306] In some embodiments, the first or second ATPS composition is a micellar-based ATPS composition comprising 34%-50% decyltetraethylene glycol ether (C10E4) (w / v).

[0307] In some embodiments, the first or second ATPS composition is a micellar-based ATPS composition comprising 1%-10% Pluronic F68 (MW 8,400Da, EO 82 -PO 31 -EO 82 )(w / v) and 14%-20% potassium phosphate (w / v).

[0308] In some embodiments, the first or second ATPS composition is a micellar-based ATPS composition comprising 10%-20% Pluronic F68 (w / v) and 7%-14% potassium phosphate (w / v).

[0309] In some embodiments, the first or second ATPS composition is a micellar-based ATPS composition comprising 20%-30% Pluronic F68 (w / v) and 1%-7% potassium phosphate (w / v).

[0310] In some embodiments, the first or second ATPS composition is a micellar-based ATPS composition comprising 5%-21% Pluronic 17R4 (MW 2,650 Da, PO 14 -EO 24 -PO 14 )(w / v) and 7%-10% potassium phosphate (w / v).

[0311] In some embodiments, the first or second ATPS composition is a micellar-based ATPS composition comprising 21%-42% Pluronic 17R4 (w / v) and 3%-7% potassium phosphate (w / v).

[0312] In some embodiments, the first or second ATPS composition is a micellar-based ATPS composition comprising 42%-60% Pluronic 17R4 (w / v) and 1%-3% potassium phosphate (w / v).

[0313] In some embodiments, the first or second ATPS composition is a micellar-based ATPS composition comprising 2%-21% Pluronic L-35 (MW 1,900Da, EO 10 -PO 16 -EO 10 )(w / v) and 14%-20% sodium sulfate (w / v).

[0314] In some embodiments, the first or second ATPS composition is a micellar-based ATPS composition comprising 21%-42% Pluronic L-35 (w / v) and 7%-14% sodium sulfate (w / v).

[0315] In some embodiments, the first or second ATPS composition is a micellar-based ATPS composition comprising 42%-60% Pluronic L-35 (w / v) and 1%-7% sodium sulfate (w / v).

[0316] In some embodiments, the first ATPS composition and / or the second ATPS composition is selected from Table 1.0.

[0317] In some embodiments, the first ATPS composition or the second ATPS composition further comprises 0.5-2 mM ethylenediaminetetraacetic acid (EDTA).

[0318] In some embodiments, the method further comprises a step of 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 PCR-based assays.

[0319] In some embodiments, the analyte of interest is a biomarker that indicates the presence or risk of a medical condition or disease in a patient, wherein the medical condition or disease is an infectious disease, cancer, or a genetic disease.

[0320] In some embodiments, the clinical biological sample is a large fluid sample with a volume >10 mL, and prior to step (a), the following steps are also included: dividing the large fluid sample into at least two samples of sample solution; wherein in step (a), each sample is individually combined with the first ATPS, and optionally, in step (c), the collected target enriched phase from step (b) is individually combined with the second ATPS to form a target enriched phase for each sample.

[0321] In some embodiments, the target enriched phase from each sample of step (b) or the second target enriched phase from each sample of step (c) are combined together to form a final target enriched phase for step (d).

[0322] In some embodiments, the bulk fluid sample is urine.

[0323] In some embodiments, a method for treating cancer or an infectious disease in a patient in need thereof is provided, the method comprising the following steps: (i) obtaining a clinical biological sample from the patient; (ii) concentrating 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 the information obtained from the target analyte indicates that the patient has cancer or is at risk of developing cancer.

[0324] 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 infectious disease is HPV.

[0325] In some embodiments, the target analyte is renal cFDNA or ctDNA, and wherein the cancer is a systemic cancer.

[0326] In some embodiments, the target analyte is urogenital tract cFDNA or ctDNA, and wherein the cancer is urogenital tract cancer.

[0327] In some embodiments, the target analyte is bladder cancer DNA and the medical condition or disease is bladder cancer.

[0328] In some embodiments, the target analyte is HPV viral RNA or HPV viral DNA and the medical condition or disease is HPV.

[0329] In some embodiments, a kit is provided, the kit comprising an ATPS composition comprising a polymer from any of the above embodiments, a salt component comprising at least one salt, a binding buffer, and a solid phase medium.

[0330] In some embodiments, a kit is provided, the kit comprising a first ATPS composition according to any of the above embodiments, a second ATPS composition, a binding buffer, and a solid phase medium.

[0331] In some embodiments, the polymer is dissolved in the aqueous solution at a concentration of 0.5%-80% (w / v); and the salt is dissolved in the aqueous solution at a concentration of 0.1%-80% (w / v).

[0332] In some embodiments, the first ATPS composition comprises the polymer dissolved in an aqueous solution at a concentration of 5%-80% (w / v) and the salt dissolved in an aqueous solution at a concentration of 0.1%-80% (w / v); and wherein the second ATPS composition comprises the polymer dissolved in an aqueous solution at a concentration of 0.5%-30% (w / v) and the salt dissolved in an aqueous solution at a concentration of 5%-60% (w / v).

[0333] In some embodiments, the concentration of the polymer is 0.5%-80% (w / v) of the first ATPS and / or the second ATPS. In some embodiments, the concentration of the polymer is 0.5%-30% (w / v) of the first ATPS and / or the second ATPS. In some embodiments, the concentration of the polymer is 5%-60% (w / v) of the first ATPS and / or the second ATPS. In some embodiments, the concentration of the polymer is 12%-50% (w / v) of the first ATPS and / or the second ATPS.

[0334] In some embodiments, the concentration of the salt is 0.1%-80% (w / v) of the first ATPS and / or the second ATPS. In some embodiments, the concentration of the salt is 5%-60% (w / v) of the first ATPS and / or the second ATPS. In some embodiments, the concentration of the salt is 0.1%-50% (w / v) of the first ATPS and / or the second ATPS. In some embodiments, the concentration of the salt is 0.1%-20% (w / v) of the first ATPS and / or the second ATPS. In some embodiments, the concentration of the salt is 0.01%-30% (w / v). In some embodiments, the concentration of the salt is 0.01%-10% (w / v) of the first ATPS and / or the second ATPS.

[0335] In some embodiments, the concentration of the surfactant is 0.1%-90% (w / v) or 0.1%-50% (w / v) of the first ATPS and / or the second ATPS. In some embodiments, the concentration of the surfactant is 0.01%-10% (w / v) of the first ATPS and / or the second ATPS.

[0336] 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).

[0337] In some embodiments, the second ATPS composition comprises at least one polymer at a concentration of 0.5%-30% (w / v) and at least one salt at a concentration of 5%-60% (w / v). In some embodiments, the second ATPS composition comprises at least one polymer at a concentration of 1%-6% (w / v) and at least one salt at a concentration of 10%-50% (w / v). In some embodiments, the second ATPS composition further comprises at least one surfactant at a concentration of 0.01%-10% (w / v).

[0338] In some embodiments, the first ATPS composition is a polymer-salt system comprising at least one polymer at a concentration of 0.5%-30% (w / v) and at least one salt at a concentration of 5%-60% (w / v). In some embodiments, the first ATPS composition comprises at least one polymer at a concentration of 1%-6% (w / v) and at least one salt at a concentration of 10%-50% (w / v). In some embodiments, the first ATPS composition further comprises at least one surfactant at a concentration of 0.01%-10% (w / v).

[0339] In some embodiments, the second ATPS composition comprises at least one polymer at a concentration of 5%-80% (w / v) and at least one salt at a concentration of 0.1%-80% (w / v). In some embodiments, the second ATPS composition comprises at least one polymer at a concentration of 5%-60% (w / v) and at least one salt at a concentration of 0.5%-50% (w / v). In some embodiments, the second ATPS composition comprises at least one polymer at a concentration of 12%-50% (w / v) and at least one salt at a concentration of 0.1%-20% (w / v). In some embodiments, the second ATPS composition further comprises at least one surfactant at a concentration of 0.01%-10% (w / v).

[0340] In some embodiments, the first ATPS composition is a polymer-polymer system comprising at least two polymers, and each polymer is dissolved in an aqueous solution at a concentration of 0.2%-50% (w / v). In some embodiments, the first ATPS composition further comprises at least one salt at a concentration of 0.01%-10% (w / v). In some embodiments, the first ATPS composition further comprises at least one surfactant at a concentration of 0.01%-10% (w / v).

[0341] In some embodiments, the first ATPS composition is a micellar system comprising at least two surfactants, and each surfactant is dissolved in an aqueous solution at a concentration of 0.1%-90% (w / v). In some embodiments, the first ATPS composition further comprises at least one salt at a concentration of 0.01%-30% (w / v).

[0342] In some embodiments, the polymer of the first ATPS composition or the second ATPS composition is dissolved in an aqueous solution at a concentration of 0.5%-80% (w / v), 0.5%-60% (w / v), 5%-80% (w / v), 5%-60% (w / v), 12%-50% (w / v) or 0.5%-30% (w / v).

[0343] In some embodiments, the salt component of the first ATPS composition or the second ATPS composition is dissolved in an aqueous solution at a concentration of 0.1%-80% (w / v), 0.5%-50% (w / v), 0.1%-20% (w / v), 5%-60% (w / v) or 10%-50% (w / v).

[0344] In some embodiments, the binding buffer comprises a 2M-7M guanidine solution. In some embodiments, the binding buffer further comprises a polymer at a concentration of 5%-20% (w / v).

[0345] In some embodiments, provided kits include an ATPS composition comprising a polymer from any of the above embodiments, a salt component comprising at least one salt, a binding buffer, and a solid phase medium.

[0346] Although the description refers to specific embodiments, this disclosure should not be construed as being limited to the embodiments set forth herein.

[0347] Other example embodiments are also discussed herein. Examples

[0348] The examples of some embodiments of the present disclosure are described in more detail herein. 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 given below and elsewhere in this application are hereby included herein by reference. Example workflow for concentration and purification of one or more target analytes from clinical biosamples

[0349] Provided herein is an example workflow for concentrating and purifying one or more target analytes (e.g., target nucleic acids) from clinical biological samples using an aqueous two-phase system (ATPS) and a solid phase medium. Table 1.0 summarizes example ATPS compositions for the first ATPS and / or second ATPS steps. These examples are for illustrative purposes only and are not intended to be an exhaustive list of all possible embodiments of the present invention. Table 1.0: Example ATPS compositions. Unless otherwise stated, all concentrations are shown as w / v ratios. Example 1a Bead purification

[0350] Reference now Figure 1A , which shows an example workflow of bead purification (also referred to in some embodiments as "bead purification", "magnetic bead workflow" or "magnetic bead purification") integrated with the previous ATPS step. In this embodiment, in the first optional lysis step 111, a suitable lysis buffer is added to the sample (e.g., blood, plasma, serum, cerebrospinal fluid, urine, saliva, feces, tears, sputum, nasopharyngeal mucus, vaginal secretions, or penile secretions) to lyse cells in the sample and release biomolecules into the lysis buffer solution. The lysed sample passes through two consecutive two-phase aqueous systems (ATPS) to separate and concentrate DNA.

[0351] In step 112, the lysed sample is mixed with the first ATPS. The mixture is vortexed vigorously and then centrifuged to separate into a top phase and a bottom phase. In some embodiments, the top phase is a target-enriched phase (also referred to as a "first target-enriched phase"). In some embodiments, the bottom phase is a target-enriched phase containing a target analyte.

[0352] In step 113, all target enrichment phases from the DNA partitioning in step 112 are transferred to the second ATPS (2 ndATPS) and mix thoroughly. The mixture is vortexed and then centrifuged to separate it into a top phase and a bottom phase. In some embodiments, the top phase is a second target enriched phase containing the target analyte. In some embodiments, the bottom phase is a second target enriched phase containing the target analyte.

[0353] In step 114, the second target enrichment phase of the target DNA distribution from step 113 is extracted into an empty microcentrifuge tube. Binding buffer (such as those described in Examples 2a-5c below) is added to the tube and fully mixed with the top phase from step 113 and multiple beads (such as those described in Table 1.1), which are also added to the tube to form a solid phase extraction complex (i.e., the beads-analyte complex in this example). The mixture containing the beads-analyte complex is incubated for a certain period of time, then centrifuged and the beads-analyte complex is fixed. The supernatant is discarded without interfering with the beads-analyte complex. In certain embodiments, the beads-analyte complex is fixed to the tube wall by a magnetic rack.

[0354] In step 115, the bead-analyte complex is further purified by washing with an appropriate solvent and discarding the supernatant containing impurities. In some embodiments, the appropriate solvent is a binding buffer or a wash buffer. Step 115 is optionally repeated.

[0355] In step 116, the bead-analyte complex is then dried with the lid open. The bead-analyte complex is then resuspended in a suitable elution solution (e.g., elution buffer) and mixed thoroughly to release the beads from the bead-analyte complex. The beads are then immobilized and the final solution containing the purified target DNA is collected for further use. Table 1.1: Examples of magnetic beads. Example 1b Column purification

[0356] Reference now Figure 1B , which shows an example workflow of column purification integrated with the previous ATPS step (also referred to as "spin column purification" or "spin column workflow" in some embodiments). In this embodiment, in the first optional lysis step 121, a suitable lysis buffer is added to the sample (e.g., blood, plasma, serum, cerebrospinal fluid, urine, saliva, feces, tears, sputum, nasopharyngeal mucus, vaginal secretions, or penile secretions) to lyse cells in the sample and release biomolecules into the lysis buffer solution. The lysed sample passes through two consecutive two-phase aqueous systems (ATPS) to separate and concentrate DNA.

[0357] In step 122, the lysed sample is mixed with the first ATPS. The mixture is vortexed vigorously and then centrifuged to separate into a top phase and a bottom phase. In some embodiments, the top phase is a target-enriched phase (also referred to as a "first target-enriched phase"). In some embodiments, the bottom phase is a target-enriched phase containing a target analyte.

[0358] In step 123, all target enrichment phases from the DNA partitioning in step 122 are transferred to the second ATPS (2 nd ATPS) and mix thoroughly. The mixture is vortexed and then centrifuged to separate it into a top phase and a bottom phase. In some embodiments, the top phase is a second target enriched phase containing the target analyte. In some embodiments, the bottom phase is a second target enriched phase containing the target analyte.

[0359] In step 124, the second target enriched phase (also referred to as "second target enriched phase") of the target DNA distribution from step 123 is extracted into an empty microcentrifuge tube. A binding buffer (such as those described in Examples 2a-5c below) is added to the tube and mixed thoroughly with the top phase from step 123 to form a mixed solution. The mixed solution is transferred to a centrifuge column to form a solid phase extraction complex, which is then centrifuged. The flow-through liquid (also referred to as "supernatant") of the centrifuge column is discarded. Step 124 is repeated until all samples pass through the centrifuge column.

[0360] In step 125, the solid phase extraction complex is further purified by washing with an appropriate solvent and discarding the supernatant containing impurities. In some embodiments, the appropriate solvent is a binding buffer or a wash buffer. Step 125 is optionally repeated.

[0361] In step 126, the solid phase extraction complex is then dried by centrifugation. A suitable elution buffer is added to the solid phase extraction complex, which is then incubated for a certain period of time to form a final solution. The final solution containing the purified target DNA is collected into a collection tube by centrifuge for further use. Example 2a: Comparison of the use of magnetic beads to extract DNA from blood with and without a previous aqueous two-phase system (ATPS) step Efficiency of DNA recovery from plasma

[0362] In this example, the efficiency of recovering DNA from plasma using magnetic beads (also referred to as "magnetic beads") is compared (i) with a prior phase separation using an ATPS system (also referred to as an "ATPS step," "ATPS extraction," or "ATPS workflow") according to the methods of the present disclosure; and (ii) without using a prior ATPS step.

[0363] Material

[0364] In this example, mixed gender pooled human plasma, citrated (Cat. No. PR-100) was purchased from TCS Biosciences Ltd. The aqueous two-phase system (ATPS) includes a polymer-salt based ATPS component (also referred to as a "polymer-salt system"). The binding buffer consists of 1-5 M guanidine salt, Tris-HCl, and EDTA. Unless otherwise stated, in the DNA recovery test, 145 bp DNA oligonucleotides and / or 2000 bp DNA oligonucleotides were used as spikes. All other chemicals were analytical grade.

[0365] TaqMan for PCR TM Fast Advanced Master Mix (2X) and Custom TaqMan TM Assay provided by ThermofisherScientific. Custom TaqMan TM The assay consisted of the following: forward primer, reverse primer, and FAM dye-labeled TaqMan MGB probe.

[0366] Plasma cell lysis

[0367] Spike 100fg 145bp DNA + 1.5uL 2000bp DNA (TATAA) stock solution into each 2mL human pooled plasma. Add 160uL of appropriate lysis buffer and 60uL proteinase K (28.5mg / mL) into each 2mL spiked plasma. Vortex the mixture thoroughly and then lyse in a preheated 60°C heating block for 15 minutes.

[0368] Extraction process

[0369] One set of lysed plasma samples (sample #1) was treated with two consecutive ATPS steps (also referred to as "double ATPS", "two-step ATPS" or "first and second ATPS" in some embodiments) to isolate and concentrate DNA prior to magnetic bead purification, while the other sets of lysed plasma (samples #2 and #3) were directly subjected to magnetic bead purification without a prior ATPS step.

[0370] For sample #1, ATPS 67 described in Table 1.0 is used as the first ATPS, and ATPS 94 described in Table 1.0 is used as the second ATPS. The sample #1 of 2.22mL cracking is transferred to 1.170mL first ATPS and vortex mixed. The mixture is centrifuged at 2,300rcf for 6 minutes. All salt-rich bottom phases (about 1mL) (also referred to as "target enriched phase" in some embodiments) are transferred to 265uL second ATPS and fully mixed. Then the mixture is centrifuged at 7,000rcf for 1 minute. The top phase (about 150uL) (also referred to as "second target enriched phase" in some embodiments) rich in polymer is carefully extracted into an empty microcentrifuge tube for further purification.

[0371] DNA purification

[0372] 800uL of binding buffer was added to the extracted top phase of sample #1 and the lysed sample #2 without ATPS, respectively. For sample #3, 11.43mL of binding buffer was added to the lysed sample #3, so that the ratio of lysed sample: binding buffer was the same as the ratio of top phase: binding buffer in sample #1. Samples #1, #2, and #3 are summarized in Table 1.2 below. 12uL of magnetic beads was added to each tube. The mixture was then incubated on a rotator for 5 minutes to prevent the beads from settling. The tube was then briefly centrifuged to settle and placed on a magnetic stand for 2 minutes to fix the beads to the tube wall. The supernatant was discarded without disturbing the beads. 800uL of binding buffer was added to each tube and the tube was slowly rotated 720° on the magnetic stand for a total of 720°. Pipette again and discard the supernatant. 800μL of wash buffer (70% ethanol, 0.001M EDTA, 0.01MTris-HCl) was added to the sample and the tube was rotated 720° on the stand for a total of 720°. The supernatant was discarded. The washing step was performed twice. To improve the drying effect, the tube was briefly centrifuged and settled using a benchtop microcentrifuge with the hinge facing outward to collect any remaining wash buffer. The beads were then dried on a magnetic stand with the lid open for 7 minutes. The bead complex was resuspended in 40 μL elution buffer (0.01M Tris-HCl, 0.001M EDTA) by continuous pipetting mixing and then gently vortexed. The tube was then placed on a magnetic stand 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 used for detection. Table 1.2: Summary of experimental conditions for plasma extraction and purification.

[0373] DNA testing

[0374] 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 (per reaction) consisted of 5 μL TaqMan Fast Advanced Master Mix (Applied Biosystems, Ref: 4444557), 0.5 μL 20x custom premixed custom oligonucleotide PSI-145FAM Dental, 0.4 μL Universal Spike II Primer (TATAA, DS25SII), 0.2 μL Universal Spike II Probe (TATAA, DSSII), 1.9 μL ultrapure water.

[0375] 8 μL of PCR reaction mixture was dispensed into each PCR well and 2 μL of purified DNA sample was added. qPCR was performed using QuantStudio TM 3 Real-time PCR system (Thermofisher Scientific). Thermal cycling consisted of 50°C for 2 min, then 95°C for 2 min, and finally 40 cycles of 95°C for 1 sec and 60°C for 20 sec. QuantStudio TM The design and analysis software determined the cycle threshold (CT) where the fluorescence signal of the amplification reaction was above background fluorescence. Data analysis of the raw CT was performed in Excel and GraphPad Prism.

[0376] All results are expressed as average CT values. The lower the average CT value, the higher the amount of target DNA in the extracted sample, and the higher the CT value, the lower the amount of target DNA in the extracted sample.

[0377] result

[0378] DNA recovery

[0379] Reference now Figure 2A-B, shows the average CT values ​​of 145bp and 2000bp DNA oligonucleotides recovered from plasma purified using magnetic beads (sample #1: using a dual ATPS step, sample #2: without an ATPS step and unscaled binding buffer volume, and sample #3: without an ATPS step and scaled binding buffer volume, according to Table 1.2). The results are summarized in Table 1.3. For 145bp DNA, plasma sample #1 had a high recovery (average CT value of 27.21), but plasma sample #2 had a low recovery (average CT value of 34.83). For sample #3, the volume of binding buffer was increased proportionally, but the recovery (average CT value of 32.07) was only slightly improved compared to sample #2, but was still significantly lower than the recovery of sample #1. Similar results were observed for 2000bp DNA recovery. The results show that when ATPS is added during the extraction process before the magnetic bead binding step, the recovery of 145bp and 2000bp DNA oligonucleotides is significantly improved. Surprisingly, the significant improvement in DNA recovery is still seen even when the volume of binding buffer is proportionally increased compared to samples without the ATPS step. Table 1.3: qPCR results of 145 bp and 2000 bp DNA oligonucleotides recovered from plasma using magnetic beads with or without two-step ATPS.

[0380] Overall, the recovery results show that, surprisingly, higher DNA recoveries can be achieved when ATPS is incorporated into the plasma extraction workflow prior to magnetic bead purification, even though additional steps of ATPS concentration and separation are involved (a potential source of target analyte loss during sample processing). By significantly improving DNA recovery using ATPS prior to magnetic bead purification, smaller amounts of magnetic beads are required to shift the binding equilibrium to achieve better yields, thereby reducing the amount of magnetic beads (which are expensive) and hazardous binding buffers required to achieve effective purification. Example 2b: Plasma extraction performance with different polymers and salts in ATPS compositions

[0381] In this example, the ability to recover DNA from plasma samples using ATPS with different polymer molecular weights and component concentrations in combination with magnetic beads, as well as the ability to recover DNA from plasma samples using ATPS with different polymer and salt chemistries in combination with magnetic beads, was tested.

[0382] The reagents used in this example are similar or identical to those discussed in the previous examples.

[0383] Plasma cell lysis

[0384] To digest unwanted proteins and cells in plasma, 160 μL of appropriate lysis buffer and 60 μL of proteinase K (28.57 mg / mL) were added to 2 mL of aliquot of citrate (TCS) plasma. 100 fg of 145 bp DNA and 100 ng of GR1kb+ standard were spiked into the above sample. After vortexing for 15 seconds, the sample was incubated at 60°C for 15 minutes. The lysate was used for further extraction.

[0385] Two-phase system

[0386] The extraction procedure involves two consecutive aqueous two-phase systems (ATPS), including a first ATPS and a second ATPS composition for separation, purification and concentration of DNA from plasma samples. In the first ATPS, DNA is partitioned into the bottom phase (also called the "target-rich phase") and protein is partitioned into the top phase (also called the "target-poor phase"). About 1 mL of the bottom phase is carefully extracted and transferred to the second ATPS. DNA is partitioned into the top phase of the second ATPS (also called the "second target-rich phase"), and the top phase is then carefully extracted for further purification and detection.

[0387] In this example, the second ATPS composition was varied from the corresponding first ATPS composition to achieve a small top phase volume of approximately 150 μL.

[0388] Extraction process

[0389] In this example, an ATPS-based separation and purification method for cfDNA is introduced. 2.22 mL of lysate was added to the first ATPS. A dye was added to visualize the two phases, resulting in a colored top phase. In this example, the vast majority of DNA was partitioned into the bottom phase, while proteins were partitioned into the top phase. The solution was made turbid by vigorous vortexing for 15 seconds. Phase separation was then promoted by centrifugation at 2300 rcf for 6 minutes. After phase separation, the volume of the bottom phase was about 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 produce a turbid solution and centrifuged at 7000 rcf for 1 minute to achieve phase separation. The volume of the top phase in the resulting solution was about 150 μL, which was then carefully extracted for further purification and detection of DNA.

[0390] DNA purification

[0391] Purification of DNA was accomplished by magnetic bead extraction. The top 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 with tilt rotation for 5 minutes. The tube was then briefly centrifuged to settle and placed on a magnetic stand for 2 minutes to fix the beads to the tube wall. The supernatant was pipetted and discarded without disturbing the magnetic beads. 800 μL of binding buffer was added to the sample. The tube was rotated on the stand for a total of 720°. The supernatant was pipetted and discarded. 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 on the stand for a total of 720°. The supernatant was pipetted and discarded. Wash twice and place the tube on the stand with the lid open to dry the beads. Add 40 μL of elution buffer (0.01M Tris-HCl, 0.001M EDTA) to the sample. Resuspend the bead complex by continuous pipetting and then vortex gently. Incubate the sample at room temperature for 3 minutes and centrifuge briefly to settle. Then place the tube on the magnetic stand for 1 minute. Carefully collect the supernatant without disturbing the magnetic beads used for detection.

[0392] Extraction efficiency

[0393] Using a second ATPS after the first ATPS can further concentrate the DNA for detection. Both the first and second ATPS contain a mixture of polymer and salt, where the resulting top phase is rich in polymer and the bottom phase is rich in salt.

[0394] In this example, the extraction efficiency of DNA in different ATPS compositions was studied while keeping the volume ratio of target-rich phase to target-poor phase in the first and second ATPS constant. To evaluate the DNA recovery performance, a sample containing 100 ng of DNA standard in 2 mL of human plasma was prepared, assuming a 100% recovery, which is 2.5 ng per μL of eluate.

[0395] DNA testing

[0396] The steps for DNA testing of each sample are the same or similar to those discussed above with respect to Example 1a. For the sake of brevity and simplicity of this disclosure, the discussion of the testing steps will not be repeated here. The results are expressed as average CT values.

[0397] result

[0398] Reference now Figure 2C, comparing the average CT values ​​for the recovery of 145 bp DNA using the first and second ATPS compositions according to 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 their respective 145 bp DNA recovery results are shown in Table 1.4. The results show that all systems with different polymer molecular weights in the first ATPS can recover detectable levels of DNA from plasma when combined with the magnetic bead workflow. Table 1.4: Recovery of DNA from plasma using ATPS and magnetic beads with different polymer molecular weights.

[0399] Reference now Figure 2D , comparing the average CT values ​​for the recovery of 145 bp DNA using the first and second ATPS compositions according to conditions 9, 10, and 11 in Table 1.5. Conditions 9, 10, and 11 correspond to three different polymer and salt combinations, and their respective 145 bp DNA recovery results are reported in Table 1.5. The results show that all systems with various polymer and salt combinations in the first or second ATPS recovered satisfactory DNA levels (e.g., sufficient for downstream use, such as for detection purposes in diagnostic applications). Table 1.5: Recovery of DNA from plasma using ATPS with different polymer and salt combinations and magnetic beads.

[0400] In summary, for systems with the same polymer and salt type but different polymer sizes (conditions 5, 7, 6 and 8, Table 1.4), satisfactory DNA amounts were recovered at 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), the recovery of DNA was also satisfactory. Example 2c: Performance of plasma extraction using various ATPS systems and magnetic beads

[0401] In this example, the performance of DNA extraction from plasma using different classes of ATPS systems (polymer-salt, polymer-polymer, micelles) in combination with magnetic beads is demonstrated. The materials used in this example are the same 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.

[0402] Plasma lysis

[0403] In this example, K2 EDTA plasma (TCS Biosciences, lot 23014500) was lysed according to protocols optimized for their respective downstream ATPS systems. The samples were spiked with 100 fg 145 bp DNA and 1.5 uL stock 2000 bp DNA.

[0404] For polymer-polymer based ATPS system, 500 uL of appropriate lysis buffer and 30 uL of proteinase 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.

[0405] For micelle-based ATPS system, 300uL of appropriate lysis buffer was added to 1mL K2 EDTA plasma, vortexed thoroughly and incubated at room temperature for 3 minutes. Then 112.5uL protein precipitation buffer A (PPt A) (20% ZnCl w / v, 1% acetic acid w / v) was added, vortexed thoroughly until homogeneous, and centrifuged at 12000rcf for 3 minutes. Other suitable protein precipitation buffers can be used in place of protein precipitation buffer A in the above method. The supernatant was extracted for further downstream processing, while the precipitate was discarded.

[0406] For polymer-salt based ATPS systems, add 80 uL of appropriate lysis buffer and 30 uL of proteinase K (28.57 mg / mL) to 1 mL of K2 EDTA plasma, vortex thoroughly and incubate in a preheated 60 °C water bath for 15 minutes.

[0407] Extraction process

[0408] Different kinds of aqueous two-phase systems (ATPS) were prepared to efficiently extract cfDNA from plasma samples. Table 1.6 summarizes the three different kinds of ATPS and the subsequent binding steps. Table 1.6: Summary of experimental conditions with alternative ATPS classes.

[0409] The sample lysate was added to its respective ATPS system: 1530uL of sample lysate was added to 500uL of polymer-polymer based system, and 1110uL of sample lysate was added to 1.17mL of the first polymer-salt based system. For the micellar system, 1200uL of extracted supernatant after protein precipitation was added to 500mg of the micellar system. In some embodiments, the first ATPS system is a single ATPS system, which is not followed by a second ATPS.

[0410] All ATPS tubes were vortexed thoroughly after adding sample lysates and vortexed thoroughly.

[0411] The polymer-polymer ATPS system was centrifuged at 2300 rcf for 6 minutes. The target cfDNA was strongly partitioned into the polymer-rich bottom phase. All of the bottom phase (approximately 300 uL) was extracted and transferred to a new 2 mL centrifuge tube for further processing.

[0412] The micellar ATPS system was centrifuged at 12000 rcf for 5 minutes. The target cfDNA was strongly partitioned into the surfactant-rich top phase. All of the top phase (approximately 280 uL) was extracted and transferred to a new 2 mL centrifuge tube for further processing.

[0413] For the polymer-salt ATPS system, the extraction process is the same or similar to the method discussed in the previous example. The first polymer-salt ATPS system was centrifuged at 2300rcf for 6 minutes. The target cfDNA was strongly partitioned into the salt-rich bottom phase. All of the bottom phase (approximately 1000uL) was extracted and transferred to a second ATPS tube containing 265uL of the second ATPS composition, which was vortexed thoroughly and then centrifuged at 7000rcf for 1 minute. The target cfDNA was partitioned and concentrated into a polymer-rich top phase of approximately 120uL. It was then extracted and transferred to a new tube for further processing.

[0414] DNA purification

[0415] After extracting the phase containing the target cfDNA from its respective ATPS system, downstream processing includes the addition of solid phase media and binding buffer for separating the nucleic acids from the liquid medium, followed by washing away unwanted ions and molecules before the target nucleic acids are eluted by elution buffer.

[0416] 1500uL binding buffer (3-7M guanidine, 50mM pH 7 Na2HPO4 / NaH2PO4) was added to the extracted polymer-rich bottom phase of the polymer-polymer system. 500uL binding buffer (3-7M guanidine) was added to the extracted surfactant-rich top phase of the micelle system. 800uL binding buffer (3-7M guanidine) was added to the extracted polymer-rich top phase of the second polymer-salt ATPS system. 6uL magnetic beads (MagQu) were then added to all centrifuge tubes containing the extraction phase and binding buffer mixture and placed on a rotator for incubation for 5 minutes. During the incubation, the target cfDNA binds to the magnetic beads. After incubation, all tubes were briefly centrifuged and then placed on a magnetic stand, where the applied magnetic field attracted the cfDNA-bound magnetic beads. The supernatant was then taken and discarded, leaving the magnetic beads bound to the inner wall of the centrifuge tube. A second binding buffer wash step was performed for the polymer-polymer and polymer-salt systems. Add 800uL of binding buffer (3-7M guanidine, 50mM pH7 Na2HPO4 / NaH2PO4) to the magnetic beads from the polymer-polymer system, and add 800uL of binding buffer (3-7M guanidine) to the magnetic beads from the polymer-salt system. Then vortex the centrifuge tube thoroughly and centrifuge briefly before returning it to the magnetic stand. Once all the magnetic beads are bound to the inside of the centrifuge wall, discard the supernatant.

[0417] 800uL of original wash buffer (70% v / v EtOH, 1mM EDTA, 10mM Tris-HCl) was added to the magnetic beads from the polymer-polymer and polymer-salt systems, while 500uL of RPE wash buffer (80% v / v EtOH, 0.1M NaCl, 10mM 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 stand. Repeat the procedure for a second wash. The centrifuge tube was then briefly centrifuged and placed back on the magnetic stand, 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.

[0418] After the beads have dried, resuspend the beads in 20uL of elution buffer (10mM Tris-HCl, 1mM EDTA) and incubate at room temperature for 3 minutes. Elute the target cfDNA from the beads into the elution buffer. Then place the tube back on the magnetic stand and extract the elution buffer once all the beads are bound.

[0419] DNA testing

[0420] The steps for DNA testing of each sample are the same or similar to those discussed above with respect to Example 2a. For the sake of brevity and simplicity of this disclosure, the discussion of the testing steps will not be repeated here. The results are expressed as average CT values.

[0421] result

[0422] distribute

[0423] The first ATPS from polymer-polymer (PP) and polymer-salt (PS) based classes effectively partitioned the target free DNA (cfDNA) from the lysed sample into the bottom phase of the first ATPS, while the unwanted excess protein partitioned into the top phase of the ATPS system. The opposite trend was seen in the micellar ATPS system, where cfDNA partitioned into the top phase. The PS second ATPS was used to condense the large bottom phase of the first ATPS into a more concentrated smaller top phase volume for more user-friendly downstream processing.

[0424] DNA recovery

[0425] Reference now Figure 2E-2F , shows the average CT values ​​for the recovery of 145bp DNA and 2000bp DNA from plasma using three different types of ATPS systems (i.e., polymer-polymer system, micelle system, and polymer-salt system). For the recovery of 145bp DNA, the efficiency of these three systems is comparable, because the differences in the average CT values ​​between the polymer-polymer system (average CT value = 24.81), the micelle system (average CT value = 24.93), and the polymer-salt system (average CT value = 24.73) are all within 1CT value (see Table 1.7). Similar results were observed in the recovery of 2000bp DNA. The results show that the recovery of 145bp and 2000bp DNA by different ATPS categories is comparable. Table 1.7: qPCR results of 145 bp and 2000 bp DNA oligonucleotides recovered from plasma by different ATPS classes using magnetic beads. Example 3a: Comparison of the use of spin columns to extract Efficiency of DNA recovery from plasma

[0426] In this example, the efficiency of recovering DNA from plasma using a spin column is compared (i) with prior phase separation using an ATPS system according to the methods of the present disclosure; and (ii) without the prior ATPS step. The materials used in this example are the same 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.

[0427] Material

[0428] The materials used in this example are the same or similar to those discussed in the previous examples, except that spin columns (EconoSpin) were used instead of magnetic beads in DNA purification. For the sake of brevity and simplicity of this disclosure, the discussion of materials will not be repeated here.

[0429] EconoSpin DNA / RNA mini spin columns were purchased from Epoch Life Sciences, Universal DNA spike II template was purchased from TATAABiocenter, and GeneRuler 1kb+DNA standard was purchased from ThermoFisher.

[0430] Plasma cell lysis

[0431] Spike 100fg 145bp double-stranded DNA (dsDNA) oligonucleotide, 1.5uL stock 2000bp dsDNA oligonucleotide and 100ng 1kb+ standard into each 2mL human pooled plasma. Add 160uL of appropriate lysis buffer and 60uL proteinase K (28.5mg / mL) to each 2mL spiked plasma. Vortex the mixture thoroughly and then lyse in a preheated 60°C heating block for 15 minutes.

[0432] Extraction process

[0433] One set of lysed plasma samples (sample #4) was treated with two consecutive ATPS to isolate and concentrate DNA before spin column purification, while the other sets of lysed plasma (samples #5 and #6) were not subjected to a previous ATPS step but directly subjected to spin column purification.

[0434] The steps for preparing samples #4-6 in this example are the same or similar to the materials discussed with respect to Example 2a. For the sake of brevity and simplicity of this disclosure, the discussion of the two-phase system steps will not be repeated here.

[0435] DNA purification

[0436] In this example, a binding buffer containing 1-5 M guanidine was used.

[0437] 800uL binding buffer is added to the top phase of the extraction of sample #4 and the cleavage sample #5 without ATPS respectively.For sample #6, 11.43mL binding buffer is added to cleavage sample #6, so that the ratio of cleavage sample: binding buffer is the same as the top phase in sample #4: the ratio of binding buffer.Sample #4-6 is summarized in the following table 2.0.Before all samples are applied to EconoSpin posts, fully vortex, and in 800uL, centrifuge for 30 seconds at 12,000rcf, until all sample lysates pass through the post.Then discard the flow through liquid (also referred to as "supernatant").800uL washing buffer (70% ethanol, 0.001M EDTA, 0.01M Tris-HCl) is added to the centrifugal column, and centrifuge for 30 seconds at 12,000rcf.Discard the flow through liquid, the centrifugal column is centrifuged for 2 minutes at 16,000rcf to remove excessive washing buffer. 40uL of elution buffer (0.01M Tris-HCl, 0.001M EDTA) was added to the spin column and incubated for 3 minutes. The spin column was centrifuged at 12,000rcf for 1 minute to elute the buffer containing the target DNA into a DNase-free centrifuge tube. Table 2.0: Summary of experimental conditions for plasma extraction and purification.

[0438] DNA testing

[0439] The steps for DNA testing of each sample are the same or similar to those discussed above with respect to Example 1a. For the sake of brevity and simplicity of this disclosure, the discussion of the testing steps will not be repeated here. The results are expressed as average CT values.

[0440] result

[0441] DNA recovery

[0442] Reference now Figure 3A-3B, showing the average CT values ​​of 145 bp and 2000 bp DNA oligonucleotides recovered from plasma purified using spin columns (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, according to Table 2.0). For 145 bp DNA, plasma sample #4 had high recovery (average CT value of 27.15), but plasma sample #5 had low recovery (average CT value of 37.49). For sample #6, the volume of binding buffer was increased proportionally, and the recovery (average CT value of 31.71) was improved compared to sample #5, but still significantly lower than the recovery of sample #4. Similar results were observed for 2000 bp DNA recovery. The data show that by using a two-step ATPS step to concentrate and purify the plasma sample before combining it with the column binding step, DNA recovery is improved, and surprisingly, DNA recovery is still significantly improved compared to the sample without the ATPS step, even when the volume of binding buffer is increased proportionally. Table 2.1: qPCR results of 145 bp and 2000 bp DNA oligonucleotides recovered from plasma using spin columns with or without two-step ATPS.

[0443] Overall, the recovery results show that, surprisingly, higher DNA recoveries can be achieved when ATPS is incorporated into the plasma extraction workflow prior to purification using spin columns, even though additional steps involving ATPS concentration and isolation, which are potential sources of target analyte losses during sample processing, are involved. Example 3b: Plasma extraction performance with different polymers and salts in ATPS compositions

[0444] In this example, the ability to recover DNA from plasma samples using ATPS with different polymer molecular weights and component concentrations in combination with a spin column, as well as the ability to recover DNA from plasma samples using ATPS with different polymer and salt chemistries in combination with a spin column, is demonstrated. The materials used in this example are the same 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.

[0445] Plasma cell lysis

[0446] In this example, 160 μL of the appropriate lysis buffer and 60 μL of proteinase K (28.57 mg / mL) were added to a 2 mL sample of EDTA (K2 TCS) plasma. 100 fg of 145 bp DNA and 100 ng of GR 1 kb+ standard were spiked into the above sample. After vortexing for 15 seconds, the sample was incubated at 60°C for 15 minutes. The lysate was used for further extraction.

[0447] Two-phase system

[0448] The extraction procedure involves two sequential aqueous two-phase systems (ATPS) to separate, purify and concentrate DNA from plasma samples. The steps for performing ATPS extraction in this example are the same or similar to those discussed with respect to Example 2b.

[0449] Extraction process

[0450] In this example, an ATPS-based separation and purification method for cfDNA is introduced. 2.22 mL of lysate was added to the first ATPS. A dye was added to visualize the two phases, resulting in a colored top phase. In this example, the vast majority of DNA was partitioned into the bottom phase, while proteins were partitioned into the top phase. The solution was made turbid by vigorous vortexing for 15 seconds. Phase separation was then promoted by centrifugation at 2300 rcf for 6 minutes. After phase separation, the volume of the bottom phase was about 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 produce a turbid solution and centrifuged at 7000 rcf for 1 minute to achieve phase separation. The volume of the top phase in the resulting solution was about 150 μL, which was then carefully extracted for further purification and detection of DNA.

[0451] DNA purification

[0452] DNA purification is completed by centrifugal column extraction. The top phase from the second ATPS is transferred to a test tube containing 800 μL binding buffer (1-5M guanidine salt). Fully mixed, 800 μL solution is transferred to a centrifugal column (EconoSpin) and centrifuged at 12,000 rcf for 30 seconds. The flow-through liquid is discarded. Repeat the process until all samples pass through the centrifugal column. Add 800 μL washing buffer (70% ethanol, 0.001M EDTA, 0.01M Tris-HCl) to the centrifugal column and centrifuge at 12,000 rcf for 30 seconds. Wash twice, and the tube is centrifuged at 16,000 rcf for 2 minutes to dry. Add 40 μL elution buffer (0.01M Tris-HCl, 0.001M EDTA) to the sample. The sample is incubated at room temperature for 3 minutes. The eluate is collected in a collection tube for detection by centrifugation at 12,000 rcf for 1 minute.

[0453] Extraction efficiency

[0454] The extraction efficiency of DNA in different ATPS compositions was studied while keeping the volume ratio in the first and second ATPS constant. To evaluate the DNA recovery performance, samples containing 100 ng of DNA standard in 2 mL of human plasma were prepared, assuming a 100% recovery, which would be 2.5 ng per μL of eluate.

[0455] DNA testing

[0456] The steps for DNA testing of each sample are the same or similar to those discussed above with respect to Example 1a. For the sake of brevity and simplicity of this disclosure, the discussion of the testing steps will not be repeated here. The results are expressed as average CT values.

[0457] result

[0458] Reference now Figure 3C , comparing the average CT values ​​for the recovery of 145 bp DNA from plasma using the first and second ATPS compositions according to conditions 5, 7, and 8 in Table 2.3. Conditions 5, 7, and 8 correspond to the same polymer and salt combination with different polymer molecular weights, and their respective 145 bp DNA recovery results are shown in Table 2.3. The results show that all systems with alternative polymer molecular weights can recover detectable levels of DNA when combined with a spin column workflow. Table 2.3: Recovery of DNA from plasma using ATPS and spin columns with different polymer molecular weights.

[0459] Reference now Figure 3D , comparing the average CT values ​​for the recovery of 145 bp DNA from plasma using the first and second ATPS compositions according to conditions 9, 22, 10, and 11 in Table 2.4. Conditions 9, 10, and 11 correspond to three different polymer and salt combinations, and their respective 145 bp DNA recovery results are reported in Table 2.4. The results show that all systems with alternative polymers and salts are able to recover detectable DNA when combined with a spin column workflow. Table 2.4: Recovery of DNA from plasma using ATPS and spin columns with different ranges of polymers and salts. Example 3c: Performance of plasma extraction using spin columns by different ATPS systems

[0460] In this example, the performance of different classes of ATPS systems (polymer salt, polymer-polymer, micelles) used with spin columns is demonstrated. The materials used in this example are the same 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.

[0461] Plasma lysis

[0462] In this example, K2 EDTA plasma (TCS Biosciences, lot number 23014500) was lysed in 3 different ways to optimize for its respective downstream ATPS system. The lysate samples used in this example are the same or similar to those discussed in Example 2c. For the sake of brevity and simplicity of this disclosure, the discussion of lysate samples will not be repeated here.

[0463] Extraction process

[0464] The extraction process and the 3 different classes of ATPS systems prepared are the same as those discussed in Example 2c and Table 1.6: 1. Polymer-polymer (PP) based; 2. Micellar; and 3. Bis-ATPS-polymer-salt (PS) based. For the sake of brevity and simplicity of this disclosure, the discussion of the preparation and extraction process of the above ATPS compositions will not be repeated here. Table 2.5 summarizes the 3 different classes of ATPS and the subsequent binding steps. Table 2.5: Summary of experimental conditions with alternative ATPS classes.

[0465] DNA purification

[0466] After extracting the phase containing the target cfDNA from its respective ATPS system, downstream processing includes adding a binding buffer to a spin column containing a porous membrane for separating the nucleic acid from the liquid medium, followed by washing away 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.

[0467] 1500uL binding buffer (3-7M guanidine, 50mM pH 7Na2HPO4 / NaH2PO4) is added to the bottom phase of the polymer-rich extraction of the polymer-polymer system. 500uL binding buffer (3-7M guanidine) is added to the top phase of the surfactant-rich extraction of the micellar system. 800uL binding buffer (1-5M guanidine) is added to the top phase of the polymer-rich extraction of the second polymer-salt ATPS system. Then 800uL of each sample is added to a separate EconoSpin column to obtain DNA and centrifuged at 12,000rcf for 30 seconds. The target cfDNA will be bound to the centrifugal column and retained, while the sample lysate flowing through is discarded through the vacuum manifold. Discard the flow-through and repeat the process until all samples flow through the centrifugal column. 800uL washing buffer (70% EtOH v / v, 1mM EDTA, 10mM Tris-HCl) is added to the centrifugal column of polymer-polymer and polymer-salt system, and 400uL RPE washing buffer (80% EtOH, 100mM NaCl, 10mMTris-HCl) is added to the centrifugal column of micellar system simultaneously.All centrifugal columns are then centrifuged for 30 seconds at 12000rcf.This washing step removes the impurities (like protein and other ions) combined with silica centrifugal column membrane.Discard the flow-through liquid, and the centrifugal column is further centrifuged for 2 minutes at 16000rcf to remove any excessive RPE washing buffer.Then the centrifugal column is placed in a new 1.5mL centrifuge tube, 80uL elution buffer (0.01M Tris-HCl, 1mM EDTA) is directly pipetted onto the silica membrane, and incubated at room temperature for 3 minutes. The spin column was centrifuged at 12000 rcf for 1 minute to elute the target cfDNA into a 1.5 mL centrifuge tube.

[0468] DNA testing

[0469] The steps for DNA testing of each sample are the same or similar to those discussed above with respect to Example 1a. For the sake of brevity and simplicity of this disclosure, the discussion of the testing steps will not be repeated here. The results are expressed as average CT values.

[0470] DNA recovery

[0471] Reference now Figure 3E-3FThe average CT values ​​of 145 bp DNA and 2000 bp DNA recovered from plasma were comparable among the three different types of ATPS systems, and the acceptable recovery rates were all within a difference of about 1 CT value, as shown in Table 2.6. This suggests that different types of ATPS classes can recover DNA to a similar extent when optimized. Table 2.6: qPCR results of 145 bp and 2000 bp DNA oligonucleotides recovered from plasma by different ATPS classes using spin columns. Example 4a: Comparison of the use of magnetic beads to extract urine from urine with and without a previous aqueous two-phase system (ATPS) step Efficiency of DNA recovery in liquid

[0472] In this example, the efficiency of recovering DNA from urine using magnetic beads is compared (i) with prior phase separation using an ATPS system (also referred to as an "ATPS step," "ATPS extraction," or "ATPS workflow") according to the methods of the present disclosure; and (ii) without the prior ATPS step. The materials used in this example are the same 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.

[0473] Urine lysis

[0474] Urine samples were collected from 3 different donors. The samples from each donor were aliquoted into 40 mL tubes and divided into 2 groups, each containing 1 sample from each donor.

[0475] All urine samples from both groups were pre-treated with 200uL of 0.1M EDTA per 10mL urine sample, vortexed thoroughly and centrifuged at 3000rcf for 10 minutes. The supernatant was transferred to a new tube while the pellet was discarded.

[0476] Unwanted proteins and cells present in the pre-treated urine samples were lysed by adding 1200uL (28.57mg / mL) proteinase K and 4mL of appropriate lysis buffer to each donor 40mL sample. The samples were then vortexed thoroughly until homogenous and then incubated in a preheated 37°C water bath for 15 minutes.

[0477] Extraction process

[0478] Two different aqueous two-phase systems (ATPS) were prepared to efficiently extract cfDNA from urine samples. The first ATPS was used for the initial extraction of urine samples, where the expected cfDNA strongly partitioned into the salt-rich bottom phase. The bottom phase from the first ATPS was then extracted and added to the second ATPS for concentrating the target cfDNA into a small volume (400uL-600uL) to allow for user-friendly downstream processing.

[0479] In this example, the first ATPS consisted of the ATPS 38 composition according to Table 1.0, containing 22600 uL of lysed urine sample.

[0480] The second ATPS consisted of the ATPS 71 composition according to Table 1.0 containing 3.5 mL to 5 mL of the first ATPS bottom phase.

[0481] For urine sample set 1 (sample #1), the urine sample from each donor was split in half and added to 2 first ATPS tubes. The first ATPS was vortexed thoroughly and then centrifuged at 2300 rcf for 6 minutes. The salt-rich bottom phases from both first ATPS (same donor) were then extracted, reconstituted and added to the second ATPS tube, which was vortexed thoroughly and centrifuged to allow phase separation. The polymer-rich top phase of the second ATPS system was extracted and placed into a new tube.

[0482] Urine sample set 2 (Sample #2) was pre-treated and processed but not subjected to the dual ATPS system for concentration and purification. The urine sample set is summarized in Table 3.0 below. Table 3.0: Summary of experimental conditions for urine extraction and purification.

[0483] DNA purification

[0484] The target cfDNA in the urine sample is partitioned into the top phase of the polymer-rich phase in the second ATPS and concentrated to 400uL-600uL. A binding buffer containing 3-7M guanidine is used.

[0485] 2 mL of binding buffer was added to the extracted top phase of sample #1 and lysed sample #2 without ATPS. 24 uL of magnetic beads were added to each tube. The mixture was then incubated on a rotator for 5 minutes to prevent the beads from settling. The tubes were then briefly centrifuged and placed on a magnetic stand for 2 minutes to fix the beads to the tube walls. The supernatant was discarded without disturbing the beads. 2 mL of binding buffer was added to each tube and the tubes were slowly rotated on the magnetic stand for a total of 720°. The supernatant was pipetted again and 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 stand for a total of 720°. The supernatant was discarded. The washing step was performed twice. To improve the drying effect, the tubes were briefly centrifuged using a tabletop microcentrifuge with the hinge facing outward to collect any remaining wash buffer. The beads were then dried on the magnetic stand for 7 minutes with the lid open. The bead-analyte complex was resuspended in 80 μL of elution buffer (0.01 M Tris-HCl, 0.001 M EDTA) by continuous pipetting mixing followed by gentle vortexing. The tube was then placed on a magnetic stand for 1 minute. The supernatant was carefully collected into a DNA low binding tube without disturbing the magnetic beads for detection.

[0486] DNA testing

[0487] The steps for DNA testing of each sample are the same or similar to those discussed above with respect to Example 2a. For the sake of brevity and simplicity of this disclosure, the discussion of the testing steps will not be repeated here. The results are expressed as average CT values.

[0488] result

[0489] DNA recovery

[0490] Reference now Figure 4A-4B , showing the recovery of 145 bp DNA from urine using magnetic beads with and without a previous ATPS step (sample #1) ( Figure 3A ) and 2000 bp DNA ( Figure 4B ). High recovery rates of 145 bp DNA (average CT value of 27.47) and 2000 bp DNA (average CT value of 27.09) were shown from urine sample #1, while no detectable recovery of target DNA was observed from urine sample #2 (as shown in Table 3.1). The results show that under the same conditions, the recovery rate of DNA from large-volume samples such as urine can be significantly improved by adding a parallel ATPS step before magnetic bead purification. Table 3.1: qPCR results of 145 bp and 2000 bp DNA oligonucleotides recovered from urine using magnetic beads with or without two-step ATPS.

[0491] Overall, the recovery results show that, surprisingly, higher DNA recoveries can be achieved when ATPS is incorporated into the plasma extraction workflow prior to purification using spin columns, even though additional steps involving ATPS concentration and isolation, which are potential sources of target analyte losses during sample processing, are involved. Example 4b: Urine extraction performance with different polymers and salts in ATPS compositions

[0492] In this example, the ability to use ATPS with different polymer molecular weights and component concentrations in combination with magnetic beads to recover DNA from urine samples was demonstrated. The materials used in this example are the same 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.

[0493] Urine lysis

[0494] Urine samples were pretreated with 200uL of 0.1M EDTA per 10mL urine sample, vortexed thoroughly and centrifuged at 3000rcf for 10 minutes. The supernatant was transferred to a new tube and the pellet was discarded. To digest unwanted proteins and cells, 60μL of proteinase K (28.57mg / mL) and 200uL of appropriate lysis buffer were added to 2mL of sample. 100fg 145bp oligonucleotide 100ng 1kb+DNA standard was spiked into the above sample. The sample was then vortexed thoroughly until homogenous and then incubated in a preheated 37°C water bath for 15 minutes.

[0495] Two-phase system

[0496] The extraction procedure involved two consecutive aqueous two-phase systems (ATPS) to isolate, purify and concentrate DNA from urine samples. To evaluate the compatibility of such a system, the phase separation properties and DNA extraction efficiency of various polymers and salts in the first ATPS were investigated. In the first ATPS, DNA partitioned into the bottom phase and proteins partitioned into the top phase. About 1 mL of the bottom phase was carefully extracted and transferred to the second ATPS. Since the composition of the ATPS bottom phase strongly depends on the components of the ATPS, the bottom phase composition of the first ATPS differed 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 top phase volume of about 150 μL. DNA partitioned into the top phase of the second ATPS, and the top phase was carefully extracted for further purification and detection.

[0497] Extraction process

[0498] 2.26mL of lysate was added to the first ATPS. Dyes were added to visualize the two phases, resulting in a colored top phase. Due to the optimized combination of interactions with the ATPS components, the vast majority of DNA was distributed to the bottom phase, while proteins were distributed to the top phase. The solution was made turbid by vigorous vortexing for 15 seconds. Phase separation was then promoted by centrifugation at 2300rcf for 6 minutes. After phase separation, the volume of the bottom phase was about 1mL. The entire bottom phase was carefully extracted and transferred to the second ATPS. The composition of the second ATPS was formulated to allow DNA to be distributed to the top phase. The mixture was vortexed for 15 seconds to produce a turbid solution and centrifuged at 7000rcf for 1 minute to achieve phase separation. The volume of the top phase in the resulting solution was about 150μL. The small volume ratio of the top phase to the bottom phase is beneficial to the concentration of DNA in the top phase. The top phase was then carefully extracted for further purification and detection of DNA.

[0499] DNA purification

[0500] Purification of DNA was accomplished by magnetic bead extraction (also known as the "magnetic bead workflow"). The top 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 rotation for 5 minutes. The tube was then briefly centrifuged to settle and placed on a magnetic stand for 2 minutes to fix the beads to the tube wall. The supernatant was pipetted and discarded 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 stand. The supernatant was pipetted and discarded. 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 stand. The supernatant was pipetted and discarded. The wash step was performed twice and the tube was placed on the stand with the lid open to dry the beads. Add 40 μL of elution buffer (0.01M Tris-HCl, 0.001M EDTA) to the sample. Resuspend the bead complex by continuous pipetting and then gently vortex. Incubate the sample at room temperature for 3 minutes and centrifuge briefly. Then place the tube on a magnetic stand for 1 minute. Carefully collect the supernatant without disturbing the magnetic beads for detection.

[0501] Extraction efficiency

[0502] Both the first and second ATPS contained a mixture of polymer and salt, where the resulting top phase was polymer-rich and the bottom phase was salt-rich. To investigate the compatibility of the current system with different reagents, the extraction efficiency of DNA in different ATPS compositions was investigated while keeping the volume ratio of the first and second ATPS constant. To evaluate the DNA recovery performance, samples containing 100 fg of 145 bp dsDNA were prepared, assuming a 100% recovery, which was 2.5 ng per μL of eluate.

[0503] DNA testing

[0504] The steps for DNA testing of each sample were the same or similar to those discussed above with respect to Example 1a. For the sake of brevity and simplicity of this disclosure, the discussion of the testing steps will not be repeated here. The recovery rate was quantified by qPCR and the results are summarized in Figure 3C-3D The results are expressed as the mean CT value.

[0505] result

[0506] Reference now Figure 4C , comparing the average CT values ​​for the recovery of 145 bp DNA using the first and second ATPS compositions according to conditions 33, 41, 34, and 35 in Table 3.3. Conditions 33, 41, 34, and 35 correspond to the same polymer and salt combination with different polymer molecular weights, and their respective 145 bp DNA recovery results are shown in Table 3.3. The results show that all systems with alternative polymer molecular weights are able to recover detectable levels of DNA from urine when combined with the magnetic bead workflow. Table 3.3: Recovery of DNA from urine using ATPS and magnetic beads with different polymer molecular weights.

[0507] Reference now Figure 4D , comparing the average CT values ​​for the recovery of 145 bp DNA from urine using the first and second ATPS compositions according to conditions 36, 6, 38, and 39 in Table 3.4. Conditions 36, 6, 38, and 39 correspond to four different polymer and salt combinations, and their respective 145 bp DNA recovery results are reported in Table 3.4. 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, indicating successful DNA extraction using multiple reagents. Table 3.4: Recovery of DNA from urine using ATPS and magnetic beads with different ranges of polymers and salts. Example 4c: Urine extraction performance using magnetic beads by various ATPS systems

[0508] In this example, the ability to recover DNA from urine samples using different classes of ATPS systems (polymer-salt, polymer-polymer, micelles) in combination with magnetic beads is demonstrated. The materials used in this example are the same 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.

[0509] Urine lysis

[0510] The urine samples to be extracted with the polymer-polymer and polymer-salt ATPS systems were pretreated with 200uL of 0.1M EDTA per 10mL urine sample, vortexed thoroughly and centrifuged at 3000rcf for 10 minutes. The supernatant was transferred to a new tube and the precipitate was discarded. Unwanted proteins and cells present in the pretreated urine samples were lysed by adding 60μL of proteinase K (28.57mg / mL) and 200μL of appropriate lysis buffer to 2mL of sample. The sample was then vortexed thoroughly until homogenous and then incubated in a preheated 37°C water bath for 15 minutes.

[0511] For samples extracted with micelle-based ATPS system, urine samples were pretreated with 200uL of 0.1M EDTA per 10mL urine sample and centrifuged at 3000rcf for 10 minutes to collect the supernatant. 300uL of suitable lysis buffer was added to 1mL of pretreated urine sample, vortexed thoroughly and incubated at room temperature for 3 minutes. 60uL 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 uniform, and centrifuged at 12000rcf for 3 minutes. Other suitable protein precipitation buffers can be used in place of protein precipitation buffer A in the above method. The supernatant was extracted for further downstream processing, while the precipitate was discarded.

[0512] Extraction process

[0513] Different varieties of aqueous two-phase systems (ATPS) similar to those discussed in Example 2c were prepared to efficiently extract cfDNA from urine samples. Table 3.5 summarizes the three different varieties of ATPS systems prepared. In the polymer-polymer and polymer-salt based ATPS systems, the target cfDNA was partitioned from the lysed sample to the bottom phase, while the unwanted excess protein was partitioned to the top phase. The opposite trend was seen in the micelle-based ATPS system. Table 3.5: Summary of experimental conditions with alternative ATPS classes.

[0514] Add the sample lysate to their respective ATPS systems: 1130uL of sample lysate to the polymer-polymer based system and 1360uL of extracted supernatant to the micelle based system. All ATPS tubes were vortexed thoroughly after adding the sample lysate and vortexed thoroughly.

[0515] The polymer-polymer ATPS system was centrifuged at 2300 rcf for 6 minutes. The target cfDNA was strongly partitioned into the polymer-rich bottom phase. All of the bottom phase (approximately 300 uL) was extracted and transferred to a new 2 mL centrifuge tube for further processing.

[0516] The micellar ATPS system was centrifuged at 12000 rcf for 5 minutes. The target cfDNA was strongly partitioned into the surfactant-rich top phase. All of the top phase (approximately 280 uL) was extracted and transferred to a new 2 mL centrifuge tube for further processing.

[0517] The first polymer-salt ATPS system was centrifuged at 2300rcf for 6 minutes. The target cfDNA was strongly distributed to the salt-rich bottom phase. All the bottom phase (about 1000uL) was extracted and transferred to the second ATPS, which was vortexed thoroughly and then centrifuged at 7000rcf for 1 minute. The target cfDNA was distributed and concentrated into the polymer-rich top phase of about 120uL. It was then extracted and transferred to a new tube for further processing.

[0518] DNA purification

[0519] After extracting the phase containing the target cfDNA from its respective ATPS system, downstream processing includes the addition of solid phase media and binding buffer for separating the nucleic acids from the liquid medium, followed by washing away unwanted ions and molecules before the target nucleic acids are eluted by elution buffer.

[0520] 1500uL binding buffer (3-7M guanidine, 50mM pH 7 Na2HPO4 / NaH2PO4) was added to the extracted polymer-rich bottom phase of the polymer-polymer system. 500uL binding buffer (3-7M guanidine) was added to the extracted surfactant-rich top phase of the micellar system. 800uL binding buffer (1-5M guanidine) was added to the extracted polymer-rich top phase of the second polymer-salt ATPS system. 6uL magnetic beads were then added to all centrifuge tubes containing the extraction phase and binding buffer mixture and placed on a rotator for incubation for 5 minutes. The target cfDNA will bind to the magnetic beads to form a bead-analyte complex. After incubation, all tubes were briefly centrifuged and then placed on a magnetic stand, where the applied magnetic field attracted the cfDNA-bound magnetic beads. The supernatant was then taken and discarded, leaving the magnetic beads bound to the inner wall of the centrifuge tube. A second binding buffer wash step was performed for the polymer-polymer and polymer-salt systems. Add 800uL of binding buffer (3-7M guanidine, 50mM pH7 Na2HPO4 / NaH2PO4) to the magnetic beads from the polymer-polymer system, and add 800uL of binding buffer (1-5M guanidine) to the magnetic beads from the polymer-salt system. Then vortex the centrifuge tube thoroughly and centrifuge briefly before returning it to the magnetic stand. Once all the magnetic beads are bound to the inside of the centrifuge wall, discard the supernatant.

[0521] 800uL of original wash buffer (70% v / v EtOH, 1mM EDTA, 10mM Tris-HCl) was added to the magnetic beads from the polymer-polymer and polymer-salt systems, while 500uL of RPE wash buffer (80% v / v EtOH, 0.1M NaCl, 10mM 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 stand. Repeat the procedure for a second wash. The centrifuge tube was then briefly centrifuged and placed back on the magnetic stand, 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.

[0522] After the beads have dried, resuspend the beads in 20uL of elution buffer (10mM Tris-HCl, 1mM EDTA) and incubate at room temperature for 3 minutes. Elute the target cfDNA from the beads into the elution buffer. Then place the tube back on the magnetic stand and extract the elution buffer once all the beads are bound.

[0523] DNA testing

[0524] The steps for DNA testing of each sample are the same or similar to those discussed above with respect to Example 1a. For the sake of brevity and simplicity of this disclosure, the discussion of the testing steps will not be repeated here. The results are expressed as average CT values.

[0525] result

[0526] Reference now Figure 4E According to Table 3.6, the average CT values ​​for the recovery of 145 bp DNA from urine when extracted using different types of ATPS systems are shown. The results show that the DNA recovery efficiency of the three systems is comparable, as the differences in the average CT values ​​between the polymer-polymer system (average CT value = 28.73), the micelle system (average CT value = 27.99), and the polymer-salt system (average CT value = 27.85) are all within 1 CT value. This shows that different types of ATPS classes can recover DNA to a similar extent when optimized. Table 3.6: qPCR results of 145 bp DNA recovery from urine using magnetic beads by different ATPS classes. Example 5a: Comparison of the use of spin columns to extract Efficiency of DNA recovery from urine

[0527] In this example, the efficiency of recovering DNA from urine using a spin column is compared (i) with prior phase separation using an ATPS system according to the methods of the present disclosure; and (ii) without the prior ATPS step. The materials used in this example are the same 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.

[0528] Urine lysis

[0529] Urine samples were collected from 4 different donors. The samples from each donor were aliquoted into 40 mL tubes and divided into 2 groups, each containing 1 sample from each donor.

[0530] All urine samples from both groups were pre-treated with 200uL of 0.1M EDTA per 10mL urine sample, vortexed thoroughly and centrifuged at 3000rcf for 10 minutes. The supernatant was transferred to a new tube while the pellet was discarded.

[0531] Unwanted proteins and cells present in the pre-treated urine samples were lysed by adding 1200uL (28.57mg / mL) proteinase K and 4mL of appropriate lysis buffer to each donor 40mL sample. The samples were then vortexed thoroughly until homogenous and then incubated in a preheated 37°C water bath for 15 minutes.

[0532] Extraction process

[0533] Two different aqueous two-phase systems (ATPS) were prepared to effectively extract cfDNA from urine samples. The steps for performing ATPS extraction to prepare urine sample #3 and urine sample #4 in this example are the same or similar to the steps discussed in 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 group is summarized in Table 4.0 below. Table 4.0. Summary of experimental conditions for urine extraction and purification.

[0534] DNA purification

[0535] The target cfDNA in the urine sample is partitioned into the top phase of the polymer-rich in the second ATPS and concentrated to 400uL-600uL. The top phase is separated for further processing.

[0536] 3-7M guanidine solution was used as binding buffer. 2mL of 3-7M guanidine was added (about 400uL-600uL) to the top phase of the second ATPS-rich polymer extracted from urine group 3 (sample #3) and vortexed thoroughly. Urine sample group 4 (sample #4) that was not purified and concentrated by ATPS was mixed with 2mL 3-7M guanidine and vortexed thoroughly. Each urine sample was then added to an EconoSpin column to obtain DNA connected to a QIAvac 24Plus vacuum manifold with an appropriate extender (3mL and 20mL). A pressure of 900mbar was applied to the vacuum manifold, and the sample lysate was flowed through the centrifugal column. The target cfDNA will be bound to the centrifugal column and retained, while the sample lysate (also referred to as "supernatant") that flows through the centrifugal column is discarded. After all possible sample lysates flow through the centrifugal column, the extender is removed and discarded. The centrifugal column is removed from the manifold and inserted into a 2mL waste tube. 500uL RPE wash buffer (80% v / v EtOH, 0.1M NaCl, 0.01M Tris-HCl) was added to the centrifuge column and centrifuged at 12000rcf for 30 seconds. The flow-through was discarded and the centrifuge column was further centrifuged at 16000rcf for 2 minutes to remove any excess RPE wash buffer. The centrifuge column was then placed in a new 1.5mL centrifuge tube, and 80uL elution buffer (0.01M Tris-HCl, 1mM EDTA) was directly pipetted onto the silica membrane and incubated at room temperature for 3 minutes. The centrifuge column was centrifuged at 12000rcf for 1 minute to elute the target cfDNA into a 1.5mL centrifuge tube.

[0537] DNA testing

[0538] The steps for DNA testing of each sample are the same or similar to those discussed above with respect to Example 1a. For the sake of brevity and simplicity of this disclosure, the discussion of the testing steps will not be repeated here. The results are expressed as average CT values.

[0539] result

[0540] DNA recovery

[0541] In urine sample #3, all lysate samples flowed through the spin column within 1 minute. In urine sample #4, only 25% of the sample lysate passed through the spin column within the first 60 minutes. In the absence of parallel ATPS, an increased flow time (1 hour) was required to allow the untreated lysate to completely pass through the column.

[0542] Reference now Figure 5A-5B , showing the average CT values ​​of 145bp and 2000bp DNA recovered from urine using a spin column with and without a previous ATPS step (sample #4). According to Table 4.1, for 145bp DNA, urine sample #3 had a high recovery (average CT value of 25.36 ± 0.08), but urine sample #4 had a low recovery (average CT value of 38.15 ± 2.10), and similar results were observed in the recovery of 2000bp DNA. As shown in the results, concentrating urine lysates by parallel ATPS before centrifugal column extraction increased the efficiency of downstream processing and significantly improved the recovery of 145bp and 2000bp DNA. Table 4.1: qPCR results of 145 bp and 2000 bp DNA oligonucleotides recovered from urine using spin columns with or without two-step ATPS.

[0543] Overall, the recovery results show that, surprisingly, higher DNA recoveries can be achieved when ATPS is incorporated into the plasma extraction workflow prior to purification using spin columns, even though additional steps involving ATPS concentration and isolation, which are potential sources of target analyte losses during sample processing, are involved. Example 5b: Urine extraction performance with different polymers and salts in ATPS compositions

[0544] In this example, the ability to recover DNA from urine samples using ATPS with different polymer MW and component concentrations in combination with a spin column is demonstrated. The materials used in this example are the same 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.

[0545] Urine lysis

[0546] In this example, urine samples were pretreated with 200uL of 0.1M EDTA per 10mL urine sample, vortexed thoroughly and centrifuged at 3000rcf for 10 minutes. The supernatant was transferred to a new tube and the pellet was discarded. In order to digest the unwanted proteins and cells present in the pretreated urine sample, 1200μL of proteinase K (28.57mg / mL) and 4mL of appropriate lysis buffer were added to the 40mL sample. 100fg 145bp DNA and 100ng GR 1kb+ standard were spiked into the above sample. The sample was then vortexed thoroughly until homogenous and then incubated in a preheated 37°C water bath for 15 minutes.

[0547] Two-phase system

[0548] The extraction procedure involved two consecutive aqueous two-phase systems (ATPS) to isolate, purify and concentrate DNA from urine samples, similar to those described in previous examples. In the first ATPS, DNA partitioned into the bottom phase and proteins partitioned into the top phase. About 1 mL of the bottom phase was carefully extracted and transferred to the second ATPS. Since the composition of the ATPS bottom phase strongly depends on the components of the ATPS, the bottom phase composition of the first ATPS differed 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 top phase volume of about 150 μL. DNA partitioned into the top phase of the second ATPS, and the top phase was carefully extracted for further purification and detection.

[0549] Extraction process

[0550] 2.22mL of lysate was added to the first ATPS. Dye was added to visualize the two phases, resulting in a colored top phase. In this example, the vast majority of DNA was distributed into the bottom phase, while proteins were distributed into the top phase. The solution was made turbid by vigorous vortexing for 15 seconds. Phase separation was then promoted by centrifugation at 2300rcf for 6 minutes. After phase separation, the volume of the bottom phase was about 1mL. The entire bottom phase was carefully extracted and transferred to the second ATPS. The second ATPS mixture was vortexed for 15 seconds to produce a turbid solution and centrifuged at 7000rcf for 1 minute to achieve phase separation. The volume of the top phase in the resulting solution was about 150μL, which was then carefully extracted for further purification and detection of DNA.

[0551] DNA purification

[0552] DNA purification is carried out by centrifugal column extraction, similar to the step discussed in Example 5a. The top phase from the second ATPS is transferred to a test tube containing 800 μL binding buffer (1-5M guanidine salt). Fully mixed, 800 μL solution is transferred to a centrifugal column (EconoSpin) and centrifuged at 12,000 rcf for 30 seconds. The flow-through liquid is discarded. Repeat the process until all samples pass through the centrifugal column. Add 800 μL washing buffer (70% ethanol, 0.001M EDTA, 0.01M Tris-HCl) to the centrifugal column and centrifuge at 12,000 rcf for 30 seconds. Wash twice, and the tube is centrifuged at 16,000 rcf for 2 minutes to dry. Add 40 μL elution buffer (0.01M Tris-HCl, 0.001M EDTA) to the sample. The sample is incubated at room temperature for 3 minutes. The eluate is collected in a collection tube for detection by centrifugation at 12,000 rcf for 1 minute.

[0553] Extraction efficiency

[0554] Both the first and second ATPS contain a mixture of polymer and salt, where the resulting top phase is polymer-rich and the bottom phase is salt-rich. In this example, the extraction efficiency of DNA in different ATPS compositions was investigated while keeping the volume ratio in the first and second ATPS constant. To evaluate the DNA recovery performance, a sample containing 100 ng of DNA standard in 2 mL of urine was prepared, assuming a 100% recovery, which is 2.5 ng per μL of eluate.

[0555] DNA testing

[0556] The steps for DNA testing of each sample are the same or similar to those discussed above with respect to Example 1a. For the sake of brevity and simplicity of this disclosure, the discussion of the testing steps will not be repeated here. The results are expressed as average CT values.

[0557] result

[0558] Reference now Figure 5C, comparing the average CT values ​​of 145 bp DNA recovered from urine according to the first and second ATPS compositions of conditions 33, 34, and 35 in Table 4.3. Conditions 33, 34, and 35 correspond to the same polymer and salt combinations with three different polymer molecular weights, and their respective 145 bp DNA recovery results are shown in Table 4.3. The results show that all systems with alternative polymer molecular weights can recover detectable DNA from urine when combined with a spin column workflow. Table 4.3: Recovery of DNA from urine using ATPS and spin columns with different polymer molecular weights.

[0559] Reference now Figure 5D , comparing the average CT values ​​of 145 bp DNA recovered from urine using the first and second ATPS compositions according to conditions 36-39 in Table 4.4. Conditions 36, 37, 38, and 39 correspond to four different polymer and salt combinations, and their respective 145 bp DNA recovery results are reported in Table 4.4. The results show that all systems with alternative polymers and salts recover detectable DNA from urine using spin columns. Table 4.4: Recovery of DNA from urine using ATPS and spin columns with different ranges of polymers and salts. Example 5c: Urine extraction performance using spin columns by different ATPS systems

[0560] In this example, the ability to recover DNA from urine samples using different classes of ATPS systems (polymer-salt, polymer-polymer, micelles) in combination with spin columns is demonstrated. The materials used in this example are the same 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.

[0561] Urine lysis

[0562] In this example, urine samples to be extracted with polymer-polymer and polymer-salt ATPS systems were pretreated with 200uL of 0.1M EDTA per 10mL urine sample, vortexed thoroughly and centrifuged at 3000rcf for 10 minutes. The supernatant was transferred to a new tube and the precipitate was discarded. Unwanted proteins and cells present in the pretreated urine sample were lysed by adding 1200μL of proteinase K (28.57mg / mL) and 4mL of a suitable lysis buffer to 40mL of sample. The sample was then vortexed thoroughly until uniform and then incubated in a preheated 37°C water bath for 15 minutes.

[0563] For samples extracted with micelle-based ATPS system, urine samples were pretreated with 200uL of 0.1M EDTA per 10mL urine sample and centrifuged at 3000rcf for 10 minutes to collect the supernatant. 300uL of appropriate lysate was added to 1mL pretreated urine sample, vortexed thoroughly and incubated at room temperature for 3 minutes. 112.5uL protein precipitation buffer A (PPt A) (20% ZnCl w / v, 1% acetic acid w / v) was then added to the sample, vortexed thoroughly until uniform, and centrifuged at 12000rcf for 3 minutes. Other suitable protein precipitation buffers can be used in place of protein precipitation buffer A in the above method. The supernatant was extracted for further downstream processing, while the precipitate was discarded.

[0564] Extraction process

[0565] The extraction process and the 3 different types of ATPS systems prepared are the same as those discussed in Example 4c: 1. Polymer-polymer (PP) based; 2. Micellar; and 3. Bis-ATPS-polymer-salt (PS) based. For the sake of brevity and simplicity of this disclosure, the discussion of the preparation and extraction process of the above ATPS compositions will not be repeated here.

[0566] Table 4.5 summarizes the three different types of ATPS used in this example and the subsequent binding steps. Table 4.5: Summary of experimental conditions with alternative ATPS classes.

[0567] Add the sample lysate to their respective ATPS systems: 1130uL of sample lysate to the polymer-polymer based system and 1360uL of extracted supernatant to the micellar system. All ATPS tubes were vortexed thoroughly after adding the sample lysate and vortexed thoroughly.

[0568] The polymer-polymer ATPS system was centrifuged at 2300 rcf for 6 minutes. The target cfDNA was strongly partitioned into the polymer-rich bottom phase. All of the bottom phase (approximately 300 uL) was extracted and transferred to a new 2 mL centrifuge tube for further processing.

[0569] The micellar ATPS system was centrifuged at 12000 rcf for 5 minutes. The target cfDNA was strongly partitioned into the surfactant-rich top phase. All of the top phase (approximately 280 uL) was extracted and transferred to a new 2 mL centrifuge tube for further processing.

[0570] The first polymer-salt ATPS system was centrifuged at 2300rcf for 6 minutes. The target cfDNA was strongly distributed to the salt-rich bottom phase. All the bottom phase (about 1000uL) was extracted and transferred to the second ATPS, which was vortexed thoroughly and then centrifuged at 7000rcf for 1 minute. The target cfDNA was distributed and concentrated into the polymer-rich top phase of about 120uL. It was then extracted and transferred to a new tube for further processing.

[0571] DNA purification

[0572] After extracting the phase containing the target cfDNA from its respective ATPS system, downstream processing includes the addition of solid phase media and binding buffer for separating the nucleic acids from the liquid medium, followed by washing away unwanted ions and molecules before the target nucleic acids are eluted by elution buffer.

[0573] 1500uL binding buffer (3-7M guanidine, 50mM pH 7Na2HPO4 / NaH2PO4) is added to the bottom phase of the polymer-rich extraction of the polymer-polymer system. 500uL binding buffer (3-7M guanidine) is added to the top phase of the surfactant-rich extraction of the micellar system. Then 800uL of each sample is added to a separate EconoSpin column to obtain DNA and centrifuged at 12,000rcf for 30 seconds. The target cfDNA will be bound to the centrifugal column and retained, while the sample lysate flowing through is discarded through the vacuum manifold. Discard the flow-through and repeat the process until all samples flow through the centrifugal column. 500uL RPE wash buffer (80% v / v EtOH, 0.1M NaCl, 0.01M Tris-HCl) is added to the centrifugal column and centrifuged at 12000rcf for 30 seconds. The flow-through was discarded and the spin column was further centrifuged at 16000 rcf for 2 minutes to remove any excess RPE wash buffer. The spin column was then placed in a new 1.5 mL centrifuge tube, and 80 uL 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 spin column was centrifuged at 12000 rcf for 1 minute to elute the target cfDNA into a 1.5 mL centrifuge tube.

[0574] DNA testing

[0575] The steps for DNA testing of each sample are the same or similar to those discussed above with respect to Example 2a. For the sake of brevity and simplicity of this disclosure, the discussion of the testing steps will not be repeated here. The results are expressed as average CT values.

[0576] DNA recovery

[0577] result

[0578] Reference now Figure 5E According to Table 4.6, the average CT values ​​of 145 bp DNA recovered from urine using different types of ATPS systems (polymer-polymer, micelle, polymer-salt) are shown. The results show that all three systems can effectively provide satisfactory DNA recovery from urine. Table 4.6: qPCR results for recovery of 145 bp DNA from urine by different ATPS classes using spin columns. Example 6: Merge results Magnetic beads as solid phase

[0579] Reference now Fig. 6A , showing a combined data representation of the CT values ​​of 145 bp DNA recovered from plasma and urine using magnetic beads (combining Examples 2a-c and 4a-c). Fig. 6A As shown, all conditions with a previous ATPS step achieved higher DNA recovery compared to the condition without a previous ATPS step, i.e. Fig. 6A , both of which resulted in Ct values ​​close to 40 (see also 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. Spin columns as solid phase

[0580] Reference now Figure 6B , showing a combined data representation of the CT values ​​of 145 bp DNA recovered from plasma and urine using spin columns (combining Examples 3a-c and 5a-c). Similarly, Figure 6B As shown, all conditions with a previous ATPS step achieved higher DNA recovery compared to the condition without a previous ATPS step, i.e. Figure 6B The ATPS step was incorporated into the plasma extraction prior to the spin column purification to achieve satisfactory DNA recovery.

[0581] In summary, adding an ATPS step prior to the solid phase extraction workflow provides the following benefits compared to non-ATPS treated samples:

[0582] Significantly reduced reagent consumption

[0583] As can be seen from the examples presented herein, in some instances, the addition of one or more ATPS steps significantly reduces the amount of binding buffer required for untreated lysates. For example, the addition of a two-step ATPS system prior to solid phase extraction reduces the binding buffer required for a 40 mL bulk fluid sample to only 2 mL. This is due to the small target enrichment phase produced by the first or second ATPS. The magnitude of the reduction in reagent consumption becomes even more pronounced as the sample input volume increases, since larger input volumes require exponentially more binding buffer, while the ATPS process can be modified to keep the top phase volume constant.

[0584] In some instances, high DNA recovery was achieved without the need for higher amounts of magnetic beads to shift the binding equilibrium for better yields, thereby reducing the use of expensive magnetic beads and hazardous binding buffers.

[0585] Column flow time is significantly reduced

[0586] 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.

[0587] Simplified laboratory setup

[0588] In some instances, since the sample lysate volume is much smaller, a custom large volume extender will not be required when applying ATPS sample lysate concentration. No specific vacuum manifold is required, and a centrifuge can be used to pass the sample lysate.

[0589] Overall, the results demonstrate that a system combining one or more ATPS extraction workflows with different solid phase media can be used with surprising efficiency for the purification of analytes from different clinical biospecimens. Example 7: Comparison of total DNA recovery using the disclosed method with commercially available extraction kits

[0590] Example 7a

[0591] DNA was extracted from urine using the exemplary method and kit described in Example 5a (referred to herein as "the present extraction method" or "Phase") and compared with the Zymo Rapid DNA Urine Kit ("Zymo"), NextPrep-Mag Urine cfDNA Isolation Kit ("NextPrep Kit"), Norgen Urine DNA Isolation Kit-Spin Column ("Norgen"), and Wizard Plus Mini Prep DNA Purification System ("Wizard"), all of which are commercially available. For each competitor's kit, the maximum urine sample input specified by the manufacturer was used and the extraction was performed according to the manufacturer's instructions. The commercial kits (Conditions AD in Table 6) and the present extraction method (Condition E in Table 6) were compared using cell-free urine. In addition, as a comparison, we performed a batch of 40 mL extractions using crude urine (unrotated urine with cells) with the current extraction method (Condition F in Table 6) to evaluate whether the current extraction method can perform equally well in the presence of cells. Urine samples were provided by 4 men and 4 women (n=8 for each kit). The input and elution volumes for each kit were normalized to a 100:1 ratio and the extraction times are compared in Table 6. Table 6: Comparison of yield and efficiency at normalized input:elution volume ratios.

[0592] Reference now Fig. 7A , which shows the recovery of 145bp DNA spike (copies / uL) using conditions AF according to Table 6. 145bp spike was detected by Droplet digital PCR ddPCR. Fig. 7AAs shown, the recovery efficiency of 140bp spiked DNA using the present extraction method (condition E) is comparable to, if not superior to, the NextPrep (condition A) and Wizard (condition D) extraction kits, and significantly superior to 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, the present extraction method can handle larger input volumes, but the extraction time is comparable, if not shorter. Compared with Zymo (condition B) with the same input volume, the present extraction method has a shorter extraction time, significantly higher yield, and more consistent performance between samples. When using urine with cells (condition F), the average DNA recovery rate of the present extraction method is significantly better than all competitors, even in the presence of cells, with satisfactory precision (476.1±32.2 copies / uL). This shows that the present extraction method performs well in recovering target DNA with crude urine as well as processed centrifuged urine. In summary, the overall objective DNA extraction performance (in terms of yield, input volume, and extraction time) using the disclosed methods was surprisingly better compared to industry standard commercially available extraction kits.

[0593] Example 7b

[0594] Further comparison of DNA recovery from urine between the present extraction method 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-Spin Columns ("Norgen"), and WizardPlus Mini Prep DNA Purification System ("Wizard")) was performed using the present extraction method (also referred to herein as "Phase") and a maximum urine sample input volume of 160 mL, while the commercial extraction kits used the maximum sample input volume and optimal output volume recommended by the manufacturer, and the extraction was performed according to the manufacturer's instructions. Urine samples were provided by 4 males and 4 females (n=8 for each kit). The conditions are summarized in Table 7. Table 7: Comparison of yield and efficiency using recommended input and output volumes.

[0595] Reference now Figure 7B , which shows the average concentration (copies / uL) of DNA recovered using the kits and conditions according to Table 7. 140bp spike-in detection by Droplet digital PCR ddPCR. The results show that the present extraction method is able to significantly outperform all competitor kits due to the ability to process high input volumes and concentrate them to low output volumes.

[0596] In summary, the experiments in Examples 7a and 7b show that the total DNA recovery rate of the present extraction method is significantly greater than all other commercially available kits due to the increased allowed sample input volume and low output / elution volume of the present extraction method and the higher recovery efficiency of the target DNA.

[0597] The exemplary embodiments of the present invention are thus fully described. Although the description refers to specific embodiments, it will be clear to those skilled in the art that the present invention can be implemented by changes in these specific details. Therefore, the present invention should not be interpreted as being limited to the embodiments set forth herein. Numbered Example 1

[0598] Embodiment 1. A method for concentrating and purifying one or more target analytes from a large volume of fluid sample, the method comprising the following steps: (a) preparing a first aqueous two-phase system (ATPS) composition, wherein the first ATPS composition comprises a polymer, a salt, a surfactant, or a combination thereof dissolved in an aqueous solution to form a first phase solution and a second phase solution; (b) adding a sample solution prepared from the large volume of fluid sample containing the one or more target analytes to the first ATPS composition, so that the one or more target analytes are distributed into the first phase solution; (c) collecting the first phase solution and mixing the first phase solution with a second ATPS composition, wherein the second The ATPS composition comprises a polymer, a salt, a surfactant or a combination thereof dissolved in an aqueous solution to form a third phase solution and a fourth phase solution, so that the one or more target analytes are distributed into the third phase solution and concentrated in the third phase solution; (d) collecting the third phase solution and mixing the third phase solution with a binding buffer to form a mixed solution, wherein the binding buffer comprises at least one discrete agent; (e) loading the mixed solution onto an extraction column configured to selectively extract and purify the one or more target analytes; (f) eluting and collecting the one or more target analytes from the extraction column to obtain a final solution containing the concentrated and purified one or more target analytes.

[0599] Embodiment 2. A method according to any one of the preceding embodiments, wherein the sample solution is prepared by dividing the large amount of fluid sample containing the one or more target analytes into at least two samples of the sample solution, and the first ATPS composition is divided into at least two samples, wherein step (b) further comprises the following steps: (i) adding each sample of the sample solution prepared from the large amount of fluid sample containing the one or more target analytes to each sample of the first ATPS composition so that the one or more target analytes are distributed into the first phase solution; and (ii) collecting and merging the first phase solutions of the at least two samples of the first ATPS composition to form a first phase solution for step (c).

[0600] Embodiment 3. A method according to any one of the preceding embodiments, wherein the extraction column is a centrifugal column, and wherein step (e) further comprises the following steps: (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 the above steps (i) and (ii) until all of the mixed solution passes through the extraction column.

[0601] Embodiment 4. The method according to any one of the preceding embodiments, further comprising the following steps: (g) subjecting the final solution to a diagnostic assay to detect and quantify the one or more target analytes.

[0602] Embodiment 5. A method for concentrating and purifying one or more target analytes from a large number of fluid samples, the method comprising the following steps: (a) dividing the large number of fluid samples containing the one or more target analytes into at least two samples of sample solutions; (b) preparing a first aqueous two-phase system (ATPS) composition of the at least two samples, wherein the first ATPS composition comprises a polymer, a salt, a surfactant, or a combination thereof dissolved in an aqueous solution to form a first phase solution and a second phase solution; (c) adding each sample of the sample solution containing the one or more target analytes to each sample of the first ATPS composition, so that the one or more target analytes are distributed into the first phase solution; (d) collecting the first ATPS composition; (e) collecting the third phase solution and mixing the third phase solution with a binding buffer to form a mixed solution, wherein the binding buffer comprises at least one discrete 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.

[0603] Embodiment 6. A method according to any of the preceding embodiments, wherein the plurality of fluid samples is selected from the group consisting of blood, plasma, serum, cerebrospinal fluid, urine, saliva, feces, tears, sputum, nasopharyngeal mucus, vaginal secretions, and penile secretions.

[0604] Embodiment 7. The method according to any one of the preceding embodiments, wherein the bulk fluid sample is urine.

[0605] Embodiment 8. The method according to any of the preceding embodiments, wherein the volume of the plurality of fluid samples is 40 mL or greater.

[0606] Embodiment 9. The method according to any one of the preceding embodiments, wherein the volume of each aliquot of the sample solution is at most 40 ml.

[0607] Embodiment 10. The method according to any one of the preceding embodiments, wherein the target analyte is selected from the group consisting of nucleic acids, proteins, antigens, biomolecules, sugar moieties, lipids, sterols, and combinations thereof.

[0608] Embodiment 11. The method according to any one of the preceding embodiments, wherein the target analyte is DNA.

[0609] Embodiment 12. A method according to any one of the preceding embodiments, wherein the target analyte is free DNA or circulating tumor DNA.

[0610] Embodiment 13. The method of any one of the preceding embodiments, wherein the polymer is dissolved in the aqueous solution at a concentration of 4%-84% (w / w).

[0611] Embodiment 14. The method according to any of the preceding embodiments, wherein the polymer is selected from the group consisting of polyalkylene glycols, such as hydrophobically modified polyalkylene glycols, poly(oxyalkylene) polymers, poly(oxyalkylene) copolymers, such as hydrophobically modified poly(oxyalkylene) copolymers, polyvinyl pyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, polyvinyl methyl ether, alkoxylated surfactants, alkoxylated starch, alkoxylated cellulose, alkyl hydroxyalkyl cellulose, silicon-modified polyethers, and poly-N-isopropylacrylamide and copolymers thereof. The method according to any of the preceding embodiments, wherein the polymer is selected from the group consisting of polyethers, polyimines, polyalkylene glycols, vinyl polymers, alkoxylated surfactants, polysaccharides, alkoxylated starch, alkoxylated cellulose, alkyl hydroxyalkyl cellulose, polyether-modified silicones, polyacrylamides, polyacrylic acids and copolymers thereof. According to the method described in any one of the preceding 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), polyvinyl pyrrolidone, 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, polyethylene imine, poly-N-isopropyl acrylamide and copolymers thereof. According to the method described in any one of the preceding 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), polyvinyl pyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, polyvinyl methyl ether and poly-N-isopropyl acrylamide. According to the method described in any one of the preceding embodiments, the polymer is selected from the group consisting of polyacrylamide, polyacrylic acid and copolymers thereof. The method according to any of the preceding embodiments, wherein the polymer is selected from the group consisting of dextran, carboxymethyl dextran, dextran sulfate, hydroxypropyl dextran and starch. The method according to any of the preceding embodiments, wherein the average molecular weight of the polymer is in the range of 200-1,000Da, 200-35,000Da, 425-2,000Da, 400-35,000Da, 980-12,000Da or 3,400-5,000,000Da. The method according to any of the preceding embodiments, wherein the polymer comprises ethylene oxide and propylene oxide units, and the polymer has an EO:PO ratio of 90:10 to 10:90.

[0612] Example 15.

[0613] Embodiment 16. The method according to any one of the preceding embodiments, wherein the salt is dissolved in the aqueous solution at a concentration of 1%-55% (w / w).

[0614] Embodiment 17. The method according to any one of the preceding embodiments, wherein the salt is dissolved in the aqueous solution at a concentration of 8%-55% (w / w).

[0615] Embodiment 18. A method according to any of the preceding embodiments, wherein the salt is selected from the group consisting of kosmotropic salts, discrete salts, inorganic salts containing cations, such as linear or branched trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium and tetrabutylammonium, and inorganic salts containing anions, such as 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 phosphates and ammonium sulfate.

[0616] Embodiment 19. The method according to any one of the preceding embodiments, wherein the surfactant is dissolved in the aqueous solution at a concentration of 0.05%-10% (w / w).

[0617] Embodiment 20. The method according to any one of the preceding embodiments, wherein the surfactant is dissolved in the aqueous solution at a concentration of 0.05%-9.8% (w / w).

[0618] Embodiment 21. The method according to any of the preceding embodiments, wherein the surfactant is selected from the group consisting of Triton-X, Triton-114, Igepal CA-630 and Nonidet P-40, anionic surfactants (such as carboxylates, sulfonates, petroleum sulfonates, alkylbenzene sulfonates, naphthalene sulfonates, olefin sulfonates, alkyl sulfates, sulfates, sulfated natural oils, sulfated natural fats, sulfated esters, sulfated alkanolamides, ethoxylated alkylphenols and sulfated alkylphenols), nonionic surfactants (such as ethoxylated fatty alcohols, polyoxyethylene surfactants, carboxylic acid esters, polyethylene glycol esters, sorbitan esters, fatty acid glycol esters, carboxylic acid amides, monoalkanolamine concentrates, The invention relates to a group consisting of a polyoxyethylene fatty acid amide), a cationic surfactant (such as a quaternary ammonium salt, an amine with an amide bond, a polyoxyethylene alkyl and alicyclic amine, a n,n,n',n' tetrasubstituted ethylenediamine, a 2-alkyl 1-hydroxyethyl 2-imidazoline), and a zwitterionic surfactant (such as n-coco 3-aminopropionic acid / sodium salt, n-tallow 3-iminodipropionate, disodium salt, n-carboxymethyl n-dimethyl n-9 octadecenyl ammonium hydroxide, n-cocoamidoethyl n-hydroxyethylglycine and its sodium salt).

[0619] Embodiment 22. An ATPS composition selected from the group consisting of A1, A2, A3, A4, AA1, AA2, AA3 and AA4.

[0620] Embodiment 23. A kit comprising a first ATPS composition selected from the group consisting of A1, A2, A3 and A4; a second ATPS composition selected from the group consisting of AA1, AA2, AA3 and AA4; and a binding buffer selected from the group consisting of Bl, B2 and B3.

[0621] Embodiment 24. The kit according to any one of the preceding embodiments, further comprising an extraction column. Numbered Example 2

[0622] Example 1. A method for concentrating and purifying one or more target analytes from a sample solution, the method comprising the following steps: (a) adding a sample solution containing one or more target analytes to a first aqueous two-phase system (ATPS) to form a mixture separated into a first phase and a second phase, wherein the one or more target analytes are concentrated in the first phase; (b) separating the first phase containing the concentrated one or more target analytes to obtain a concentrated solution; (c) applying magnetic beads to the concentrated solution so that the magnetic beads bind to the one or more target analytes to form a bead-analyte complex; and (d) recovering the one or more target analytes from the bead-analyte complex to obtain a final solution containing the concentrated and purified one or more target analytes.

[0623] Embodiment 2. A method according to any one of the preceding embodiments, wherein the step (b) further comprises the following steps: (i) adding the separated first phase containing the concentrated one or more target analytes to a second ATPS to form a second mixture divided into a third phase and a fourth phase, wherein the one or more target analytes are concentrated in the third phase; and (ii) separating the third phase containing the concentrated one or more target analytes to form a concentrated solution for step (c) in step (b).

[0624] Embodiment 3. A method according to any one of the preceding embodiments, wherein the concentrated solution of step (b) is mixed with a binding buffer, wherein the binding buffer comprises at least one dispersant selected from the group consisting of n-butanol, ethanol, guanidine chloride, guanidine thiocyanate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea and urea, thereby obtaining the concentrated solution for step (c).

[0625] Embodiment 4. A method according to any one of the preceding embodiments, wherein the step (d) further comprises the following steps: (i) mixing the bead-analyte complex with a grading buffer comprising a polymer, a salt, a surfactant, a dispersant or a combination thereof to form a grading solution, so that the one or more target analytes below the target size are released from the bead-analyte complex into the grading solution; (ii) fixing the bead-analyte complex using a magnetic support; and (iii) separating the one or more target analytes below the target size in the grading solution from the fixed bead-analyte complex.

[0626] Embodiment 5. A method according to any one of the preceding embodiments, wherein step (d) further comprises the following steps: (iv) adding the one or more target analytes separated below the target size to a second binding buffer, wherein the second binding buffer comprises at least one dispersant selected from the group consisting of 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 a mixture of the one or more target analytes separated below the target size and the second binding buffer, wherein the magnetic beads bind to the one or more target analytes below the target size to form a second bead-analyte complex; and (vi) recovering the one or more target analytes from the second bead-analyte complex.

[0627] Embodiment 6. The method according to any one of the preceding embodiments, further comprising the following steps: (e) subjecting the final solution to a diagnostic assay to detect and quantify the one or more target analytes.

[0628] Embodiment 7. The method according to any of the preceding embodiments, 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.

[0629] Embodiment 8. The method according to any one of the preceding embodiments, wherein the one or more target analytes are DNA.

[0630] Embodiment 9. A method according to any one of the preceding embodiments, wherein the one or more target analytes are free DNA or circulating tumor DNA.

[0631] Embodiment 10. The method according to any one of the preceding embodiments, wherein 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 a combination thereof.

[0632] Embodiment 11. The method according to any one of embodiments 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 is dissolved in an aqueous solution, wherein the second ATPS component is selected from the group consisting of a polymer, a salt, a surfactant, and a combination thereof.

[0633] Embodiment 12. The method of embodiment 10 or 11, wherein the polymer is dissolved in the aqueous solution at a concentration of 4%-84% (w / w).

[0634] Embodiment 13. The method according to any one of embodiments 10-12, wherein the polymer is selected from the group consisting of polyalkylene glycols, such as hydrophobically modified polyalkylene glycols, poly(oxyalkylene) polymers, poly(oxyalkylene) copolymers, such as hydrophobically modified poly(oxyalkylene) copolymers, polyvinyl pyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, polyvinyl methyl ether, alkoxylated surfactants, alkoxylated starch, alkoxylated cellulose, alkyl hydroxyalkyl cellulose, silicon-modified polyethers, and poly-N-isopropylacrylamide and copolymers thereof. The method according to any one of the preceding embodiments, wherein the polymer is selected from the group consisting of polyethers, polyimines, polyalkylene glycols, vinyl polymers, alkoxylated surfactants, polysaccharides, alkoxylated starch, alkoxylated cellulose, alkyl hydroxyalkyl cellulose, polyether-modified silicones, polyacrylamides, polyacrylic acids and copolymers thereof. According to the method described in any one of the preceding 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), polyvinyl pyrrolidone, 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, polyethylene imine, poly-N-isopropyl acrylamide and copolymers thereof. According to the method described in any one of the preceding 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), polyvinyl pyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, polyvinyl methyl ether and poly-N-isopropyl acrylamide. According to the method described in any one of the preceding embodiments, the polymer is selected from the group consisting of polyacrylamide, polyacrylic acid and copolymers thereof. The method according to any of the preceding embodiments, wherein the polymer is selected from the group consisting of dextran, carboxymethyl dextran, dextran sulfate, hydroxypropyl dextran and starch. The method according to any of the preceding embodiments, wherein the average molecular weight of the polymer is in the range of 200-1,000Da, 200-35,000Da, 425-2,000Da, 400-35,000Da, 980-12,000Da or 3,400-5,000,000Da. The method according to any of the preceding embodiments, wherein the polymer comprises ethylene oxide and propylene oxide units, and the polymer has an EO:PO ratio of 90:10 to 10:90.

[0635] Example 14.

[0636] Embodiment 15. The method of any one of embodiments 10-14, wherein the salt is dissolved in the aqueous solution at a concentration of 1%-80% (w / w).

[0637] Embodiment 16. The method of any one of embodiments 10-15, wherein the salt is dissolved in the aqueous solution at a concentration of 8%-80% (w / w).

[0638] Embodiment 17. A method according to any one of embodiments 10-16, wherein the salt is selected from the group consisting of kosmotropic salts, discrete salts, inorganic salts containing cations, such as linear or branched trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium and tetrabutylammonium, and inorganic salts containing anions, such as 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 phosphates and ammonium sulfate.

[0639] Embodiment 18. The method of any one of embodiments 10-17, wherein the surfactant is dissolved in the aqueous solution at a concentration of 0.05%-10% (w / w).

[0640] Embodiment 19. The method of any one of embodiments 10-18, wherein the surfactant is dissolved in the aqueous solution at a concentration of 0.05%-9.8% (w / w).

[0641] Embodiment 20. The method according to any one of embodiments 10-19, wherein the surfactant is selected from Triton-X, Triton-114, Igepal CA-630 and Nonidet P-40, anionic surfactants (such as carboxylates, sulfonates, petroleum sulfonates, alkylbenzene sulfonates, naphthalene sulfonates, olefin sulfonates, alkyl sulfates, sulfates, sulfated natural oils, sulfated natural fats, sulfated esters, sulfated alkanolamides, ethoxylated alkylphenols and sulfated alkylphenols), nonionic surfactants (such as ethoxylated fatty alcohols, polyoxyethylene surfactants, carboxylates, polyethylene glycol esters, sorbitan esters, fatty acid glycol esters, carboxylic acid amides, monoalkanolamine concentrates, The invention relates to a group consisting of a polyoxyethylene fatty acid amide), a cationic surfactant (such as a quaternary ammonium salt, an amine with an amide bond, a polyoxyethylene alkyl and alicyclic amine, a n,n,n',n' tetrasubstituted ethylenediamine, a 2-alkyl 1-hydroxyethyl 2-imidazoline), and a zwitterionic surfactant (such as n-coco 3-aminopropionic acid / sodium salt, n-tallow 3-iminodipropionate, disodium salt, n-carboxymethyl n-dimethyl n-9 octadecenyl ammonium hydroxide, n-cocoamidoethyl n-hydroxyethylglycine and its sodium salt).

[0642] Embodiment 21. A method according to any one of the preceding embodiments, wherein step (a) further comprises the following steps: (i) embedding a component capable of forming a first ATPS into a porous material; and (ii) contacting the sample solution with the porous material with the embedded component, wherein the component forms a first phase and a second phase when the sample solution passes through the porous material. Numbered Example 3

[0643] Embodiment 1. A method for concentrating and purifying at least one target analyte from a clinical biological sample, the method comprising the following steps: (a) combining the clinical biological sample with a first aqueous two-phase system (ATPS) composition to form a target enriched phase solution and a target lean phase solution, the 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; (b) collecting the target enriched phase; (c) optionally adding the target enriched phase to a second ATPS composition to form a second target enriched phase solution and a second target lean phase solution, and collecting the second target enriched phase, the second ATPS composition comprising a polymer dissolved in an aqueous solution; (d) optionally mixing the target enriched phase from step (b) or the second target enriched phase from step (c) with a binding buffer to form a mixed solution; (e) contacting the target enriched phase from step (b), the second target enriched phase from step (c) or the mixed solution from step (d) with a solid phase medium, the solid phase medium being configured to selectively bind the target analyte, so that the solid phase medium binds the target analyte; and (f) eluting and collecting the target analyte from the solid phase medium with an elution solution to obtain a final solution containing a concentrated and purified target analyte.

[0644] Embodiment 2. According to any one of the preceding embodiments, the method further comprises a step of washing the solid phase medium with one or more appropriate solvents to remove impurities after step (e) and before step (f).

[0645] Embodiment 3. The method according to any one of the preceding embodiments, wherein the suitable solvent is a binding buffer.

[0646] Embodiment 4. The method according to any one of the preceding embodiments, further comprising a step of treating the clinical biological sample with a lysis composition before step (a).

[0647] Embodiment 5. A method according to any of the preceding embodiments, wherein the binding buffer comprises a dispersant comprising an anion selected from the group consisting of thiocyanate, isothiocyanate, perchlorate, acetate, trichloroacetate, trifluoroacetate, chloride and iodide.

[0648] Embodiment 6. A method according to any of the preceding embodiments, wherein the binding buffer comprises a discrete 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.

[0649] Embodiment 7. The method according to any one of the preceding embodiments, wherein the binding buffer comprises a 2M to 7M solution of the dispersant.

[0650] Embodiment 8. The method according to any one of the preceding embodiments, wherein the binding buffer further comprises a polymer at a concentration of 5-20% (w / v).

[0651] Embodiment 9. A method according to any of the preceding embodiments, wherein the clinical biological sample is blood, plasma, urine, saliva, feces, cerebrospinal fluid (CSF), lymph fluid, serum, sputum, peritoneal fluid, sweat, tears, nasal swab, vaginal swab, endocervical swab, semen or breast milk.

[0652] Embodiment 10. The method according to any one of the preceding embodiments, wherein the clinical biological sample is <500 mL.

[0653] Embodiment 11. The method according to any one of the preceding embodiments, wherein the target analyte is selected from the group consisting of nucleic acids, proteins, antigens, biomolecules, sugar moieties, lipids, sterols, exosomes, and combinations thereof.

[0654] Embodiment 12. A method according to any one of the preceding embodiments, wherein the target analyte is a nucleic acid, and the nucleic acid is gDNA, cDNA, plasmid DNA, mitochondrial DNA, cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), circulating fetal DNA, microbial cell-free DNA, microRNA (miRNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), circular RNA, long non-coding RNA (lncRNA) or a combination thereof.

[0655] Embodiment 13. A method according to any one of the preceding embodiments, wherein the nucleic acid is cfDNA, fetal free DNA, mitochondrial DNA, microbial free DNA or ctDNA.

[0656] Embodiment 14. A method according to any one of the preceding embodiments, wherein step (e) comprises the following steps: (i) contacting a portion of the mixed solution with the solid phase medium so that the target analyte binds to the solid phase medium to form a solid phase extraction complex; (ii) disturbing the solid phase extraction complex and discarding the flow-through liquid; and (iii) optionally repeating steps (i) and (ii).

[0657] Embodiment 15. The method according to any one of the preceding embodiments, wherein the solid phase medium is a plurality of beads.

[0658] Embodiment 16. The method of any one of the preceding embodiments, wherein the beads are selected from the group consisting of magnetic beads, silica-based beads, carboxyl beads, hydroxyl beads, and amine-coated beads.

[0659] Embodiment 17. The method of any preceding embodiment, wherein the solid phase extraction complex is a bead-analyte complex; the perturbation is rotation; and the flow-through is supernatant.

[0660] Embodiment 18. A method according to any one of the preceding embodiments, wherein 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; and the elution solution is a graded buffer which, when in contact with the beads during the elution step (f), causes the target analyte to be released without releasing the other nucleic acids, thereby obtaining a final solution containing one or more concentrated and purified target analytes.

[0661] Embodiment 19. The method according to any one of the preceding embodiments, wherein the grading buffer comprises a polymer, a discretizing agent, or any combination thereof.

[0662] Embodiment 20. The method according to any one of the preceding embodiments, wherein the solid phase medium is an extraction column.

[0663] Embodiment 21. The method according to any one of the preceding embodiments, wherein the extraction column is a centrifugal column.

[0664] Embodiment 22. A method according to any one of the preceding embodiments, wherein step (e) comprises the following steps: (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 the above steps (i) and (ii) one or more times until all of the mixed solution passes through the extraction column.

[0665] Embodiment 23. A method according to any of the preceding embodiments, wherein the 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.

[0666] Embodiment 24. A method according to any of the preceding embodiments, 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(hydroxymethylaminomethane).

[0667] Embodiment 25. A method according to any of the preceding embodiments, wherein the polymer is selected from the group consisting of polyethers, polyimines, polyacrylates, polyalkylene glycols, vinyl polymers, alkoxylated surfactants, polysaccharides, alkoxylated starches, alkoxylated celluloses, alkyl hydroxyalkyl celluloses, polyether-modified silicones, polyacrylamides, polyacrylic acids, and one or more copolymers thereof.

[0668] Embodiment 26. A method according to any of the preceding embodiments, 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), polyvinyl pyrrolidone, 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, polyethylene imine, poly-N-isopropylacrylamide and copolymers thereof.

[0669] Embodiment 27. The method according to any one of the preceding embodiments, wherein 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); wherein the nonionic surfactant is an ethoxylated fatty alcohol, a polyoxyethylene surfactant Surfactant, carboxylic acid ester, polyethylene glycol ester, sorbitan ester, fatty acid glycol ester, carboxamide, monoalkanolamine condensate or polyoxyethylene fatty acid amide; the cationic surfactant is a quaternary ammonium salt, an amine with an amide bond, a polyoxyethylene alkylamine, a polyoxyethylene alicyclic amine, n,n,n',n'tetrasubstituted ethylenediamine or 2-alkyl 1-hydroxyethyl 2-imidazoline; and the amphoteric surfactant is n-coconut 3-aminopropionic acid or its sodium salt, n-tallow 3-iminodipropionic acid or its disodium salt, n-carboxymethyl n-dimethyl n-9 octadecenyl ammonium hydroxide or n-coconut amide ethyl n-hydroxyethyl glycine or its sodium salt.

[0670] Embodiment 28. The method according to any one of the preceding embodiments, wherein the surfactant is TritonX-100, Triton X-114, Triton X-45, Tween 20, Igepal CA630, Brij 58, Brij O10, BrijL23, Pluronic L-61, Pluronic F-127, sodium dodecyl sulfate, sodium cholate, sodium deoxycholate, sodium N-lauroyl sarcosinate (NLS), hexadecyltrimethylammonium bromide or span 80.

[0671] Embodiment 29. The method of any of the preceding embodiments, wherein the average molecular weight of the polymer is in the range of 200-1,000 Da, 200-35,000 Da, 300-35,000 Da, 400-2,000 Da, 400-35,000 Da, 2,500-2,500,000 Da, 1,250-4,000,000 Da, or 6,000-5,000,000 Da.

[0672] Embodiment 30. The method of any one of the preceding embodiments, wherein the polymer of the first ATPS composition or the second ATPS composition is dissolved in an aqueous solution at a concentration of 0.5%-80% (w / v).

[0673] Embodiment 31. The method according to any one of the preceding embodiments, wherein the polymer of the first ATPS composition or the second ATPS composition is dissolved in an aqueous solution at a concentration of 0.2%-50% (w / v).

[0674] Embodiment 32. The method according to any one of the preceding embodiments, wherein the salt component of the first ATPS composition or the second ATPS composition is dissolved in an aqueous solution at a concentration of 0.1% to 80% (w / v).

[0675] Embodiment 33. The method according to any one of the preceding embodiments, wherein the surfactant of the first ATPS composition or the second ATPS composition is dissolved in an aqueous solution at a concentration of 0.1%-90% (w / v).

[0676] Embodiment 34. The method according to any one of the preceding embodiments, wherein the first ATPS composition or the second ATPS composition is a polymer-salt system, a polymer-polymer system, or a micellar system.

[0677] Embodiment 35. A method according to any of the preceding embodiments, wherein the first ATPS composition or the second ATPS composition is a polymer-salt system; the polymer is dissolved in an aqueous solution at a concentration of 0.5%-80% (w / v); and the salt component is dissolved in an aqueous solution at a concentration of 0.1%-80% (w / v).

[0678] Embodiment 36. A method according to any one of the preceding embodiments, wherein the polymer of the first ATPS composition is dissolved in an aqueous solution at a concentration of 5%-80% (w / v), and the salt component of the first ATPS composition is dissolved in an aqueous solution at a concentration of 0.1%-80% (w / v); and the polymer of the second ATPS composition is dissolved in an aqueous solution at a concentration of 0.5%-30% (w / v), and the salt component of the second ATPS composition is dissolved in an aqueous solution at a concentration of 5%-60% (w / w).

[0679] Embodiment 37. The method according to any of the preceding embodiments, wherein the first ATPS composition or the second ATPS composition further comprises at least one salt at a concentration of 0.01%-10% (w / v), or at least one surfactant at a concentration of 0.01%-10% (w / v).

[0680] Embodiment 38. The method according to any one of the preceding embodiments, wherein the first ATPS composition or the second ATPS composition is a polymer-polymer system comprising at least two polymers, and each polymer is dissolved in an aqueous solution at a concentration of 0.2%-50% (w / v).

[0681] Embodiment 39. The method according to any of the preceding embodiments, wherein the first ATPS composition or the second ATPS composition further comprises at least one salt at a concentration of 0.01%-10% (w / v), or at least one surfactant at a concentration of 0.01%-10% (w / v).

[0682] Embodiment 40. The method according to any of the preceding embodiments, wherein 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 sodium N-lauroyl sarcosinate (NLS); the nonionic surfactant is ethoxylated fatty alcohol, polyoxyethylene surfactant, carboxylic acid ester, polyethylene glycol ester, dehydrated sorbitan ester, fatty acid glycol ester, carboxamide, monoalkanolamine condensate or polyoxyethylene fatty acid amide; the cationic surfactant is quaternary ammonium salt, amine with amide bond, polyoxyethylene alkylamine, polyoxyethylene alicyclic amine, n,n,n',n'tetrasubstituted ethylenediamine or 2-alkyl 1-hydroxyethyl 2-imidazoline; and the amphoteric surfactant is n-coconut 3-aminopropionic acid or its sodium salt, n-tallow 3-iminodipropionic acid or its disodium salt, n-carboxymethyl n-dimethyl n-9 octadecenyl ammonium hydroxide or n-coconut amide ethyl n-hydroxyethyl glycine or its sodium salt.

[0683] Embodiment 41. The method according to any one of the preceding embodiments, wherein the first ATPS composition or the second ATPS composition is a micellar system comprising at least one surfactant, and each surfactant is dissolved in an aqueous solution at a concentration of 0.1%-90% (w / v).

[0684] Embodiment 42. The method according to any one of the preceding embodiments, wherein the first ATPS composition or the second ATPS composition further comprises at least one salt at a concentration of 0.01%-30% (w / v).

[0685] Embodiment 43. A method according to any of the preceding embodiments, wherein 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.

[0686] Embodiment 44. The method according to any of the preceding embodiments, wherein the first ATPS composition or the second ATPS composition is selected from Table 1.0.

[0687] Embodiment 45. The method according to any one of the preceding embodiments, wherein the first ATPS composition or the second ATPS composition further comprises 0.5-2 mM ethylenediaminetetraacetic acid (EDTA).

[0688] Embodiment 46. The method according to any of the preceding embodiments, further comprising a step of 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 PCR-based assays.

[0689] Embodiment 47. A method according to any of the preceding embodiments, wherein the target analyte is a biomarker that indicates the presence or risk of a medical condition or disease in the patient, wherein the medical condition or disease is an infectious disease, cancer, or a genetic disease.

[0690] Embodiment 48. A method according to any of the preceding embodiments, wherein the clinical biological sample is a large fluid sample with a volume >10 mL, and further comprising the following steps before step (a): dividing the large fluid sample into at least two samples of sample solution; wherein in step (a) each sample is individually combined with the first ATPS, and optionally, in step (c) the collected target enriched phase from step (b) is individually combined with the second ATPS to form a target enriched phase for each sample.

[0691] Embodiment 49. A method according to any of the preceding embodiments, wherein the target enriched phase from each sample of step (b) or the second target enriched phase from each sample of step (c) is combined together to form a final target enriched phase for step (d).

[0692] Embodiment 50. A method according to any of the preceding embodiments, wherein the plurality of fluid samples is urine.

[0693] Embodiment 51. A method for 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) concentrating and purifying at least one target analyte from the clinical biological sample according to the method of any of the preceding embodiments; (iii) analyzing the final solution; and (iv) treating the patient if the information obtained from the target analyte indicates that the patient has cancer or is at risk of developing cancer.

[0694] Embodiment 52. A method according to any of the preceding embodiments, 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.

[0695] Embodiment 53. A method according to any of the preceding embodiments, wherein the target analyte is renal cFDNA or ctDNA, and wherein the cancer is a systemic cancer.

[0696] Embodiment 54. A method according to any of the preceding embodiments, wherein the target analyte is urogenital tract cFDNA or ctDNA, and wherein the cancer is urogenital tract cancer.

[0697] Embodiment 55. The method of any one of the preceding embodiments, wherein the target analyte is bladder cancer DNA and the medical condition or disease is bladder cancer.

[0698] Embodiment 56. A method according to any of the preceding embodiments, wherein the target analyte is HPV viral RNA or HPV viral DNA, and the medical condition or disease is HPV.

[0699] Embodiment 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 any one of the preceding embodiments.

Claims

1. A method for concentrating and purifying at least one target analyte from a clinical biological sample, the method comprising the following steps: (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 lean phase solution; (b) collecting the target enriched 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 lean phase solution, and collecting the second target rich phase; (d) optionally mixing the target enriched phase from step (b) or the second target enriched phase from step (c) with a binding buffer to form a mixed solution; (e) contacting the target enriched phase from step (b), the second target enriched phase from step (c), or the mixed solution from step (d) with a solid phase medium configured to selectively bind the target analyte, such that the solid phase medium binds the target analyte; as well as (f) eluting and collecting the target analyte from the solid phase medium with an elution solution to obtain a final solution containing concentrated and purified target analyte.

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

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

4. The method according to claim 1, further comprising a step of treating the clinical biological sample with a lysis composition before step (a).

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

6. The method of claim 1, wherein the binding buffer comprises a discrete 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 2M to 7M solution of the dispersant.

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

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

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

11. The method of claim 1, wherein the target analyte is selected from the group consisting of nucleic acids, proteins, antigens, biomolecules, sugar moieties, lipids, sterols, exosomes, and any combination thereof.

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, cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), circulating fetal DNA, microbial cell-free DNA, microRNA (miRNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), circular RNA, long noncoding RNA (lncRNA), or a combination thereof.

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

14. The method of claim 1, wherein step (e) comprises the following steps: (i) contacting a portion of the mixed solution with the solid phase medium, so that the target analyte binds to the solid phase medium to form a solid phase extraction complex; (ii) disturbing the solid phase extraction complex and discarding the flow-through; as well as (iii) optionally repeating steps (i) and (ii).

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

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

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

18. The method according to claim 15, wherein 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; The elution solution is a fractionation buffer which, when contacted with the beads during the elution step (f), results in the release of the target analyte without releasing other nucleic acids, resulting in a final solution containing concentrated and purified one or more target analytes.

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

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

22. The method of claim 20, wherein step (e) comprises the steps of: (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; as well as (iii) optionally repeating the above steps (i) and (ii) one or more times until all of the mixed solution passes through the extraction column.

23. 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, linear or branched trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium, and tetrabutylammonium.

24. 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.

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

26. 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), polyvinyl pyrrolidone, 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, polyethylene imine, poly-N-isopropylacrylamide, and copolymers thereof.

27. The method of claim 1, wherein The surfactant is selected from the group consisting of anionic surfactants, nonionic surfactants, cationic surfactants and amphoteric surfactants; in 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 carboxamide, 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-coconut 3-aminopropionic acid or its sodium salt, n-tallow 3-iminodipropionate or its disodium salt, n-carboxymethyl n-dimethyl n-9 octadecenyl ammonium hydroxide or n-coconut amide ethyl n-hydroxyethyl glycine or its sodium salt.

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-lauroyl sarcosinate (NLS), hexadecyltrimethylammonium bromide, or span 80.

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

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

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

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

33. The method of claim 1, 25 or 26, wherein the surfactant of the first ATPS composition or the second ATPS composition is dissolved in an aqueous solution at a concentration of 0.1%-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 micellar system.

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

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

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%-10% (w / v), or at least one surfactant at a concentration of 0.01%-10% (w / v).

38. 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 dissolved in an aqueous solution at a concentration of 0.2%-50% (w / v).

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%-10% (w / v), or at least one surfactant at a concentration of 0.01%-10% (w / v).

40. The method of claim 39, 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, 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 carboxamide, 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-coconut 3-aminopropionic acid or its sodium salt, n-tallow 3-iminodipropionate or its disodium salt, n-carboxymethyl n-dimethyl n-9 octadecenyl ammonium hydroxide or n-coconut amide ethyl n-hydroxyethyl glycine or its sodium salt.

41. 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 dissolved in an aqueous solution at a concentration of 0.1%-90% (w / v).

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%-30% (w / v).

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, linear or branched trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium, and tetrabutylammonium.

44. The method of claim 1, wherein the first ATPS composition and / or the second ATPS composition is selected from Table 1.

0.

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

46. ​​The method of claim 1, further comprising the step of 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 PCR-based assays.

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

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

49. The method of claim 48, wherein the target enriched phase from each sample of step (b) or the second target enriched phase from each sample of step (c) are combined together to form a final target enriched phase for step (d).

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

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) concentrating and purifying at least one target analyte from the clinical biological sample according to the method of claim 1; (iii) analyzing the final solution; as well as (iv) treating the patient if the information obtained from the target analyte indicates that the patient has cancer or is at risk of developing cancer.

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

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

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

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

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

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