Method and kit for isolating target nucleic acid below target size from sample

Abstract

The present disclosure relates to methods, compositions and kits for isolating a target nucleic acid below a target size from a sample comprising nucleic acid components. In some embodiments, the methods involve one or more aqueous two phase system (ATPS) compositions, at least one solid phase medium, and at least one buffer. Some embodiments provide kits comprising one or more ATPS compositions, at least one solid phase medium, and at least one buffer. Other embodiments provide methods of diagnosing a disease or condition using the methods described herein.

Description

Cross - Reference to Related Applications

[0001] This application claims the benefit and priority of U.S. Provisional Application Serial No. 63 / 381,933, filed Nov. 2, 2022. The entire content of the foregoing application is hereby incorporated by reference in its entirety for all purposes. Technical Field

[0002] This application relates to methods and kits for separating target nucleic acids. More specifically, this application relates to methods and kits for separating target nucleic acids that are smaller than a target size from a sample. Background Art

[0003] Effectively concentrating and separating target nucleic acids that are smaller than a certain size from a sample is a challenge, especially separating, purifying, and concentrating very rare and sparse nucleic acid fragments in the context of a complex biological matrix. Typically, the yield of the relevant fragments is so low that subsequent analysis may not have sufficient diagnostic sensitivity and specificity. For some applications, such as non - invasive prenatal testing (NIPT) and circulating tumor DNA enrichment, size differences are an important criterion for distinguishing target nucleic acids from non - target nucleic acids. Therefore, there is a need for improved methods that are simple, stable, robust, and effective for separating target nucleic acids that are smaller than a certain size from a sample. Summary of the Invention

[0004] Novel methods and kits are disclosed herein for separating, concentrating, and / or purifying target analytes (such as nucleic acids) that are smaller than a target size using a solid - phase medium (such as beads or columns).

[0005] In some embodiments, a method for separating a target nucleic acid that is smaller than a target size from a sample comprising a nucleic acid component is provided; the method comprising the steps of: (a) preparing a sample solution from the sample; (b) contacting a plurality of beads with the sample solution, wherein the nucleic acid component binds to the plurality of beads to form a bead - analyte complex; (c) mixing the bead - analyte complex with a fractionation buffer comprising at least one dissociating agent to form a bulk fractionation solution, wherein the target nucleic acid that is smaller than the target size is released from the bead - analyte complex into the bulk fractionation solution; (d) immobilizing the bead - analyte complex; and (e) separating the bulk fractionation solution comprising the separated target nucleic acid that is smaller than the target size from the immobilized bead - analyte complex.

[0006] In some embodiments, a kit for separating target nucleic acids smaller than a target size from a sample comprising a nucleic acid component is provided, the kit comprising: (a) at least one ATPS component selected from the group consisting of polymers, salts, surfactants, and combinations thereof; (b) a plurality of beads; (c) a fractionation buffer comprising at least one chaotropic agent selected from the group consisting of thiocyanate, isothiocyanate, perchlorate, acetate, trichloroacetate, trifluoroacetate, chloride, and iodide; and (d) a binding buffer comprising at least one chaotropic agent selected from the group consisting of thiocyanate, isothiocyanate, perchlorate, acetate, trichloroacetate, trifluoroacetate, chloride, and iodide.

[0007] In some embodiments, a method for concentrating and purifying one or more target analytes from a sample solution is provided, the method comprising the following steps: (a) Adding a sample solution containing the one or more target analytes to a first aqueous two-phase system (ATPS) to form a mixture that separates into a first phase and a second phase, 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 complexes to obtain a final solution comprising the concentrated and purified one or more target analytes.

[0008] In some embodiments, corresponding kits can be advantageously used in conjunction with and for performing methods according to various aspects of the present invention. In some embodiments, the kit can include the components described in the various embodiments, but can additionally include containers accessible by syringes or pipettes for storage, packaging, and / or reaction and optionally devices for manipulating aqueous solutions. Such containers and devices can include columns, test tubes, capillaries, plastic test tubes, falcon tubes, culture tubes, microtiter plates, pipettes, cuvettes, etc.

[0009] Other example embodiments are discussed herein.

[0010] Advantages

[0011] The various embodiments of the present disclosure have many advantages. For example, the methods and kits of the present disclosure surprisingly and effectively concentrate and purify target analytes below a target size from a sample (e.g., a clinical / biological sample). These methods and kits are particularly effective in purifying target analytes present in very low concentrations in biological samples (e.g., cell-free DNA). As shown in the examples, the methods of the present disclosure allow precise selection of the size of the recovered DNA molecules.

[0012] In some embodiments, the methods of the present disclosure (referred to in some embodiments as "reverse fractionation") utilize complete binding of the nucleic acid components in a sample, followed by selective release of target nucleic acids below a target size from a solid-phase medium (e.g., a magnetic bead-analyte complex or a solid-phase extraction column). Compared to methods that utilize a first binding step to selectively bind and remove unwanted larger nucleic acids, the disclosed methods achieve surprisingly improved nucleic acid size fractionation, leaving the target nucleic acids below the target size in the supernatant for further purification by the solid-phase medium.

[0013] In certain embodiments, the disclosed methods can accommodate variations in sample volume and can surprisingly achieve stable and effective DNA size fractionation across different sample volumes, with stable DNA retention values and DNA recovery, particularly for small DNA fragments. In certain embodiments, the disclosed methods are compatible with different sample types (e.g., plasma and urine) and demonstrate stable and effective DNA size fractionation across different sample types. This allows for a wide range of applications and analyses, such as the diagnosis of diseases or disorders that require different types of clinical / biological samples.

[0014] The purified nucleic acids obtained by the disclosed methods can be used in a wide range of downstream applications, such as nucleic acid detection or analysis in forensic, diagnostic, or therapeutic applications, as well as laboratory procedures such as sequencing, amplification, reverse transcription, labeling, digestion, blotting procedures, etc. The disclosed methods can improve the performance of downstream characterization or processing of nucleic acids.

[0015] In certain embodiments, the disclosed methods and kits can be used in a variety of applications. For example, the methods and kits of the present disclosure can be used for size-selective fractionation of DNA during the preparation of sequencing libraries, i.e., separating DNA molecules of a desired size or size range for subsequent sequencing applications, such as next-generation sequencing (NGS). In certain embodiments, the disclosed methods and kits can be used to enrich the fetal fraction in maternal samples for non-invasive prenatal testing (NIPT) by effectively separating fetal nucleic acids from maternal nucleic acids. In certain embodiments, the disclosed methods and kits can be used to increase the ratio of circulating tumor DNA:cell-free DNA and / or the variant allele frequency (VAF) in clinical samples for further analysis, such as cancer diagnostic assays.

[0016] These and other features and characteristics, as well as the operating methods and functions of the related components, will become more apparent upon consideration of the following detailed description and the appended claims in reference to the accompanying drawings, all of which form a part of this specification, where like reference numerals represent corresponding parts in the various figures. However, it should be clearly understood that the drawings are for illustrative and descriptive purposes only and are not intended as a definition of the limitations of the claims. Description of the Drawings

[0017] Figure 1A An exemplary workflow of direct fractionation is shown according to an exemplary embodiment.

[0018] Figure 1B An exemplary workflow of reverse fractionation is shown according to an exemplary embodiment.

[0019] Figure 2A An electropherogram of DNA oligonucleotide recovery from plasma extracted from different volumes of the top phase in the 2nd ATPS using direct fractionation (expected DNA retention value of about 150 bp) is shown according to an exemplary embodiment.

[0020] Figure 2B An electropherogram of DNA oligonucleotide recovery from plasma extracted from different volumes of the top phase in the 2nd ATPS using reverse fractionation (expected DNA retention value of about 150 bp) is shown according to an exemplary embodiment.

[0021] Figure 3A An electropherogram of DNA oligonucleotide recovery from plasma extracted from different volumes of the top phase in the 2nd ATPS using direct fractionation (expected DNA retention value of about 300 bp) is shown according to an exemplary embodiment.

[0022] Figure 3B An electropherogram of DNA oligonucleotide recovery from plasma extracted from different volumes of the top phase in the 2nd ATPS using reverse fractionation (expected DNA retention value of about 300 bp) is shown according to an exemplary embodiment.

[0023] Figure 4A An electropherogram of DNA oligonucleotide recovery for different sample types using direct fractionation (expected DNA retention value of about 150 bp) is shown according to an exemplary embodiment.

[0024] Figure 4B An electropherogram of DNA oligonucleotide recovery for different sample types using reverse fractionation (expected DNA retention value of about 150 bp) is shown according to an exemplary embodiment.

[0025] Figure 5A An electropherogram of DNA oligonucleotide recovery for different sample types using direct fractionation (expected DNA retention value of about 300 bp) is shown according to an exemplary embodiment.

[0026] Figure 5B An electropherogram showing the DNA oligonucleotide recovery of different sample types using reverse fractionation (expected DNA retention value of approximately 300 bp) is shown according to an exemplary embodiment.

[0027] Figure 6A-6H An electropherogram showing the DNA oligonucleotide recovery of plasma samples using reverse fractionation with different reverse fractionation buffer formulations (buffer R-015 to buffer R-021, respectively) is shown according to an exemplary embodiment. Detailed Description

[0028] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0029] As used herein and in the claims, the term "comprising" (or any related form such as "comprise" and "comprises"), "including" (or any related form such as "include" or "includes"), "containing" (or any related form such as "contain" or "contains"), or "having" (or any related form such as "have" or "has") means including the following elements but not excluding other elements. It should be understood that for each embodiment in which the term "comprising" (or any related form such as "comprise" and "comprises"), "including" (or any related form such as "include" or "includes"), or "containing" (or any related form such as "contain" or "contains") is used, this disclosure / application also includes alternative embodiments in which the term "comprising", "including", "containing", or "having" is replaced by "consisting essentially of" or "consisting of". These alternative embodiments using "consisting of" or "consisting essentially of" are understood to be embodiments of a narrower scope of the "comprising", "including", or "containing" embodiments.

[0030] For example, alternative embodiments of "a solution comprising A, B, and C" would be "a solution consisting of A, B, and C" and "a solution consisting essentially of A, B, and C". Even if the latter two embodiments are not explicitly written out, this disclosure / application includes those embodiments. Additionally, it should be understood that the scopes of the three embodiments listed above are different.

[0031] For clarity, "comprising", "including", and "containing" and any related forms are open-ended terms that allow additional elements or features in addition to the specified essential elements, while "consisting of" is a closed-ended term that is limited to the elements recited in the claim and does not include any elements, steps, or components not specified in the claim.

[0032] "Consisting essentially of" limits the scope of the claim to specific materials, components, or steps ("essential elements") that do not materially affect the (multiple) essential features of the claimed invention. In some embodiments, the essential features are one or more basic and novel features of the claimed invention.

[0033] As used herein, the singular forms "a / an" and "the" are intended to also include the plural forms, unless the context clearly indicates otherwise. In some embodiments, the term "a / an" may be interchangeable with terms such as "at least one" and "one or more".

[0034] 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 stated, a range is intended to include all values belonging to the range, including integers, fractions, parts, etc. For example, the range 1-7 described in a claim refers to a range that includes values and sub-ranges, such as 1, 1.5, 2-3, 6, and 7.

[0035] As used herein, the term "about" is understood to be within the normal tolerances in 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 therebetween. As used herein, the phrase "about" a particular value also includes the particular value, e.g., about 50 includes 50.

[0036] As used herein, "aqueous" refers to a characteristic property of a solvent / solute system in which the solvating substance predominantly has hydrophilic characteristics. Examples of aqueous solvent / solute systems include those in which water or an aqueous composition is the major solvent. Polymer and / or surfactant components (the use of which is described in the examples) are "aqueous" because they form an aqueous phase when combined with a solvent such as water. Further, as will be understood by one of ordinary skill in the art, herein the term liquid "mixture" refers only to combinations of components as defined herein.

[0037] As used herein, an aqueous two-phase system (ATPS) means a liquid-liquid separation system by which separation or concentration of an analyte can be achieved by partitioning, where two phases, portions, regions, components, etc. interact with at least one analyte to which they are exposed and optionally in which they are dissolved in different ways. An ATPS is formed when two immiscible phases of a certain concentration form components, such as salts and polymers, or two incompatible polymers (e.g., PEG and dextran) are mixed in an aqueous solution. ATPS methods are relatively inexpensive and scalable because they use two-phase partitioning to separate analytes (e.g., nucleic acids) from contaminants.

[0038] As used herein, the term "isolated" means removing an analyte from its original environment and thus changing it from its original environment. For example, an isolated nucleic acid provided typically has fewer non-nucleic acid components (e.g., proteins, lipids) compared to the amount of the component present in the source sample. A composition comprising an isolated analyte (e.g., a sample nucleic acid) can be substantially isolated (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% free of non-analyte components (e.g., non-nucleic acid components)).

[0039] As used herein, "concentrate" means that the mass ratio of the analyte in question to the solution in which the analyte is suspended is higher than the mass ratio of the analyte in its pre-concentrated solution. For example, it can be slightly higher, or more preferably at least two-fold, ten-fold or one-hundred-fold higher.

[0040] As used herein, "biological sample" means any tangible substance obtained directly or indirectly from a living organism (e.g., virus, bacterium, plant, animal or human). Examples of biological samples include but are not limited to nucleic acids, proteins, cells, organelles, tissue extracts, tissues, organs, biological fluids (e.g., blood, plasma, urine, saliva, feces, cerebrospinal fluid (CSF), lymph fluid, serum, sputum, peritoneal fluid, sweat, tears, nasal swabs, vaginal swabs, endocervical swabs, semen, breast milk and other body fluids).

[0041] As used herein, "clinical sample" refers to any sample obtained directly or indirectly from a subject (e.g., a human). In some embodiments, the subject is a human patient. Examples of clinical samples include, but are not limited to, blood, plasma, urine, saliva, feces, cerebrospinal fluid (CSF), lymph fluid, serum, sputum, peritoneal fluid, sweat, tears, nasal swabs, vaginal swabs, endocervical swabs, semen, breast milk, and other body fluids.

[0042] As used herein, "nucleic acid component" generally refers to nucleic acids extracted from a given sample, regardless of their size. In some embodiments, the nucleic acid component includes DNA, RNA, or a combination thereof. Examples of nucleic acid components include, but are not limited to, 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.

[0043] In some embodiments, "target nucleic acid", "target analyte", or "small DNA fragment" refers to a nucleic acid fragment below a selected size, e.g., a nucleic acid containing fewer than 1000 base pairs (e.g., fewer than 1000 bp, 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 450 bp, 400 bp, 350 bp, 300 bp, 250 bp, 200 bp, 150 bp, 100 bp, or 50 bp). In some embodiments, the target nucleic acid / analyte is a single-stranded nucleic acid, while in other embodiments, the target nucleic acid / analyte is a double-stranded nucleic acid. In some embodiments, the target nucleic acid / analyte is DNA or RNA. Examples of target nucleic acid / analyte include, but are not limited to, 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.

[0044] As used herein, "cell-free DNA" (cfDNA) is DNA that exists extracellularly, e.g., DNA present in a sample obtained from a subject (e.g., blood, plasma, serum, or urine).

[0045] As used herein, the term "polymer" refers to any polymer comprising at least one substituted or unsubstituted monomer. Examples of "polymers" include, but are not limited to, homopolymers, copolymers, terpolymers, random copolymers, and block copolymers. Block copolymers include, but are not limited to, block, graft, dendrimer, and star polymers.

[0046] As used herein, a "copolymer" refers to a polymer derived from two monomer species; similarly, a terpolymer refers to a polymer derived from three monomer species. Polymers also include various morphologies, including but not limited to linear polymers, branched polymers, random polymers, crosslinked polymers, and dendrimer systems. In some embodiments, the polymer also includes its chemically modified equivalents, such as hydrophobically modified or silicone-modified. For example, a polyacrylamide polymer refers to any polymer comprising at least one substituted or unsubstituted acrylamide unit, such as a homopolymer, copolymer, terpolymer, random copolymer, block copolymer, or terblock copolymer of polyacrylamide; the polyacrylamide can be a linear polymer, branched polymer, random polymer, crosslinked polymer, or dendrimer of polyacrylamide; the polyacrylamide can be a hydrophobically modified polyacrylamide, or a silicone-modified polyacrylamide.

[0047] In some embodiments, examples of polymers include, but are not limited to, polyethers, polyimines, polyalkylene glycols, alkoxylated surfactants, polysaccharides, polyether-modified silicones, polyacrylamides, polyacrylic acids, and their copolymers. In some embodiments, the polymer is hydrophobically modified or silicone-modified.

[0048] Examples of polyalkylene glycols (also referred to as "PAG" or "poly(alkylene oxide)" or "poly(epoxyalkane)") include, but are not limited to, hydrophobically modified polyalkylene glycols, poly(alkylene oxide) polymers, poly(alkylene oxide) copolymers, hydrophobically modified poly(alkylene oxide) copolymers, dipropylene glycol, tripropylene glycol, polyethylene glycol (also referred to as "PEG"), and polypropylene glycol (also referred to as "PPG"). In some embodiments, examples of copolymers of PAG include, but are not limited to, poly(ethylene glycol-propylene glycol) (also referred to as "PEG-PPG" or "UCON") and poly(ethylene glycol-random-propylene glycol) (also referred to as "PEG-random-PPG"). In some embodiments, PEG-PPG comprises a random copolymer, a block copolymer, or a combination thereof. In some embodiments, PEG-PPG comprises a random copolymer and a block copolymer. In some embodiments, PEG-PPG is PEG-random-PPG.

[0049] As used herein, "vinyl polymers" refers to a group of polymers derived from substituted vinyl (H2C=CHR) monomers. Examples of vinyl polymers include, but are not limited to, polyvinylpyrrolidone, polyvinyl alcohol, polyvinylcaprolactam, and polyvinyl methyl ether.

[0050] Examples of polysaccharides include, but are not limited to, dextran, carboxymethyl dextran, dextran sulfate, hydroxypropyl dextran, starch, carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl hydroxyethyl cellulose, and maltodextrin. In some embodiments, the polysaccharide is an alkoxylated starch, alkoxylated cellulose, or alkyl hydroxyalkyl cellulose.

[0051] Examples of polyacrylamides include, but are not limited to, poly N-isopropylacrylamide.

[0052] Examples of polyimines include, but are not limited to, polyethyleneimine.

[0053] 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-lauroylsarcosinate (NLS), ethoxylated fatty alcohols, polyoxyethylene surfactants, carboxylic esters, polyethylene glycol esters, sorbitan esters, fatty acid glycol esters, carboxamides, single alkanolamine condensates, and polyoxyethylene fatty acid amides.

[0054] In some embodiments, the polymer has an average molecular weight of about 200 - 1,000 Da, 200 - 35,000 Da, 300 - 35,000 Da, 400 - 2,000 Da, or 400 - 35,000 Da. Examples thereof include, but are not limited to, polyalkylene glycol (PAG) having an average molecular weight of about 400 Da, 500 Da, 600 Da, 700 Da, 800 Da, 900 Da, 1,000 Da, 2,000 Da, 3,000 Da, 4,000 Da, 5,000 Da, 6,000 Da, 7,000 Da, 8,000 Da, 9,000 Da, 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, and 35,000 Da. In some embodiments, the PAG has an average molecular weight within the range between any two of the above molecular weights.

[0055] Examples of PAGs include, but are not limited to, PEG 200, PEG 300, PEG 400, PEG 500, PEG 600, PEG 700, PEG 800, PEG 900, PEG 1000, PEG 2000, PEG 3000, PEG 4000, PEG 5000, PEG 6000, PEG7000, PEG 8000, PEG 9000, PEG 10000, PEG 15000, PEG 20000, PEG 25000, PEG 30000, PEG35000, PPG 425, PPG 725, PPG 900, PPG 1000, and PPG 2000. In some embodiments, the PEG has an average molecular weight in the range between any two of the above PEG molecular weights. In some embodiments, the PPG has an average molecular weight in the range between any two of the above PPG molecular weights.

[0056] 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 the range between any two of the above ratios.

[0057] In some embodiments, the polymer is a PAG having an average molecular weight of about 980 - 12,000 Da and an EO:PO ratio of 50:50 to 75:25. Examples thereof include, but are not limited to, PEG-PPGs having an average molecular weight of about 980 Da, 1,230 Da, 1,590 Da, 2,470 Da, 2,660 Da, 3,380 Da, 3,930 Da, 6,950 Da, and 12,000 Da. In some embodiments, the PEG-PPG has an average molecular weight in the range between any two of the above 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-random-PPG having an average molecular weight of about 2,500 or 12,000 Da and an EO:PO ratio of about 75:25.

[0058] In some embodiments, the polymer is a vinyl polymer having an average molecular weight of about 2,500 - 2,500,000 Da. Examples thereof include, but are not limited to, 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 the range between any two of the above molecular weights.

[0059] In some embodiments, the polymer is a polysaccharide and has an average molecular weight of about 6,000 - 5,000,000 Da. Examples thereof include, but are not limited to, dextran having an average molecular weight of about 6,000 Da, 12,000 Da, 25,000 Da, 60,000 Da, 70,000 Da, 80,000 Da, 150,000 Da, 270,000 Da, 410,000 Da, 450,000 Da, 550,000 Da, 650,000 Da, 670,000 Da, 1,500,000 Da, 2,000,000 Da, 2,800,000 Da, 4,000,000 Da, and 5,000,000 Da. In some embodiments, the dextran has an average molecular weight within the range between any two of the above molecular weights.

[0060] In some embodiments, the polymer is a polyether and has an average molecular weight of about 200 - 35,000 Da. Examples thereof include, but are not limited to, silicone-modified polyethers (or “polyether-modified silicones”) having an average molecular weight of about 200 - 35,000 Da.

[0061] In some embodiments, the polymer is a polyacrylamide and has an average molecular weight of 1,000 - 5,000,000 Da. Examples thereof include, but are not limited to, polyacrylamide or poly(N-isopropylacrylamide) having an average molecular weight of 1,000 Da, 2,000 Da, 5,000 Da, 10,000 Da, 40,000 Da, 85,000 Da, and 5,000,000 Da. In some embodiments, the polyolefin has an average molecular weight within the range between any two of the above molecular weights.

[0062] In some embodiments, the polymer is a polyacrylic acid and has an average molecular weight of about 1,250 - 4,000,000 Da. Examples thereof include, but are not limited to, polyacrylic acid having an average molecular weight of 1,200 Da, 2,100 Da, 5,100 Da, 8,000 Da, 8,600 Da, 8,700 Da, 16,000 Da, and 83,000 Da. In some embodiments, the polyolefin has an average molecular weight within the range between any two of the above molecular weights.

[0063] As used herein, the term "salt" refers to a substance containing a cation and an anion. Examples of salts include, but are not limited to, salts in which the cation is sodium, potassium, calcium, ammonium, lithium, magnesium, aluminum, cesium, barium, straight-chain or branched trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium, or tetrabutylammonium, and / or in which the anion is phosphate, hydrogen phosphate, dihydrogen phosphate, sulfate, sulfide, sulfite, bisulfate, carbonate, bicarbonate, acetate, nitrate, nitrite, sulfite, chloride, fluoride, chlorate, perchlorate, chlorite, hypochlorite, bromide, bromate, hypobromite, iodide, iodate, cyanate, thiocyanate, isothiocyanate, oxalate, formate, chromate, dichromate, permanganate, hydroxide, hydrogen ion, citrate, borate, or tris(hydroxymethyl)aminomethane. In some embodiments, the salt is a lyophilic salt, a discrete salt, or an inorganic salt.

[0064] In some embodiments, examples of "surfactants" include, but are not limited to, anionic surfactants, nonionic surfactants, cationic surfactants, zwitterionic surfactants, and amphoteric surfactants.

[0065] Examples of anionic surfactants include, but are not limited to, carboxylates, sulfonates, petroleum sulfonates, alkylbenzene sulfonates, naphthalene sulfonates, olefin sulfonates, alkyl sulfates, sulfates, sulfated natural oils and fats, sulfated esters, sulfated alkanolamides, ethoxylated alkylphenols, and sulfated alkylphenols.

[0066] Examples of nonionic surfactants include, but are not limited to, ethoxylated fatty alcohols, polyoxyethylene surfactants, carboxylic esters, polyethylene glycol esters, sorbitan esters, fatty acid glycol esters, carboxamides, monoolamine condensates, and polyoxyethylene fatty acid amides.

[0067] Examples of cationic surfactants include, but are not limited to, quaternary ammonium salts, amines having an amide bond, polyoxyethylene alkyl and alicyclic amines, N,N,N',N'-tetrasubstituted ethylenediamines, and 2-alkyl-1-hydroxyethyl-2-imidazolines.

[0068] Examples of amphoteric surfactants include, but are not limited to, N-cocoyl-3-aminopropionic acid / sodium salt, N-tallow-3-iminodipropionate disodium salt, N-carboxymethyl-N,N-dimethyl-N-9-octadecenyl ammonium hydroxide, N-cocoylamidoethyl-N-hydroxyethyl glycine and sodium salt, and sodium N-lauroylsarcosinate (NLS).

[0069] In some embodiments, the surfactant comprises a polymer such as PAG. In some embodiments, the surfactant has EO x -PO y -EO xThe structure, where EO refers to ethylene oxide units, PO refers to propylene oxide units, and x and y are the number of monomers, respectively. In some embodiments, x = 2 - 136. In some embodiments, y = 16 - 62. In some embodiments, examples of surfactants include but are not limited to (C2H4O) n C 14 H 22 O, where n = 4 - 10 (e.g., Triton X-100, Triton X-114, Triton X-45, Tween 20, Igepal CA630), Brij58, Brij O10, Brij L23, EOx-POy-EOx, where x = 2 - 136 and y = 16 - 62 (e.g., Pluronic L-61, Pluronic F-127), sodium dodecyl sulfate, sodium cholate, sodium cholate, sodium deoxycholate, sodium N-lauroylsarcosinate (NLS), cetyltrimethylammonium bromide, or span 80.

[0070] As used herein, the term "disrupting agent" refers to a substance that disrupts the hydrogen bond network between water molecules in a solution. For example, the disrupting agent can include anions selected from thiocyanate, isothiocyanate, perchlorate, acetate, trichloroacetate, trifluoroacetate, chloride, or iodide. The disrupting agent can include cations selected from sodium, guanidinium salts, lithium, or magnesium. Unless otherwise specified, the disrupting agent defined by a cation or an anion includes all compounds having the appropriate conjugate anion or cation, respectively. For example, 5M guanidinium salt includes guanidinium hydrochloride (GHCl), guanidinium thiocyanate, guanidinium isothiocyanate (GITC), etc. Examples of disrupting agents include but are not limited to guanidinium hydrochloride (GHCl), guanidinium thiocyanate, guanidinium isothiocyanate (GITC), sodium thiocyanate, sodium iodide, sodium perchlorate, sodium trichloroacetate, sodium trifluoroacetate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, thiourea, urea, etc.

[0071] The DNA sizes and cut-off values indicated herein with reference to base pairs "bp" refer to the chain lengths of DNA molecules and are thus used to describe the lengths of single-stranded as well as double-stranded DNA molecules. Thus, if the DNA is a single-stranded DNA molecule, it should be understood that the above indication of size or length in "bp" refers to the size or length of the nucleotides in the single-stranded DNA molecule. Embodiments of the present invention

[0072] Example 1

[0073] On the one hand, a method for concentrating and purifying one or more target analytes from a sample solution is provided, the method comprising the following steps: (a) Add a sample solution containing the one or more target analytes to a first aqueous two-phase system (ATPS) to form a mixture that separates into a first phase and a second phase, wherein the one or more target analytes are concentrated in the first phase; (b) Separate the first phase containing the concentrated one or more target analytes to obtain a concentrated solution; (c) Apply 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) Recover the one or more target analytes from the bead - analyte complexes to obtain a final solution containing the concentrated and purified one or more target analytes.

[0074] In some embodiments, step (b) further comprises the steps of: (i) Add the separated first phase containing the concentrated one or more target analytes to a second ATPS to form a second mixture that separates into a third phase and a fourth phase, wherein the one or more target analytes are concentrated in the third phase; and (ii) Separate the third phase containing the concentrated one or more target analytes to form the concentrated solution for step (c) in step (b).

[0075] In some embodiments, the concentrated solution of step (b) is mixed with a binding buffer, wherein the binding buffer comprises at least one chaotropic agent selected from the following: 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).

[0076] In some embodiments, step (d) further comprises the steps of: (i) Mix the bead - analyte complexes with a fractionation buffer comprising a polymer, a salt, a surfactant, a chaotropic agent, or a combination thereof to form a fractionation solution such that one or more target analytes smaller than the target size are released from the bead - analyte complexes into the fractionation solution; (ii) Fix the bead - analyte complexes using a magnetic holder; and (iii) Separate the one or more target analytes smaller than the target size in the fractionation solution from the fixed bead - analyte complexes.

[0077] In some embodiments, step (d) further comprises the steps of: (iv) Add the separated one or more target analytes that are below the target size to a second binding buffer, wherein the second binding buffer comprises at least one chaotropic agent 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) Apply magnetic beads to a mixture of the separated one or more target analytes that are below the target size and the second binding buffer, wherein the magnetic beads bind to the one or more target analytes that are below the target size to form a second bead-analyte complex; and (vi) Recover the one or more target analytes from the second bead-analyte complex.

[0078] In some embodiments, the method further comprises the steps of: (e) Perform a diagnostic assay on the final solution to detect and quantify the one or more target analytes.

[0079] 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.

[0080] In some embodiments, the one or more target analytes are DNA.

[0081] In some embodiments, the one or more target analytes are cell-free DNA or circulating tumor DNA.

[0082] In some embodiments, the first ATPS comprises a first ATPS component that is capable of forming a first phase and a second phase when dissolved in an aqueous solution, wherein the first ATPS component is selected from the group consisting of polymers, salts, surfactants, and combinations thereof.

[0083] In some embodiments, the second ATPS comprises a second ATPS component that is capable of forming a third phase and a fourth phase when dissolved in an aqueous solution, wherein the second ATPS component is selected from the group consisting of polymers, salts, surfactants, and combinations thereof.

[0084] In some embodiments, the polymer is dissolved in an aqueous solution at a concentration of 4% - 84% (w / w).

[0085] In some embodiments, the salt is dissolved in an aqueous solution at a concentration of 1% - 80% (w / w). In some embodiments, the salt is dissolved in an aqueous solution at a concentration of 8% - 80% (w / w).

[0086] In some embodiments, the surfactant is dissolved in an aqueous solution at a concentration of 0.05%-10% (w / w). In some embodiments, the surfactant is dissolved in an aqueous solution at a concentration of 0.05%-9.8% (w / w).

[0087] In some embodiments, step (a) further comprises the steps of: (i) embedding the porous material in components capable of forming a first ATPS; and (ii) contacting the sample solution with the porous material embedded with the components, wherein the components form a first phase and a second phase when the sample solution passes through the porous material.

[0088] In one aspect, there is provided a method for concentrating and purifying one or more target analytes from a sample solution, the method comprising the steps of: (a) adding the sample solution containing the one or more target analytes to a first aqueous two-phase system (ATPS) to form a mixture that separates into a first phase and a second phase, 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 that separates 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 chaotropic agent selected from the following: 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 complexes with a fractionation buffer comprising a polymer, a salt, a surfactant, a chaotropic agent, or a combination thereof to form a fractionation solution such that one or more target analytes smaller than the target size are released from the bead-analyte complexes into the fractionation solution; (h) fixing the bead-analyte complexes using a magnetic support; (i) separating the one or more target analytes smaller than the target size in the fractionation solution from the fixed bead-analyte complexes; (j) Add one or more separated target analytes below the target size to a second binding buffer, wherein the second binding buffer contains at least one chaotropic agent selected from the following: n-butanol, ethanol, guanidine chloride, guanidine thiocyanate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea; (k) Apply magnetic beads to a mixture of one or more separated target analytes below the target size and the second binding buffer, wherein the magnetic beads bind to one or more target analytes below the target size to form a second bead-analyte complex; (l) Recover one or more target analytes below the target size from the second bead-analyte complex, thereby obtaining a final solution containing the concentrated and purified one or more target analytes below the target size; and (m) Perform a diagnostic assay on the final solution to detect and quantify the one or more target analytes below the target size.

[0089] Various ATPS systems useful in various embodiments of the present invention include, but are not limited to, polymer-polymer, polymer-salt, polymer-surfactant, salt-surfactant, surfactant, surfactant-surfactant, or polymer-salt-surfactant.

[0090] In one embodiment, the first and / or second ATPS comprises a polymer. In some embodiments, possible polymers that can be used include, but are not limited to, polyalkylene glycols (PAGs) (e.g., hydrophobically modified polyalkylene glycols), poly(oxyalkylene) polymers, poly(oxyalkylene) copolymers (e.g., hydrophobically modified poly(oxyalkylene) copolymers), polyvinylpyrrolidone, polyvinyl alcohol, polyvinylcaprolactam, polyvinyl methyl ether, alkoxylated surfactants, alkoxylated starches, alkoxylated celluloses, alkyl hydroxyalkyl celluloses, silicone-modified polyethers, and poly N-isopropylacrylamide and its copolymers. In some embodiments, the polymer is selected from the group consisting of: polyethers, polyimines, polyalkylene glycols, vinyl polymers, alkoxylated surfactants, polysaccharides, alkoxylated starches, alkoxylated celluloses, alkyl hydroxyalkyl celluloses, polyether-modified silicones, polyacrylamides, polyacrylic acids, and their copolymers. In some embodiments, the polymer is selected from the group consisting of: dipropylene glycol, tripropylene glycol, polyethylene glycol, polypropylene glycol, poly(ethylene glycol-propylene glycol), poly(ethylene glycol-ran-propylene glycol), polyvinylpyrrolidone, polyvinyl alcohol, polyvinylcaprolactam, polyvinyl methyl ether, dextran, carboxymethyl dextran, dextran sulfate, hydroxypropyl dextran, starch, carboxymethyl cellulose, polyacrylic acid, hydroxypropyl cellulose, methyl cellulose, ethyl hydroxyethyl cellulose, maltodextrin, polyethyleneimine, poly N-isopropylacrylamide, and their copolymers. In some embodiments, the polymer is selected from the group consisting of: dipropylene glycol, tripropylene glycol, polyethylene glycol, polypropylene glycol, poly(ethylene glycol-propylene glycol), poly(ethylene glycol-random-propylene glycol), polyvinylpyrrolidone, polyvinyl alcohol, polyvinylcaprolactam, polyvinyl methyl ether, and poly N-isopropylacrylamide. In some embodiments, the polymer is selected from the group consisting of: polyacrylamides, polyacrylic acids, and their copolymers. In some embodiments, the polymer is selected from the group consisting of: dextran, carboxymethyl dextran, dextran sulfate, hydroxypropyl dextran, and starch. In some embodiments, the polymer has an average molecular weight in the range of 200 - 1,000 Da, 200 - 35,000 Da, 425 - 2,000 Da, 400 - 35,000 Da, 980 - 12,000 Da, or 3,400 - 5,000,000 Da. In some embodiments, the polymer comprises ethylene oxide and propylene oxide units, and the polymer has an EO:PO ratio of 90:10 to 10:90.

[0091] In one embodiment, the polymer concentration of the first and / or second ATPS is in the range of from about 4% to about 84% (w / w) by weight of the total weight of the aqueous solution. In various embodiments, the polymer solution is selected from about 4% w / w, about 4.5% w / w, about 5% w / w, about 5.5% w / w, about 6% w / w, about 6.5% w / w, about 7% w / w, about 7.5% w / w, about 8% w / w, about 8.5% w / w, about 9% w / w, about 9.5% w / w, about 10% w / w, about 10.5% w / w, about 11% w / w, about 11.5% w / w, about 12% w / w, about 12.5% w / w, about 13% w / w, about 13.5% w / w, about 14% w / w, about 14.5% w / w, about 15% w / w, about 15.5% w / w, about 16% w / w, about 16.5% w / w, about 17% w / w, about 17.5% w / w, about 18% w / w, about 18.5% w / w, about 19% w / w, about 19.5% w / w, about 20% w / w, about 20.5% w / w, about 21% w / w, about 21.5% w / w, about 22% w / w, about 22.5% w / w, about 23% w / w, about 23.5% w / w, about 24% w / w, about 24.5% w / w, about 35% w / w, about 35.5% w / w, about 36% w / w, about 36.5% w / w, about 37% w / w, about 37.5% w / w, about 38% w / w, about 38.5% w / w, about 39% w / w, about 39.5% w / w, about 40% w / w, about 40.5% w / w, about 41% w / w, about 41.5% w / w, about 42% w / w, about 42.5% w / w, about 43% w / w, about 43.5% w / w, about 44% w / w, about 44.5% w / w, about 45% w / w, about 45.5% w / w, about 46% w / w, about 46.5% w / w, about 47% w / w, about 47.5% w / w, about 48% w / w, about 48.5% w / w, about 49% w / w, about 49.5% w / w, about 50% w / w, about 50.5% w / w, about 51% w / w, about 51.5% w / w, about 52% w / w, about 52.5% w / w, about 53% w / w, about 53.5% w / w, about 54% w / w, about 54.5% w / w, about 55% w / w, about 55.5% w / w, about 56% w / w, about 56.5% w / w, about 57% w / w, about 57.5% w / w, about 58% w / w, about 58.5% w / w, about 59% w / w, about 59.5% w / w, about 60% w / w, about 60.5% w / w, about 61% w / w, about 61.5% w / w, about 62% w / w, about 62.5% w / w, about 63% w / w, about 63.5% w / w, about 64% w / w, about 64.5% w / w, about 65% w / w, about 65.Polymer solutions of 5% w / w, about 66% w / w, about 66.5% w / w, about 67% w / w, about 67.5% w / w, about 68% w / w, about 68.5% w / w, about 69% w / w, about 69.5% w / w, about 70% w / w, about 70.5% w / w, about 71% w / w, about 71.5% w / w, about 72% w / w, about 72.5% w / w, about 73% w / w, about 73.5% w / w, about 74% w / w, about 74.5% w / w, about 75% w / w, about 75.5% w / w, about 76% w / w, about 76.5% w / w, about 77% w / w, about 77.5% w / w, about 78% w / w, about 78.5% w / w, about 79% w / w, about 79.5% w / w, about 80% w / w, about 80.5% w / w, about 81% w / w, about 81.5% w / w, about 82% w / w, about 82.5% w / w, about 83% w / w, about 83.5% w / w and about 84% w / w.

[0092] In one embodiment, the first and / or second ATPS contains salt and thus forms a salt solution. In some embodiments, the salt includes but is not limited to lyotropic salts, discrete salts, inorganic salts containing cations and anions, the cations being, for example, linear or branched trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium and tetrabutylammonium, and the anions being, for example, phosphate, sulfate, nitrate, chloride and bicarbonate. In another embodiment, the salt comprises NaCl, Na3PO4, K3PO4, Na2SO4, potassium citrate, (NH4)2SO4, sodium citrate, sodium acetate and combinations thereof. Other salts such as ammonium acetate can also be used. In another embodiment, the salt is selected from magnesium salts, lithium salts, sodium salts, potassium salts, cesium salts, zinc salts and aluminum salts. In some embodiments, the salt is 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 ranges from about 0.01% to about 90%. Those skilled in the art will understand that the amount of salt required to form the aqueous two-phase system will be affected by the molecular weight, concentration and physical state of the polymer.

[0094] In various embodiments, the salt concentration is about 1%-80% w / w. In various embodiments, the salt concentration is about 1% w / w, about 1.5% w / w, about 2% w / w, about 2.5% w / w, about 3% w / w, about 3.5% w / w, about 4% w / w, about 4.5% w / w, about 5% w / w, about 5.5% w / w, about 6% w / w, about 6.5% w / w, about 7% w / w, about 7.5% w / w, about 8% w / w, about 8.5% w / w, about 9% w / w, about 9.5% w / w, about 10% w / w, about 10.5% w / w, about 11% w / w, about 11.5% w / w, about 12% w / w, about 12.5% w / w, about 13% w / w, about 13.5% w / w, about 14% w / w, about 14.5% w / w, about 15% w / w, about 15.5% w / w, about 16% w / w, about 16.5% w / w, about 17% w / w, about 17.5% w / w, about 18% w / w, about 18.5% w / w, about 19% w / w, about 19.5% w / w, about 20% w / w, about 20.5% w / w, about 21% w / w, about 21.5% w / w, about 22% w / w, about 22.5% w / w, about 23% w / w, about 23.5% w / w, about 24% w / w, about 24.5% w / w, about 35% w / w, about 35.5% w / w, about 36% w / w, about 36.5% w / w, about 37% w / w, about 37.5% w / w, about 38% w / w, about 38.5% w / w, about 39% w / w, about 39.5% w / w, about 40% w / w, about 40.5% w / w, about 41% w / w, about 41.5% w / w, about 42% w / w, about 42.5% w / w, about 43% w / w, about 43.5% w / w, about 44% w / w, about 44.5% w / w, about 45% w / w, about 45.5% w / w, about 46% w / w, about 46.5% w / w, about 47% w / w, about 47.5% w / w, about 48% w / w, about 48.5% w / w, about 49% w / w, about 49.5% w / w, about 50% w / w, about 50.5% w / w, about 51% w / w, about 51.5% w / w, about 52% w / w, about 52.5% w / w, about 53% w / w, about 53.5% w / w, about 54% w / w, about 54.5% w / w, about 55% w / w, about 55.5% w / w, about 56% w / w, about 56.5% w / w, about 57% w / w, about 57.5% w / w, about 58% w / w, about 58.5% w / w, about 59% w / w, about 59.5% w / w, about 60% w / w, about 60.5% w / w, about 61% w / w, about 61.5% w / w, about 62% w / w, about 62.5% w / w, about 63% w / w, about 63.5% w / w, about 64% w / w, about 64.5% w / w, about 65% w / w, about 65.5% w / w, about 66% w / w, about 66.5% w / w, about 67% w / w, about 67.5% w / w, about 68% w / w, about 68.5% w / w, about 69% w / w, about 69.5% w / w, about 70% w / w, about 70.5% w / w, about 71% w / w, about 71.5% w / w, about 72% w / w, about 72.5% w / w, about 73% w / w, about 73.5% w / w, about 74% w / w, about 74.5% w / w, about 75% w / w, about 75.5% w / w, about 76% w / w, about 76.5% w / w, about 77% w / w, about 77.5% w / w, about 78% w / w, about 78.5% w / w, about 79% w / w, about 79.5% w / w or about 80% w / w.

[0095] In one embodiment, the first and / or second ATPS 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 and fats, sulfated esters, sulfated alkanolamides, alkylphenols), ethoxylated and sulfated nonionic surfactants (such as ethoxylated fatty alcohols, polyoxyethylene surfactants, carboxylic esters, polyethylene glycol esters, sorbitan esters, fatty acid ethylene glycol esters, carboxylic acid amides, monoalkanolamine concentrates, polyoxyethylene fatty acid amides), cationic surfactants (such as quaternary ammonium salts, amines with amide bonds, polyoxyethylene alkyl and cycloaliphatic amines, n,n,n',n'-tetrasubstituted ethylenediamine, 2-alkyl-1-hydroxyethyl-2-imidazoline), and amphoteric surfactants (such as n-cocoyl 3-aminopropionic acid / sodium salt, n-tallow 3-iminodipropionate, disodium salt, n-carboxymethyl n-dimethyl n-9-octadecenyl ammonium hydroxide, n-cocoylamidoethyl n-hydroxyethyl glycine and sodium salt).

[0096] In one embodiment, the surfactant concentration of the first and / or second ATPS 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, about 2.4% w / w, about 2.5% w / w, about 2.6% w / w, about 2.7% w / w, about 2.8% w / w, about 2.9% w / w, about 3% w / w, 3.1% w / w, about 3.2% w / w, about 3.3% w / w, about 3.4% w / w, about 3.5% w / w, about 3.6% w / w, about 3.7% w / w, about 3.8% w / w, about 3.9% w / w, about 4% w / w, about 4.1% w / w, about 4.2% w / w, about 4.3% w / w, about 4.4% w / w, about 4.5% w / w, about 4.6% w / w, about 4.7% w / w, about 4.8% w / w, about 4.9% w / w, about 5% w / w, about 5.1% w / w, about 5.2% w / w, about 5.3% w / w, about 5.4% w / w, about 5.5% w / w, about 5.6% w / w, about 5.7% w / w, about 5.8% w / w, about 5.9% w / w, about 6% w / w, 6.1% w / w, about 6.2% w / w, about 6.3% w / w, about 6.4% w / w, about 6.5% w / w, about 6.6% w / w, about 6.7% w / w, about 6.8% w / w, about 6.9% w / w, about 7% w / w, about 7.1% w / w, about 7.2% w / w, about 7.3% w / w, about 7.4% w / w, about 7.5% w / w, about 7.6% w / w, about 7.7% w / w, about 7.8% w / w, about 7.9% w / w, about 8% w / w, about 8.1% w / w, about 8.2% w / w, about 8.3% w / w, about 8.4% w / w, about 8.5% w / w, about 8.6% w / w, about 8.7% w / w, about 8.8% w / w, about 8.9% w / w, about 9% w / w, 9.1% w / w, about 9.2% w / w, about 9.3% w / w, about 9.4% w / w, about 9.5% w / w, about 9.6% w / w, about 9.7% w / w, about 9.8% w / w, about 9.9% w / w or about 10% w / w.

[0097] In one embodiment, the binding buffer (including the second binding buffer) contains a chaotropic agent. In some embodiments, possible chaotropic agents that can be used 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 chaotropic agent in the binding buffer ranges from about 0.1 M to 8 M. In various embodiments, the concentration of the chaotropic agent is about 0.1 M, about 0.2 M, about 0.3 M, about 0.4 M, about 0.5 M, about 0.6 M, about 0.7 M, about 0.8 M, about 0.9 M, about 1 M, about 1.1 M, about 1.2 M, about 1.3 M, about 1.4 M, about 1.5 M, about 1.6 M, about 1.7 M, about 1.8 M, about 1.9 M, about 2 M, about 2.1 M, about 2.2 M, about 2.3 M, about 2.4 M, about 2.5 M, about 2.6 M, about 2.7 M, about 2.8 M, about 2.9 M, about 3 M, about 3.1 M, about 3.2 M, about 3.3 M, about 3.4 M, about 3.5 M, about 3.6 M, about 3.7 M, about 3.8 M, about 3.9 M, about 4 M, about 4.1 M, about 4.2 M, about 4.3 M, about 4.4 M, about 4.5 M, about 4.6 M, about 4.7 M, about 4.8 M, about 4.9 M, about 5 M, about 5.1 M, about 5.2 M, about 5.3 M, about 5.4 M, about 5.5 M, about 5.6 M, about 5.7 M, about 5.8 M, about 5.9 M, about 6 M, about 6.1 M, about 6.2 M, about 6.3 M, about 6.4 M, about 6.5 M, about 6.6 M, about 6.7 M, about 6.8 M, about 6.9 M, about 7 M, about 7.1 M, about 7.2 M, about 7.3 M, about 7.4 M, about 7.5 M, about 7.6 M, about 7.7 M, about 7.8 M, about 7.9 M, or about 8 M.

[0099] In some embodiments, the fractionation buffer includes a polymer, a salt, a surfactant, a chaotropic agent, or a combination thereof. In some embodiments, possible polymers, salts, surfactants, and chaotropic agents that can be used include, but are not limited to, those described above.

[0100] In some embodiments, possible magnetic beads that can be used include, but are not limited to, those listed in Table 1.1 below. Table 1.1: Examples of Magnetic Beads

[0101] Example 2

[0102] In some embodiments, provided is a method for separating a target nucleic acid smaller than a target size from a sample comprising a nucleic acid component; the method comprising the steps of: (a) preparing a sample solution from the sample; (b) contacting a plurality of beads with the sample solution, wherein the nucleic acid component binds to the plurality of beads to form a bead-analyte complex; (c) mixing the bead-analyte complex with a fractionation buffer comprising at least one dissociating agent to form a bulk fractionation solution, wherein the target nucleic acid smaller than the target size is released from the bead-analyte complex into the bulk fractionation solution; (d) immobilizing the bead-analyte complex; and (e) separating the bulk fractionation solution comprising the separated target nucleic acid smaller than the target size from the immobilized bead-analyte complex.

[0103] In some embodiments, step (a) further comprises (a1) adding the sample to a first aqueous two-phase system (ATPS) to form a mixture that separates into a first target-rich phase and a first target-depleted phase, wherein the nucleic acid component is concentrated in the first target-rich phase; and (a2) separating the first target-rich phase containing the concentrated nucleic acid component to obtain a sample solution.

[0104] In some embodiments, after step (a2), step (a) further comprises the steps of: (a3) adding the sample solution from step (a2) to a second ATPS to form a second mixture that separates into a second target-rich phase and a second target-depleted phase, wherein the nucleic acid component is concentrated in the second target-rich phase; and (a4) separating the second target-rich phase containing the concentrated nucleic acid component to form the sample solution in step (a).

[0105] In some embodiments, before step (b), a plurality of beads and the sample solution of step (a) are mixed with a binding buffer, wherein the binding buffer comprises at least one dissociating agent.

[0106] In some embodiments, step (e) further comprises the steps of: (e1) mixing the bulk fractionation solution with a target binding buffer and a plurality of second beads such that the plurality of second beads bind the target nucleic acid smaller than the target size to form a second bead-analyte complex, wherein the target binding buffer comprises at least one dissociating agent; and (e2) recovering the target nucleic acid smaller than the target size from the second bead-analyte complex.

[0107] In some embodiments, the plurality of beads are magnetic beads, silica-based beads, carboxyl beads, hydroxyl beads, amine-coated beads, or any combination thereof.

[0108] In some embodiments, the plurality of second beads are magnetic beads, silica-based beads, carboxyl beads, hydroxyl beads, amine-coated beads, or any combination thereof.

[0109] In some embodiments, the plurality of beads are magnetic beads, and step (b) further comprises the steps of: (b1) fixing the bead - analyte complex by applying a magnetic field to separate the bead - analyte complex from the bulk supernatant; (b2) removing the bulk supernatant; and (b3) removing the magnetic field and proceeding to step (c).

[0110] In some embodiments, the plurality of second beads are magnetic beads, and the target nucleic acid recovery of step (e2) further comprises the steps of: (i) fixing the second bead - analyte complex by applying a first magnetic field to separate the second bead - analyte complex from the first supernatant; (ii) removing the first supernatant; (iii) washing the fixed second bead - analyte complex with a wash buffer; (iv) discarding the wash buffer; (v) removing the first magnetic field; (vi) mixing the second bead - analyte complex with an elution buffer to form a bulk elution solution, wherein the target nucleic acid below the target size is separated from the magnetic beads in the second bead - analyte complex and released into the bulk elution solution; (vii) fixing the magnetic beads by applying a second magnetic field; (viii) collecting the bulk elution solution comprising the separated target nucleic acid below the target size.

[0111] In some embodiments, a method is provided that further comprises the step of: (f) performing a diagnostic assay on the separated target nucleic acid for detection, quantification, characterization, or a combination thereof of the target nucleic acid.

[0112] In some embodiments, at least one chaotropic agent of the fractionation buffer is selected from the group consisting of: thiocyanate, isothiocyanate, perchlorate, acetate, trichloroacetate, trifluoroacetate, chloride, and iodide.

[0113] In some embodiments, at least one chaotropic agent of the fractionation buffer is selected from the group consisting of: guanidine hydrochloride (GHCl), guanidine thiocyanate, guanidine isothiocyanate (GITC), sodium thiocyanate, sodium iodide, sodium perchlorate, sodium trichloroacetate, sodium trifluoroacetate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2 - propanol, thiourea, and urea.

[0114] In some embodiments, the at least one chaotropic agent has a concentration of about 1.5 - 8 M in the fractionation buffer.

[0115] In some embodiments, the at least one chaotropic agent is present in the fractionation buffer at a concentration of about 1.8 - 3.9 M.

[0116] In some embodiments, the at least one chaotropic agent is present in the fractionation buffer at a concentration of about 1.8 - 3.0 M.

[0117] In some embodiments, the fractionation buffer further comprises at least one polymer selected from the group consisting of: polyvinyl alcohol, polyethylene glycol, polypropylene glycol, dextran, poly(ethylene glycol-random-propylene glycol), pluronic, polyvinylpyrrolidone, and polyacrylate.

[0118] In some embodiments, the at least one polymer is present in the fractionation buffer at a concentration of about 0.1% - 15% (w / w).

[0119] In some embodiments, the at least one polymer is present in the fractionation buffer at a concentration of about 1.0% - 5.0% (w / w).

[0120] In some embodiments, the at least one polymer is a polymer having an average molecular weight range of 100 Da to 35,000 Da.

[0121] In some embodiments, the fractionation buffer further comprises one or more of a pH buffer, a metal chelator, or a combination thereof.

[0122] Examples of pH buffers include, but are not limited to, phosphate buffer, acetic acid-sodium acetate buffer, citrate-sodium citrate buffer, citrate-NaOH-HCl buffer, borate buffer, carbonate buffer, HEPES buffer, MOPS buffer, TAE buffer, TBST buffer, Tris-HCl buffer, TE buffer, and TEN buffer.

[0123] Examples of metal chelators include, but are not limited to, 2,2'-bipyridine, dimercaprol, ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), ionophore nitrilotriacetic acid (NTA), salicylic acid, and triethanolamine (TEA).

[0124] In some embodiments, the nucleic acid component and / or the target nucleic acid is DNA, RNA, or a combination thereof.

[0125] In some embodiments, the nucleic acid component and / or the target nucleic acid is cDNA, plasmid DNA, cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), circulating fetal DNA, microRNA (miRNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), or a combination thereof.

[0126] In some embodiments, the first ATPS comprises a first ATPS component that is capable of forming the first target-rich phase and the first target-depleted phase when dissolved in an aqueous solution, wherein the first ATPS component is selected from the group consisting of: polymers, salts, surfactants, and combinations thereof.

[0127] In some embodiments, the second ATPS includes a second ATPS component that, when dissolved in an aqueous solution, is capable of forming the second target-rich phase and the second target-lean phase, wherein the second ATPS component is selected from the group consisting of: polymers, salts, surfactants, and combinations thereof.

[0128] In some embodiments, the polymer is dissolved in an aqueous solution at a concentration of 0.5% - 80% (w / v).

[0129] In some embodiments, the polymer is selected from the group consisting of: polyethers, polyimines, polyalkylene glycols, vinyl polymers, alkoxylated surfactants, polysaccharides, alkoxylated starches, alkoxylated celluloses, alkyl hydroxyalkyl celluloses, polyether-modified silicones, polyacrylamides, polyacrylic acids, and copolymers thereof. In some embodiments, the polymer is hydrophobically modified or silicone-modified.

[0130] In some embodiments, the polymer is dipropylene glycol, tripropylene glycol, polyethylene glycol, polypropylene glycol, poly(ethylene glycol - propylene glycol), poly(ethylene glycol - ran - propylene glycol), polyvinylpyrrolidone, polyvinyl alcohol, polyvinylcaprolactam, polyvinyl methyl ether, dextran, carboxymethyl dextran, dextran sulfate, hydroxypropyl dextran, starch, carboxymethyl cellulose, polyacrylic acid, hydroxypropyl cellulose, methyl cellulose, ethyl hydroxyethyl cellulose, maltodextrin, polyethyleneimine, poly(N - isopropylacrylamide), or a copolymer thereof.

[0131] In some embodiments, the polymer is dipropylene glycol, tripropylene glycol, polyethylene glycol, polypropylene glycol, poly(ethylene glycol - propylene glycol), poly(ethylene glycol - random - propylene glycol), polyvinylpyrrolidone, polyvinyl alcohol, polyvinylcaprolactam, polyvinyl methyl ether, or poly(N - isopropylacrylamide).

[0132] In some embodiments, the polymer is polyacrylamide, polyacrylic acid, or a copolymer thereof.

[0133] 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.

[0134] In some embodiments, the polymer has an average molecular weight in the range of 200 - 1,000 Da, 200 - 35,000 Da, 425 - 2,000 Da, 400 - 35,000 Da, 980 - 12,000 Da, or 3,400 - 5,000,000 Da. In some embodiments, the polymer comprises ethylene oxide and propylene oxide units. In some embodiments, the polymer has an EO:PO ratio of 90:10 to 10:90.

[0135] In some embodiments, the polymer has an average molecular weight in the range of 980 - 12,000 Da or 3,400 - 5,000,000 Da.

[0136] In some embodiments, the polymer has an average molecular weight in the range of 100 to 10,000 Da.

[0137] In some embodiments, the salt is dissolved in an aqueous solution at a concentration of 0.1% - 80% (w / w).

[0138] In some embodiments, the salt is dissolved in an aqueous solution at a concentration of 0.1% - 50% (w / w).

[0139] In some embodiments, the salt includes cations 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.

[0140] In some embodiments, the salt includes anions selected from the group consisting of phosphate, hydrogen phosphate, dihydrogen phosphate, sulfate, sulfide, sulfite, bisulfate, carbonate, bicarbonate, acetate, nitrate, nitrite, sulfite, chloride, fluoride, chlorate, perchlorate, chlorite, hypochlorite, bromide, bromate, hypobromite, iodide, iodate, cyanate, thiocyanate, isothiocyanate, oxalate, formate, chromate, dichromate, permanganate, hydroxide, hydrogen ion, citrate, borate, and tris(hydroxymethyl)aminomethane.

[0141] 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.

[0142] In some embodiments, the salt is selected from the group consisting of: 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, and ammonium acetate.

[0143] In some embodiments, the salt is selected from the group consisting of: (NH4)3PO4, sodium formate, ammonium formate, K2CO3, KHCO3, Na2CO3, NaHCO3, MgSO4, MgCO3, CaCO3, CsOH, Cs2CO3, Ba(OH)2, and BaCO3.

[0144] In some embodiments, the salt is selected from the group consisting of: NH4Cl, NH4OH, tetramethylammonium chloride, tetrabutylammonium chloride, tetramethylammonium hydroxide, and tetrabutylammonium hydroxide.

[0145] In some embodiments, the surfactant is dissolved in an aqueous solution at a concentration of 0.05% - 10% (w / w).

[0146] 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 carboxylate, sulfonate, petroleum sulfonate, alkylbenzene sulfonate, naphthalene sulfonate, olefin sulfonate, alkyl sulfate, sulfate, sulfated natural oil, sulfated natural fat, sulfated ester, sulfated alkanolamide, sulfated alkylphenol, ethoxylated alkylphenol, or sodium N-lauroylsarcosinate (NLS); wherein the nonionic surfactant is ethoxylated fatty alcohol, polyoxyethylene surfactant, carboxylic acid ester, polyethylene glycol ester, sorbitan ester, fatty acid diol ester, carboxamide, single-chain alkanolamine condensate, or polyoxyethylene fatty acid amide; wherein 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; wherein the amphoteric surfactant is n-cocoyl 3-aminopropionic acid or its sodium salt, n-tallow 3-iminodipropionate or its disodium salt, n-carboxymethyl n-dimethyl n-9-octadecenyl ammonium hydroxide, or n-cocoylamidoethyl n-hydroxyethyl glycine or its sodium salt.

[0147] In some embodiments, the surfactant is selected from the group consisting of: 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, cetyltrimethylammonium bromide, and span 80.

[0148] In some embodiments, at least one dissociating agent of the binding buffer comprises an anion selected from the group consisting of: thiocyanate, isothiocyanate, perchlorate, acetate, trichloroacetate, trifluoroacetate, chloride, and iodide.

[0149] In some embodiments, at least one dissociating agent of the binding buffer is selected from the group consisting of: guanidine hydrochloride (GHCl), guanidine thiocyanate, guanidine isothiocyanate (GITC), sodium thiocyanate, sodium iodide, sodium perchlorate, sodium trichloroacetate, sodium trifluoroacetate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, thiourea, and urea.

[0150] In some embodiments, the binding buffer comprises guanidine and optionally further comprises at least one polymer.

[0151] In some embodiments, at least one dissociating agent of the target binding buffer comprises an anion selected from the group consisting of: thiocyanate, isothiocyanate, perchlorate, acetate, trichloroacetate, trifluoroacetate, chloride, and iodide.

[0152] In some embodiments, at least one dissociating agent of the target binding buffer is selected from the group consisting of: guanidine hydrochloride (GHCl), guanidine thiocyanate, guanidine isothiocyanate (GITC), sodium thiocyanate, sodium iodide, sodium perchlorate, sodium trichloroacetate, sodium trifluoroacetate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, thiourea, and urea.

[0153] In some embodiments, the target binding buffer comprises guanidine and optionally further comprises at least one polymer.

[0154] In some embodiments, the 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.

[0155] In some embodiments, step (a) comprises the steps of: preparing a DNA library from the sample to obtain a sample solution.

[0156] Some embodiments provide a method for diagnosing or determining the risk of cancer in a subject, comprising the steps of: (i) isolating a target nucleic acid from a biological sample of the subject using the methods described herein; (ii) measuring the presence of the target nucleic acid and determining whether the subject has the cancer or is at risk of having the cancer.

[0157] In some embodiments, the target nucleic acid is cell-free DNA and circulating tumor DNA, whereby the method increases the ratio of circulating tumor DNA:cell-free DNA, and / or the variant allele frequency (VAF) in the sample for cancer diagnostic assays.

[0158] Some embodiments provide a method for treating cancer in a subject, the method comprising diagnosing the cancer in the subject by the above steps (i) and (ii), and further comprising treating the subject if it is determined that the subject has the cancer or is at risk of having the cancer.

[0159] Some embodiments provide a method for diagnosing or determining the risk of a genetic disease or disorder in a fetus, comprising the steps (i) isolating a target nucleic acid from a biological sample of the fetus's mother using the methods described herein; (ii) measuring the presence of the target nucleic acid and determining whether the fetus has a genetic disease or disorder or is at risk of having the genetic disease or disorder.

[0160] In some embodiments, the target nucleic acid is circulating fetal DNA, whereby the method enriches the fetal fraction in the sample for non-invasive prenatal testing. For clarity, the fetal fraction refers to the fraction of all DNA derived from the fetus that circulates in the mother's blood.

[0161] Some embodiments provide a method for treating a genetic disease or disorder in a fetus, the method comprising diagnosing the genetic disease or disorder in the fetus, including the above steps (i) and (ii), and further comprising treating the fetus or the fetus's mother if it is determined that the fetus has the genetic disease or disorder or is at risk of having the genetic disease or disorder.

[0162] Methods for measuring the presence of a target nucleic acid and determining the presence, risk or absence of a disease (such as cancer and fetal genetic diseases and disorders) are known to those of skill in the art.

[0163] In some embodiments, a kit for separating a target nucleic acid of a size smaller than a target size from a sample comprising a nucleic acid component is provided. The kit includes: (a) at least one ATPS component selected from the group consisting of polymers, salts, surfactants, and combinations thereof; (b) a plurality of beads; (c) a fractionation buffer comprising at least one chaotropic agent selected from the group consisting of thiocyanate, isothiocyanate, perchlorate, acetate, trichloroacetate, trifluoroacetate, chloride, and iodide; and (d) a binding buffer comprising at least one chaotropic agent selected from the group consisting of thiocyanate, isothiocyanate, perchlorate, acetate, trichloroacetate, trifluoroacetate, chloride, and iodide.

[0164] In some embodiments, the plurality of beads are magnetic beads, silica-based beads, carboxyl beads, hydroxyl beads, amine-coated beads, or any combination thereof.

[0165] In some embodiments, at least one chaotropic agent of the fractionation buffer is selected from the group consisting of guanidine hydrochloride (GHCl), guanidine thiocyanate, guanidine isothiocyanate (GITC), sodium thiocyanate, sodium iodide, sodium perchlorate, sodium trichloroacetate, sodium trifluoroacetate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, thiourea, and urea.

[0166] In some embodiments, the at least one chaotropic agent has a concentration of about 1.5 - 8 M in the fractionation buffer.

[0167] In some embodiments, the fractionation buffer further comprises at least one polymer selected from the group consisting of polyvinyl alcohol, polyethylene glycol, polypropylene glycol, dextran, poly(ethylene glycol-random-propylene glycol), pluronic, polyvinylpyrrolidone, and polyacrylate.

[0168] In some embodiments, the at least one polymer is present in the fractionation buffer at a concentration of about 0.1% - 15% (w / w).

[0169] In some embodiments, the at least one polymer has an average molecular weight ranging from 100 Da to 35,000 Da.

[0170] In some embodiments, the fractionation buffer further comprises one or more of a pH buffer, a metal chelator, or a combination thereof.

[0171] In some embodiments, a method for separating a target nucleic acid smaller than a target size from a sample comprising a nucleic acid component is provided; the method comprising the steps of: (a) preparing a sample solution from the sample; (b) contacting the sample solution with a solid-phase medium configured to selectively bind the nucleic acid component such that the nucleic acid component binds to the solid-phase medium to form a medium-analyte complex; (c) adding a fractionation buffer comprising at least one dissociating agent to the medium-analyte complex to form a bulk fractionation solution, wherein the target nucleic acid smaller than the target size is released from the solid-phase medium into the bulk fractionation solution; and (d) separating the bulk fractionation solution comprising the separated target nucleic acid smaller than the target size from the solid-phase medium.

[0172] In some embodiments, the solid-phase medium is a solid-phase extraction column.

[0173] In some embodiments, the solid-phase extraction column is a spin column.

[0174] In some embodiments, the solid-phase medium is a plurality of beads.

[0175] In some embodiments, the plurality of beads are magnetic beads, silica-based beads, carboxyl beads, hydroxyl beads, amine-coated beads, or any combination thereof.

[0176] 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.

[0177] 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.

[0178] In some embodiments, the concentration of the surfactant is 0.1%-50% (w / v) of the first ATPS and / or the second ATPS. In some embodiments, the concentration of the surfactant is 0.01%-10% (w / v) of the first ATPS and / or the second ATPS.

[0179] In some embodiments, the first ATPS composition is polymer-salt based, which comprises at least one polymer at a concentration of 5%-80% (w / v) and at least one salt at a concentration of 0.1%-80% (w / v). In some embodiments, the first ATPS composition comprises at least one polymer at a concentration of 5%-60% (w / v) and at least one salt at a concentration of 0.5%-50% (w / v). In some embodiments, the first ATPS composition comprises at least one polymer at a concentration of 12%-50% (w / v) and at least one salt at a concentration of 0.1%-20% (w / v). In some embodiments, the first ATPS composition further comprises at least one surfactant at a concentration of 0.01%-10% (w / v).

[0180] 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).

[0181] In some embodiments, the first ATPS composition is polymer-salt based, which comprises at least one polymer at a concentration of 0.5%-30% (w / v) and at least one salt at a concentration of 5%-60% (w / v). In some embodiments, the first ATPS composition comprises at least one polymer at a concentration of 1%-6% (w / v) and at least one salt at a concentration of 10%-50% (w / v). In some embodiments, the first ATPS composition further comprises at least one surfactant at a concentration of 0.01%-10% (w / v).

[0182] 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).

[0183] In some embodiments, the first ATPS composition is polymer-polymer based and comprises at least one polymer at a concentration of 0.2%-50% (w / v). In some embodiments, the first ATPS composition further comprises at least one salt at a concentration of 0.01%-10% (w / v). In some embodiments, the first ATPS composition further comprises at least one surfactant at a concentration of 0.01%-10% (w / v).

[0184] In some embodiments, the first ATPS composition is surfactant based and comprises at least one surfactant at a concentration of 0.1%-50% (w / v). In some embodiments, the first ATPS composition further comprises at least one salt at a concentration of 0.01%-30% (w / v).

[0185] Although the description refers to specific embodiments, the present disclosure should not be construed as limited to the embodiments set forth herein. Examples

[0186] Examples are provided herein that more particularly describe certain embodiments of the present disclosure. The examples provided herein are for illustrative purposes only and are not meant to limit the scope of the invention in any way. All references given below and elsewhere in this application are hereby incorporated herein by reference. Example 1: Protocol for Concentrating DNA without Using a Gradient Buffer

[0187] 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.

[0188] The protocol steps are performed as follows: 1. Add a desired volume of the processed biological sample (e.g., plasma) (e.g., 2 mL - 3 mL) to the first ATPS (Solution B) to form Solution B'. Methods of processing the biological sample include, but are not limited to, lysis to form a sample lysate. 2. Vortex solution B' thoroughly (e.g., for about 10 seconds) until homogeneous, and then centrifuge at 2,300 x g for 6 minutes. 3. Transfer the bottom phase of solution B' to a second ATPS (solution C) to form solution C'. 4. Vortex solution C’ thoroughly for 10 seconds until homogeneous, and then centrifuge at 7,000 x g for 1 minute. 5. Add 800 μL of binding buffer (e.g., binding buffer BB1, BB2, or BB3) to a new 2 mL microcentrifuge tube. 6. Transfer the top phase of solution C', which contains the concentrated target analyte, from step 5 to the microcentrifuge tube. 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 so that the magnetic beads bind to the target analyte to form a bead - analyte complex. 8. Incubate the microcentrifuge tube by tilting and rotating for 5 minutes, and then place it on a magnetic stand for 2 minutes to immobilize the bead - analyte complex on 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 μL 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 immobilize the bead - analyte complex on 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 μL) to the microcentrifuge tube, and then rotate it on the magnetic stand, rotating 120 degrees each time for a total of 720 degrees. After rotation, without disturbing the bead - analyte complex, pipette and discard the supernatant in the microcentrifuge tube. 13. Repeat step 12 at least once. 14. Then briefly rotate the microcentrifuge tube with the hinge facing outwards to collect any remaining wash buffer in the tube. 15. Place the microcentrifuge tube back on the magnetic stand for 1 minute to immobilize the bead - analyte complex on the tube wall. 16. Without disturbing the bead - analyte complex, carefully discard all the supernatant (e.g., 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 known to those skilled in the art (e.g., 40 μL) directly to the bead - analyte complex (in a microcentrifuge tube). 20. Resuspend the bead - analyte complex by continuous stirring using a pipette, and then pipette up and down 5 times. 21. Vortex the microcentrifuge tube gently (e.g., for 15 seconds). 22. Incubate the microcentrifuge tube at room temperature for 3 minutes. 23. Place the microcentrifuge tube on a magnetic stand for 1 minute. 24. Without disturbing the magnetic beads, carefully collect the supernatant containing the purified target analyte into a clean maximum recovery tube. 25. The purified target analyte is ready for immediate use or long - term storage at - 20 °C or below. Example 2: Protocol for Concentrating DNA Using a Gradient Buffer

[0189] The following is another exemplary method for concentrating and separating a target analyte according to the present disclosure. In this example, the target analyte is DNA.

[0190] The protocol steps are performed as follows: 1. Add a required volume of the treated biological sample (e.g., plasma) (e.g., 2 mL - 3 mL) to the first ATPS (Solution B) to form Solution B'. The treatment of the biological sample includes, but is not limited to, lysis to form a sample lysate. 2. Vortex Solution B' thoroughly for 10 seconds until homogeneous, and then centrifuge at 2,300 x g for 6 minutes. 3. Transfer the bottom phase of Solution B' to the second ATPS (Solution C) to form Solution C'. 4. Vortex Solution C’ thoroughly for 10 seconds until homogeneous, and then centrifuge at 7,000 x g for 1 minute. 5. Add 800 μL of binding buffer (e.g., 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 the binding buffer. 7. Vortex the provided magnetic beads (e.g., magnetic beads selected from Table 1, 12 μL) before use, and then add them from step 6 to the microcentrifuge tube such that the magnetic beads bind to the target analyte to form a bead - analyte complex. 8. Incubate the microcentrifuge tube by tilting and rotating for 5 minutes, and then place it on a magnetic stand for 2 minutes to immobilize the bead - analyte complex on 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 300 uL of fractionation buffer (e.g., fractionation buffer F1, F2, or F3) to the microcentrifuge tube. Vortex the microcentrifuge tube for 20 seconds, incubate with tilting and rotation for 5 minutes, and then place it on the magnetic stand for 2 minutes to immobilize the bead - analyte complex on the tube wall, such that the target analyte below the target size is released from the bead - analyte complex into the supernatant. 11. Add 600 uL of the second binding buffer (e.g., 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 well - 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 such 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 tilting and rotation for 5 minutes, and then place it on the magnetic stand for 4 minutes to immobilize the second bead - analyte complex on 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 it on the magnetic stand, rotating 120 degrees each time for a total of 720 degrees. After rotation, without disturbing the second bead - analyte complex, pipette and discard the supernatant in the microcentrifuge tube. 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 known to those skilled in the art (e.g., 40 μL) directly to the second bead - analyte complex (in the microcentrifuge tube). 22. Use a pipette to resuspend the second bead - analyte complex by continuous stirring, and then pipette it 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. Without disturbing the magnetic beads, carefully collect the supernatant containing the purified target analyte smaller than the target size into a clean maximum recovery tube. 27. The purified target analyte smaller than the target size is ready for immediate use or long-term storage at -20 °C or below. Example 3: Performance Evaluation of the Example Method

[0191] The performance of the methods and kits disclosed below can be evaluated according to the following steps: (i) Prepare several magnetic bead extraction kit components by varying the following components: a. Solution B i. Polymer ii. Salt iii. Surfactant b. Solution C i. Polymer ii. Salt c. Magnetic beads d. Binding buffer i. Dispersant ii. Polymer e. Fractionation buffer i. Dispersant ii. Polymer (ii) Prepare a sample solution to evaluate and spike in a known amount of target DNA. (iii) Use the variants of the magnetic bead extraction kit prepared in step 1 above and an industry standard extraction kit using its designated procedure for extraction. (iv) Quantify the target DNA using standard qPCR or ddPCR procedures. Table 2

[0192] In various exemplary embodiments, Solution B, Solution C, the binding buffer, and the fractionation buffer are selected from the embodiments shown below in various different combinations. Example 4a: Direct Gradient and Reverse Gradient

[0193] In some embodiments, two different protocols / methods are provided herein to separate target nucleic acids (e.g., DNA) into larger and smaller fragments according to their size using a fractionation buffer, namely direct fractionation and reverse fractionation.

[0194] Direct Gradient

[0195] Now refer to Figure 1A, which shows the general workflow of a direct fractionation scheme according to an exemplary embodiment. In this embodiment, in a first optional lysis step 111, a suitable lysis buffer is added to a sample (such as blood, plasma, serum, cerebrospinal fluid, urine, saliva, feces, tears, sputum, nasopharyngeal mucus, vaginal secretion or penile secretion) to lyse the cells in the sample and release biomolecules into the lysis buffer solution. The lysed sample is passed through two consecutive aqueous two-phase systems (ATPS) to separate and concentrate DNA. In step 112, the lysed sample is mixed with the first ATPS. The mixture is centrifuged to separate it into a top phase and a bottom phase. In step 113, all the bottom phases (also referred to as the "first target-rich phase") to which DNA may be partitioned are transferred into the second ATPS (2nd ATPS) and thoroughly mixed. The mixture is centrifuged to separate it into a top phase and a bottom phase. In step 114, the top phase (also referred to as the "second target-rich phase") from the second ATPS to which the target DNA has been partitioned is extracted into an empty microcentrifuge tube. A direct fractionation buffer (such as those described in Examples 5-6 below) is added to the tube and thoroughly mixed with the top phase from the 2nd ATPS and magnetic beads also added to the tube, such that the magnetic beads bind only to larger DNA fragments. The mixture is incubated for a certain period of time, then spun down and placed on a magnetic rack to immobilize the beads on the tube wall. The large DNA fragments bind to the beads, while the smaller DNA fragments remain in the supernatant, and then, without disturbing the beads, the supernatant is extracted and transferred to a new microcentrifuge tube (see step 115). Further in step 115, a binding buffer (such as binding buffer A as described in Example 5 below) is added to the extracted supernatant. Then magnetic beads are added to the supernatant-binding buffer mixture and incubated for a certain period of time to bind the smaller DNA fragments. Then the tube is spun down and placed on a magnetic rack to immobilize the bead-analyte complex. The supernatant is discarded without disturbing the bead-analyte complex. In step 116, the bead-analyte complex can be further purified by several washing steps (optional) using a suitable washing buffer. Then the bead-analyte complex is resuspended in a suitable elution buffer, thoroughly mixed, and then placed on a magnetic rack to immobilize the magnetic beads. The supernatant containing the purified DNA sample is collected for further use.

[0196] Reverse Gradient

[0197] Now refer to Figure 1B, which shows the general workflow of the reverse fractionation scheme according to an exemplary embodiment. In this embodiment, steps 121, 122, and 123 are the same as or similar to steps 111, 112, and 113 described in the direct fractionation scheme above. In step 124, the top phase of the second ATPS to which the target DNA will be assigned (also referred to as the "second target-rich phase") is extracted into a microcentrifuge tube containing a binding buffer (such as binding buffer A described in Example 5 below). Magnetic beads are added to the mixture and incubated for a period of time. Then the tube is spun down and placed on a magnetic rack to immobilize the beads on the tube wall. DNA of all sizes should bind to the magnetic beads, while proteins and contaminants remain in the supernatant, which is discarded. In step 125, a reverse fractionation buffer (such as the buffer described in Examples 5-7 below) is then added to the beads and mixed well. After incubation for a period of time, the tube is spun down and placed on a magnetic rack. Large DNA fragments bind to the beads, while smaller DNA fragments remain in the supernatant, which is then extracted and transferred to a new microcentrifuge tube without disturbing the beads (see step 126). Further, in step 126, a target binding buffer (e.g., binding buffer B in Example 5 below) and magnetic beads are added to the supernatant to capture the fractionated DNA fragments onto the beads. The mixture is incubated for a period of time, then spun down and placed on a magnetic rack to immobilize the bead-analyte complex. The supernatant is pipetted off and discarded. In step 127, the bead-analyte complex can be further purified by several washing steps (optional) using a suitable washing buffer. Then the bead-analyte complex is resuspended in a suitable elution buffer, mixed well, and then placed on a magnetic rack to immobilize the magnetic beads. The supernatant containing the purified DNA sample is collected for further use.

[0198] In some embodiments, possible magnetic beads that can be used include, but are not limited to, those listed in Table 1.2 below. Table 1.2: Magnetic Bead Examples Example 4b: Calculation of DNA Retention Value

[0199] In some embodiments, the DNA retention value of a given sample is calculated by the method described below.

[0200] Using a size fractionation procedure, samples with DNA ladders were extracted according to any of the methods described in this disclosure. To estimate the DNA retention value, the extracted DNA samples and positive control conditions (representing 100% recovery conditions for all sized DNA) were analyzed using the Agilent Bioanalyzer High Sensitivity DNA Kit (part number 5067-4626). The concentration of each peak was measured by the Agilent 2100 analysis software and exported to a spreadsheet. First, for the positive control conditions, the concentration of all DNA fragments greater than 100 bp was divided by the concentration of the 100 bp DNA fragment to calculate the ratio (Conc / Conc_a) (as shown in Table I). This 100 bp DNA size fragment (also known as the "non-size-selected internal 100 bp control") was chosen because it is not affected by size fractionation and thus can serve as a normalization value for 100% recovery conditions in each experiment. Table I: Results for Positive Control Conditions Positive Control

[0201] The same ratio calculation was performed on the extracted DNA samples (referred to as "Example A"), as shown in Table II. The % recovery was then calculated by dividing the "Ratio" for "Example A" in Table II by the "Ratio" for "Positive Control" in Table I. Due to artifacts of the bioanalyzer and fluctuations in the baseline signal, values greater than 100% may occur. All values greater than 100% were assumed to be 100% recovery. The bp size giving 70% recovery compared to the non-size-selected internal 100 bp control was estimated by finding two % recovery values across 70% and performing a linear regression between these two points. The resulting value was considered the DNA retention value for the said example. Table II: Results for Example A Example 5

[0202] The following studies were conducted to compare the stability and robustness of direct fractionation and reverse fractionation schemes for extracting DNA from different volume plasma samples (specifically, different volumes of the top phase of the 2ATPS added to the fractionation step). The ability of the direct fractionation and reverse fractionation schemes to accommodate sample-to-sample variability was evaluated.

[0203] Materials and Methods

[0204] Plasma Cell Lysis

[0205] Spike 100 fg of 145 bp dsDNA oligonucleotide, 80 ng of 20 bp ladder (Jena Bioscience, catalog number M212), and 40 ng of 50 bp ladder (Jena Bioscience, catalog number M-213) into every 2 mL of plasma. Add 160 μL of the appropriate lysis buffer and 60 μL of proteinase K (28.5 mg / mL) to every 2 mL of spiked plasma. Vortex the mixture thoroughly and then incubate it in a preheated 60 °C heating block for 15 minutes.

[0206] Two-Phase System

[0207] The lysed plasma samples were passed through two consecutive aqueous two-phase systems (ATPS) to separate and concentrate the DNA. Transfer 2.22 mL of the lysed sample to the first ATPS (polymer, salt, and / or surfactant) and vortex to mix. Centrifuge the mixture at 2,300 rcf for 6 minutes. Transfer all of the salt-rich bottom phase (about 1 mL) (also called the "first target-rich phase") where the DNA may be partitioned to the second ATPS (polymer, salt, and / or surfactant) and mix well. Then centrifuge the mixture at 7,000 xg for 1 minute. Carefully extract the top phase (about 150 μL) (also called the "second target-rich phase") where the target DNA will be partitioned into an empty microcentrifuge tube for further purification. Three replicates were performed, and different top phase volumes (100 μL, 150 μL, and 180 μL) of the second ATPS were extracted. One set of samples with each top phase volume will undergo direct fractionation, while the other set will undergo a reverse fractionation protocol.

[0208] Direct Gradient

[0209] For each top phase volume of the ATPS, two direct fractionation buffer formulations with different expected DNA retention sizes were used: (i) fractionation buffer D1 for a smaller (about 150 bp) expected retention size, i.e., 3 - 7 M guanidine salt, 0.1% - 15% (w / v) polymer, 0.01 M - 0.5 M pH buffer, and 0.01 M - 0.5 M metal chelator; and (ii) fractionation buffer D2 for a larger (about 300 bp) expected retention size, i.e., 3 - 7 M guanidine salt, 0.1% - 15% (w / v) polymer, 0.01 M - 0.5 M pH buffer, and 0.01 M - 0.5 M metal chelator.

[0210] For each sample, the top phase of the second ATPS was transferred to an empty microcentrifuge tube. 80 μL of direct fractionation buffer (fractionation buffer D1 or D2) was added to the tube and mixed well with 12 μL of magnetic beads that were also added to the tube. The magnetic beads (Cat#MF-SIL-5024) were purchased from MagQu Co., Ltd. The mixture was incubated with tilting and rotation for 5 minutes. Then the tube was briefly rotated to sediment and placed on a magnetic rack for 2 minutes to immobilize the beads on the wall of the tube. Large DNA fragments bound to the beads while smaller DNA fragments remained in the supernatant, and then the supernatant was extracted and transferred to a new microcentrifuge tube without disturbing the beads. 600 μL of binding buffer A (3 - 7 M guanidine salt and 0.1% - 15% (w / v) polymer) was added to the extracted supernatant, which provided the necessary discrete salts for salt bridge formation between the DNA and the solid-phase magnetic beads. Then 6 μL of magnetic beads was added to the supernatant-binding buffer mixture and incubated with tilting and rotation for an additional 5 minutes. Then the tube was rotated to sediment and placed on the magnetic rack for 4 minutes. The supernatant was discarded without disturbing the bead-analyte complex. Then 600 μL of binding buffer A was added to the bead-analyte complex again and the tube was rotated on the rack for a total of 720°. The supernatant was discarded. Then the bead-analyte complex was further purified.

[0211] Reverse Gradient

[0212] For each top phase volume, two reverse fractionation buffer formulations were applied for different expected DNA retention sizes: (i) fractionation buffer R1 for a smaller (about 150 bp) expected retention size, namely 3 - 7 M guanidine salt, 0.1% - 15% (w / v) polymer, 0.01 M - 0.5 M pH buffer, and 0.01 M - 0.5 M metal chelator; and (ii) fractionation buffer R2 for a larger (about 300 bp) expected retention size, namely 0.5 - 5.0 M guanidine, 0.01 M - 0.5 M pH buffer, and 0.01 M - 0.5 M metal chelator.

[0213] Transfer each top-phase volume from the second ATPS into a microcentrifuge tube containing 800 μL of binding buffer A (3 - 7 M guanidine salt and 0.1% - 15% (w / v) polymer). Add 12 μL of magnetic beads to the mixture and incubate with tilting rotation for 5 minutes. Spin the tube down and place on a magnetic rack for 2 minutes to immobilize the beads on the tube wall. DNA of all sizes should bind to the magnetic beads, while proteins and contaminants remain in the supernatant, which is discarded. Then add 300 μL of reverse fractionation buffer (fractionation buffer R1 or R2) to the beads. After vortex mixing, incubate the mixture on a rotator for 5 minutes. Spin the tube down and place on the magnetic rack for an additional 2 minutes. Larger DNA fragments bind to the beads, while smaller DNA fragments remain in the supernatant. Then extract the supernatant and transfer it to a new microcentrifuge tube. Add 600 μL of binding buffer B (3 - 7 M guanidine salt and 0.1% - 15% (w / v) polymer, also referred to as "target binding buffer" in some embodiments) and 6 μL of magnetic beads to the supernatant to capture the fractionated DNA fragments onto the beads. Incubate the mixture on a rotator for an additional 5 minutes, then spin down and place on the magnetic rack for 4 minutes. Pipette and discard the supernatant again. Then further purify the bead - analyte complex.

[0214] Purification of DNA

[0215] Add 800 μL of wash buffer (70% ethanol, 0.001 M EDTA, 0.01 M Tris - HCl) to each processed sample that has undergone the direct fractionation or reverse fractionation protocol as described above, and rotate the tube a total of 720° on the magnetic rack. Discard the supernatant. The wash step is performed twice. Briefly centrifuge the tube in a benchtop microcentrifuge with the hinge facing outwards to collect any remaining wash buffer. Then dry the bead - analyte complex on the magnetic holder with the lid open for 7 minutes. Resuspend the bead - analyte complex in 40 μL of elution buffer (0.01 M Tris - HCl, 0.001 M EDTA) by mixing with a continuous pipette, then perform a gentle vortex. Then place the tube on the magnetic rack for 1 minute to immobilize the magnetic beads. Carefully collect the supernatant containing the purified DNA sample into a DNA low - binding tube for detection without disturbing the magnetic beads.

[0216] Detection of DNA

[0217] Quantify the recovery of DNA oligonucleotides of different sizes in the extracted samples by electrophoresis. The gel mixture is from Agilent TMPrepared using a high-sensitivity DNA kit (Agilent, 5067-4626). 9 μL of the gel dye mixture was dispensed into each well on the microfluidic chip, and 1 μL of the purified DNA sample was added. Electrophoresis on the chip was performed using an Agilent TM 2100 Bioanalyzer. The fluorescence signal of the reaction was collected and analyzed using the software Agilent TM Technologies 2100 Expert. The actual DNA cut-off value was estimated by the calculation method described in Example 4b, i.e., calculating the value of the base pairs of the purified DNA sample where the DNA recovery % was 70%.

[0218] The conditions of the sample sets used in this study and the composition of the fractionation buffer formulations (for direct and reverse fractionation) are summarized in Table 3 below. Table 3: Conditions of the sample sets and composition of the fractionation buffer formulations

[0219] Results

[0220] Recovery of DNA

[0221] Differences in sample volume, such as the volume of the top phase extracted in the 2nd ATPS directly used for subsequent fractionation (direct fractionation or reverse fractionation) protocols, may affect the efficiency of DNA fractionation. To address such differences, different top phase volumes (100 μL, 150 μL, and 180 μL) of the 2nd ATPS were used in the fractionation protocols for both direct and reverse fractionation to study the stability and robustness of the two fractionation types.

[0222] Figure 2A and 2B show the electrophoretograms of DNA oligonucleotide recovery from plasma extracted from different volumes of the top phase in the 2nd ATPS using direct fractionation (using fractionation buffer D1) and reverse fractionation (using fractionation buffer R1), respectively, where the expected DNA cut-off value is small (about 150 bp). The actual DNA cut-off values of the purified DNA extracted from different top phase volumes of the 2nd ATPS using direct fractionation (using fractionation buffer D1) or reverse fractionation (using fractionation buffer R1) are shown in Table 4. Table 4: Actual DNA cut-off values of the purified DNA extracted from different top phase volumes of the 2nd ATPS using direct fractionation (using fractionation buffer D1) or reverse fractionation (using fractionation buffer R1)

[0223] See Figure 2AAnd Table 4, using direct fractionation, when the top phase volume is small (Condition 1, 100 μL), the recovery of larger DNA fragments (>100 bp) is significantly reduced, while when the top phase volume is large (Condition 3, 180 μL), the recovery of larger DNA oligonucleotides (100 bp - 200 bp) is significantly increased. These results show that when the top phase volume deviates from the standard (Condition 2, 150 μL) in direct fractionation, the cut-off value shifts significantly, indicating that the change in sample volume greatly affects the stability and efficiency of DNA fractionation in direct fractionation.

[0224] Now refer to Figure 2B and Table 4, using reverse fractionation, the difference in the size of DNA fragments recovered using reverse fractionation with different top phase volumes (100 μL, 150 μL, and 180 μL) is significantly smaller compared to direct fractionation, and the shift in the DNA cut-off value is minimal, i.e., within the acceptable range of 131 bp - 175 bp. As Figure 2B shown, there is a large overlap in the electrophoretograms of Conditions 4 - 6 using reverse fractionation with different top phase volumes.

[0225] Figure 3A and 3B respectively show the electrophoretograms of DNA oligonucleotide recovery from plasma extracted from different volumes of the top phase in the 2nd ATPS using direct fractionation (using fractionation buffer D2) and reverse fractionation (using fractionation buffer R2), where a larger DNA cut-off value (about 300 bp) is expected. The actual DNA cut-off values of the purified DNA extracted in different top phase volumes of the 2nd ATPS using direct fractionation (using fractionation buffer D2) or reverse fractionation (using fractionation buffer R2) are shown in Table 5. Table 5: Actual DNA cut-off values of purified DNA extracted in different top phase volumes of the 2nd ATPS using direct fractionation (using fractionation buffer D2) or reverse fractionation (using fractionation buffer R2)

[0226] Refer to Figure 3A and Table 5, using direct fractionation, when the top phase volume is small (Condition 7, 100 μL), the cut-off value shifts significantly towards a smaller value (i.e., 229 bp). It is worth noting that when the top phase volume is large (Condition 9, 180 μL), the recovery of small DNA fragments (<100 bp) is low (i.e., 31% relative to the 150 μL condition), even though the recovery of larger DNA fragments is similar to that using a top phase volume of 150 μL (Condition 8).

[0227] Now refer to Figure 3BAnd Table 5 show that, in the case of reverse fractionation, the differences in the sizes of DNA fragments recovered by reverse fractionation using different top-phase volumes (100 μL, 150 μL, and 180 μL) are significantly smaller compared to direct fractionation, and the shift in the DNA retention value is minimal, i.e., within the acceptable range of 281 bp - 356 bp. As Figure 3B shown, there is a large overlap in the electrophoretograms of Conditions 10 - 12 of reverse fractionation using different top-phase volumes.

[0228] Overall, the results indicate that reverse fractionation is unexpectedly more stable with respect to different top-phase volumes compared to direct fractionation, both in terms of the DNA retention value and the recovery of DNA, especially for small DNA fragments. The high stability and efficiency of DNA fractionation using the reverse fractionation method are beneficial for separating small DNA fragments below the target size. Example 6

[0229] The following studies were conducted to compare the stability and robustness of direct and reverse fractionation schemes for DNA extraction from different sample types (i.e., plasma and urine). The stability and robustness of fractionation are important for a wide range of applications. One of the variables is the sample type, as different components in the sample may affect the performance of fractionation to varying degrees. To investigate the tolerance of direct and reverse fractionation to different sample types, plasma and urine samples were studied using the same direct and reverse fractionation buffers and protocols as described in Example 5, and their performance differences were investigated.

[0230] Materials and Methods

[0231] Plasma Cell Lysis

[0232] 100 fg of 145 bp dsDNA oligonucleotide, 80 ng of 20 bp ladder (Jena Bioscience, catalog number M212), and 40 ng of 50 bp ladder (Jena Bioscience, catalog number M - 213) were spiked into each 2 mL of plasma. 160 μL of the appropriate lysis buffer and 60 μL of proteinase K (28.5 mg / mL) were added to each 2 mL of spiked plasma. The mixture was vortexed thoroughly and then lysed in a preheated 60 °C heating block for 15 minutes.

[0233] Urine Pretreatment and Lysis

[0234] The urine samples were pretreated with 200 μL of 0.1 M EDTA per 10 mL of urine sample, vortexed thoroughly and centrifuged at 3000 x g for 10 minutes. This preserves the cell-free DNA (cfDNA) present in the sample and prevents its degradation over time. The supernatant was transferred to a new tube while discarding the pellet. 100 fg of 145 bp dsDNA oligonucleotide, 80 ng of 20 bp ladder (Jena Bioscience, cat. no. M212), and 40 ng of 50 bp ladder (Jena Bioscience, cat. no. M-213) were spiked into every 2 mL of urine. The unwanted proteins and cells present in the pretreated urine samples were lysed by adding 1200 μL of proteinase K (28.57 mg / mL) and 4 mL of a suitable lysis buffer solution to 40 mL of the sample. The sample was then vortexed thoroughly until homogeneous and then incubated in a pre-warmed 37 °C water bath for 15 minutes.

[0235] Two-Phase System

[0236] The lysed plasma and urine samples were passed through two consecutive aqueous two-phase systems (ATPS) to separate and concentrate the DNA. 2.22 mL of the lysed plasma or 2.26 mL of the lysed urine was transferred to the first ATPS (polymer, salt, and / or surfactant) and vortexed. The mixture was centrifuged at 2,300 rcf for 6 minutes. All of the salt-rich bottom phase (≈1 mL) to which the DNA may be partitioned (also referred to as the “first target-rich phase”) was transferred to the second ATPS (polymer, salt, and / or surfactant) and mixed well. The mixture was then centrifuged at 7,000 x g for 1 minute. The polymer-rich top phase (≈150 μL) to which the target DNA will be partitioned (also referred to as the “second target-rich phase”) was carefully aspirated into an empty microcentrifuge tube for further purification. One sample set with plasma or urine samples will undergo direct fractionation, while the other set will undergo a reverse fractionation protocol.

[0237] Direct Gradient

[0238] For each sample type, two direct fractionation buffer formulations with different expected DNA retention sizes were used: (i) fractionation buffer D1 for a smaller (≈150 bp) expected retention size, i.e., 3–7 M guanidine salt, 0.1%–15% (w / v) polymer, 0.01 M–0.5 M pH buffer, and 0.01 M–0.5 M metal chelator; and (ii) fractionation buffer D2 for a larger (≈300 bp) expected retention size, i.e., 1–5 M guanidine salt, 0.1%–15% (w / v) polymer, 0.01 M–0.5 M pH buffer, and 0.01 M–0.5 M metal chelator.

[0239] The steps for directly fractionating plasma and urine samples to separate and isolate smaller DNA fragments from larger DNA fragments are the same as or similar to the steps discussed above for Example 5. For the sake of brevity and simplicity of this disclosure, the discussion of the direct fractionation steps is not repeated here.

[0240] Reverse Gradient

[0241] For different expected DNA retention sizes, two reverse fractionation buffer formulations were applied to each sample type: (i) Fractionation buffer R1 for a smaller (about 150 bp) expected retention size, namely 1 - 5 M guanidine salt, 0.1% - 15% (w / v) polymer, 0.01 M - 0.5 M pH buffer, and 0.01 M - 0.5 M metal chelator; and (ii) Fractionation buffer R2 for a larger (about 300 bp) expected retention size, namely 0.5 - 5.0 M guanidine, 0.01 M - 0.5 M pH buffer, and 0.01 M - 0.5 M metal chelator.

[0242] The steps for reverse fractionating plasma and urine samples to separate and isolate smaller DNA fragments from larger DNA fragments are the same as or similar to the steps discussed above for Example 5. For the sake of brevity and simplicity of this disclosure, the discussion of the reverse fractionation steps is not repeated here.

[0243] Purification of DNA

[0244] The steps for DNA purification of each sample are the same as or similar to the steps discussed above for Example 5. For the sake of brevity and simplicity of this disclosure, the discussion of the purification steps is not repeated here.

[0245] Detection of DNA

[0246] The recovery of DNA oligonucleotides of different sizes in the extracted samples was quantified by electrophoresis. The gel mixture was prepared from the Agilent TM High Sensitivity DNA Kit (Agilent Technologies, 5067 - 4626). 9 μL of the gel dye mixture was dispensed into each well on the microfluidic chip, and 1 μL of the purified DNA sample was added. Electrophoresis on the chip was performed by the Agilent TM 2100 Bioanalyzer. The fluorescence signals of the reaction were collected and analyzed using the software Agilent TM Technologies 2100 Expert. The actual DNA retention value was estimated by the calculation method described in Example 4b, i.e., calculating the value of the base pairs of the purified DNA sample where the DNA recovery % was 70%.

[0247] The conditions for the sample sets used in this study and the composition of the fractionation buffer formulations (for direct and reverse fractionation) are summarized in Table 6 below. Table 6: Conditions for the sample sets and composition of the fractionation buffer formulations

[0248] Results

[0249] Recovery of DNA

[0250] Figure 4A and 4B respectively show the electrophoretograms of DNA oligonucleotide recovery for different sample types (i.e., plasma and urine) using direct fractionation (using fractionation buffer D1) and reverse fractionation (using fractionation buffer R1), where the expected DNA retention value is small (about 150 bp). The actual DNA retention values of the purified DNA extracted from different sample types using direct fractionation (using fractionation buffer D1) or reverse fractionation (using fractionation buffer R1) are shown in Table 7. Table 7: Actual DNA retention values of the purified DNA extracted from different sample types using direct fractionation (using fractionation buffer D1) or reverse fractionation (using fractionation buffer R1)

[0251] Refer to Figure 4A and Table 7. In the case where the expected retention value is small (about 150 bp), direct fractionation performs well for plasma samples, but only a small amount of small (<50 bp) DNA was recovered in urine samples, indicating that direct fractionation cannot adapt to various sample type inputs.

[0252] Now refer to Figure 4B and Table 7. In the case of reverse fractionation, compared with direct fractionation, the difference in the sizes of the DNA fragments recovered using reverse fractionation with plasma and urine is significantly smaller, and the difference in DNA retention values (147 bp for plasma and 125 bp for urine) is small, indicating the stability of reverse fractionation and its wider availability for various sample types.

[0253] Figure 5A and 5B respectively show the electrophoretograms of DNA oligonucleotide recovery from plasma extracted from different sample types using direct fractionation (using fractionation buffer D2) and reverse fractionation (using fractionation buffer R2), where the expected DNA retention value is large (about 300 bp). The actual DNA retention values of the purified DNA extracted from different sample types using direct fractionation (using fractionation buffer D2) or reverse fractionation (using fractionation buffer R2) are shown in Table 8. Table 8: Actual DNA retention values of purified DNA extracted in different sample types using direct fractionation (using fractionation buffer D2) or reverse fractionation (using fractionation buffer R2)

[0254] Reference Figure 5A And Table 8, when urine was used in place of plasma, with direct fractionation, the retention values shifted significantly to much smaller values (i.e., 138 bp), indicating that direct fractionation may result in excessive removal of target DNA when changing the sample type, which may be detrimental to downstream assays.

[0255] Reference Figure 5B , in the case of reverse fractionation, there was a large overlap in the electrophoretograms of the DNA fragments recovered using reverse fractionation with different sample types (i.e., plasma and urine), indicating that size fractionation and the target DNA extracted are generally consistent in different sample types. Although the DNA retention values in urine samples shifted to slightly larger values, this did not result in excessive removal of target DNA (compared to direct fractionation), and thus it is more suitable for downstream assays.

[0256] Overall, these results indicate that reverse fractionation is more stable for different sample types and has a wider range of applications compared to direct fractionation. Example 7

[0257] The following studies were conducted to evaluate the DNA retention sizes estimated using reverse fractionation with different fractionation buffer formulations.

[0258] Materials and Methods

[0259] Plasma Cell Lysis

[0260] 100 fg of 145 bp dsDNA oligonucleotide and 80 ng of 20 bp ladder (Jena Bioscience, catalog number M212) were spiked into each 2 mL of plasma. 160 μL of the appropriate lysis buffer and 60 μL of proteinase K (28.5 mg / mL) were added to each 2 mL of spiked plasma. The mixture was vortexed thoroughly and then lysed in a preheated 60 °C heating block for 15 minutes.

[0261] Two-Phase System

[0262] The lysed plasma samples were passed through two consecutive aqueous two-phase systems (ATPS) to separate and concentrate the DNA. The steps for performing consecutive ATPS on the plasma samples were the same as or similar to those discussed above for Example 5. For the sake of brevity and simplicity of this disclosure, the discussion of the consecutive ATPS steps will not be repeated here. The samples collected after the ATPS steps will be subjected to a reverse fractionation protocol with different reverse fractionation buffer formulations.

[0263] Reverse Gradient

[0264] Different reverse fractionation buffer formulations were used and are summarized in Table 9 below. The steps for reverse fractionation of plasma samples to separate and isolate smaller DNA fragments from larger DNA fragments are the same as or similar to the steps discussed above with respect to Example 5. For the sake of brevity and simplicity of this disclosure, the discussion of the reverse fractionation steps will not be repeated here. Table 9: Reverse Fractionation Buffer Formulations

[0265] Purification of DNA

[0266] The steps for DNA purification of each sample are the same as or similar to the steps discussed above with respect to Example 5. For the sake of brevity and simplicity of this disclosure, the discussion of the purification steps will not be repeated here.

[0267] Detection of DNA

[0268] The recovery of DNA oligonucleotides of different sizes in the extracted samples was quantified by electrophoresis. The steps for DNA detection of each sample are the same as or similar to the steps discussed above with respect to Example 5. For the sake of brevity and simplicity of this disclosure, the discussion of the detection steps will not be repeated here. The recovery % and the estimated DNA cut-off value (also referred to as the "actual DNA cut-off value" in some embodiments) were calculated by the method described in Example 4b, i.e., calculating the value of the base pairs of the purified DNA sample in which the DNA recovery % was 70%.

[0269] Results

[0270] Recovery of DNA

[0271] Figure 6A-6H Electrophorograms showing the DNA oligonucleotide recovery of plasma samples by reverse fractionation using different reverse fractionation buffer formulations R-015 to R-021 are shown. The estimated DNA cut-off values and recovery % of DNA of different sizes extracted using different reverse fractionation formulations are shown in Table 10. Table 10: Estimated DNA Cut-off Values and Recovery % of DNA of Different Sizes Extracted Using Different Reverse Fractionation Buffer Formulations

[0272] Referring to FIGS. 6A - 6H and Table 10, the results show that the reverse fractionation method can be carried out by using a wide range of reverse fractionation buffer formulations. The results also indicate that by varying the concentrations of the chaotropic agent, pH buffer, and / or metal chelator in the fractionation buffer, the DNA retention value (e.g., from about 100 bp to about 500 bp) can be controlled, thereby allowing precise size selection of the recovered DNA molecules. Numbered embodiments Group 1:

[0273] Example 1. A method for concentrating and purifying one or more target analytes from a sample solution, the method comprising the steps of: (a) adding the sample solution containing one or more target analytes to a first aqueous two - phase system (ATPS) to form a mixture that separates into a first phase and a second phase, 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 complexes to obtain a final solution containing the concentrated and purified one or more target analytes.

[0274] Example 2. The method according to Example 1, wherein step (b) further comprises the steps of: (i) adding the separated first phase containing the concentrated one or more target analytes to a second ATPS to form a second mixture that separates into a third phase and a fourth phase, wherein the one or more target analytes are concentrated in the third phase; and (ii) separating the third phase containing the concentrated one or more target analytes to form the concentrated solution for step (c) in step (b).

[0275] Example 3. The method according to any one of the preceding examples, wherein the concentrated solution of step (b) is mixed with a binding buffer, wherein the binding buffer comprises at least one chaotropic agent selected from: n - butanol, ethanol, guanidine chloride, guanidine thiocyanate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2 - propanol, sodium dodecyl sulfate, thiourea, and urea, to obtain the concentrated solution for step (c).

[0276] Example 4. The method according to any one of the preceding examples, wherein step (d) further comprises the steps of: (i) mixing the bead - analyte complex with a fractionation buffer comprising a polymer, a salt, a surfactant, a chaotropic agent, or a combination thereof to form a fractionation solution such that one or more target analytes below the target size are released from the bead - analyte complex into the fractionation solution; (ii) immobilizing the bead - analyte complex using a magnetic support; and (iii) separating the one or more target analytes below the target size in the fractionation solution from the immobilized bead - analyte complex.

[0277] Example 5. The method according to Example 4, wherein step (d) further comprises the steps of: (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 chaotropic agent 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.

[0278] Example 6. The method according to any one of the preceding examples, the method further comprising the step of: (e) performing a diagnostic assay on the final solution to detect and quantify the one or more target analytes.

[0279] Example 7. The method according to any one of the preceding examples, wherein the one or more target analytes are selected from the group consisting of nucleic acids, proteins, antigens, biomolecules, sugar moieties, lipids, sterols, and combinations thereof.

[0280] Example 8. The method according to any one of the preceding examples, wherein the one or more target analytes are DNA.

[0281] Example 9. The method according to any one of the preceding examples, wherein the one or more target analytes are cell - free DNA or circulating tumor DNA.

[0282] Example 10. The method according to any one of the preceding examples, wherein the first ATPS comprises a first ATPS component that is capable of forming a first phase and a second phase when dissolved in an aqueous solution, wherein the first ATPS component is selected from the group consisting of polymers, salts, surfactants, and combinations thereof.

[0283] Example 11. The method according to any one of Examples 2-10, wherein the second ATPS comprises a second ATPS component that is capable of forming a third phase and a fourth phase when dissolved in an aqueous solution, and wherein the second ATPS component is selected from the group consisting of polymers, salts, surfactants, and combinations thereof.

[0284] Example 12. The method according to Example 10 or 11, wherein the polymer is dissolved in the aqueous solution at a concentration of 4% - 84% (w / w).

[0285] Example 13. The method according to any one of Examples 10 - 12, wherein the polymer is selected from the group consisting of: polyalkylene glycol (PEG), such as hydrophobically modified polyalkylene glycol; poly(oxyalkylene) polymer; poly(oxyalkylene) copolymer, such as hydrophobically modified poly(oxyalkylene) copolymer; polyvinylpyrrolidone; polyvinyl alcohol; polyvinylcaprolactam; polyvinyl methyl ether; alkoxylated surfactant; alkoxylated starch; alkoxylated cellulose; alkyl hydroxyalkyl cellulose; silicone-modified polyether; and poly(N-isopropylacrylamide) and its copolymers. The method according to any one of the foregoing examples, wherein the polymer is selected from the group consisting of: polyether, polyimine, polyalkylene glycol, vinyl polymer, alkoxylated surfactant, polysaccharide, alkoxylated starch, alkoxylated cellulose, alkyl hydroxyalkyl cellulose, polyether-modified silicone, polyacrylamide, polyacrylic acid and its copolymers. The method according to any one of the foregoing examples, wherein the polymer is selected from the group consisting of: dipropylene glycol, tripropylene glycol, polyethylene glycol, polypropylene glycol, poly(ethylene glycol - propylene glycol), poly(ethylene glycol - random - propylene glycol), polyvinylpyrrolidone, polyvinyl alcohol, polyvinylcaprolactam, polyvinyl methyl ether, dextran, carboxymethyl dextran, dextran sulfate, hydroxypropyl dextran, starch, carboxymethyl cellulose, polyacrylic acid, hydroxypropyl cellulose, methyl cellulose, ethyl hydroxyethyl cellulose, maltodextrin, polyvinylimine, poly(N-isopropylacrylamide) and its copolymers. The method according to any one of the foregoing examples, wherein the polymer is selected from the group consisting of: dipropylene glycol, tripropylene glycol, polyethylene glycol, polypropylene glycol, poly(ethylene glycol - propylene glycol), poly(ethylene glycol - random - propylene glycol), polyvinylpyrrolidone, polyvinyl alcohol, polyvinylcaprolactam, polyvinyl methyl ether and poly(N-isopropylacrylamide). The method according to any one of the foregoing examples, wherein the polymer is selected from the group consisting of: polyacrylamide, polyacrylic acid and its copolymers. The method according to any one of the foregoing examples, wherein the polymer is selected from the group consisting of: dextran, carboxymethyl dextran, dextran sulfate, hydroxypropyl dextran and starch. The method according to any one of the foregoing examples, wherein the polymer has an average molecular weight in the range of 200 - 1,000 Da, 200 - 35,000 Da, 425 - 2,000 Da, 400 - 35,000 Da, 980 - 12,000 Da or 3,400 - 5,000,000 Da. The method according to any one of the foregoing examples, wherein the polymer comprises ethylene oxide and propylene oxide units, and the polymer has an EO:PO ratio of 90:10 to 10:90.

[0286] Example 14.

[0287] Example 15. The method according to any one of Examples 10-14, wherein the salt is dissolved in an aqueous solution at a concentration of 1%-80% (w / w).

[0288] Example 16. The method according to any one of Examples 10-15, wherein the salt is dissolved in an aqueous solution at a concentration of 8%-80% (w / w).

[0289] Example 17. The method according to any one of Examples 10-16, wherein the salt is selected from the group consisting of: lyophilic salts, discrete salts, inorganic salts containing cations and anions, NaCl, Na3PO4, K3PO4, Na2SO4, potassium citrate, (NH4)2SO4, sodium citrate, sodium acetate, ammonium acetate, magnesium salts, lithium salts, sodium salts, potassium salts, cesium salts, zinc salts, aluminum salts, bromide salts, iodide salts, fluoride salts, carbonates, sulfates, citrates, carboxylates, borates, phosphates, potassium phosphate, and ammonium sulfate, the cations are, for example, linear or branched trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium, and tetrabutylammonium, and the anions are, for example, phosphate, sulfate, nitrate, chloride, and bicarbonate.

[0290] Example 18. The method according to any one of Examples 10-17, wherein the surfactant is dissolved in an aqueous solution at a concentration of 0.05%-10% (w / w).

[0291] Example 19. The method according to any one of Examples 10-18, wherein the surfactant is dissolved in an aqueous solution at a concentration of 0.05%-9.8% (w / w).

[0292] Example 20. The method according to any one of Examples 10-19, wherein the surfactant is selected from the group consisting of: Triton-X, Triton-114, Igepal CA-630, and Nonidet P-40, anionic surfactants (such as carboxylates, sulfonates, petroleum sulfonates, alkylbenzene sulfonates, naphthalene sulfonates, olefin sulfonates, alkyl sulfates, sulfates, sulfated natural oils, sulfated natural fats, sulfated esters, sulfated alkanolamides, ethoxylated alkylphenols, and sulfated alkylphenols), nonionic surfactants (such as ethoxylated fatty alcohols, polyoxyethylene surfactants, carboxylic acid esters, polyethylene glycol esters, sorbitan esters, fatty acid ethylene glycol esters, carboxylic acid amides, monoalkanolamine concentrates, polyoxyethylene fatty acid amides), cationic surfactants (such as quaternary ammonium salts, amines with amide bonds, polyoxyethylene alkyl and cycloaliphatic amines, n,n,n',n'-tetrasubstituted ethylenediamine, 2-alkyl-1-hydroxyethyl-2-imidazoline), and amphoteric surfactants (such as n-cocoyl 3-aminopropionic acid / sodium salt, n-tallow 3-iminodipropionate, disodium salt, n-carboxymethyl-n-dimethyl-n-9-octadecenyl ammonium hydroxide, n-cocoylamidoethyl-n-hydroxyethyl glycine and its sodium salt).

[0293] Example 21. The method according to any one of the foregoing examples, wherein step (a) further comprises the steps of: (i) embedding the porous material in a component capable of forming a first ATPS; and (ii) contacting the sample solution with the porous material embedded with the component, wherein the component forms a first phase and a second phase when the sample solution passes through the porous material.

[0294] The exemplary embodiments of the present invention have thus been fully described. Although the description refers to specific embodiments, those skilled in the art will appreciate that the present invention can be implemented with variations of these specific details. Therefore, the present invention should not be construed as limited to the embodiments set forth herein.

Claims

1. A method for separating a target nucleic acid of a size smaller than a target size from a sample containing nucleic acid components; The method comprises the following steps: (a) Preparing a sample solution from the sample; (b) Contacting a plurality of beads with the sample solution, wherein the nucleic acid component binds to the plurality of beads to form a bead - analyte complex; (c) Mixing the bead - analyte complex with a fractionation buffer containing at least one dissociating agent to form a bulk fractionation solution, wherein the target nucleic acid below the target size is released from the bead - analyte complex into the bulk fractionation solution; (d) Fixing the bead - analyte complex; and (e) Separating the bulk fractionation solution containing the separated target nucleic acid below the target size from the fixed bead - analyte complex.

2. The method according to claim 1, wherein step (a) further comprises (a1) Adding the sample to a first aqueous two - phase system (ATPS) to form a mixture that separates into a first target - rich phase and a first target - poor phase, wherein the nucleic acid component is concentrated in the first target - rich phase; and (a2) Separating the first target - rich phase containing the concentrated nucleic acid component to obtain the sample solution.

3. The method according to claim 2, wherein after step (a2), step (a) further comprises the following steps: (a3) Adding the sample solution from step (a2) to a second ATPS to form a second mixture that separates into a second target - rich phase and a second target - poor phase, wherein the nucleic acid component is concentrated in the second target - rich phase; and (a4) Separating the second target - rich phase containing the concentrated nucleic acid component to form the sample solution in step (a).

4. The method according to any one of the preceding claims, wherein before step (b), the plurality of beads and the sample solution of step (a) are mixed with a binding buffer, wherein the binding buffer contains at least one dissociating agent.

5. The method according to any one of the preceding claims, wherein step (e) further comprises the following steps: (e1) Mixing the bulk fractionation solution with a target binding buffer and a plurality of second beads such that the plurality of second beads bind to the target nucleic acid below the target size to form a second bead - analyte complex, wherein the target binding buffer contains at least one dissociating agent; and (e2) Recovering the target nucleic acid below the target size from the second bead - analyte complex.

6. The method according to any one of the preceding claims, wherein the plurality of beads are magnetic beads, silica - based beads, carboxyl beads, hydroxyl beads, amine - coated beads, or any combination thereof.

7. The method according to claim 5, wherein the plurality of second beads are magnetic beads, silica - based beads, carboxyl beads, hydroxyl beads, amine - coated beads, or any combination thereof.

8. The method according to any one of the preceding claims, wherein the plurality of beads are magnetic beads, and step (b) further comprises the following steps: (b1) Fixing the bead - analyte complex by applying a magnetic field to separate the bead - analyte complex from the bulk supernatant; (b2) Removing the bulk supernatant; and (b3) Remove the magnetic field and proceed to step (c).

9. The method according to claim 5, wherein the plurality of second beads are magnetic beads, and the target nucleic acid recovery in step (e2) further comprises the following steps: (i) Fix the second bead - analyte complex by applying a first magnetic field to separate the second bead - analyte complex from the first supernatant; (ii) Remove the first supernatant; (iii) Wash the immobilized second bead - analyte complex with a wash buffer; (iv) Discard the wash buffer; (v) Remove the first magnetic field; (vi) Mix the second bead - analyte complex with an elution buffer to form a bulk elution solution, wherein the target nucleic acid below the target size is separated from the magnetic beads in the second bead - analyte complex and released into the bulk elution solution; (vii) Fix the magnetic beads by applying a second magnetic field; (viii) Collect the bulk elution solution containing the separated target nucleic acid below the target size.

10. The method according to any one of the preceding claims, further comprising the following step: (f) Perform a diagnostic assay on the separated target nucleic acid for detection, quantification, characterization, or a combination thereof of the target nucleic acid.

11. The method according to any one of the preceding claims, wherein the at least one chaotropic agent of the fractionation buffer is selected from the group consisting of thiocyanate, isothiocyanate, perchlorate, acetate, trichloroacetate, trifluoroacetate, chloride, and iodide.

12. The method according to any one of the preceding claims, wherein the at least one chaotropic agent of the fractionation buffer is selected from the group consisting of guanidine hydrochloride (GHCl), guanidine thiocyanate, guanidine isothiocyanate (GITC), sodium thiocyanate, sodium iodide, sodium perchlorate, sodium trichloroacetate, sodium trifluoroacetate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2 - propanol, thiourea, and urea.

13. The method according to any one of the preceding claims, wherein the at least one chaotropic agent has a concentration of about 1.5 - 8 M in the fractionation buffer.

14. The method according to any one of the preceding claims, wherein the at least one chaotropic agent is present in the fractionation buffer at a concentration of about 1.8 - 3.9 M.

15. The method according to any one of the preceding claims, wherein the at least one chaotropic agent is present in the fractionation buffer at a concentration of about 1.8 - 3.0 M.

16. The method according to any one of the preceding claims, wherein the fractionation buffer further comprises at least one polymer selected from the group consisting of polyvinyl alcohol, polyethylene glycol, polypropylene glycol, dextran, poly(ethylene glycol - random - propylene glycol), pluronic, polyvinylpyrrolidone, and polyacrylate.

17. The method according to claim 16, wherein the at least one polymer is present in the fractionation buffer at a concentration of about 0.1% - 15% (w / w).

18. The method according to claim 16, wherein the at least one polymer is present in the fractionation buffer at a concentration of about 1.0% - 5.0% (w / w).

19. The method according to any one of claims 16 - 18, wherein the at least one polymer has an average molecular weight in the range of 100 to 35,000 Da.

20. The method according to any one of the preceding claims, wherein the fractionation buffer further comprises one or more of a pH buffer, a metal chelator, or a combination thereof.

21. The method according to any one of the preceding claims, wherein the nucleic acid component and / or the target nucleic acid is DNA, RNA, or a combination thereof.

22. The method according to any one of the preceding claims, wherein the nucleic acid component and / or the target nucleic acid is cDNA, plasmid DNA, cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), circulating fetal DNA, microRNA (miRNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), or a combination thereof.

23. The method according to any one of the preceding claims, wherein the first ATPS comprises a first ATPS component that is capable of forming the first target-rich phase and the first target-depleted phase when dissolved in an aqueous solution, wherein the first ATPS component is selected from the group consisting of polymers, salts, surfactants, and combinations thereof.

24. The method according to any one of claims 3 - 23, wherein the second ATPS comprises a second ATPS component that is capable of forming the second target-rich phase and the second target-depleted phase when dissolved in an aqueous solution, wherein the second ATPS component is selected from the group consisting of polymers, salts, surfactants, and combinations thereof.

25. The method according to claim 23 or 24, wherein the polymer is dissolved in the aqueous solution at a concentration of 0.5% - 80% (w / v).

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

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

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

29. The method according to any one of the preceding claims, wherein the polymer is selected from the group consisting of: polyacrylamide, polyacrylic acid, and copolymers thereof.

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

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

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

90.

33. The method according to any one of claims 23 - 32, wherein the salt is dissolved in the aqueous solution at a concentration of 0.1% - 80% (w / w).

34. The method according to claim 33, wherein the salt is dissolved in the aqueous solution at a concentration of 0.1% - 50% (w / w).

35. The method according to claim 33, 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.

36. The method according to claim 33, wherein the salt comprises an anion selected from the group consisting of: phosphate, hydrogen phosphate, dihydrogen phosphate, sulfate, sulfide, sulfite, bisulfate, carbonate, bicarbonate, acetate, nitrate, nitrite, sulfite, chloride, fluoride, chlorate, perchlorate, chlorite, hypochlorite, bromide, bromate, hypobromite, iodide, iodate, cyanate, thiocyanate, isothiocyanate, oxalate, formate, chromate, dichromate, permanganate, hydroxide, hydrogen ion, citrate, borate, and tris(hydroxymethyl)aminomethane.

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

38. The method according to any one of the preceding claims, wherein the 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, and ammonium acetate.

39. The method according to any one of the preceding claims, wherein the salt is selected from the group consisting of: (NH4)3PO4, sodium formate, ammonium formate, K2CO3, KHCO3, Na2CO3, NaHCO3, MgSO4, MgCO3, CaCO3, CsOH, Cs2CO3, Ba(OH)2, and BaCO3.

40. The method according to any one of the preceding claims, wherein the salt is selected from the group consisting of: NH4Cl, NH4OH, tetramethylammonium chloride, tetrabutylammonium chloride, tetramethylammonium hydroxide, and tetrabutylammonium hydroxide.

41. The method according to any one of claims 23 - 40, wherein the surfactant is dissolved in the aqueous solution at a concentration of 0.05% - 10% (w / w).

42. The method according to claim 41, wherein the surfactant is selected from the group consisting of: anionic surfactants, nonionic surfactants, cationic surfactants, and amphoteric surfactants; wherein the anionic surfactant is carboxylate, sulfonate, petroleum sulfonate, alkylbenzene sulfonate, naphthalene sulfonate, olefin sulfonate, alkyl sulfate, sulfate, sulfated natural oil, sulfated natural fat, sulfated ester, sulfated alkanolamide, sulfated alkylphenol, ethoxylated alkylphenol, or sodium N - lauroyl sarcosinate (NLS); wherein the nonionic surfactant is ethoxylated fatty alcohol, polyoxyethylene surfactant, carboxylic acid ester, polyethylene glycol ester, sorbitan ester, fatty acid diol ester, carboxamide, monochain alkanolamine condensate, or polyoxyethylene fatty acid amide; wherein the cationic surfactant is quaternary ammonium salt, amine with amide bond, polyoxyethylene alkylamine, polyoxyethylene alicyclic amine, n,n,n',n' - tetra - substituted ethylenediamine, or 2 - alkyl - 1 - hydroxyethyl - 2 - imidazoline; wherein the amphoteric surfactant is 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, or n - cocoamidoethyl - n - hydroxyethyl glycine or its sodium salt.

43. The method according to claim 41, wherein the surfactant is selected from the group consisting of: Triton X-100, Triton X-114, Triton X-45, Tween 20, Igepal CA630, Brij 58, Brij O10, Brij L23, Pluronic L-61, Pluronic F-127, sodium dodecyl sulfate, sodium cholate, sodium deoxycholate, sodium N-lauroylsarcosinate, cetyltrimethylammonium bromide, and span 80.

44. The method according to claim 4, wherein the at least one chaotropic agent of the binding buffer comprises an anion selected from the group consisting of: thiocyanate, isothiocyanate, perchlorate, acetate, trichloroacetate, trifluoroacetate, chloride, and iodide.

45. The method according to claim 4, wherein the at least one chaotropic agent of the binding buffer is selected from the group consisting of: guanidine hydrochloride (GHCl), guanidine thiocyanate, guanidine isothiocyanate (GITC), sodium thiocyanate, sodium iodide, sodium perchlorate, sodium trichloroacetate, sodium trifluoroacetate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, thiourea, and urea.

46. The method according to claim 5, wherein the at least one chaotropic agent of the target binding buffer comprises an anion selected from the group consisting of: thiocyanate, isothiocyanate, perchlorate, acetate, trichloroacetate, trifluoroacetate, chloride, and iodide.

47. The method according to claim 5, wherein the at least one chaotropic agent of the target binding buffer is selected from the group consisting of: guanidine hydrochloride (GHCl), guanidine thiocyanate, guanidine isothiocyanate (GITC), sodium thiocyanate, sodium iodide, sodium perchlorate, sodium trichloroacetate, sodium trifluoroacetate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, thiourea, and urea.

48. The method according to any one of the preceding claims, wherein the 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.

49. The method according to any one of the preceding claims, wherein said step (a) comprises the steps of: Preparing a DNA library from the sample to obtain the sample solution.

50. The method according to any one of the preceding claims, wherein the target nucleic acid is cell-free DNA and circulating tumor DNA, whereby the method increases the ratio of circulating tumor DNA:cell-free DNA, and / or the variant allele frequency (VAF) in the sample for cancer diagnostic assays.

51. The method according to any one of the preceding claims, wherein the target nucleic acid is circulating fetal DNA, whereby the method enriches the fetal fraction in the sample for non-invasive prenatal testing.

52. A kit for isolating a target nucleic acid of a size smaller than a target size from a sample comprising a nucleic acid component, the kit comprising: (a) at least one ATPS component selected from the group consisting of: polymers, salts, surfactants, and combinations thereof; (b) Multiple beads; (c) A fractionation buffer comprising at least one chaotropic agent selected from the group consisting of thiocyanate, isothiocyanate, perchlorate, acetate, trichloroacetate, trifluoroacetate, chloride, and iodide; and (d) A binding buffer comprising at least one chaotropic agent selected from the group consisting of thiocyanate, isothiocyanate, perchlorate, acetate, trichloroacetate, trifluoroacetate, chloride, and iodide.

53. The kit according to claim 52, wherein the multiple beads are magnetic beads, silica-based beads, carboxyl beads, hydroxyl beads, amine-coated beads, or any combination thereof.

54. The kit according to any one of claims 52-53, wherein the at least one chaotropic agent of the fractionation buffer is selected from the group consisting of guanidine hydrochloride (GHCl), guanidine thiocyanate, guanidine isothiocyanate (GITC), sodium thiocyanate, sodium iodide, sodium perchlorate, sodium trichloroacetate, sodium trifluoroacetate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, thiourea, and urea.

55. The kit according to any one of claims 52-54, wherein the at least one chaotropic agent has a concentration of about 1.5-8 M in the fractionation buffer.

56. The kit according to any one of claims 52-55, wherein the fractionation buffer further comprises at least one polymer selected from the group consisting of polyvinyl alcohol, polyethylene glycol, polypropylene glycol, dextran, poly(ethylene glycol-random-propylene glycol), pluronic, polyvinylpyrrolidone, and polyacrylate.

57. The kit according to claim 56, wherein the at least one polymer is present in the fractionation buffer at a concentration of about 0.1%-15% (w / w).

58. The kit according to any one of claims 52-57, wherein the fractionation buffer further comprises one or more of a pH buffer, a metal chelator, or a combination thereof.

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