Phase separation behavior modifiers for aqueous two-phase separation in porous materials

Abstract

The present invention relates to a method and / or device for improving the separation behavior and performance of an aqueous two-phase system (ATPS) for separating and / or concentrating one or more target analytes from a sample. In one embodiment, the methods and devices of the present invention include an ATPS component in a porous material and one or more phase separation behavior modifiers that improve the separation behavior and performance characteristics of the ATPS, increasing the stability or reducing fluctuations of ATPS by adjusting the total volume of a sample solution subjected to phase separation, the volume ratio of ATPS two phases, the fluid flow rate, and the concentration of ATPS components is included, but not limited to.

Description

[0001] This application is a divisional application of the invention patent divisional application with application number 202210384453.3, application date June 1, 2018, and invention name "Phase separation behavior modifier for double aqueous phase separation in porous materials". The application number of the original application of the divisional application is 201880027462.9 and the application date is June 1, 2018.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 513,994, filed on June 1, 2017, and U.S. Provisional Application No. 62 / 599,001, filed on December 14, 2017. The entire contents and disclosures of the foregoing applications are incorporated into this application by reference.

[0004] Various publications are cited throughout this application. The entire disclosures of these publications are incorporated herein by reference in their entirety to more fully describe the state of the art to which this invention pertains. Technical Field

[0005] The present invention relates to a method and / or device for improving the behavior and performance of an aqueous two-phase system (ATPS) in a porous material for separating and / or concentrating one or more target analytes from a sample solution. The method and device involve using one or more phase separation behavior modifiers to improve the separation behavior and performance characteristics of ATPS components in a porous material. The modifier can increase the stability of the ATPS or reduce its fluctuations by affecting the volume and volume ratio of the two phases after separation, the fluid flow rate and / or the concentration of the ATPS components. In one embodiment, when the modifier is added to the ATPS, the concentration range of the ATPS components used for phase separation is significantly expanded, thereby increasing the yield of the purified target analyte. The present invention also provides a device for implementing the method of the present invention. Background Art

[0006] The concentration of target analytes is a critical parameter in a variety of research and diagnostic applications. The purified target analytes must be of high quality and sufficient quantity for various downstream applications, including detection, clinical diagnosis, etc. Obtaining the target analytes in a purified form is a complex task due to the presence of a large number of microporous materials and macromolecules (e.g., proteins and carbohydrates) (also known as "polymers") in samples such as urine, blood, plasma, serum, saliva, and other biological fluids.

[0007] For target analytes present at very low concentrations in biological fluids such as urine and blood, a large amount of biological fluid is required to obtain sufficient amounts of the target analytes for subsequent detection by molecular techniques.

[0008] Detecting the presence of an analyte at very low concentrations is a huge challenge. The analyte can be a biomarker of disease, such as cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), or a protein present in a sample, such as a patient's saliva, blood, urine, and other body fluids. If the analyte concentration is too low, multiple existing diagnostic or detection methods may falsely report that the analyte is not present. For example, diagnostic gold standards such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) may produce false-negative results if the target analyte is present at very low levels, outside the detection limit.

[0009] Although new biomarkers for a variety of diseases have been discovered and developed in recent years, diagnostic tests based on these new biomarkers, such as cfDNA testing, have not yet been adopted in routine clinical procedures due to lack of sensitivity and specificity. When biomarkers are present at low levels, accurate detection depends on separation methods that can enrich the biomarker from the background. Depending on the separation method, this challenge can be complicated by differences in fragment size and the effects of test inhibitors.

[0010] Methods for concentrating target analytes are described in the literature. Patent publication WO2017041030 discloses a method for concentrating analytes by dehydrating a porous material with ATPS. However, in order to meet practical needs, the concentration multiple is quite small. Although ATPS is popular, it is still not widely used in the industry due to its limitations. For example, it is limited by the maximum sample volume that the system can handle, the minimum and maximum concentrations of ATPS components, and the yield of the purified target analyte. Specifically, due to fluctuations in sample volume, concentration and ratio of ATPS components, etc., the performance of ATPS is not very stable during phase separation, thereby affecting the purification efficiency, purity and yield of the target analyte.

[0011] To overcome these limitations, the present invention provides an improved method and apparatus for concentrating and purifying target analytes from a sample for further analysis. By using ATPS components and one or more phase separation behavior modifiers embedded in a porous material, the method and apparatus of the present invention can simultaneously perform multiple tasks, including separation of target analytes, removal of non-target analytes and impurities, and concentration of target analytes with high yields under any suitable conditions, so that the method and apparatus can be implemented using various concentrations of ATPS components, thereby achieving stable phase separation at different flow rates throughout the process without fluctuations or with minimal fluctuations. In short, the present invention significantly improves the behavior and performance of ATPS. The present invention also provides a device for implementing the method of the present invention. Summary of the invention

[0012] The foregoing background and the following detailed description may be better understood when read in conjunction with the accompanying drawings, which are intended to be illustrative rather than limiting. The present disclosure is not limited to the exact arrangements and examples shown herein.

[0013] The present invention relates to a method and / or device for improving the behavior and performance of an aqueous two-phase system (ATPS) in a porous material for separating or concentrating a target analyte from a sample solution. In one embodiment, the method and / or device of the present invention includes an ATPS component in a porous material, and one or more phase separation behavior modifiers, which improve the separation behavior and performance characteristics of the ATPS, including but not limited to increasing the stability of the ATPS or reducing its fluctuations by adjusting parameters, including but not limited to the total volume of the sample solution undergoing phase separation, the volume ratio, the fluid flow rate, and the concentration of the ATPS component.

[0014] In one embodiment, the methods and devices of the present invention include a porous material having an ATPS component embedded therein and one or more phase separation behavior modifiers that can expand the concentration range of the ATPS component that is effective for phase separation.

[0015] In one embodiment, the methods and devices of the present invention include a porous material having an ATPS component embedded therein and one or more phase separation behavior modifiers that can increase the yield of the purified target analyte.

[0016] In one embodiment, the methods and devices of the present invention include a porous material embedding an ATPS component and one or more phase separation behavior modifiers that improve the stability of the ATPS during phase separation by minimizing fluctuations in the volumes and / or volume ratios of the two phases.

[0017] In one embodiment, the methods and devices of the present invention include a porous material having an ATPS component embedded therein and one or more phase separation behavior modifiers to remove contaminants from a sample solution.

[0018] In one embodiment, the present invention provides a method and / or apparatus for concentrating a target analyte in a solution using an ATPS component and one or more phase separation behavior modifiers embedded in a porous material.

[0019] In one embodiment, the phase separation behavior modifier is a compound that can induce or promote phase separation by changing the temperature, pH, salt concentration, hydrophobicity / hydrophilicity, surface tension and / or ionic strength of the ATPS. Thus, the target analyte is preferably separated into one of the two phases according to its characteristics. In one embodiment, the phase separation behavior modifier includes a compound carrying an acidic functional group, an amine functional group and / or a hydrophilic group and a hydrophobic group.

[0020] In one embodiment, the present invention provides a device for separating or concentrating one or more target analytes from a sample solution, the device comprising:

[0021] (1) Porous materials;

[0022] (2) components capable of forming a first phase solution and a second phase solution of an aqueous two-phase system (ATPS); and

[0023] (3) one or more phase separation behavior modifiers;

[0024] wherein the components and the phase separation behavior modifier are embedded in the porous material, wherein when a solution containing the target analyte flows through the porous material, a two-phase aqueous system comprising the first phase solution and the second phase solution is generated, wherein the phase separation behavior modifier changes the separation behavior of the two-phase solution, and the target analyte is separated or concentrated on the porous material.

[0025] In one embodiment, the present invention provides a device for separating or concentrating one or more target analytes, the device comprising:

[0026] (1) Porous materials;

[0027] (2) components capable of forming a first phase solution and a second phase solution of an aqueous two-phase system (ATPS); and

[0028] (3) one or more phase separation behavior modifiers;

[0029] wherein one or more of the components and the phase separation behavior modifiers are embedded in the porous material, wherein the remainder of the components and the phase separation behavior modifiers are mixed with a solution containing the target analyte to form a mixed solution, wherein when the mixed solution flows through the porous material, a two-phase aqueous system comprising the first phase solution and the second phase solution is generated, wherein the phase separation behavior modifier changes the separation behavior of the two-phase solution, and the target analyte is separated or concentrated on the porous material.

[0030] In one embodiment, the present invention provides a method for separating or concentrating one or more target analytes from a sample solution, the method comprising:

[0031] (1) obtaining a sample solution containing a target analyte; and

[0032] (2) contacting the sample solution with a porous material having one or more phase separation behavior modifiers embedded therein and components of a first phase solution and a second phase solution capable of forming an aqueous two-phase system (ATPS), wherein when a solution containing the target analyte flows through the porous material, a two-phase system comprising the first phase solution and the second phase solution is generated,

[0033] wherein the modifier changes the separation behavior of the two-phase solution;

[0034] The target analyte is separated or concentrated on the porous material. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Several examples are provided showing how a phase separation behavior modifier can be distributed in a rectangular porous material. The phase separation behavior modifier can be distributed in a plurality of different locations within the porous material and can be distributed at different concentrations at each location.

[0036] Figure 2 Phase separation diagrams (double node curves) at different flow rates are shown, comparing the separation behavior and characteristics of ATPS components with and without behavior modifiers, illustrating the benefits of phase separation behavior modifiers on the phase separation characteristics of ATPS components. In some cases, these phase separation diagrams represent the time course of fluid flow through a porous material in which ATPS components are embedded, thereby causing the dehydrated ATPS components in the material to dissolve again. As time goes by, more ATPS components will enter the solution. The curves are double node curves that represent the relative concentrations of ATPS components that are effective for phase separation, thereby setting the boundary between a single-phase system and a two-phase system. According to the double node curve theory, phase separation will only occur when the relative concentrations of the ATPS components reach a certain value. Any point outside the double node curve represents a single-phase system (no phase separation), while any point inside the double node curve represents a two-phase system (phase separation). The horizontal solid line refers to the tie line, which represents the condition where the two phases exist in equilibrium with each other at a specific temperature. At a given temperature, all systems on the tie line produce the same two phases, and the point where the tie line intersects the double node curve represents the composition of the two phases that exist in equilibrium with each other at that temperature. The volume ratio of the ATPS solution can be determined by comparing the length of the double node curve to the left of the tie line with the length of the double node curve to the right of the tie line (called the "lever arm rule"). For example, Figure 2 In A, when the relative concentration of ATPS components is n, the volume ratio of the two-phase solution is 1:5, and Figure 2 In B, when the relative concentration of the ATPS component is 2n, the volume ratio is 5:1. In contrast, when the relative concentration of the ATPS component is 3n (beyond the tie line), the two-phase solution does not phase separate but remains a single phase ( Figure 2C in the figure). Therefore, the distribution coefficient of the target analyte along the tie line is the same regardless of the relative concentrations of the ATPS components. Since the yield of the target analyte under ATPS depends on the distribution coefficient of the target analyte and the volume of the target phase recovered, the yield will depend on the tie line and the position of the ATPS on the tie line. In general, the longer the tie line, the higher the yield. Figure 2 The phase diagram of DF in FIG. 5 shows that the addition of a phase separation behavior modifier to the ATPS shifts the position of the double node curve downward, thereby increasing the length of the tie line at any given temperature, thereby allowing phase separation to occur within a wider relative concentration range of the two-phase components and improving the distribution coefficient. Therefore, the ATPS concentration range that can phase separate is significantly expanded. In addition, in certain embodiments where the ATPS component and the phase separation behavior modifier are dehydrated in the porous material, the phase separation volume ratio becomes more stable relative to fluctuations in flow rate and ATPS component concentration. Along the same tie line, an increase in the concentration of the ATPS component will lead to a more extreme volume ratio. According to the double node curve theory, if the operating relative concentration of the ATPS component is too close to either side of the double node curve, even a slight change in the volume ratio of the two-phase solution (such as a slight dilution or concentration of the solution) will cause the operating relative concentration to move to the other side of the double node curve where only one phase exists. However, in the case where the porous material is embedded with the dehydrated form of the ATPS component and the modifier, the increase in the concentration of the ATPS component is also associated with the increase in the phase separation behavior modifier, resulting in a relative change in the ratio of the volume ratio of the two-phase solution to the other side of the double node curve. Figure 2 Compared with the phase diagram in AC, the double node curve is proportionally shifted downward, and the tie line at temperature T is longer. Therefore, when the modifier is present, the same relative concentration of ATPS components has less effect on the volume ratio, making the system more stable and more robust. It should be noted that although Figure 2 A temperature-dependent ATPS with a single ATPS component is illustrated, but the above concepts are equally applicable to temperature-independent ATPS and ATPS with multiple ATPS components. DETAILED DESCRIPTION

[0037] Several embodiments of the present invention are described below. For the purpose of explanation, specific configurations and details are set forth to provide a thorough understanding of the embodiments. In addition, for the plural or singular form of words and the directional descriptions of the embodiments such as "upper", "lower", "front", "back", "left", "right", etc., these expressions are to help the reader understand the embodiments and do not mean physical limitations. For those skilled in the art, it is obvious that the present invention can be implemented without specific details. By referring to the subsequent examples, the present invention will be better understood, but those skilled in the art will readily understand that the specific examples are only for illustrative purposes and should not limit the scope of the present invention as defined by the subsequent claims. It should be noted that the transitional term "comprising", which is synonymous with "comprising" or "characterized by", is inclusive or open-ended and does not exclude additional, unreferenced elements or method steps.

[0038] The present invention relates to a method and / or device for improving the behavior and performance of an aqueous two-phase system (ATPS) in a porous material for separating or concentrating a target analyte from a sample solution. The present invention also provides a device for implementing the method of the present invention.

[0039] In one embodiment, the methods and / or devices of the present invention utilize phase separation behavior modifiers to improve one or more phase separation behaviors and performance characteristics of the ATPS components, among a variety of different possibilities contemplated. In one embodiment, it is also contemplated that improvements in the phase separation behavior and performance characteristics of the ATPS components can improve the functionality and integration of a variety of other devices, methods, or practices used in conjunction with the porous materials or devices of the present invention.

[0040] In one embodiment, the present invention provides a method and apparatus for separating or concentrating target analytes in a solution using ATPS components embedded in a porous material and one or more phase separation behavior modifiers. In one embodiment, the phase separation behavior modifier is a compound that can induce or promote phase separation by changing the temperature, pH, salt concentration and / or ionic strength of the ATPS. Thus, the target analyte is preferentially separated into one phase or another phase according to its characteristics.

[0041] Accordingly, the present invention provides an aqueous two-phase system (ATPS) that is capable of separating or concentrating a target analyte from a sample over a wide range of ATPS component concentrations. The present invention overcomes the limitations of the prior art, such as restrictions on sample size, minimum and maximum concentrations of ATPS components, and yield of purified target analytes. The present invention facilitates faster and more efficient distribution of target analytes in an aqueous two-phase system without the need for complex instrumentation, and allows purification of target analytes with high yields from large volumes and complex biological materials while avoiding target analyte contamination.

[0042] In one embodiment, the present invention provides a device for separating or concentrating one or more target analytes from a sample solution, the device comprising:

[0043] (1) Porous materials;

[0044] (2) components capable of forming a first phase solution and a second phase solution of an aqueous two-phase system (ATPS); and

[0045] (3) one or more phase separation behavior modifiers;

[0046] wherein the components and the phase separation behavior modifier are embedded in the porous material, wherein when a solution containing the target analyte flows through the porous material, a two-phase aqueous system comprising the first phase solution and the second phase solution is generated, wherein the phase separation behavior modifier changes the separation behavior of the two-phase solution, and the target analyte is separated or concentrated on the porous material.

[0047] In one embodiment, the present invention provides a device for separating or concentrating one or more target analytes, the device comprising:

[0048] (1) Porous materials;

[0049] (2) components capable of forming a first phase solution and a second phase solution of an aqueous two-phase system (ATPS); and

[0050] (3) one or more phase separation behavior modifiers;

[0051] wherein one or more of the components and the phase separation behavior modifiers are embedded in the porous material, wherein the remainder of the components and the phase separation behavior modifiers are mixed with a solution containing the target analyte to form a mixed solution, wherein when the mixed solution flows through the porous material, a two-phase aqueous system comprising the first phase solution and the second phase solution is generated, wherein the phase separation behavior modifier changes the separation behavior of the two-phase solution, and the target analyte is separated or concentrated on the porous material.

[0052] In one embodiment, the present invention provides a method for separating or concentrating one or more target analytes from a sample solution, the method comprising:

[0053] (1) obtaining a sample solution containing a target analyte; and

[0054] (2) contacting the sample solution with a porous material having one or more phase separation behavior modifiers embedded therein, and components of a first phase solution and a second phase solution capable of forming an aqueous two-phase system (ATPS), wherein when the solution containing the target analyte flows through the porous material, a two-phase system comprising the first phase solution and the second phase solution is generated, wherein the phase separation behavior modifier changes the separation behavior of the two-phase solution;

[0055] The target analyte is separated or concentrated on the porous material.

[0056] In one embodiment, when ATPS is applied to a porous material (e.g., paper), the two phases of ATPS separate from each other as the mixed phase solution flows through the porous material. The resulting phase solutions have different physicochemical properties, and each phase passes through the porous matrix at a different rate. Different molecules in the mixture will be differentially distributed in the two-phase solution due to their different properties, and ATPS can be used to separate and concentrate target molecules through simple settings and human intervention.

[0057] The present invention has the advantage that high concentration and high yield of target analytes can be obtained in a simple manner and is compatible with downstream application analysis without further purification or concentration.

[0058] The methods and devices provided herein are highly robust and are inexpensive, simple, easy to operate, safe, user-friendly and rapid. The methods and devices of the present invention are capable of purifying and concentrating target analytes, thereby ensuring that the performance of downstream applications using the purified and concentrated analytes is not affected by impurities in the original sample.

[0059] Furthermore, the present invention is applicable to samples containing very small amounts or volumes of target analytes and is amenable to automation, including high-throughput screening systems.

[0060] In one embodiment, the present invention provides a method and / or device including an ATPS as a concentration module connected to a lateral flow immunoassay (LFA) component as a detection module. In one embodiment, the detection module is contained in a plastic housing having an observation window and a gold nanoprobe. As the sample is siphoned upward along the device, the ATPS components phase separate, and the analyte is substantially concentrated at the leading edge. The concentrated analyte is then detected by the LFA module, producing a visual test result.

[0061] Type of sample and target analyte

[0062] In one embodiment, the methods and devices of the present invention can separate target analytes in a sample from non-target molecules (e.g., small molecules and large molecules, typically of natural origin, which may interfere with the detection or quantification of the target analytes), thereby achieving more accurate detection and diagnosis.

[0063] In one embodiment, the methods and devices of the present invention are applicable to any type of sample. In one embodiment, samples include, but are not limited to, food, blood, plasma, serum, tissue, bacteria, virus, RNA virus, smear specimens, bacterial culture, cell culture (e.g., cell suspension and adherent cells), urine, saliva, feces and body excretions (e.g., tears, sputum, nasopharyngeal mucus, vaginal secretions and penile secretions), polymerase chain reaction (PCR) mixtures and in vitro nucleic acid modification reaction mixtures.

[0064] In one embodiment, target analytes include, but are not limited to, proteins, nucleic acids, carbohydrates, lipids, bacteria, viruses, pathogens, food allergens, nanoparticles, and the like.

[0065] In one embodiment, the target analyte is a food allergen, including but not limited to plant protein and animal protein. Lupin protein is more preferred in the present invention.

[0066] In one embodiment, the target analyte is a nucleic acid derived from a subject or an exogenous agent, or both, which can be of various types (e.g., DNA including cDNA, RNA including mRNA and rRNA), various forms (e.g., single-stranded, double-stranded, coiled, as a plasmid, non-coding or coding), and various lengths (e.g., oligonucleotides, genes, chromosomes, and genomic DNA).

[0067] In one embodiment, the target analyte is a protein (peptide or polypeptide), including intact protein molecules, degraded protein molecules and digested fragments of protein molecules. In one embodiment, biomarkers include but are not limited to antigens, receptors and antibodies derived from subjects or exogenous agents or both.

[0068] In one embodiment, the target analyte is a small molecule, such as a metabolite. In one embodiment, the metabolite is a disease-related metabolite, which indicates the presence or extent of a disease or a healthy state. In one embodiment, the metabolite is a drug-related metabolite, such as a drug byproduct, whose level changes in a subject taking the drug.

[0069] In one embodiment, the target analyte is derived from the subject himself (e.g., a molecule derived or released from any organ, tissue or cell of the subject), an exogenous source (e.g., a pathogen associated with a specific disease, such as a virus or bacteria), or a food allergen (protein) from food or medicine ingested by the subject. Mutans Streptococcus is a preferred bacterium in the present invention.

[0070] In one embodiment, the target analyte is not normally present in healthy subjects. In one embodiment, the target analyte is a molecule normally present in healthy subjects, but its levels are indicative of a particular disease or health condition.

[0071] ATPS (Aqueous Two-Phase System) in Porous Materials

[0072] In one embodiment, the present invention provides the use of ATPS components in porous materials. Various ATPS systems can be used in the present invention, including but not limited to polymer-polymer (e.g., PEG-PVP), polymer-salt (e.g., PEG-salt) and micelles. The porous material can be made of any suitable porous material capable of absorbing and transferring liquids. Porous materials suitable for use in the present invention include but are not limited to hydrogels, glass fiber paper, cotton-based paper, other types of paper, polymer foams, cellulose foams, other types of foams, rayon fabrics, cotton fabrics, other types of fabrics, wood, stone, and any other material capable of absorbing and transferring liquids.

[0073] In one embodiment, the present invention provides the use of ATPS (aqueous two-phase system) for separation and / or concentration of target analytes in a sample solution, wherein the phase separation characteristics are changed by one or more phase separation modifiers, which improve the behavior and performance of ATPS, thereby inducing or promoting phase separation of ATPS.

[0074] In one embodiment, the ATPS comprises a mixed phase solution comprising a first phase solution and a second phase solution, wherein components of the first phase solution and components of the second phase solution are embedded in the porous material at a concentration or loading sufficient to undergo phase separation when the mixed phase solution flows through the porous material. In one embodiment, the phase separation characteristics are altered by one or more phase separation modifiers to induce, stabilize and / or promote ATPS phase separation.

[0075] In one embodiment, before adding the sample containing the target analyte to the porous material, the components of the first phase solution of ATPS and / or the components of the second phase solution are embedded in the porous material and then dehydrated.

[0076] In one embodiment, components of the first phase solution of the ATPS and / or components of the second phase solution are combined with a sample containing a target analyte to produce a mixture prior to adding the mixture to the porous material.

[0077] In one embodiment, before adding the mixture to the porous material, some components of the first phase solution and / or components of the second phase solution of the ATPS are embedded in the porous material and then dehydrated, while the remaining components of the first phase solution and / or components of the second phase solution are combined with a sample containing the target analyte to produce a mixture.

[0078] In one embodiment, a two-component ATPS (aqueous two-phase system) in a porous material is provided for concentration of one or more target analytes and / or purification of a sample solution. The target analyte is contacted with a mixed phase solution comprising a first phase solution and a second phase solution, and is separated into the first phase solution, the second phase solution, or the interface (or intermediate phase) between the first phase solution and the second phase solution.

[0079] In one embodiment, a two-component ATPS (aqueous two-phase system) in a porous material is provided for removing one or more contaminants from a sample to obtain a purified sample of a target analyte. In one embodiment, the one or more contaminants are contacted with a mixed phase solution comprising a first phase solution and a second phase solution, wherein the contaminants are separated into the first phase solution, the second phase solution, or the interface (or intermediate phase) between the first phase solution and the second phase solution.

[0080] In one embodiment, the porous material and ATPS are selected so that the first phase solution flows through the porous matrix at a first rate and the second phase solution flows through the porous matrix at a second rate, wherein the first rate and the second rate are different.

[0081] In one embodiment, the porous material is commercially available or manufactured in-house.

[0082] Phase separation behavior modifiers

[0083] In one embodiment, the method and apparatus of the present invention include a porous material in which an ATPS component is embedded and one or more phase separation behavior modifiers, wherein the phase separation behavior modifiers change the phase separation behavior of the ATPS, thereby inducing or promoting phase separation. In one embodiment, the phase separation behavior modifiers of the present invention can expand the concentration range of the ATPS component effective for phase separation.

[0084] In such Figure 1 In one embodiment shown, it is further contemplated that the methods and devices of the present invention can use different types of phase separation behavior modifiers in various combinations. In one embodiment, it is also contemplated that the phase separation behavior modifier can be distributed at multiple different locations within the porous material of the device, and can be distributed at different concentrations at each location.

[0085] The double node curve represents the conditions (such as temperature and phase component concentration) under which two different phases can coexist, thus showing the conditional boundary for phase separation to occur. According to the double node curve theory, any point outside the double node curve represents a single-phase system (no phase separation), while any point inside the double node curve represents a two-phase system (phase separation).

[0086] Figure 2Phase separation diagrams (double node curves of temperature and relative concentrations of the components that form the first and second phases of the ATPS) at different flow rates are shown, comparing the separation behavior and characteristics of the ATPS components with and without the addition of a behavior modifier to the system. Figure 2 AC in Figure 2), at a given temperature, two points can be observed on the double node curve (except for the extreme value of the double node curve), indicating the relative concentration range of the two-phase components at which the solution will undergo phase separation. The solution of a certain component with a relative concentration outside this range will exist as a single phase. Figure 2 In DF), a phase separation behavior modifier is added to ATPS, and similarly, two points can be observed on the double node curve at a given temperature and modifier concentration (in addition to the extreme value of the double node curve). Figure 2 In the example, the solid horizontal line ("tie line") within the boundary of the double node curve represents the condition where two phases exist in equilibrium with each other at a given temperature. At a given temperature, all systems on a particular tie line produce the same two phases, and the intersection of the tie line with the double node curve represents the composition of the two phases that exist in equilibrium with each other at that temperature. The volume ratio of the ATPS solution can be determined by comparing the length of the double node curve from the point on the tie line to the left with the length of the double node curve from the point on the tie line to the right (called the "lever arm rule"). For example, at Figure 2 In A, when the relative concentration of ATPS components is n, the volume ratio of the two-phase solution is 1:5, and Figure 2 In B, when the relative concentration of the ATPS component is 2n, the volume ratio is 5:1. In contrast, when the relative concentration of the ATPS component is 3n (beyond the tie line), the two-phase solution does not phase separate but remains a single phase ( Figure 2 C in the figure). Therefore, the partition coefficient of the target analyte along the tie line is the same regardless of how the relative concentrations of the ATPS components vary. Since the yield of the target analyte recovered by the ATPS depends on the partition coefficient of the target analyte and the volume of the target phase recovered, the yield will depend on the tie line and the position of the ATPS on that tie line. In general, the longer the tie line, the higher the yield. Figure 2The phase diagram of DF in FIG. 5 shows that the addition of a phase separation behavior modifier to the ATPS shifts the position of the double node curve downward, thereby increasing the length of the tie line at any given temperature, thereby allowing phase separation to occur over a wider relative concentration range of the two-phase components and improving the partition coefficient. Regardless of the flow rate, the range covered by the two points on the double node curve is always wider in the presence of the modifier. In addition, in certain embodiments of dehydration of ATPS components and phase separation behavior modifiers in porous materials, the phase separation volume ratio becomes more stable relative to fluctuations in flow rate and ATPS component concentration. Along the same tie line, an increase in the concentration of the ATPS component will lead to a more extreme volume ratio. According to the double node curve theory, if the operating relative concentration of the ATPS component is too close to either side of the double node curve, even a slight change in the volume ratio of the two-phase solution (such as a slight dilution or concentration of the solution) will cause the operating relative concentration to move to the other side of the double node curve where only one phase exists. However, in the case of porous materials embedded with dehydrated forms of ATPS components and modifiers, an increase in the concentration of the ATPS component is also associated with an increase in the phase separation behavior modifier, resulting in Figure 2 Compared with the phase diagram in AC, the double node curve is proportionally shifted downward, and the tie line at temperature T is longer. Therefore, when the modifier is present, the same relative concentration of ATPS components has less effect on the volume ratio, making the system more stable and more robust. It should be noted that although Figure 2 A temperature-dependent ATPS with a single ATPS component is illustrated, but the above concepts are equally applicable to temperature-independent ATPS and ATPS with multiple ATPS components.

[0087] Figure 2 The double node curve theory shown in is generally applicable to all types of modifiers, including the modifiers described herein. It is believed that phase separation behavior modifiers are able to change the properties of one or both of the phase components, such as temperature, pH, hydrophobicity / hydrophilicity, surface tension and / or ionic strength, and allow phase separation to occur over a wide range of relative concentrations of the two phase components, thereby initiating or promoting ATPS phase separation.

[0088] In one embodiment, adding a phase separation behavior modifier to the ATPS component in the porous material can improve the separation behavior and performance characteristics of the ATPS component, including but not limited to increasing the stability of the ATPS or reducing its fluctuations by adjusting parameters, including but not limited to the total volume of the sample solution undergoing phase separation, volume ratio, fluid flow rate, and concentration of the ATPS component.

[0089] In one embodiment, the methods and devices of the present invention include a porous material embedded with an ATPS component and one or more phase separation behavior modifiers to increase the yield of a purified target analyte.

[0090] In one embodiment, the methods and devices of the present invention include a porous material in which an ATPS component is embedded and one or more phase separation behavior modifiers that improve the stability of the ATPS during phase separation by minimizing fluctuations in the ATPS.

[0091] In one embodiment, the methods and devices of the present invention include a porous material having an ATPS component embedded therein and one or more phase separation behavior modifiers to remove contaminants from a sample solution.

[0092] In one embodiment, the phase separation behavior modifier is combined with a sample containing a target analyte and / or one or more ATPS components prior to adding the mixture to the porous material to produce the mixture. In one embodiment, the phase separation behavior modifier is combined sequentially with a sample containing a target analyte and / or one or more ATPS components prior to adding to the porous material.

[0093] In one embodiment, the phase separation behavior modifier is embedded in the porous material containing the ATPS component and then dehydrated before the sample is added to the porous material.

[0094] In one embodiment, some of the phase separation behavior modifier is embedded in the porous material containing the ATPS component and then dehydrated before adding the mixture to the porous material, while the remaining phase separation behavior modifier is combined with a sample containing the target analyte to produce a mixture.

[0095] In one embodiment, some of the phase separation behavior modifier is embedded in a porous material containing a component of the ATPS first or second phase solution before the mixture is added to the porous material and then dehydrated, while the remaining phase separation behavior modifier is combined with a component of the ATPS other phase solution to form the mixture.

[0096] In one embodiment, before adding the mixture to the porous material, some of the phase separation behavior modifier is embedded in the porous material without ATPS components and then dehydrated, while the remaining phase separation behavior modifier is combined with the components of the first phase solution of ATPS and the components of the second phase solution of ATPS to produce the mixture.

[0097] In one embodiment, the phase separation behavior modifier is present in dry form in some regions of the porous material. In one embodiment, the phase separation behavior modifier is embedded in some regions of the porous material. In another embodiment, the phase separation behavior modifier is embedded in the entire region of the porous material.

[0098] In one embodiment, the phase separation behavior modifier is present in dry form and at a uniform concentration in all regions of the porous material. In another embodiment, the phase separation behavior modifier is present in dry form and at different concentrations in different regions of the porous material.

[0099] In one embodiment, the separation behavior modifiers of the present invention include but are not limited to:

[0100] a. A compound carrying an acidic functional group in an aqueous solution selected from dextran, sucrose, sugars, polysaccharides, sorbates, polysorbates, carboxylates, polycarboxylates, phosphates, potassium phosphates, polyphosphates, sulfates and polysulfates, and polyols;

[0101] b. a compound carrying an amine functional group in an aqueous solution selected from amines, polyamines, ammonium salts and polyammonium salts;

[0102] c. A compound carrying a hydrophilic group and a hydrophobic group, selected from lipids, surfactants, nucleic acids, hormones, proteins, and amino acids;

[0103] d. Chaotropic agents; and

[0104] e. Lyophilic agent.

[0105] In one embodiment, the phase separation behavior modifiers of the present invention include but are not limited to sucrose, potassium phosphate, dextran, Tween-20 and Triton-X114.

[0106] Regulate or stabilize the total volume of either or both phases

[0107] In one embodiment, the phase separation behavior modifiers of the present invention are selected to:

[0108] (a) Reducing fluctuations in the total volume of either or both phases during and after phase separation (increasing stability). For example, when ATPS components are embedded in a porous material and dehydrated, and redissolve during fluid flow, the continued redissolution of the ATPS components changes the total volume of the first phase and / or the second phase. Changes in flow rate result in changes in the total volume of the first phase and / or the second phase. In one embodiment, a modifier such as a compound carrying hydrophilic and hydrophobic groups is used, which interacts with the dehydrated ATPS components within the porous material, thereby reducing fluctuations in the total volume of the first phase and / or the second phase during fluid flow within the porous material. For a more robust device, it is desirable to reduce fluctuations in concentration multiples; or

[0109] (b) Increasing the total volume of the sample solution that undergoes phase separation. When the ATPS components are dehydrated in the porous material and redissolved during fluid flow, the continued redissolution of the ATPS components increases the total volume of the first phase and / or the second phase. In one embodiment, a compound carrying an acidic functional group in the aqueous solution is used as a modifier to reduce the pH of the first phase and / or the second phase solution, thereby promoting the redissolution of the first phase and / or the second phase solution and increasing the total volume of the phase solution. In general, compounds carrying acidic functional groups will cause phase separation to occur earlier in the porous material and / or at a lower ATPS concentration, which will cause a larger volume of fluid to undergo phase separation when flowing through the porous material. This may be beneficial in concentrating more of the target from a given solution in the relevant phase; or

[0110] (c) Reducing the total volume of the sample solution that undergoes phase separation. In one embodiment, a compound carrying an amine functional group in an aqueous solution is used as a modifier to increase the pH of the first phase and the second phase, thereby hindering the redissolution of the two phases. When the ATPS components are dehydrated in the porous material and redissolved during fluid flow, the total volume of the first phase and / or the second phase will be reduced due to the hindrance of redissolution of the ATPS components. Therefore, the phases will separate later and / or when the concentration of the ATPS components in the porous material is higher, which will reduce the total volume of the fluid that undergoes phase separation when flowing through the material. This may be beneficial to concentrate more of the target from a given solution in the relevant phase.

[0111] Adjust or stabilize the volume ratio of the first phase and the second phase

[0112] In one embodiment, the phase separation behavior modifiers of the present invention are selected to:

[0113] (a) Reducing fluctuations in the volume ratio between the first and second phases of the ATPS during or after phase separation (increasing stability). When the ATPS components are dehydrated in a porous material and redissolved when a fluid flows in the porous material, the continued redissolution of the ATPS components will change the volume ratio of the two phases. Changes in flow rate result in changes in the volume ratio. In one embodiment, by adding a modifier, such as a compound carrying hydrophilic and hydrophobic groups, the agent will interact with the dehydrated ATPS components through the hydrophilic and hydrophobic groups, reducing fluctuations in the volume ratio when the fluid flows within the porous material. Reduced fluctuations in the volume ratio are desirable for a more robust device; or

[0114] (b) Increasing the volume ratio between the first phase and the second phase of the ATPS. In one embodiment, by adding a modifier such as a compound carrying an acidic functional group in aqueous solution to the ATPS (depending on which phase the relevant phase is), the pH of the relevant phase is reduced, thereby promoting the redissolution of the relevant phase. The increase in the volume ratio may help to further increase the target concentration, improve fluid flow, or ideally adjust the volume of the relevant phase. This may be beneficial to concentrate more of the target from a given solution in the relevant phase; or

[0115] (c) Reducing the volume ratio between the first and second phases of the ATPS. In one embodiment, by adding a modifier such as a compound carrying an amine functional group in aqueous solution to the ATPS (depending on which phase the relevant phase is), the pH of the relevant phase will increase, thereby hindering the redissolution of the relevant phase. The reduction in the volume ratio may help to further increase the target concentration, improve the flow of the fluid, or ideally adjust the volume of the relevant fluid phase. This may be beneficial to concentrate more of the target from a given solution in the relevant phase.

[0116] Adjust the concentration of the first and second phase components

[0117] In one embodiment, the phase separation behavior modifiers of the present invention are selected to:

[0118] (a) Reducing fluctuations in the concentration of first phase components and / or second phase components in the first phase or the second phase. When ATPS components are embedded in a porous material and dehydrated, and redissolved during fluid flow, the continued redissolution of the ATPS components will change the concentration of the first phase components and / or the second phase components. Changes in flow rate result in changes in the concentration of components in the first phase and / or the second phase. In one embodiment, by adding a modifier to the solution or porous material, such as a chemical carrying hydrophilic and hydrophobic groups, the agent will interact with the ATPS components through the hydrophilic and hydrophobic groups, reducing fluctuations in concentration when the fluid flows in the porous material. For a more robust device, it is desirable to reduce fluctuations in concentration multiples; or

[0119] (b) Increasing the concentration of one ATPS phase component within the same phase. In one embodiment, when the ATPS component is dehydrated in a porous material, by adding a modifier such as a chemical carrying an acidic functional group in an aqueous solution directly to a solution containing the target analyte, or by dehydrating the modifier in a porous material, the pH of the solution in that phase will decrease, thereby promoting the redissolution of the ATPS component in that phase, resulting in a higher concentration. This may be beneficial to increase the concentration factor, thereby increasing the concentration of the target analyte by increasing the volume ratio, thereby resulting in a larger volume of phase separation. This may be beneficial to concentrate more of the target from a given solution in the relevant phase; or

[0120] (c) Reducing the concentration of one ATPS component in the same phase. In one embodiment, by adding a modifier such as a compound carrying an amine functional group in an aqueous solution directly to a solution containing the target analyte, or by dehydrating the modifier in a porous material, the pH of the ATPS component of the phase is increased and its redissolution rate is reduced, resulting in a lower concentration of one ATPS component in the same phase during the redissolution process. This may be beneficial for producing a more sustained release of the ATPS component during the flow of the fluid in the porous material, making the ATPS concentration more uniform and producing a larger volume of phase separation. This may be beneficial for concentrating more of the target from a given solution in the relevant phase; or

[0121] (d) Increasing the concentration of a component of one phase in another phase. In one embodiment, when the ATPS components are dehydrated in the porous material, by directly adding a compound carrying an acidic functional group in an aqueous solution to a solution containing the target analyte, or by dehydrating the modifier in the porous material, the pH of the first phase component in the second phase will be reduced, thereby promoting the redissolution of the first phase component in the second phase solution, resulting in a higher concentration of the first phase component in the second phase. This may be beneficial to increase the volume ratio, produce a larger volume of phase separation, and / or increase the concentration of the target analyte. This may be beneficial to concentrate more of the target from a given solution in the relevant phase; or

[0122] (e) Reducing the concentration of one ATPS component in another phase. In one embodiment, by adding a modifier such as a compound carrying an amine functional group in aqueous solution directly to a solution containing the target analyte, or by dehydrating the modifier in a porous material, the pH and redissolution rate of the phase component will increase, resulting in a lower concentration of one ATPS component in another phase during the redissolution process. This may be beneficial to produce a more sustained release of the ATPS component during the flow of the fluid in the porous material, making the ATPS concentration more uniform and producing a larger volume of phase separation. This may be beneficial to concentrate more of the target from a given solution in the relevant phase; or

[0123] (f) Any combination of ae.

[0124] Inducing phase separation earlier in porous materials and / or at lower ATPS component concentrations

[0125] In one embodiment, by causing phase separation to occur earlier in the porous material and / or at a lower concentration of the ATPS component, this results in a larger volume of fluid undergoing phase separation when flowing through the porous material. In one embodiment, this can be achieved by adding a modifier, such as a compound carrying an acidic functional group in an aqueous solution, to reduce the pH of the first and / or second phase, thereby promoting the redissolution of the phase components. When the ATPS component is dehydrated in the porous material and redissolved during fluid flow, the continued redissolution of the ATPS component increases the total volume of the first phase and / or the second phase. This may be beneficial for concentrating more of the target from a given solution. In one embodiment, the phase separation behavior modifier is selected to induce initial phase separation at a lower concentration of the ATPS component, otherwise the concentration of the ATPS component is too low to induce phase separation in the absence of the phase separation behavior modifier. In one embodiment where the ATPS comprises a micellar solution, when the concentration of the ATPS component is insufficient to cause phase separation, one or more modifiers can be added to change the parameters of the ATPS component, such as temperature, pH, or ionic strength, to allow phase separation at a lower concentration. This will benefit situations where the temperature is too low to diagnose, by extending the operating temperature range, or where downstream diagnostic components are sensitive to ATPS species, requiring that the ATPS species concentration be kept low.

[0126] In one embodiment, by causing phase separation to occur earlier in the porous material and / or at a lower concentration of the ATPS component, a larger volume of fluid will be subjected to phase separation as it flows through the porous material. In one embodiment, this can be achieved by adding a modifier, such as a compound carrying an acidic functional group in an aqueous solution, to reduce the pH of the first phase and / or the second phase, thereby promoting the redissolution of the components. As the ATPS components and / or modifiers dehydrate in the porous material and redissolve as the fluid flows, the continued redissolution of the ATPS components will increase the total volume of the first phase and / or the second phase. This may be beneficial in concentrating more target analytes from a given solution. In the case where the ATPS components dehydrate in the porous material and need to be redissolved, phase separation will only occur when the concentration reaches a certain point. By adding a modifier that induces phase separation at a lower concentration of the ATPS component, phase separation will begin earlier at a location further upstream in the porous material. This may be beneficial in increasing the total volume subjected to phase separation, thereby concentrating more target analytes for downstream applications.

[0127] In such Figure 1 In one embodiment shown, the present method and / or device can use multiple different types and various combinations of phase separation behavior modifiers. In one embodiment, the phase separation behavior modifier can be distributed at multiple different locations within the porous material of the device and / or distributed at different concentrations at each location.

[0128] Tuning the rate of domain aggregation of ATPS

[0129] Domain aggregation is the process by which small droplets of one phase of ATPS aggregate together to form a larger volume of that phase. In one embodiment, the phase separation behavior modifier of the present invention is selected to:

[0130] (a) reducing the fluctuation of the domain aggregation rate of one or two phases of the ATPS. In one embodiment, by adding self-interacting charged surfactants or proteins to the solution, these agents will be partitioned to the interface between the domains of one of the phases and increase the inter-domain interactions, thereby reducing the fluctuation of the domain aggregation rate; or

[0131] (b) increasing the rate of domain aggregation in one or both phases of the ATPS. In one embodiment, by adding self-interacting charged surfactants or proteins to the solution, these agents will partition to the interface between the domains of one of the phases and increase the inter-domain interactions, thereby increasing the rate of domain aggregation. This may be beneficial in speeding up the run time of the diagnostic; or

[0132] (c) reducing the rate of domain aggregation in one or both phases of the ATPS. In one embodiment, the surface tension between the two phases is reduced by adding a surfactant to the solution. As the surface tension decreases, the thermodynamic force driving the domain aggregation becomes smaller. This may be beneficial in cases where the target analyte partitions slowly and thus requires more time to reach the appropriate phase, which is achieved by slowing aggregation and keeping the domains small in the initial stage;

[0133] Adjust the flow rate of the fluid

[0134] In one embodiment, the flow rate in the porous material can be increased or decreased by adding a reagent that changes the viscosity and / or hydrophobicity of the solution. Typically, the flow rate of a fluid is inversely proportional to its viscosity. As the hydrophobicity of the fluid becomes more similar to the porous material, the flow rate also increases. In one embodiment, the phase separation behavior modifier of the present invention is selected to:

[0135] (a) reducing the fluctuation of the flow rate in the porous matrix of the first phase or the second phase. In one embodiment, by adding a modifier, such as a compound carrying hydrophilic and hydrophobic groups, the agent will interact with the ATPS component through the hydrophilic and hydrophobic groups to reduce the fluctuation of the flow rate; or

[0136] (b) increasing the flow rate within the porous matrix of the first phase or the second phase. In one embodiment, the flow rate is increased by adding a modifier, such as a compound carrying a hydrophilic group, which will interact with the ATPS component through the hydrophilic group to reduce the ionic strength of the system, thereby reducing the viscosity and hydrophobicity of the solution; or

[0137] (c) reducing the flow rate within the porous matrix of the first phase or the second phase. In one embodiment, by adding a modifier, such as a compound carrying a hydrophobic group, the agent will interact with the ATPS component through the hydrophobic group to increase the ionic strength of the system. As a result, the viscosity and hydrophobicity of the solution increase and the flow rate decreases; or

[0138] (d) Any combination of ac.

[0139] Adjust the partitioning of target analytes or contaminants into first or second phase components

[0140] In one embodiment, the phase separation behavior modifiers of the present invention are selected to:

[0141] (a) reducing fluctuations in the partitioning of target analytes and / or contaminants between the first phase and the second phase; or

[0142] (b) Increase the partitioning of target analytes and / or contaminants to the relevant phases.

[0143] Target partitioning can be driven by a variety of forces / principles, including but not limited to size exclusion, hydrophobic / hydrophilic interactions, electrostatic interactions. In one embodiment, the partitioning of target analytes to the relevant phase can be increased by adding modifiers that change the environment within the phase. For example, the addition of a chaotropic agent will make the hydrophobic phase more hydrophobic by disrupting hydrogen bonds in the relevant phase, thereby increasing the partitioning of hydrophobic analytes or contaminants to that phase. This may be beneficial to further increase the concentration of the target analyte or contaminant; or

[0144] (c) reduce the partitioning of target analytes and / or contaminants into the associated phase. In one embodiment, the addition of a lyophile will make the hydrophobic phase more hydrophilic by increasing the interaction between the two phases, thereby reducing the partitioning of hydrophobic analytes or contaminants into that phase. This may be desirable if one is trying to separate two targets into different phases; or

[0145] (d) Any combination of ac.

[0146] In one embodiment, the phase separation behavior modifier is selected to perform any combination of the above functions.

[0147] Adjustment of the concentration factor

[0148] In one embodiment, the relative amounts of the ATPS components in the porous material may be varied.

[0149] In one embodiment, by varying the amount of ATPS components dehydrated on the porous material, thereby varying the volume ratio of the two phases, the target analyte can be preferentially concentrated in one phase. In one embodiment, the target analyte is retained at the leading edge of the ATPS and then collected and optionally further analyzed using appropriate techniques.

[0150] In one embodiment, the order of the ATPS components on the porous material can be adjusted to achieve a desired effect or phenomenon. In one embodiment, one or more ATPS components can be dehydrated and embedded in the porous material.

[0151] In one embodiment, to better quantify the phenomena associated with the present invention, an analytical method was developed to evaluate the correlation between the relative amounts of ATPS components dehydrated on a porous material and the concentration multiples achieved. In one embodiment, the concentration factor can be controlled and fine-tuned by adjusting the relative amounts of the ATPS components as desired.

[0152] In one embodiment, to integrate the ATPS components into the porous material, the ATPS components are dissolved in water (or a suitable buffer) and a predetermined amount of the solution / suspension is applied to the porous material. The porous material is then placed in a freeze dryer or equivalent solvent removal device to remove the solvent (e.g., water), resulting in the dehydration of the ATPS components directly on the porous material. Upon introduction of the sample solution into the porous material, the ATPS components are immediately rehydrated, thereby separating the components or molecules in the sample as they flow through the porous material and concentrating the target analyte in front of the fluid flow. In one embodiment, no external power or device is required to provide the driving force.

[0153] In one embodiment, the present invention provides a device comprising a porous glass fiber paper impregnated with a polymer / polymer ATPS, such as PEG-PVPATPS. When a sample containing multiple analytes is applied to one end of the device, the impregnated porous glass fiber paper causes the ATPS component containing the analyte to flow preferentially over other ATPS components. Thus, the target analyte is concentrated in the ATPS component containing the analyte at the front of the fluid flow.

[0154] In one embodiment, the present invention provides a device comprising a porous glass fiber paper pretreated with a polymer-salt ATPS, such as a PEG-salt ATPS. When a sample containing multiple analytes is applied to one end of the device, the pretreated porous glass fiber paper causes the ATPS component containing the analyte to flow preferentially over other ATPS components. Thus, the target analyte is concentrated in the ATPS component containing the analyte at the front of the fluid flow.

[0155] In one embodiment, the present invention provides a device comprising a porous glass fiber paper impregnated with micellar ATPS. When a sample containing multiple analytes is applied to one end of the device, the impregnated porous glass fiber paper causes the ATPS component containing the analyte to flow preferentially over another ATPS component. Thus, the target analyte is concentrated in the ATPS component containing the analyte at the front of the fluid flow.

[0156] In one embodiment, the sample solution suitable for the device / method disclosed in the present invention is a buffer solution, which includes but is not limited to phosphate buffered saline (PBS) and Tris-EDTA (TE) buffer. In the present invention, preferably, the buffer solution is PBS buffer (8mM sodium chloride, 0.2mM potassium chloride, 1.15mM sodium monohydrogen phosphate, 0.2mM potassium dihydrogen phosphate solution, pH 7.35-7.65). In one embodiment, the buffer solution is TE buffer containing 2% bovine serum albumin (BSA), 0.1% PEG and 20mM Tris, pH 7.5.

[0157] In one embodiment, multiple ATPS components are incorporated into the porous material to achieve control over the concentration factor of the target analyte.

[0158] In one embodiment, optimized ratios of ATPS components are implemented in polymer / salt ATPS, polymer / polymer ATPS, or micellar ATPS to control the concentration factor.

[0159] In one embodiment, the ratios of the ATPS components are adjusted to achieve a concentration factor between 10-fold and 100-fold.

[0160] In one embodiment, there are multiple ATPS, including but not limited to polymer-polymer (e.g., PEG-PVP), polymer-salt (e.g., PEG-salt) and micelles. The first and / or second component includes a polymer. The polymer includes but is not limited to polyalkylene glycols such as hydrophobically modified polyalkylene glycols, poly(oxyalkylene) polymers, poly(oxyalkylene) copolymers such as hydrophobically modified poly(oxyalkylene) copolymers, polyvinyl pyrrolidone (PVP), polyvinyl alcohol, polyvinyl caprolactam, polyvinyl methyl ether, alkoxylated starch, alkoxylated cellulose, alkyl hydroxyalkyl cellulose, silicone-modified polyethers and poly-N-isopropylacrylamide and copolymers thereof. In another embodiment, the first polymer includes polyethylene glycol, polypropylene glycol.

[0161] In one embodiment, the polymer concentration of the first or second phase is about 0.01% to about 90% (w / w) of the total weight of the aqueous solution. In various embodiments, the polymer solution is selected from about 0.01% w / w, about 0.05% w / w, about 0.1% w / w, about 0.15% w / w, about 0.2% w / w, about 0.25% w / w, about 0.3% w / w, about 0.35% w / w, about 0.4% w / w, about 0.45% w / w, about 0.5% w / w, about 0.55% w / w, about 0.6% w / w, about 0.65% w / w, about 0.7% w / w, about 0.75% w / w, about 0.8% w / w, about 0.85% w / w, about 0.9% w / w, about 0.95% w / w, or about 1% w / w polymer solution. In some embodiments, the polymer solution is selected from about 1% w / w, about 2% w / w, about 3% w / w, about 4% w / w, about 5% w / w, about 6% w / w, about 7% w / w, about 8% w / w, about 9% w / w, about 10% w / w, about 11% w / w, about 12% w / w, about 13% w / w, about 14% w / w, about 15% w / w, about 16% w / w, about 17% w / w, about 18% w / w, about 19% w / w, about 20% w / w, about 21% w / w, about 22% w / w, about 23% w / w, about 24% w / w, about 25% w / w, about 26% w / w, about 27% w / w, about 28% w / w, about 29% w / w, about 30% w / w, about 31% w / w, about 32% w / w, about 33% w / w, about 34% w / w, about 35% w / w, about 36% w / w, about 37% w / w, about 38% w / w, about 39% w / w, about 40% w / w, about 41% w / w, about 42% w / w About 26% w / w, about 27% w / w, about 28% w / w, about 29% w / w, about 30% w / w, about 31% w / w, about 32% w / w, about 33% w / w, about 34% w / w, about 35% w / w, about 36% w / w, about 37% w / w, about 38% w / w, about 39% w / w, about 40% w / w, about 41% w / w, about 42% w / w, about 43% w / w, about 44% w / w, about 45% w / w, about 46% w / w, about 47% w / w, about 48% w / w, about 49% w / w and about 50% w / w polymer solutions.

[0162] In one embodiment, the first and / or second phase comprises a salt, including but not limited to an inorganic salt containing a cation such as linear or branched trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium and tetrabutylammonium, and an anion such as sulfate, nitrate, chloride and bicarbonate. In another embodiment, the salt is selected from the group consisting of NaCl, Na2S04, potassium citrate, (NH4)2S04, sodium citrate, sodium acetate, sodium cholate and combinations thereof. Other salts, such as ammonium acetate, may also be used.

[0163] In one embodiment, the total salt concentration is in the range of 0.001 mM to 100 mM. One skilled in the art will appreciate that the amount of salt required to form a two-phase aqueous system will be affected by the molecular weight, concentration, and physical state of the polymer.

[0164] In various embodiments, the salt phase is selected from a salt solution having a weight ratio of about 0.001% to 90%. In various embodiments, the salt solution is selected from a salt solution of about 0.01% w / w, about 0.05% w / w, about 0.1% w / w, about 0.15% w / w, about 0.2% w / w, about 0.25% w / w, about 0.3% w / w, about 0.35% w / w, about 0.4% w / w, about 0.45% w / w, about 0.5% w / w, about 0.55% w / w, about 0.6% w / w, about 0.65% w / w, about 0.7% w / w, about 0.75% w / w, about 0.8% w / w, about 0.85% w / w, about 0.9% w / w, about 0.95% w / w, or about 1% w / w. In some embodiments, the saline solution is selected from about 1% w / w, about 2% w / w, about 3% w / w, about 4% w / w, about 5% w / w, about 6% w / w, about 7% w / w, about 8% w / w, about 9% w / w, about 10% w / w, about 11% w / w, about 12% w / w, about 13% w / w, about 14% w / w, about 15% w / w, about 16% w / w, about 17% w / w, about 18% w / w, about 19% w / w, about 20% w / w, about 21% w / w, about 22% w / w, about 23% w / w, about 24% w / w, about 25% w / w, about and about 50% w / w polymer solutions.

[0165] In one embodiment, the first and / or second phase of the ATPS comprises a solvent that is immiscible with water. In some embodiments, the solvent comprises a non-polar organic solvent. In some embodiments, the solvent comprises an oil. In some embodiments, the solvent is selected from pentane, cyclopentane, benzene, 1,4-dioxane, ether, dichloromethane, chloroform, toluene, and hexane.

[0166] In one embodiment, the first phase and / or the second phase of the ATPS comprises a micellar solution.

[0167] In one embodiment, one phase of the ATPS comprises a micellar solution and the other phase comprises a liquid phase containing a polymer. In one embodiment, one phase of the liquid phase comprises a micellar solution and the other phase of the liquid phase comprises a salt solution. In one embodiment, one phase comprises a first polymer and the other phase comprises a second polymer. In one embodiment, the first / second polymer is selected from polyethylene glycol and PVP. In one embodiment, one phase comprises a polymer and the other phase comprises a salt. In some embodiments, one phase comprises PVP and the other phase comprises sodium cholate. In one embodiment, one phase comprises a salt and the other phase comprises another salt.

[0168] In one embodiment, the volume ratio of the first phase to the second phase is in the range of 1:1 to 1:1000. In some embodiments, the ratio of the first phase to the second phase is selected from a ratio of about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, and about 1:10. In some embodiments, the ratio of the first phase to the second phase is selected from a ratio of about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, and about 1:100. In some embodiments, the ratio of the first phase to the second phase is selected from a ratio of about 1:200, about 1:300, about 1:400, about 1:500, about 1:600, about 1:700, about 1:800, about 1:900, and about 1:1000.

[0169] In one embodiment, the volume ratio of the second phase to the first phase is selected from a ratio of about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, and about 1:10. In some embodiments, the ratio of the second phase to the first phase is selected from a ratio of about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, and about 1:100. In some embodiments, the ratio of the second phase to the first phase is selected from a ratio of about 1:200, about 1:300, about 1:400, about 1:500, about 1:600, about 1:700, about 1:800, about 1:900, and about 1:1000.

[0170] Downstream processing of analytes after separation

[0171] In various embodiments, the present invention may be integrated with a second device at a downstream region of the porous material of the first device such that the guiding fluid within the porous material is transferred to the second device.

[0172] In various embodiments, the present invention can be integrated with a second device in a downstream region of the porous material of the first device so that the guiding fluid in the porous material is transferred to the second device. A phase separation behavior modifier is selected to directly or indirectly improve the performance of the second device integrated in the downstream region through a combination of any means described in the present invention.

[0173] The analytes and / or analyte-containing solutions obtained by the present invention can be used in a wide range of downstream applications, such as detection or analysis of analytes in forensic, diagnostic or therapeutic applications, and laboratory procedures such as sequencing, amplification, reverse transcription, labeling, enzyme digestion, blotting, etc. The present invention can improve the performance of downstream characterization or processing of analytes.

[0174] In one embodiment, the downstream application of the analyte includes but is not limited to any detection, analysis or diagnostic procedure involving the detection or quantification of the purified analyte. In one embodiment, the detection, analysis or diagnostic procedure combined with the present invention includes but is not limited to any such procedure performed in a commercial clinical laboratory, and laboratory procedures such as sequencing, amplification (e.g., PCR, RT-PCR, real-time PCR and real-time RT-PCR), reverse transcription, labeling, enzyme digestion, blotting, ELISA, RIA, immunoassay, enzymatic assay, GC / MS, proteomic-based methods, etc.

[0175] In one embodiment, the analyte and / or analyte-containing solution obtained by the present invention can be analyzed by lateral flow immunoassay (LFA). LFA has many desirable characteristics, including ease of use and wide applicability to a variety of analytes. However, due to its detection limitations, LFA can generally only provide qualitative results. For example, the detection limit of LFA for pathogens (Streptococcus mutans) is 10 6 In the present invention, the modifier is used to improve the phase separation of ATPS, and the detection limit of LFA for Streptococcus mutans can be increased to as low as 10 4 CFU / ml, a 100-fold improvement. Together with the 100-fold improvement in concentration multiple, the present invention is suitable for providing quantitative results in a wider range.

[0176] In one embodiment, the partitioning of the target analyte to the relevant phase can be enhanced by adding a modifier to the ATPS. In one embodiment, the concentration is increased by a factor of 100. Improving the partitioning of the target analyte also helps to increase the yield of the target analyte.

[0177] If the target analyte concentration is very low, a false negative result may be generated. In one embodiment, the detection limit of the LFA is increased by increasing the concentration of the target analyte by adding one or more modifiers to the ATPS. Thus, the reproducibility of the test is improved.

[0178] In one embodiment, by adding one or more modifiers, ATPS phase separation can be induced to occur earlier and / or at a lower concentration of ATPS components, thereby significantly improving the efficiency or speed of the entire detection or diagnosis process.

[0179] In various embodiments, the present invention may be used in conjunction with one or more processes or reagents for washing and eluting analytes remaining in a porous matrix, or for post-separation processing of the remaining analytes.

[0180] The first or second device is designed to be able to manually and / or automatically extract the first phase solution and / or the second phase solution from the porous material of the first or second device, and then use the extracted solution in another method or process.

[0181] In various embodiments, the phase separation behavior modifiers of the present invention are selected to directly or indirectly improve the performance of the method or process using the extraction phase in combination by any of the means described herein.

[0182] In one embodiment, after the solution containing the analyte is contacted with the ATPS, one or more wash buffers are applied to the ATPS to wash away non-target analytes or impurities from the porous matrix. The wash buffer may include solutions of different ionic strengths, pH values, or solutions containing additives such as detergents. The wash buffer includes, but is not limited to, a solution containing 20%-50% ethanol and 20%-50% isopropanol; a solution containing about 0.1-4.0 M guanidine hydrochloride, a detergent, and up to about 80% ethanol; or a solution containing about 80% ethanol.

[0183] In one embodiment, the target analyte enters the porous matrix of ATPS and flows from one end to the other end. In one embodiment, the target analyte separated by the present invention is eluted from the porous matrix using a suitable elution buffer or deionized water. In one embodiment, the separated target analyte is not eluted, but stored in the porous matrix for standby use. For example, after using the present invention to separate the analyte, the porous material (e.g., paper) containing the target analyte (e.g., DNA) is dried and stored. In one embodiment, the analyte remaining on the porous matrix can be eluted for further analysis or processing. The choice of elution buffer may depend on the intended use of the purified biomarker. Examples of elution buffers suitable for nucleic acid type analytes include, but are not limited to, Tris-EDTA (TE) buffer, double distilled water, and PCR buffer. In one embodiment, the purified analyte on the porous paper is eluted in a tube containing TE buffer (10mM TrisCl, 1mM EDTA solution, pH 7.5), and the purified analyte is recovered in a relatively small volume, for example, less than 100 μl.

[0184] In one embodiment, the present invention provides a device for separating or concentrating one or more target analytes, the device comprising:

[0185] (1) Porous materials;

[0186] (2) components capable of forming a first phase solution and a second phase solution of an aqueous two-phase system (ATPS); and

[0187] (3) one or more phase separation behavior modifiers;

[0188] wherein the component and the phase separation behavior modifier are embedded in the porous material, wherein when the solution containing the target analyte flows through the porous material, a two-phase aqueous system comprising the first phase solution and the second phase solution is generated, wherein the phase separation behavior modifier changes the separation behavior of the two-phase solution, and the target analyte is separated or concentrated on the porous material, or

[0189] wherein one or more of the components and the phase separation behavior modifiers are embedded in the porous material, wherein the remainder of the components and the phase separation behavior modifiers are mixed with a solution containing the target analyte to form a mixed solution, wherein when the mixed solution flows through the porous material, a two-phase aqueous system comprising the first phase solution and the second phase solution is generated, wherein the phase separation behavior modifier changes the separation behavior of the two-phase solution, and the target analyte is separated or concentrated on the porous material.

[0190] In one embodiment, one or more phase separation behavior modifiers are present in some regions of the porous material and absent in other regions of the porous material.

[0191] In one embodiment, one or more phase separation behavior modifiers are uniformly distributed on the porous material, or are present in an amount that gradually increases from one end to the other end of the porous material.

[0192] In one embodiment, one or more phase separation behavior modifiers are present in different amounts in different regions of the porous material.

[0193] In one embodiment, one or more target analytes partition into the first phase solution or the second phase solution, or accumulate at the interface between the first phase solution and the second phase solution.

[0194] In one embodiment, the porous material and the components are selected so that the first phase solution and the second phase solution flow through the porous material at different rates.

[0195] In one embodiment, the one or more phase separation behavior modifiers are selected from the group consisting of:

[0196] (a) a compound carrying one or more acidic functional groups;

[0197] (b) compounds carrying one or more amine functional groups; and

[0198] (c) Compounds carrying hydrophilic and hydrophobic groups.

[0199] In one embodiment, the compound carrying one or more acidic functional groups is selected from the group consisting of saccharides, polysaccharides, sorbates, polysorbates, carboxylates, polycarboxylates, phosphates, polyphosphates, sulfates, polysulfates and polyols.

[0200] In one embodiment, the compound carrying one or more amine functional groups is selected from the group consisting of amines, polyamines, ammonium salts and polyammonium salts.

[0201] In one embodiment, the compound carrying hydrophilic and hydrophobic groups is selected from the group consisting of lipids, surfactants, nucleic acids, hormones, proteins and amino acids.

[0202] In one embodiment, the phase separation behavior modifier is a chaotropic agent. In one embodiment, the phase separation behavior modifier is a kosmotropic agent.

[0203] In one embodiment, one or more phase separation behavior modifiers are selected to:

[0204] (a) reducing fluctuations in the volume of the first phase solution, the second phase solution, or the total volume of both during or after phase separation;

[0205] (b) increasing or decreasing the total volume of the solution containing the target analyte that is subjected to phase separation;

[0206] (c) reducing fluctuations in the volume ratio between the first phase solution and the second phase solution; or

[0207] (d) Increasing or decreasing the volume ratio between the first phase solution and the second phase solution.

[0208] In one embodiment, one or more phase separation behavior modifiers are selected to:

[0209] (a) reducing fluctuations in the concentration of one or more components in the first phase solution;

[0210] (b) reducing fluctuations in the concentration of one or more components in the second phase solution;

[0211] (c) increasing or decreasing the concentration of one or more components in the first phase solution; or

[0212] (d) Increasing or decreasing the concentration of one or more components in the second phase solution.

[0213] In one embodiment, one or more phase separation behavior modifiers are selected to induce phase separation when the concentration of the components is insufficient to induce phase separation in the absence of the modifiers.

[0214] In one embodiment, one or more phase separation behavior modifiers are selected to:

[0215] (a) reduce fluctuations in the rate at which domains aggregate; or

[0216] (b) Increase or decrease the domain aggregation rate.

[0217] In one embodiment, one or more phase separation behavior modifiers are selected to:

[0218] (a) reducing the fluctuation of the flow rate of the first phase solution in the porous material;

[0219] (b) reducing the fluctuation of the flow rate of the second phase solution in the porous material;

[0220] (c) increasing the flow rate of the first phase solution in the porous material;

[0221] (d) reducing the flow rate of the first phase solution in the porous material;

[0222] (e) increasing the flow rate of the second phase solution within the porous material; or

[0223] (f) Reducing the flow rate of the second phase solution within the porous material.

[0224] In one embodiment, one or more phase separation behavior modifiers are selected to:

[0225] (a) reducing the fluctuation of the distribution of the target analyte between the first phase solution and the second phase solution;

[0226] (b) increasing the partitioning of the target analyte into the first phase solution;

[0227] (c) reducing the partitioning of the target analyte into the first phase solution;

[0228] (d) increasing the partitioning of the target analyte into the second phase solution; or

[0229] (e) Reduce the partitioning of target analytes into the second phase solution.

[0230] In one embodiment, the device is further integrated with a second device such that the guiding fluid within the porous material is transferred into the second device.

[0231] In one embodiment, the present invention also provides a method for separating or concentrating one or more target analytes from a sample solution using the device.

[0232] In one embodiment, the target analytes separated or concentrated on the porous material are extracted to produce an extract solution, which is then used in another method or process.

[0233] Throughout this application, it should be noted that the transitional term "comprising," which is synonymous with "including" or "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0234] By referring to the following examples, the present invention will be better understood. However, those skilled in the art will readily appreciate that the examples provided are only for illustrative purposes and are not intended to limit the scope of the present invention, which is defined by the following claims.

[0235] Examples

[0236] Reality Example 1 - Use of dextran to reduce volume in a device for detecting pathogens (Streptococcus mutans) in patient samples The product ratio fluctuates.

[0237] Preparation of sample pads for LFA: Glass fiber porous paper was cut into rectangles of 0.5 cm x 4 cm. The prepared ATPS components, 20% (w / w) PVP and 18.5% (w / w) sodium cholate were pipetted onto the glass fiber porous paper. The above porous paper with ATPS was then dried in a freeze dryer for 2 hours. 10% (w / w) dextran was then pipetted onto the glass fiber porous paper as a phase separation behavior modifier on the paper segment. PEG solution (in DI H20) was added to each porous material. 50 μl of Tris-buffered solution (20 mM Tris, pH 7.5) containing 2% bovine serum albumin (BSA) and 0.1% PEG was added immediately after the first solution.

[0238] The porous paper containing ATPS and phase separation behavior modifiers was then dried in a freeze dryer for 2 hours. The ATPS in the porous paper was then placed in a buffer solution containing an indicator (colloidal gold) in PBS (total pH 7.4) to induce capillary action-mediated flow. Dextran solution was applied to one end of the porous paper to generate a gradient concentration along the diffusion direction:

[0239] The purpose of the dextran (phase separation behavior modifier) ​​is to stabilize (i.e. reduce fluctuations) the volume ratio of the ATPS during variable fluid flow rates, making the test more robust. The gradient of the dextran is important so that when the fluid flow reaches the end of the porous material, the volume ratio is still large enough. The paper segments are placed under a low pressure vacuum for 2 hours to dehydrate. Multiple layers of porous paper with dehydrated ATPS components and phase separation behavior modifiers are combined together.

[0240] Preparation of LFA test strips: (1) Anti-mutans Streptococcus antibody at a concentration of 1 mg / mL and (2) protein (bovine serum albumin, BSA) at a concentration of 0.2 mg / mL were added to the test strips. Colloidal gold nanoparticles were conjugated to anti-mutans Streptococcus antibody according to the manufacturer's instructions. The conjugate was then dried onto a conjugate pad material using a lyophilizer. The absorbent pad consisted of untreated paper.

[0241] The LFA test strip is integrated with the sample pad, the porous device / component having the dehydrated ATPS component and the phase separation behavior modifier. The porous device / component and the LFA component are placed in a suitable housing so that the components are held in place.

[0242] Using LFA detection: Glass fiber porous paper dehydrated with ATPS components and phase separation behavior modifiers, blank paper (no ATPS or modifiers) and control paper (ATPS without modifiers) were immersed in the same saliva solution, which was in PBS buffer solution with a pH of 7.4. After the sample solution flowed through the porous material and passed through the LFA test for 2 minutes, it took another 2 minutes to form the test result. The diagnosis result was determined by visually observing the presence or absence of the test line. The results are summarized in Table 1 below:

[0243] Table 1 - Test line strength

[0244]

[0245] Table 1 clearly shows that the addition of phase separation behavior modifiers to ATPS can increase the intensity of the test line, indicating a significant increase in the concentration of the target analyte.

[0246] Example 2 - Use of polysorbates and sucrose to increase the partitioning of a food allergen (lupin) in a test device

[0247] Preparation of sample pads for LFA: Cut glass fiber porous paper into 0.5 cm × 4 cm rectangles. Pipette the prepared ATPS components, 20% (w / w) PVP and 18.5% (w / w) sodium cholate onto the glass fiber porous paper. Pipette 5% (w / w) polysorbate and 5% (w / w) sucrose onto the porous glass fiber paper as phase separation behavior modifiers. Add PEG solution (in DI H20) to each porous material. Add 50 μl of Tris-buffered solution (20 mM Tris, pH 7.5) containing 2% bovine serum albumin (BSA) and 0.1% PEG immediately after the first solution.

[0248] The above porous paper containing the dehydrated ATPS component and the phase separation behavior modifier was then dried in a freeze dryer for 1 hour. The ATPS dehydrated porous paper was then placed in a buffer solution containing an indicator (colloidal gold) in PBS (total pH 7.4) to induce capillary action mediated flow. Polysorbate and sucrose were applied to evenly distribute the components on the porous material. Multiple layers of the porous paper with the dehydrated ATPS component and the phase separation behavior modifier were combined together.

[0249] The purpose of polysorbate and sucrose is to increase the partitioning of food allergen targets into Phase 1. The paper segments were then dehydrated in an oven at 37 degrees Celsius for 28 hours.

[0250] Preparation of LFA test strips: (1) Anti-lupin antibody at a concentration of 1 mg / mL and (2) protein (bovine serum albumin, BSA) at a concentration of 0.2 mg / mL were added to the test strips. Colloidal gold nanoparticles were conjugated to the anti-lupin antibody according to the manufacturer's instructions. The conjugate was then dried onto the conjugate pad material using a lyophilizer. The absorbent pad consisted of untreated paper.

[0251] The LFA test strip is first integrated with the sample pad (i.e., the porous material for testing the food allergen lupin) and then integrated with the food matrix grinder / processor. The integrated device should be assembled so that the sample solution flows downward from the food matrix grinder / processor into the porous component and then flows further forward through the porous material, where the ATPS component and the phase separation behavior modifier are redissolved, and the phase separation behavior modifier will induce phase separation and promote the partitioning of the food allergen into the first advanced phase, and finally enter the LFA component. All components are integrated into a suitable box / housing so that the components are kept in place.

[0252] Food allergens were detected using LFA. The relevant food sample was placed in the food matrix grinder / processor portion of the device. The food matrix grinder / processor was manually operated to break down the food matrix. Glass fiber porous paper containing dehydrated ATPS components and phase separation behavior modifiers, blank paper (without ATPS or modifiers), and control paper (ATPS without modifiers) were immersed in the food matrix sample in PBS buffer solution at pH 7.4. After the sample solution flowed through the remaining device portion for 2 minutes, an additional 2 minutes were required to form the test result. The diagnostic result was determined by visually observing the presence or absence of the test line. The results are summarized in Table 2 below:

[0253] Table 2 - Test line strength

[0254]

[0255] Table 2 clearly shows that the addition of phase separation behavior modifiers to ATPS can increase the intensity of the test line, indicating a significant increase in the concentration of the target analyte.

[0256] Example 3 - Use of potassium phosphate to increase the total phase separation volume in a sample solution purification and DNA amplification device

[0257] The porous hydrogel material was cut into a cylindrical shape with a radius of 2 cm and a height of 6 cm. The porous hydrogel component was integrated into a suitable box / housing. The device can expose the bottom and top of the hydrogel.

[0258] The ATPS components, 20% (w / w) PVP and 18.5% (w / w) sodium cholate, and 10% (w / w) potassium phosphate as a phase separation behavior modifier, were mixed with a blood sample solution containing the relevant DNA fragments in a PBS buffer solution at pH 7.4. The purpose of the potassium phosphate is to induce macroscopic phase separation at an earlier point (further upstream) in the process of the solution flowing through the porous material, so that a larger volume of solution undergoes phase separation and a larger volume of solution can be collected at the end of the process. The final volume should be approximately 120% of the volume that the hydrogel can accommodate. The sample solution is placed in an open container or test tube that can hold the conical porous device in an upright position. A blank hydrogel material (without ATPS components or modifiers) and a control hydrogel material (ATPS without modifiers) are used for comparison.

[0259] The bottom and top of the device are opened to expose the bottom and top of the conical porous hydrogel. The device is placed in a mixture of a sample solution and ATPS components. The liquid of the mixture contacts the bottom of the porous hydrogel.

[0260] Extraction of sample solution: After the solution flowed to the top of the porous hydrogel for 2 minutes, the purified sample solution was extracted by removing the top area of ​​the porous hydrogel with a pipette. The extracted sample solution can then be used for DNA amplification. The yield of the purified sample was analyzed by UV-Vis spectrophotometry. The results are summarized in Table 3 below.

[0261] Table 3 DNA concentration multiples using the present invention

[0262]

Claims

1. A method for separating or concentrating one or more target analytes from a sample solution, the method comprising the following steps: (a) providing one or more phase separation behavior modifiers and two-phase aqueous system components, wherein the two-phase aqueous system components form a first phase solution and a second phase solution; (b) embedding one or more phase separation behavior modifiers and at least a portion of the components of the two-phase aqueous system in a porous material; (c) combining the one or more phase separation behavior modifiers and the remaining portion of the two-phase aqueous system components with a sample solution containing the target analyte to produce a mixed solution; as well as (d) adding the mixed solution to the porous material; wherein, when the mixed solution flows through the porous material, a two-phase aqueous system comprising the first phase solution and the second phase solution is generated, wherein the phase separation behavior modifier changes the separation behavior of the two-phase solution; and Wherein, the target analyte is separated or concentrated on the porous material.

2. The method according to claim 1, wherein: Step (b) comprises the following steps: (i) embedding a portion of the phase separation behavior modifier in the porous material containing the first phase solution and the second phase solution of the two-phase aqueous system components; and (ii) dehydrating the porous material; And step (c) comprises the following steps: (iii) combining the remaining portion of the phase separation behavior modifier with the sample containing the target analyte to produce the mixed solution.

3. The method according to claim 1, wherein: Step (b) comprises the following steps: (i) embedding a portion of the phase separation behavior modifier in the porous material containing the first phase solution of the two-phase aqueous system components; and (ii) dehydrating the porous material; And step (c) comprises the following steps: (iii) combining the remaining portion of the phase separation behavior modifier with the components of the second phase solution of the two-phase aqueous system components to produce the mixed solution.

4. The method according to claim 1, wherein: Step (b) comprises the following steps: (i) embedding a portion of the phase separation behavior modifier in the porous material containing the second phase solution of the two-phase aqueous system components; and (ii) dehydrating the porous material; And step (c) comprises the following steps: (iii) combining the remaining portion of the phase separation behavior modifier with the components of the first phase solution of the two-phase aqueous system components to produce the mixed solution.

5. The method according to claim 1, wherein: Step (b) comprises the following steps: (i) embedding a portion of the phase separation behavior modifier in the porous material; and (ii) dehydrating the porous material; And step (c) comprises the following steps: (iii) combining the remaining portion of the phase separation behavior modifier with the components of the first phase solution and the components of the second phase solution of the two-phase aqueous system components to produce the mixed solution.

6. The method according to claim 1, further comprising the steps of: (f) extracting the target analyte separated or concentrated on the porous material to produce an extract; and (g) subjecting the extract to a detection, analysis or diagnostic procedure involving the detection or quantification of the target analyte.

7. The method of claim 1, wherein the one or more phase separation behavior modifiers are present in some regions of the porous material and absent in other regions of the porous material. 8 . The method according to claim 1 , wherein the one or more phase separation behavior modifiers are uniformly distributed on the porous material, or exist in an amount gradually increasing from one end to the other end of the porous material.

9. The method of claim 1, wherein the one or more phase separation behavior modifiers are present in different amounts in different regions of the porous material.

10. The method of claim 1, wherein the one or more phase separation behavior modifiers are selected from the group consisting of: (a) a compound carrying one or more acidic functional groups; (b) compounds carrying one or more amine functional groups; (c) compounds carrying hydrophilic and hydrophobic groups; (d) Chaotropic agents; as well as (e) Lyophilic agents.

11. The method of claim 10, wherein the compound carrying one or more acidic functional groups is selected from the group consisting of saccharides, polysaccharides, sorbates, polysorbates, carboxylates, polycarboxylates, phosphates, polyphosphates, sulfates, polysulfates and polyols.

12. The method of claim 10, wherein the compound carrying one or more amine functional groups is selected from the group consisting of: amines, polyamines, amine salts and polyamine salts.

13. The method of claim 10, wherein the compound carrying hydrophilic and hydrophobic groups is selected from the group consisting of lipids, surfactants, nucleic acids, hormones, proteins and amino acids.

14. The method of claim 1, wherein the phase separation behavior modifier comprises sucrose, potassium phosphate, dextran, Tween-20 or Triton-X114.

Images