Device for integrated extraction, enrichment and detection of analytes - Patents.com

Abstract

To provide a device for integrating extraction, condensation and detection of an analyte.SOLUTION: In the present application, disclosed herein are devices and methods that use an aqueous two-phase system and lateral flow assay to detect targeted analyte in a sample. These devices and methods may be used to diagnose a disease or illness in a biological sample such as a blood or serum and the like. In addition, these devices and methods may be used to detect contaminants in water such as allergen in a food sample or toxin in an environment. Components of the device and kit may be conveniently assembled in a portable container, and these are adapted to an action in most of environments. The present device is simple to use, and a non-trained operator is required to simply add a sample to the device. Conveniently, a time to detect a target analyte takes very short. Thus, the devices and methods to be disclosed herein provide novel and useful means for point-of-care.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 61 / 949,887, filed March 7, 2014, and U.S. Provisional Application No. 61 / 953,870, filed March 16, 2014, which are incorporated by reference in their entireties. [Background technology]

[0002] The identification of unknowns, including substances and materials on the one hand, and diseases and disorders on the other, is of paramount importance to many different industries. Enzymes and antibodies are used in a wide variety of contexts, including, but not limited to, medical diagnostics, food testing, and environmental contamination (e.g., toxins, pathogens). Summary of the Invention [Means for solving the problem]

[0003] Described herein are methods, devices, kits, and systems including tests performed at the point of care (POC) or site of use as useful tools for medical, consumer, and other applications. Point of care or site of use locations in certain applications can include medical facilities, mobile clinics, workplaces, factories, farms, and homes. The subject matter described herein is also useful for a variety of other applications, including, by way of non-limiting example, identification of air pollutants, toxins, ingredients in food and medicines, as well as industrial, military, and space-related applications. Among many other aspects, provided herein are tests that are quick, simple, and easy to use. The tests can be performed by individuals with little, if any, training in clinical diagnostics. Also described herein are methods for obtaining test results in a cost-effective and time-efficient manner. These tests need to be reliable and sensitive, especially for small amounts of biomolecules. Furthermore, the tests described herein are adaptable to many different environments, such as environments with large variations in temperature and / or humidity. As such, the point-of-care or point-of-use devices described herein can require a minimal amount of equipment and are stable in a wide variety of environmental conditions.

[0004] Also disclosed herein are devices and methods that include lateral flow assay (LFA) technology and aqueous multiphase systems for the detection of analytes of interest. In certain embodiments, these devices and methods utilize the concentration capabilities of aqueous multiphase systems in combination with LFAs to provide at least about 100-fold to about 1000-fold improvement over the detection limits of previous LFA assays, approaching or matching, and in some cases exceeding, the sensitivity of laboratory assays such as enzyme-linked immunosorbent assays (ELISAs).

[0005] Generally, in certain applications, a sample of interest containing an analyte of interest is applied to an aqueous two-phase system (ATPS). As described herein, methods and devices have been developed for concentrating and extracting the analyte of interest from a single phase or interface of the ATPS and then detecting the analyte of interest on an LFA, where the presence of the analyte can be detected and / or quantified. Alternatively or additionally, the analyte of interest can be concentrated and detected and / or quantified on an integrated ATPS-LFA system. Thus, extraction can be bypassed by seamlessly integrating the ATPS with downstream LFA detection. In certain applications described herein, these devices require the user to only add the sample of interest. In various embodiments, these devices can be used to detect the analyte of interest in biological samples such as serum, saliva, urine, blood or swab samples.

[0006] In certain embodiments, the devices disclosed herein are conveniently assembled to form a portable diagnostic device. The various devices and methods described herein prove to be robust, versatile, scalable, inexpensive, sensitive, simple and accurate, and require minimal training, power, and equipment. It should be noted that although the following description generally refers to aqueous two-phase systems (ATPS), three- or four-phase systems, and even more multiphase systems, can be implemented as well.

[0007] Examples of these techniques are described herein to illustrate how these concepts work. These examples are intended to be illustrative and non-limiting. Using the teachings provided herein, numerous other systems integrating ATPS and lateral flow detection can be readily realized. It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications and variations in light of these examples and embodiments will be suggested to those skilled in the art, which modifications and variations are to be encompassed within the spirit and scope of this application and the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0008] One aspect of the subject matter described herein, including the methods, devices, kits, and systems described herein, is a device for detection and / or quantification of an analyte of interest in a sample, the device comprising: (a) a lateral flow assay (LFA); and (b) an aqueous two-phase system (ATPS). In certain applications, the ATPS comprises a mixed phase solution that is partitioned into a first phase solution and a second phase solution. In some embodiments, the partitioning of the mixed phase into the first phase solution and the second phase solution occurs within the LFA. In certain embodiments, the partitioning of the mixed phase into the first phase solution and the second phase solution does not occur within the LFA. In certain applications, the analyte of interest contacts the mixed phase solution, and when / after the first phase solution or the second phase solution separates, the analyte of interest partitions into the first phase solution or the second phase solution. In some embodiments, the analyte of interest contacts the mixed phase solution and when / after the first or second phase solutions separate, the analyte of interest partitions to the interface between the first and second phase solutions, hi some embodiments, the analyte of interest is concentrated upon partitioning.

[0009] In certain embodiments, the first phase solution comprises a micellar solution and the second phase solution comprises a polymer. In certain embodiments, the first phase solution comprises a polymer and the second phase solution comprises a micellar solution. In certain applications, the first phase solution comprises a micellar solution and the second phase solution comprises a salt. In certain applications, the first phase solution comprises a salt and the second phase solution comprises a micellar solution. In some embodiments, the micellar solution comprises a non-ionic surfactant. In some embodiments, the nonionic surfactant is selected from the group consisting of cetomacrogol, cetostearyl alcohol, cetyl alcohol, cocamide, decyl glucoside, IGEPAL, isoceteth, lauryl glucoside, monolaurin, Nonidet®, nonoxynol, NP-40, octyl glucoside, oleyl alcohol, poloxamer, pentaethylene glycol monododecyl ether, polysorbate, polyglycerol, sorbitan, stearyl alcohol, Triton-X, and Tween. In certain applications, the micellar solution is a Triton-X solution. In some embodiments, the Triton-X solution is selected from a Triton-X-100 solution and a Triton-X-114 solution.

[0010] In some embodiments, the first phase solution comprises a first polymer and the second phase solution comprises a second polymer. In some embodiments, the first / second polymers are selected from polyethylene glycol and dextran. In some embodiments, the first phase solution comprises a polymer and the second phase solution comprises a salt. In some embodiments, the first phase solution comprises a salt and the second phase solution comprises a polymer. In some embodiments, the first phase solution comprises polyethylene glycol and the second phase solution comprises potassium phosphate. In some embodiments, the first phase solution comprises potassium phosphate and the second phase solution comprises polyethylene glycol. In certain embodiments, the first phase solution is selected from Component 1 in Table 1 and the second phase solution is selected from Component 2 in Table 1 as described herein. In certain embodiments, the second phase solution is selected from Component 1 in Table 1 and the first phase solution is selected from Component 2 in Table 1 as described herein.

[0011] In some embodiments, the analyte of interest is selected from a protein, an antigen, a biomolecule, a sugar moiety, a lipid, a nucleic acid, a sterol, and combinations thereof. In certain applications, the analyte of interest is from an organism selected from the group consisting of a plant, an animal, a virus, a fungus, a protozoan, and a bacterium. In one embodiment, the device further comprises a probe that interacts with the analyte of interest.

[0012] Another aspect of the methods, devices, kits, and systems described herein is an LFA device comprising one or more probes that interact with at least one analyte of interest, or at least two different analytes of interest, or at least three different analytes of interest, or at least four different analytes of interest, or at least five different analytes of interest, or at least seven different analytes of interest, or at least ten different analytes of interest, or at least fifteen different analytes of interest, or at least twenty different analytes of interest, or more. In some embodiments, at least two different probes, or at least three different probes, or at least four different probes, or at least five different probes, or at least seven different probes, or at least ten different probes, or at least fifteen different probes, or at least twenty different probes are described herein. In certain embodiments, provided herein is a probe comprising a material selected from the group consisting of synthetic polymers, metals, minerals, glass, quartz, ceramics, biological polymers, plastics, and combinations thereof. In certain embodiments, provided herein are probes comprising a polymer selected from the group consisting of polyethylene, polypropylene, cellulose, chitin, nylon, polyoxymethylene (Delrin®), polytetrafluoroethylene (Teflon®), polyvinyl chloride, and combinations thereof. In some embodiments, the polypropylene is polypropylene glycol. In some embodiments, the polyethylene is polyethylene glycol. In certain embodiments, provided are probes comprising a biological polymer selected from the group consisting of dextran and polyethylene glycol, and combinations thereof. In certain embodiments, provided are probes comprising a metal (e.g., a metal selected from the group consisting of gold, silver, titanium, stainless steel, aluminum, platinum, and / or alloys thereof).

[0013] In certain aspects of the methods, devices, kits, and systems described herein, a probe is provided that includes nanoparticles (e.g., gold nanoparticles). In certain embodiments, the probe includes a coating. In various embodiments, the coating can include polyethylene glycol or other polymers (e.g., polypropylene glycol), or a graft copolymer, such as poly(L-lysine)-graft-dextran (PLL-g-dex, a graft copolymer with dextran side chains on a poly(L-lysine) backbone, etc.). In one exemplary, but non-limiting embodiment, the coating includes dextran. In another embodiment, the coating includes a hydrophilic protein. In certain applications, the coating includes serum albumin. In certain embodiments, the coating has an affinity for the first phase solution or the second phase solution. In certain embodiments, the probe includes a binding moiety that binds to an analyte of interest. In certain embodiments, the binding moiety is selected from the group consisting of an antibody, a lectin, a protein, a glycoprotein, a nucleic acid, a small molecule, a polymer, and a lipid, and / or a combination thereof. In one embodiment, the binding moiety is an antibody or an antibody fragment. In certain embodiments, the probe comprises magnetic particles. In certain embodiments, the methods, devices, kits, and systems include a magnet. In certain embodiments, the magnet is configured to facilitate and / or increase partitioning of the analyte of interest into the first phase solution or the second phase solution. In one embodiment, the magnet is configured to facilitate and / or increase the flux of the analyte of interest through the LFA. In certain embodiments, the magnet is attachable to and / or detachable from the device. In certain applications of the methods, devices, kits, and systems provided herein, a collector configured to be placed in contact with an ATPS is provided, where the analyte of interest partitions at the interface between the collector and the first phase solution and / or the second phase solution.In some embodiments, the collector comprises a material selected from a plastic, a mesoporous material, silica, a polymer (e.g., polypropylene, polyoxymethylene (Delrin®), polytetrafluoroethylene (Teflon®), etc.), a magnet, a material with pores, a material with grooves, and any combination thereof.

[0014] In some embodiments, the methods, devices, kits, and systems described herein comprise a probe comprising a detectable label. In some embodiments, the detectable label is selected from the group consisting of a chromogenic label, a fluorescent label, an enzymatic label, a heat-generating label, a radioactive label, and combinations thereof. In some embodiments, the LFA comprises a porous matrix. In certain applications, the porous matrix is ​​sufficiently porous to allow the mixed phase solution, the first phase solution, the second phase solution, and / or the analyte of interest to flow through the LFA. In certain embodiments, provided herein is a porous matrix that is sufficiently long and / or deep for the mixed phase solution, the first phase solution, the second phase solution, and / or the analyte of interest to flow vertically and / or horizontally, and any combination thereof, through the LFA. In some embodiments, a method, device, kit, or system is provided, wherein the first phase solution flows through the porous matrix at a first velocity and the second phase solution flows through the porous matrix at a second velocity, the first velocity and the second velocity being different. In certain embodiments, the porous matrix comprises a material selected from cellulose, fiberglass, nitrocellulose, polyvinylidene fluoride, nylon, charge modified nylon, polyethersulfone, and combinations thereof. In some embodiments, an LFA is provided that includes a capture moiety for an analyte of interest that interacts with the analyte of interest. In certain embodiments, an LFA is provided that includes a competitive assay. In certain embodiments, an LFA is provided that includes an analyte of interest. In certain embodiments, a method, device, kit, and / or system is provided that utilizes an LFA that includes a sandwich assay. In certain embodiments, the LFA includes a probe capture moiety that interacts with a probe or a component thereof. In certain embodiments, a mixed phase solution is dehydrated on and / or in the LFA strip, and upon addition of a sample, the mixed phase solution is partitioned into a first phase solution and a second phase solution.

[0015] In certain embodiments of the methods, devices, kits, and / or systems described herein, an LFA is provided that includes a well having a volume sufficient to accommodate a solution. In certain applications, the solution is selected from the group consisting of at least a portion of the ATPS, at least a portion of the first phase solution, at least a portion of the second phase solution, a resuspension solution of the analyte of interest, and combinations thereof. In further or additional embodiments, an LFA is provided that includes a well located at a location of the LFA selected from a corner, an end, a center, a junction, an off-center site, and a bend. In some embodiments, the well includes one or more pads selected from a filter pad, a buffer pad, a surfactant pad, a salt pad, a probe pad, a polymer pad, and combinations thereof. In certain embodiments, an LFA is provided that includes a strip. In further or additional embodiments, a strip is provided that is configured according to a structure selected from the structures shown in Figures 16, 20, and 62-70. In further or additional embodiments, an LFA is provided where the strip includes multiple channels. In certain applications, the LFA further includes a dry receiving paper. In some embodiments, the device comprises a running buffer. In some embodiments, a device is provided that comprises a port for input of a sample (e.g., a biological sample) into the device. In certain applications, a port connected to an ATPS is provided. In some embodiments, an LFA is provided in which the ATPS and the LFA are integrated and the port is connected to the LFA. In certain embodiments, the port comprises a structure selected from a tube, a funnel, a valve, a syringe, a straw, a fluid path, a plunger, a piston, a gravity feed, a pump, and combinations thereof. In some embodiments, the device does not require a power source. In certain embodiments, the ATPS and the LFA are integrated prior to use of the device. In yet further or additional embodiments, the ATPS and the LFA are separated prior to use of the device.In some embodiments, the device is configured to insert an LFA into the ATPS.

[0016] In certain aspects, the methods, devices, systems, and / or kits described herein comprise or utilize a device comprising (a) a first component comprising a chamber for housing an ATPS, and (b) a second component comprising an LFA. In one embodiment of this aspect, a device is provided comprising an actuator for delivering the sample and / or analytes of interest, or both, into the ATPS. In certain embodiments, a device is provided further comprising an actuator for delivering a solution to the LFA. In certain applications, an LFA is provided comprising a solution selected from the group consisting of a mixed phase solution, a first phase solution, and a second phase solution, and combinations thereof. In certain embodiments, the ATPS and the LFA are contained within a single housing. In certain applications, a portable LFA device is provided.

[0017] In certain aspects, methods, devices, systems, and / or kits are provided for detecting and / or quantifying an analyte of interest in a sample, the detecting and / or quantifying comprising (a) applying the sample to a device described herein, and (b) detecting the presence or absence of the analyte of interest on the LFA. In certain embodiments, the method comprises applying the sample to an ATPS. In certain applications, the method comprises applying the sample to an LFA, where the LFA and the ATPS are integrated. In certain embodiments, the method comprises concentrating the analyte of interest in the ATPS. In one embodiment, the method comprises concentrating the analyte of interest in the LFA. In some embodiments, the method utilizes one or more samples selected from the group consisting of tissue / fluid from biological organisms (e.g., plants, animals, algae, fungi, etc.), food samples, chemical samples, drug samples, environmental samples (e.g., water samples, soil samples, etc.), and combinations thereof. In some embodiments, a sample is provided that is selected from the group consisting of a blood sample, a serum sample, a plasma sample, a urine sample, a saliva sample, a bronchoalveolar lavage fluid, a nasal sample, a fecal sample, a sample from a wound or a surgical site, and combinations thereof. In certain embodiments, the analyte of interest comprises a biological molecule. In certain embodiments, the biological molecule is selected from the group consisting of a nucleic acid, a protein, a lipid, a small molecule, a sugar, an antibody, an antigen, an enzyme, and combinations thereof. In certain embodiments, the sample is derived from a source selected from the group consisting of a vertebrate (e.g., a mammal, a bird, etc.), a bacterium, a virus, a protozoan, an algae, a fungus, a protozoan, a drug, a pathogen, a toxin, an environmental pollutant, and components thereof, and combinations thereof.

[0018] In certain aspects, methods, devices, kits, and / or systems are provided that utilize a paper-fluidic device for the detection of an analyte of interest in a sample, said paper-fluidic device being used in combination with an aqueous two-phase system (ATPS). In certain embodiments, the device comprises a porous matrix having an ATPS or a component thereof disposed therein, the porous matrix being configured and having sufficient porosity to allow the ATPS or a component thereof to flow through the porous matrix when the ATPS or a component thereof is in a fluid phase.

[0019] In certain embodiments, the paper fluidic device includes the ATPS or components thereof. In certain embodiments, the ATPS or components thereof are selected from the group consisting of a first phase solution, a second phase solution, and a mixed phase solution comprising a mixture of a first phase solution and a second phase solution, and / or combinations thereof. In certain embodiments, the ATPS and / or components thereof are dehydrated on and / or in at least a first portion of a porous matrix. In certain applications, paper fluidic devices, methods, systems, and / or kits are provided in which a first portion of a porous matrix has a width different from a width of a second portion of the porous matrix. In further or additional embodiments, paper fluidic devices, methods, systems, and / or kits are provided that utilize a sample to hydrate the ATPS, thereby providing the ATPS or components thereof in a fluid phase. In some embodiments, the paper fluidic device further comprises a well for containing a content selected from the group consisting of a mixed phase solution, a first phase solution, a second phase solution, a sample, and combinations thereof. In certain applications, the paper fluidic device comprises an actuator for releasing the content of the well into and / or onto the porous matrix. In certain embodiments, the well comprises one or more pads selected from a filter pad, a buffer pad, a surfactant pad, a salt pad, a probe pad, a polymer pad, and combinations thereof. In some embodiments, the first phase solution and the second phase solution flow through the porous matrix at different rates. In one embodiment, the first phase solution and the second phase solution flow through the porous matrix in different directions. In certain embodiments, the first phase solution comprises a micellar solution and the second phase solution comprises a polymer. In further certain embodiments, the first phase solution comprises a micellar solution and the second phase solution comprises a salt. In certain applications, a paper fluidic device is provided that comprises a Triton-X (or other surfactant) micellar solution. In some embodiments, the first phase solution comprises a polymer and the second phase solution comprises a polymer.In certain embodiments, the first phase solution comprises a polymer and the second phase solution comprises a salt. In certain embodiments, a paper fluid composition is provided in which the first phase solution comprises polyethylene glycol and the second phase solution comprises potassium phosphate.

[0020] In one aspect, devices, methods, systems and / or kits are provided that comprise a paper fluidic device in which a first phase solution is selected from component 1 in Table 1 and a second phase solution is selected from component 2 in Table 1. In certain applications, devices, methods, systems and / or kits are provided that are configured according to the structures shown in Figures 16, 20 and 62-70. In certain applications, a porous matrix is ​​provided that comprises a first pathway and a second pathway.

[0021] In another aspect, methods, devices, systems, and / or kits are provided that include or utilize a paper fluidic device in which a first phase solution flows preferentially through a first path and a second phase solution flows preferentially through a second path. In some embodiments, a paper fluidic device is provided in which the composition / device includes a probe that binds to an analyte of interest to generate a probe-analyte complex. In certain embodiments, a paper fluidic composition is provided in which, during use, an analyte of interest binds to the probe in a probe-analyte complex. In some embodiments, a probe is provided that includes magnetic particles. In certain embodiments, the device includes and / or the method utilizes a magnetic field oriented to attract magnetic particles to a portion of a porous matrix, the force of the magnetic field on the magnetic particles enhancing the flow of the probe-analyte complex toward the portion of the porous matrix. In certain embodiments, a probe is provided that includes a polymer selected from the group consisting of polyethylene, polypropylene, nylon, polyoxymethylene (Delrin®), polytetrafluoroethylene (Teflon®), dextran, and polyvinyl chloride, and combinations thereof. In some embodiments, the polyethylene is polyethylene glycol. In some embodiments, the polypropylene is polypropylene glycol. In one embodiment, a probe is provided that comprises a biological polymer selected from the group consisting of collagen, cellulose, and chitin. In certain applications, a paper fluidic device is provided, wherein the probe comprises a metal selected from the group consisting of gold, silver, titanium, stainless steel, aluminum, platinum, alloys thereof, and / or combinations thereof. In one embodiment, a probe is provided that comprises nanoparticles (e.g., gold nanoparticles). In certain applications, the probe comprises a coating. In certain embodiments, the coating comprises polyethylene glycol. In one embodiment, the coating comprises dextran. In one embodiment, the coating comprises polypropylene. In one embodiment, the coating comprises polypropylene glycol.In certain embodiments, the coating comprises a hydrophilic protein. In certain embodiments, the coating comprises serum albumin. In certain applications, a paper-fluid composition / device is provided in which the probe has a coating with an affinity for the first phase solution or the second phase solution.

[0022] In certain aspects of the methods, devices, systems, and / or kits described herein, a paper-fluidic composition / device is provided that includes a probe further comprising a binding moiety that binds to the analyte of interest. In some embodiments, a paper-fluidic device is provided that includes a binding moiety selected from the group consisting of an antibody, a lectin, a protein, a glycoprotein, a nucleic acid, a small molecule, a polymer, a lipid, and / or a combination thereof. In certain embodiments, a probe is provided that includes a binding moiety that includes an antibody or an antibody fragment. In certain embodiments, a paper-fluidic device is provided that includes a probe that includes a detectable label. In one embodiment, the detectable label is selected from the group consisting of a chromogenic label, a fluorescent label, an enzymatic label, a pyrogenic label, a radioactive label, and combinations thereof. In certain applications, a paper-fluidic composition is provided that further includes a dry receiving paper, where the first phase solution or the second phase solution preferentially flows through the porous matrix toward the dry receiving paper.

[0023] In certain embodiments, the methods, devices, systems, and / or kits described herein comprise a paper fluidic device / composition that comprises a running buffer during use, and the first phase solution and / or the second phase solution flows faster through the porous matrix when in contact with the running buffer. In certain applications of the subject matter described herein, a paper fluidic device is provided that comprises a porous matrix comprising a material selected from cellulose, fiberglass, nitrocellulose, polyvinylidene fluoride, nylon, charge modified nylon, polyethersulfone, and / or combinations thereof. In one embodiment, a paper fluidic device is provided that comprises a porous matrix comprising a capture moiety of an analyte of interest that interacts with the analyte of interest or a component thereof. In one embodiment, the paper fluidic device further comprises an analyte of interest. In one embodiment, the porous matrix comprises a probe capture moiety that interacts with a probe or a component thereof. In certain embodiments, a LFA is provided that comprises or facilitates a competitive assay. In certain embodiments, the LFA comprises a sandwich assay. In certain embodiments, a paper-fluid composition is provided in which the porous matrix is ​​configured to concentrate an analyte of interest as the analyte flows through the porous matrix, In certain applications, the paper-fluidic device includes a control analyte, and quantification of the analyte of interest is performed by comparison of the control analyte to the analyte of interest on the porous matrix.

[0024] In certain embodiments, a method for detecting an analyte of interest in a sample is provided, the method comprising (a) applying the sample to a device described and / or claimed herein, and (b) detecting the presence or absence of the analyte of interest.

[0025] The foregoing summary, as well as the following detailed description, can be better understood when read in conjunction with the accompanying drawings, which are intended to be illustrative and not restrictive, and it is understood that the present disclosure is not limited to the precise arrangements, examples and instrumentalities shown in the drawings. [Brief description of the drawings]

[0026] [Figure 1] (Top diagram) Schematic diagram of a typical lateral flow immunoassay test strip, showing two examples of mechanisms utilized by LFAs: sandwich assay (bottom left diagram) and competitive assay (bottom right diagram). [Diagram 2] Schematic diagram of analyte enrichment using capture probes with an ATPS. The probes capture the analytes of interest and partition them predominantly into one of the phases. The phase containing the enriched probes is then collected and can then be analyzed by an LFA. [Diagram 3] Schematic diagram for analyte enrichment using magnetic capture probes with an ATPS. The probes capture the analytes of interest and partition into one phase. A magnet is applied, which further concentrates the magnetic probes into a smaller volume. [Figure 4] FIG. 1 shows an exemplary, but non-limiting, design of a handheld device that utilizes a piston and plunger to transfer concentrated analytes from an ATPS to an LFA test strip. The device is composed of a concentration chamber and a detection chamber. In this example, the analytes are concentrated in the lower bulk phase of the ATPS. To collect a sample, (1) the plunger is depressed. The sample is mixed with the ATPS solution. Then (2) the tip of the device is inserted into the sample to draw in the solution, followed by (3) releasing the plunger and (4) sealing the PEN device with a cap. (5) After the analyte is concentrated in the lower bulk phase, the detection step is initiated by (6) rotating the plunger to the "test" position, which lowers the piston, and (7) introducing the concentrated analyte through the check valve into the detection chamber. (8) A catch fits into a groove, ensuring that the movement of the piston is controlled and only a controlled volume is introduced into the detection chamber. [Diagram 5] FIG. 1 shows an exemplary, but non-limiting, design of a portable device that mechanically inserts an LFA test strip into the upper phase containing concentrated analytes. To draw in the sample that has been premixed with the ATPS solution, (1) a button on the side of the device is pressed. After phase separation is complete and the analytes are concentrated in the upper bulk phase, (2) a detection button is pressed, which lowers the LFA test strip and initiates the detection step by contacting the sample pad of the strip with the concentrated analyte. Finally, (3) the results can be viewed through a result window. [Figure 6A] 6A-D show an exemplary, but non-limiting, design of a portable device incorporating a sealed well (A). An ATPS solution is mixed into the well. After phase separation is complete, flow of the well can be triggered by puncturing the seal at the bottom of the well, and the lower phase containing the concentrated analyte flows toward detection. This approach can be incorporated into devices that concentrate analytes in either the upper or lower phase. If the analyte is concentrated in the lower phase, the well is depressed into the device chamber, thereby triggering flow of the lower phase toward detection. Alternatively, the well can be loaded from below and a button can be attached to the well incorporated on the device. When the button is pressed, it brings the test strip into contact with the well. FIG. 6B and FIG. 6C show the results of 1:1 volume PEG:salt ATPS and 9:1 volume PEG:salt ATPS on a test strip. FIG. 6D illustrates exemplary mechanisms for puncturing a well and initiating flow of ATPS solution(s) through the LFA, i.e., by pressing a button in contact with the well (lower diagram) or by pressing the well itself (upper diagram). [Figure 6B] See Figure 6A. [Figure 6C] See Figure 6A. [Figure 6D] See Figure 6A. [Figure 7]FIG. 1 shows an exemplary, but non-limiting, design of a handheld device. This device is similar to the device shown in FIG. 4, but utilizes a magnetic probe. During the formation of the lower phase, a magnet close to the cap of the pen further concentrates the magnetic probe into a smaller volume. When the button on the pen is pressed, the magnetic particles are flushed through a check valve to initiate detection. [Figure 8] Schematic diagram showing the phenomenon of concentration at an interface. Due to the specific properties of the probe and the ATPS, the probe partitions to the interface in a highly concentrated state, which can then be collected and subsequently analyzed by the LFA. [Figure 9] FIG. 1 shows observation of condensation at the solid-liquid interface. The probe was found to be present in the polypropylene wall of the tube containing the PEG-salt ATPS. [Figure 10A] FIG. 10A-B: (A) Experimental collection of analyte-probe complexes using polypropylene sticks in a PEG-salt ATPS. (B) Schematic of enrichment at a solid-liquid interface. Probes bind to the analyte of interest in homogeneous solution. Due to the glass walls of the tube, the probes preferentially attach to a polypropylene collector inserted into the ATPS. The collector containing the enriched probes can be removed and the probes resuspended for subsequent detection by LFA. Note that these particles only attach to the solid propylene if the two-phase system of interest is present. [Figure 10B] See Figure 10A. [Figure 11] Schematic of a handheld device that utilizes solid-liquid interface enrichment: ATPS sample is added to the enrichment well and a collection stick is inserted to collect the probes. After the probes attach to the collection stick, they are transferred to the detection well where they are resuspended and flow towards the LFA is initiated for detection. [Figure 12A]FIG. 12A-C show: (a) The paper allows phase separation of the ATPS as it flows. In this example, a polymer-salt ATPS is used. The hydrophilic, less viscous phase, containing the concentrated analyte of interest, flows faster through the paper while the bulky components of the hydrophobic, more viscous phase are retarded. (b) The 1:1 polymer-salt ATPS phase separates in the paper and flows through it within 5 minutes. (c) The 9:1 PEG-salt ATPS also flows through the paper within 5 minutes, but the capture probe in the polymer-poor phase is not easily visible due to its smaller volume. [Figure 12B] See Figure 12A. [Figure 12C] See Figure 12A. [Figure 13A] Figures 13A-C show: (A) Paper wells allow for more efficient ATPS phase separation. The bulky components of the hydrophobic, more viscous phase are retained by the paper and remain in the top layer of the well, while the hydrophilic, less viscous phase containing the analytes of interest flows faster to the bottom layer. To improve analyte collection, the addition of a running buffer can be used to flash the sample out of the paper well and onto the membrane while maintaining phase integrity. Figure 13B shows that the 1:1 PEG-salt ATPS phase separates in the paper layer and flows through the membrane to be detected. Figure 13C shows that the 9:1 PEG-salt ATPS flows more slowly through the membrane due to the PEG-rich phase containing a high concentration of large PEG molecules. The concentrated salt phase then becomes the leading edge. [Figure 13B] See Figure 13A. [Figure 13C] See Figure 13A. [Figure 14A]Figures 14A-D illustrate: (A) A variation of the three-dimensional paper well where we use both a concentration well and a mixing well. To start the assay, a sample mixed with an ATPS solution is applied to the concentration well. (B) A buffer is then added to the concentration well to facilitate phase separation. (C) Once the concentrated analyte reaches the mixing well, (D) another buffer is added to the mixing well to facilitate flow, allowing the concentrated analyte to reach the downstream LFA test strip. [Figure 14B] Figures 14B to 14D are shown. [Figure 15A] 15A-B show (A) a mechanism by which dehydration of the ATPS-required components in a paper strip leads to phase separation after the components are rehydrated with a sample solution. In this experiment, the sample solution consists of a blue dye that prefers the polymer-rich phase and a red-colored nanoparticle indicator for the LFA that prefers the polymer-poor phase. FG refers to fiberglass paper. (B) Control condition without dehydrated ATPS components. [Figure 15B] See Figure 15A. [Figure 16A] Figures 16A-E illustrate various paper device configurations using dehydrated PEG-salt ATPS components. (a) Simple configuration with separate PEG and salt segments. (b) Simple configuration with combined PEG and salt segments. (c) Example incorporating multiple repeating segments to increase concentration efficiency. (d) Repeat of example c, where the PEG segment is not in the direct flow path of the device. (e) Example configuration with a phase separation site located at the end of the flow path. An LFA test strip, not shown in this schematic, is placed in the downstream region of the device. [Figure 16B] See Figure 16A. [Figure 16C] See Figure 16A. [Figure 16D] See Figure 16A. [Figure 16E] See Figure 16A. [Figure 17A]17A-B show: (A) a method to further concentrate the leading fluid using offset dehydrated PEG segments in a PEG-salt paper only diagnostic device by drawing the solution into the dehydrated PEG segments to form a PEG-rich phase that excludes the analyte of interest based on size exclusion, and (B) an experiment verifying the concentration effect. [Figure 17B] See Figure 17A. [Figure 18A] 18A-B show: (A) a schematic of the introduction of a running buffer to drive a lag phase containing concentrated analyte towards an LFA test strip, and (B) an exemplary alternative design for the introduction of a running buffer to drive a leading phase containing concentrated analyte towards an LFA test strip. [Figure 18B] See Figure 18A. [Figure 19] Schematic of the flow rerouting design using three-dimensional paper wells with different paper materials. The polymer-rich phase is more hydrophobic and flows preferentially into the more hydrophobic paper, while the polymer-poor phase flows preferentially through the more hydrophilic paper. An LFA test strip can be attached to the downstream region of this device for detection. [Figure 20] FIG. 14 illustrates another example of a paper device design that incorporates the use of a magnetic probe to enhance enrichment performance, where the position of the magnet exerts a force on the particles that are bound to the analyte of interest, effectively concentrating the particles to the side of the paper that contains the detection zone of the LFA. [Figure 21A] Figures 21A-C show that GMP was found to partition heavily into the lower PEG-poor phase (A), allowing the rapid recovery of most of the PGMs from the ATPS solution (C) using a small magnet (B). [Figure 21B] See Figure 21A. [Figure 21C] See Figure 21A. [Figure 22]13A-13D are images showing LFA strips used to detect Tf without (top) and with (bottom) a prior ATPS enrichment step. [Figure 23A] Figures 23A-D are images showing PGNPs and PP straws in solutions containing (A) PEG only, (B) salt only, or (C) 1:1 ATPS. PGNPs partitioned heavily into the PP straw only in the presence of ATPS. (D) The PP straw was pulled out from (C), and this figure shows that the concentrated PGNPs can be easily collected by pulling out the straw and then subjected to a detection assay. [Figure 23B] See Figure 23A. [Figure 23C] See Figure 23A. [Figure 23D] See Figure 23A. [Figure 24] 1 shows images of LFA strips used to detect transferrin (Tf) without (top) and with (bottom) a prior ATPS enrichment step. [Diagram 25] FIG. 1 is a schematic diagram showing the proposed portable device. [Figure 26] FIG. 1 is a schematic showing DGNPs and their role in the approach to improve the detection limit of LFAs using ATPS. [Figure 27] FIG. 1 is a schematic diagram showing positive and negative results for the LFA competition test. [Figure 28A] 28A-C show normalized visible absorption spectra (400-700 nm) of (a) citrate-capped or bare gold nanoparticles, (b) PEGylated gold nanoparticles, and (c) dextran-coated gold nanoparticles in various % (w / w) potassium phosphate solutions. [Figure 28B] See Figure 28A. [Figure 28C] See Figure 28A. [Figure 29A] 29A-C show the partitioning behavior of DGNPs in our PEG / salt ATPS at (A) 0 min, (B) 30 min, and (C) 12 h. [Figure 29B]See Figure 29A. [Figure 29C] See Figure 29A. [Diagram 30] 1 shows the experimentally measured concentration coefficients of Tf in PEG / salt ATPS at various time points within a 12 hour period. Error bars represent the standard deviation from triplicate measurements. [Figure 31A] 31A-B are images showing LFA strips used to detect Tf without (A) and with (B) a prior ATPS concentration step using PEG / salt ATPS. [Figure 31B] See Figure 31A. [Diagram 32] 1 shows the results of LFA quantification using MATLAB®. Error bars represent the standard deviation from measurements with triplicate LFA test strips. [Diagram 33] Figures 33A-D show that Brilliant Blue FCF dye and dextran-coated gold nanoparticles partition heavily into the upper PEG-rich and lower PEG-poor phases, respectively. (A) At 25°C, the mixed 1:1 volumetric ratio ATPS phases separated to form (B) two equal volume phases. (C) At 25°C, the mixed 9:1 volumetric ratio ATPS phases separated to form (D) a more PEG-rich phase and a less PEG-poor phase. Equal amounts of Brilliant Blue FCF and dextran-coated gold nanoparticles were added to both ATPS. The darker purple color of the 9:1 volumetric ratio ATPS indicates a significant increase in the concentration of gold nanoparticles in the lower PEG-poor phase. [Figure 34A] Figures 34A-B show that the paper membrane can induce phase separation of ATPS as it flows. The PEG-rich domains were retained near the beginning of the paper membrane, while the PEG-poor domains moved rapidly to the front. (a) The 1:1 volumetric ratio ATPS phases separated and flowed through the paper membrane within 5 min. (b) The 9:1 volumetric ratio ATPS phase also separated and flowed through the membrane within 5 min, but phase separation was less efficient, as indicated by the less distinct front at 300 s. [Figure 34B] See Figure 34A. [Figure 35A] Figures 35A-B show that 3D paper wells can further enhance ATPS phase separation. After addition of the mixed ATPS with dye, the PEG-rich domains were retained in the top layer of the wells, while the PEG-poor domains containing concentrated gold nanoparticles quickly flowed to the bottom layer. (a) ATPS with a 1:1 volume ratio showed enhanced phase separation in the 3D paper wells, with the PEG-poor phase flowing through the membrane within 5 minutes. (b) ATPS with a 9:1 volume ratio also improved phase separation, with the PEG-poor phase clearly visible. In contrast to Figure 34, the leading edge was clear and dark purple, indicating the enrichment of dextran-coated gold nanoparticles. [Figure 35B] See Figure 35A. [Figure 36A] Figures 36A-C show that three-dimensional paper wells were combined with PEG / salt ATPS and LFA for Tf detection. (a) The paper well device was combined with a Tf competitive assay on nitrocellulose paper. Samples that did not contain Tf were correctly diagnosed by visual test and control lines when using (b) 1:1 or (c) 9:1 volume ratio ATPS solutions. [Figure 36B] See Figure 36A. [Figure 36C] See Figure 36A. [Figure 37A] Figures 37A-B show that using a 1:1 volume ratio of PEG / salt ATPS in a 3D paper well can improve the detection limit of Tf by 2-fold, while a 9:1 volume ratio of ATPS can improve it by 10-fold. (a) Conventional LFA detects Tf at 1 ng / μL, but cannot detect Tf at 0.5 ng / μL, leading to false negatives. Using a 1:1 volume ratio of ATPS in a 3D paper well successfully detects Tf at 0.5 ng / μL. (b) Conventional LFA detects Tf at 1 ng / μL, but cannot detect Tf at 0.1 ng / μL, leading to false negatives. Using a 9:1 volume ratio of ATPS in a 3D paper well successfully detects Tf at 0.1 ng / μL. [Figure 37B] See Figure 37A. [Figure 38]1 illustrates an exemplary ATPS-LFA system. [Figure 39] FIG. 1 is a schematic diagram showing the LFA sandwich assay. A positive result (top panel) is indicated by two bands at the test and control lines, while a negative result (bottom panel) is indicated by a single band at the control line. [Diagram 40] Figures 40A-C show the aqueous two-phase PEG-salt system after (A) 10 min, (B) 30 min, and (C) 24 h incubation in a 37°C water bath. White paper was filled with the letter "X" to help visualize the turbidity of each phase. Arrows indicate the location of the macroscopic interface. [Diagram 41] Experimentally determined concentration coefficients of M13 in PEG / salt ATPS at various time points are shown, with error bars representing standard deviation from triplicate measurements. [Diagram 42] Figure 1 shows the experimentally determined concentration coefficients of M13 in PEG / salt ATPS at various volume ratios. Symbols correspond to the measured data and error bars represent the standard deviation from triplicate measurements. The solid line corresponds to the prediction from equation (6). [Diagram 43] Figures 43A-F show the LFA for detecting M13 without a prior concentration step with PEG / salt ATPS. Figure (A) shows the negative control where no M13 was added. The remaining solutions contained M13 at concentrations of (B) 1x1010, (C) 3.2x109, (D) 1x109, (E) 3.2x108, and (F) 1x108 pfu / mL. [Diagram 44] Figures 44A-F show LFAs for detecting M13 with a prior concentration step. Figure (A) shows a negative control where no M13 was added. The remaining solutions initially contained M13 at concentrations of (B) 1x109, (C) 3.2x108, (D) 1x108, (E) 3.2x107, and (F) 1x107 pfu / mL. [Diagram 45] With a prior concentration step ( [ka] ) and without ( [ka] ), the signal intensity relative to M13 is shown. Error bars represent the standard deviation of at least three measurements. [Figure 46] A schematic diagram of the ATPS-LFA system in PBS is shown. [Figure 47] FIG. 1 is a schematic diagram showing competition-based LFA and interpretation of positive and negative results. [Figure 48A] Figure 48A-D shows the surface modification of GNPs to affect their partitioning behavior in the ATPS. (A) Schematic of GNPs and the function of each component. To demonstrate that the partitioning behavior of GNPs in our PEG-salt ATPS can be customized, various amounts of PEG were conjugated to the GNPs to manipulate their partitioning behavior. (B) When a 5000:1 molar ratio of PEG:GNPs was used during conjugation, the resulting GNPs partitioned preferentially into the PEG-rich upper phase. (C) When a 1000:1 molar ratio of PEG:GNPs was used during conjugation, the GNPs partitioned into the PEG-poor lower phase, but aggregated due to the high salt concentration of that phase. These aggregated GNPs could not be used in the subsequent detection assay. (D) When a 3000:1 molar ratio of PEG:GNPs was used during conjugation, the resulting GNPs partitioned heavily to the interface. For (B), (C), and (D), the red color observed at the top of the liquid-air interface was due to reflection and not due to the presence of the nanoprobe. [Figure 48B] See Figure 48A. [Figure 48C] See Figure 48A. [Figure 48D] See Figure 48A. [Figure 49] The results of the LFA for detection of Tf in PBS are shown without (top panel) and with (bottom panel) a prior concentration step using a PEG / salt ATPS interface extraction step. [Figure 50] The results of the LFA for detection of Tf in FBS are shown without (top panel) and with (bottom panel) a prior enrichment step using a PEG / salt ATPS interface extraction step. [Figure 51]1 shows the results of an LFA for the detection of Tf in synthetic urine without (top panel) and with (bottom panel) a prior enrichment step using a PEG / salt ATPS interfacial extraction step. [Figure 52] FIG. 1 shows simultaneous concentration and detection of an analyte of interest using a micellar ATPS. [Figure 53A] Figure 53A-B shows a comparison of the time required to reach phase separation for a 1:9 volume ratio Triton X-114 system in PBS. Brilliant blue FCF dye and cherry BSA-GN were color indicators for the lower micelle-rich and upper micelle-poor phases, respectively. (a) At 25°C, Triton X-114 ATPS reached macroscopic phase separation equilibrium in 8 hours in a test tube. (b) When a three-dimensional paper core was applied to the mixed ATPS, phase separation was observed within the core already at 30 seconds, as the micelle-rich domains were retained near the bottom of the core and the micelle-poor domains were able to flow more freely upwards through the core. Once outside the core, the distinct phases remained separated from each other, while the micelle-poor phase remained concentrated in a small volume at the front, showing a more complete separation at 180 seconds (3 minutes). [Figure 53B] See Figure 53A. [Figure 54A] Figures 54A-B illustrate the integration of a paper core and Triton X-114 micellar ATPS with an LFA. (a) The integrated diagnostic strip consists of a concentration zone where phase separation occurs, followed by a detection zone containing immobilized test and control line components. (b) A true negative test was confirmed within 20 minutes when analyzing a solution containing no pLDH. [Figure 54B] See Figure 54A. [Figure 55] We show that the paper-based micellar ATPS with a 1:9 volume ratio achieved a 10-fold improvement in the detection limit of pLDH in PBS at 25 °C. Standard LFA detected pLDH at 10 ng μL-1, but could not accurately detect pLDH at 1 ng μL-1. The integrated diagnostic strip successfully detected pLDH at 1 ng μL-1. [Figure 56] We show that the paper-based micellar ATPS with a 9:1 volume ratio achieved a 10-fold improvement in the detection limit of pLDH in undiluted FBS at 25 °C. Standard LFA detected pLDH at 10 ng μL-1, but could not accurately detect pLDH at 1 ng μL-1. The integrated diagnostic strip successfully detected pLDH at 1 ng μL-1. [Figure 57A] Figures 57A-D show phase separation results using (A) PEG 4600 and potassium phosphate on fiberglass, (B) PPG 425 and potassium phosphate on fiberglass, (C) PEG 8000 and dextran 9000 on cellulose, and (D) PEG 4600 and dextran 35000 on fiberglass. [Figure 57B] See Figure 57A. [Figure 57C] See Figure 57A. [Fig. 57D] See Figure 57A. [Figure 58A] 58A-B show phase separation on different paper types with (A) a salt phase as the preceding fluid or (B) a PEG phase as the preceding fluid. [Figure 58B] See Figure 58A. [Figure 59A] Figures 59A-D show various designs for dehydration of the components of a polymeric ATPS, including placing a hydrophobic polymer (e.g., PEG) phase in (A) a narrow LFA region, (B) the center of a wide LFA region, (C) a region of LFA extending from the narrow LFA region to the wide LFA region, and (D) primarily a wide LFA region. [Figure 59B] See Figure 59A. [Figure 59C] See Figure 59A. [Fig. 59D] See Figure 59A. [Figure 60] Figure 1 shows time-lapse images of phase separation on a dehydrated polymer ATPS-LFA system. [Figure 61] Figure 1 shows time-lapse images of phase separation on a dehydrated polymer ATPS-LFA system. [Figure 62]A design is shown in which the dehydrated nanoprobe paper segment is wider than the width of the sensing membrane (width is defined as the dimension perpendicular to the direction of flow but in the plane of the flow). [Figure 63A] 63A-B show successful phase separation in a wide design, (A) without ATPS or (B) with ATPS. [Figure 63B] See Figure 63A. [Figure 64] 13 is an image of an experiment where continuous flow was not possible due to the viscosity of the PEG-rich phase of the polymer ATPS, which significantly slowed flow within the sensing membrane. [Figure 65] We show that the PEG-rich phase, which significantly slows down flow within the sensing membrane, can be countered by leaving a blank spacer that does not contain PEG. [Figure 66] 1 shows a dehydrated LFA design with a constant gradient of fluid from the source to the LFA membrane to reduce the amount of nanoprobes left behind. [Figure 67] 1 illustrates a dehydrated ATPS-LFA system using PEG / salt ATPS. [Figure 68] 1 illustrates the design of a three-dimensional paper well having a paper pad containing components of a dehydrated polymeric ATPS. [Figure 69A] Figures 69A-B show separations on three-dimensional paper wells with paper pads containing components of dehydrated polymeric ATPS located at (A) the beginning of the LFA and (B) a location not at the beginning of the LFA. [Figure 69B] See Figure 69A. [Figure 70] An exemplary two-well configuration design is shown in which there are two separate three-dimensional wells separated by a length of horizontal paper. The first well contains the dehydrated components of the ATPS. The second well allows more time and space for phase separation. [Figure 71] This shows that MF1 paper is compatible with blood samples in PEG-salt solutions. [Figure 72]1 shows that filtration of blood samples on MF1 paper can be slow. [Figure 73A] 73A, B show the results of phase separation of blood samples and PEG-salt solutions using (A) a 3-layer 3D-well or (B) a 5-layer 3D-well on filter paper. [Figure 73B] See Figure 73A. [Figure 74] 1 shows the results of phase separation of blood samples and PEG-salt solutions using three-dimensional wells on paper. [Figure 75] 1 shows the results of phase separation of blood samples and PEG-salt solutions using three-dimensional wells with layers of paper for filtration and concentration. [Figure 76] Detection of C. trachomatis in viral transport medium (VTM) using a sandwich format LFA. [Figure 77] Detection of S. mutans using sandwich format LFA and micellar ATPS. [Figure 78] Detection of S. mutans using sandwich format LFA and PEG / salt ATPS. [Figure 79] 1 shows detection of troponin using competitive format LFA and PEG / salt ATPS. [Figure 80A] Figure 80A-C shows the schematic (A) and results of the addition of mixed ATPS onto a paper membrane, where both phases of the ATPS were able to completely separate in the glass well and then flowed through the paper. The PEG-poor phase (open arrow) containing purple dextran-coated gold nanoparticles was observed to flow quickly through the paper, while the PEG-rich phase (solid arrow) containing blue dye was retained at the origin of the paper membrane. Enhanced phase separation within the paper membrane was evident when using (B) a 1:1 volume ratio of ATPS or (C) a 9:1 volume ratio of ATPS. [Figure 80B] See Figure 80A. [Fig. 80C] See Figure 80A. [Figure 81]Detection of pLDH in 0.1 ng / mL FBS samples using a 9:1 volume ratio of Triton-X-PBS ATPS and three-dimensional LFA. [Figure 82] A schematic diagram of probe-analyte complexes added to an LFA in a mixed ATPS solution for simultaneous concentration and detection, or in one phase of the ATPS after phase separation and concentration of the probe-analyte complexes in one phase is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Provided herein are methods, devices, kits, and systems that can be performed at the point of care (POC) or site of use and provide useful tools for medical, consumer, and other applications. The tests provided herein (assay devices and / or systems) are quick, simple, and easy to use and can be easily performed by individuals with little, if any, training in clinical diagnostics. The tests are highly sensitive and reliable. In certain embodiments, the devices and methods utilize the concentration capabilities of aqueous multiphase systems in combination with LFAs to provide unexpected and surprising sensitivity, e.g., at least about 10-fold to about 100-fold improvement over the detection limits of previous LFA assays, which approaches or matches, and in some cases exceeds, the sensitivity of laboratory assays such as enzyme-linked immunosorbent assays (ELISAs).

[0028] I. Device Disclosed herein is a device for detection and / or quantification of an analyte of interest in a sample that utilizes (e.g., is configured to provide) a lateral flow assay (LFA) and an aqueous two-phase system (ATPS), the ATPS including a mixed phase solution that separates into a first phase solution and a second phase solution, and the LFA includes a test line and a control line for detecting the analyte of interest. In some embodiments, the separation of the mixed phase into a first phase solution and a second phase solution occurs within an LDA. In some embodiments, the separation of the mixed phase into a first phase solution and a second phase solution does not occur within an LDA. In some embodiments, the first phase solution flows through the LFA faster than the second phase solution and is referred to as a leading fluid, while the second phase solution is referred to as a lag fluid. In some embodiments, the second phase solution flows through the LFA faster than the first phase solution and is referred to as a leading fluid, while the first phase solution is referred to as a lag fluid. In some embodiments, the analyte of interest is distributed / concentrated in the leading fluid. In some embodiments, the analyte of interest partitions / concentrates in the lag fluid.

[0029] Configuration and Operation In some embodiments, the device comprises a port for introduction of an ATPS and / or a sample into the device. In some embodiments, the device comprises a first component comprising an ATPS and a second component comprising an LFA. In some embodiments, the first component and the second component are provided as separate components. In some embodiments, the first component and the second component are mated together by a user. In some embodiments, the first component has a first port connected to the ATPS. In some embodiments, the first component comprises a chamber for the ATPS. In some embodiments, the first port is connected to the chamber. In some embodiments, the first port is configured for introduction of a sample and / or an analyte of interest into the first component. In some embodiments, the first port is configured for introduction of an ATPS or a component thereof into the first component. In some embodiments, the second port is configured for introduction of a sample and / or an analyte of interest into the second component. In some embodiments, the second port is configured for introduction of an ATPS or a component thereof into the second component.

[0030] In some embodiments, the device comprises multiple chambers. In some embodiments, the first component comprises a first chamber that holds the sample and / or ATPS solution and allows phase separation to occur. In some embodiments, the second component comprises a second chamber (e.g., a detection chamber) that houses the LFA and facilitates application of the analyte of interest to the LFA after phase separation has occurred. In some embodiments, additional chambers may be added to facilitate flow, such as by using a pressure differential or creating a reduced pressure. In some embodiments, the device collects the analyte of interest from the ATPS and transfers it to the LFA using a mechanical component.

[0031] In some embodiments, the ATPS and LFA are integrated (ATPS-LFA), i.e., components of the ATPS are connected to components of the LFA, either directly or through connectors. In certain embodiments, the ATPS and LFA are provided as separate components, e.g., assembled together by the user, or the ATPS and LFA are provided as a single integrated unit. In some embodiments, the device includes a connection between the ATPS and the LFA (e.g., an ATPS-LFA connector). Illustrative, but non-limiting examples of ATPS-LFA connectors include tubes, ports, valves, funnels, gates, pumps, holes, channels, filters, combinations thereof, and the like. In some embodiments, the integrated device includes components of the ATPS dehydrated on an LFA. In some embodiments, the device includes a single port connected to the ATPS-LFA. In some embodiments, the single port is configured for introduction of a sample and / or an analyte of interest into the ATPS-LFA. In some embodiments, the single port is configured for introduction of the ATPS or components thereof into the ATPS-LFA. In some embodiments, the port comprises a structure selected from, but not limited to, a tube, a funnel, a valve, a syringe, a straw, a fluid passage, a plunger, a piston, a pump, combinations thereof, and the like.

[0032] In some embodiments, the device comprises an LFA strip. In some embodiments, the device is configured to insert said LFA strip into an ATPS. In some embodiments, the ATPS and the LFA are contained within a single housing.

[0033] In some embodiments, the device comprises an actuator that delivers the sample and / or analytes of interest into the ATPS, hi some embodiments, the device further comprises an actuator that delivers solutions to the LFA.

[0034] Portability In some embodiments, the device is a portable device. In some embodiments, the device weighs less than about 100 ounces, less than about 90 ounces, less than about 80 ounces, less than about 70 ounces, less than about 60 ounces, less than about 50 ounces, less than about 40 ounces, less than about 32 ounces, less than about 24 ounces, less than about 16 ounces, less than about 8 ounces, less than about 4 ounces, less than about 2 ounces, less than about 1 ounce. In some embodiments, multiple devices are packed in a portable container. In some embodiments, the maximum length of the device is about 1 inch, about 2 inches, about 3 inches, about 4 inches, about 6 inches, about 7 inches to about 8 inches, about 9 inches, about 10 inches, about 12 inches, about 14 inches, about 16 inches, about 18 inches, about 19 inches, about 20 inches, about 24 inches, about 26 inches, about 28 inches, or about 30 inches. In some embodiments, the device folds to a size smaller than its unfolded size so that it can be conveniently transported and / or stored.

[0035] FIG. 4 illustrates a handheld device that can combine an ATPS with an LFA using a piston and plunger system. In one illustrative embodiment, the enrichment chamber and the detection chamber are connected by a check valve or similar mechanism that ensures that the LFA strip does not come into premature contact with the unenriched sample. The piston and plunger system can be used to draw the sample into the enrichment chamber. Enrichment can be performed prior to sampling with the device by premixing the sample with the ATPS solution prior to extraction of the analyte, or enrichment can be performed within the device by preloading the ATPS solution inside the enrichment chamber, either as a liquid or in dehydrated form. After phase separation has occurred, the user can use the piston / plunger to redirect a predefined volume of enriched analyte from the enrichment chamber through the check valve and into the detection chamber to begin the detection process. The check valve can be repositioned to transfer the desired bulk phase (top or bottom phase) in which the analyte resides to the LFA strip.

[0036] FIG. 5 illustrates a device design demonstrating collection of the upper bulk phase for application to an LFA strip. In the illustrated embodiment, a sample premixed with an ATPS solution is drawn into the device. After phase separation, the user can press a button to initiate the detection step by contacting the LFA test strip(s) with the concentrated analyte in the upper phase of the ATPS. In various embodiments, the components of the ATPS solution can be preloaded into the device, either as liquids or in dehydrated form.

[0037] Figure 6 presents an illustrative, but non-limiting example of a portable cassette-type device. Sample and ATPS solution can be first loaded into the sample well on the cassette device. The well may be designed as a removable component that can be attached to the cassette after loading with the sample. After phase separation has occurred, the detection step can be initiated by mechanical movement of either the LFA test strip or the sample well.

[0038] Figure 7 illustrates one configuration in which a magnetic capture probe can be applied to the integration of an ATPS and an LFA in a portable device. The device shown can utilize a plunger and piston system to transfer concentrated analytes captured by an external magnet to a detection chamber, similar to the device shown in Figure 4. In some embodiments, the device does not require a power source (e.g., electrical power).

[0039] Detection Time In some embodiments, the device has a detection time. In some embodiments, the detection time comprises a phase separation time. In some embodiments, the detection time comprises a flow time.

[0040] In some embodiments, the detection time (e.g., the time between application of the sample to the device and detection of the analyte of interest in the control / test) is less than about 2 hours, less than about 1.5 hours, less than about 1 hour, or less than about 0.5 hours. In some embodiments, the detection time is less than about 30 minutes, less than about 25 minutes, less than about 20 minutes, less than about 15 minutes, less than about 10 minutes, or less than about 5 minutes. In some embodiments, the detection time is less than about 5 minutes, or less than about 4 minutes, less than about 3 minutes, less than about 2 minutes, or less than about 1 minute. In some embodiments, the detection time is less than about 30 seconds, less than about 20 seconds, or less than about 10 seconds.

[0041] In some embodiments, the phase separation time (e.g., the time it takes for the first / second phase solution to separate from the mixed phase solution of the ATPS) is less than about 2 hours, less than about 1.5 hours, less than about 1 hour, or less than about 0.5 hours. In some embodiments, the detection time is less than about 30 minutes, or less than about 25 minutes, less than about 20 minutes, less than about 15 minutes, less than about 10 minutes, or less than about 5 minutes. In some embodiments, the phase separation time is less than about 5 minutes, less than about 4 minutes, less than about 3 minutes, less than about 2 minutes, or less than about 1 minute. In some embodiments, the phase separation time is less than about 30 seconds, less than about 20 seconds, or less than about 10 seconds.

[0042] In some embodiments, the flow time (e.g., the time it takes for a solution containing the analyte of interest to travel from the sample pad of the LFA to the test and control lines) is less than about 2 hours, less than about 1.5 hours, less than about 1 hour, or less than about 0.5 hours. In some embodiments, the flow time is less than about 30 minutes, or less than about 25 minutes, or less than about 20 minutes, or less than about 15 minutes, or less than about 10 minutes, or less than about 5 minutes. In some embodiments, the detection time is less than about 5 minutes, or less than about 4 minutes, or less than about 3 minutes, or less than about 2 minutes, or less than about 1 minute. In some embodiments, the flow time is less than about 30 seconds, or less than about 20 seconds, or less than about 10 seconds.

[0043] Detection limit In some embodiments, the device has a detection limit, which is the minimum amount of analyte of interest that must be present in a sample in order to be detected.

[0044] In some embodiments, the minimum amount of the analyte of interest in the sample is about 0.01 ng / ml, about 0.05 ng / ml, about 0.1 ng / ml, about 0.15 ng / ml, about 0.20 ng / ml, about 0.25 ng / ml, about 0.3 ng / ml, about 0.35 ng / ml, about 0.40 ng / ml, about 0.45 ng / ml, about 0.5 ng / ml, about 0.55 ng / ml, about 0.60 ng / ml, about 0.65 ng / ml, about 0.7 ng / ml, about 0.75 ng / ml, about 0.80 ng / ml, about 0.85 ng / ml, about 0.9 ng / ml, about 0.95 ng / ml, or about 1 ng / ml. In some embodiments, the minimum amount of the analyte of interest in the sample is about 1 ng / ml, about 10 ng / ml, about 20 ng / ml, about 30 ng / ml, about 40 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml, about 100 ng / ml, about 110 ng / ml, about 120 ng / ml, about 130 ng / ml, about 140 ng / ml, about 150 ng / ml, about 160 ng / ml, about 170 ng / ml, about 180 ng / ml, about 190 ng / ml, about 200 ng / ml, about 210 ng / ml, about 220 ng / ml, about 230 ng / ml, about 240 ng / ml, about 250 ng / ml, about 260 ng / ml, about 270 ng / ml, about 280 ng / ml, about 290 ng / ml, about 300 ng / ml, about 310 ng / ml, about 320 ng / ml, about 330 ng / ml, about 340 ng / ml, about 350 ng / ml, about 360 ng / ml, about 370 ng / ml, about 380 ng / ml, about 390 ng / ml, about 400 ng / ml, about 410 ng / ml, about 420 ng / ml, about 430 ng / ml, about 440 ng / ml, about 450 ng / ml, about 460 ng / ml, about 470 ng / ml, about 480 ng / ml, about 490 ng / ml, about 5 , about 180ng / ml, about 190ng / ml, about 200ng / ml, about 250ng / ml, about 300ng / ml, about 350ng / ml, about 400ng / ml, about 450ng / ml, about 500ng / ml, about 550ng / ml, about 600ng / ml, about 650ng / ml, about 700ng / ml, about 750ng / ml, about 800ng / ml, about 850ng / ml, about 900ng / ml, about 980ng / ml, or about 1000ng / ml.

[0045] In some embodiments, the minimum amount of the analyte of interest is about 1 μg / ml, about 2 μg / ml, about 3 μg / ml, about 4 μg / ml, about 5 μg / ml, about 6 μg / ml, about 7 μg / ml, about 8 μg / ml, about 9 μg / ml, about 10 μg / ml, about 11 μg / ml, about 12 μg / ml, about 13 μg / ml, about 14 μg / ml, about 15 μg / ml, about 16 μg / ml, about 17 μg / ml, about 18 μg / ml, about 19 μg / ml / ml or about 20 μg / ml. In some embodiments, the minimum amount of analyte of interest is about 10 μg / ml, about 20 μg / ml, about 30 μg / ml, about 40 μg / ml, about 50 μg / ml, about 60 μg / ml, about 70 μg / ml, about 80 μg / ml, about 90 μg / ml, or about 100 μg / ml. In some embodiments, the minimum amount of analyte of interest is about 10 μg / ml.

[0046] In some embodiments, the minimum amount of analyte of interest is about 1 part per trillion, about 10 parts per trillion, or about 100 parts per trillion. In some embodiments, the minimum amount of analyte of interest is about 1 part per billion, about 10 parts per billion, about 100 parts per billion, about 1000 parts per billion, about 10,000 parts per billion, or about 100,000 parts per billion. In some embodiments, the minimum amount of analyte of interest is about 1 part per thousand, about 10 parts per thousand, or about 100 parts per thousand.

[0047] In some embodiments, the analyte of interest is present on or derived from a cell. In some embodiments, the sample has a minimum concentration of cells for the analyte of interest present on or derived from a cell to be detected. In some embodiments, the minimum concentration of cells is about 1 cell per ml of sample, about 10 cells per ml of sample, about 20 cells per ml of sample, about 30 cells per ml of sample, about 40 cells per ml of sample, about 50 cells per ml of sample, about 60 cells per ml of sample, about 70 cells per ml of sample, about 80 cells per ml of sample, about 90 cells per ml of sample, or about 100 cells per ml of sample.

[0048] In some embodiments, the minimum concentration of cells is about 1×10 0 cells / mL, approximately 1×10 1 cells / mL, approximately 1×10 2 cells / mL, approximately 1×10 3 cells / mL, approximately 1×10 4 cells / mL, approximately 1×10 5 cells / mL, approximately 1×10 6 cells / mL, approximately 1×10 7 cells / mL, approximately 1×10 8 cells / mL or approximately 1 x 10 9 In some embodiments, the minimum concentration of cells is about 1×10 6 In cells / mL.

[0049] In some embodiments, the analyte of interest is a viral particle and is present on or derived from a virus. In some embodiments, the sample has a minimum concentration of viral particles for the analyte of interest present on or derived from a virus to be detected. In some embodiments, the minimum concentration of viral particles in a sample is about 1×10 0 pfu / mL, approximately 1×10 1 pfu / mL, approximately 1×10 2 pfu / mL, approximately 1×10 3 pfu / mL, approximately 1×10 4 pfu / mL, approximately 1×105 pfu / mL, approximately 1×10 6 pfu / mL, approximately 1×10 7 pfu / mL, approximately 1×10 8 pfu / mL, approximately 1×10 9 pfu / mL, approximately 1×10 10 pfu / mL, approximately 1×10 11 pfu / mL, or approximately 1 x 10 12 pfu / mL.

[0050] In some embodiments, the minimum concentration of viral particles in the sample is about 1×10 6 pfu / mL, 2 x 10 6 pfu / mL, 3 × 10 6 pfu / mL, 4 × 10 6 pfu / mL, 5 × 10 6 pfu / mL, 6 × 10 6 pfu / mL, 7 × 10 6 pfu / mL, 8 × 10 6 pfu / mL, 9 × 10 6 pfu / mL, approximately 1×10 7 pfu / mL, approximately 2×10 7 pfu / mL, approximately 3×10 7 pfu / mL, approximately 4×10 7 pfu / mL, approximately 5×10 7 pfu / mL, approximately 6×10 7 pfu / mL, approximately 7×10 7 pfu / mL, approximately 8×10 7 pfu / mL, approximately 9×10 7 pfu / mL, approximately 1×10 8 pfu / mL, approximately 2×10 8 pfu / mL, approximately 3×10 8 pfu / mL, approximately 4×10 8 pfu / mL, approximately 5×10 8 pfu / mL, approximately 6×10 8 pfu / mL, approximately 7×10 8 pfu / mL, approximately 8×10 8 pfu / mL, approximately 9×10 8 pfu / mL, approximately 1×10 9 pfu / mL, approximately 2×10 9 pfu / mL, approximately 3×109 pfu / mL, approximately 4×10 9 pfu / mL, approximately 5×10 9 pfu / mL, approximately 6×10 9 pfu / mL, approximately 7×10 9 pfu / mL, approximately 8×10 9 pfu / mL, approximately 9×10 9 pfu / mL, approximately 1×10 10 pfu / mL, approximately 2×10 10 pfu / mL, approximately 3×10 10 pfu / mL, approximately 4×10 10 pfu / mL, approximately 5×10 10 pfu / mL, approximately 6×10 10 pfu / mL, approximately 7×10 10 pfu / mL, approximately 8×10 10 pfu / mL, approximately 9×10 10 pfu / mL, or approximately 1 x 10 11 pfu / mL. In some embodiments, the minimum concentration of viral particles in a sample is about 1×10 7 pfu / mL. In some embodiments, the minimum concentration of viral particles in a sample is about 1×10 8 pfu / mL. In some embodiments, the minimum concentration of viral particles in a sample is about 1×10 9 pfu / mL.

[0051] temperature In some embodiments, the device operates regardless of the temperature surrounding the device, provided that the temperature is, of course, below the ignition / melting / flash point of the material of the device, below the temperature at which the components of the device volatilize, sublime, or break down, and above the freezing point of the components of the device during the operation of the assay. In some embodiments, the device operates at room temperature. In some embodiments, the device operates at a temperature of about -50 degrees Celsius to about 60 degrees Celsius. In some embodiments, the device operates at a temperature of about -10 degrees Celsius to about 45 degrees Celsius. In some embodiments, the device operates at a temperature of about 10 degrees Celsius to about 30 degrees Celsius.

[0052] Aqueous two-phase system (ATPS) In certain embodiments, the device is configured to support an aqueous two-phase system (ATPS) method, and assays using such devices are provided herein. In some embodiments, the ATPS includes a phase solution. The term "phase solution" generally refers to a first phase solution or a second phase solution of the ATPS. In some embodiments, the phase solution is in a mixed solution (e.g., with a first / second phase solution). In some embodiments, the phase solution is a first / second phase solution after being dispensed from a mixed solution of the ATPS. In some embodiments, the phase solution is a first / second phase solution after being dispensed from a mixed solution in the LFA. The phase solution can refer to the second phase solution while it is in a mixed state (e.g., with a first phase solution). In some embodiments, the phase solution is a leading fluid in the LFA. In some embodiments, the phase solution is a lagging fluid in the LFA.

[0053] In some embodiments, the ATPS comprises two aqueous solutions, a first phase solution and a second phase solution (e.g., a mixed phase solution), that are initially mixed. In some embodiments, the mixed phase solution is a homogenous solution. In some embodiments, the first phase solution and the second phase solution are immiscible. In some embodiments, the immiscibility is driven by a change in temperature and / or a change in the concentration of different components, such as salts. In some embodiments, the first / second phase solutions comprise components, such as micelles, salts, and / or polymers. In some embodiments, the analytes of interest that contact the ATPS are preferentially distributed, partitioned, and / or concentrated in the first phase solution relative to the second phase solution, or vice versa, based on their physical and chemical properties, such as size, shape, hydrophobicity, and charge. In some embodiments, the analytes of interest (e.g., components of mammalian cells, bacteria, or viruses) partition predominantly (or excessively) into the first phase solution or the second phase solution of the ATPS and are therefore concentrated in the ATSP. In some embodiments, the analyte of interest is concentrated by adjusting the volume ratio between the first and second phase solutions. In some embodiments, the analyte of interest is concentrated by reducing the volume of the phase into which the analyte partitions. Illustratively, in some embodiments, by using a volume ratio of first phase solution to second phase solution of, for example, 1:9, the volume of the phase into which the analyte partitions is 1 / 10 of the total volume, so that the analyte of interest is concentrated 10-fold in the first phase solution.

[0054] In some embodiments, other concentrations are obtained by using other ratios. Thus, in some embodiments, the ratio of the first phase solution to the second phase solution is selected from the following ratios: 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 solution to the second phase solution is selected from the following ratios: about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, about 1:100. In some embodiments, the ratio of the first phase solution to the second phase solution is selected from the following ratios: 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.

[0055] In some embodiments, the ratio of the second phase solution to the first phase solution is selected from the ratios 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 solution to the first phase solution is selected from the ratios 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 solution to the first phase solution is selected from the ratios 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.

[0056] In some embodiments, the analyte is partitioned substantially equally between the first and second phase solutions, preventing concentration of the analyte. In such systems, concentration of the analyte of interest is achieved by introducing an additional component, such as a probe that captures the analyte of interest and partitions predominantly into one of the phases, thereby improving the partitioning behavior of the analyte of interest and allowing concentration. In some embodiments, the first / second phase solution containing the concentrated analyte is collected and applied to the LFA. The use of such additional components is not limited to substantially equal partitioning systems. It will be appreciated that even when the analyte partitions predominantly into one of the phases, in some embodiments, such partitioning is improved by the use of the additional components (e.g., probes) described above.

[0057] In some embodiments, the first / second phase solution comprises a micellar solution. In some embodiments, the micellar solution comprises a non-ionic surfactant. In some embodiments, the micellar solution comprises a detergent. In some embodiments, the micellar solution comprises Triton-X. In some embodiments, the micellar solution comprises a polymer similar to Triton-X, such as, by way of non-limiting examples, Igepal CA-630 and Nonidet P-40. In some embodiments, the micellar solution consists essentially of Triton-X.

[0058] In some embodiments, the micellar solution has a viscosity (at room temperature, about 25 degrees Celsius) of about 0.01 centipoise to about 5000 centipoise, about 0.01 centipoise to about 4500 centipoise, about 0.01 centipoise to about 4000 centipoise, about 0.01 centipoise to about 3500 centipoise, about 0.01 centipoise to about 3000 centipoise, about 0.01 centipoise to about 2500 centipoise, about 0.01 centipoise to about 2000 centipoise, about 0.01 centipoise to about 1500 centipoise, about 0.01 centipoise to about 1000 centipoise, or about 0.01 centipoise to about 500 centipoise. In some embodiments, the micellar solution has a viscosity (at room temperature, about 25 degrees Celsius) of about 0.01 centipoise to about 450 centipoise, about 0.01 centipoise to about 400 centipoise, about 0.01 centipoise to about 350 centipoise, about 0.01 centipoise to about 300 centipoise, about 0.01 centipoise to about 250 centipoise, about 0.01 centipoise to about 200 centipoise, about 0.01 centipoise to about 150 centipoise, or about 0.01 centipoise to about 100 centipoise.

[0059] In some embodiments, the first / second phase solution includes a polymer (e.g., a polymer solution). In certain embodiments, the polymer is polyethylene glycol (PEG). In various embodiments, the PEG may have a molecular weight of 1000 to 100,000. In certain embodiments, the PEG is selected from PEG-4600, PEG-8000, and PEG-20,000. In certain embodiments, the polymer is polypropylene glycol (PPG). In various embodiments, the PPG may have a molecular weight of 100 to 10,000. In certain embodiments, the PPG is selected from PPG 425. In certain embodiments, the polymer is dextran. In various embodiments, the dextran may have a molecular weight of 1000 to 1,000,000. In certain embodiments, the dextran is selected from dextran 6000, dextran 9000, dextran 35,000, and dextran 200,000.

[0060] In some embodiments, the polymer solution is selected from a polymer solution that is 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 polymer solution is 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 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, about 50% w / w, about 51% w / w, about 52% w / w, about 53% w / w, about 54% w / w, about 55% w / w, about 56% w / w, about 57% w / w The polymer solution is selected from the group consisting of 6% 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. In some embodiments, the polymer solution is selected from a polymer solution that is about 10% w / w, about 20% w / w, about 30% w / w, about 40% w / w, about 50% w / w, about 60% w / w, about 70% w / w, about 80% w / w, or about 90% w / w. In some embodiments, the polymer solution is selected from a polymer solution that is about 10% w / w to about 80% w / w. In some embodiments, the polymer solution is selected from a polymer solution that is about 10% w / w to about 25% w / w.

[0061] In some embodiments, the first / second phase solution comprises a salt (e.g., the first / second phase solution is a salt solution). In some embodiments, the analyte of interest and / or the probe-analyte complex partitions into the salt solution. In some embodiments, the analyte of interest and / or the probe-analyte complex partitions into a salt solution that comprises a kosmotropic salt. In some embodiments, the analyte of interest and / or the probe-analyte complex partitions into a salt solution that comprises a chaotropic salt. In some embodiments, the salt is selected from magnesium salts, lithium salts, sodium salts, potassium salts, cesium salts, zinc salts, and aluminum salts. In some embodiments, the salt is selected from bromides, iodides, fluorides, carbonates, sulfates, citrates, carboxylates, borates, and phosphates. In some embodiments, the salt is potassium phosphate. In some embodiments, the salt is ammonium sulfate.

[0062] In some embodiments, the salt solution is selected from a salt solution that is 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 salt solution is 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 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, about 50% w / w, about 51% w / w, about 52% w / w, about 53% w / w, about 54% w / w, about 55% w / w, about 56% w / w, about 57% w / w The salt solution is selected from a salt solution that is about 6% 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. In some embodiments, the salt solution is selected from a salt solution that is about 0.1% w / w to about 10%. In some embodiments, the salt solution is selected from about 1% w / w to about 10% salt solution.

[0063] In some embodiments, the first / second phase solution comprises a water immiscible solvent. 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, diethyl ether, dichloromethane, chloroform, toluene, and hexane.

[0064] In some embodiments, the first phase solution comprises a micellar solution and the second phase solution comprises a polymer. In some embodiments, the second phase solution comprises a micellar solution and the first phase solution comprises a polymer. In some embodiments, the first phase solution comprises a micellar solution and the second phase solution comprises a salt. In some embodiments, the second phase solution comprises a micellar solution and the first phase solution comprises a salt. In some embodiments, the micellar solution is a Triton-X solution. In some embodiments, the first phase solution comprises a first polymer and the second phase solution comprises a second polymer. In some embodiments, the first / second polymer is selected from polyethylene glycol and dextran. In some embodiments, the first phase solution comprises a polymer and the second phase solution comprises a salt. In some embodiments, the second phase solution comprises a polymer and the first phase solution comprises a salt. In some embodiments, the first phase solution comprises polyethylene glycol and the second phase solution comprises potassium phosphate. In some embodiments, the second phase solution comprises polyethylene glycol and the first phase solution comprises potassium phosphate. In some embodiments, the first phase solution comprises a salt and the second phase solution comprises a salt. In some embodiments, the first phase solution comprises a kosmotropic salt and the second phase solution comprises a chaotropic salt. In some embodiments, the second phase solution comprises a kosmotropic salt and the first phase solution comprises a chaotropic salt.

[0065] In some embodiments, the first phase solution is selected from Component 1 in Table 1 and the second phase solution is selected from Component 2 in Table 1. In some embodiments, the second phase solution is selected from Component 1 in Table 1 and the second phase solution is selected from Component 2 in Table 1.

[0066] In some embodiments, the components of Table 1 are suspended or dissolved in a buffer. In some embodiments, the components of Table 1 are dissolved / suspended in a buffer that is compatible with the biological system from which the sample is derived. In some embodiments, the components of Table 1 are dissolved / suspended in saline. In some embodiments, the components of Table 1 are dissolved / suspended in PBS. In some embodiments, the components of Table 1 are dissolved / suspended in water. [Table 1]

[0067] In some embodiments, the device further comprises a collector configured to be placed in contact with the ATPS, and the analyte of interest is distributed to the interface between the collector and the first phase solution and / or the second phase solution. In some embodiments, the collector comprises a material selected from plastic, mesoporous material, silica, polypropylene, magnet, magnetic particles, paramagnetic particles, materials with pores, materials with grooves, and any combination thereof. In some embodiments, the collector comprises polypropylene. In some embodiments, the collector is optimized to increase collection of the analyte of interest. In some embodiments, the collector comprises pores that maximize surface area. In some embodiments, the width of the pore is about 1 μm, about 5 μm, about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 45 μm, about 50 μm, about 55 μm, about 60 μm, about 65 μm, about 70 μm, about 75 μm, about 80 μm, about 85 μm, about 90 μm, about 95 μm, or about 100 μm. In some embodiments, the width of the pore is about 100 μm, about 200 μm, about 300 μm, about 400 μm, about 500 μm, about 600 μm, about 700 μm, about 800 μm, about 900 μm, or about 1 mm. In some embodiments, the pores have a depth of about 1 μm, about 5 μm, about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 45 μm, about 50 μm, about 55 μm, about 60 μm, about 65 μm, about 70 μm, about 75 μm, about 80 μm, about 85 μm, about 90 μm, about 95 μm, or about 100 μm. In some embodiments, the pores have a depth of about 100 μm, about 200 μm, about 300 μm, about 400 μm, about 500 μm, about 600 μm, about 700 μm, about 800 μm, about 900 μm, or about 1 mm.

[0068] Lateral Flow Assay (LFA) In certain embodiments, the devices and systems described herein are configured to provide a lateral flow assay (LFA) for the detection of an analyte of interest in a sample, the LFA being used in combination with an aqueous two-phase system (ATPS). In some embodiments, the LFA comprises a porous matrix in which the ATPS or its components are disposed, the porous matrix being configured and sufficiently porous to allow the ATPS or its components to flow through the porous matrix when the ATPS or its components are in a fluid phase. Such porous LFA devices are referred to herein as paper devices or paper-fluidic devices, the terms being used interchangeably. As noted above, paper as used herein is not limited to thin sheets derived from wood pulp or other fibrous plant materials. However, in certain embodiments, the use of such papers in the LFA devices described herein is contemplated.

[0069] In some embodiments, the porous matrix is ​​sufficiently porous to allow the mixed phase solution, the first and / or second phase solutions of the ATPS, and / or the analytes of interest to flow through the LFA. In some embodiments, the porous matrix is ​​sufficiently long and / or deep to allow the mixed phase solution, the first and / or second phase solutions, and / or the analytes of interest to flow vertically and / or horizontally through the LFA. In some embodiments, the first phase solution flows through the porous matrix at a first velocity and the second phase solution flows through the porous matrix at a second velocity, the first velocity and the second velocity being different. In some embodiments, the porous matrix comprises a material selected from sintered glass ceramics, minerals, cellulose, fiberglass, nitrocellulose, polyvinylidene fluoride, nylon, charge modified nylon, polyethersulfone, and combinations thereof.

[0070] Focus in the moment Previously, phase separation in ATPS has been performed under no-flow conditions. However, we have recently found that ATPS phase separates when it flows through paper, which we term "concentration on flow". This finding is exciting and intriguing, as the paper was found to significantly reduce the time of the concentration process. Based on this underlying phenomenon, we designed a paper-fluidic device to fully integrate the necessary components for combined ATPS and LFA diagnostics. Studying ATPS when it flows through different types of paper under different conditions was important for us to be able to develop a more complex paper-flow detection device. From our experimental data using different ATPS, such as polymer-salt ATPS and micellar ATPS, we realized that when a homogeneous ATPS solution is applied to a particular paper material, phase separation and concentration of the analyte will occur as the solution flows. The inventors have also demonstrated that this phenomenon persists even when ATPSs are fabricated with varying volume ratios, e.g., the volume of the upper phase divided by the volume of the lower phase (see, e.g., FIG. 12).

[0071] In some embodiments, the LFA comprises paper. In some embodiments, the paper comprises a plurality of sheets of porous material through which fluid can flow. In some embodiments, the paper comprises a material selected from cellulose, fiberglass, nitrocellulose, polyvinylidene fluoride, charge modified nylon, polyethersulfone, and the like. In some embodiments, the paper is a HI-FLOW PLUS® membrane.

[0072] In some embodiments, the paper is a woven paper. In some embodiments, the paper is a Whatman paper. In some embodiments, the Whatman paper is selected from Whatman S17, Whatman MF1, Whatman VF1, Whatman Fusion 5, Whatman GF / DVA, Whatman LF1, Whatman CF1, and Whatman CF4.

[0073] In some embodiments, the paper concentrates the analyte of interest as it flows through the LFA (e.g., a "flow-to-focus" based device). In some embodiments, the paper concentrates the analyte of interest as it flows horizontally through the LFA. In some embodiments, the paper concentrates the analyte of interest as it flows vertically through the LFA. In some embodiments, the paper concentrates the analyte of interest as it flows vertically through the LFA by gravity. In some embodiments, the paper concentrates the analyte of interest as it flows vertically through the LFA by capillary action. In some embodiments, the paper has properties that affect which phase solution becomes the leading fluid. As a non-limiting example, when using a PEG-salt ATPS, adding the solution to fiberglass paper will cause the salt phase to become the leading solution, while using cellulose paper will cause the PEG phase to become the leading solution. In some embodiments, phase separation within the paper facilitates phase separation. As a non-limiting example, micellar ATPS typically requires hours to phase separate in ATPS without flow, but when applied to a paper strip, this phase separation occurs within minutes. This speeds up the diagnostic process by making ATPS, the rate-limiting step in traditional diagnostic processes, a viable option for our rapid paper diagnostic assays. In some embodiments, the "flow focusing" device comprises a PEG-salt ATPS. In some embodiments, the "flow focusing" device comprises a micellar ATPS. In some embodiments, the LFA of the "flow focusing" device comprises fiberglass paper. In some embodiments, the LFA of the "flow focusing" device comprises nitrocellulose paper.

[0074] In some embodiments, the LFA comprises a filter that removes blood cells or other particles, a sample pad where a sample containing an analyte of interest is applied to the LFA, a detection zone (e.g., test line and control line) where the analyte of interest binds and is detected, and an absorbent pad (e.g., dry receiving paper) that absorbs excess sample and / or solution applied to the LFA (see, e.g., FIG. 1, top). In some embodiments, the control line and / or test line are areas or spots rather than lines in the proper sense.

[0075] In some embodiments, the LFA comprises an LFA strip. The terms "LFA" and "LFA strip" are used interchangeably herein. In some embodiments, the LFA strip has a length greater than its width and depth. In some embodiments, the LFA is rectangular. In some embodiments, the LFA has a shape selected from the group consisting of circular, oval, square, polygonal, and irregular shapes. In some embodiments, the LFA comprises a plurality of pathways and / or connections. In some embodiments, the LFA strip comprises a sample pad, a detection zone, and an absorbent pad. In some embodiments, the detection zone is located between the sample pad and the absorbent pad, which absorbs and expels a sample containing an analyte of interest from the sample pad toward the detection zone.

[0076] Sandwich assay In some embodiments, the LFA is configured to provide or perform a sandwich assay (see, e.g., FIG. 1, bottom left diagram). In some embodiments, the sandwich assay includes a capture moiety that binds to the analyte of interest. In some embodiments, the device includes a probe. In some embodiments, the probe includes a detectable property (chromogenic, fluorescent, radioactive, etc.). In some embodiments, the probe includes a binding moiety (e.g., an antibody) that interacts with the analyte of interest. In some embodiments, the probe is added to the sample, and the probe binds to the analyte of interest to form a probe-analyte complex. In some embodiments, the probe-analyte complex is applied to a sample pad and flows through the LFA toward an absorbent pad. In some embodiments, the analyte of interest in the probe-analyte complex binds to the capture moiety. In some embodiments, the capture moiety is immobilized on a test line, leaving the probe-analyte complex immobilized on the test line. In some embodiments, the probe is chromogenic, and accumulation of the probe-analyte complex at the test line causes the test line to exhibit a strong color (e.g., a detectable signal), indicating a positive result. In some embodiments, the absence of the analyte of interest in the sample, the probe of the probe-analyte complex does not interact with the capture moiety, and the absence of a test line indicates a negative result. In some embodiments, the LFA includes a probe capture moiety at the control line that directly interacts with the probe and / or the binding moiety, such that regardless of the presence of the analyte of interest in the sample, the probe / binding moiety binds to the probe capture moiety and accumulates at the control line. In some embodiments, the probe capture moiety is a secondary antibody that binds to the binding moiety, and the binding moiety is a primary antibody that binds to the analyte of interest. In some embodiments, the probe becomes immobilized and detected at the control line, indicating that the test is valid. In some embodiments, a detectable signal at the test line and the control line indicates a positive result (e.g., the analyte of interest is present in the sample).In some embodiments, a negative result is indicated by a detectable signal at the control line.

[0077] Competition assay In some embodiments, the LFA is configured to provide a competitive assay (see, for example, the diagram at the bottom right of FIG. 1). In some embodiments, a probe is added to the sample, and the probe binds to the analyte of interest to form a probe-analyte complex. In some embodiments, the LFA includes the analyte of interest immobilized at the test line. In some embodiments, the probe is saturated with the analyte of interest in the sample, and the probe does not bind to the analyte of interest immobilized at the test line. In some embodiments, the absence of a detectable signal at the test line indicates a positive result. In some embodiments, the analyte of interest is not present in the sample, and the probe binds to the analyte of interest at the test line, indicating a negative result. In some embodiments, the LFA includes a probe capture moiety on the control line that directly interacts with the probe, and the probe binds to the probe capture moiety and accumulates at the control line, regardless of the presence of the analyte of interest in the sample. In some embodiments, the probe becomes immobilized and detected at the control line, indicating that the test is valid. In some embodiments, the absence of a detectable signal at the test line but the presence of a detectable signal at the control line indicates a positive result (e.g., the analyte of interest is present in the sample), in some embodiments, detectable signals at both the test line and the control line indicates a negative result.

[0078] Well In some embodiments, the sample pad comprises a well, hi some embodiments, the well has a volume sufficient to accommodate a solution selected from the group consisting of at least a portion of the ATPS, at least a portion of the first phase solution, at least a portion of the second phase solution, a resuspension solution for the analyte of interest, and combinations thereof.

[0079] In certain embodiments, the well volume is in the range of about 1 μL to about 10 μL. In certain embodiments, the well volume is in the range of about 1 μL to about 100 μL. In certain embodiments, the well volume is in the range of about 1 μL to about 1000 μL. In certain embodiments, the well volume is in the range of about 1 μL to about 5000 μL. In certain embodiments, the well volume is in the range of about 1 mL to about 10 mL. In certain embodiments, the well volume is in the range of about 1 mL to about 100 mL. In certain embodiments, the well volume is in the range of about 1 mL to about 1000 mL.

[0080] In some embodiments, the resuspension solution comprises a buffer for resuspending concentrated / extracted analytes of interest from the ATPS.

[0081] In some embodiments, the well is located at a location on the LFA selected from a corner, an end, a center, a junction, an off-center, and a bend in the LFA. In some embodiments, the well comprises one or more pads selected from a salt pad, a probe pad, a polymer pad, and combinations thereof. In some embodiments, the well comprises a plurality of pads. In some embodiments, the first / second phase solutions separate and / or the analytes of interest concentrate as they flow through the plurality of pads. In some embodiments, the first / second phase solutions separate and / or the analytes of interest concentrate as they flow vertically through the plurality of pads. In some embodiments, the first / second phase solutions separate and / or the analytes of interest concentrate as they flow vertically through the plurality of pads by gravity. In some embodiments, the first / second phase solutions separate and / or the analytes of interest concentrate as they flow vertically through the plurality of pads by capillary action. In some embodiments, the well is a paper well. In some embodiments, the paper well is a three-dimensional paper structure that holds a larger volume of sample compared to typical paper strips used in LFAs. In some embodiments, the paper well is made of a paper material that allows phase separation to occur, allowing subsequent concentration of the analyte in the preceding fluid. In some embodiments, the flow of the preceding fluid is directed to an absorbent pad that allows detection of the analyte (see, e.g., FIG. 13).

[0082] In some embodiments, the device utilizes a "concentration on flow" mechanism while utilizing gravity to further facilitate flow and macroscopic phase separation in the well. In some embodiments, the well provides sufficient cross-sectional area to facilitate phase separation since the first and second phase solutions may flow at different rates due to the difference in viscosity of the phase solutions and their affinity for the paper material. In some embodiments, the well enhances or promotes phase separation and / or concentration of the analyte of interest as the phase solution(s) travel through the well and emerge in the preceding fluid. In some embodiments, the LFA test strip is directly connected to the well at a downstream location so that the concentrated analyte in the preceding fluid first contacts the LFA strip and the detection step occurs simultaneously with the concentration process, further reducing the overall assay time.

[0083] In some embodiments, the LFA comprises multiple wells. In some embodiments, the LFA comprises a mixing well. In some embodiments, the LFA comprises a concentration well. In some embodiments, the ATPS is applied to the concentration well where phase separation and / or concentration of the analyte of interest occurs. In some embodiments, the concentrated analyte is removed from the mixing well after phase separation and applied to the concentration well. In some embodiments, the concentration well contains a running buffer that enhances / promotes phase separation in the concentration well and / or LFA (see, e.g., FIG. 14).

[0084] In some embodiments, the device comprises an actuator for releasing the contents of the well into and / or onto the porous matrix, hi some embodiments, the actuator comprises a mechanism for puncturing the well.

[0085] Dehydrated ATPS in LFA In some embodiments, the ATPS or its components are dehydrated on and / or within at least a first portion of the porous matrix. In some embodiments, application of a sample to the device hydrates the ATPS, thereby converting the ATPS or its components to a fluid phase. Dehydration can make the device easier to handle, since the user only needs to add a sample (e.g., saliva) to the device. In some embodiments, the user only needs to apply a sample solution to the strip to detect the presence / absence of an analyte of interest. In some embodiments, the sample solution flows through the LFA, resolubilizing the ATPS and triggering phase separation within the LFA and subsequent concentration of the analyte of interest (see, e.g., FIG. 15).

[0086] In some embodiments, all the necessary components for a given ATPS are mixed to form a homogenous solution, which is applied to the paper and then dehydrated. When a sample solution is added to the dehydrated paper strip, the ATPS components will be rehydrated as the sample flows, resulting in phase separation. In some ATPSs, where the phase containing the concentrated analyte is less viscous and potentially contains less polymer or micelles, the phase will flow faster and the concentrated analyte will appear in the leading fluid and reach the LFA to initiate detection. Also, the length and concentration of the component segments of the dehydrated ATPS can be adjusted for different applications.

[0087] In some embodiments, the ATPS is dehydrated on the LFA. In some embodiments, a component of a first ATPS is dehydrated on an LFA. In some embodiments, a component of a second ATPS is dehydrated on an LFA. In some embodiments, a mixed phase solution is dehydrated on an LFA. In some embodiments, a component of a first phase solution and / or a component of a first ATPS is dehydrated on a first portion of an LFA. In some embodiments, a component of a second phase solution and / or a component of a second ATPS is dehydrated on a second portion of an LFA. In some embodiments, the first and second portions are the same. In some embodiments, the first and second portions are different. As a non-limiting example, in a PEG-salt ATPS, PEG and salt solutions are dehydrated separately in different portions or segments of the paper (see, e.g., FIG. 16). In some embodiments, dehydrating the first / second phase solution and / or components of the ATPS on different portions of the LFA provides a more uniform concentration of the first / second phase solution components and / or components of the ATPS. In some embodiments, dehydrating the first / second phase solution components and / or components of the ATPS on different portions allows the first phase solution or components of the ATPS to flow in a first direction after hydration and the second phase solution and / or components of the ATPS to flow in a second direction after hydration, where the first direction is different from the second direction. In some embodiments, the analyte of interest is concentrated in the first direction but not in the second direction. In some embodiments, the analyte of interest is concentrated in the second direction but not in the first direction. In some embodiments, dehydrating the first / second phase components and / or the ATPS components at different locations allows the analyte of interest to flow in a first / second direction without requiring the sample to flow in the first / second direction (see, e.g., Figures 16D, 16E, and 17). In some embodiments, dehydrating the first / second phase components and / or the ATPS components at different locations allows the analyte of interest to flow faster and, therefore, be detected sooner.In some embodiments, dehydrating the first / second phase components and / or the ATPS components in different portions allows for increased reliability of the results. In some embodiments, dehydrating the first / second phase components and / or the ATPS components in different portions prevents aggregation of the first / second phase solution components and / or the ATPS components (e.g., PEG-salt ATPS). In some embodiments, the first / second phase components and / or the ATPS components that contain salt solutions are dehydrated in multiple segments. In some embodiments, the first / second phase components and / or the ATPS components that do not contain hydrophobic polymers (e.g., PEG) are dehydrated in multiple segments. In some embodiments, the dehydrated PEG is not located near the detection zone because the PEG-rich phase retards flow through the detection membrane (see, e.g., FIG. 64). In some embodiments, the LFA strip comprises a blank spacer near the detection zone that does not contain PEG or salt (see, e.g., FIG. 65).

[0088] In some embodiments, the probe is provided in a probe buffer. In some embodiments, the probe buffer is dehydrated on an LFA. In some embodiments, the LFA comprises a probe and a dehydrated probe buffer. In some embodiments, the probe buffer improves flux of the probe-analyte complex through the LFA.

[0089] In some embodiments, dehydration of the ATPS components improves the detection limit compared to devices in which the ATPS components are added in liquid form. In some embodiments, adding the liquid ATPS components dilutes the patient-derived sample solution. In some embodiments, dehydration of the ATPS components allows for the development of a distinguishable first phase solution and / or a distinguishable second phase solution during flow, concentrating the analyte of interest or probe-analyte complexes that will reach the test and control lines in a small volume in front of the leading fluid. In some embodiments, concentrating the analyte of interest and / or probe-analyte complexes in front of the leading fluid reduces the time required for detection.

[0090] LFA Design / Construction In some embodiments, the LFA strip has a width that does not change from the first end to the second end. In some embodiments, the width is defined as the dimension perpendicular to the direction of flow in the LFA and in the plane of length. In some embodiments, a first portion of the LFA strip has a first width and a second portion of the LFA strip has a second width, the first width and the second width being different. In some embodiments, the first width is greater than the second width, while in other embodiments, the first width is less than the second width. In certain embodiments, the LFA strip is contemplated to have more than two widths, for example, the strip may be continuously narrow or progressively narrow in three or more positions. In some embodiments, the first portion comprises a sample pad and the second portion comprises a detection zone. In some embodiments, the first portion comprises a dehydrated probe buffer and the second portion comprises a detection zone (see, e.g., FIG. 62). In some embodiments where the first portion comprises a dehydrated probe buffer and the second portion comprises a detection zone, the detection limit is improved compared to an LFA strip where the width of the portion comprising the sample pad is the same as the width of the portion comprising the detection zone. In some embodiments, the wider sample pad segment allows more of the analyte of interest in the sample to bind to the probe compared to an LFA strip where the width does not change. In some embodiments, the wider sample pad segment allows a larger sample volume and therefore more of the analyte of interest to bind to the probe compared to an LFA strip where the width does not change (see, e.g., FIG. 63).

[0091] In some embodiments, the LFA comprises a gradient (e.g., a change in the depth of the LFA along the length of the LFA). In some embodiments where the LFA does not comprise a gradient, some of the probe-analyte complexes remain in the sample pad. In some embodiments where the LFA comprises a gradient, more probe-analyte complexes flow through the LFA than an LFA without a gradient (see, e.g., FIG. 66).

[0092] In some embodiments, the LFA comprises a section containing dehydrated probes and / or probe buffer that allows for a sample with a larger volume that resolubilizes at a slower rate and more completely saturates the binding sites on the probes compared to when the probe / probe buffer is resolubilized at a faster rate, resulting in improved detection limits in competitive assays. In some embodiments, the dehydrated ATPS or components thereof is placed downstream of the section containing dehydrated probes and / or probe buffer to allow the probes and analytes of interest time to bind to each other before they reach the ATPS or components thereof. In some embodiments, the ATPS (components) concentrates the probe-analyte complexes in a smaller volume. In some embodiments, the smaller volume contains more probe-analyte complexes that pass through the test and control lines than in other embodiments where not all of the probe-analyte complexes reach the test and control lines due to distribution over a larger volume. In some embodiments, concentrating the probe-analyte complexes in a smaller leading phase increases the sensitivity and / or reliability of the device. In some embodiments, detection time is reduced by concentrating the probe-analyte complex into a smaller leading phase, see, e.g., Figure 67, which shows a schematic of one possible design using a PEG / salt ATPS.

[0093] In some embodiments, the LFA is configured according to the structure shown in Figure 16. In some embodiments, the LFA is configured according to the structure shown in Figure 20. In some embodiments, the LFA is configured according to the structure shown in Figures 62-70.

[0094] In some embodiments, the LFA is designed for use with a probe that contains or complexes with magnetic / paramagnetic particles. In some embodiments, the LFA comprises a paper strip with a fork at the end of the paper strip that is used to split the flow of the first and second phase solutions of the ATPS. In some embodiments, the detection zone of the LFA is placed on the prong and a magnet is placed near or at the prong (see, e.g., FIG. 20). The magnet concentrates the probe / probe-analyte complex in the fluid flowing into the LFA detection zone, thereby increasing the sensitivity of the diagnosis. Conversely, in some embodiments, the probe comprises a magnet or magnetic field and the device comprises magnetic or paramagnetic particles placed near or at the prong.

[0095] In some embodiments, the LFA comprises a three-dimensional structure. In some embodiments, the LFA comprises a layer of a porous matrix that provides the three-dimensional structure. In some embodiments, the three-dimensional structure integrates the LFA with the ATPS. In some embodiments, the ATPS has a long phase separation time (e.g., a micellar ATPS), and the phase separation time is improved by using a three-dimensional structure (e.g., increasing the height of the LFA strip). In some embodiments, as the mixed phase solution flows vertically through the LFA (e.g., through a layer of a porous matrix), the mixed phase solution separates into a first phase solution and a second phase solution.

[0096] In some embodiments, the LFA has a thickness (e.g., height, depth, or vertical dimension). In some embodiments, the thickness is about 0.1 mm to about 30 cm. In some embodiments, the thickness is about 0.1 mm to about 1 mm, about 0.1 mm to about 10 mm, or about 0.1 mm to about 1 cm. In some embodiments, the thickness is about 1 mm to about 10 mm, about 1 mm to about 1 cm, about 1 mm to about 1.5 cm, about 1 mm to about 3 cm, about 1 mm to about 3.5 cm, about 1 mm to about 4 cm, about 1 mm to about 4.5 cm, about 1 mm to about 5 cm, about 1 mm to about 5.5 cm, about 1 mm to about 6 cm, about 1 mm to about 6.5 cm, about 1 mm to about 7 cm, about 1 mm to about 7.5 cm, about 1 mm to about 8 cm, about 1 mm to about 8.5 cm, about 1 mm to about 9 cm, about 1 mm to about 9.5 cm, or about 1 mm to about 10 cm. In some embodiments, the thickness is from about 0.5 cm to about 5 cm.

[0097] Multiple Pathways In some embodiments, the LFA comprises a first and a second path, or at least a first and a second path. In some embodiments, the first phase solution flows preferentially through the first path and the second phase solution flows preferentially through the second path. In some embodiments, the analyte of interest may not be concentrated in the leading fluid during flow through the paper. In some embodiments, the LFA is designed to redirect flow such that the leading fluid is the first fluid to pass through the detection zone at the appropriate time. In some embodiments, the redirection of flow comprises the introduction of a new flow path. In some embodiments, the redirection of flow comprises the incorporation of a three-dimensional paper structure. In some embodiments, the redirection of flow comprises providing or integrating multiple paths where there is preferential flow depending on the phase of the flow. In some embodiments, the redirection of flow comprises combining multiple methods for redirecting flow as described above. In some embodiments, the redirection of flow enhances phase separation and / or induces the analyte of interest to flow through the detection zone.

[0098] In some embodiments, the LFA is configured to redirect flow by including an LFA segment containing a running buffer and an LFA segment containing dry receiving paper that are introduced after the sample is applied to the LFA. In some embodiments, the LFA is configured to redirect flow by including an LFA segment containing a running buffer and an LFA segment containing dry receiving paper that are introduced after the sample begins to flow through the LFA. In some embodiments, the target is concentrated in the retarding fluid, in which case the running buffer will redirect the retarding fluid (as the leading fluid) to the detection zone (see, e.g., FIG. Error! Reference source not found. A). In some embodiments, the running buffer and dry receiving paper segment push the leading fluid containing the concentrated analyte to the detection zone (FIG. 18B).

[0099] In some embodiments, there are multiple analytes of interest, the device is configured to distribute a first analyte of interest in a first phase solution and a second analyte of interest in a second phase solution, the LFA includes a first pathway that directs the first phase solution to a first detection zone, and the LFA includes a second pathway that directs the second phase solution to a second detection zone. In some embodiments, the first pathway is comprised of a first type of porous matrix and the second pathway is comprised of a second type of porous matrix, the first type of porous matrix and the second type of porous matrix being different (e.g., different porosity, charge, hydrophobicity, three-dimensional structure, etc.). In some embodiments, the first type of porous matrix is ​​more hydrophobic / hydrophilic than the second type of porous matrix (see, e.g., FIG. 19). In some embodiments, the polymer-rich phase solution may preferentially flow through the more hydrophobic strip, while the polymer-poor phase solution containing the concentrated analyte may flow through the more hydrophilic paper. In some embodiments, the analytes of interest are concentrated by "purifying" the polymer-rich phase, which may contain contaminants that may affect the performance of downstream LFAs. In some embodiments, one or more of a plurality of analytes of interest are partitioned into different phases of the ATPS.

[0100] In some embodiments, the probe comprises magnetic particles and the probe-analyte complex is redirected by one or more magnets. In some embodiments, the one or more magnets are positioned along a paper strip to concentrate the probe at / towards a specific location (e.g., detection zone) of the LFA. Alternatively, in some embodiments, the probe comprises a magnet and the probe-analyte complex is redirected by one or more magnetic / paramagnetic particles. In some embodiments, the one or more magnetic / paramagnetic particles are positioned along a paper strip to concentrate the probe at / towards a specific location (e.g., detection zone) of the LFA.

[0101] probe In certain embodiments, the systems and / or devices described herein include a probe and / or the methods described herein utilize a probe that includes a binding moiety that binds to an analyte of interest to form a probe-analyte complex. As used herein, the terms "analyte of interest" and "probe-analyte complex" are used interchangeably unless otherwise specified.

[0102] In some embodiments, the analyte of interest partitions solely and preferentially into the first phase solution or the second phase solution or the interface between the first and second phase solutions, hi some embodiments, the analyte of interest partitions solely and predominantly into the first phase solution or the second phase solution or the interface between the first and second phase solutions.

[0103] In some embodiments, the analyte of interest does not partition solely and preferentially into the first phase solution or the second phase solution or the interface between the first and second phase solutions, hi some embodiments, the analyte of interest does not partition solely and predominantly into the first phase solution or the second phase solution or the interface between the first and second phase solutions.

[0104] In some embodiments, the probe-analyte complex preferentially partitions into the first phase solution, or the second phase solution, or the interface between the first and second phase solutions, such that the analyte of interest (of the probe-analyte complex) preferentially partitions into the first phase solution, or the second phase solution, or the interface between the first and second phase solutions.

[0105] In some embodiments, the probe-analyte complex partitions predominantly into the first phase solution, or the second phase solution, or the interface between the first and second phase solutions, such that the analyte of interest (of the probe-analyte complex) partitions predominantly into the first phase solution, or the second phase solution, or the interface between the first and second phase solutions.

[0106] In some embodiments, the term "preferentially partitioned" when used in reference to partitioning of an analyte of interest (or a probe-analyte complex) into the first / second phase solutions of an ATPS refers to a state in which a greater amount of the analyte of interest is located in a preferred phase solution than in another phase solution of the ATPS.

[0107] In some embodiments, the term "extremely partitioned" when used in reference to partitioning of a target analyte (or probe-analyte complex) into the first and second phase solutions of an ATPS refers to about 90% or more of the target analyte being located in a phase solution that is preferred over another phase solution of the ATPS.

[0108] In some embodiments, a greater amount of the analyte of interest is distributed into the first phase solution. In some embodiments, more than about 50%, or more than about 55%, or more than about 60%, or more than about 65%, or more than about 70%, or more than about 75%, or more than about 80%, or more than about 85%, or more than about 90%, or more than about 95%, or more than about 98%, or more than about 99% of the analyte of interest is distributed into the first phase solution. In some embodiments, more than about 99%, or more than about 99.1%, or more than about 99.2%, or more than about 99.3%, or more than about 99.4%, or more than about 99.5%, or more than about 99.6%, or more than about 99.7%, or more than about 99.8%, or more than about 99.9% of the analyte of interest is distributed into the first phase solution.

[0109] In some embodiments, a greater amount of the analyte is distributed into the second phase solution. In some embodiments, more than about 50%, or more than about 55%, or more than about 60%, or more than about 65%, or more than about 70%, or more than about 75%, or more than about 80%, or more than about 85%, or more than about 90%, or more than about 95%, or more than about 98%, or more than about 99% of the analyte of interest is distributed into the second phase solution. In some embodiments, more than about 99%, or more than about 99.1%, or more than about 99.2%, or more than about 99.3%, or more than about 99.4%, or more than about 99.5%, or more than 99.6%, or more than about 99.7%, or more than about 99.8%, or more than about 99.9% of the analyte of interest is distributed into the second phase solution.

[0110] In some embodiments, a greater amount of the analyte is distributed at the interface between the first and second phase solutions. In some embodiments, more than about 50%, or more than about 55%, or more than about 60%, or more than about 65%, or more than about 70%, or more than about 75%, or more than about 80%, or more than about 85%, or more than about 90%, or more than about 95%, or more than about 98%, or more than about 99% of the analyte of interest is distributed at the interface. In some embodiments, more than about 99%, or more than about 99.1%, or more than about 99.2%, or more than about 99.3%, or more than about 99.4%, or more than about 99.5%, or more than 99.6%, or more than about 99.7%, or more than about 99.8%, or more than about 99.9% of the analyte of interest is distributed at the interface.

[0111] In some embodiments, the probe-analyte complex is extracted and / or collected in an LFA (see, eg, FIG. 2).

[0112] In some embodiments, the probe and / or probe-analyte complex are resuspended in a solution (e.g., a resuspension solution). In some embodiments, the resuspension solution comprises water. In some embodiments, the resuspension solution comprises a saline solution. In some embodiments, the resuspension solution comprises a buffer. In some embodiments, the resuspension solution comprises a phosphate buffered saline (PBS) solution. In some embodiments, the buffer comprises acetic acid. In some embodiments, the buffer comprises Tris. In some embodiments, the buffer comprises ethylenediaminetetraacetic acid (EDTA). In some embodiments, the buffering agent comprises boric acid. In some embodiments, the buffer is a borate buffer. In some embodiments, the buffer is a lithium borate buffer. In some embodiments, the buffer is a sodium borate buffer.

[0113] In some embodiments, the resuspension solution has a pH of about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5 or about 12. In some embodiments, the resuspension solution has a pH of about 9.

[0114] In some embodiments, the device comprises or is configured to utilize one probe, and / or the performance of the assay on the device utilizes one probe, In some embodiments, the device comprises or is configured to utilize at least two different probes, or at least three different probes, or at least four different probes, or at least five different probes, or at least seven different probes, or at least ten different probes, or at least fifteen different probes, or at least twenty different probes, and / or the performance of the assay on the device utilizes a plurality of such different probes.

[0115] In some embodiments, the probe comprises a material selected from the group consisting of synthetic polymers, metals, minerals, glass, quartz, ceramics, biological polymers, plastics, and combinations thereof. In some embodiments, the probe comprises a polymer selected from the group consisting of polyethylene, polypropylene, nylon (Delrin®), polytetrafluoroethylene (Teflon®), dextran, and polyvinyl chloride. In some embodiments, the polyethylene is polyethylene glycol. In some embodiments, the polypropylene is polypropylene glycol. In some embodiments, the probe comprises a biological polymer selected from the group consisting of collagen, cellulose, and chitin. In some embodiments, the probe comprises a metal selected from the group consisting of gold, silver, platinum, titanium, stainless steel, aluminum, and alloys thereof. In some embodiments, the probe comprises nanoparticles (e.g., gold nanoparticles, silver nanoparticles, etc.).

[0116] In some embodiments, the probe further comprises a coating. In some embodiments, the coating comprises polyethylene glycol or polypropylene glycol. In some embodiments, the coating comprises polypropylene. In some embodiments, the coating comprises polypropylene glycol. In some embodiments, the coating comprises dextran. In some embodiments, the coating comprises a hydrophilic protein. In some embodiments, the coating comprises serum albumin. In some embodiments, the coating has an affinity for the first phase solution or the second phase solution.

[0117] In some embodiments, the amount of an analyte of interest in a sample is very low, so that the analyte needs to be substantially concentrated to enable detection by an LFA. In certain embodiments, substantial concentration is achieved at the interface, since the degree of concentration of an analyte depends on the volume of the phase in which the analyte is distributed or concentrated, and the "volume" at the interface is very small compared to the bulk phase.

[0118] In some embodiments, the probe partitions preferentially (or excessively) at the interface to drive the analyte of interest toward the interface. In some embodiments, the probe partitions preferentially (or excessively) at the interface due to its surface chemistry. As a non-limiting example, to drive the probe-analyte complex to the interface of a polymer-salt ATPS system, such as a polyethylene glycol-potassium phosphate (PEG / salt) system, the probe is conjugated to PEG (or PEGylated) to promote PEG-PEG interactions with the PEG-rich phase and / or modified with hydrophilic proteins to promote hydrophilic interactions with the PEG-poor phase. Using such optimized probes modified with specific antibodies or other molecules capable of binding to the target, the analyte of interest is captured and collected at the interface. Because the volume of the interface is very small, the analyte is highly concentrated and applied to the subsequent LFA (FIG. 8).

[0119] In some embodiments, gold nanoprobes (GNPs) are prepared that can partition to the PEG / salt ATPS interface and the operating conditions are optimized to allow for rapid phase separation times with very high GNP / analyte recovery. As a non-limiting example, a 100-fold improvement in the detection of transferrin (Tf) was demonstrated after combination with interfacial extraction from the ATPS using an LFA (see Example 5).

[0120] In some embodiments, the probe-analyte complex partitions to a solid-liquid interface in the ATPS. In some embodiments, the solid is a wall of a chamber housing the ATPS. In some embodiments, the solid is a collector. In some embodiments, the solid comprises a solid polymer. In some embodiments, the solid polymer is selected from polyethylene, cellulose, chitin, nylon, polyoxymethylene (Delrin®), polytetrafluoroethylene (Teflon®), polyvinyl chloride, and combinations thereof. In some embodiments, the solid polymer comprises polypropylene (see, e.g., FIG. 9). In some embodiments, the probe-analyte complex adheres to the solid and is highly concentrated because it is in a small volume at the solid-liquid interface and is not diluted by the bulk phase solution. In some embodiments, the bulk phase is removed without disturbing the concentrated analyte, which is recovered by washing and then applied to the LFA (FIG. 10). In some embodiments, this approach significantly concentrates the analyte and allows for collection without the use of external forces (e.g., magnets). Alternatively, the probes include magnetic materials and the method is performed using magnets. In some embodiments, these probes are modified to concentrate at the interface for extreme analyte enrichment. As mentioned above, this approach can use magnets to further separate the analytes of interest from other contaminants that are non-specifically concentrated by ATPS. In some embodiments, ATPS enrichment allows the magnetic probes to work more efficiently since they are initially concentrated in a very small volume at a specific location (the interface). Thus, smaller magnets or weaker magnetic fields are needed to capture the concentrated analytes. In some embodiments, the combination of ATPS interface enrichment and magnetic probes allows the development of more effective, faster, and cheaper devices compared to the current state of the art.

[0121] joining part In some embodiments, the binding moiety is a molecule that binds to the analyte of interest. In some embodiments, the binding moiety is a molecule that specifically binds to the analyte of interest. In some embodiments, "specifically binds" refers to the molecule binding preferentially to the analyte of interest over other molecules or binding with greater affinity to the analyte of interest. As a non-limiting example, an antibody will selectively bind to the antigen against which it was raised. Also, as a non-limiting example, a DNA molecule will bind to a substantially complementary sequence under stringent conditions and not to unrelated sequences. In some embodiments, "specific binding" can refer to a binding reaction capable of determining the presence of an analyte of interest in a heterogeneous population of molecules (e.g., proteins and other biological materials). In some embodiments, the binding moiety binds to a specific analyte of interest and does not bind in significant amounts to other molecules present in the sample.

[0122] In some embodiments, the binding moiety is selected from the group consisting of an antibody, a lectin, a protein, a glycoprotein, a nucleic acid, a monomeric nucleic acid, a polymeric nucleic acid, an aptamer, an aptazyme, a small molecule, a polymer, a lectin, a carbohydrate, a polysaccharide, a sugar, a lipid, and any combination thereof, hi some embodiments, the binding moiety is a molecule capable of binding to an analyte molecule of interest.

[0123] In some embodiments, the binding moiety is an antibody or an antibody fragment. Antibody fragments include Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab') 2 These include, but are not limited to, fragments, Fv fragments, Fv' fragments, Fd fragments, Fd' fragments, scFv fragments, hsFv fragments, cameloid antibodies, bispecific antibodies, and other fragments described below.

[0124] In some embodiments, an "antibody" refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. As used herein, the terms "antibody" and "immunoglobulin" are used interchangeably unless otherwise specified. In some embodiments, the immunoglobulin gene is an immunoglobulin constant region gene. In some embodiments, the immunoglobulin gene is, by way of non-limiting example, a kappa, lambda, alpha, gamma, delta, epsilon, or mu constant region gene. In some embodiments, the immunoglobulin gene is an immunoglobulin variable region gene. In some embodiments, the immunoglobulin gene comprises a light chain. In some embodiments, the light chain is selected from a kappa light chain, a lambda light chain, or a combination thereof. In some embodiments, the immunoglobulin gene comprises a heavy chain. In some embodiments, the heavy chain is classified as gamma, mu, alpha, delta, or epsilon, which correspond in order to the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively.

[0125] In some embodiments, the immunoglobulin comprises a tetramer. In some embodiments, the tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) chain and one "heavy" (about 50-70 kDa) chain. In some embodiments, the N-terminus of each chain defines a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The variable light chain (V L ) and variable heavy chain (V H ) refer to these light and heavy chains respectively.

[0126] In some embodiments, the antibody comprises an intact immunoglobulin. In some embodiments, the antibody is selected from a number of well-characterized fragments produced by digestion with various peptidases. In some embodiments, the peptidase is pepsin. In some embodiments, the pepsin digests disulfide bonds in the hinge region, itself forming V fragments through disulfide bonds. H -C H 1 and is a dimer of Fab, F(ab)' 2 In some embodiments, the F(ab)' 2 is reduced under mild conditions to cleave the disulfide bond in the hinge region, thereby forming F(ab)' 2 The dimer is converted to a Fab' monomer. In some embodiments, the Fab' monomer consists essentially of a Fab including a portion of the hinge region. In some embodiments, the Fab' fragment is synthesized de novo, either chemically or by utilizing recombinant DNA technology. In some embodiments, the antibody fragment is produced by modification of a whole antibody. In some embodiments, the antibody fragment is synthesized de novo using recombinant DNA technology. In some aspects, the antibody comprises a single chain antibody (an antibody that exists as a single polypeptide chain). In some embodiments, the antibody comprises a single chain Fv antibody (sFv or scFv) in which a variable heavy chain and a variable light chain are linked together (directly or via a peptide linker) to form a contiguous polypeptide. In some embodiments, the antibody comprises a single chain Fv antibody. In some embodiments, the antibody comprises a VFv antibody, either directly linked or linked by a peptide-encoded linker. H - and V L - a covalently linked V that can be expressed from a nucleic acid containing a coding sequence H -V L In some embodiments, the V H and V L are connected together as a single peptide chain, and V H and VL The domains are non-covalently associated. In some embodiments, Fabs are displayed on phage, where one chain (heavy or light) is fused to the g3 capsid protein and a complementary chain that is exported to the periplasm as a soluble molecule. In some embodiments, the two chains can be encoded on the same or different replicons. In some embodiments, the two antibody chains in each Fab molecule are post-translationally associated, and the dimer is incorporated into the phage particle via binding of one chain, for example, to g3p. In some embodiments, antibodies are displayed on phage.

[0127] In some embodiments, the binding moiety comprises an aptamer. In some embodiments, the aptamer comprises an antibody analog formed from a nucleic acid. In some embodiments, the aptamer does not require the binding of a label to be detected in some assays where rearrangement is directly detected, such as nano-CHEM-FET. In some embodiments, the binding moiety comprises an aptazyme. In some embodiments, the aptazyme comprises an antibody analog formed from a nucleic acid. In some embodiments, the aptazyme acts to change its configuration and capture a specific molecule only in the presence of a second specific analyte.

[0128] The term "small molecule" or "small organic molecule" refers to a molecule comparable in size to organic molecules commonly used in pharmaceuticals. The term does not include biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

[0129] In some embodiments, the antibody fragments are derived via proteolytic digestion of intact antibodies. In some embodiments, the antibody fragments are produced directly by recombinant host cells. In some embodiments, the Fab, Fv or ScFv antibody fragments are expressed in and secreted from E. coli, thus allowing the facile production of large amounts of these fragments. In some embodiments, the antibody fragments are isolated from an antibody phage library. In some embodiments, the Fab'-SH fragments are directly recovered from E. coli and chemically coupled to form F(ab') 2 In some embodiments, F(ab') fragments can be formed. 2 The fragments are directly isolated from recombinant host cell culture. In some embodiments, Fab and F(ab') 2 The fragments have increased in vivo half-life. In some embodiments, Fab and F(ab') 2 The fragment contains the salvage receptor binding epitope residues. Other techniques for the production of antibody fragments will be apparent to those skilled in the art. In certain embodiments, the antibody of choice is a single chain Fv fragment. In some embodiments, the Fv or sFv has a complete binding site that lacks constant regions, and thus is suitable for reduced non-specific binding during in vivo use. In some embodiments, the antibody fragment is a "linear antibody". In some embodiments, the linear antibody fragment is monospecific. In some embodiments, the linear antibody fragment is bispecific.

[0130] In some embodiments, the antibody fragment is a diabody. In some embodiments, the diabody is an antibody fragment having two antigen-binding sites that may be bivalent or bispecific.

[0131] In some embodiments, the antibody fragment is a single domain antibody. In some embodiments, the single domain antibody is an antibody fragment that includes all or a portion of the heavy chain variable domain or all or a portion of the light chain variable region of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody.

[0132] In some embodiments, the probe comprises a detectable label. In some embodiments, the probe has a detectable property. Detectable labels / detectable properties include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Exemplary useful labels include fluorescent nanoparticles (e.g., quantum dots (Qdots)), fluorescent dyes (e.g., fluorescein, Texas Red, rhodamine, green fluorescent protein, etc., see, e.g., Molecular Probes, Inc., Eugene, OR), radioactive labels (e.g., 3 H, 125 I, 35 S, 14 C. 32 P, 99 Tc, 203 Pb, 67 Ga, 68 Ga, 72 As, 111 In, 113m In, 97 Ru, 62 Cu, 641Cu, 52 Fe, 52m Mn, 51 Cr, 186 Re, 188 Re, 77 As, 90 Y, 67 Cu, 169 Er, 121 Sn, 127 Te, 142 Pr, 143 Pr, 198 Au, 199 Au, 161 Tb, 109 Pd, 165 Dy, 149 Pm, 151Pm, 153 Sm, 157 Gd, 159 Gd, 166 Ho, 172 Tm, 169 Yb, 175 Yb, 177 Lu, 105 Rh, 111 Ag, etc.), enzymes (e.g., horseradish peroxidase, alkaline phosphatase and others commonly used in ELISA), various chromogenic labels, magnetic or paramagnetic labels (e.g., magnetic and / or paramagnetic nanoparticles), spin labels, radio-opaque labels, and the like.

[0133] Alternatively or additionally, the probe is attached to another particle that includes a detectable label. Alternatively or additionally, the probe is attached to another particle that has a detectable property. In some embodiments, the probe provides a detectable signal (e.g., a test line, a control line) at the detection zone. In some embodiments, the detectable label / characteristic is selected from the group consisting of a chromogenic label / characteristic, a fluorescent label / characteristic, an enzymatic label / characteristic, a heat-generating label / characteristic, and a radioactive label / characteristic. In some embodiments, the probe is a gold nanoparticle and the detectable property is a color. In some embodiments, the color is selected from orange, red, and purple.

[0134] In some embodiments, the probe comprises magnetic and / or paramagnetic particles. In some embodiments, the magnetic particles comprise a material selected from the group consisting of iron, nickel, cobalt, and combinations thereof. In some embodiments, the magnetic particles comprise alnico, an aluminum-nickel-cobalt alloy. In some embodiments, the magnetic particles comprise an alloy of rare earth metals. In some embodiments, the rare earth metals are selected from scandium, yttrium, lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, or combinations thereof. In some embodiments, the magnetic component comprises a rare earth mineral. In some embodiments, the rare earth mineral comprises a natural magnet. In some embodiments, the magnetic particles comprise iron. In some embodiments, the magnetic particles comprise a ferromagnetic material. In some embodiments, the ferromagnetic material is a soft ferromagnetic material. In some embodiments, the soft ferromagnetic material comprises annealed iron. In some embodiments, the soft ferromagnetic material comprises zinc, nickel, manganese, or a combination thereof. In some embodiments, the soft ferromagnetic material comprises manganese-zinc ferrite (Mn a Zinc (l-A) Fe 2 O 4 ) and nickel-zinc ferrite (Ni a Zinc (l-a) Fe 2 O 4 In some embodiments, the ferromagnetic material is a hard ferromagnetic material. In some embodiments, the hard ferromagnetic material comprises alnico or ferrite. In some embodiments, the hard ferromagnetic material is selected from strontium ferrite, SrFe 12 O 19 (SrO 6Fe 2 O 3 ), barium ferrite, BaFe 12 O 19 (BaO·6Fe 2 O 3 ) and cobalt ferrite, CoFe2 O 4 (CoO Fe 2 O 3 In some embodiments, the magnetic component comprises a ferrite. In some embodiments, the ferrite is selected from hematite (Fe 2 O 3 In some embodiments, the magnetic component comprises a ferrite. In some embodiments, the ferrite is magnetite (Fe 3 O 4 In some embodiments, the magnetic particles are selected from Fe 3 O 4 In some embodiments, the magnetic particles include Fe 2 O 3 In some embodiments, the magnetic particles consist essentially of Fe3O4. In some embodiments, the magnetic particles consist essentially of Fe 2 O 3 It essentially consists of:

[0135] In some embodiments, the magnetic particles are of a shape selected from, but not limited to, spheres, cubes, ellipsoids, rods, capsule-shaped, tablet-shaped, featureless and random shapes.

[0136] In some embodiments, the magnetic particles have an average diameter of about 1 nm to about 100 μm. In some embodiments, the magnetic particles have an average diameter of about 20 nm to about 1 μm. In some embodiments, the magnetic particles have an average diameter of about 10 nm to about 50 nm. In some embodiments, the magnetic particles have an average diameter of about 100 nm to about 750 nm. In some embodiments, the magnetic particles have an average diameter of about 500 nm. In some embodiments, the magnetic particles have an average diameter of about 1 μm to about 15 μm. In some embodiments, the magnetic particles have an average diameter of about 2 μm to about 10 μm. In some embodiments, the magnetic particles have an average diameter of about 1 μm to about 5 μm. In some embodiments, the magnetic particles have an average diameter of about 1 μm to about 1 μm. In some embodiments, the magnetic particles have an average diameter at their widest point of about 0.5 μm to about 20 μm.

[0137] FIG. 21 shows an illustrative, but non-limiting, example of a method for using a magnetic probe to enrich an analyte in an ATPS. Using an antibody (or other binding moiety) specific for the analyte of interest, the target is captured and enriched by the magnetic probe in one of the bulk phases in the ATPS. In a complex sample containing a variety of molecules, the ATPS may non-specifically enrich molecules with similar physicochemical properties to the analyte of interest or the probe. However, by use of a magnet or an external magnetic field, further enrichment and isolation of the analyte of interest bound to the magnetic capture probe is achieved prior to detection.

[0138] In some embodiments, the device comprises a magnetic material. In some embodiments, the magnetic material is a source of a magnetic field. In some embodiments, the magnet comprises a material selected from magnetite, lodestone, cobalt, nickel, manganese, aluminum, gadolinium, dysprosium, ceramic (e.g., ferrite), and alnico. In some embodiments, the magnet comprises an ore. In some embodiments, the magnet comprises iron ore. In some embodiments, the magnet comprises a rare earth magnet. In some embodiments, the rare earth magnet is selected from samarium-cobalt magnets and neodymium-iron-boron magnets (NIB). In some embodiments, the magnet has a magnetic field strength of about 0.1 Gauss, about 0.2 Gauss, about 0.3 Gauss, about 0.4 Gauss, about 0.5 Gauss, about 0.6 Gauss, about 0.7 Gauss, about 0.8 Gauss, about 0.9 Gauss, or about 1 Gauss. In some embodiments, the magnet has a magnetic field strength of about 1 Gauss, about 2 Gauss, about 3 Gauss, about 4 Gauss, about 5 Gauss, about 6 Gauss, about 7 Gauss, about 8 Gauss, about 9 Gauss, or about 10 Gauss. In some embodiments, the magnet has a magnetic field strength of about 10 Gauss, about 20 Gauss, about 30 Gauss, about 40 Gauss, about 50 Gauss, about 60 Gauss, about 70 Gauss, about 80 Gauss, about 90 Gauss, about 100 Gauss, about 110 Gauss, about 120 Gauss, about 130 Gauss, about 140 Gauss, about 150 Gauss, about 160 Gauss, about 170 Gauss, about 180 Gauss, about 190 Gauss, about 200 Gauss, about 250 Gauss, about 300 Gauss, about 350 Gauss, about 400 Gauss, about 450 Gauss, or about 500 Gauss. In some embodiments, the magnet has a magnetic field strength of about 100 Gauss.

[0139] In some embodiments, the magnet is configured to facilitate and / or increase partitioning of the analyte of interest into the first phase solution or the second phase solution. In some embodiments, the magnet is configured to facilitate and / or increase flux of the analyte of interest through the LFA. In some embodiments, the magnet is attachable to and / or detachable from the device. In some embodiments, the magnet is disposed in / on the collector.

[0140] In some embodiments, the devices are configured to utilize and / or comprise one or more probes that interact with at least one analyte of interest, or at least two different analytes of interest, or at least three different analytes of interest, or at least four different analytes of interest, or at least five different analytes of interest, or at least seven different analytes of interest, or at least ten different analytes of interest, or at least fifteen different analytes of interest, or at least twenty different analytes of interest.

[0141] Analyte / sample of interest In various embodiments, the devices, systems and / or methods described herein detect and / or quantitate one or more analytes of interest. In some embodiments, the analytes of interest are selected from proteins, antigens, biomolecules, sugar moieties, lipids, nucleic acids, sterols, small organic molecules, and combinations thereof. In some embodiments, the analytes of interest are derived from an organism selected from the group consisting of plants, animals, viruses, fungi, protozoans, and bacteria.

[0142] In some embodiments, the analyte of interest comprises a biological molecule. In some embodiments, the biological molecule is selected from the group consisting of a nucleic acid, a protein, a lipid, a small molecule, a metabolite, a sugar, an antibody, an antigen, an enzyme, and combinations thereof. In some embodiments, the sugar is lactose.

[0143] The terms "protein," "peptide," and "polypeptide" are used interchangeably herein. In some embodiments, a protein refers to a polymer of amino acid residues. In some embodiments, a protein refers to an amino acid polymer in which one or more amino acid residues are artificial chemical analogues of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

[0144] The terms "nucleic acid" or "oligonucleotide" are used interchangeably herein. In some embodiments, the nucleic acid comprises at least two nucleotides covalently linked to each other. In some embodiments, the nucleic acid of the invention is single-stranded. In some embodiments, the nucleic acid is double-stranded. In some embodiments, the nucleic acid is triple-stranded. In some embodiments, the nucleic acid comprises a phosphodiester bond. In some embodiments, the nucleic acid comprises a nucleic acid analog. In some embodiments, the nucleic acid analog has a backbone that comprises bonds other than and / or in addition to phosphodiester bonds, such as, but not limited to, phosphoramide, phosphorothioate, phosphorodithioate, or O-methyl phosphoramidite bonds. In some embodiments, the nucleic acid analog is selected from nucleic acid analogs having a backbone selected from cationic backbones, nonionic backbones, and non-ribose backbones. In some embodiments, the nucleic acid contains one or more carbocyclic sugars. In some embodiments, the nucleic acid comprises modifications of its ribose-phosphate backbone. In some embodiments, these modifications are made to facilitate the addition of additional moieties, such as labels. In some embodiments, these modifications are made to increase the stability and half-life of such molecules in physiological environments.

[0145] In some embodiments, the biological molecule is derived from a virus. In some embodiments, the biological molecule is a virus particle. In some embodiments, the virus particle is derived from a virus selected from the group consisting of influenza virus, immunodeficiency virus, and respiratory virus. In some embodiments, the virus is selected from adenovirus, herpesvirus, papillomavirus, poxvirus, parvovirus, astrovirus, calicivirus, picornavirus, coronavirus, flavivirus, togavirus, hepevirus, retrovirus, orthomyxovirus, arenavirus, bunyavirus, filovirus, paramyxovirus, rhabdovirus, and reovirus. In some embodiments, the virus is selected from rotavirus, orbivirus, coltivirus, marburg virus, rubella virus, norwalk virus, rhinovirus, epstein-barr virus, and cytomegalovirus. In some embodiments, the virus is smallpox virus. In some embodiments, the virus is hepatitis virus. In some embodiments, the virus is rabies virus. In some embodiments, the virus is respiratory syncytial virus. In some embodiments, the virus is a mumps virus. In some embodiments, the virus is a measles virus. In some embodiments, the virus is an Ebola virus. In some embodiments, the virus is a polio virus. In some embodiments, the virus is selected from seasonal H1N1, swine H1N1, and H3N2 influenza. In some embodiments, the virus is a bacteriophage. In some embodiments, the bacteriophage is an M13 bacteriophage.

[0146] In some embodiments, the biological molecule is from a bacterium. In some embodiments, the biological molecule is a bacterial particle. In some embodiments, the bacterium is of a genus selected from Streptococcus, Chlamydia, Mycobacterium, and Neisseria. In some embodiments, the bacterium is Streptococcus mutans. In some embodiments, the bacterium is of a genus selected from the group consisting of Bacillus, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydophila, Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Treponema, Vibrio, and Yersinia. In some embodiments, the bacterium is Streptococcus pneumoiae. In some embodiments, the bacterium is Chlamydia trachomatis. In some embodiments, the bacterium is Neisseria gonorrhoeae. In some embodiments, the bacterium is Mycobacterium tuberculosis.

[0147] In some embodiments, the bacterium is Staphylococcus aureus. In some embodiments, the bacterium is Staphylococcus saprophyticus. In some embodiments, the bacterium is Escherichia coli. In some embodiments, the bacterium is Bacillus anthracis. In some embodiments, the bacterium is Clostridium botulinum. In some embodiments, the bacterium is Helicobacter pylori.

[0148] In some embodiments, the bacteria is an oral bacterium. In some embodiments, the oral bacterium causes periodontal disease. In some embodiments, the oral bacterium is selected from Porphyromonas gingivalis, Bacteroides forsythus, Treponema denticola, Prevotella intermedia, Fusobacterium nucleatum, Microaerophile bacteria Actinomyces actinomycetemcomitans, Campylobacter rectus, Eikenella corrodens, and Eubacterium species.

[0149] In some embodiments, the sample or analyte of interest is derived from a protozoan. In some embodiments, the protozoan causes a disease selected from the group consisting of amebiasis, giardiasis, toxoplasmosis, cryptosporidiosis, trichomoniasis, Chagas disease, leishmaniasis, sleeping sickness, amebic dysentery, acanthamoeba keratitis, and primary amebic meningoencephalitis. In some embodiments, the protozoan is of the genus Plasmodium, known to cause malaria. In some embodiments, the analyte of interest is Plasmodium lactate dehydrogenase (pLDH).

[0150] In some embodiments, the analyte of interest is a food allergen. In some embodiments, the food allergen is a plant biomolecule. In some embodiments, the plant biomolecule is a plant protein. In some embodiments, the plant protein is gluten. In some embodiments, the food allergen is a plant biomolecule. In some embodiments, the plant biomolecule is a plant protein. In some embodiments, the plant protein is gluten. In some embodiments, the food allergen is an animal biomolecule. In some embodiments, the animal biomolecule is a polysaccharide. In some embodiments, the polysaccharide is lactose.

[0151] In some embodiments, the analyte of interest is a protein. In some embodiments, the protein is present in and / or derived from a biological sample (e.g., blood, plasma, serum, urine, saliva, tears / ocular fluid, swab bodily fluids). In some embodiments, the protein is a biomarker. In some embodiments, the protein is a biomarker for a disease or disorder. In some embodiments, the analyte of interest is troponin or a fragment thereof. In some embodiments, the analyte of interest is transferrin or a fragment thereof.

[0152] In some embodiments, the device is configured to detect / quantify multiple analytes in a sample. In some embodiments, the ATPS concentrates multiple analytes with similar physicochemical properties. In some embodiments, the LFA can test for multiple analytes of interest. In some embodiments, the LFA comprises multiple test lines. In some embodiments, the LFA comprises a first test line having a first capture moiety specific for a first analyte of interest and a second test line having a second capture moiety specific for a second analyte of interest, the first capture moiety being different from the second capture moiety. The device can comprise about 2 test lines, about 3 test lines, about 4 test lines, about 5 test lines, about 6 test lines, about 7 test lines, about 8 test lines, about 9 test lines, or about 10 test lines for about 1 test line.

[0153] In some embodiments, the analyte of interest is present in a sample. In some embodiments, the analyte of interest is derived from the sample. In some embodiments, the analyte of interest is isolated from the sample. In some embodiments, the analyte of interest is purified from the sample.

[0154] In some embodiments, the sample is selected from the group consisting of tissue / fluid from a biological organism, a food sample, a chemical sample, a drug sample, an environmental sample, and combinations thereof.

[0155] In some embodiments, the food sample is selected from nuts, dairy products, wheat products, soy products, and egg products. In some embodiments, the food sample is beans. In some embodiments, the food is fruit.

[0156] In some embodiments, the sample is selected from the group consisting of a blood sample, a serum sample, a plasma sample, a urine sample, a saliva sample, a tear / ocular fluid sample, a swab sample, and combinations thereof.

[0157] In some embodiments, the sample is from a source selected from the group consisting of bacteria, viruses, protozoa, algae, fungi, drugs, pathogens, toxins, environmental contaminants, and components thereof, and combinations thereof. In some embodiments, the sample is a biological warfare agent. In some embodiments, the sample is a water sample. In some embodiments, the water sample is from a water testing facility. In some embodiments, the water sample is from a water purification facility.

[0158] In some embodiments, the sample is from a building, an inanimate structure, or a chemical solution, hi some embodiments, the sample is from carpet, paint, treated wood, deodorant, cleaning product, cosmetics, clothing, insect repellent, deodorant, floor, wall, ceiling, linoleum, plastic, insect spray, and dental gel / paste.

[0159] II. Method Disclosed herein are methods for detecting one or more analytes of interest in a sample. Also provided are methods for quantifying one or more analytes of interest in a sample, comprising applying the sample to any one of the devices disclosed herein and quantifying the analyte(s) of interest on a lateral flow assay. Also provided are methods for concentrating an analyte of interest in a sample. In various embodiments, the method comprises applying a sample to any one of the devices disclosed herein and detecting the presence or absence of the analyte of interest on a lateral flow assay. In various embodiments, the method comprises applying a sample to any one of the devices disclosed herein and concentrating the analyte(s) of interest in an aqueous two-phase system and / or on a lateral flow assay. Disclosed herein are methods for partitioning / separating one or more analytes of interest in a sample. In various embodiments, the method includes applying a sample to any one of the devices disclosed herein and partitioning / separating the analytes of interest in an aqueous two-phase system and / or on a lateral flow assay. In some embodiments, the method(s) include applying the sample to an ATPS. In some embodiments, the method(s) include applying the sample to an LFA. In some embodiments, the method includes applying the sample to an LFA integrated with an ATPS.

[0160] In some embodiments, the method includes applying the sample to an ATPS, hi some embodiments, the method includes applying the sample to an LFA, where the ATPS is dehydrated on the LFA.

[0161] In some embodiments, the analyte of interest is concentrated in the phase solutions or at the interface of the ATPS. In some embodiments, the method includes extracting the concentrated analyte of interest from the phase solutions or at the interphase and applying the analyte to the LFA. In some embodiments, the method includes contacting the ATPS with a collector, e.g., as described herein, where the analyte of interest, probe and / or probe-analyte complex have an affinity for and interact with the collector. In some embodiments, the method includes detecting the analyte of interest on the collector when the collector is inserted into the detection well (FIG. 11).

[0162] In some embodiments, the method includes extracting the analyte of interest from the sample. In some embodiments, extracting the analyte of interest may further include using an ATPS. In some embodiments, extracting the analyte of interest may include crushing, lysing, grinding, blending, chopping, stirring, centrifuging, or cutting the sample. In some embodiments, to test for food allergens from solid foods, the sample is first ground. In some embodiments, the analyte of interest is extracted from the ground sample by an extraction buffer. In some embodiments, the ground sample is mixed with an ATPS liquid to extract the analyte of interest from the sample.

[0163] In some embodiments, the method further comprises mixing the sample and / or analyte of interest with a probe. In some embodiments, the method further comprises mixing the sample and / or analyte of interest with a probe to form a probe-analyte complex prior to applying the sample and / or analyte of interest to the ATPS and / or LFA. In some embodiments, the method further comprises washing the probe-analyte complex prior to applying the sample and / or analyte of interest to the ATPS and / or LFA.

[0164] III.Use In some embodiments, the methods and devices described herein are used to detect an analyte of interest in a sample. In some embodiments, the methods and devices described herein are used to quantify an analyte of interest in a sample. In some embodiments, the methods and devices described herein are used to detect and quantify an analyte of interest in a sample.

[0165] In some aspects, the methods described herein include diagnosing a disease or disorder. In some embodiments, the methods may include observing the results on the LFA of the device disclosed herein and determining a diagnosis based on the results. In certain cases, the results of the assays determine the direction of the diagnosis, while in other contexts, the results of the assays may be evaluated in the context of differential diagnosis. In some embodiments, diagnosis of a disease or disorder requires further testing with a further device.

[0166] infectious disease In some embodiments, the disease or disorder is an infectious disease. In some embodiments, the infectious disease is a respiratory infection. In some embodiments, the infectious disease is influenza. In some embodiments, the infectious disease is a tropical disease.

[0167] In some embodiments, the infectious disease is selected from the common cold, cervical cancer, herpes, smallpox, chickenpox, shingles, hepatitis, rabies, mumps, and polio. In some embodiments, the infectious disease is tuberculosis. In some embodiments, the infectious disease is Ebola. In some embodiments, the infectious disease is malaria. In some embodiments, the infectious disease is measles. In some embodiments, the infectious disease is whooping cough. In some embodiments, the infectious disease is tetanus. In some embodiments, the infectious disease is meningitis. In some embodiments, the infectious disease is syphilis. In some embodiments, the infectious disease is hepatitis B.

[0168] sexually transmitted diseases In some embodiments, the disease or disorder is a sexually transmitted disease, hi some embodiments, the sexually transmitted disease is selected from chlamydia, gonorrhea, herpes (HSV-1, HSV-2), human papillomavirus infection, syphilis, hepatitis B, hepatitis C, hepatitis A, bacterial vaginosis, pubic lice, scabies, trichomoniasis, amebiasis, cryptosporidium, giardiasis, candidiasis, and shigellosis.

[0169] Periodontal disease In some embodiments, the disease or disorder is periodontal disease. In some embodiments, the periodontal disease is gingivitis. In some embodiments, the periodontal disease is periodontitis. In some embodiments, the periodontal disease is caused by the presence of the bacterium Streptococcus mutans in the oral cavity.

[0170] Allergens / Toxins In some embodiments, the analyte of interest is a contaminant in a liquid or food sample. In some embodiments, the analyte of interest is an allergen in a liquid or food sample. In some embodiments, the allergen is selected from dairy allergy, egg allergy, soy allergy, wheat allergy, shellfish allergy, and nut allergy. In some embodiments, the nut allergy is peanut allergy. In some embodiments, the disease or disorder is an intolerance or sensitivity to a food or food component. In some embodiments, the disease or disorder is gluten intolerance.

[0171] In some embodiments, the analyte of interest is an environmental toxin. The environmental toxin is selected from pesticides and herbicides. In some embodiments, the environmental toxin is selected from molds and fungi. In some embodiments, the environmental toxin is selected from polychlorinated biphenyls (PCBs) and phthalates. In some embodiments, the environmental toxin is selected from volatile organic compounds, dioxins, asbestos, heavy metals, chloroform, and chlorine.

[0172] Various embodiments contemplated herein include, but are not necessarily limited to, one or more of the following, and combinations thereof:

[0173] Embodiment 1: A device for detection and / or quantification of an analyte of interest in a sample comprising: a. a lateral flow assay (LFA); and b. an aqueous two-phase system (ATPS) comprising a mixed phase solution that separates into a first phase solution and a second phase solution.

[0174] Embodiment 2: The device of embodiment 1, wherein said separation of said mixed phase into a first phase solution and a second phase solution occurs within said LFA.

[0175] Embodiment 3: The device of embodiment 1, wherein said separation of said mixed phase into a first phase solution and a second phase solution does not occur within said LFA.

[0176] Embodiment 4: The device of any one of embodiments 1-3, wherein the analyte of interest contacts the mixed phase solution and the analyte of interest partitions into the first phase solution or the second phase solution.

[0177] Embodiment 5: The device of any one of embodiments 1-3, wherein the analyte of interest contacts the mixed phase solution and the analyte of interest partitions at the interface between the first phase solution and the second phase solution.

[0178] Embodiment 6: The device of embodiment 4 or 5, wherein said analyte of interest is concentrated upon dispensing.

[0179] Embodiment 7: The device of any one of embodiments 1-6, wherein the first phase solution comprises a micellar solution and the second phase solution comprises a polymer.

[0180] Embodiment 8: The device of any one of embodiments 1-6, wherein the first phase solution comprises a micellar solution and the second phase solution comprises a salt.

[0181] Embodiment 9: The device of embodiment 7 or 8, wherein the micellar solution comprises a surfactant.

[0182] Embodiment 10: The device of embodiment 9, wherein the surfactant is selected from the group consisting of ionic surfactants, zwitterionic surfactants, and non-ionic surfactants.

[0183] Embodiment 11: A device described in any one of embodiments 7 to 10, wherein the micellar solution contains Triton-X.

[0184] Embodiment 12: The device of any one of embodiments 1-6, wherein the first phase solution comprises a first polymer and the second phase solution comprises a second polymer.

[0185] Embodiment 13: The device of embodiment 12, wherein the first / second polymer is selected from polyethylene glycol, polypropylene glycol, and dextran.

[0186] Embodiment 14: The device of any one of embodiments 1-6, wherein the first phase solution comprises a polymer and the second phase solution comprises a salt.

[0187] Embodiment 15: The device of embodiment 14, wherein the first phase solution comprises polyethylene glycol and the second phase solution comprises potassium phosphate.

[0188] Embodiment 16: The device of any one of embodiments 1-6, wherein the first phase solution is selected from Component 1 in Table 1 and the second phase solution is selected from Component 2 in Table 1.

[0189] Embodiment 17: The device of any one of embodiments 1 to 6, wherein the analyte of interest is selected from the group consisting of proteins, antigens, biomolecules, small organic molecules, sugar moieties, lipids, nucleic acids, sterols, and combinations thereof.

[0190] Embodiment 18: The device of embodiment 17, wherein said analyte of interest is derived from an organism selected from the group consisting of a plant, an animal, a virus, a protozoa, a fungus, and a bacterium.

[0191] Embodiment 19: The device of any one of embodiments 1 to 17, further comprising a probe that interacts with said analyte of interest.

[0192] Embodiment 20: The device of embodiment 19, comprising one or more probes that interact with at least one analyte of interest, or at least two different analytes of interest, or at least three different analytes of interest, or at least four different analytes of interest, or at least five different analytes of interest, or at least seven different analytes of interest, or at least ten different analytes of interest, or at least fifteen different analytes of interest, or at least twenty different analytes of interest.

[0193] Embodiment 21: The device of embodiment 19 or 20, comprising at least 2 different probes, or at least 3 different probes, or at least 4 different probes, or at least 5 different probes, or at least 7 different probes, or at least 10 different probes, or at least 15 different probes, or at least 20 different probes.

[0194] Embodiment 22: The device of embodiment 19, wherein the probe comprises a material selected from the group consisting of synthetic polymers, metals, minerals, glass, quartz, ceramics, biological polymers, plastics, and combinations thereof.

[0195] Embodiment 23: The device of embodiment 19, wherein the probe comprises a polymer selected from the group consisting of polyethylene, polypropylene, cellulose, chitin, nylon, polyoxymethylene, polytetrafluoroethylene, polyvinyl chloride, and combinations thereof.

[0196] Embodiment 24: The device of embodiment 19, wherein said probe comprises a biological polymer selected from the group consisting of dextran, polypropylene, polyethylene glycol, and combinations thereof.

[0197] Embodiment 25: The device of embodiment 19, wherein the probe comprises a metal selected from the group consisting of gold, silver, platinum, and combinations thereof.

[0198] Embodiment 26: The device of embodiment 19, wherein the probe comprises a nanoparticle.

[0199] Embodiment 27: The device of claim 19, wherein the nanoparticles are gold nanoparticles.

[0200] Embodiment 28: The device of any one of embodiments 17 to 27, wherein the probe comprises a coating.

[0201] Embodiment 29: The device of embodiment 28, wherein the coating comprises a polymer selected from polypropylene glycol and polyethylene glycol.

[0202] Embodiment 30: The device of embodiment 28, wherein the coating comprises dextran.

[0203] Embodiment 31: The device of embodiment 28, wherein the coating comprises a hydrophilic protein.

[0204] Embodiment 32: The device of embodiment 28, wherein the coating comprises serum albumin.

[0205] Embodiment 33: The device of any one of embodiments 28 to 32, wherein the coating has an affinity for the first phase solution or the second phase solution.

[0206] Embodiment 34: The device of any one of embodiments 28 to 33, wherein the probe further comprises a binding moiety that binds to the analyte of interest.

[0207] Embodiment 35: The device of embodiment 34, wherein the binding moiety is selected from the group consisting of an antibody, a lectin, a protein, a glycoprotein, a nucleic acid, a small molecule, a polymer, a lipid, and combinations thereof.

[0208] Embodiment 36: The device of embodiment 34, wherein the binding moiety is an antibody or an antibody fragment.

[0209] Embodiment 37: A device of any one of embodiments 18 to 35, wherein the probe comprises magnetic particles.

[0210] Embodiment 38: The device of embodiment 37, further comprising a magnet.

[0211] Embodiment 39: The device of embodiment 38, wherein the magnet is configured to facilitate and / or increase partitioning of the analyte of interest into the first phase solution or the second phase solution.

[0212] Embodiment 40: The device of embodiment 38, wherein the magnet is configured to facilitate and / or increase the flow of the analyte of interest through the LFA.

[0213] Embodiment 41: The device of any one of embodiments 38 to 40, wherein the magnet is attachable to and / or detachable from the device.

[0214] Embodiment 42: The device of any one of embodiments 1 to 41, further comprising a collector configured to be placed in contact with the ATPS, wherein the analyte of interest is distributed at an interface between the collector and the first phase solution and / or the second phase solution.

[0215] Embodiment 43: The device of embodiment 42, wherein the collector comprises a material selected from plastic, mesoporous material, silica, polypropylene, magnet, material with pores, material with grooves, and combinations thereof.

[0216] Embodiment 44: The device of any one of embodiments 19 to 43, wherein the probe comprises a detectable label.

[0217] Embodiment 45: The device of embodiment 44, wherein the detectable label is selected from the group consisting of a chromogenic label, a fluorescent label, an enzymatic label, a heat-generating label, a radioactive label, and combinations thereof.

[0218] Embodiment 46: The device of any one of embodiments 1 to 45, wherein the LFA comprises a porous matrix.

[0219] Embodiment 47: The device of embodiment 46, wherein the porous matrix is ​​sufficiently porous to allow the mixed phase solution, the first phase solution, the second phase solution, and / or the analyte of interest to flow through the LFA.

[0220] Embodiment 48: The device of embodiment 46 or 47, wherein the porous matrix is ​​sufficiently long and / or deep for the mixed phase solution, first phase solution, second phase solution and / or analytes of interest to flow vertically and / or horizontally through the LFA.

[0221] Embodiment 49: The device of any one of embodiments 46 to 48, wherein a first phase solution flows through the porous matrix at a first velocity and a second phase solution flows through the porous matrix at a second velocity, the first velocity and the second velocity being different.

[0222] Embodiment 50: The device of any one of embodiments 46 to 49, wherein the porous matrix comprises a material selected from cellulose, fiberglass, nitrocellulose, polyvinylidene fluoride, nylon, charge-modified nylon, polyethersulfone, and combinations thereof.

[0223] Embodiment 51: The device of any one of embodiments 1 to 50, wherein said LFA comprises a capture moiety for an analyte of interest that interacts with said analyte of interest.

[0224] Embodiment 52: The device of any one of embodiments 1 to 51, wherein, when a competitive assay is used, the LFA is configured to provide a competitive assay.

[0225] Embodiment 53: The device of any one of embodiments 1 to 51, wherein said LFA comprises said analyte of interest.

[0226] Embodiment 54: The device of embodiment 53, wherein when a sandwich assay is used, the LFA is configured to provide a sandwich assay.

[0227] Embodiment 55: The device of any one of embodiments 18 to 54, wherein the LFA comprises a probe capture moiety that interacts with the probe or a component thereof.

[0228] Embodiment 56: The device of any one of embodiments 1 to 55, wherein the components of the first phase solution and / or the components of the second phase solution are dehydrated on and / or in the LFA strip, and upon addition of the sample, the mixed phase solution is distributed into the first phase solution and the second phase solution.

[0229] Embodiment 57: The device of any one of embodiments 1 to 56, wherein the LFA comprises a well having a volume sufficient to accommodate a solution selected from the group consisting of at least a portion of the ATPS, at least a portion of the first phase solution, at least a portion of the second phase solution, a resuspension solution of the analyte of interest, and combinations thereof.

[0230] Embodiment 58: The device of embodiment 57, wherein the sufficient volume is from about 1 nanoliter to about 5 milliliters.

[0231] Embodiment 59: The device of embodiment 57, wherein the well is located at a location of the LFA selected from a corner, an end, a center, a connection, an off-center portion, and a bend.

[0232] Embodiment 60: A device of any one of embodiments 56 to 58, wherein the well comprises a pad.

[0233] Embodiment 61: The device of embodiment 60, wherein said pad is selected from the group consisting of a salt pad, a buffer pad, a filter pad, a surfactant pad, a probe pad, a polymer pad, and combinations thereof.

[0234] Embodiment 62: A device of any one of embodiments 1 to 61, wherein the LFA is configured according to a structure selected from the structures shown in Figures 16, 20 and 62 to 70.

[0235] Embodiment 63: A device of any one of embodiments 1 to 62, wherein the LFA comprises multiple paths.

[0236] Embodiment 64: The device of any one of embodiments 1 to 63, wherein the LFA comprises a dry receiving paper.

[0237] Embodiment 65: A device of any one of embodiments 1 to 64, comprising a running buffer.

[0238] Embodiment 66: A device according to any one of embodiments 1 to 65, comprising a port for introduction of a sample into the device.

[0239] Embodiment 67: The device of embodiment 66, wherein the port is connected to an ATPS.

[0240] Embodiment 68: The device of embodiment 66, wherein the ATPS and the LFA are integrated and the port is connected to the LFA.

[0241] Embodiment 69: The device of any one of embodiments 66 to 68, wherein the port comprises a structure selected from the group consisting of a tube, a funnel, a valve, a syringe, a straw, a flow path, a plunger, a piston, a pump, and combinations thereof.

[0242] Embodiment 70: A device of any one of embodiments 1 to 69, which does not require a power source.

[0243] Embodiment 71: The device of any one of embodiments 1 to 70, wherein the ATPS and the LFA are integrated prior to use of the device.

[0244] Embodiment 72: The device of any one of embodiments 1 to 70, wherein the ATPS and the LFA are separated prior to use of the device.

[0245] Embodiment 73: The device of embodiment 72, configured to insert the LFA into the ATPS.

[0246] Embodiment 74: A device of embodiment 72 or 73, comprising: a: a first component comprising a chamber for accommodating the ATPS; and b: a second component comprising the LFA.

[0247] Embodiment 75: A device of any one of embodiments 1 to 74, comprising an actuator for delivering the sample and / or analytes of interest into the ATPS.

[0248] Embodiment 76: A device of any one of embodiments 1 to 75, comprising an actuator that delivers a solution to the LFA.

[0249] Embodiment 77: The device of embodiment 76, wherein the solution is selected from the group consisting of a mixed phase solution, a first phase solution, a second phase solution, and combinations thereof.

[0250] Embodiment 78: A device of any one of embodiments 1 to 77, wherein the ATPS and the LFA are contained within a single housing.

[0251] Embodiment 79: A device of any one of embodiments 1 to 78, which is a portable device.

[0252] Embodiment 80: A method for detecting and / or quantifying a target analyte in a sample, comprising: a: applying the sample to a device of any one of embodiments 1 to 78; and b: detecting the presence and / or quantifying the target analyte on the LFA.

[0253] Embodiment 81: The method of embodiment 80, comprising applying the sample to the ATPS.

[0254] Embodiment 82: The method of embodiment 80, comprising applying the sample to the LFA integrated with the ATPS.

[0255] Embodiment 83: The method of any one of embodiments 80 to 82, comprising concentrating said analyte of interest in said ATPS.

[0256] Embodiment 84: The method of any one of embodiments 80 to 83, comprising concentrating said analyte of interest in said LFA.

[0257] Embodiment 85: The method of any one of embodiments 80 to 84, wherein the sample is selected from the group consisting of tissue / fluid from a biological organism, a food sample, a chemical sample, a drug sample, an environmental sample, and combinations thereof.

[0258] Embodiment 86: The method of any one of embodiments 80 to 85, wherein the sample is selected from the group consisting of a blood sample, a swab sample, a serum sample, a plasma sample, a urine sample, a saliva sample, and combinations thereof.

[0259] Embodiment 87: The method of any one of embodiments 80 to 86, wherein the sample is derived from a source selected from the group consisting of bacteria, viruses, protozoa, algae, fungi, drugs, pathogens, mammals, toxins, environmental pollutants, and components thereof, and combinations thereof.

[0260] Embodiment 88: The method of any one of embodiments 80 to 87, wherein said analyte of interest comprises a biological molecule.

[0261] Embodiment 89: The method of embodiment 88, wherein the biological molecule is selected from the group consisting of nucleic acids, proteins, metabolites, lipids, small molecules, sugars, antibodies, antigens, enzymes, and combinations thereof.

[0262] Embodiment 90: A paper fluidic device for detection of an analyte of interest in a sample comprising a porous matrix configured: a: to receive and / or contain an ATPS or a component thereof; and b: configured and having sufficient porosity to allow the ATPS or a component thereof to flow through the porous matrix when the ATPS or a component thereof is in a fluid phase.

[0263] Embodiment 91: A paper fluidic device of embodiment 90, containing the ATPS or a component thereof.

[0264] Embodiment 92: The paper fluidic device of embodiment 90 or 91, wherein the ATPS or a component thereof is selected from the group consisting of a first phase solution, a second phase solution, and a mixed phase solution comprising a mixture of a first phase solution and a second phase solution.

[0265] Embodiment 93: The paper fluidic device of embodiment 91 or 92, wherein said ATPS or components thereof are dehydrated on and / or in at least a first portion of said porous matrix.

[0266] Embodiment 94: The paper fluidic device of embodiment 93, wherein the first portion of the porous matrix has a different width than the second portion of the porous matrix.

[0267] Embodiment 95: The paper-fluidic device of embodiment 94, configured such that application of the sample to the device hydrates the ATPS, thereby providing the ATPS or a component thereof in a fluid phase.

[0268] Embodiment 96: The paper fluidic device of any one of embodiments 92 to 95, further comprising a well for containing a solution selected from the mixed phase solution, the first phase solution, the second phase solution, the sample, the probe, and combinations thereof.

[0269] Embodiment 97: The paper fluidic device of embodiment 96, wherein the well comprises an actuator for releasing the contents of the well into and / or onto the porous matrix.

[0270] Embodiment 98: The device of embodiment 96 or 97, wherein the well comprises a pad.

[0271] Embodiment 99: The device of embodiment 98, wherein said pad is selected from the group consisting of a salt pad, a buffer pad, a filter pad, a surfactant pad, a probe pad, a polymer pad, and combinations thereof.

[0272] Embodiment 100: The paper fluidic device of any one of embodiments 92 to 99, wherein during use, the first phase solution and the second phase solution flow through said porous matrix at different rates.

[0273] Embodiment 101: The paper fluidic device of any one of embodiments 92 to 100, wherein during use, the first phase solution and the second phase solution flow through the porous matrix in different directions.

[0274] Embodiment 102: The paper fluidic device of any one of embodiments 92 to 101, wherein the first phase solution comprises a micellar solution and the second phase solution comprises a polymer.

[0275] Embodiment 103: The paper fluidic device of any one of embodiments 92 to 101, wherein the first phase solution comprises a micellar solution and the second phase solution comprises a salt.

[0276] Embodiment 104: The paper fluidic device of embodiment 102 or 103, wherein the micellar solution comprises a surfactant.

[0277] Embodiment 105: The paper fluidic device of embodiment 104, wherein the surfactant is selected from the group consisting of non-ionic surfactants, surfactants containing zwitterions, and ionic surfactants.

[0278] Embodiment 106: The paper fluidic device of embodiment 105, wherein said micellar solution comprises Triton-X.

[0279] Embodiment 107: The paper fluidic device of any one of embodiments 93 to 101, wherein the first phase solution comprises a polymer and the second phase solution comprises a polymer.

[0280] Embodiment 108: The paper fluidic device of any one of embodiments 92 to 101, wherein the first phase solution comprises a polymer and the second phase solution comprises a salt.

[0281] Embodiment 109: The paper fluidic device of embodiment 108, wherein the first phase solution comprises polyethylene glycol and the second phase solution comprises potassium phosphate.

[0282] Embodiment 110: The paper fluidic device of any one of embodiments 92 to 101, wherein the first phase solution is selected from component 1 in Table 1, and the second phase solution is selected from component 2 in Table 1.

[0283] Embodiment 111: A paper fluidic device according to any one of embodiments 90 to 110, constructed according to a structure selected from the structures shown in Figures 16, 20 and 62 to 70.

[0284] Embodiment 112: A paper fluidic device according to any one of embodiments 90 to 111, wherein the porous matrix comprises a first passage and a second passage.

[0285] Embodiment 113: The paper fluidic device of embodiment 112, wherein the first phase solution flows preferentially through the first path and the second phase solution flows preferentially through the second path.

[0286] Embodiment 114: A paper fluidic device according to any one of embodiments 90 to 113, comprising a probe that binds to said analyte of interest to form a probe-analyte complex.

[0287] Embodiment 115: The paper fluidic device of any one of embodiments 90 to 114, wherein said analyte of interest is bound to the probe in a probe-analyte complex.

[0288] Embodiment 116: The paper fluidic device of embodiment 114 or 115, wherein the probe comprises magnetic particles.

[0289] Embodiment 117: The paper fluidic device of embodiment 116, further comprising a magnetic field oriented to attract the magnetic particles to a portion of the porous matrix, the force of the magnetic field acting on the magnetic particles enhancing the flow of the probe-analyte complex toward the portion of the porous matrix.

[0290] Embodiment 118: The paper fluidic device of any one of embodiments 114 to 117, wherein the probe comprises a polymer selected from the group consisting of polyethylene, polypropylene, nylon, polyoxymethylene, polytetrafluoroethylene (Teflon (registered trademark)), dextran, polyvinyl chloride, and combinations thereof.

[0291] Embodiment 119: The paper fluidic device of any one of embodiments 114 to 118, wherein the probe comprises a biological polymer selected from the group consisting of cellulose and chitin.

[0292] Embodiment 120: A paper fluidic device of any one of embodiments 114 to 119, wherein the probe comprises a metal selected from the group consisting of gold, silver, titanium, stainless steel, aluminum, platinum, and alloys thereof, and combinations thereof.

[0293] Embodiment 121: A paper fluidic device of any one of embodiments 114 to 120, wherein the probe comprises nanoparticles.

[0294] Embodiment 122: The paper fluidic device of embodiment 121, wherein the nanoparticles are gold nanoparticles.

[0295] Embodiment 123: A paper fluidic device of any one of embodiments 114 to 122, wherein the probe comprises a coating.

[0296] Embodiment 124: The paper fluidic device of embodiment 123, wherein the coating comprises a polymer selected from polypropylene glycol and polyethylene glycol.

[0297] Embodiment 125: The paper fluidic device of embodiment 123, wherein the coating comprises dextran.

[0298] Embodiment 126: The paper fluidic device of embodiment 123, wherein the coating comprises a hydrophilic protein.

[0299] Embodiment 127: The paper fluidic device of embodiment 123, wherein the coating comprises serum albumin.

[0300] Embodiment 128: The paper fluidic device of any one of embodiments 123 to 127, wherein the coating has an affinity for the first phase solution or the second phase solution.

[0301] Embodiment 129: The paper fluidic device of any one of embodiments 114 to 128, wherein said probe comprises a binding moiety that binds to said analyte of interest.

[0302] Embodiment 130: The paper fluidic device of embodiment 129, wherein the binding moiety is selected from the group consisting of antibodies, lectins, proteins, metabolites, glycoproteins, nucleic acids, small molecules, polymers, and lipids.

[0303] Embodiment 131: The paper fluidic device of embodiment 129, wherein the binding moiety is an antibody or an antibody fragment.

[0304] Embodiment 132: The paper fluidic device of any one of embodiments 114 to 131, wherein the probe comprises a detectable label.

[0305] Embodiment 133: The paper fluidic device of embodiment 132, wherein the detectable label is selected from the group consisting of a chromogenic label, a fluorescent label, an enzymatic label, a pyrogenic label, a radioactive label, and combinations thereof.

[0306] Embodiment 134: A paper fluidic device of any one of embodiments 90 to 133, comprising a dry receiving paper, wherein the first phase solution or the second phase solution preferentially flows through the porous matrix toward the dry receiving paper.

[0307] Embodiment 135: A paper fluidic device of any one of embodiments 90 to 134, comprising a running buffer, wherein the first phase solution and / or the second phase solution flows faster through the porous matrix upon contact with the running buffer.

[0308] Embodiment 136: A paper fluidic device of any one of embodiments 90 to 135, wherein the porous matrix comprises a material selected from cellulose, fiberglass, nitrocellulose, polyvinylidene fluoride, nylon, charge-modified nylon, polyethersulfone, and combinations thereof.

[0309] Embodiment 137: A paper fluidic device of any one of embodiments 90 to 136, wherein the porous matrix comprises a probe capture portion, which during use interacts with the probe or a component thereof.

[0310] Embodiment 138: A paper fluidic device of any one of embodiments 90 to 137, wherein the porous matrix comprises a capture portion for an analyte of interest, and in use, the capture portion for the analyte of interest interacts with the analyte of interest or a component thereof.

[0311] Embodiment 139: The paper fluidic device of embodiment 138, wherein the LFA is configured to provide a sandwich assay when the paper fluidic device is in use.

[0312] Embodiment 140: A paper fluidic device of any one of embodiments 90 to 137, wherein the porous matrix contains the analyte of interest.

[0313] Embodiment 141: The paper fluidic device of embodiment 140, wherein the LFA is configured to provide a competitive assay when the paper fluidic device is in use.

[0314] Embodiment 142: A paper fluidic device of any one of embodiments 90 to 141, wherein the porous matrix is ​​configured to concentrate the analyte of interest as it flows through the porous matrix.

[0315] Embodiment 143: A paper fluidic device of any one of embodiments 90 to 142, further comprising a control analyte, and quantification of the analyte of interest is performed by comparing the control analyte with the analyte of interest on the porous matrix.

[0316] Embodiment 144: A method for detecting or quantifying a target analyte in a sample, comprising: a: applying the sample to a device of any one of embodiments 90 to 143; and b: detecting the presence and / or quantifying the target analyte. EXAMPLES

[0317] The following specific non-limiting examples should be construed as merely illustrative and should not be construed as limiting the present disclosure in any way. Without further elaboration, it is believed that one skilled in the art can utilize the present disclosure to its fullest extent based on the description herein. All publications cited herein are incorporated herein by reference in their entirety.

[0318] Example 1. ATPS using PEGylated gold nanoprobes for concentration by magnet or solid in two-phase systems Two approaches were investigated for integrating aqueous two-phase systems (ATPS) with lateral flow immunoassays (LFA) to improve the sensitivity of LFA. The first approach utilized magnets to collect magnetic nanoprobes that could capture target proteins from the ATPS solution. The second approach utilized a newly discovered phenomenon where the surface chemistry was engineered to allow nanoprobes to be distributed at the solid-liquid interface between the ATPS and a polypropylene surface, allowing for fast and easy collection without the use of a syringe or magnet.

[0319] It was speculated that in order to efficiently collect magnetic nanoparticles, the size of the magnetic nanoparticles needs to be large enough to respond to a magnetic field, but if the size of the particles is too large, the particles may have difficulty moving through the LFA test strip.

[0320] To address this issue, we first used an ATPS together with gold-coated magnetic nanoprobes (GMPs) to capture target biomolecules in the sample solution. To avoid using expensive bulky magnets to collect particles or large nanoprobes in solution that may respond to a weak magnetic field, we first used the ATPS to passively concentrate the GPMs in a small volume into one of the two bulky phases of the APTS. Subsequently, we used an inexpensive, mobile magnet to rapidly recover the GPMs and capture the biomolecules. Because the gold coating of the GMPs also acts as a chromogenic indicator for LFAs, we applied the GMPs directly to the IFA strips.

[0321] In addition to improving the magnetic extraction of GMP using ATPS, it was found that the phase separation of ATPS benefited from the use of GMP and a magnetic field. The PEG-salt ATPS phases were completely separated in the order of hours. Because the concentration of nanoprobes in the lower PEG-poor phase remained constant as a result of minimal entrainment of PEG-rich domains in the macroscopic PEG-poor phase, extraction of concentrated samples could occur before the system reached equilibrium, but 30 min was still required to achieve extraction of sufficient amounts of the lower phase for subsequent LFA. Alternatively, GMP in the ATPS was partitioned to the PEG-poor domains extremely and almost instantly once phase separation was induced. The PEG-poor domains containing GMP can find each other and coalesce more easily in the presence of a magnetic field, presumably due to GMP pulling on the PEG-poor domains in response to the magnetic field. This force, together with gravity, further drives the coalescence of the PEG-poor domains, thus resulting in accelerated phase separation, resulting in sufficient amounts of the lower phase for extraction within only 10 min.

[0322] In this study, GMPs were first prepared that were small enough to migrate through the LFA test strip without flow problems. These GMPs were then used to capture the model protein transferrin (Tf) in a PEG salt ATPS solution resulting in a volume ratio of 9:1 (top phase: bottom phase). A small magnet (1 / 8" diameter x 1 / 32" thick) was placed in the solution to accelerate phase separation. The GMPs and captured protein were collected after 10 minutes, the small magnet was removed, and the GMP-Tf complex was applied directly to the LFA for detection.

[0323] The aforementioned approach effectively concentrated and detected Tf using an ATPS with a magnetic nanoprobe, but still required a small magnet. The following approach explored the possibility of extracting the target nanoprobe complex without using an external magnetic field, but instead by manipulating the surface chemistry of PEGylated gold nanoprobes (PGNPs) to partition excessively at the solid-liquid interface between the ATPS and a polypropylene (PP) surface. This unique phenomenon was found to be ATPS-specific, since PGNPs did not partition at the solid-liquid interface when an aqueous solution composed of only polymer or salt was used. Furthermore, this phenomenon was also found to be material-specific, since PGNPs partition excessively on the polypropylene (PP) surface of our ATS, but not on the glass surface.

[0324] To promote the collection of target PGNP complexes at the PP-ATPS solid-liquid interface, anti-Tf antibody decorated PGNPs were added to an ATPS solution containing Tf, followed by submerging a commercial PP straw into the ATPS. The PGNPs first captured the target protein in the ATPS and then preferentially distributed onto the PP surface. After 10 min, the PP straw was removed from the ATPS, and a significant amount of the protein-GNP complexes was still adsorbed onto the surface. This effectively eliminated the need to manually extract the concentrated Tf-PGNPs using a syringe.

[0325] In this study, PGNPs were distributed at the PP-liquid interface in ATPS. These PGNPs were then used to capture Tf in a PEG salt ATPS solution resulting in a 1:1 volume ratio. A PP straw was submerged in the ATPS solution, and the PGNPs and captured protein were collected by pulling the straw out of the solution after 10 min. The PGNP-Tf complex on the straw was washed with running buffer and directly applied to the LFA for detection. Because of the minimal volume associated with the PP-ATPS interface and the small volume required to wash the PGNPs off the straw, the PGNPs were highly concentrated and the detection limit of Tf in the LFA was improved by 50-fold.

[0326] To prepare the LFA strip, an LFA test was performed that utilizes a competitive mechanism. In a competitive assay, the target of interest is immobilized on a nitrocellulose membrane to form a test line. An immobilized secondary antibody against the primary antibody on the nanoprobe (GMP or PGNP) constitutes the control line. In an LFA, when the sample first comes into contact with the nanoprobe, if the target molecule is present in the sample, it will bind to the specific antibody decorated on the nanoprobe. If the target molecules present in the sample saturate the antibodies on the nanoprobe, these nanoprobes will no longer be able to bind to the target molecules immobilized on the test strip. As a result, the nanoprobe will not form a visible band at the test line, which indicates a positive result. On the other hand, if the sample does not contain the target molecule in a concentration that can saturate the antibodies on the nanoparticles, these antibodies on the nanoparticles will be able to bind to the target molecule immobilized on the test strip and form a visible band at the test line. This indicates a negative result. Furthermore, regardless of the presence of the target molecule in the sample, the antibody on the nanoprobe will bind to the immobilized secondary antibody at the control line, which indicates that enough sample has been absorbed through the test line to reach the control line. The presence of a visible control line indicates a valid test.

[0327] To prepare GNPs, iron oxide magnetic nanoparticles with a diameter of 50 nm were coated with gold. Briefly, the iron oxide particles were diluted with 0.01 M sodium citrate and stirred for 10 minutes. Subsequently, four additions of 1% w / v gold chloride solution were applied to the iron oxide nanoparticle solution with 10 minutes of stirring intervals. The solution turned from black to maroon after the addition of gold chloride. The gold-coated magnetic nanoparticles were then collected by using a magnet.

[0328] A collector was constructed by attaching a small neodymium magnet (1 / 8" diameter x 1 / 32" thick, KJ Magnetics, Philadelphia, PA) to the end of a drinking straw. The magnet was wrapped in parafilm to prevent direct contact with the GMP in solution.

[0329] To determine whether GMP partitions excessively into the lower PEG-poor phase of the PEG-salt ATPS, 30 μL of GMP was added to 2 mL of ATPS solution resulting in a volume ratio of 9:1. To determine whether the presence of a magnet accelerates phase separation, partitioning experiments were performed with and without a magnetic collector. The volume of the lower phase was measured at 10 and 30 minutes in each experiment.

[0330] Next, we tested whether GMP could be incorporated into ATPS and LFA. First, 2 mL of 9:1 ATPS solutions spiked with various concentrations of Tf were prepared. 30 μL of GMP was applied to each solution, followed by the addition of a magnetic collector. The solutions were placed in a 25° C. water bath and incubated for 10 minutes. After the incubation period, the collector was removed from the solution and transferred to a tube containing 50 μL of running buffer. The small magnet was removed from the straw, and the GMP collected on the straw was washed off by shaking the straw in running buffer for a few seconds. The straw was then removed, and an LFA test strip was immersed in the running buffer containing the collected GMP. After 10 minutes, the test strip was removed from the tube, and an image of the test strip was taken using a Canon EOS 1000D camera (Canon USA, Inc., Lake Success, NY).

[0331] Intact gold nanoparticles were prepared to produce a clear, cherry-colored solution with a particle size of approximately 25–30 nm in diameter. To prepare PPGNPs, 320 mg of goat anti-Tf antibody was incubated with 20 mL of colloidal gold solution for 30 min, followed by the addition of thiolated PEG2000 using a 5000:1 molar ratio of PEG:NPs and further incubation for 30 min. To prevent nonspecific binding of other proteins to the surface of colloidal gold, 2 mL of 10% bovine serum albumin (BSA) solution was added to the mixture and mixed for another 15 min. The resulting solution was mixed gently on a shaker for the duration of the incubation. To remove free unbound antibody, PEG and BSA, the mixture was subsequently centrifuged at 4 °C and 9,000 g for 30 min. The pellet of PGNPs was washed with 1% BSA solution, and this washing step was repeated twice. Finally, the recovered PGNPs were resuspended in 3 mL of 0.1 M sodium borate buffer, pH 9.0.

[0332] PEG salt ATPS solutions containing various Tf concentrations resulting in a 1:1 volume ratio were first prepared in glass tubes. 5 μL of GNP solution was added to the Tf-ATPS solution to make the final volume 1 mL. The ATPS solutions were mixed thoroughly and a PP straw was inserted into each ATPS solution. The solutions were then incubated at 25° C. for 15 min. For each mixture, the PP straw was carefully removed from the ATPS solution. Assuming that the total volume of the PP-ATPS interface was approximately 1 μL, 49 μL of test buffer was used to wash the adsorbed Tf-GNP complexes from the portion of the straw that was immersed in the ATPS solution to achieve a total volume of 50 μL. The inner and outer surfaces of the straw were washed for a total of 10 seconds. The LFA test strip was then immersed vertically such that the sample pad was in contact with the mixture. The test strips were removed from the mixture after 10 minutes and images of the test strips were immediately taken using a Canon EOS 1000D camera (Canon USA, Inc., Lake Success, NY).

[0333] The GMP developed in this study achieved four functions: (1) capture the target protein in the sample, (2) partition highly into one of the bulky phases of the ATPS, (3) be collected by an inexpensive handheld magnet, and (4) directly serve as a colorimetric indicator of the LFA after extraction and application to an LFA paper strip. Functions (2) and (3) were demonstrated by placing the GMP in a PEG salt ATPS solution. As shown in Figure 21A, it was observed that the GMP partitioned highly into the lower PEG-poor phase due to the repulsive, steric, and volumetrically excluded interactions operating between the GMP and the greater number of PEG molecules in the PEG-rich phase. This allowed the GMP to concentrate in a small volume, which allowed collection by a smaller and lighter magnet (Figure 21B). Furthermore, because these GMPs were highly responsive when in close proximity to the magnet, the particles could be rapidly retrieved and transferred for subsequent detection (Figure 21C). Last but not least, by using this technique, GMP could be collected in a very small volume, which allowed for extreme enrichment of the target protein.

[0334] To determine whether GMP could improve the sensitivity of the LFA when used with the ATPS, a preliminary LFA study was performed with and without a preconcentration step (Figure 22).

[0335] The second approach to integrate ATPS with LFA was to guide PEGylated gold nanoprobes (PGNPs) to the solid-liquid interface of ATPS and polypropylene (PP) solid. Similar to the first approach, this approach no longer requires an external magnet. This mechanism requires ATPS. As shown in Figures 23A and B, PGNPs did not partition to the solid-liquid interface when aqueous solutions of only PEG or only salt were used. Instead, PGNPs appeared to be uniformly distributed throughout each aqueous solution without preferring the solid-liquid interface. Since PGNPs were found to partition to the solid-liquid interface only in the presence of phase-separated ATPS, ATPS with a 1:1 volume ratio was used in this study because this ATPS phase separates the fastest compared to ATPS with other volume ratios. Figure 23C demonstrated the extreme partitioning behavior of PGNPs on the PP straw after 10 min. The PP straw also allowed for easy extraction of the concentrated target PGNP complex. After removing the straw, the collected PGNPs were easily washed off using running buffer and directly applied to the LFA (Figure 23D).

[0336] The results of the LFA are shown in FIG. 24. Each test showed the presence of a control line, indicating that the fluid flowed completely through the strip, confirming the validity of the test. In the negative control, where Tf was absent, a test line appeared, thus indicating a true negative result. At high Tf concentrations (10 ng / μL), no test line appeared, thus indicating a true positive result. However, at low Tf concentrations without the preconcentration step, a visible test line appeared, suggesting that an insufficient amount of Tf was present in the sample to saturate the antibody decorated on the PGNPs. Thus, these tests showed false negative results. The results of the LFA using this technique are shown in the upper panel (FIG. 24, top). As false negative results appeared at 1 ng / μL in the LFA study without preconcentration, this indicated that the detection limit of Tf in the LFA is 10 ng / μL.

[0337] To improve the performance of the LFA, the same amount of PGNPs used in the single LFA test was placed in an ATPS solution with a Tf concentration of 1 ng / μL. By increasing the volume of this ATPS solution by 50 times compared to the volume used for the LFA strip, the PGNPs were able to interact with 50 times more Tf and therefore achieve a higher saturation level. However, since the PGNPs were also diluted by 50 times, they needed to be concentrated and collected before detection. By placing the PP straw in the ATPS solution, the PGNPs were highly distributed on the surface of the straw. After incubating the ATPS solution at room temperature for 10 minutes, the PP straw was removed from the solution. Unlike other adsorption phenomena where materials on solid surfaces are typically not recoverable, the PGNPs were easily recovered by rinsing the straw with running buffer. This buffer containing the PGNPs was then applied directly to the LFA. The results of the LFA using this technique are shown in the lower panel (Figure 24, bottom). The detection limit of Tf in the LFA improved from 10 ng / μL to 0.2 ng / μL, a 50-fold improvement, as 0.2 ng / μL is the lowest concentration that demonstrates a true positive result. Handheld devices are used for these two techniques, allowing the user to operate the assay with minimal steps (Figure 25).

[0338] Example 2. ATPS and dextran-coated gold nanoparticles Traditional citrate-capped or bare gold nanoparticles typically do not aggregate in aqueous solution due to electrostatic repulsive interactions resulting from the negative charge of the surface citrate ions. However, they aggregate in solutions of higher ionic strength, where van der Waals attractive interactions are greater than the electrostatic repulsive interactions. Dextran-coated gold nanoparticles were successfully made using dextran, which is both a reducing agent and a stabilizing agent, instead of citrate. The stability of dextran-coated gold nanoparticles was compared with traditional citrate-capped gold nanoparticles and commonly used PEG-coated gold nanoparticles. The phase separation behavior of the PEG-salt ATPS was optimized to allow for rapid extraction of DGNPs at room temperature. The partitioning behavior of DGNPs was then investigated in the PEG-salt ATPS. Transferrin (Tf), a common serum protein for iron transport, was selected as a model protein biomarker. The detection limit of Tf in LFA was then determined with and without a preconcentration ATPS step using DGNPs. Qualitative results of the LFA were interpreted visually, and computerized image analysis was also performed for further quantitative validation of the LFA results.

[0339] Traditional citrate-capped or bare gold nanoparticles were prepared. Briefly, 10 mL of ddHO was added to the 100-mL well of 1000 mM NaCl. 2 The 0 was heated to 100°C with stirring. Once the heated solution began to boil, 100 μL of a 1% w / v gold(III) chloride solution was added. The solution was stirred and boiled for 1 minute, after which 25 μL of a 6% w / v sodium citrate solution was added. The solution rapidly changed from dark purple to a clear dark cherry color, then cooled to room temperature and stored at 4°C.

[0340] Prior to preparing the PEG-coated (PEGylated) gold nanoparticles, the concentration of the previously prepared neat gold nanoparticle solution was determined. This concentration, used to determine the amount of PEG needed for any given PEG:gold nanoparticle molar ratio, was calculated using Beer's law with a path length of 1 cm.

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[0341] Methoxy polyethylene glycol (PEG) thiol (molecular weight 5000, Nanocs Inc., New York, NY) was used to decorate the gold nanoparticle surface with a PEG layer. Briefly, 5 mL of neat gold nanoparticle solution was first adjusted to pH 9.0 using 1 N NaoH solution. Methoxy PEG thiol was added to the particle solution at a PEG:nanoparticle molar ratio of 50,000:1 to ensure that the surface of the gold nanoparticles was sufficiently covered. The mixture was then reacted at room temperature for 30 min to allow the thiol groups of PEG to form dative bonds with the gold nanoparticles. The free PEG was removed by centrifugation at 9000 g for 30 min. The resulting PEGylated gold nanoparticles were then washed with 500 μL of ddH2O3 solution. 2 Resuspended in O.

[0342] Dextran-coated gold nanoparticles were prepared. Briefly, 12 g of dextran (molecular weight 15,000-25,000) was dissolved in 160 mL of UltraPure sterile water (Rockland Immunochemicals Inc., Gilbertsville, PA) and the solution was heated to 100 °C with stirring. When the heated solution began to boil, 2.16 mL of 1% w / v gold(III) chloride solution was added. The solution was stirred and boiled for 20 min until the color of the solution changed to blue-purple. The solution was then cooled to room temperature and stored at 4 °C. Excess dextran was then removed by centrifugation at 9000 g for 30 min.

[0343] Neat (i.e., citrate-capped), PEGylated and dextran-coated gold nanoparticle solutions were mixed separately with various concentrations of stock potassium phosphate solutions. The mixed solutions were incubated at room temperature for 30 min. The absorbance spectrum of each sample was determined using a UV-Visible spectrophotometer. The critical coagulation concentration (CCC) was identified as the salt concentration at which the absorbance spectrum of the GNP solution near the peak absorbance wavelength significantly decreased in intensity.

[0344] Stock potassium phosphate solution (5:1 dibasic:monobasic) was prepared using Dulbecco's Phosphate Buffered Saline (PBS) (Invitrogen, pH 7.4, 1.47 mM KH 2 PO 4 , 8.10 mM Na 2 HPO 4 , 138 mM NaCl, 2.67 mM KCl and 0.495 mM MgCl 2 A stock polyethylene glycol (MW 8000, VWR, Brisbane, Calif.) solution was then prepared in PBS. Using the stock solution, 2 mL of PEG-salt ATPS solution in PBS was prepared at a specific concentration of PEG and salt resulting in a PEG-rich (salt-poor):PEG-poor (salt-rich) volume ratio of 9:1. This volume ratio was measured after incubating the ATPS solution at room temperature for a minimum of 12 hours.

[0345] Iodine-125( 125I) was used to radiolabel the tyrosine residues of goat anti-Tf antibody (Bethyl Laboratories, Montgomery, TX). 125 I (MP Biomedicals, Irvine, CA) was activated with IODO-BEADS (Pierce Biotechnology, Rockford, IL). 125 I was reacted with goat anti-Tf antibody for 15 min. Radiolabeled antibody was released by using a Sephadex G15 size exclusion column. 125 The antibodies were purified from I. The specific activity and concentration of the radiolabeled antibodies were determined by phosphotungstic acid assay.

[0346] To prepare dextran-coated gold nanoparticles (DGNPs), first 10 mL of the previously prepared dextran-coated gold nanoparticle solution was adjusted to pH 9.0 using 1N NaOH. 80 μg of goat anti-Tf antibody was then added to the dextran-coated gold nanoparticle solution. The mixture was then reacted at room temperature for 30 minutes to allow the antibody to form a dative bond with the particles. Free unbound antibody was removed by centrifugation at 9000 g for 30 minutes. The collected DGNPs were further washed with 1% bovine serum albumin (BSA) solution to allow BSA to cover all free surfaces of the DGNPs to prevent nonspecific binding. Excess BSA and additional unbound antibody were removed by centrifugation at 9000 g for 30 minutes. The collected DGNPs were resuspended in 1 mL of 0.1 M sodium borate buffer (pH 9.0).

[0347] To test the partitioning behavior of DGNPs in PEG-salted ATPS, the concentration of DGNPs in each phase of the ATPS solution was quantified. Radiolabeled DGNPs were prepared by conjugating radiolabeled goat anti-Tf antibodies to dextran-coated gold nanoparticles. For each partitioning experiment, 200 μL of radiolabeled DGNP solution was added to three identical PEG-salted ATPS solutions in PBS, each with a final volume of 2 mL. The mixtures were incubated at 25°C for 30 min, 2 h, 4 h, and 12 h. The PEG-rich, salt-poor upper phase and the PEG-poor, salt-rich lower phase were carefully extracted, and the volume of each phase was measured. The amount of radiolabeled DGNPs in each phase was quantified by measuring the radioactivity of the radiolabeled antibodies bound to DGNPs using a Cobra Series Auto-Gamma Counter.

[0348] Competitive LFA was utilized in this test. A detailed diagram can be found in Figure 27. First, Tf protein was immobilized on a nitrocellulose membrane to form a test line. A secondary anti-IgG antibody, which can bind to the primary antibody of the DGNP, was also immobilized downstream of the immobilized Tf protein to form a control line. If Tf protein is not present in the sample solution, the DGNP will bind to the Tf printed on the strip and form a visible band on the test line. On the other hand, if Tf protein is present in the sample, the Tf protein will first bind to the specific antibody on the surface of the DGNP. If the DGNP is saturated with Tr protein, the DGNP will bypass the Tf printed on the strip, resulting in the absence of a visible band on the test line. Excess DGNP will still bind to the control line, indicating that fluid has actually flowed through the strip and that the results are valid. In summary, a negative test was indicated by two visible bands, one at the test line and one at the control line, while a positive test was indicated by only one visible band at the control line.

[0349] LFA without the PEG salt ATPS preconcentration step was performed before combining with ATPS. In this test, PBS solutions containing various concentrations of Tf were prepared and each Tf concentration was tested in triplicate. In each LFA strip, 10 μL of the corresponding Tf solution was mixed with 10 μL of DGNP solution and 30 μL of test buffer (0.2% BSA, 0.3% Tween 20, 0.2% sodium azide, 0.1% polyethylene glycol, 0.1 m Trizma base, pH 8) used to aid the flow of the sample in the test strip. The LFA test strip was dipped vertically into each mixture so that the sample pad was in contact with the mixture. The strip was removed after 10 minutes and the results were read visually and quantified using MATLAB®.

[0350] Subsequently, LFA with a PEG salt ATPS preconcentration step was performed. In these experiments, PBS solutions containing various Tf concentrations were first prepared, and each Tf concentration was tested in triplicate. Since the volume of the lower phase was approximately 200 μL, 100 μL of DGNP solution was added to the Tf / ATPS solution to bring the final volume to mL to achieve a similar DGNP concentration as the LFA test performed without the ATPS step. The ATPS solution was mixed thoroughly and incubated at 25° C. for 30 min. In each mixture, 20 μL of the PEG-starved lower phase was extracted and mixed with 30 μL of test buffer to match the volume used for the test without the ATPS step. The LFA test strip was immersed vertically, with the sample pad in contact with the mixture. The strip was removed after 10 min, and the results were read visually and quantified using MATLAB®.

[0351] A custom MATLAB script was written to quantitatively analyze the images obtained. Immediately after the LFA test was completed, images of the test strips were taken using a Canon EOS 1000D camera (Canon USA, Inc., Lake Success, NY). To ensure image consistency, lighting was controlled and each strip was oriented in the same direction. These images were cropped and converted to an 8-bit grayscale matrix. Intensity was averaged along the axis perpendicular to the flow and parallel to the test and control lines to generate a one-dimensional intensity map. Two maxima were identified at the control and test lines, and the distance between these two lines was calibrated using a negative control (with strong test and control lines).

[0352] To obtain the test line intensity from a set of test data, the control line was located and the test line location was determined using a pre-calibrated distance. The test line area was set to be 15 pixels wide, centered at the test line location. The baseline for this measurement was then determined by averaging the signal of two 25 pixel wide areas located on either side of the test line. The first baseline area started 25 pixels upstream of the test line location and continued 25 pixels upstream, and the other started 25 pixels downstream of the test line location and continued 25 pixels downstream. The test line intensity was then calculated as the area under the curve of the test line area to capture the effect of all DGNPs bound to the test line.

[0353] The critical coagulation concentration (CCC) values ​​of neat, PEGylated, and dextran-coated gold nanoparticle samples were measured to determine whether the dextran coating provided the necessary steric stability for gold nanoparticles in the PEG salt ATPS solution. The peak absorbance wavelength of the gold nanoparticle absorbance spectrum was approximately 535 nm. Thus, the absorbance spectrum near 535 nm for each type of gold nanoparticle was observed over a range of potassium phosphate concentrations, since potassium phosphate is the salt component of the PEG salt ATPS. The results are shown in Figure 28. In each plot, the peak absorbance value was assigned a value of 1.0 (or 100% normalized absorbance), and the absorbance values ​​for the remainder of the plot were adjusted accordingly. From these plots, it was determined that the CCC for neat gold nanoparticles was 0.3-3.4% w / w potassium phosphate, the CCC for PEGylated gold nanoparticles was 7.5-10% w / w potassium phosphate, and the CCC for the dextran-coated gold nanoparticle solution was greater than 30% w / w potassium phosphate. These results indicated that the dextran-coated gold nanoparticles were more stable than the PEGylated gold nanoparticles in the potassium phosphate salt solution. Furthermore, since 30% w / w potassium phosphate is greater than the potassium phosphate concentration in the bottom phase (approximately 10% w / w), the dextran-coated gold nanoparticles were predicted to remain stable and functional in our PEG salt ATPS solution. This was confirmed by their successful use in the studies described herein.

[0354] The partitioning behavior of radiolabeled DGNPs was first observed visually in PEG-salt ATPS with a 9:1 PEG-rich:PEG-poor volume ratio. Initially, the PEG-salt ATPS solution containing DGNPs appeared an opaque light purple color (Figure 29A). A dark purple lower phase then appeared, indicating that DGNPs partitioned heavily into the PEG-poor lower phase (Figure 29B). Over time, the lower phase increased in volume as the DGNP-rich, PEG-poor domains migrated to the bottom of the ATPS solution. In contrast, DGNPs were found to have shown minimal partitioning into the upper PEG-rich phase, as there was little purple color in the upper phase. After a 12-hour equilibration period, the volumes of the upper and lower phases no longer changed, and the upper phase became clear, suggesting that all the DGNP-rich, PEG-poor domains had reached the bottom phase of the ATPS solution (Figure 29C). After approximately 30 min, the color intensity of the lower phase did not change over time, suggesting that the DGNP concentration in the lower phase remained relatively constant from 30 min to 12 h.

[0355] The partitioning behavior of radiolabeled DGNP into the two phases was then evaluated using the partition coefficient (K DGNP ) was quantified.

number

[0356] To demonstrate that DGNPs can be predictively concentrated in PEG-salt ATPS, the concentration factor or concentration-fold improvement of DGNPs was measured in a 9:1 PEG-salt ATPS solution. The concentration factor is the concentration of DGNPs in the lower phase, C DGNP The lower part is the initial DGNP concentration, C DGNP It is defined as the division by the initial value.

number

[0357] The concentration coefficient equation can be derived starting from the molar balance equation of DGNP in ATPS solution:

number

number

[0358] Since the partition coefficient of DGNP is much less than Vlower / Vupper, the concentration ratio can be further simplified to the following:

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[0359] To demonstrate that DGNPs can be used as a chromogenic indicator for LFAs, LFAs were performed to detect Tf without a preconcentration ATPS step. The results of this test are shown in Figure 31A. Since bands were observed in the LAF strips, it was concluded that DGNPs functioned as a chromogenic indicator. Each test showed the presence of a control line, indicating that fluid flowed completely through the strips, confirming the validity of the test. In the negative control without Tf, the appearance of a test line indicated a true negative result. When high Tf concentrations were used (3.16 ng / μL and 1.0 ng / μL), the test line was absent, indicating a true positive result. However, at low concentrations such as 0.316 ng / μL, a visible test line appeared, thus indicating a false negative result. This suggested that the detection limit of Tf in the LFA without a preconcentration step was 1 ng / μL.

[0360] Utilizing a 9:1 PEG salt volume ratio solution, it was demonstrated that the detection limit of the LFA could be improved 10-fold by concentrating the DGNPs saturated with Tf. The results of this study are shown in Figure 31B. After incubating the ATPS solution at room temperature for 30 minutes, the concentrated DGNPs in the PEG-starved lower phase were extracted and applied to the LFA. As in the previous experiment, both the control and test lines appeared in the absence of Tf, indicating a valid negative result. Although no test lines appeared at higher Tf concentrations (0.316 ng / μL and 0.1 ng / μL), a visible test line first appeared at 0.0316 ng / μL, indicating that the detection limit of Tf in the LFA with a preconcentration step was 0.1 ng / μL. This corresponds to a 10-fold improvement in the detection limit of the LFA when DGNP and PEG salt ATPS were used.

[0361] To further verify the visual interpretation of the LFA results, images of the test strips were taken and the intensity of the test lines was quantified. When a preconcentration step was used, the test line signal of each LFA was weaker when the same Tf concentration was tested, indicating that Tf had concentrated and saturated the DGNPs, which were unable to bind to the test line and indicated a positive result. The signal intensities of both the control and test lines were similar in the negative controls with and without ATPS combination. This indicated that the amount of DGNPs that migrated down the LFA strips in both experiments was similar. MATLAB® analysis of the LFA panel also demonstrated the improvement of the detection limit (Figure 32). Notably, the two curves in Figure 32 are separated by a 10-fold difference in initial Tf concentration with nearly identical test line signals.

[0362] Example 3. Direct addition of mixed PEG salt ATPS to LFA In this example, the sensitivity, speed and ease of use of a next-generation integrated device with both ATPS phase separation and downstream detection capabilities were optimized. Instead of applying concentrated samples after ATPS phase separation, the mixed ATPS could be added directly to the paper-based device. The solution separated into two phases as it flowed toward the detection zone, allowing the concentration and detection steps to occur simultaneously and the overall time to result to be further reduced. The paper membrane was suggested to speed up the rate of macroscopic phase separation of the ATPS. To further exploit this phenomenon, the paper device was stretched vertically, thereby increasing the cross-sectional area of ​​flow and utilizing the effects of capillary action and / or gravity on macroscopic separation. In addition to accelerating phase separation, this three-dimensional component also has the ability to process larger, more dilute volumes of sample, resulting in greater enrichment fold improvement. The ATPS and LFA in the novel three-dimensional paper structure successfully provided a 10-fold improvement in the detection limit of the model protein Tf, while reducing the overall time to result and maintaining ease of use. This device offers a significant improvement over traditional LFA testing and can be modified for the detection of a variety of diseases with low levels of characteristic biomarkers. This novel platform technology is highly sensitive, low-cost, rapid, and instrument-free, and therefore has the potential to revolutionize the status quo of diagnostic medicine within resource-poor regions.

[0363] Determination of polymer salt ATPS solution volume ratio All materials, chemicals, and reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated. Polyethylene glycol 8000 (PEG, VWR, Brisbane, CA) and potassium phosphate salts (5:1 dibasic:monobasic ratio) were dissolved in Dulbecco's phosphate buffered saline (PBS) (Invitrogen, Grand Island, NY, pH 7.4, 1.47 mM KH 2 PO 4 , 8.1 mM Na 2 HPO 4 , 137.92 mM NaCl, 2.67 mM KCl and 0.49 mM MgCl2 The equilibrium volume ratio (volume of the upper phase divided by the volume of the lower phase) was obtained by varying the w / w composition of PEG and salt along the same tie line. ATPS with 1:1 and 9:1 volume ratios were found and used for further experiments.

[0364] Preparation of antibody-decorated dextran-coated gold nanoprobes (DGNPs) Dextran-coated gold nanoparticles were synthesized. Briefly, 6 g of dextran (Mw. 15,000-25,000) from Leuconostoc species was dissolved in 80 mL of filtered UltraPure sterile water (Rockland Immunochemicals Inc., Gilbertsville, PA). The solution was stirred and heated to boiling, after which 1080 μL of 1% w / v gold(III) chloride hydrate solution was added. The color of the reaction mixture turned reddish blue-purple and it was stirred and boiled for approximately 20 min. The newly formed dextran-coated gold nanoparticles were centrifuged to remove free dextran and resuspended in 70 mL of water. To form the functionalized DGNPs, the pH of the dextran-coated gold nanoparticle solution was adjusted to 9.0 using 1.5 M NaOH. For every 1 mL of dextran-coated gold nanoparticle solution, 8 μg of anti-Tf antibody (Bethyl Laboratories, Montgomery, TX) was added. The reaction mixture was placed on a shaker for 30 min to promote the formation of dative bonds between the antibody and the dextran-coated gold nanoparticles. Free antibody was removed by centrifugation. The pellet was resuspended in 100 μL of 0.1 M sodium borate buffer, pH 9.0.

[0365] Visualization of ATPS To visualize the two phases of the ATPS, dextran-coated gold nanoparticles, which are purple due to surface plasmon resonance and brilliant blue FCF dye (The Kroger Co., Cincinnati, OH), were added to a total of 3 g of PBS solutions containing predetermined concentrations of PEG and salt at volume ratios of 1:1 and 9:1. These solutions were mixed thoroughly by vortexing and incubated at 25°C. Images of the solutions were taken when the ATPS reached equilibrium. All images were taken with the use of a Canon EOS 1000D camera (Canon USA, Inc., Lake Success, NY).

[0366] The two phases of ATPS were then visualized as they flowed along the paper membrane. Two 8 × 30 mm strips of glass fiber paper were laser cut by a VersaLASER 3.50 (Universal Laser Systems, Scottsdale, AZ). 50 mg of mixed ATPS containing brilliant blue FCF dye and dextran-coated gold nanoparticles (corresponding to a 1:1 or 9:1 equilibrium volume ratio) was then added dropwise to one end of the strip using a pipette. Images of the resulting flow were taken at 0, 30, 105, and 300 seconds. Videos were also taken with an 8-megapixel camera on a commercial smartphone (Apple Inc., Cupertino, CA).

[0367] To visualize the phase separation of the ATPS in the three-dimensional paper wells, 140 mg of mixed ATPS containing brilliant blue FCF dye and dextran-coated gold nanoparticles was added to the paper wells. The three-dimensional paper wells were formed by stacking nine 8×10 mm laser-cut strips of glass fiber paper onto one edge of an 8×60 mm laser-cut strip of glass fiber. After the mixed ATPS was applied to the three-dimensional paper wells, 50 μL of running buffer (0.2% bovine serum albumin (BSA), 0.3% Tween 20, 0.1 mL Trizma base, pH 8) was added to the three-dimensional paper wells. The running buffer was added to help the sample flow from the paper wells to the rest of the device. Videos were recorded and images were taken at 0 and 30 seconds, at the time of addition of the running buffer, and after flow was completed.

[0368] Detection of Tf LFA test for Tf detection An LFA test strip was constructed that utilizes a competitive assay format. Briefly, DGNPs decorated with anti-Tf antibodies were first added to the sample solution and allowed to bind to any Tf present in the sample to form DGNP / Tf complexes. To verify the detection limit of Tf by the LFA, 30 μL of running buffer and 20 μL of sample solution consisting of 15 μL of a known amount of Tf in PBS and 5 μL of DGNPs were mixed in a test tube. The LFA test strip was inserted vertically into the tube to submerge the sample pad in the sample and the fluid was absorbed and drained by the strip towards the absorbent pad. If Tf is present, the DGNP / Tf complexes that run through the LFA stop will not be able to bind to the Tf immobilized at the test line, indicating a positive result by the presence of a single band at the control line. Alternatively, if Tf is not present, the antibodies on the DGNPs can bind to Tf at the test line. As these probes exhibit a purplish red color, DGNPs will accumulate at the test line to form a visible band, indicating a negative result. Regardless of the presence of Tf, the antibody on the DGNPs always binds to the secondary antibody immobilized at the control line. The band at the control line means that the sample has completely flowed through the strip, indicating a valid test. A negative result is therefore indicated by two bands, one at the test line and one at the control line, in contrast to a positive result indicated by a single band at the control line. Each Tf concentration was tested in triplicate. A representative LFA strip was imaged after 10 min with a Canon EOS 1000D camera under controlled lighting conditions.

[0369] Detection of Tf by three-dimensional paper wells The LFA components of the paper-based device were slightly modified from the previously described setup. Specifically, the cellulose sample pad was replaced with a 5×20 mm glass fiber paper, which was connected to a nitrocellulose membrane containing test and control lines. A three-dimensional paper well composed of multiple strips of glass fiber paper was used at the beginning of the sample pad. For experiments using a 1:1 volume ratio ATPS, the well was composed of five layers of glass fiber paper (four 5×7 mm strips + lower sample pad). To start the test, 40 μL of a 1:1 volume ratio mixed ATPS containing a known concentration of Tf was added to the paper well, followed by 50 μL of running buffer. Images were taken after 10 min. For experiments using a 9:1 volume ratio ATPS, 20 layers of paper (19 5×7 mm strips + lower sample pad) were used to form the paper well. 200 μL of 9:1 volumetric ratio mixed ATPS containing known concentrations of Tf were added to the paper wells and incubated for 10 min, followed by the addition of 100 μL of running buffer. After another 10 min, images were taken with a Canon EOS 1000D camera under a controlled lighting environment. Each Tf concentration was tested in triplicate.

[0370] Visualization of ATPS The integration of ATPS and LAF could significantly improve the detection limit of traditional LFA tests without sacrificing its benefits. To achieve this, it was first necessary to identify an ATPS that could be visualized while the phases were passing through the paper membrane. Specifically, a PEG-salt ATPS was used, which forms a more hydrophobic PEG-rich phase at the top and a denser, more hydrophilic PEG-poor phase at the bottom. Partitioning of biomolecules in the ATPS is determined primarily by relative hydrophilicity (as biomolecules tend to prefer the layer with which they experience the greatest attractive interactions) and size (as large biomolecules typically do not remain in the PEG-rich phase because they experience large sterically excluded volumetric repulsive interactions with the majority of PEG molecules in the PEG-rich phase). Brilliant blue FCF dye was added to the mixed ATPS, and because it is small and hydrophobic, the dye partitions heavily into the PEG-rich phase. Purple dextran-coated gold nanoparticles were also added to the mixed ATPS, and because they are large (approximately 50 nm in diameter as measured by dynamic light scattering) and hydrophilic, they partition heavily into the PEG-poor phase. Images of 1:1 and 9:1 ATPS volumes were taken before and after phase separation (Figure 33). The amounts of Brilliant Blue FCF and dextran-coated gold nanoparticles were kept constant in the 1:1 and 9:1 ATPS volumes. As a result, after phase separation, the upper phase of the 9:1 ATPS volume was larger in volume compared to the upper phase of the 1:1 ATPS volume, and therefore less concentrated in blue dye. In addition, the lower phase of the 9:1 ATPS volume was significantly smaller in volume than the lower phase of the 1:1 ATPS volume, and showed a darker purple color, demonstrating that the shrinkage of the lower phase can effectively concentrate the dextran-coated gold nanoparticles in the ATPS. The 9:1 ATPS sample was predicted to concentrate the nanoparticles 10-fold, since the volume of the lower phase was 1 / 10 of the volume of the entire sample solution. Note that the more extreme the volume ratio, the greater the concentration fold improvement that can be obtained, but the longer the system will take to perform the separation.

[0371] Visualization of ATPS on Paper After adding the mixed ATPS to the paper membrane, it was observed that the PEG-poor phase containing the purple dextran-coated gold nanoparticles flowed rapidly through the paper. Meanwhile, the PEG-rich phase containing the blue dye was retained at the origin of the paper membrane (Figure 34). This was similar to the case where both phases of the ATPS were allowed to completely separate in the glass well before flow was induced into the paper (Figure 80). Enhanced phase separation occurring within the paper membrane was evident when a 1:1 volume ratio of ATPS was used (Figure 34A), where the 1:1 volume ratio ATPS phase separated almost immediately within the paper. As shown in Figure 33, the concentration factor of the purple dextran-coated gold nanoparticles achieved using ATPS is a function of the volume ratio. Specifically, since the particles are heavily partitioned into the PEG-poor phase, a 1:1 volume ratio should result in a 2-fold concentration of the particles since half of the initial volume flows as the flow front. One possible explanation for the phase separation behavior in the paper film is that the PEG-rich domains have more interactions with the paper and are less mobile. In addition, the PEG-rich domains are also more viscous and therefore may have more difficulty moving through the tortuous paper network. In contrast, the PEG-poor domains have less interactions with the paper, are less viscous, and can move quickly through the paper network and coalesce at the front. In the 9:1 volume ratio ATPS, the PEG-poor domains comprise only one-tenth of the total volume, making it more difficult to coalesce and flow ahead of the PEG-rich domains. Specifically, the time required for macroscopic phase separation to occur in the paper was longer than the time required for the fluid to be absorbed and drained by the paper, and good separation was not observed (Figure 34B).

[0372] It was also observed that some PEG-poor domains did not reach the flow front when using both 1:1 and 9:1 volume ratios of ATPS. This indicated that not all PEG-poor domains could escape from the PEG-rich domains, and revealed why the equilibrium volume ratio measured in the test tube did not match the absorption-excretion distance ratio of the paper membrane. To improve the LFA, most of the PEG-poor domains needed to reach the macroscopic PEG-poor phase and be separated from it before reaching the detection zone. Therefore, an additional component was needed to further improve the phase separation phenomenon.

[0373] Visualization of ATPS in a three-dimensional paper-well device Paper wells utilizing a three-dimensional paper structure were designed to further enhance the phase separation behavior. The use of three-dimensional paper wells also allows gravity, which normally induces phase separation in test tubes, to act to aid ATPS phase separation within the paper. The three-dimensional paper structure also increases the cross-sectional area perpendicular to the direction of flow. Thus, more volume can be simultaneously imbibed into and drained from the paper, preventing more PEG-rich domains from interacting with the paper and allowing the PEG-poor domains to coalesce more easily. As shown in Figure 35, the three-dimensional paper wells contributed to a greater phase separation efficiency than the two-dimensional paper membranes previously shown. The PEG-rich domains were retained in the upper layer of the paper well, while the PEG-poor domains containing concentrated dextran-coated gold nanoparticles flowed toward the lower layer of the paper well. In addition, the PEG-poor domains were initially released from the paper wells, effectively separating them from the PEG-rich domains. A running buffer was also added to further induce fluid flow and help flush any remaining PEG-poor domains out of the paper wells, and it is contemplated that this addition may be automated in the future. Note that flow was slow (the solution does not reach the end of the strip after 390 seconds) when a 9:1 volume ratio of ATPS was used, due to the presence of a large volume of the more viscous PEG-rich phase.

[0374] Detection of Tf using a three-dimensional paper well device After visualizing the improved ATPS phase separation and flow behavior in the 3D paper well device, we investigated whether an LFA could be combined with this technique to improve the detection limit. The brilliant blue FCF dye was no longer used, and functionalized anti-Tf DGNPs were used instead of dextran-coated gold nanoparticles. The DGNPs partition similarly to dextran-coated gold nanoparticles in the ATPS, but can also capture biomarkers of interest in the sample and act as colorimetric indicators for the LFA.

[0375] The detection limit of the LFA-only control, which did not incorporate the ATPS and three-dimensional paper well device, was identified. To improve the detection limit of the LFA relative to the control in a competitive format, the antibody decorated on the DGNPs needs to bind more Tf. This can be achieved by exposing the same number of DGNPs to a larger number of Tf molecules, and at a fixed concentration of Tf, the total volume of the solution needs to be increased. The DGNPs can be saturated by increasing the total volume, but are diluted by doubling the volume. Since each LFA strip can only handle 20 μL of sample, dilution of the DGNPs results in an invalid test because insufficient DGNPs bind to the control line. However, using the ATPS and three-dimensional paper well device, the DGNPs saturated with Tf molecules are distributed excessively to the PGE-poor phase and therefore collect at the front of the flow in the paper, which results in a valid test. Thus, the sample volume was increased 2-fold to 40 μL when using ATPS with a 1:1 volume ratio, predicted to concentrate DGNPs 2-fold, resulting in the same number of DGNPs entering the detection zone when compared to the control. Similarly, the sample volume was increased 10-fold to 200 μL when using ATPS with a 9:1 volume ratio, predicted to concentrate DGNPs 10-fold. Since only the lower phase of the ATPS with 1:1 and 9:1 volume ratios, which contains DGNPs, passes through the detection zone, the volumetric throughput of the device was fine-tuned by varying the number of layers containing the three-dimensional paper wells. To accommodate this increase in sample volume, the three-dimensional paper well device used for the detection of Tf in the ATPS with a 1:1 volume ratio was constructed from five layers of paper, whereas the ATPS with a 9:1 volume ratio was increased to 20 layers of paper (FIGS. 36B and C) to accommodate the increased volume. Despite the significant increase in the number of layers, the ATPS three-dimensional paper-well device with a 9:1 volume ratio still remained relatively small compared to a dime.

[0376] When the ATPS solution containing DGNPs was added to the paper wells, the DGNPs were absorbed through the Debye and rapidly concentrated at the front of the solution. The DGNPs reached the detection area of ​​the device ahead of the rest of the solution held in the paper wells. In a competitive assay, the presence of a test line indicates a negative result, while the absence of a test line indicates a positive result. When using a negative control that did not contain the model protein Tf, in less than 25 minutes, both setups using 1:1 and 9:1 volume ratios of ATPS resulted in visible bands at both the test and control lines, respectively, indicating the absence of Tf and a valid test (Figure 36). After verifying that the three-dimensional paper wells could be combined with IFA to accurately assess the absence of Tf in the negative control ATPS samples, the detection limits were found when using 1:1 and 9:1 volume ratios of ATPS solutions with varying Tf concentrations. These experiments demonstrated a 2-fold and 10-fold improvement in the detection limit of Tf over conventional LFAs, respectively (Figure 37). In the 9:1 volumetric ratio ATPS experiments using 3D paper wells, the samples were incubated in the device for an additional 10 min to allow sufficient time for the DGNPs to capture the target proteins and macroscopically phase separate before adding the running buffer. The incubation time was not necessary in the 1:1 volumetric ratio ATPS because the DGNPs are more concentrated in the mixed ATPS, facilitating the DGNPs to explore the entire solution. The results of these experiments showed that while the conventional LFA detected Tf at a concentration of 1 ng / μL (a concentration at which no test line appeared), the 3D paper diagnostic device was able to detect Tf at 0.5 ng / μL (a 2-fold improvement in the detection limit) when using the 1:1 volumetric ratio ATPS. Similarly, the 3D paper diagnostic device was able to detect Tf at 0.1 ng / μL (a 10-fold improvement in the detection limit) when using the 9:1 volumetric ratio ATPS. These results suggest that ATPS solutions with desirable volume ratios combined with appropriately sized three-dimensional paper wells can significantly and predictably improve biomarker detection using LFAs.

[0377] Example 4. Detection of M13 bacteriophage in PEG salt ATPS To increase the feasibility of combining ATPS with LFA in a point-of-need (PON) environment, the following modifications were made to the system: (1) polyethylene glycol (PEG) and potassium phosphate (salt) ATPS were investigated with short-term phase separation, (2) the kinetic behavior of biomolecule partitioning was examined leading to a significant further reduction in extraction time, (3) compatibility of the gold nanoparticle indicators used in the LFA with the salt-rich phase of the PEG-salt ATPS was ensured by decorating the gold surface with PEG, and (4) a custom MATLAB script was generated to quantify the observed LFA signal intensity. A visual representation summarizing these changes is shown in Figure 38.

[0378] Cultivation of bacteriophage M13 Escherichia coli (E. coli) bacteria strain ER2738 (American Type Culture Collection, ATCC, Manassas, VA) was incubated in 8 mL of Luria medium (Sigma Aldrich, St. Louis, MO) in an incubator shaker for 6 hours at 37°C. To cultivate the model virus bacteriophage M13 (ATCC), a small amount of frozen M13 was added to the bacterial culture. The bacterial culture was then placed in the incubator shaker for 14 hours at 37°C. The solution was centrifuged at 4°C and 6000g for 15 minutes to isolate M13 in the supernatant from the bacteria in the pellet. The supernatant was extracted from the solution and filtered through a 0.22 μm syringe filter (Millipore, Billerica, MA). Dilutions of this stock M13 solution were calculated using the concentration of M13 determined by plaque assay. M13 concentrations are therefore reported in plaque forming units per mL (pfu / mL). All reagents and materials were purchased from Sigma-Aldrich (St. Louis, Mo.) unless otherwise indicated.

[0379] Volume ratio headings for PEG salt systems For the polymer salt ATPS, polyethylene glycol 8000 (PEG, VWR, Brisbane, CA) and potassium phosphate (5:1 dibasic:monobasic) were dissolved in Dulbecco's PBS (Invitrogen, Grand Island, NY, pH 7.4, 1.47 mM KH 2 PO 4 , 8.1 mM Na 2 HPO 4 , 137.92 mM NaCl, 2.67 mM KCl and 0.49 mM MgCl 2 A final solution containing 2% (w / w) Luria medium was made with a total mass of 5 g, consistent with the experiments with M13 samples containing Luria medium. The equilibrium volume ratio (volume of the upper macroscopic phase divided by the lower macroscopic phase) was measured after a minimum of 12 hours (well past equilibrium) by marking the interface and the top of the solution on the outside of the tube. The tube was then emptied, dried and weighed on a balance. Water was added up to the mark and the volume of both phases was measured using 1 g / mL as the density of water. Different equilibrium volume ratios were found by varying the initial PEG concentration, initial salt concentration and keeping the temperature constant at 37° C. It should be noted that these volume ratios were chosen at the same tie line to ensure similar distribution of biomolecules, and the specific operating conditions used are presented in Table 2. [Table 2]

[0380] Partitioning and concentration of M13 For each concentration experiment, four identical 5 g PEG salt solutions were prepared. Of these solutions, three were used for the concentration step (n=3), while one was used as a control representing the initial M13 concentration. To ensure that each solution was one phase prior to phase separation, all solutions were equilibrated at 4°C and mixed homogeneously. M13 was then added to the four solutions to obtain an overall initial concentration of 1×10 8pfu / mL. To determine how M13 concentration varies with time, the 9:1 volume ratio solutions were incubated in a 37°C water bath for 30 minutes, 1 hour, 4 hours, and 24 hours. To determine how M13 concentration varies with volume ratio, the 9:1, 6:1, 3:1, and 1:1 volume ratio solutions were incubated in a 37°C water bath for 30 minutes. In each experiment, the lower phase of the three solutions was carefully extracted by using a syringe. The control solutions underwent the same incubation steps to mimic the same conditions as the test solutions. However, after incubation, the controls were again equilibrated to 4°C and mixed homogeneously before an aliquot was withdrawn to represent the initial concentration of M13 in the ATPS. The M13 concentrations of the extracted lower phase and control solutions were determined by plaque assay.

[0381] Preparation of gold nanoparticles Colloidal gold nanoparticles were prepared. The diameter of the gold nanoparticles was then determined by using dynamic light scattering (DLS) using a Zetasizer Nano SZ particle analyzer (Malvern Instruments Inc., Westborough, MA). The concentration of the gold nanoparticles was then determined using Beer's law. The absorbance values ​​used were the peak absorbance and then the molar extinction coefficient taken from the data sheet provided by BBInternational Life Sciences (see Table 3). [Table 3]

[0382] After quantification of the gold nanoparticles, a mouse monoclonal antibody against the M13 coating protein pVIII (Abcam Inc., Cambridge, MA) was added to the nanoparticle solution at a ratio of 16 μg anti-M13 per mL of colloidal gold to form gold nanoprobes. The pH of the solution was adjusted to 9.0 using 0.1 M NaOH to facilitate dative binding of the antibody to the gold, and the solution was mixed on a shaker for 30 min. To provide steric colloidal stability when the gold nanoprobes are added to the PEG-poor salt-rich phase prior to the LFA, PEG-2000-SH (Nanocs, Boston, MA) molecules were conjugated to the gold nanoparticles at a molar ratio of 5000 PEG-2000-SH molecules per gold nanoprobe. The solution was mixed for an additional 30 min. To prevent non-specific binding, 100 μL of 10% bovine serum albumin (BSA) solution was added to 1 mL of colloidal gold solution to block remaining excess surface, and the solution was mixed for 30 min. Finally, the solution was centrifuged at 4 °C and 8600 g for 30 min to remove free antibody, PEG, and BSA. The decorated gold nanoprobes were resuspended in 150 μL of 0.1 M sodium borate buffer, pH 9.0.

[0383] Combination of ATPS and LFA To combine the ATPS preconcentration step with the LFA, various M13 concentrations were added to the PEG salt solution in a 9:1 volume ratio. After 30 min of incubation at 37° C., the concentrated lower phase was extracted by using a syringe. For consistency between immunoassays performed with and without a preconcentration step, M13 was added to the lower phase of samples in 9:1 solution that did not initially contain M13, thereby forming a control solution. Then, 30 μL of the lower phase sample and 10 μL of the anti-M13 decorated colloidal gold probe solution were mixed together with 30 μL of running buffer. The running buffer, which aids in the flow of the sample through the test strip, was prepared using ddH2SO4. 2The buffer consisted of 0.2% w / v BSA, 0.3% w / v Tween 20, 0.2% w / v sodium azide, 0.1% PEG-8000, and 0.1 M Trizma base in O, adjusted to pH 8. The resulting solution was mixed and incubated for 5 minutes before adding the LFA strips to bind the antibody to M13. The LFA strips were assembled and the sandwich assay was performed as shown diagrammatically in Figure 39. After 10 minutes, the LFA strips were removed and the results were obtained by visually inspecting the test and control lines on the LFA strips.

[0384] Quantitative analysis of LFA Images of the LFA strips were taken in a controlled lighting environment with a Canon DSLR camera immediately after the test was completed. A custom MATLAB script was written to quantify the LFA results. The images were cropped and converted to an 8-bit grayscale matrix. The matrix was then split in half, one half of which contained the control line and the other half contained the test line. In each half of the matrix, the minimum intensity (darkest spot) was located along a vector perpendicular to the control or test line. The location of the average of these minima was used as the center of a 15-pixel-high rectangular region spanning the length of the control or test line. The average grayscale intensity (I 対照 The same procedure was applied to the other half of the image containing the test lines to calculate the average grayscale intensity of the test line region (I 強度 ) was obtained by calculating the average grayscale intensity (I 基準 ) was used to normalize the intensities of the test and control lines as follows:

number

number

[0385] Partitioning of Biomolecules A 10-fold improvement in the LFA virus detection limit can be achieved by combining the Triton X-114 micellar ATPS with the LFA. The Triton X-114 micellar system takes more than 6 hours for the volumes of both phases to equilibrate. In contrast, the polymer salt ATPS in this study has a more rapid phase separation. In addition, a 30-minute early extraction time was used for the 9:1 volume ratio.

[0386] Aqueous solutions composed of PEG, a polymer that undergoes significant salt and hydrogen bonding interactions, undergo macroscopic phase separation. A PEG-rich, salt-poor phase forms at the top, while a PEG-poor, salt-rich phase forms at the bottom. Large hydrophilic biomolecules partition heavily into the lower, PEG-poor phase. In this example, bacteriophage M13, a model virus similar in size and shape to Ebola and Marburg viruses, was investigated. Since M13 has a length of 900 nm and a relatively hydrophilic protein shell, it was predicted to partition heavily into the lower, PEG-poor phase due to the fact that it experiences fewer repulsive, steric, and volumetrically excluded interactions with the PEG polymer compared to the PEG-rich phase. In an aqueous two-phase micellar system, M13 was observed to partition heavily into the macroscopic micellar-poor phase, where M13 virus particles also experience fewer volumetrically excluded interactions.

[0387] As the solution is heated and phase separation first begins, PEG-rich (M13-poor) and PEG-poor (M13-rich) domains form throughout the solution. The virus concentration in these microscopic domains is assumed to reach equilibrium values ​​very quickly, and similar domains begin to coalesce with one another. As these domains grow larger, density differences generate non-uniform gravitational and buoyant forces, which cause the PEG-rich domains to rise and the PEG-poor domains to sink. This results in the formation of a macroscopic upper PEG-rich (M13-poor) phase and a macroscopic lower PEG-poor (M13-rich) phase. The kinetics of this process and the effect of entrainment or capture domains are further described below.

[0388] Since viruses are expected to partition predominantly into the lower macroscopic phase, our only interest was in the concentration of the biomolecule in the lower phase at the time of extraction. We therefore defined the concentration factor as the concentration of the virus in the lower macroscopic phase, C ウイルス、下部 is the initial overall concentration, C ウイルス、初期 It is defined as being divided by.

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[0389] One potential concern that could negatively affect the concentration coefficient is the presence of PEG-rich (M13-poor) domains entrained in the macroscopic lower phase. Not all domains had enough time to migrate to the corresponding macroscopic phase before equilibration. However, this is not an issue when using an equilibration volume ratio of 9:1. Due to the large volume associated with the PEG-rich domains in the 9:1 volume ratio, the PEG-rich domains are easier to coalesce and form a continuous phase. The PEG-rich domains have a small distance to travel to reach the macroscopic upper phase because the macroscopic lower phase is very small. As a result, the entrainment of the PEG-rich (M13-poor) domains in the macroscopic lower phase is also minimal, and the concentration of the biomolecule is predicted to be close to the equilibrium value. However, the PEG-poor (M13-rich) domains are predicted to still be in the macroscopic upper phase. Due to the small volume associated with the PEG-poor domains, it is more difficult for these domains to coalesce. They also need to travel an additional distance through the more viscous PEG-rich macroscopic phase to reach the macroscopic lower phase. However, since the main concern is the concentration of biomolecules in the lower phase and only small volumes are required for the LFA, it is possible to extract at the 30 minute time point and still achieve the predicted equilibrium concentration factor.

[0390] The phase separation of the 9:1 volume ratio solution as a function of time was visually inspected. Initially, PEG-rich and PEG-poor domains form throughout the solution. The PEG-rich domains rapidly coalesce and form a continuous phase. The solution appears turbid as the entrained PEG-poor domains scatter light (Figure 40A). A macroscopic lower PEG-poor phase forms from the bottom up as the PEG-poor domains coalesce and sink to the bottom due to their high density. After 30 minutes, there is a turbid macroscopic upper PEG-rich phase that is separated from the clear macroscopic lower PEG-poor phase (Figure 40B). While the upper phase contains entrained PEG-poor (M13-rich) domains, the main concern is the turbidity of the lower phase as it contains concentrated biomolecules. After an extended period of time, all or most of the PEG-poor domains migrate to the macroscopic lower phase and the system is near equilibrium as indicated by the two clear phases (Figure 40C). Note that the lower phase at 30 min was already clear due to the absence of significant amounts of entrained PEG-rich (M13-poor) domains, suggesting that the measured concentration of virus in that phase should already reflect the equilibrium value.

[0391] In order to extract the lower phase at 30 minutes, it was desirable to ensure that the macroscopic lower PEG-poor (M13-rich) phase did not contain significant amounts of PEG-rich (M13-poor) domains. Thus, concentration coefficients were determined as a function of time that showed that the measured concentration of M13 in the lower phase was equal to the equilibrium value even after a short time. Specifically, M13 partitioning experiments were performed using the same temperature and initial PEG and salt concentrations (i.e., conditions that yielded a 9:1 equilibrium volume ratio) used in Figure 40, and extracted from the lower phase at 30 minutes, 1 hour, 4 hours, and 24 hours (well past equilibrium). As shown in Figure 41, the concentration coefficients remained constant as a function of extraction time from 30 minutes to 24 hours, demonstrating that entrainment of PEG-rich (M13-poor) domains in the macroscopic lower PEG-poor (M13-rich) phase was indeed minimal. Thus, in subsequent tests, a 30 minute extraction time was used as the short time required for PON applications.

[0392] Distribution of M13 in ATPS The concentration coefficient in equation (9) can be shown to be a function of the volume ratio. Using a molar balance to equate the initial moles of virus in the single homogenous phase with the total moles of virus in the two resulting phases, we obtain the following equation:

number

number

[0393] The partition coefficient of M13 was measured (the equilibrium concentration in the upper phase divided by the equilibrium concentration in the lower phase) using a 9:1 volume ratio of ATPS and was found to be less than 0.0001. 下部 / V 上部 Since this is much smaller than 1 / 9 of the value, we can approximate this equation as follows:

number

[0394] Thus, a linear relationship is predicted for the concentration coefficient as a function of the volume ratio. The concentration coefficient increases with increasing volume ratio since the biomolecule partitions heavily into the lower phase, and increasing volume ratio corresponds to a shrinkage of the phase into which M13 partitions heavily. Specifically, when a 9:1 volume ratio is used, M13 is predicted to be 10-fold concentrated since the volume of the phase into which M13 partitions heavily is 1 / 10 of the initial volume. This was subsequently tested using a 30-minute extraction time since short times are necessary for PON applications. M13 partitioning experiments were performed at 37° C. with initial concentrations of PEG and salt that resulted in 9:1, 6:1, 3:1, and 1:1 equilibrium volume ratios. Figure 42 shows the results of an experiment testing the concentration coefficient as a function of the volume ratio. To statistically determine whether the experimental data matched the model predictions, the coefficient of determination or R-squared value was calculated. The R-squared value was found to be 0.858, suggesting that the experimental data was in reasonable agreement with the predicted values, further confirming that the simple model was adequate even for a 30 min extraction time.

[0395] Detection of M13 via LFA After demonstrating that M13 can be concentrated in 30 minutes via ATPS, colloidal gold probes and LFA strips were prepared. PEG decorated on the surface of the gold probe provided steric stability and prevented aggregation of the gold probe in the environment of the extracted salt-rich phase. Figure 43 shows the results of the LFA with a sample that did not undergo a preconcentration step. M13 concentrations were selected at equally spaced points along the logarithmic scale. The presence of a control line or upper band containing polyclonal anti-IgG antibodies indicates a valid test since it confirms that the sample has run through the entire strip. The presence of a test line or lower band containing antibodies to the M13 coating protein pVIII indicates the presence of M13. The negative control shown in Figure 43A does not contain M13 and, as expected, there is no visible test line. In the remaining panels, the intensity of the test line is greatest at the highest M13 concentration shown in Figure 43B, and the intensity of the line decreases as the M13 concentration decreases. The test line is 3.2 x 10 8 pfu / mL (Figure 43E), with a detection limit of approximately 1 × 109 pfu / mL (Figure 43D).

[0396] Concentration of M13 prior to LFA 1×10 9 With the detection limit established in pfu / mL, possible improvements in the detection limit were investigated using ATPS. A 9:1 volume ratio solution was used, which was predicted to produce a concentration factor of approximately 10 based on equation (12). After 30 min incubation at 37°C, the lower phase was extracted and the samples were prepared for LFA. Figure 44 shows the results of the LFA with a pre-concentration step. Again, the test line was absent in the negative control containing no M13 (Figure 44a). In the remaining panels, the initial M13 concentration is 10 times less than that shown in Figure 6, but the intensity of the LFA test line is consistent with the sample without the pre-concentration step. The intensity of the test line decreased with decreasing concentration of M13 to 3.2 x 10 7 pfu / mL (Figure 44E), decreasing until the detection limit was approximately 1 x 10 8 pfu / mL (Figure 44d), indicating a 10-fold improvement in the detection limit when the ATPS enrichment step was applied.

[0397] To confirm the conclusions from visual interpretation of the LFA strips, the LFA images were converted to grayscale and the test line intensity was checked against background using our custom MATLAB® script. Results with and without the ATPS concentration step are shown in Figure 45. Note that the two curves are separated by a 10-fold difference in initial M13 concentration with nearly identical test line signals, further confirming our 10-fold concentration improvement.

[0398] Example 5. Detection of transferrin in biological serum by interfacial extraction of PEG salt ATPS The enrichment of biomolecules using a single ATPS step was optimized by directing the biomolecules of interest toward the interface of the two bulky phases. Because the interfacial region represents an extremely small volume region that can form independent of the volume ratio, this new approach allows the concentration of the target to be independent of the extreme volume ratio (volume ratios much above or below 1) that have long phase separation times. Instead, the volume ratio that can reach equilibrium the fastest was selected, which reduced the extraction time to within 10 minutes in phosphate buffered saline (PBS), a significant improvement over previous approaches. This approach is toward the maximum concentration multiple that can be achieved in a single ATPS step, since the interfacial volume is much smaller than the two macroscopic bulky phases, and thus the biomolecules can be enriched even more extremely. Last but not least, an increase in sample volume desirably increases the total number of biomolecules of interest that can be enriched at the interface and detected by the LFA. This is also true for enriching the molecules of interest in the bulky phase using an extreme volume ratio, but an increase in sample volume also increases the phase separation time. In the proposed interfacial extraction approach, the extreme volume ratio is no longer necessary. Figure 46 diagrammatically compares interfacial extraction with the extraction of one of the two bulky phases.

[0399] To guide the target biomolecules towards the interface, a capture mechanism involving GNPs was utilized. Due to the specific antibodies decorating the particle surface, the GNPs first captured the target proteins in the sample. The surface chemistry of the GNPs was optimized so that the particles partitioned to the interface upon phase separation. The proteins captured by the GNPs were then extracted from the interface. As the interfacial volume was very small, the proteins were highly concentrated and subsequently applied to the LFA detection strip. This example provides a summary of the prepared GNPs capable of partitioning to the interface of PEG-salt ATPS. The volume ratio at which the phases separated the fastest and also allowed maximum recovery of GNPs was then investigated. The model protein transferrin (Tf) was then used to demonstrate a 100-fold improvement of LFA over Tf in combination with an ATPS interfacial extraction step. The study was then expanded to get closer to real-world applications and the system was re-optimized for fetal bovine serum (FAB) and synthetic urine, as well as the small volumes favored for blood sampling. The data show that even in more complex systems, ATPS interfacial extraction can be performed within 15-25 min and can result in a 100-fold improvement in the detection limit of Tf in LFAs.

[0400] This example provides the development of a nanoprobe that can localize at interfaces and also function as a chromogenic indicator of LFAs. This approach differs from previously investigated extraction methods in that it uses functionalized magnetic or gold particles to concentrate and collect the biomolecules of interest, which required equipment that is not suitable for point-of-care devices. This method is an effective and rapid method to improve the detection limit of LFAs by 100 times and close the sensitivity gap between laboratory and paper-based immunoassays. Improved LFAs with enhanced sensitivity will have a significant impact on the field of diagnostics, coming closer to providing point-of-care solutions that are not currently available. On the other hand, although it has been demonstrated that this approach can improve the performance of LFA detection, this preconcentration procedure can also be applied to other detection methods.

[0401] Radiolabeling of anti-Tf antibodies All reagents and materials were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated. Iodine-125 ( 125 I) was used to radiolabel the tyrosine residues of goat anti-human Tf polyclonal antibody (catalog number A80-128A, Bethyl Laboratories, Montgomery, TX). 125 I (MP Biomedicals, Irvine, CA) was activated with IODO-BEADS (Pierce Biotechnology, Rockford, IL). 125 I was reacted with goat anti-Tf antibody for 15 min. Radiolabeled protein was purified and released. 125 I was removed using a Sephadex G15 size exclusion column. The radioactivity and concentration of radiolabeled protein was quantified using the phosphotungstic acid assay.

[0402] Preparation of GNPs Intact gold nanoparticles were prepared, resulting in a clear, cherry-colored solution with a particle size of approximately 25-30 nm in diameter. To prepare GNPs, 320 mg of goat anti-Tf antibody was incubated with 20 mL of colloidal gold solution for 30 min, followed by the addition of thiolated PEG5000 using a 3000:1 molar ratio of PEG:GNPs and further incubation for 30 min. To prevent non-specific binding of other proteins to the surface of colloidal gold, 2 mL of 10% bovine serum albumin (BSA) solution was added to the mixture and mixed for another 10 min. The resulting solution was mixed gently on a shaker for the duration of the incubation. To remove free (unbound) antibody, PEG and BSA, the mixture was subsequently centrifuged at 4 °C and 9,000 g for 30 min. The pellet of GNPs was washed twice with 1% BAS solution. Finally, the recovered GNPs were resuspended in 2 mL of 0.1 M sodium borate buffer, pH 9.0.

[0403] Distribution of GNP GNPs decorated with radiolabeled anti-Tf antibodies were partitioned in ATPS under different conditions to determine the volume ratio that would result in the fastest and highest GNP recovery. In each partitioning experiment, three identical PEG salt solutions in Dulbecco's phosphate buffered saline (PBS, Invitrogen, pH 7.4, ionic strength 154 mM) were prepared to a total volume of 5000 μL. Three different volume ratios (1:1, 6:1, and 1:6) of PEG salt ATPS solutions were prepared using specific concentrations of PEG and potassium phosphate. Subsequently, 10 μL of radiolabeled anti-Tf antibody decorated GNPs were added to each ATPS solution. The solutions were equilibrated at 0°C to ensure that the solutions were homogenous. Once equilibrated at 0°C, the solutions were incubated at 37°C in a water bath to induce phase separation, and the GNPs were found to partition into two coexisting phases. The GNPs at the interface were carefully withdrawn using a pipette, and 30 μL of the interface solution was withdrawn to collect most, if not all, of the GNPs at the interface. The two coexisting phases were also withdrawn separately using a pipette. The amount of GNPs at the interface and the two coexisting phases was quantified by measuring the amount of radioactivity in each region using a Cobra Series Auto-Gamma Counter, as the GNPs were bound to the radiolabeled anti-Tf antibody. The quantified amount of GNPs in each of the three regions was used to calculate the recovery of GNPs at the interface using a mass balance.

[0404] Preparation of LFA test strips An LFA test utilizing a competitive mechanism was carried out in this study (Figure 47). In a competitive assay, the target of interest is immobilized on a nitrocellulose membrane to form a test line. An immobilized secondary antibody against the primary antibody on the GNP constitutes the control line. When performing an LFA, the sample is first contacted with the GNPs, and if the target molecule is present in the sample, it binds to the specific antibody decorated on the GNPs. If the target molecule present in the sample saturates the antibodies on the GNPs, these GNPs can no longer bind to the target molecule immobilized on the test strip. As a result, the GNPs do not form a visible band at the test line, which indicates a positive result. On the other hand, if the sample does not contain the target molecule at a concentration that can saturate the antibodies on the GNPs, these antibodies on the GNPs can bind to the target molecule immobilized on the test strip and form a visible band at the test line. This indicates a negative result. Furthermore, regardless of the presence of the target molecule in the sample, the antibody on the GNPs binds to the immobilized secondary antibody at the control line, which indicates that enough sample has been absorbed through the test line to reach the control line. The presence of a visible control line indicates a valid test.

[0405] Performing LFA with Tf but without preconcentration Tf stock solutions containing various concentrations of Tf were prepared with PBS. Then, 20 μL of each Tf stock solution was mixed with 10 μL of GNP suspension and 20 μL of test buffer (0.2% BSA, 0.3% Tween 20, 0.2% sodium azide, 0.1% PEG, 0.1 M Trizma base, pH 8) used to aid the flow of the sample in the test strip. A total of five sample solutions (50 μL each) with various concentrations of Tf were prepared (0 (negative control), 0.001, 0.01, 0.1 and 1 ng / μL). The test strips were immersed vertically into each sample solution such that the sample pad was in contact with the solution. After 10 min, the test strips were removed and images of each strip were immediately taken with a Canon EOS 1000D camera (Canon USA, Inc., Lake Success, NY).

[0406] For experiments performed in FBS (HyClone, characterized, pH 7.4), a more concentrated GNP suspension was used so that the volume of GNPs could be appropriately scaled down for low volume experiments. The concentration of Tf in the FBS stock solution was adjusted to be the same as the final Tf concentration used for the PBS experiments by adding 5 μL of Tf stock solution to 5 μL of GNP suspension followed by 40 mL of test buffer. Similarly, experiments were performed with the use of synthetic urine.

[0407] Combining ATPS interface extraction with LFA for Tf A volume ratio of 1:1 was used for the tests carried out in PBS based on findings in the GNP partitioning experiments. By utilizing anti-Tf antibodies, the GNPs first captured Tf in the sample, and then all Tf-GNPS complexes were concentrated at the interface. The same protocol was used as described in the GNP partitioning section, except that various concentrations of Tf were also added to the ATPS solution. Briefly, 10 μL of GNP suspension was added to 4990 μL of Tf-spiked ATPS solution, resulting in a 1:1 volume ratio and containing various Tf concentrations (0 (negative control), 0.001, 0.01, and 0.1 ng / μL). The solution was equilibrated at 0°C to ensure that the solution was homogenous. Once equilibration was achieved, the solution was placed in a water bath at 37°C to induce phase separation. After 10 minutes, 30 μL of interface solution containing concentrated GNPs and Tf was withdrawn. This interface solution was mixed with 20 μL of test buffer to form 50 μL of sample solution. An LFA test strip was dipped vertically into each sample solution such that the sample pad was in contact with the solution. After 10 minutes, the test strips were removed and an image of each strip was immediately taken with a Canon EOS 1000D camera.

[0408] In the tests performed in FBS and synthetic urine, the concentrations of PEG and potassium phosphate had to be adjusted first to achieve a 1:1 volume ratio. ATP in FBS also phase separated more slowly, and instead of the 10 min incubation used in the PBS system, the solution was kept in a 37° C. water bath for 25 min. In addition, as mentioned above, the volume was reduced. Thus, rather than showing an increase in the detection limit using 5000 μL (100 times more volume than the 50 μL of Tf stock solution used in the LFA-only experiment), the tests performed in FBS and synthetic urine showed a comparable improvement using 1000 μL (100 times more volume than the 10 μL of Tf stock solution used in the LFA-only experiment). The protocol already described in PBS was modified for the lower volume, whereby 5 μL of the more concentrated gold suspension was added to 995 μL of the Tf-spiked ATPS solution in FBS or synthetic urine. 20 μL of the interface area was extracted, followed by the addition of 30 μL of gold buffer. Each LFA strip was immersed in the suspension for 15 minutes before being removed and imaged.

[0409] To combine ATPS interface extraction with a paper-based LFA detection assay, the GNPs developed in this example had three functions. First, the decorated specific antibodies on the surface of the GNPs captured the target proteins present in the sample. Second, the optimized formulation of PEG and protein on the surface of the GNPs allowed the GNPs to partition at the interface and not into the bulky phase. Finally, the GNPs directly acted as a chromogenic indicator for the LFAs, and thus the subsequent detection assay could be performed immediately without extra washing or other preparation steps. A schematic diagram of the GNPs is shown in Figure 48A. The GNPs have three main constituent elements: PEG polymer, gold nanoparticles, and anti-Tf antibody. Each component itself directs the nanoparticles to one of the two bulky phases. First, the decorated PEG directs the nanoparticles to the upper PEG-rich phase due to the favorable PEG-PEG interaction between the polymer on the particle surface and the abundant polymer in the upper phase (Figure 48B). Specifically, increasing the molar ratio of PEG:GNPs changes the conformation of the bound PEG to more closely resemble a "brush" conformation, expanding the amount of exposed surface area and increasing the PEG-PEG interactions. On the other hand, larger sized gold nanoparticles cause partitioning of the nanoparticles to the lower PEG-poor phase, where the nanoparticles experience less repulsive excluded volume interactions with the PEG polymer. Hydrophilic proteins (anti-Tf AB and BSA) on the GNPs increase the hydrophilicity of the GNPs and also cause partitioning of the GNPs to the lower PEG-poor phase, which is more hydrophilic than the upper PEG-rich phase. However, since the lower PEG-poor phase is also salt-rich, the nanoparticles aggregate when there is not enough PEG on the surface of the nanoparticles to provide sufficient steric stability (Figure 48C). Combined, the three components of the GNPs can be altered and delicately balanced to ultimately guide the GNPs to the interface of the ATPS (Figure 48D).

[0410] In competitive LFA, successful detection of a protein of interest in a sample relies on the antibody on the GNPs being saturated with the target. If the antibody is not saturated, the GNPs may bind to the immobilized target on the LFA to form a test line, indicating a false negative result. One way to detect a sample with a low concentration of target is to increase the sample volume. This increases the total number of molecules of interest, which potentially results in saturation of the antibody for a given fixed amount of GNPs. However, since only a small volume can flow through the LFA test strip and the GNPs are diluted in this approach, if not enough GNPs flow through the test strip, the control line will not be visible, indicating an invalid result. Since the use of ATP interface extraction provides a rapid and effective means of collecting GNPs that are saturated with small amounts of the protein of interest, this approach can result in the detection of low concentrations of the protein of interest by allowing the analysis of a larger sample volume.

[0411] Three volume ratios were tested to determine the optimal volume ratio that could recover the majority of GNPs within the shortest incubation time, and the results are shown in Table 4. [Table 4]

[0412] The 1:1 volume ratio phase separated the fastest and allowed the greatest recovery of GNPs. When phase separation is induced by increasing the temperature, microscopic PEG-rich and PEG-poor domains are formed, and similar domains find each other and coalesce. As the domains coalesce, they migrate and eventually form a macroscopic PEG-rich salt-poor phase at the top and a macroscopic PEG-poor salt-rich phase at the bottom due to the difference in interfacial tension and density of the two phases. The 1:1 volume ratio phase separates faster than the 6:1 or 1:6 volume ratios because it is easier for the domains to find each other and coalesce when each phase is present in significant amounts. With more heterogeneous volume ratios, the domains of the smaller volume phase may be entrained in the larger continuous phase, as it is difficult for the domains to coalesce. Furthermore, the 6:1 volume ratio phase separates more slowly than the 1:6 volume ratio because the PEG-rich phase is the 6:1 volume ratio continuous phase and it is more difficult for the PEG-poor domains to find each other and progress into the corresponding macroscopic phase of the more viscous PEG-continuous phase. [Table 5]

[0413] Since the GNPs do not partition into either domain, they remain between the domains as they coalesce. Eventually, the GNPs appear as a thin red film at the interface once phase separation is complete. GNP recovery is efficient using the 1:1 volume ratio because entrainment is minimal at this volume ratio and therefore less GNPs are lost at the interface between the entrained domains and the continuous phase. As the 1:1 volume ratio phase separated the fastest while producing the highest GNP recovery, the 1:1 volume ratio phase was used in subsequent experiments.

[0414] To demonstrate the enhancement of LFA by incorporating an ATPS interface extraction step, a model protein, transferrin (Tf), was utilized. Tf is a serum protein for iron transport. To establish the detection limit of Tf in LFA, a series of LFA tests with various Tf concentrations without any preconcentration step were performed. When the sample contained enough Tf molecules to saturate the anti-Tf antibodies decorated on the GNPs, these anti-Tf antibodies did not bind to the immobilized Tf on the nitrocellulose membrane at the test line and thus did not form a visible band at the test line. This indicated a positive result, which was observed when a sample with a Tf concentration of 1 ng / μL was tested (Figure 49, top panel). On the other hand, when insufficient Tf was present in the sample to saturate the anti-Tf antibodies or no Tf was present in the sample to saturate the anti-Tf antibodies, these anti-Tf antibodies successfully bound to the immobilized Tf on the nitrocellulose membrane and thus formed a visible band at the test line. This indicated a negative result, which was observed when testing samples with Tf concentrations below 1 ng / μL, which indicated that the detection limit for Tf was approximately 1 ng / μL when performing the LFA without a preconcentration step, since 1 ng / μL was the lowest Tf concentration that showed a true positive result.

[0415] To demonstrate whether the ATPS interface extraction step can improve the detection limit of Tf using the LFA by 100-fold, the same amount of GNPs was applied to an ATPS solution with a Tf concentration (0.01 ng / μL) 100-fold lower than the detection limit of the LFA. The sample volume was increased 100-fold from 50 μL to 5000 μL to keep the total number of Tf molecules the same. Since only a limited amount of sample (50 μL) could be applied to the LFA test strip, the diluted GNPs in this large volume of sample solution had to be concentrated and applied to the LFA. To recover these GNPs saturated with the protein of interest, the solution was placed in a water bath at 37° C. and the GNPs were collected at the interface within 10 min. The GNPs were then extracted and applied directly to the LFA test strip. The results of this study are shown in the bottom panel of FIG. 49. A true positive result was obtained at 0.01 ng / μL, which represented a 100-fold improvement in the detection limit. The test line intensity of the false negative result at 0.001 ng / μL using this method was lighter than without the preconcentration step when comparing samples with the same Tf concentration, indicating that more Tf was captured, making it...

Claims

1. 1. A paper fluidic device for enrichment of an analyte of interest in a sample, comprising: A porous matrix; a probe that binds to the analyte to form a probe-analyte complex; An aqueous two-phase system (ATPS) or a component thereof disposed on or in a porous matrix; Including, the porous matrix is ​​configured and has sufficient porosity to allow the ATPS or a component thereof to flow through the porous matrix when the ATPS or a component thereof is in a fluid phase; the ATPS, when hydrated, comprises a mixed phase that separates into a first phase solution and a second phase solution as it passes through a porous matrix; Paper fluidic devices.

2. the probe comprises a binding moiety that binds to the analyte of interest; The device of claim 1 , wherein the binding moiety is selected from the group consisting of an antibody or antibody fragment, a lectin, a protein, a metabolite, a glycoprotein, a nucleic acid, a small molecule, a polymer, and a lipid.

3. The device of claim 2 , wherein the binding moiety comprises an antibody that binds to the analyte of interest.

4. The device of any one of claims 1 to 3, wherein the probe comprises a material selected from the group consisting of synthetic polymers, metals, minerals, glass, quartz, ceramics, biological polymers, plastics, and combinations thereof.

5. the probe comprises a biological polymer selected from the group consisting of dextran, polypropylene glycol, polyethylene glycol, and combinations thereof; or The device of claim 4 , wherein the probe comprises a metal selected from the group consisting of gold, silver, platinum, and combinations thereof.

6. The device of claim 4 , wherein the probe comprises a nanoparticle.

7. The device of any one of claims 4 to 6, wherein the probe comprises a coating.

8. The device of claim 7 , wherein the coating comprises polypropylene glycol or polyethylene glycol.

9. The device of claim 7 , wherein the coating has an affinity for the first phase solution or the second phase solution.

10. The device of any one of claims 1 to 9, wherein the probe comprises a detectable label.

11. 11. The device of claim 10, wherein the probe comprises a detectable label, the detectable label being selected from the group consisting of a chromogenic label, a fluorescent label, an enzymatic label, a heat-generating label, a radioactive label, and combinations thereof.

12. The device of any one of claims 1 to 3, wherein the probe comprises a magnetic particle.

13. The device of claim 12 comprising a magnet with a magnetic field oriented to attract the probe to a portion of the porous matrix.

14. The device of claim 12, comprising a magnet with a magnetic field, the force of the magnetic field enhancing the flow of the probe-analyte complexes toward the portion of the porous matrix.

15. The device according to any one of claims 1 to 14, wherein the ATPS or components thereof are dehydrated on and / or in at least a first portion of the porous matrix.

16. The device of claim 1 , wherein a first portion of the porous matrix has a different width than a second portion of the porous matrix.

17. A device according to any one of claims 1 to 16, wherein application of the sample to the device hydrates the ATPS, thereby providing the ATPS or a component thereof in a fluid phase.

18. 18. The device of any one of claims 1 to 17, wherein the ATPS and porous matrix are configured such that, in use, first phase solution and second phase solution flow through the porous matrix at different rates.

19. 18. A device according to any one of claims 1 to 17, wherein the ATPS and porous matrix are configured such that, in use, first phase and second phase solutions flow through the porous matrix in different directions.

20. The device of any one of claims 1 to 19, wherein the first phase solution comprises a micellar solution and the second phase solution comprises a polymer.

21. The device of any one of claims 1 to 19, wherein the first phase solution comprises a micellar solution and the second phase solution comprises a salt.

22. The device of claim 20 , wherein the micellar solution comprises a surfactant.

23. The device of claim 22, wherein the micellar solution comprises Triton-X.

24. The device of any one of claims 1 to 19, wherein the first phase solution comprises a polymer and the second phase solution comprises a polymer.

25. The device of any one of claims 1 to 19, wherein the first phase solution comprises a polymer and the second phase solution comprises a salt.

26. 26. The device of claim 25, wherein the first phase solution comprises polyethylene glycol and the second phase solution comprises potassium phosphate.

27. The first phase solution comprises a component selected from the group consisting of ethylhydroxyethylcellulose, methoxypolyethylene glycol, methylcellulose, polyethylene glycol (PEG), polypropylene glycol (PPG), polyvinyl alcohol, and polyvinylpyrrolidone; and the second phase solution comprises a component selected from the group consisting of aluminum sulfate, dextran, ficoll, hydroxypropyl dextran, hydroxypropyl starch, magnesium sulfate, maltodextrin, polyvinyl alcohol, polyvinylpyrrolidone, potassium phosphate, sodium citrate, and sodium sulfate; A device according to any one of the preceding claims.

28. or the ATPS comprises a PEG and a salt ATPS, the PEG and the salt being located in the same region in the porous matrix; or the ATPS comprises a PEG and a salt ATPS, the PEG and the salt being disposed in distinct regions in the porous matrix, the porous matrix comprising a single region comprising the salt and a single region comprising PEG, the region comprising PEG being disposed downstream from the region comprising the salt; or the ATPS comprises a PEG and a salt ATPS, the PEG and the salt being disposed in distinct regions in the porous matrix, the porous matrix comprising a first region comprising the salt, a second region comprising PEG, a third region comprising the salt, and a fourth region comprising the PEG, the second region being disposed downstream from the first region, the third region being disposed downstream from the second region, and the fourth region being disposed downstream from the third region; or the ATPS comprises a PEG and a salt ATPS, the PEG and the salt being disposed in distinct regions in the porous matrix, the porous matrix comprising a first region comprising the salt, a second region comprising PEG, a third region additionally comprising PEG, and a fourth region additionally comprising PEG, the second region being disposed downstream from the first region, the third region being disposed downstream from the second region, and the fourth region being disposed downstream from the third region; Downstream refers to the direction of flow of analyte through the porous matrix in use. A device according to any one of the preceding claims.

29. 20. The device of any one of claims 1 to 19, wherein the porous matrix comprises a material selected from cellulose, glass fiber, nitrocellulose, polyvinylidene fluoride, nylon, charge modified nylon, polyethersulfone, and combinations thereof.

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