RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No.

61/375,532, filed on August 20, 2010, which is hereby incorporated by reference in its entirety.

This application is also related to the following applications, filed concurrently herewith, the entire contents of which are incorporated herein by reference:

[0002] PCT Patent Application No. (TBA), filed on August 22, 2011 , entitled

"MULTIPHASE SYSTEMS AND USES THEREOF," identified by attorney docket number

0042697.251WO1;

[0003] PCT Patent Application No. (TBA), filed on August 22, 2011 , entitled "DENSITY- BASED SEPARATION OF BIOLOGICAL ANALYTES USING MULTIPHASE SYSTEMS," identified by attorney docket number 0042697.251W02; and

[0004] PCT Patent Application No. (TBA), filed on August 22, 2011 , entitled

"MULTIPHASE SYSTEMS FOR ANALYSIS OF SOLID MATERIALS," identified by attorney docket number 0042697.251W04.

INCORPORATION BY REFERENCE

[0005] All non-patent literature, patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described herein.

BACKGROUND

[0006] It is known that the aqueous mixtures of two polymers such as poly(ethylene glycol) (PEG) and dextran can separate spontaneously into two aqueous phases, called aqueous two-phase systems. Phase separation in aqueous solutions of polymers is an extraordinary and underexplored phenomenon. When two aqueous solutions of polymers are mixed, the resulting system is not homogeneous; rather, two discrete phases, or layers, form. These layers are ordered according to density and arise from the mutual immiscibility of the polymers for one another. In these systems, each phase predominantly consists of water (upwards of 70 - 90% (w/v)), while the polymer component is present in concentrations ranging from micromolar to millimolar. A low interfacial tension and rapid mass transfer of water-soluble molecules across the boundary characterize the interface between layers. [0007] Previous studies of partitioning between aqueous phases have been limited to biphasic systems of immiscible polymers or inorganic salts and have focused on applications in protein chemistry, cell partitioning, and manufacturing. These Aqueous Two-Phase Systems ("ATPS") are exemplified by the poly(ethylene glycol)-dextran, dextran-Ficoll systems, and a poly(ethylene glycol) system comprising (NH ₄ ) ₂ S0 ₄ .

[0008] Although numerous biphasic systems have been reported, there have been relatively few reports of polymer systems that exhibit multiphase, e.g., more than two phases, separation: (i) poly(ethylene glycol)-dextran-Ficoll, (ii) Triton X100-poly(ethylene glycol)-dextran, and (iii) poly(propylene glycol)-poly(ethylene glycol)-dextran-Ficoll. The four-phase system, however, is not entirely an aqueous system because the liquid poly(propylene glycol) that was employed in the assay is insoluble in water. Thus, while polymeric, this "four" phase system is equivalent to the incorporation of an organic solvent or perfluorinated alkane into a three-phase system.

SUMMARY

[0009] Described herein are methods of separating or analyzing analytes of interest using multi-phase systems ("MPS") comprising two or more phases having different densities, and a second phase property. In combination, the density and the second property of the multi-phase system are used to investigate, analyze and/or separate analytes in a sample of interest.

[0010] As used herein, a sample comprising one or more analyte is a mixture of species that can be characterized, and differentiated, by a set of physical properties (e.g., density, size, or potentially others). A multi-phase system can be designed to separate the analyte mixture into multiple populations at the interfaces between phases. These populations can be pure (a single species; ideal) or mixed. If mixed, the compositions of the species captured at each interface share at least one feature. For example, their densities are greater than that of the phase above but lesser than that of the phase below.

[0011] A method of analyzing or separating a sample comprising one or more analytes of interest using a multi-phase system includes a) providing a multi-phase system comprising two or more phases including at least a first phase and a second phase which optionally share a common solvent and are in contact with and phase-separated from each other to define upper and lower boundaries for each phase, wherein each of the first and second phases comprises a phase component selected from the group consisting of a polymer, a surfactant, and combinations thereof, and at least one of the first and second phases comprises a polymer;

each of the two or more phases has a different density so that the multi-phase system establishes a density gradient; and

each of the two or more phases has a second characteristic property;

b) introducing a sample comprising one or more analytes of interest into the multi-phase system; and

c) allowing the analyte to migrate to a location in the multiphase system that is characteristic both of its density and the second characteristic property of the two or more phases, wherein during migration the sample contacts one or more of the two or more phases sequentially.

[0012] The phase component is selected from the group consisting of a polymer, a surfactant and combinations thereof. The phase "combination" refers to the combination of a polymer and a surfactant, a combination of two or more polymers, a combination of two or more surfactants, or a combination of any number of polymers and any number of surfactants.

[0013] In some embodiments, the second characteristic property is one or more properties selected from the group consisting of viscosity, refraction index, optical absorbance, ionic strength, electrical conductivity, electrical susceptibility, magnetic susceptibility, X-ray opacity, and chemical potential. The common solvent of the first and second phases can be aqueous and organic.

[0014] As used herein, MPS refers to a multi-phase system. When two or more solutions containing a phase component are mixed, the resulting system is not homogeneous; rather, two or more discrete phases, or layers, form. These layers are ordered according to density and arise from the exhibit limited interaction of the phase components with one another. The two or more phases or solutions, which exhibit limited interaction and form distinct phase boundaries between adjacent phases. Each phase can be aqueous or non-aqueous. The nonaqueous phase comprises an organic liquid or an organic solvent.

[0015] As used herein, AMPS refers to an aqueous multi-phase polymer system. ATPS refers to an aqueous two-phase polymer system.

[0016] As used herein, an aqueous multi-phase polymer system comprises two or more polymer aqueous solutions or phases, which are phase-separated and in which at least two aqueous solutions each comprises a polymer. In some embodiments, the aqueous multi-phase polymer system can be combined with one or more immiscible organic phases to form a multiphase system.

[0017] As used herein, the use of the phrase "polymer" should include at least a block of the polymer composition, including, but are not limited to, the homopolymer, copolymer, terpolymer, random copolymer, and block copolymer of that polymer. As used herein, copolymer refers to a polymer derived from two monomeric species; similarly, a terpolymer refers to a polymer derived from three monomeric species. The polymer should also include various morphology, including, but are not limited to, linear polymer, branched polymer, crosslinked polymer, and dendrimer system. As an example, polyacrylamide refers to any polymer including a block of polyacrylamide, e.g. homopolymer, copolymer, terpolymer, block copolymer of polyacrylamide. Polyacrylamide can be linear polymer, branched polymer, crosslinked polymer, or dendrimer of polyacrylamide.

[0018] In one aspect, a method of analyzing or separating a sample comprising one or more analytes of interest using a multi-phase system is described, comprising:

a) providing a multi-phase system comprising two or more phases including at least a first and a second phases in contact with and phase-separated from each other to define upper and lower boundaries for each phase, wherein

each of the first and second phases comprises a phase component selected from the group consisting of a polymer, a surfactant, and combinations thereof and at least one of the first and second phases comprises a polymer;

each of the two or more phases has a different density so that the multi-phase system establishes a density gradient; each of the two or more phases has a second characteristic property;

b) introducing a sample comprising one or more analytes of interest into the multi-phase system; and

c) allowing the analyte to migrate to a location in the multiphase system that is characteristic both of its density and the second characteristic property of the two or more phases, wherein during migration the sample contacts one or more of the two or more phases sequentially.

[0019] In any of the preceding embodiments, the second characteristic property is one or more properties selected from the group consisting of viscosity, refractive index, optical absorbance, ionic strength, electrical conductivity, thermal conductivity, electrical susceptibility, magnetic susceptibility, X-ray opacity, and chemical potential.

[0020] In any of the preceding embodiments, the second characteristic property of each of the two or more phases is different.

[0021] In any of the preceding embodiments, the second characteristic properties for at least two of the two or more phases are the same.

[0022] In any of the preceding embodiments, the second characteristic property is viscosity.

[0023] In any of the preceding embodiments, the density of the one or more analytes is greater than the density of at least one of the two or more phases, and wherein the migration of the one or more analytes through the at least one phase of lesser density is a function of the size and morphology of the analyte.

[0024] In any of the preceding embodiments, the density of the one or more analytes is greater than all of the two or more phases.

[0025] In any of the preceding embodiments, the analyte/multi-phase system is centrifuged to affect migration of the analyte.

[0026] In any of the preceding embodiments, the analyte migrates under gravitational forces.

[0027] In any of the preceding embodiments, the location of the analyte is determined before the analyte reaches an equilibrium location.

[0028] In any of the preceding embodiments, at least one of the phases comprises a paramagnetic solution and the second characteristic is magnetic susceptibility.

[0029] In any of the preceding embodiments, the one or more analytes are diamagnetic.

[0030] In any of the preceding embodiments, each of the first and second phases comprises a paramagnetic solution.

[0031] In any of the preceding embodiments, the concentration of the paramagnetic solutions are the same.

[0032] In any of the preceding embodiments, the concentration of the paramagnetic solutions are different. [0033] In any of the preceding embodiments, the sample comprises two or more of the analytes and the method further comprises:

allowing the one or more analytes to migrate to an equilibrium boundary location of one of the two or more phases, wherein the equilibrium boundary location is based on density and wherein the equilibrium boundary location of the two or more analytes is the same or different; and

subjecting the density separated multiphase system to a magnetic field, wherein the two or more analytes at the equilibrium boundary location migrate to a second location that is a function of the magnetic field.

[0034] In any of the preceding embodiments, the two or more analytes have the same equilibrium boundary location and different second locations after subjection to the magnetic field.

[0035] In any of the preceding embodiments, the two or more phases comprise a magnetic additive.

[0036] In any of the preceding embodiments, distribution of the magnetic additive between phase is restricted due to preferential interaction of the magnetic additive with a phase component.

[0037] In any of the preceding embodiments, the phase component in the first phase of the two or more phases has a higher affinity towards the magnetic additive than the phase component in the second phase of the two or more phases.

[0038] In any of the preceding embodiments, there is a first buoyant density gradient within the first phase and a second buoyant density gradient within the second phase, and the first buoyant gradient is steeper than the second buoyant gradient.

[0039] In any of the preceding embodiments, the magnetic additive is equally distributed in the first and second phases.

[0040] In any of the preceding embodiments, there is a first buoyant density gradient within the first phase and a second buoyant density gradient within the second phase, and the first and second buoyant gradients are about the same.

[0041] In any of the preceding embodiments, the second characteristic is electric potential. [0042] In any of the preceding embodiments, the sample comprises at least two analytes having substantially similar densities and the location of the analytes in the multi-phase system is a function of the second characteristic.

[0043] In any of the preceding embodiments, the densities of the at least two analytes differ by less than 10%.

[0044] In any of the preceding embodiments, the densities of the at least two analytes differ by less than 5%.

[0045] In any of the preceding embodiments, the densities of the at least two analytes differ by less than 1%.

[0046] In any of the preceding embodiments, the ordering of the two or more phases based on the value of the secondary characteristic is different than the ordering of the two or more phases based on density.

[0047] In any of the preceding embodiments, the ordering of the two or more phases based on the value of the secondary characteristic is the same as the ordering of the two or more phases based on density.

[0048] In any of the preceding embodiments, the two or more analytes migrate into a phase interior.

[0049] In any of the preceding embodiments, the two or more analytes migrate into the interior of different phases of the multi-phase systems.

[0050] In any of the preceding embodiments, the two or more analytes migrate into the interior of the same phase of the multi-phase systems.

[0051] In any of the preceding embodiments, the first and second phases share a common solvent which is an aqueous solvent.

[0052] In any of the preceding embodiments, the first and second phases share a common solvent which is an organic solvent.

[0053] In any of the preceding embodiments, the phase separated system comprises three or more phases.

[0054] In any of the preceding embodiments, the phase separated system comprises three or more phases, the first and second phases share a common solvent which is water and additional phase separated phases are selected from the group consisting of organic solutions. [0055] In any of the preceding embodiments, the analyte is selected from the group consisting of solid particles, an aggregate of particles, a liquid or gel immiscible in the solvent, a liquid crystal, crystalline materials.

[0056] In any of the preceding embodiments, the analyte is selected from the group consisting of gem, bead, metal, glass, rock, mineral, crystal, plastic, bone, rubber, paper, fabric, coal, polymer particles, and a combination thereof.

[0057] In any of the preceding embodiments, the polymer is selected from the group of homopolymers, random copolymers, block copolymers, graft copolymers, ter-polymers, dendrimers, star polymers and combinations thereof.

[0058] In any of the preceding embodiments, the polymer is linear, branched and/or cross- linked.

BRIEF DESCRIPTION OF THE DRAWING

[0059] The subject matter is described with reference to the following figures, which are presented for the purpose of illustration only and are not intended to be limiting of the invention.

[0060] Fig. 1 illustrates the effect of time on the penetration of nanoparticles to a MPS composed of Brij 35, PEOZ and Ficoll at 16000g (The scale bar is 3 cm).

[0061] Fig. 2A shows an image of a three-phase aqueous multiphase system composed of Brij 35, PEOZ and Ficoll after separating the reaction products of a gold nanoparticle synthesis for 8 minutes. Fig. 2B shows TEM images of stock solution and samples collected from the layers as shown in (A), the scale bar in each image corresponds to 200 nm.

[0062] Fig. 3 shows the UV-Vis spectra on samples collected from the top, middle and bottom phases from Fig. 2 as well as the stock solution.

[0063] Fig. 4 shows the penetration of nanoparticles into single phase PEOZ with different densities and viscosities at different time intervals.

[0064] Fig. 5 is a schematic illustration of a magnetic levitation device applied to a single phase with three solid sphere added.

[0065] Fig. 6 illustrates an ATPS with a density step from the top phase to the bottom phase, a buoyant density gradient within the top and bottom phase under a magnetic field (the two gradients have similar slopes) [0066] Fig. 7 illustrates an ATPS with a density step from the top phase to the bottom phase, a buoyant density gradient within the top and bottom phase under a magnetic field (the two gradients have different slopes).

DETAILED DESCRIPTION

Multi-Phase Systems

[0067] A multi-phase system comprising two or more phases is described, wherein each phase comprises a phase component selected from the group consisting of a polymer, a surfactant, and a combination thereof. Each of the two or more phases has a different density and the phases, taken together, represent a density gradient, with the density of the phases increasing from the top phase to the bottom phase of the MPS.

[0068] In some embodiments, the MPS includes at least two phases with a common solvent. In some embodiments, the multi-phase system comprises at least three phases. In some embodiments, the multi-phase system comprises at least four phases. In some embodiments, the multi-phase polymer system comprises at least five phases. In some embodiments, the multi-phase polymer system comprises at least six phases. Multi-phase system with more phases are contemplated. When more than two phases are used, it is possible to include phases using different solvents. It is also possible to include phases that do not include a phase component, such as aqueous or organic solvents, liquid polymers, liquid metals, e.g., mercury, and ionic liquids. Such variety improves the ability of the system to separate complex samples. For example, the additional phases can extend the density range of the sample, making it possible to separate or distinguish samples of higher or lower density. Non-limiting examples of liquid polymers include poly(propylene glycol) (PPG), poly(ethylene glycol) (PEG), Pluronic L121 (PL), polydimethylsiloxane (PDMS), poly(ethyl vinyl ether) (PEVE), polybutadiene (PBD).

[0069] Each of the phases of the multi-phase system comprises a phase component. The phase component is selected from the group consisting of a polymer, a surfactant, and combinations thereof.

[0070] Non-limiting examples of polymer include dextran, polysucrose (herein referred to by the trade name "Ficoll"), poly( vinyl alcohol), poly(2-ethyl-2-oxazoline), poly(methacrylic acid), poly(ethylene glycol), polyacrylamide, polyethyleneimine, hydroxyethyl cellulose, polyvinylpyrrolidone, carboxy-polyacrylamide, poly(acrylic acid), poly(2-acrylamido-2- methyl- 1-propanesulfonic acid), dextran sulfate, diethylaminoethyl-dextran, chondroitin sulfate A, poly(2-vinylpyridine-N-oxide), poly(diallyldimethyl ammonium chloride), poly(styrene sulfonic acid), polyallylamine, alginic acid, poly(bisphenol A carbonate), polydimethylsiloxane , polystyrene, poly(4-vinylpyridine), polycaprolactone, polysulfone, poly(methyl methacrylate- co-methacrylic acid), poly(methyl methacrylate), poly(tetrahydrofuran), poly(propylene glycol), and poly( vinyl acetate). As used herein, a polymer includes its homopolymer, copolymer, terpolymer, block copolymer, random polymer, linear polymer, branched polymer, crosslinked polymer, and/or dendrimer system.

[0071] Non-limiting examples of surfactants include polysorbate (herein referred to by the trade name "Tween"), 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate

(CHAPS), polyoxyethylene-polyoxypropylene (herein referred to by the trade name

"Pluronic"), nonylphenol polyoxyethylene, l-O-Octyl- -D-glucopyranoside, 4-(l, 1,3,3- Tetramethylbutyl)phenyl-polyethylene glycol (herein referred to by the trade name "Triton X- 100"), 2-(Perfluoroalkyl)ethyl methacrylate (herein referred to by the trade name "Zonyl"), Ν,Ν-dimethyldodecylamine N-oxide, polyethylene glycol dodecyl ether (herein referred to by the trade name "Brij"), sodium dodecyl sulfate, sodium cholate, benzylalkonium chloride and dodecyltrimethylammonium chloride. In some specific embodiments, surfactant phases comprising Pluronic and CHAPS are selected to form an aqueous multi-phase polymer system with one or more aqueous polymer phases. Non- limiting examples of the polymer used in these embodiments include poly(methacrylic acid), poly(2-ethyl-2-oxazoline), dextran, Ficoll, polyacrylamide, and polyethyleneimine. The use of the surfactant can provide additional aqueous phases and facilitate the formation of the multi-phase systems. Other appropriate surfactants to accomplish this objective can be selected by the persons of ordinary skills in the art.

[0072] The phase components are selected so that the resulting phases are phase-separated from each other. As used herein, phase-separation refers to the phenomena where two ore more solutions, each comprising a phase component, when mixed together, form the same number of distinct phases where each phase has clear boundaries and is separated from other phases. Each phase component used in the solution is selected to be soluble in the solvent of the phase, so that each resulting phase is a distinct solution of the phase component and that each phase is phase-separated from other adjacent phase(s). When the multi-phase polymer system is designed, each phase component is selected to predominantly reside in one particular phase of the multi-phase system. It should be noted that in the resulting multi-phase system, every phase can contain varying amounts of other phase components from other phases in the MPS, in addition to the selected desired phase component in that phase. Unless otherwise specified, the phase component composition in each phase of the multi-phase system recited herein generally refers to the starting phase component composition of each phase, or to the predominant phase component composition of each phase. In some embodiments, the phase component composition on a phase component comprises predominantly one phase component and small amount of one or more other phase components. In some embodiments, the phase component composition in a phase comprises more than 50% and more typically more than about 70% of one phase component. In some embodiments, the phase component composition in a polymer phase comprises more than about 75% of one phase component. In some embodiments, the phase component composition in a phase comprises more than about 80% of one phase component. In some embodiments, the phase component composition on a phase comprises more than about 85% of one phase component by weight. In some embodiments, the phase component composition on a phase comprises more than about 90% of one phase component by weight. In some embodiments, the phase component composition in a phase comprises more than about 95% of one phase component. In some embodiments, the phase component composition in a phase comprises more than about 99% of one phase component by weight. Other combinations of phase component compositions are contemplated.

[0073] In some embodiments, the concentration of the phase component in the phase is from about 0.1% to about 50% (wt/vol). In some embodiments, the concentration of the phase component in the phase is from about 0.5% to about 40% (wt/vol). In some embodiments, the concentration of the phase component in the phase is from about 1% to about 20% (wt/vol). In some embodiments, the concentration of the phase component in the phase is from about 5% to about 10% (wt/vol). In some embodiments, the concentration of the phase component in the phase is about 10% (wt/vol). In some embodiments, the concentration of the phase component in the phase is about 15% (wt/vol). In some embodiments, the composition or density of the resulting phases in the multi-phase system could be affected by the starting concentration of the phase component phases.

[0074] In some embodiments, the multi-phase system is aqueous and each phase of the MPS comprises a phase component soluble in an aqueous solvent. Non-limiting examples of aqueous solvent include water, D ₂ 0, seawater, buffered water, cell culture medium, mine effluent, and irrigation water and mixtures thereof. Seawater could be used in studying small ocean organisms to keep buoyant densities close to what they are in nature. Irrigation water or mine effluent could be used to study the density effects on micro-organisms when exposed to these liquids. The analytes may also include particulate matter that could be suspended in these aqueous solutions.

[0075] In some embodiments, the aqueous multi-phase systems can comprise additional one or more organic phases comprising organic solvents. The organic phase is immiscible with and phase-separated from the aqueous polymer phases.

[0076] In some embodiments described herein, the multi-phase may comprise at least one aqueous phase. In some embodiments described herein, the multi-phase may comprise at least one organic phase. In some embodiments described herein, the multi-phase may comprise all organic phases or all aqueous phases.

[0077] In some embodiments, the aqueous multi-phase systems may be combined with one or more phases comprising organic solvents. Suitable organic solvents are those that are immiscible with water and will phase-separate from the aqueous phases.

[0078] In some embodiments, the multi-phase system is organic and each phase of the MPS comprises a phase component dissolved in an organic solvent.

[0079] In some specific embodiments, the different phases of the MPS comprise the same organic solvent. In other specific embodiments, the different phases of the MPS comprise different organic solvent.

[0080] Non-limiting examples of organic solvent include organic solvent selected from the groups consisting of liquid polymer, non-polar organic solvent, polar aprotic or protic solvent. Non-limiting examples of non-polar organic solvent include hexane and xylene. Non-limiting examples of polar aprotic organic solvent include dichloromethane and chloroform. Non- limiting examples of polar protic solvent include ethanol and methanol. Other suitable examples of organic solvents include supercritical fluid, fuel, oils, and fluorinated solvents, and combinations thereof. In some embodiments, non-limiting examples of suitable organic solvents include chloroform, dichloromethane ether, ethyl acetate, dimethylformamide, benzene, toluene, xylene, hexanes, acetonitrile, diethylether, trichloroethane, benzyl alcohol, acetone, aniline, mineral oil, perfluorinated solvents, and oils, tetrahydrofuran (THF), or water miscible solvents such as ethanol and methanol, supercritical C0 ₂ , complex hydrocarbons such as fuel, and hydrophobic, high viscosity fluids such as lubricants.

[0081] In other embodiments, the MPS comprises a liquid polymer as one of the phases. Non-limiting examples of liquid polymers include polyethyleneimine, polybutadiene, polydimethylsiloxane, poly(propylene glycol), poly(ethyl vinyl ether), cis(polyisoprene) and Tween (surfactant).

[0082] In some embodiments, the multi-phase system comprises at least an aqueous phase and at least an organic phase. Each phase may comprise a phase component and the mixture of aqueous and organic phases, taken together, represent a density gradient.

[0083] In some embodiments, one or more of the phases of the MPS are degassed to remove residual amount of gas dissolved in the phases. In some embodiments, the phases are degassed to remove oxygen from the phase to avoid possible oxidation of the sample applied onto the MPS. For example, highly viscous phases of MPS are degassed to remove any bubbles entrapped in phases or at interfaces. Degassing can also remove H ₂ , N ₂ , CH ₄ , NH ₃ , Ar, and other trace gases such as H ₂ S, and NOx. Other gases may be added (e.g., ammonia), to perform chemistry on separated species.

[0084] In some embodiments, one or more salt can be added to an aqueous multi-phase phase component system. The salts dissolve in the phase resulting in change of the phase density and typically do not partition between phases. Salts can also change the ionic strength of the solution. Non-limiting examples of salts include light or heavy salts, NaCl, NaBr, LiBr, KBr, RbBr, CsBr, and some phosphate salts. Non-limiting examples of salt also include sodium chloride, potassium chloride, sulfates, phosphates, nitrites, and citrates. Other salts could be sodium metatungstate or manganese chloride. The addition of salts can help the phase- separation process. In some embodiments, salt(s) can be added to the phase component systems in order to adjust the density, pH, and/or osmolality of the multiphase systems. In some embodiments, small molecules can be added for some specific functions. In some specific embodiments, heparin or sodium EDTA is added as an anticoagulant. In some other embodiments, sodium benzoate is added as a preservative. In other embodiments, paramagnetic salts are added to exploit magnetic properties.

[0085] In one or more embodiments, particularly multi-phase systems designed for use with more than two phase components, one or more polymers or surfactants that do not phase separate with each of the other phase components can be used as additives to modify the density, viscosity, osmolality, or refractive index of the phase component in which the additive resides. The polymers or surfactants are added to the various phases of the multi-phase system in addition to the phase components at concentrations less than is required to phase separate into a separate phase. In this instance, the surfactant performs the functions that are typical of surfactants, such as modify the surface tension of the solution. [0086] Non-limiting examples of other additives that can be included in the phases include those used in formulations to produce aggregation include, organic additives such as dyes and reactive or non-reactive dissolved gasses and cosolvents. In addition, the phases additives can be colloids or micelles.

[0087] Various types of form factors of the MPS can be used. In some embodiments, the MPS is contained in a tube or container, such as a test tube or flexible plastic tubing. In still other embodiments, the MPS is deposited on cloth or string. For example, a string or porous filament can be held in a test tube during the formation of the multi-phase system, such that the phase separated domains are absorbed into the porous filament. The filament is then removed from the MPS and contains a thin layer of phase-separated domains along the length of the filament.

[0088] In some embodiments, the MPS is deposited on paper. In some embodiments, the MPS is deposited on patterned paper. Paper can be patterned using hydrophobic barrier substantially permeating the thickness of the paper, thus defining one or more hydrophilic regions on the paper. In some specific embodiments, the paper can be patterned following the procedures described in PCT Publication No. 2008/049083, the content of which is

incorporated in its entirety by reference. In some specific embodiments, the MPS is aqueous and the aqueous phases of the MPS are deposited on a plurality of the hydrophilic regions on paper. By way of example, phase separation bands from an MPS column can be individually spotted on patterned regions of paper. The individual spotted regions can be stacked to recreate the density gradient in the MPS column. Alternatively, the individual spotted regions can be stacked to provide a density variation that is different from the original MPS column.

[0089] Generally speaking, if a combination of multiple phase component phases results in a phase-separated MPS, any sub-combination of the multiple phase component phases will also result in a phase-separated MPS. Thus, if a five -phase component MPS phase-separates, any four-polymer aqueous system selected from the five phase component phases can also phase- separate. Likewise, any two- or three- phase component MPS selected from the five phase component phases can also phase-separate. Other suitable combinations of polymers are contemplated.

[0090] As used herein, a MPS can be identified by its phase components in the phases of the MPS. For instance, a Ficoll— dextran— poly(2-ethyl-2-oxazoline) system refers to a three- phase MPS, wherein the phase components in each phases of the MPS are Ficoll, dextran, and poly(2-ethyl-2-oxazoline),but not necessarily in that order. Each phase includes a suitable solvent capable of dissolving the phase components. In some instances, a liquid polymer is used and the liquid polymer forms a phase with no solvent added.

[0091] According to one aspect, the analyte of interest of a size is suitable for density- based separation by MPS. The sample should be insoluble in the phases of the MPS and should be of a size that its interactions in solution are predominantly gravity driven. When analytes are sufficiently small, the molecular forces (electrostatic, van der Waals, etc) are sufficient strong relative to the size and mass of the analyte that the interaction with the solvent is dictated by these forces. Such analytes are capable therefore of partitioning selectively into one or another phase due to favorable molecular interactions that effectively disregard the densities of the analytes.

[0092] In most instances, the analyte has at least one dimension that is greater than 100 nm, greater than 200 nm, or greater than 500 nm, or greater than 1 μιη, which is sufficient for gravitation forces to predominate.

[0093] More detailed descriptions of the composition, method of preparation of the same, and method of using same can be found in the following applications, filed concurrently herewith, the entire contents of which are incorporated herein by reference:

PCT Patent Application No. (TBA), filed on August 22, 2011, entitled "MULTIPHASE SYSTEMS AND USES THEREOF," identified by attorney docket number 0042697.251 WO 1; PCT Patent Application No. (TBA), filed on August 22, 2011, entitled "DENSITY-BASED SEPARATION OF BIOLOGICAL ANALYTES USING MULTIPHASE SYSTEMS," identified by attorney docket number 0042697.251W02; and

PCT Patent Application No. (TBA), filed on August 22, 2011, entitled "MULTIPHASE SYSTEMS FOR ANALYSIS OF SOLID MATERIALS," identified by attorney docket number 0042697.251W04.

Method of analyzing or separating one or more analyte of interest based on density and non- density second characteristic property

[0094] Solutions containing different polymers and/or surfactants will naturally have different densities. As disclosed above, selection of phase components that are mutually immiscible will give rise to phase separated systems ordered on the basis of density. In addition, such systems can also have other properties, or can be engineered to possess other properties that can be used to obtain useful information regarding a test sample. An MPS system possessing a second characteristic property can be used to analyze, separate or isolate analytes of interest. [0095] In one aspect, a method of analyzing or separating a sample comprising one or more analytes of interest using a multi-phase system includes:

a) providing a multi-phase system comprising two or more phases including at least a first phase and a second phase which are in contact with and phase-separated from each other to define upper and lower boundaries for each phase, wherein

each of the first and second phases comprises a phase component selected from the group consisting of a polymer, a surfactant, and combinations thereof, and at least one of the first and second phases comprises a polymer;

each of the two or more phases has a different density so that the multi-phase system establishes a density gradient; and each of the two or more phases has a second characteristic property;

b) introducing a sample comprising one or more analytes of interest into the multi-phase system; and

c) allowing the analyte to migrate to a location in the multiphase system that is characteristic both of its density and the second characteristic property of the two or more phases, wherein during migration the sample contacts one or more of the two or more phases sequentially.

[0096] In some embodiments, the second characteristic property is one or more physical properties selected from the group consisting of viscosity, refraction index, optical absorbance, ionic strength, electrical conductivity, electrical susceptibility, magnetic susceptibility, thermal conductivity, X-ray opacity, and chemical potential. In some specific embodiments, the second characteristic is electric potential.

[0097] In some embodiments, an example using density and thermal properties is illustrated. Two materials with similar densities but different melting temperatures or rates of melting at a given temperature are isolated at an interface. The phases are heated above one material's transition temperature. This material melts, and the second component, remaining solid, is easily removed from the system. Thus, the second solid component can be easily recovered from the interface selectively.

[0098] In some embodiments, the second characteristic property of each of the two or more phases is different. For example, when the second characteristic is viscosity, each of the phases can have a different viscosity and the separation of analytes can be conducted on the basis of both density and the size, shape and morphology of the analyte. In some embodiments, the second characteristic properties for at least two of the two or more phases are the same. For example, when the second characteristic is the paramagnetism of the solution, each sample can have a paramagnetic solution having a different concentration of paramagnetic salt. The different magnetic strengths augment the sensitivity of the analyte to separation by density.

[0099] In some embodiments, the sample comprises at least two analytes having substantially similar densities and the location of the analytes in the multi-phase system is a function of the second characteristic. In some embodiments, the densities of the at least two analytes differ by less than 10%. In some embodiments, the densities of the at least two analytes differ by less than 5%. In some embodiments, the densities of the at least two analytes differ by less than 1%.

[0100] In some embodiments, the second characteristic property of the first and the second phases are substantially similar. For example, it may be useful to use phases that are electrically or ionically conductive. If such a property benefits separation across all phases, then the second characteristic property, e.g., ionic conductivity, can be constant across all phases. In other embodiments, the second property can vary greatly from phase to phase. For example, solution viscosity is a highly variable property and the phase to phase variation can be great. In other instance, the property does not vary greatly from phase to phase, or not at all.

[0101] The first and second phases can optionally share a common solvent. In some embodiments, the ordering of the two or more phases based on the value of the secondary characteristic is different than the ordering of the two or more phases based on density. As described herein, the densities of the phases in a MPS increase from the top phase to the bottom phase, i.e., the top phase of a MPS has the smallest density and the bottom phase has the highest density. Because the secondary characteristic of the phases is not required to be linked to the densities of the phases, the trend in the second property also will not be required to trend in the same way as density. In comparison, in some embodiments, the second characteristic property of the phase can increase from the top phase to the bottom or decrease from the top phase to the bottom phase. In some embodiments, the second characteristic property of the phase can first increase from the top phase to a middle phase, and then decrease from the middle phase to the bottom. In other instances, the secondary characteristic property can be arranged in no particular order or pattern. Taking the example of viscosity as the secondary characteristic property, the top and bottom phase of a three-phase MPS can both be more viscous than the middle phase. In some embodiments, the top and bottom phase of a three- phase MPS can both be less viscous than the middle phase. [0102] Because it is infrequent that the density of an analyte will exactly match that of the phase in an MPS, analytes separated by density alone, typically are localized at boundaries between phases in which the densities are lower and higher than the densities of the analyte. Using the additional separation abilities of the secondary characteristic property allows separation of the analyte into the interior of a phase-separated phase. In some embodiments, the two or more analytes migrate into a phase interior. In some embodiments, the two or more analytes migrate into the interior of different phases of the multi-phase systems. In some embodiments, the two or more analytes migrate into the interior of the same phase of the multiphase systems.

[0103] In some embodiments, the common solvent is an aqueous solvent. In some embodiments, the common solvent is an organic solvent. In some embodiments, the phase separated system comprises three or more phases. In some embodiments, the phase separated system comprises three or more phases, the common solvent is water and additional phase separated solutions are selected from the group consisting of organic solutions. It is also possible to include phases that do not include a phase component, such as organic or aqueous solvent, liquid polymers, liquid metals, and ionic liquids. Such variety improves the ability of the system to separate complex samples. The solutions can be supplemented with additives that provide the secondary characteristic to the solution. For example paramagnetic salts can be added to the solution to impart magnetic properties to the solution. Electrolyte salts can be added to the solution to impart ionic conductive properties to the solution.

[0104] In other embodiments, the polymer and/or surfactant can be selected to impart the additional secondary characteristic to the phase. As noted above, viscosity is one such characteristic that can be selected by adjusting the solids content of the phase-separated phase. In other instances, for example, where the secondary characteristic property is electrical conductivity, the MPS can be prepared using electrically conductive polymers and/or surfactants.

[0105] Various types of analytes are contemplated. In some embodiments, the analyte is selected from the group consisting of solid particles, colloids, an aggregate of particles, a liquid or gel immiscible in the solvent, a liquid crystal, crystalline materials. Non-limiting examples of analyte include bone, coal, gems, glass, metals, fuels, gases, plastics, fabrics, paper, and polymer particles. Samples comprising the analyte(s) can be introduced to the MPS in the form of a solution or suspension of material. Non-limiting examples of ways in which these samples can be added to the MPS include by pour, pipette, injection, drip, siphon, capillary action, spray, aspiration followed by expulsion, and pump.

Analyzing or separating one or more analytes based on density and viscosity

[0106] In some embodiments, the second characteristic property is viscosity. Viscosity affects the migration of an analyte through the phase by providing a drag or resistance to the flow or movement of the analyte through the medium. While at equilibrium, the analyte will occupy a position dictated by its density, the time and rate of migration to that equilibrium position is affected by the solution viscosity. Thus, it is possible to separate samples of similar or identical density, based on the shape, size and/or morphology of the analyte.

[0107] In some specific embodiments, the density of the one or more analytes is greater than the density of at least one of the two or more phases, and wherein the migration of the one or more analytes through the at least one phase of lesser density is a function of the size and morphology of the analyte. For example, in a sample that includes the analytes of the same composition, i.e., density, but with different shapes, the analytes will move through the phase (alternately referred to as migration or sedimentation) at different rates. The shape and size of the analyte particles will affect its sedimentation rate, even if the particles with different morphologies have similar or substantially the same density. A round particle provides less resistance and will migrate more rapidly than a rod shaped particle, for example. Thus, two or more analyte particles of the same density can be separated based on their morphologies under dynamic conditions, i.e., before the equilibrium is reached.

[0108] In some embodiments, the density of the one or more analytes is greater than all of the two or more phases. Under this scenario, the particles will eventually settle at the bottom of the MPS under equilibrium conditions due to its density. During migration sedimentation of the heavy particles to the bottom of the density gradient, the particles will traverse phase- separated phases of differing densities. Large particles and particles with aspected or complex morphologies will sediment more slowly. As demonstrated here, it is possible to distribute particles of different morphologies so that they are located in phases of different densities at an intermediate point in the sedimentation process. At this point, each phase can be collected separately, resulting in separation particles of same composition and different morphology. In other words, the isolation and separation of the analyte occurs before the analyte reaches an equilibrium location.

[0109] In some embodiments, the sample comprises two or more of the analytes and the migration of the two or more analytes includes sedimentation and the difference in the sedimentation rates of the two or more analytes is more than about 20%, 25%, 30%>, 40%>, 50%>, 60%, 70%, 80%, 90%, 100%, 300%, or 400%.

[0110] In some embodiments, the analyte/multi-phase system is centrifuged to affect migration of the analyte. In other embodiments, the analyte migrates under gravitational forces.

[0111] In some embodiments, the two or more analytes migrate into a phase interior. In some embodiments, the two or more analytes migrate into the interior of different phases of the multi-phase systems due to viscosity. Thus, the particles with different morphology, thus different sedimentation rate, will accumulate in different phases to allow the selective collection of particles with certain morphology. In other words, particles with different morphologies can be separated. In some embodiments, the two or more analytes migrate into the interior of the same phase of the multi-phase systems.

[0112] Specifically, the ability to obtain homogeneous populations of nanoscale objects from heterogeneous mixtures is a widely investigated problem since the accurate study of nanoparticles in sensing, imaging, and electronics applications benefits from monodisperse populations. The optical, electrical, and magnetic properties of nanoparticles strongly depend on their size and shape, as well as their composition. Typical nanoparticle syntheses do not produce homogeneous populations of nanoparticles, and post-synthesis separation methods are necessary to obtain a desired population with a narrow polydispersity. For example, the seed- mediated synthesis of gold nanorods, one of the most common preparations of gold nanorods, produces a variety of shapes (e.g., spheres rather than rods comprise up to 5-20%> (w/w) of the gold nanoparticles formed).

[0113] Current techniques used to separate monodisperse nanorods from mixtures are either time-consuming and inefficient (e.g., column chromatography) or require expensive instrumentation and secondary chemical modification of the nanoparticles (e.g., capillary electrophoresis).

[0114] Size- and shape-dependent particle separation is accomplished using multi-phase systems, which are mixtures of mutually immiscible aqueous solutions of polymers or surfactants that result in spontaneous phase separation, having phases of different viscosity. The phases of a MPS order according to density. [0115] While the phases order according to density, other physical properties, such as viscosity, do not necessarily increase in correlation with an increase in density. For example, an AMPS composed of Brij 35 (a poly(ethylene oxide) surfactant), poly(2-ethyl-2-oxazoline) and Ficoll (a polysucrose) has a highly viscous middle phase bordered by phases with lower viscosities (Table 1).

Table 1. Quantification of separation of nanoparticles using an AMPS.

Length of Thickness of

P η NS% NR% NS size NR NR top 1.031 30.8 1 99 NA 36±4 11±1 middle 1.045 541.9 99 1 25±2 NA NA bottom 1.112 139.0 37 63 244±36 289±27 100±9

[0116] Densities (p, g/cm3) and viscosities (η, cP) of each phase of a three-phase system composed of Brij 35 (26% wt/vol), PEOZ (30%> wt/vol) and Ficoll (35%> wt/vol) are shown in Table 1, where NS stands for nanospheres; where NR is nanorods. NA stands for 'not applicable' since less than 25 particles were counted for the corresponding entries. In the solution of nanoparticles, the size range of length of nanorods was 32-355 nm, the thickness was 8-125 nm; while the diameter of nanospheres was in the range of 18-265 nm.

[0117] The viscosity barriers produced, in conjunction with the density steps of this MPS are used to exploit the differences in the hydrodynamic behavior of different particle shapes during centrifugation to separate the reaction products of a gold nanoparticle synthesis based on both shape and size.

[0118] In MPS, viscosity is decoupled from density. Since the layers of a MPS are produced by phase separation, different zones of viscosity can be produced that (i) are thermodynamically stable, (ii) offer distinct interfaces that facilitate sample collection after separation, (iii) can be prepared in advance of use and stored, and (iv) reform readily by centrifugation if disrupted.

[0119] Since both density and viscosity effect sedimentation rates, the ability to control each property separately between phases affords exceptional tunability in MPS and make them good candidates for the separation of analytes based on density, size, and shape.

[0120] The concept of separation of analytes can be illustrated by using gold particles with different shape and sizes as an example. The MPS can be treated as single centrifugation medium, where the phases in the MPS are thermodynamically stable layers with different densities and viscosities. The density of gold nanoparticles (p _go id= 1 .3 g/cm ³ ) is considerably greater than the phases that comprise an MPS (p < 1.8 g/cm ³ ). As a result, it would not be ideal to use a centrifugation method that separates particles by density at equilibrium (e.g., isopycnic centrifugation). A rate-dependent centrifugation method uses time as an independent variable to optimize the separation of particles through the phases of a MPS during

centrifugation.

[0121] The large library of MPS offers a wide range of viscosities at the same density. For example, at 0.2 % density range (1.038 ± 0.001 g/cm ³ ), 3-[(3- cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS) and polyethyleneimine differ in viscosity by more than 100-fold, 2.1 cP and 302.5 cP, respectively (Table 2).

[0122] Figure 1 illustrates a separation result of a sample of gold particle subjected to a three-phase MPS composed of 26% (vol/vol) Brij 35, 30% (wt/vol) PEOZ, and 35% (wt/vol) Ficoll (top: p = 1.031 g/cm ³ , η = 30.8 cP; middle: p = 1.045 g/cm ³ , η = 541.9 cP; bottom: p = 1.112 g/cm ³ , η = 139 cP). The analytes of gold particles comprises nanoparticle species:

nanorods, nanospheres or larger particles of any shape. The nano-gold particle can be centrifuged through these systems for different time intervals (2-8 minutes) in order to evaluate the relationship between layer viscosity and the duration of centrifugation (Figure 1). At a solution viscosity of 19.1 cP, the nanorods penetrated into the system in less than 4 minutes and ceased to migrate as a band of particles, instead diffusing uniformly into the PEOZ solution. In some embodiments, the experiment conditions and centrifuge time are adjusted so that each phase of the MPS would contain, ideally, only one nanoparticle species: nanorods, nanospheres or larger particles of any shape. Isolating particles in separate zones, where each zone is at equilibrium and separated by an interface, would facilitate the recovery of separated species and improve sample purity.

[0123] Nanoparticles with larger sizes have the faster sedimentation rates compared to their smaller counterparts. Nanospheres will have intermediate sedimentation rate compared to the larger particles in this experiment and nanorods will generally have a slower sedimentation rate. In this system, it takes a 25 nm diameter sphere at 16000g 3 - 4 minutes to migrate through the top phase and begin to penetrate the middle phase, while a nanorod (/ = 36 nm, d = 11 nm) would require 11-12 minutes to travel through the top phase of the MPS. Based on these estimates, the optimal separation results require between 5-10 minutes of centrifugation. Figure 2 shows the image of a three-phase aqueous multiphase system composed of Brij 35, PEOZ and Ficoll after separating the reaction products of a gold nanoparticle synthesis for 8 minutes. The solvent of the stock solution stays as a clarified layer on top of the system, small nanorods (i.e., desired product) slightly penetrate into the top phase, small nanospheres migrate to the middle phase, and large particles of both shapes sediment to the bottom. Figure 2B shows TEM images of stock solution and samples collected from the layers as shown in Figure 2A, the scale bar in each image corresponds to 200 nm.

Analyzing or separating one or more analytes based on density and magnetic susceptibility

[0124] In some embodiments, the secondary characteristic property is the magnetic properties of the solution, or its magnetic susceptibility. The density-based separation and analysis using MPS as described herein, and magnetic levitation (MagLev) are combined to create separation media that do not require the use of centrifugation, and whose steps and gradients in density are stable and time-invariant. Paramagnetic co-solutes are included in one or more phases into order to produce buoyant density gradients within each phase of the MPS upon the application of a magnetic field gradient. The step in density between phases of a MPS and the concentration of paramagnetic co-solutes in the system control the dynamic range of buoyant densities available to each phase of the MPS.

[0125] Diamagnetic materials suspended in a paramagnetic solution will experience forces exerted on the particle when the solution is exposed to a magnetic field. As discussed in greater detail below, the magnetic field causes a first buoyant density gradient within the first phase and a second buoyant density gradient within the second phase. These forces can oppose or enhance the effect of gravity on the particle. This effect can alter the 'effective density' of the material. Such an effect can be use to further refine the density-based separation in an MPS. Modifications to the system can provide further refinement to the 'effective density' of the material, for example, by adjusting the strength (concentration) of the paramagnetic solution. In some specific embodiments, the concentration of the paramagnetic solutions are the same. In some specific embodiments, the concentration of the paramagnetic solutions are different.

[0126] Two or more analytes can be separated in an MPS system in which the phase- separated phases contain a paramagnetic solution by allowing the one or more analytes to migrate to an equilibrium boundary location of one of the two or more phases, wherein the equilibrium boundary location is based on density and wherein the equilibrium boundary location of the two or more analytes is the same or different; and subjecting the density separated multiphase system to a magnetic field, wherein the two or more analytes at the equilibrium boundary location migrate to a second location that is a function of the magnetic field.

[0127] In some embodiments, the two or more analytes have the same equilibrium boundary location and different second locations after being subjected to the magnetic field.

[0128] The paramagnetic solution is provided by adding a magnetic additive to the phases. The magnetic additive can be selected to have an affinity for a particular phase component so the diffusion of the additive into other phase is discouraged or prevented. In some

embodiments, the distribution of the magnetic additive between phases is restricted due to preferential interaction of the magnetic additive with a phase component. A variety of aqueous solutions of paramagnetic salts (e.g., MnCb, MnS04, GdCb, FeCb, CuS04, etc.) and chelated paramagnetic ions (e.g., Gd(DTPA) and Mn(EDTA); both in aqueous and non-aqueous solutions) are suitable for use as magnetic additives. In some specific embodiments, the magnetic additive is selectively bound to the phase component of one of the phases, thus resulting in selective enrichment of the magnetic additive in that phase. Non- limiting examples include metal ions such as Mn, Ca, Gd, Fe which can be chelated by polymer or surfactant with multiple carboxylic groups or other polarizable or charged moieties. In these embodiments, the phase component in the first phase of the two or more phases has a higher affinity towards the magnetic additive than the phase component in the second phase of the two or more phases. By preparing media using polymers with functional groups that can coordinate multivalent cations (e.g., carboxylic acids), it is demonstrated that the generation of a step in magnetic susceptibility between phases of the MPS; i.e., a step in the gradient of buoyant density.

Different concentrations of the paramagnetic additives in the phases will provide buoyant density gradients in each phase, if desired.

[0129] In other embodiments, the magnetic additives equally distribute in the first and second phases. In these embodiments, there is a first buoyant density gradient within the first phase and a second buoyant density gradient within the second phase, and the first and second buoyant gradients are about the same.

[0130] In some embodiments, the two or more analytes migrate into a phase interior. In some embodiments, the two or more analytes migrate into the interior of different phases of the multi-phase systems due to the phase's magnetic susceptibility. Thus, the particles can accumulate in different phases to allow the selective collection of particles. In some embodiments, the two or more analytes migrate into the interior of the same phase of the multiphase systems. [0131] The step in density between phases of a MPS decouples the levitation dynamic range (i.e., the sensitivity to differences in density) of MagLev from the magnitude of the density of the levitation medium, which makes possible the analysis of density ranges that are otherwise unavailable to either technique.

[0132] Magnetic levitation ("MagLev") is a convenient and low-cost means for accurately determining the density (with a resolution of +/- 0.0002 g/cm ³ ) of a diamagnetic object. The technique involves placing diamagnetic samples into a container filled with a paramagnetic fluid, which is then placed between two permanent magnets. The vertical position of the sample, in the presence of the magnetic field, correlates with its density. This position of the sample is independent of mass or volume, and measurements of density by magnetic levitation thus do not require standardized sample sizes. Further detail can be found in US Appln. Publ. No. 2010/0285606-A1, which is incorporated in its entirety by reference.

[0133] Magnetic levitation of diamagnetic objects is well-suited for analyzing contact trace objects: i) it does not destroy the sample, ii) it is readily calibrated with a series of density standards, and iii) it is applicable to small and irregularly shaped particles. Magnetic levitation is a technique whose sensitivity can be adjusted to the application.

[0134] An exemplary magnetic levitation device is illustrated in FIG. 5. The device is small (e.g., 8 cm x 6 cm x 12 cm) and both portable and inexpensive to fabricate and does not require additional external equipment. A magnetic levitation-based density measurement can be obtained in a short period of time (seconds to minutes, depending on the size of the object). The magnetic field in the magnetic levitation system is established by aligning two magnets 500, 510, e.g., NdFeB permanent magnets, co-axially apart from one another, with like poles facing each other. Diamagnetic objects 520, 521, 522 in a paramagnetic solution 525 are place within the magnetic field generated by the two magnets. In a magnetic field gradient, suspended in a paramagnetic fluid medium, diamagnetic samples appear to be repelled from regions of high magnetic field; in actuality, the diamagnetic object displaces an equal volume of paramagnetic solution, and it is the attractive interaction between this paramagnetic volume and the regions of high magnetic field and this paramagnetic volume that results in magnetic levitation. The relative position of an object in the vertical direction when placed in the magnetic levitation device, its "levitation height", is reached when the gravitational (F _g ) and magnetic forces (Fm) acting, in opposite directions, on the object have the same magnitude. Levitation height is indicated as "h" in FIG. 5. This position correlates, linearly, with the density of the sample. The analytical expression (and the associated assumptions and approximation) for correlating the levitation height of the sample with their density are described in K. A. Mirica, S. S. Shevkoplyas, S. T. Phillips, M. Gupta, G. M. Whitesides, Journal of the American Chemical Society 2009, 131, 10049, which is incorporated in its entirety by reference. A detailed knowledge of the parameters involved in making this correlation (e.g., the density of the paramagnetic medium, magnetic susceptibility of the medium, the magnetic field at the surface of the magnets) are not necessary in practice of the method.

[0135] In the magnetic levitation experiments presented here, each sample is placed in a container, e.g., the 1 cm · 1 cm · 4.5 cm cuvette shown in FIG. 5. Magnetic levitation density analysis of these samples is achieved by placing them in a solution containing a strongly paramagnetic ion. A variety of aqueous solutions of paramagnetic salts (e.g., MnCb, MnS04, GdCb, FeCb, Q1SO4, etc.) and chelated paramagnetic ions (e.g., Gd(DTPA) and Mn(EDTA); both in aqueous and non-aqueous solutions) are suitable for magnetic levitation. In some embodiments, the paramagnetic solution includes MnCl ₂ (chosen because the solutions are transparent and because manganese salts are inexpensive). The salts used to prepare the paramagnetic solutions are mild and not aggressive to most substances. For example, prolonged exposure to a paramagnetic solution, e.g., MnCl ₂ , does not affect the density of a sample of smokeless gunpowder. Also, repeated measurements (i.e., repeated exposure to a solution of MnCl ₂ and washing and drying steps) do not affect the density of a sample of smokeless gunpowder.

[0136] In some embodiments, the analytes are diamagnetic materials. When suspended in a solution containing paramagnetic ions, diamagnetic materials (e.g., glass or plastics) will levitate within the MagLev device. Levitation is a result of a diamagnetic object, which interacts weakly with magnetic fields, displacing an equal volume of the paramagnetic solution, which is attracted to regions of high magnetic field. At equilibrium, the magnetic forces (F _m ) acting on the diamagnetic material are balanced with the gravitational force (F _g ) in the vertical direction at a height, h (m), which Applicants call the levitation height. The levitation height can be expressed as a function of the physical properties of the diamagnetic material, the paramagnetic solution, and the MagLev device using equation I:

(P _s - P _m )g ₀ d ²

h (I) [0137] In this equation, p _m (kg/m ³ ) is the density of the paramagnetic medium, p _s (kg/m ³ ) is the density of the levitating material, g is the acceleration due to gravity (m/s ² ), χ _η and χ (both unitless) are the magnetic susceptibilities of the paramagnetic medium and the suspended material, respectively, μο (T'm/A) is the magnetic permeability of free space, and B ₀ (T) is the magnetic field strength at the surface of the magnet. The MagLev device that Applicants use in this study has a magnetic field strength of 0.355 T.

[0138] Equation 1 is used to calculate the density of a diamagnetic material using the measurement of the levitation height. By this association, the linear gradient can be considered in magnetic field produced by the device to be equivalent to a linear gradient in buoyant density within the levitation medium.

[0139] In some embodiments, diamagnetic additives are incorporated into the levitation medium (e.g., sucrose or CaCl ₂ ) as a means to tune the density of the levitation medium without changing the magnetic susceptibility of the medium. Water-soluble polymers, such as those used in the preparation of ATPS, are also diamagnetic and may be used as additives to MagLev levitation media.

[0140] In some embodiments, the use of density and magnetic levitation to separate two or more analytes is illustrated with reference to Figure 6. As shown in Figure 6, a two phase MPS is used to separate a sample containing five diamagnetic beads, 601-605. The MPS has a magnetic additive evenly distributed into the top and the bottom phases. In the absence of a magnetic field gradient (B VB = 0), gravity is the only force acting on the beads. Additionally, in the absence of any magnetic field, the top phase has a density from that of the bottom phase, however, the buoyant density within the top phase at each vertical position are the same (See Figure 6A). The position of beads within the MPS is, therefore, confined to three possible positions: the top of the system, the interface between phases, or the bottom of the system. In this scenario, beads 601 and 602 rest on the top boundary of the top phase, bead 603 reside at the interface of the top and bottom phase, and beads 604 and 605 are at the bottom boundary of the bottom phase.

[0141] In comparison, when the MPS is placed within the MagLev device (B VB≠ 0), a gradient in buoyant density is produced within each phase, and the beads can now levitate within a phase and at a height that is proportional to their densities. As shown in Figure 6B, the buoyant density changes within the top phase and within the bottom phase, so that a gradient is present within each phase, and the beads 601 and 602 which have different densities, now levitate at different vertical locations within the top phase. This is so because beads 601 and 602 have different densities within the range of the buoyant density of the top phase, i.e., from about 1.03 to about 1.05 g/cm ³ . Thus, beads 601 and 602, both at the top boundary of the top phase in the absence of the magnetic field, now levitate at different locations of the MPS, i.e., within the top phases. Similarly, beads 604 and 605 now reside within bottom phase at different vertical positions. Note that in this case, the two phases have the same concentration of the paramagnetic solutions. In some embodiments, the two phases have the same concentration of a magnetic additive. As a result, the top and bottom phases have the same buoyant density slope.

[0142] In another embodiment, the use of density and magnetic levitation to separate two or more analytes is illustrated with reference to Figure 7. As shown in Figure 7, a two phase MPS is used to separate a sample containing five diamagnetic beads, 701-705. The MPS has a magnetic additive not evenly distributed into the top and the bottom phases. In the absence of a magnetic field gradient (B VB = 0), gravity is the only force acting on the beads. Additionally, in the absence of any magnetic field, the top phase has a density different from that of the bottom phase, however, the buoyant density within the top phase at each vertical position are the same (See Figure 7A). The position of beads within the MPS is, therefore, confined to three possible positions: the top of the system, the interface between phases, or the bottom of the system. In this scenario, beads 701 and 702 rest on the top boundary of the top phase, bead 703 resides at the interface of the top and bottom phase, and spheres 704 and 705 are at the bottom boundary of the bottom phase.

[0143] In comparison, when the MPS is placed within the MagLev device (B VB≠ 0), a gradient in buoyant density is produced within each phase, and the beads can now levitate within a phase and at a height that is proportional to their densities. As shown in Figure 7B, the buoyant density changes within the top phase and within the bottom phase, so that a gradient is present within each phase, and the beads 701 and 702 which have different densities, now levitate at different vertical locations within the top phase. Thus, beads 701 and 702, both at the top boundary of the top phase in the absence of the magnetic field, now levitate at different locations of the MPS, i.e., within the top phases. Similarly, bead 704 now reside within bottom phase at a different vertical position, compared with its position in Figure 7A. Note that in this case, the two phases have different concentration of the paramagnetic solutions. In some embodiments, the two phases have the different concentration of a magnetic additive. In some specific embodiments, the phase component of the bottom phase may have higher affinity towards the magnetic additive than the phase component of the top. In some specific embodiments, the top bottom may have a phase component containing multiple carboxylic acid which could chelate magnetic salt, e.g., a metal ion. As a result, the top and bottom phases have different buoyant density slopes (See Figure 7B, which shows a steeper buoyant density slope in the bottom phase (slope 2 is steeper than slope 1) due to the preferential accumulation of the magnetic additive in the bottom).

[0144] In some embodiments, the analyte is allowed to migrate based on gravity. In other embodiments, the analyte is allowed to migrate using a centrifuge. Non-limiting examples of centrifuges include motorized centrifuges or soft-centrifuges. Soft centrifugation refers the uses of soft tubing, e.g., polyethylene tubing, as the sample container and a simple device as the rotor (see, Wong et al., "Egg beater as centrifuge: isolating human blood plasma from whole blood in resource-poor setting", Lab Chip, 2008, 8, 2032-2037). In some specific embodiments, the soft centrifugation is achieved by an eggbeater centrifuge. Other methods of soft centrifugation known in the art are also contemplated.

[0145] After migration, the analyte of interest may be recovered from the MPS be extracting the analyte from one of the phases or from an interface. As a result, one analyte may preferentially accumulate in one of the phase or at an interface in the MPS, while another impurities or subjects in the sample containing the analyte may preferentially accumulate in another phase or interface of the MPS. The desired subject in the mixture can then be recovered by retrieving the phase that this subject preferentially has accumulated in, thus resulting in an improved purity of such subject.

[0146] The sample containing the analyte can be liquid, solid, gel, or liquid crystal. The sample is selected from the group consisting of forensics study sample, a sample indicative of animal health, a sample indicative of human identity used for border control, home land security, or intelligence, a sample from food processing, a sample indicative of product quality, a sample indicative of product authenticity, a sample indicative of environmental safety, a sample containing different crystal polymorphs, and a combination thereof.

[0147] The analytes in the sample include, without limitation, cells, organelles, cell extracts, viruses, tissue extracts, small molecules, large-sized molecules, e.g., biomolecules including proteins, particles, and colloids. The method disclosed herein can be used for separating small molecules and large bimolecular, e.g., proteins, extracting recombinant proteins, analyzing enzymatic digestions, and partitioning cells. Other uses known in the art are contemplated. In some embodiments, a two-phase MPS can be used for purposes disclosed herein. In some other embodiments, three or more phase systems are used. It was believed that the inclusion of additional phases may prevent the enrichment of the target molecule in a specific phase, because the target molecule may distribute into the additional phases. This belief may account for the lack of literature regarding these multi-phase polymer systems. Applicants have surprisingly found that broadening the landscape of polymers that demonstrate immiscibility in aqueous multi-phase polymer systems provides superior tunability for applications based on differences in density and affinity and finer control over the partitioning of complex mixtures of subjects.

[0148] In some embodiments, the multi-phase polymer system is provided by mixing suitable polymers or surfactants with a solvent and subjecting the mixture to centrifugation. Any type of centrifugation known in the art can be used in the formation of the MPS. In some embodiments, the MPS is formed using soft centrifugation. Soft centrifugation is described above. In some specific embodiments, the soft centrifugation is achieved by an eggbeater centrifuge. Other methods of soft centrifugation known in the art are also contemplated.

[0149] In some embodiments, analytes, e.g., cells, cell organelles, or proteins, can be separated if they have different densities.

[0150] The method Applicants report here combines the portability and simplicity of the soft centrifuge with aqueous multiphase density barriers generated from immiscible polymers or surfactants. Immiscible polymers or surfactants have numerous advantages over

discontinuous density gradients for field use: they are easily prepared, owing to the nature of their mutual immiscibility; they are stable, and thus amenable to long term storage; and they are versatile, as Applicants have previously identified a suite of multiphase systems that can be altered (composition and/or density) to suit the application.

[0151] Moreover, the soft centrifugation assay requires no more than 10 of a biosample such as whole blood (easily obtained from a single fmgerstick), and rapidly separates blood components. In some embodiments, the soft centrifugation takes 10 minutes or less.

[0152] The invention is described in the following embodiments, which are presented for the purpose of illustration only and are not intended to be limiting of the invention.

Example 1. Use of Viscosity as a Secondary Characteristic Property

Chemicals

[0153] Alginic acid sodium salt, Brij 35 , 3-[(3-cholamidopropyl) dimethylammonio]-l- propanesulfonate (CHAPS), chondroitin sulfate A, dextran sulfate sodium salt, ethyl orange, Ficoll, (hydroxypropyl)methyl cellulose, poly(2-acrylamido-2-methyl-l-propanesulfonic acid), poly(2-ethyl-2-oxazoline), polyacrylamide, poly(diallyldimethylammonium chloride), poly(ethylene glycol), polyethyleneimine, poly(methacrylic acid sodium salt), poly(propylene glycol), polyvinylpyrrolidone, sodium cholate, Tween 20, Triton X-100, Pluronic F68 and Zonyl were purchased from Sigma- Aldrich. Poly(acrylic acid), poly(allylamine hydrochloride), poly(styrenesulfonic acid sodium salt), and poly( vinyl alcohol) were obtained from

Polysciences. Dextran was purchased from Spectrum Chemical. Diethylaminoethyl-dextran hydrochloride was purchased from MP Biomedicals. Carboxy-modified polyacrylamide, hydroxyethyl cellulose, methyl cellulose were purchased from Scientific Polymer Products. N, N-dimethyldodecylamine N-oxide was purchased from Fluka. N-octyl-P-D-glucopyranoside was purchased from Calbiochem. Sodium dodecyl sulfate was purchased from J.T. Baker. Glycerol was obtained from Pierce Scientific. Polystyrene microspheres were purchased from Polysciences, Inc. All chemicals were used without further purification.

Experimental Designs

[0154] The MPS is a single centrifugation medium, where the phases in the MPS are thermodynamically stable layers with different densities and viscosities. The density of gold nanoparticles (p _go i _d = 19.3 g/cm ³ ) is considerably greater than the phases that comprise an MPS (p < 1.8 g/cm ³ ). As a result, a centrifugation method that separates particles by density at equilibrium (e.g., isopycnic centrifugation) is not useful to separate gold particles. A rate- dependent centrifugation method uses time as an independent variable to optimize the separation of particles through the phases of an MPS during centrifugation.

[0155] Gold nanorods were prepared according to a seeded growth method developed by Nikoobakht and El-Sayed with minor modifications (Nikoobakht et al., Chem. Mater., 2003, 15 (10), pp 1957-1962). This method uses cetyltrimethylammonium bromide (CTAB) to produce gold nanorods with an aspect ratio of approximately four-to-one. It is indeed possible to produce nanorods with a higher aspect ratio (up to 18: 1), but the smaller aspect ratio better demonstrates the sensitivity of our approach to discriminating nanoscale objects based on size since the difference in sedimentation rates between a sphere and a rod increases in proportion to the aspect ratio (please see eqn's 3 & 4).

[0156] Cethyl trimethylammonium bromide (CTAB) solution (2.5 mL, 0.20 M) was first mixed with 1.5 mL of 1.0 mM HAuCl ₄ . To the vigorously stirred solution, 0.60 mL of ice-cold 0.010 M NaBH ₄ was quickly added, which resulted in the immediate formation of a brownish- yellow solution. The seed solution was continued to be vigorously stirred for another 2 minutes and for aging it was kept at 25°C for about 30 minutes. [0157] 50 ml of a 0.2M CTAB solution was mixed with 5 ml of an aqueous 5mM HAuCl ₄ solution, 2.8 ml of an aqueous 4mM AgN0 ₃ solution, and 40 ml of water. Following the addition of 1 ml of an aqueous 0.8M solution of ascorbic acid, the dark yellow solution turned colorless. Finally, 1.0 ml of an aged seed solution of nanoparticles was added to the growth solution at 27-30°C. The color of the solution gradually changed within 10-20 minutes. The growth medium was kept at constant temperature (27-30°C) for 20 hours.

[0158] UV-Vis spectroscopy was carried out on a Hewlett Packard 8453

Spectrophotometer. 60 μΐ of sample was collected from each layer and directly analyzed without dilution. For the measurement of the precipitate, the entire solution above was decanted and the solid in the bottom was suspended in a 100 10 mg/ml CTAB solution. (Figure 3)

[0159] The TEM was carried on JEOL 2100; Applicants used Lacey Formvar/Carbon, 300- mesh, copper TEM grids (Ted Pella, Inc.). For TEM on layer 1 and 2; 50 μΐ of sample from each layer was diluted by adding 150 μΐ of DI water, centrifuged the solutions at 16,000g for 10 minutes, and collected 20 μΐ of sample from the bottom. Applicants put 2μ1 of this concentrated solution onto a TEM grid and allowed it to evaporate at ambient conditions. For the bottom layer, the entire solution was decanted and suspended the precipitate in 50 μΐ lOmg/ml CTAB solution and dropped 2μ1 of this solution onto a TEM grid and allowed it to evaporate at ambient conditions.

[0160] The size and number of nanoparticles was determined by analyzing 10 bright field TEM images with even illumination from each solution layer using Image J software (National Institute of Health, USA). The analyzed images were chosen to be as representative of the layer as possible. Data was from -1000 particles/layer and their average size was calculated taking the standard deviation as error of measurements.

[0161] The rhemometry measurements were carried out on a AR2-G2 stress-controlled rheometer (TA Instruments) at room temperature in the shear-rate range of 1/10 to 1/200 (1/s) at room temperature with a geometry cone/plate 1° 40 mm (S#987620).

[0162] Shown in Figure 4 is the penetration of nanoparticles into single phase PEOZ with different densities and viscosities at different time intervals.

[0163] Charged polymers and surfactants such as poly(methacrylic acid), poly(allyl amine), poly(acrylic acid), poly(diallydimethylammonium chloride), polyethyleneimine, and 3-[(3- cholamidopropyl)dimethylammonio]-l-propanesulfonate form gel-like structures and result in the precipitation of the nanoparticles from the solution. However, non-ionic chemicals such as dextran, Ficoll (a polysucrose), poly(ethylene glycol), poly(2-ethyl-2-oxazoline), poly( vinyl alcohol), Brij 35 (a poly(ethylene oxide) surfactant) and Pluronic F68 (a poly(ethylene oxide)- poly(propylene oxide) copolymer surfactant) were compatible with nanoparticles; they are also species that frequently produce multiphase systems and provide a number of potential candidates for separation media.

[0164] The large library of MPS offers a wide range of viscosities at the same density. For example, at 0.2 % density range (1.038 ± 0.001 g/cm ³ ), 3-[(3- cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS) and polyethyleneimine differ in viscosity by more than 100-fold, 2.1 cP and 302.5 cP, respectively. (Table 2)

Table 2. The viscosities of different reagents with densities 1.038 ± 0.001 g/cm ³ (± 0.2%).

reagent density (gr/cm ³ ) viscosity (cP)

CHAPS 1.037 2.1

PVP 1.038 7.4

PEG 1.037 29.8

Pluronic F68 1.039 30

PEOZ 1.039 175

PEI 1.037 302.5

[0165] UV-Vis spectroscopy was used to characterize the nanoparticle populations, both before and after separation, by quantifying the plasmonic absorption bands of the gold nanoparticles. The wavelengths absorbed by gold nanoparticles depend on the size and shape of the particle. For these wavelengths (400 - 1100 nm), the polymers and surfactants of MPS are transparent and do not contribute to the measured absorbance. Transmission electron microscopy (TEM) on these samples was used to image the contents of each layer and perform a statistical analysis of the particle size and shape distribution of the isolated population (> 1000 particles for each sample).

[0166] Following the synthesis of gold nanoparticles, the stock solution contained a mixture of rods and spheres (49% and 51%, respectively). The lengths of the rods ranged from 32 - 355 nm, and their thicknesses ranged from 8 - 125 nm. The diameters of the spheres ranged from 18 - 265 nm.

Dynamic Separation by Sedimentation Differences [0167] In order to separate objects by shape and size, the differences in viscous drag that such objects experience during centrifugation was exploited. The concept is best exemplified by the ratio of sedimentation coefficients of two different shapes, where the sedimentation coefficient S is defined as:

^ ~ ~® ί ψΐ. 1}

[0168] Where "r is the terminal velocity of the object in the medium and ^a is the acceleration provided by gravity or centrifugation. Using Stokes' Law, the sedimentation coefficient for a sphere ¾ can be found:

Is — I— i¾-¾.2>

[0169] where η (P; gcm ^' V ¹ ) is the viscosity of the medium, dn (cm) is the diameter of the sphere and p _p (g/cm ³ ) and /(g/cm ³ ) are the densities of the particle and the liquid medium, respectively. This equation can be modified to account for arbitraty shapes:

[0170] where f/f _a is the frictional coefficient of an arbitrary shape and usually numerically solved. The term d _eq (cm) is the diameter of a sphere of equivalent volume to the object.

[0171] Hubbard and Douglas reported the frictional coefficient for a rod with a diameter, d (cm), and a length, / (cm), to be equivalent to (valid for 0< l/d <8):

[0172] The aspect ratio of the target nanorod population is 3.2 (/ = 36 nm, d = \ \ nm); comparing sedimentation coefficients given by equations 2, 3 and 4, one would expect the sedimentation velocities of nanospheres (d = 25 nm) to be ~ 4.4 times faster than the nanorods in a liquid medium. All larger reaction by-products will also sediment more rapidly than the desired nanorods.

[0173] After synthesizing gold nanoparticles, a 20-fold concentrated stock solution was prepared by centrifuging the total reaction volume for 5 minutes at 16000g and collecting the sediment by pipette. This concentrated mixture of gold nanoparticles was layered on top of an MPS and centrifuged these systems at 16000g for a range of times. The effect of time on the penetration of particles to MPS was monitored visually and spectroscopically; as demonstrated in an example of visual tracking of nanoparticle penetration into MPS in Figure 1.

Separation of Reaction Products of a Nanorod Synthesis

[0174] To estimate the viscosity range of the top phase that will lead to the best separation of nanoparticles, a range of viscosities were surveyed. Ideally, the top phase should be viscous enough to delay the penetration of the nanorods into the system until the nanospheres, which have larger sedimentation velocities, migrate into the middle phase.

[0175] The stock solution of nanoparticles were overlayed onto a single phase polymer solution composed of poly(2-ethyl-2-oxazoline) (PEOZ) at viscosities of 1100, 65.1, 19.1 and 2.56 cP (Table 3). Nanoparticle reaction products were centrifuged through these systems for different time intervals (2-8 minutes) in order to evaluate the relationship between layer viscosity and the duration of centrifugation (Figure 1).

Table 3. The densities and viscosities for dilutions of PEOZ.

dilutions of PEOZ (wt/v) density (gr/cm ³ ) viscosity (cP)

35% 1.059 1100

24% 1.039 193

17.50% 1.028 65.1

12% 1.017 19.1

8% 1.012 10.8

4% 1.004 2.56

[0176] Solution viscosities of 1100 cP and 65.1 cP were too high for the effective separation of nanorods and nanospheres into distinct populations. At a solution viscosity of 19.1 cP, the nanorods penetrated into the system in less than 4 minutes and ceased to migrate as a band of particles, instead diffusing uniformly into the PEOZ solution. A target range between 20 and 65 cP for the viscosity of the top phase of an MPS was identified.

[0177] Using the desired range of viscosity and the theoretical optimum timeframe for the nanoparticle separation assay, a series of three-phase MPS were investigated as centrifugation media. A three-phase system would ensure that each phase of the MPS would contain, ideally, only one nanoparticle species: nanorods, nanospheres or larger particles of any shape. Isolating particles in separate zones, where each zone is at equilibrium and separated by an interface, would facilitate the recovery of separated species and improve sample purity. [0178] A three-phase MPS composed of 26% (vol/vol) Brij 35, 30% (wt/vol) PEOZ, and 35% (wt/vol) Ficoll (top: p = 1.031 g/cm ³ , η = 30.8 cP; middle: p = 1.045 g/cm ³ , η = 541.9 cP; bottom: p = 1.112 g/cm ³ , η = 139 cP) provided enrichment of the gold nanorod population. (Figure 2)

[0179] In this system, it takes a 25 nm diameter sphere at 16000g 3 - 4 minutes to migrate through the top phase and begin to penetrate the middle phase, while a nanorod (/ = 36 nm, d = 11 nm) would require 11-12 minutes to travel through the top phase of the MPS. Based on these estimates, optimal separation results require between 5-10 minutes of centrifugation.

[0180] The efficiency of the nanoparticle separation was quantified by transmission electron microscopy (TEM; Figure 2) and UV-Vis spectroscopy (Figure 3). In the top phase, nanorods are enriched to 99.8% and the average length and thickness of the nanorods is 36 ± 4 nm and 11 ± 1 nm, respectively. The amount of nanorods of similar size in other layers was less than 1.4%, demonstrating that the method is highly shape- and size-dependent. Similarly, the middle phase contained 98.6 % nanospheres, with average diameters 25 ± 2 nm, indicating a decrease in polydispersity.

Example 2. Use of Magnetic Properties as the Secondary Characteristic Property

[0181] The choice of paramagnetic ion and its concentration (i.e., the total magnetic susceptibility of the medium) control the slope of the density gradient within the levitation medium, while the density of the paramagnetic medium controls the range of densities that are available to the system. Salts or complexes of manganese and gadolinium can be used for MagLev experiments.

[0182] Diamagnetic additives was incorporated into the levitation medium (e.g., sucrose or CaCl ₂ [Mirica]) as a means to tune the density of the levitation medium without changing the magnetic susceptibility of the medium. Water-soluble polymers, such as those used in the preparation of ATPS, are also diamagnetic and may be used as additives to MagLev levitation media.

[0183] ATPS were prepared from solutions of immiscible water-soluble polymers whose mixtures result in spontaneous phase separation.

[0184] Two formulations of ATPS were investigated for their use in MagLev assays: (i) those composed of polymers that do not interact with multivalent cations and (ii) those composed of polymers with coordinating functional groups that may chelate multivalent cations. [0185] An ATPS was prepared from a mixture of poly(ethylene glycol) and dextran;

neither polymer interacts preferentially with Mn ²⁺ and a homogeneous distribution of paramagnetic ions throughout both phases of the ATPS is expected . An ATPS from a mixture of poly( vinyl alcohol) and carboxy-polyacrylamide is prepared; the carboxylic acid groups on carboxy-polyacrylamide are capable of serving as ligands to coordinate multivalent cations, but are in a sufficiently low density that the resulting Mn-polymer complexes are soluble. In this case, a heterogeneous distribution of paramagnetic ions between the phases of an ATPS prepared from carboxy-polyacrylamide is expected, and thus a step in the magnetic

susceptibility of the levitation medium across the phase boundary.

[0186] Beads with calibrated densities (+/- 0.0002 g/cm ³ ) were used as references to estimate the concentration of Mn ²⁺ in each phase of an ATPS. For example, the density of the top phase of an ATPS was measured and added to a selection of reference beads. By measuring the levitation height of each bead, equation 1 can be used to calculate the magnetic susceptibility of the medium and, therefore, the concentration of Mn ²⁺ in the phase.

[0187] In order to quantify the concentration of Mn ²⁺ in each phase of an ATPS, inductively coupled plasma atomic emission spectroscopy (ICP-AES) is used. The intensities of the Mn- specific wavelength (λ = 257.610 nm) were measured, in both the radial and axial modes, from aliquots of each phase at two different dilutions. These intensities were compared to those from a standard curve generated using known concentrations of MnCl ₂ in order to determine the concentration of Mn ²⁺ in each phase.

[0188] An ATPS was prepared from a mixture of 5% (wt/vol) poly(ethylene glycol), 10% (wt/vol) dextran, and 300 mM MnCl ₂ . In a poly(ethylene glycol)-dextran ATPS, Mn ²⁺ does not partition between phases, and the gradient in buoyant density generated by placing the

ATPS in a MagLev device is uniform (V p = 0.001 g/cm ³ /mm for 300 mM MnCl ₂ ); the difference in polymer concentration in each phase, however, results in a unique range of buoyant density that is available to each phase (Table 4).

Table 4. Values of buoyant densities accessible by magnetic levitation (MagLev) using a poly(ethylene glycol)- dextran aqueous two-phase polymer system (ATPS) containing 300 mM MnC¾.

buoyant density (g/cm ³ ) at levitation height phase density

ATPS phase 45 mm 22 mm 0 mm

(g/cm ³ )

top phase 1.046 1.023 1.046 1.069 bottom phase 1.073 1.050 1.073 1.096

[0189] While the gradient in buoyant density within each phase is equivalent, the dynamic range of available densities differs due to the step in density produced by the ATPS. The interface of the ATPS marks this step in density and is located at a vertical position of 22 mm within the MagLev device.

[0190] Each phase of the ATPS occupies a range of available levitation heights: the bottom phase occupies the levitation heights between 0 mm and 22 mm, while the top phase occupies the levitation heights between 22 mm and 45 mm. The total buoyant density range available to each phase, if used as a separate levitation medium, is truncated at the interface due to the step in density between phases. This truncation results in a gap of buoyant density that cannot be addressed by the ATPS that is defined by the difference in density between phases (1.046 - 1.073 g/cm ³ ).

[0191] The magnitude of the density gap can be tuned by changing the concentrations of the polymers used to produce the ATPS or through the use of an alternative two-phase system.

Separating Beads Based on Density

[0192] A series of five reference beads was added to the poly(ethylene glycol)-dextran ATPS to demonstrate how steps and gradients in density can be combined to increase the range of densities available for analysis and separation by MagLev. These colored beads have densities of 1.020 g/cm ³ (green), 1.035 g/cm ³ (blue), 1.070 g/cm ³ (clear), 1.085 g/cm ³ (amber), and 1.100 g/cm ³ (clear).

[0193] In the absence of a magnetic field gradient (B VB = 0), gravity is the only force acting on the beads. The position of beads within the ATPS is, therefore, confined to three possible positions: the top of the system, the interface between phases, or the bottom of the system (Figure 6). When the ATPS is placed within the MagLev device (BVB≠0), a gradient in buoyant density is produced within each phase, and the beads can now levitate within a phase and at a height that is proportional to their densities.

[0194] Using this approach, all five beads were isolated at different positions within the ATPS based on their differences in density and calculate the densities of those beads that levitate by measuring their levitation heights. The densities of reference beads with densities of 1.035 g/cm ³ and 1.085 g/cm ³ were calculated to be 1.037 g/cm ³ and 1.083, respectively. The error in measurement for these densities is +/- 0.002 g/cm ³ based on the uncertainties of the values of the experimental parameters (e.g., the accuracy of measuring the magnetic field strength is +/- 0.003 mT).

Steps in Magnetic Susceptibility

[0195] An ATPS was prepared from a mixture of 20% (wt/vol) poly( vinyl alcohol), 10% (wt/vol) carboxy-polyacrylamide, and 300 mM MnCl ₂ . In a poly( vinyl alcohol)-carboxy- polyacrylamide ATPS, Mn ²⁺ does partition between phases. After phase separation, the top phase had an effective MnCl ₂ concentration of 130 mM, while the bottom phase had an effective MnCl ₂ concentration of 310 mM. The partitioning of Mn ²⁺ results in dissimilar magnetic susceptibilities between phases of the ATPS and a unique gradient in buoyant density characterizes each phase: V p = 0.0005 g/cm ³ /mm for the top phase and V p = 0.0010 g/cm ³ /mm for the bottom phase. As a result, the range of buoyant density that is available to each phase differs (Table 5).

Table 5. Values of buoyant densities accessible by MagLev using a poly(vinyl alcohol)-carboxy-polyacrylamide

buoyant density (g/cm ³ ) at levitation height phase density

ATPS phase 45 mm 22 mm 0 mm

(g/cm ³ )

top phase 1.063 1.056 1.063 1.070 bottom phase 1.073 1.050 1.073 1.097

[0196] For this ATPS, Mn ²⁺ partitions preferentially into the bottom phase, generating in a step in magnetic susceptibility (χ) between phases. This step in χ produces dissimilar gradients in buoyant density between the top and bottom phase of the ATPS. A series of five reference beads were separated using the poly( vinyl alcohol)-carboxy-polyacrylamide ATPS. These colored beads have densities of 1.045 g/cm ³ (yellow), 1.060 g/cm ³ (clear), 1.065 g/cm ³ (yellow), 1.085 g/cm ³ (amber), and 1.100 g/cm ³ (clear). [0197] In the absence of a magnetic field gradient (B VB = 0), the beads are restricted to one of three locations within the ATPS, but the beads can levitate within a phase once the system is placed within the MagLev device (BVB ^ O) (Figure 7).

[0198] The densities of reference beads with densities of 1.060 g/cm ³ and 1.085 g/cm ³ were calculated to be 1.060 g/cm ³ and 1.082 g/cm ³ , respectively. The error in measurement for these densities is +/- 0.002 g/cm ³ .

[0199] Upon review of the description and embodiments of the present invention, those skilled in the art will understand that modifications and equivalent substitutions may be performed in carrying out the invention without departing from the essence of the invention. Thus, the invention is not meant to be limiting by the embodiments described explicitly above, and is limited only by the claims which follow.

[0200] What is claimed is: