WO2012094170A2 - Methods and microfluidic devices for concentrating and transporting particles - Google Patents

Abstract

Shaped hydrophilic surfaces are capable of concentrating and transporting particles in a liquid mixture, and are useful in microfluidic devices are described herein. Such devices use small amounts of samples and it is desirable that the analyte may be concentrated, and/or transported into a zone where the analyte is detected by a microfluidic device.

Description

METHODS AND MICROFLUIDIC DEVICES FOR CONCENTRATING AND

TRANSPORTING PARTICLES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 61/429,392, filed January 3, 2011, the content of which is incorporated by reference in its entirety into the present disclosure.

FIELD OF THE INVENTION

[0002] The present technology relates to concentrating and transporting particles and relates to the field of physical chemistry, chemistry, physics, nanotechnology, and micro f uidics.

BACKGROUND

[0003] Microfluidic devices are useful, e.g., for detecting analytes present in small amounts and/or in low concentrations. Such devices use small amounts of samples and it is desirable that the analyte may be concentrated, and/or transported into a zone where the analyte is detected by a microfluidic device. Employing external force via pumps or gravitation may be problematic for purposes of such concentration or transporting. There is a need for methods and microfluidic devices forces.

SUMMARY

[0004] Provided herein are methods and microfluidic devices that concentrate or transport particles in a liquid mixture of the particles, typically without requiring an external force such as a pump or gravitation to do so. Because they concentrate or transport particles without requiring external force, such methods can be performed by small devices, e.g., and without limitation, microfluidic devices. Without being bound by mechanism, the liquid mixture of the particles contain a polar liquid which is

preferentially attracted to a hydrophilic surface and the particles are transported away from the hydrophilic surface or concentrated. In accordance of the present technology, the shapes of the hydrophilic surface are such that the molecular forces acting between the hydrophilic surface and the polar liquid and between the hydrophilic surface and particles are added to provide a net transporting of the particles away from the surface or concentrating of the particles in a region away from the surface. Such methods and devices are particularly suited for detecting small amounts of analytes using microfluidic methods and devices.

[0005] In one aspect, the present technology provides a microfluidic device comprising, consisting essentially of, or consisting of a closed concentrating chamber comprising a 2- dimensional or a 3- dimensional concentrating surface, the concentrating surface comprising a hydrophilic polymer, wherein the closed concentrating chamber has a closed loop diameter of about 1 μιη to 5000 μιη.

[0006] In another aspect, the present technology provides a method of concentrating or transporting particles comprising, consisting essentially of, or consisting of contacting a liquid mixture comprising the particles with a microfluidic device of the present technology, thereby concentrating the particles.

[0007] In another aspect, the present technology provides a method of concentrating or transporting particles comprising, consisting essentially of, or consisting of contacting a liquid mixture comprising the particles with a partially enclosed surface, the surface comprising a hydrophilic polymer, thereby transporting the particles.

[0008] In another aspect, the present technology provides a microfluidic separation device comprising, consisting essentially of, or consisting of: an inlet; an inlet channel comprising an inner surface comprising a hydrophilic polymeric surface and a hydrophilic polymer free surface; a proximal outlet channel, which is proximal to the hydrophilic polymeric surface; and a distal outlet channel, which is distal to the hydrophilic polymeric surface; wherein the inlet is joined with the inlet channel, and the inlet channel is joined with the proximal outlet channel and the distal outlet channel.

[0009] In another aspect, the present technology provides a method of concentrating particles the method comprising, consisting essentially of, or consisting of using the microfluidic separation device of the present technology. In one embodiment, the method comprises introducing via the inlet a liquid mixture comprising particles into the inlet channel, contacting the liquid mixture with the hydrophilic polymer surface, withdrawing a concentrated liquid mixture at the distal outlet channel, thereby concentrating the particles in the concentrated liquid mixture. The extent of concentrating depends, e.g., on the fraction of hydrophilic polymer surface: surface free of hydrophilic polymer, the flow rate, properties of the hydrophilic repelling surface and the liquid mixture, and the design of channel geometries.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1 depicts a closed concentrating chamber including a Nafion surface.

[0011] FIGS. 2A-2C depict how fluorescent nanoparticles are transported by a asymmetrically shaped surface.

[0012] FIG. 2D schematically illustrates how a V-shaped interior hydrophilic surface transports particles.

[0013] FIG. 3A graphically illustrates the concentrating of particles occurring in a microfluidic device of the present technology.

[0014] FIGS. 3B and 3C depict schematically and by a microscopic image a

concentrating microfluidic device of the present technology.

[0015] FIG. 4 depicts a top-view image of exclusion zone with fluorescence confocal microscopy.

[0016] FIG. 5 depicts a sketch of a WATER chip.

[0017] FIG. 6 graphically illustrates experimental measurements of WATER mixing chips.

[0018] FIG. 7 depicts a schematic diagram of a WATER separation chip.

[0019] FIG. 8 depicts time-sequence images of solute-manipulation on a WATER chip.

[0020] FIG. 9 depicts concentrated human embryonic stem cells (hESc) in a WATER chip.

DETAILED DESCRIPTION

Definitions

[0021] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied ( + ) or ( - ) by increments of 1.0 or 0.1, as appropriate. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term "about". It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0022] As used, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a nanoparticle" includes a plurality of nanoparticles, including mixtures thereof.

[0023] As used herein, "amine" refers to a chemical functional group of formula -NR₂, wherein each R independently is hydrogen or a hydrocarbon radical that is alkyl, cycloalkyl, aryl, or heteroaryl, wherein each hydrocarbon radical can have up to 10 carbon atoms.

[0024] As used herein, "closed concentrating chamber" refers to a chamber can be used to contain the sample solution, for example, the triangular box showed in FIG. 1.

[0025] "Comprising" is intended to mean that the devices and methods include the recited elements, but do not exclude others. "Consisting essentially of," when used to define devices and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. "Consisting of shall mean excluding more than trace elements of other components in the devices of the present technology, and substantial method steps for performing the methods of the present technology or achieve an intended result. Embodiments defined by each of these transition terms are within the scope of this invention.

[0026] As used herein, "concentrating surface" refers to a surface which includes a hydrophilic polymer such that, if a liquid mixture containing particles is contacted with the concentrating surface, the liquid is preferentially attracted to the surface and the particles are transported away from the surface or are concentrated. As used herein, concentrated refers to an increase in the: number of particles per volume of the liquid mixture, number of particles per area of the liquid mixture, mass of particles per volume of the liquid mixture, or mass of particles per area of the liquid mixture, compared to the corresponding measurements before the liquid mixture is contacted with the concentrating surface. As used herein transported refer to displacement of the particles away from the surface. [0027] As used herein, a "liquid mixture" refers to a mixture comprising a liquid. A liquid mixture can be a colloidal dispersion, a solution, or a mixture of particles in a liquid such that th mixture is neither a solution nor a colloidal dispersion. A colloidal dispersion can be a sol or a latex.

[0028] As used herein, "nanoparticles" are solid particles ranging in size from about 1 to 1000 nm. Nanoparticles may have a diameter less than or equal to 5 nm, or

alternatively less than about 10 nm, or alternatively less than about 15 nm, or alternatively less than about 20 nm, or alternatively less than about 25 nm, or alternatively less than about 30 nm, or alternatively less than about 50 nm, or alternatively less than about 100 nm, or alternatively less than about 150 nm, or alternatively less than about 200 nm, or alternatively less than about 300 nm, or alternatively less than about 400 nm, or

alternatively less than about 500 nm. In some embodiments, the nanoparticle has a

diameter between about 100 nm to about 500 nm; or alternatively between about 50 nm to about 200 nm; or alternatively between about 150 nm to about 300 nm; or alternatively between about 200 nm to 500 nm; or alternatively between about 100 nm to about 150 nm; alternatively between about 1 nm to about 50 nm; alternatively between about 50 nm to about 100 nm; alternatively between about 150 nm to about 200 nm; alternatively

between about 200 nm to about 300 nm; alternatively between about 300 nm to about 400 nm; or alternatively between about 400 nm to about 500 nm. Drugs, bioactive or other relevant materials can be incubated with the nanoparticles, and thereby be adsorbed or attached to the nanoparticle.

[0029] The nanoparticle can comprise a variety of materials including, but not limited to, polymers such as polystyrene, silicone rubber, polycarbonate, polyurethanes, polypropylenes, polymethylmethacrylate, polyvinyl chloride, polyesters, polyethers, and polyethylene, and metal oxide nanoparticles, such as, but not limited to, Ti0₂. Biodegradable, biopolymer (e.g.

polypeptides such as BSA, polysaccharides, etc.), other biological materials (e.g. carbohydrates) and/or polymeric compounds are also suitable for use as a nanoparticle scaffold. The nanoparticles may themselves have a negative charge or alternatively a positive charge on them or may be modified with groups such as, but not limited to, amine, carboxylic acid, sulfonic acic or sulfate, groups to attach a negative charge or positive charge to the scaffold. Factors such as nanoparticle surface charge and hydrophilic/hydrophobic balance of these polymeric materials can be achieved by synthetic modification of the polymers. Such synthetic modification is well within the skill of the skilled artisan. Various methods for producing the negatively charged nanoparticles are described in US Patent No. 7,390,384; and Kim et al. (2009) Polymer Bulletin 62:23-32, which are incorporated herein by reference in their entirety.

[0030] Examples of negatively charged nanoparticles include, but are not limited to, polymer blends of poly(lactide-co-glycolide) (PLGA) and poly(styrene-co-4-styrene-sulfonate) (PSS) an< polystyrene nanoparticles modified with carboxylate, sulfate, or sulfonate. Examples of positively charged nanoparticles include, but are not limited to, polymer blends of

polyethylenimine (PEI) or cationic polymers, non- limiting examples of which include poly 3- (methacryloylamino) propyl trimethyl ammonium chloride (MAPTAC), poly vinylbenzyl trimethyl ammonium chloride (VBTMAC), and poly 2-(methacryloyloxy) trimethyl ammonium chloride (MATMAC). In some embodiments, the surface charge on the nanoparticle is from about 0.1 μQq/g to about 2000 με ^; about 1 μQq/g to about 1000 με ^; about 1 μQq/g to about 900 με /g; about 1 μeq/g to about 800 με /g; about 1 μeq/g to about 700 με /g; about 1 μeq/g to about 600 με /g; about 1 μeq/g to about 500 με /g; about 1 μeq/g to about 400 με /g; about 1 μeq/g to about 300 με /g; about 1 μeq/g to about 200 με /g; about 1 μeq/g to about 100 με /g; about 0.1 μeq/g to about 1000 με /g; or about 0.1 μeq/g to about 100 μeq/g. In some embodiments, for the carboxyl surface modified polymer, the surface charge ranges from about μeq/g to about 1000 μeq/g. In some embodiments, for the amine surface modified polymer, the surface charge ranges from about 1 μeq/g to about 100 μeq/g.

[0031] Τΐιε surface charge of the nanoparticle and the zeta potential can be determined using a Malvern Zetamaster. Zeta potential is an abbreviation for electrokinetic potential in colloidal systems. Zeta potential is the potential difference between the dispersion medium and the stationary layer of fluid attached to the dispersed particle. The value of zeta potential can be related to the stability of colloidal dispersions. The zeta potential indicates the degree of repulsion between adjacent, similarly charged particles in a dispersion. For molecules and particles that are small enough, a high zeta potential will confer stability, i.e. the solution or dispersion will resist aggregation. When the potential is low, attraction exceeds repulsion and tt dispersion will break and flocculate. So, colloids with high zeta potential (negative or positive) are electrically stabilized while colloids with low zeta potentials tend to coagulate or flocculate.

[0032] Nanoparticles comprising the above materials and having diameters less than 1 ,000 nanometers are available commercially or they can be produced from progressive nucleation in solution (e.g., by colloid reaction), or by various physical and chemical vapor deposition processes, such as sputter deposition. Plasma-assisted chemical vapor deposition (PACVD) can also be used to prepare suitable nanoparticles. PACVD functions in relatively high atmospheric pressures (on the order of one torr and greater) and is useful for generating particles having diameters of about 1000 nanometers and smaller. The PACVD system typically includes a horizontally mounted quartz tube with associated pumping and gas feed systems. A susceptor is located at the center of the quartz tube and heated using a 60 KHz radio frequency source. The synthesized particles are collected on the walls of the quartz tube. Nitrogen gas is commonly used as the carrier. A constant pressure in the reaction chamber of 10 torr is generally maintains to provide deposition and formation of the ultrafme nanoparticles. PACVD can be used to prepare a variety of suitable biodegradable nanoparticles.

[0033] As used herein, "sol" refers to a colloidal dispersion of particles in a liquid. While a so may be clear to the eye, it is not homogenous like a solution. As used herein, a latex refers to a colloidal dispersion of particles in a liquid that is not clear to the eye. Structurally, latexes contain particles of larger size than particles in a sols.

[0034] In one aspect, the present technology provides a microfluidic device comprising a closed concentrating chamber comprising a 2-dimensional or a 3-dimensional

concentrating surface, the concentrating surface comprising a hydrophilic polymer,

wherein the closed concentrating chamber has a closed loop diameter of about 1 μιη to

5000 μιη and is not necessarily limited by the 3-dimensional shape. In one embodiment, the closed loop diameter is from about 1 μιη to about 1000 μιη, or alternatively from

about 100 to 500; or alternatively from 250 to 750; or alternatively from 250 to 1000, all in μιη. For example, and without limitation, a closed concentrating chamber in

accordance with the present technology is illustrated in FIG. 1. In FIG. 1 , the closed

concentrating chamber has the shape of a triangle where the inside of the three arms

include the hydrophilic concentrating surface. When contacted with a liquid mixture, for example, and without limitation, an aqueous mixture, the aqueous part is attracted

towards the three arms of the triangle, and the particles in the liquid mixture are

concentrated at a region inside the triangle, as illustrated by the white region within the triangle in FIG. 1. As will be apparent to a skilled artisan upon reading this disclosure, a variety of other closed concentrating chambers which are 2-dimensional and 3- dimensional are also within the contemplation of the present technology. Examples of 2- dimensional chambers, include, without limitation, those shaped roughly as polygons , circles, ellipses, triangles, and the like. Examples of 3-dimensional chambers, include, without limitation, those shaped roughly as 3-dimensional polygons , spheres, an ovoid; a prolate spheroid, an oblate spheroid; a pyramid, a funnel, a cone; and the like.

[0035] In another embodiment, the concentrating surface is 2-dimensional. In another embodiment, the concentrating surface is 3- dimensional. Examples of 3-dimensional surfaces include, without limitation V-shaped, U-shaped, hollow conical, hollow hemispherical, or hollow pyramidal surfaces.

[0036] In another embodiment, the closed concentrating chamber has a closed loop diameter of about 1 μιη-about 5000 μιη. In another embodiment, the closed loop diameter is about 1 μιη, about 250 μιη, about 500 μιη, about 750 μιη, about 1000 μιη, about 2000 μιη, about 3000 μιη, about 4000 μιη, or about 5000 μιη. In another embodiment, the closed loop diameter is about 1 μιη- less than about 250 μιη, about 250 μιη-less than about 500 μιη, about 500 μιη-less than about 750 μιη, about 750 μιη-less than about 1000 μιη, about 1000 μιη-less than about 2000 μιη, about 2000 μιη-less than about 3000 μιη, about 3000 μιη-less than about 4000 μιη, or about 4000 μιη-less than about 5000 μιη.

[0037] In another aspect, the present technology provides a method of concentrating or transporting particles comprising contacting a liquid mixture comprising the particles with the micro fluidic device of the present technology, thereby concentrating the particles. In another embodiment, the concentrating occurs about 100 μιη to about 5 mm away from the concentrating surface. As used herein, "exclusion range" and "away from surface," describe the solutes being moved away from the hydrophilic surfaces. The distance away from the concentrating surface, at which the concentrating occurs, will depend, e.g. , on the nature, geometry, or surface properties of the repelling hydrophilic surface, the nature of the particles, or the nature of the liquid, including, without limitation the pH or chemical composition of the liquid.

[0038] In another embodiment, the concentrating results in a concentration of the particles, that is up to about 25%, about 50%, about 75%, about 100%), about 125%, or about 150% more in a region of maximum particle concentration than a concentration of the particles in a region of least particle concentration. Typically, the region of least particle concentration occurs nearest to the concentrating surface where the liquid mixture and the hydrophilic polymer surface are in contact. In another embodiment, the concentrating occurs in a central region of the closed concentrating chamber. In another embodiment, the concentrating results in a concentration of the particles, that is up to about 25%, about 50%, about 75%, about 100%, about 125%, or about 150%) more in the central region than a concentration of the particles in a peripheral region of the closed concentrating chamber. To control the concentrating in a particular direction or the extent of the concentration, concentration chambers of the present technology comprising various shapes of the concentrating surfaces may be used. The extent of concentrating occurring can be demonstrated following various methods described in the examples and/or known to the skilled artisan.

[0039] In another aspect, the present technology provides a method of transporting or concentrating particles comprising contacting a liquid mixture comprising the particles with a partially enclosed surface, the partially enclosed surface comprising a hydrophilic polymer, thereby transporting the particles. In another embodiment, the partially enclosed surface comprises a shaped surface, which in accordance with the present technology can repel particles away from the hydrophilic polymeric surface. The surface can be any combination of repelling and non-repelling surfaces, such that, there is an overall transporting or concentrating of the particles. In another embodiment, the partially enclosed surface is a V-shaped, a U shaped, a hollow conical, a hollow pyramidal, or a hollow hemispherical. The surface can be discrete or continuous. That is, the surface can be the combinations of repelling/non-repelling surfaces. In another embodiment, the transporting occurs away from the partially enclosed surface, e.g., and without limitation, as schematically shown below, where the arrow indicates the main direction of the transporting:

-shaped U-shaped or side view of hollow hemispherical

hollow conical hollow pyramidal [0040] For example, and without limitation, FIG. 2D schematically illustrates concentration and transport of particles inside a V-shaped surface, where the inside of the v-shaped surface includes a hydrophilic polymeric surface. The outside of the V-shaped surface, when coated with a hydrophilic surface repels particles away from the outer surface. However, when repelled by the outside surface, no net concentrating and transporting is achieved, as contemplated by the present technology.

[0041] In other embodiments, a plurality of such partially enclosed surfaces are arranged to enhance the amount of transporting, in terms of the amount of particles transported and/or the distance the particles are transported. The distance transported can be the mean distance transported, the minimum distance transported, or the maximum distance transported.

[0042] In another embodiment, the transporting does not require using a pump or gravitational force. The primary driving force in such types of transporting is the molecular interaction between the hydrophilic polymer and the particles, and the shape of the surface. In other embodiments, the transporting may be augmented by using a pump.

[0043] In another aspect, the present technology provides a microfluidic separation device comprising an inlet joined with a inlet channel, the inlet channel comprising an inner surface, the inner surface comprising a hydrophilic polymer surface and a surface free of a hydrophilic polymer, wherein the inlet channel is joined with a proximal outlet channel, which is proximal to the hydrophilic polymer surface, and a distal outlet channel, which is distal to the hydrophilic polymer surface.

[0044] In one embodiment, the hydrophilic surface, in a dimension, is about 1 mm to about 30 mm, about 1 mm to about 20 mm, or about 1 mm to about 10 mm long. In another embodiment, the dimension is about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, or about 10 mm. In another embodiment, the dimension is about 15 mm, about 20 mm, about 25 mm, or about 30 mm. In one embodiment, the microfluidic separation device further comprises a liquid mixture comprising particles.

[0045] In another aspect, the present technology provides a method of concentrating particles by using the microfluidic separation device of the present technology. In one embodiment, the method comprises introducing via the inlet a liquid mixture comprising particles into the inlet channel, contacting the liquid mixture with the hydrophilic polymer surface, withdrawing a concentrated liquid mixture at the distal outlet channel, thereby concentrating the particles in the concentrated liquid mixture. The extent of concentrating depends, e.g., on the fraction of hydrophilic polymer surface: surface free of hydrophilic polymer, the flow rate, properties of the liquid mixture, and the design of channel geometries.

[0046] Within the various aspects and embodiments of the present technology, in another embodiment, the hydrophilic polymer is a polysulfonic acid, a polycarboxylic acid, salts of each thereof, or a polyol. In one embodiment, the polysulfonic acid is Nation. In another embodiment, the polycarboxylic acid is poly acrylic acid. In another embodiment, the polyol is polyvinyl alcohol.

[0047] In another embodiment, the liquid mixture comprises a polar liquid. In another embodiment, the polar liquid comprises water, acetone, ethanol, methanol, or a mixture thereof. In another embodiment, the polar liquid is at least as polar as 30% aqueous acetone. In another embodiment, the liquid comprises up to 30%> aqueous acetone or up to 95% aqueous ethanol. In another embodiment, the liquid mixture is a dispersion or a solution. In another embodiment, the dispersion is a sol or a latex.

[0048] In another embodiment, the particles are hydrophobic particles. In another embodiment, the particles are hydrophilic particles. In another embodiment, the particles are nanoparticles. In another embodiment, the nanoparticle is a surface functionalized nanoparticle. In another embodiment, the nanoparticle that is surface functionalized includes, without limitation, polystyrene nanoparticles. In another embodiment, the surface functionalized nanoparticle is a negatively charged nanoparticle, at about pH 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In another embodiment, the negatively charged nanoparticle comprises carboxylic acid groups. In another embodiment, the surface functionalized nanoparticle is a positively charged nanoparticle, at about pH 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In another embodiment, the positively charged nanoparticle comprises amine groups.

[0049] In another embodiment, the particle is a protein, a protein conjugate,

nanoparticles, quantum dots, glucose, cholesterol, a drug, or a chemical reagent such as, without limitation, fluorescent molecules. Examples of fluorescent dyes for protein conjugation include, without limitation, fluorescein isothiocyanate (FITC) or Texas Red conjugates of proteins. Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl- coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue.TM., and Texas Red. Other suitable optical dyes are described in the Haugland (1996), Handbook of Fluorescent Probes and Research Chemicals (6^th ed. 1996). Attachment of the fluorescent label may be either directly to particle or compound or alternatively, can by via a linker. Suitable binding pairs for use in indirectly linking the fluorescent label to the intermediate include, but are not limited to, antigens/antibodies, e.g., rhodamine/anti- rhodamine, biotin/avidin and biotin/strepavidin.

[0050] Suitable proteins include, without limitation, antibodies. In another

embodiment, the particle is a human chorionic gonadotropin protein marker. In another embodiment, the human chorionic gonadotropin protein marker is a protein. In another embodiment, the human chorionic gonadotropin protein marker is a protein conjugate wherein the protein is conjugated to fluorescence molecules, nanoparticles, or quantum dots.

[0051] In another embodiment, the particle is a bacteria or another cell. In another embodiment, the particle is a carbon nanotube. In another embodiment, the particle is a Ti0₂ particle. In another embodiment, the particle is a quantum dot. Suitable examples of quantum dots include, without limitation, CdSe and ZnS quantum dots.

[0052] In another embodiment, the liquid mixture is a biological liquid, which is derived from an organism, as such, or after mixing and/or separating cells, e.g., stem cells, tissues, or fluids of the organism. In another embodiment, the liquid mixture comprises blood, urine or other biological fluid. Aqueous solution can also be used to transport the solute in accordance with the present technology.

[0053] The present technology having been disclosed in summary and detail is illustrated and not limited by the following examples. EXAMPLES

Example 1. Concentrating Mechanism For Sensing Device

[0054] To demonstrate the concentration of particles from their aqueous dispersion, the hydrophilic surfaces are designed to form a closed chamber. Due to the solute exclusion range of a few hundred micrometers, the test system operated with a closed loop diameter of around 500 μιη. In FIG. 1, the hydrophilic surfaces of the closed concentrating chamber was made of Nafion. Aqueous dispersions of fluorescent nanoparticle (500 nm) were added into the chamber. After several minutes, the nanoparticles were repelled by the hydrophilic surface and were concentrated around the center. To compare the heterogeneous particle distribution, the fluorescent intensities of the peripheral region and the central region were measured. The data showed that the fluorescent intensity of the central region was 150% higher than that of the peripheral regions, thereby indicating a concentration of the particles around the central region. By modifying the properties and orientations of the hydrophilic surface, the extent of concentration of the particles and the location of the high concentration regions may be modulated.

Example 2. Particle Transportation

[0055] To demonstrate the transporting of particles in an aqueous dispersion, the hydrophilic surfaces are set in a specific orientation to provide a shaped surface (FIGs. 2A-D). Without being bound by mechanism, it has been shown that forces that repel and thereby transport the particles away from the hydrophilic surface are normal to the hydrophilic surfaces. Taking advantage of this property, the position and orientation of the surfaces can be adjusted to optimally control the magnitude and direction of the final force vectors. The particle velocities in the aqueous mixtures are correlated to the force vectors. Here, two oriented V-shaped Nafion surfaces were used to demonstrate the transporting of particles in a microfluidic system. Fluorescent nanoparticle (500 nm) were added to the channels in between the shaped surface (FIG. 2A). After 2 minutes, the nanoparticles are repelled away from the hydrophilic surface in a specific direction. The velocity of the transported particles is around 12 micrometers per second. (FIGS. 2B-C). In this example, it was demonstrated how test nanoparticles can be transported without external energy input. With appropriate shaped hydrophilic enclosed surfaces, the flow of the concentrated particles and the diluted particles in separate microfluidic channels may be achieved.

Example 3. Microfluidic Separation And Concentration

[0056] Microfluidic channels were integrated with the Nafion separation and concentration system. A dispersion of fluorescent nanoparticles (500 nm) in water was used to demonstrate the separation/concentration in microfluidic channels (FIG. 3A). After the aqueous dispersion passed the Nafion surface, 400 μιη microfluidic channels were used to collect the concentrated nanoparticles and the diluted nanoparticles.

Fluorescence microscopy was used to analyze the separation efficiency. The intensity data showed that the concentrated output had 2.7 times higher fluorescence intensity than did the diluted nanoparticle output, thereby demonstrating a concentration of the

nanoparticles in the concentrated output. FIG. 3B shows a simple schematic model of the concentration effect created. The separation efficiency may be controlled by modifying the inlet channel dimensions, outlet channel dimensions, and channel shapes.

Example 4 - Long Distance Solute Migration

[0057] Diffusion dominates solute transportation at low Reynolds numbers. Here, Applicants report a phenomenon of long-distance solute migration within microfluidic channels. Without external energy inputs, solutes were repelled up to hundreds of microns from polymer channel walls and passed through laminar interfaces.

[0058] Microfluidic systems provide a wealth of potential applications for research and medicine. Especially for developing worlds, the low-cost and disposable systems are expected to serve critical functions in diagnosis and environmental monitoring. Their utility is often derived from the unique properties of fluids: flow is increasingly influenced by viscosity rather than inertia at the micro scale. At low Reynolds number, turbulent mixing is nonexistent and as a result diffusion dominates solute transportation. This behavior has been exploited by many microfluidic devices, such as interfacial reactors and gradient flow generators, through the use of laminar interfaces. Due to the high diffusion coefficient of small molecules, the laminar interface has been successfully used as a "molecular extractor," but an analogous application for larger solutes remains unexplored. The diffusion limitation

[0059] For large particle suspensions at low Reynolds numbers, with diameters in the micron range, manipulation and transportation necessitate elaborate experimental techniques. For instance, chaos flow has been used to mix clinical samples and reagents on diagnostic chips. (Johnson, T.J., Ross, D., Locascio, L.E. (2001) Analytical Chemistry 74:45-51 and Stroock, A.D. et al. (2002) Chaotic Mixer for Microchannels. pp. 647-651.) Antibody labeling requires a large quantity of conjugated magnetic or charged tags and a strong electromagnetic field within the channel, thereby limiting experimental configurations. For suspensions of different particles with a wide distribution of sizes, samples can be separated into individual populations by laminar flow through micron- obstacle fields. (Huang, L.R. et al. (2004) Science 304: 987-990.) Microf udic systems need to be specifically designed for addressed specimens, which may limit the flexibility of system integrations. Not only passive approaches, various active methods, such as electrokinetic, optical manipulation and ultrasonic agitation, can also be applied to transport specimens within laminar flows. (Wang, M.M. et al. (2005) Nat. Biotech.

23:83.) These systems may lack the crucial costefficiency , simplicity or fiexibility required for a convenient-using microfluidic systems. Here, overcoming the diffusion limitation for large particles, Applicants incorporate the exclusion zone (EZ) concept into microfluidics to find a solution for non-turbulent mixing of laminar flows.

The EZ zone

[0060] The molecular dynamics of hydration shell assembly and interfacial ordered water formation have been progressively appreciated but remain somewhat of a mystery. (Israelachvili, J.N. et al. (1988) Science 241 : 795; Israelachvili, H.W. (1996) Nature 379: 219; Chaplin, M. (2006) Nature Reviews Molecular Cell Biology 7: 861.) Oscillatory repulsive hydration forces and attractive hydrophobic forces demonstrate polar alignment of water layers at liquid-solid interfaces. (Israelachvili, J.N. et al.

(1988) Science 241 : 795; Israelachvili, H.W. (1996) Nature 379: 219.) The

phenomenon of solute migration away from hydrophilic interfaces has been well documented. (Israelachvili, J. et al. (1996) Nature 379:219-225.) The magnitude of this solute "exclusion zone" (EZ)— is debated (Zheng, J.M. et al. (2009) Science 332:511; Trevors, J. et al. (2005) Progress in Biophysics and Molecular Biology 89: 1; Zheng, J.M. et al. Physical Review E 68; Kofmger, J. et al. (2008) Proceedings of the National Academy of Sciences 105: 13218; Whitesides, G.M. et al. (2006) Nature 442:368; Zheng, J.M. et al. (2006) Advances in Colloid and Interface Science 127: 19; Tu, Y. et al. (2009) Proceedings of the National Academy of Sciences 106: 18120), but long distance effects (several hundred micrometers) are apparent with various hydrophilic surfaces. (Zheng, J.M. et al. (2009) Science 332:511; Zheng, J.M. et al. Physical Review E 68; Zheng, J.M. et al. (2006) Advances in Colloid and Interface Science 127: 19; Chai, B.H. et al. (2010) J. Phys. Chem. B. 114:5371; Bing-hua, C. et al. (2008) J. Phys. Chem. 112:2242; Zhao, Q. et al. (2008) Langmuir 24: 1750.) Exclusion zones in the vicinity of the hydrophilic polymer (Nafion) were shown to exclude aqueous microspheres and dye molecules within 600 μιη. (Zheng, J.M. et al. (2006) Advances in Colloid and Interface Science 127: 19.) Consistent with this observation, magnetic resonance images show EZ water in a more ordered state than bulk water and that the EZ effect persists for over 24 hrs. (Zheng, J.M. et al. (2009) Science 332: 511.) Moreover, results of molecular dynamic simulations showed interfacial water molecules forming structured layers suggestive of a pseudo-crystal lattice which may play a key role in the mechanism of EZ formation.( Israelachvili, J.N. et al. (1988) Science 241 : 795; Kofmger, J. et al. (2008) Proceedings of the National Academy of Sciences 105: 13218; Tu, Y. et al. (2009) Proceedings of the National Academy of Sciences 106: 18120; Vybiral, B. et al. (2007) Homeopathy 96:183; Hummer, G. et al. (2001) Nature 414: 188.)

[0061] From a more empirical perspective, exclusion zones have many features which can be exploited in microfluidics devices. Applicants demonstrate these features using Water Assembly and Transferred Energy for Reorganization (WATER) Chips made with selected polymers. The synthetic polymer Nafion 117 (Sigma, St. Louis, MO) was chosen to provide an effective hydrophilic interface to trigger EZ formation (ref). Microchannels were patterned either by laser cutting

(Ver5aLASER2.3o, AZ) (Selimovic et al. (2011) Lab on a Chip 11 :2325) or mechanical shearing. In order to spatially assign the EZ regions within WATER chips, sections of the Nafion can also (Selimovic et al. (2011) Lab on a Chip 11 :2325) be selectively deactivated by laser writing or metal-sputter coating. In the vicinity of the Nafion surface, confocal microscopy images revealed a 3 dimensional EZ forming within 10 seconds and solutes in this region were excluded by up to 200 μιη after several minutes (FIG. 4) Cross the interfaces between the laminar flow

[0062] In order to demonstrate that solutes can be directed through laminar interfaces by exclusion zones, a simple microchannel consisting of a straight main channel with T- shaped inlets and outlets was created. The EZ-inducing Nafion was positioned on one side of the main channel (FIG. 5). An aqueous solution containing 0.5 μιη carboxyl- functionalized fluorescent microspheres (4.37 x 10 particle/ml in ¾0, Invitrogen) was flowed from inlet 1, and pure ¾0 stream flowed from inlet 2. According to the standard diffusion model, particles with 0.5 μιη diameters take, on average, 14 hrs to travel 200 μιη, which can be performed in seconds using an EZ. In the presence of Nafion, and thus with an imposed EZ on the flow from inlet 1 , solutes were directed through the laminar interface, perpendicular to the direction of the flow (FIG. 3B). In order to quantify these results, fluorescent specimens collected from outlets 1 and 2 were measured with a spectrofluorophotometer (Shimadzu RF-15ol). The penetration efficiency was defined as the equation below and tested at various flow rates (1 μΐ/min to 50 μΐ/min) and channel lengths (10 mm; 30 mm).

Penetration Efficiency= (I _outlet2^— I outlet2 background)/ (I outletl ^— I outlet2 background)

[0063] Ioutkt represented the fluorescence measurement results of samples collected from outlets (N=3, for each flow velocity and different channel geometry), and ackground represents the diffusion across the laminar boundary measured without EZ. The penetration efficiency of Applicants' device corresponds to the linear multiplier compared with diffusion. Results showed the penetration efficiency is related to channel widths and exposure time to Nafion (which depends on channel length and flow velocity. Within the EZ, fluids exhibit distinct behavior compared to normal microfluidic phenomena. At low Reynolds numbers (Reynold number = 3-30), the Peclet number dramatically increases to — 250,000, indicating that unaided solute transportation was dominated by convection rather than diffusion.

[0064] The solute-repelling properties of dynamic exclusion zones are influenced by different solvents and solutes, similar to static exclusion zones. (ref) One of the major properties of the EZ is solute-repelling forces related to the solutes sizes; in this study, data indicated 2 μιη microspheres experienced larger repelling forces than 15 μιη ones perpendicular to Nafion surface. Namely, using EZ as driving force for particle separation, the 2 μηι microspheres can pass through the laminar interface more frequently than 15 μιη microspheres (FIG. 7). Here, different size microspheres stratified within EZ were transported by separate laminar flows. The respective microsphere streams were guided into T-shape outlet channels and the particle size distribution from each outlet was analyzed with flow cytometry (LSR II, BD, USA). Results show that there was only 2% of 15 μιη in the region away from the Nafion, about 4 times lower than the one found in the vicinity of Nafion.

Velocity-sensitive switches

[0065] A velocity-sensitive switch was designed to further investigate how solutes cross the laminar interface. The WATER switch was constructed with Nafion and acrylic polymer channel with side-reservoirs perpendicular to the flow channel opposite the Nafion interface (FIG. 8). Deionized water and the solution of fluorescent beads were flowed through the main channel as indicated. With fluid velocity slowly decreasing, at a specific velocity threshold , the fluorescent particles were pushed across the channel (N 200 μιη) and irreversibly trapped in the reservoirs. Time-lapse images were taken for 5 independent chips with varied channel widths and lengths and the fluorescence intensity within the reservoirs were measured to quantify the threshold velocity. By tuning the channel geometries, the threshold can be modified for potential applications, such as blood cell transportation for diagnostic assay or on-chip flow-rate indicator.

Conclusion

[0066] The unique advantage of an EZ-based design is the manipulation of specimens, at scales typically dominated by diffusion, in a non-turbulent fashion. Without external energy input, directional and selective solute transportation have been performed on WATER chips. By dispatching specimens according to tunable flow-rate thresholds, Applicants have also demonstrated the capacity for exclusion zones to serve as "valves" in microfluidic systems. EZ provides alternative solutions for current bioanalysis challenges in various aspects: the solute-free EZ serves as virtual cushion to prevent reactant absorption on channels ; clinical specimens (human embryonic stem cell was used here as an example) can be highly concentrated before assay (FIG. 9).

[0067] Here, Applicants emphasize the modular capabilities of Applicants' EZ-based chip designs. Due to the simplicity and compartmentalized operations carried out by each chip, multiple units can be integrated either in parallel or in series to yield amplified results. These multi-unit designs can be related to the performance of each individual unit, thereby achieving specific criteria on mixing or separation. The modular nature of Applicants' designs also allow for simple integration into existing micro fluidics technologies, and in some cases may replace some of the more complex and costly components of microfluidic systems.

[0068] In addition to the mechanistic implications of this experiment, a similar design could be used to separate a small portion of sample could be intermittently isolated during the experiment or mid-synthesis for analysis without harming the major product. For delicate samples, it could also provide non-turbulent dilution without losing total fluid volume.

[0069] In addition to PDMS providing a fundamental building block, the applications of EZ also leach the possibility of various engineered can be easily integrated, even with rigid material, into existing designs. By introducing the EZ to laminar interfaces in the micron scale, Applicants have created a low-cost, robust EZ-based polymer chip designs which holds potential for a variety of applications from environmental monitoring to point-of-care medical diagnostic devices fit for developing and established economies.

[0070] Although several embodiments of the invention are described herein in detail, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims.

[0071] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including," containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed.

[0072] Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification, improvement and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this invention. The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.

[0073] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0074] In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0075] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

Claims

WHAT IS CLAIMED IS:

1. A closed loop concentrating chamber comprising a 2-dimensional or a 3 -dimensional hydrophilic polymeric concentrating surface, having a closed loop diameter of about 1 μιη to 5000 μιη.

2. The closed loop concentrating chamber of claim 1 , wherein the closed loop diameter is from about 1 μιη to about 1000 μιη.

3. A device comprising the closed loop concentrating chamber of claim 1 or 2.

4. A method of concentrating or transporting particles comprising contacting a liquid mixture comprising the particles with the micro fluidic device of claim 3, thereby concentrating or transporting the particles.

5. The method of claim 4, wherein the concentrating occurs about 100 μιη to about 5 mm away from the concentrating surface.

6. The method of claim 4, wherein the concentrating occurs in a central region of the closed concentrating chamber.

7. The method of claim 6, wherein the concentrating results in a concentration of the particles, that is up to about 150% more in the central region than a concentration of the particles in a peripheral region of the closed concentrating chamber.

8. A method of concentrating or transporting particles comprising contacting a liquid mixture comprising the particles with a partially enclosed hydrophilic polymeric surface, thereby concentrating or transporting the particles.

9. The method of claim 8, wherein the partially enclosed surface is a V-shaped, a U

shaped, a hollow conical, a hollow pyramidal, or a hollow hemispherical surface.

10. The method of claim 8, wherein the transporting is distal from the partially enclosed surface.

11. The method of any one of claims 4-10, wherein the liquid mixture comprises a polar liquid.

12. The method of claim 11, wherein the polar liquid comprises water, acetone, ethanol, methanol, or a mixture thereof.

13. The method of claim 11 or 12, wherein the polar liquid is at least as polar as 30% aqueous acetone.

14. The method of claim 11 or 12, wherein the liquid comprises up to 30%> aqueous

acetone or up to 95% aqueous ethanol.

. Dkt. No.: 060933-6160

15. The method of any one of claims 4-10, wherein the liquid mixture is a dispersion or a solution.

16. The method of claim 15, wherein the dispersion is a sol or a latex.

17. The method of any one of claims 4-10, wherein the particle is a protein, a protein conjugate, a nanoparticle, a quantum dot, glucose, cholesterol, a cell, a drug, or a chemical reagent.

18. The method of any one of claims 4-10, wherein the particle is a nanoparticle.

19. The method of any one of claims 4-10, wherein the particle is a human chorionic gonadotropin protein marker.

20. The method of any one of claims 4-10, wherein the liquid mixture is one or more of blood, urine, plasma, a biological fluid or a cell suspension.