Methods and devices for high-throughput dielectrophoretic concentration
Disclosed herein are methods and devices for assaying and concentrating analytes in a fluid sample using dielectrophoresis. As disclosed, the methods and devices utilize substrates having a plurality of pores through which analytes can be selectively prevented from passing, or inhibited, on application of an appropriate electric field waveform. The pores of the substrate produce nonuniform electric field having local extrema located near the pores. These nonuniform fields drive dielectrophoresis, which produces the inhibition. Arrangements of electrodes and porous substrates support continuous, bulk, multi-dimensional, and staged selective concentration.
The present invention was made by employees of Sandia National Laboratories. The Government has certain rights in the invention.
BACKGROUND OF THE INVENTION1. Field of the Invention
The present invention generally relates to dielectrophoresis and its application in analytical devices and filtration technologies.
2. Description of the Related Art
Dielectrophoresis (DEP) is the motion of particles caused by the effects of conduction and dielectric polarization in non-uniform electric fields. Unlike electrophoresis, where the force acting on a particle is determined by its net charge, the dielectrophoretic force depends on the geometrical, conductive, and dielectric properties of the particle. A complex conductivity of a medium can be defined as σ*=σ+iωε, where σ is the real conductivity and ε is the permittivity of the medium, i is the square root of −1, and ω is the angular frequency of the applied electric field, E. According to well-known theory, the dielectrophoretic force is proportional to the differences in complex conductivity of the particle and suspending liquid and square of the applied electric field. Without being bound by theory, for a spherical particle of radius r, the DEP force, FDEP is given by
FDEP=2πr3εmRe[fCM]∇E2
where εm is the absolute permittivity of the suspending medium, E is the local (rms) electric field, ∇ is the del vector operator and Re[fCM] is the real part of the Clausius-Mossotti factor, defined as:
where σp* and σm* are the complex conductivities of the particle and medium respectively, as described in Hughes, et al. (1998) Biochimica et Biophysica Acta 1425:119-126, which is herein incorporated by reference. Depending on the relative conductivities of the particle and medium, the Clausuis-Mossotti factor can be positive, resulting in a force toward stronger electric fields, or negative, resulting in a force away from stronger electric fields. The particle motion toward and away from stronger electric fields is called, respectively, positive and negative DEP.
Thus, when a particle is exposed to a non-uniform electric field, it experiences dielectrophoretic forces resulting from conduction and polarization that scale with the electric field intensity. The magnitude, sign, and phase of these forces depend on the frequency of the applied field and electrical properties of the particle and medium, such as conductivity, permittivity, morphology and shape of the particle. Thus dielectrophoresis can be used to sort and move particles selectively. See Pohl, H. A., J. Appl. Phys., 22:869-871; Pohl, H. A., Dielectrophoresis, Cambridge University Press (1978); Huang Y., R. C. Gascoyne et al., Biophysical Journal, 73:1118-1129; Wang X. B., Gascoyne, R. C., Anal. Chem. 71:911-918, 1999; and U.S. Pat. No. 5,858,192, all of which are hereby incorporated by reference.
Insulator-based (electrodeless) dielectrophoresis (iDEP) has been previously described and utilized for the selective concentration and separation of analytes in microfluidic devices. See Cummings and Singh (2003) Anal. Chem. 75:4724-4731, Lapizco-Encinas, et al. (2004) Electrophoresis 25:1695-1704, and Lapizco-Encinas, et al. (2004) Anal. Chem. 76:1571-1579, which are herein incorporated by reference. These devices use spatially nonuniform insulating structures to generate the nonuniform electric field needed to drive DEP. These iDEP devices are practically limited to processing microliter volumes and require microfabrication. Prior art devices and methods employing iDEP may be used effectively for systems that process such small-volume samples, but are ineffective for real-time monitoring and analysis of large volumes and flows, e.g., flow rates greater than one liter of per hour.
Thus, a need exists for methods and devices that allow dielectrophoretic based assays of large volumes and high flow rates.
SUMMARY OF THE INVENTIONThe present invention provides a substrate comprising a plurality of pores and a nonuniform electric field having local extrema located near the pores. In some embodiments, the substrate is a membrane, a film, or a filter. In some embodiments, the substrate is a woven structure of a plurality of fibers. In some embodiments, the substrate is a non-woven structure of a plurality of fibers. In some embodiments, the substrate is a plurality of aligned fibers. In some embodiments, the substrate comprises a material with insulative properties. In some embodiments, the substrate comprises a material with conductive properties.
In some embodiments, the present invention provides a method for assaying, inhibiting, or concentrating analytes in a fluid sample which comprises passing the fluid sample through or in the vicinity of a substrate having a plurality of pores and a nonuniform electric field having local extrema located near the pores. In some embodiments, the analytes having a size larger than the pores are physically entrapped on one side of the substrate. In some embodiments, the analytes having a size smaller than the pores pass though the pores of the substrate. In some embodiments, the analytes are immobilized on or constrained in a given area from the surface of the substrate due to dielectrophoretic forces. In some embodiments, the substrate is a membrane, a film, or a filter. In some embodiments, the substrate is a woven structure of a plurality of fibers. In some embodiments, the substrate is a non-woven structure of a plurality of fibers. In some embodiments, the substrate is a plurality of aligned fibers. In some embodiments, the substrate comprises a material with insulative properties. In some embodiments, the substrate comprises a material with conductive properties.
In some embodiments, the present invention provides a device for assaying, inhibiting, or concentrating analytes in a fluid sample which comprises at least one assembly comprising at least one substrate having a plurality of pores capable of a nonuniform electric field gradient having local extrema located near the pores in the presence of an applied field and at least one electrode. In some embodiments, the analytes having a size larger than the pores are physically entrapped on one side of the substrate. In some embodiments, the analytes having a size smaller than the pores pass though the pores of the substrate. In some embodiments, the analytes are immobilized on or constrained in a given area from the surface of the substrate due to dielectrophoretic forces. In some embodiments, the substrate is a membrane, a film, or a filter. In some embodiments, the substrate is a woven structure of a plurality of fibers. In some embodiments, the substrate is a non-woven structure of a plurality of fibers. In some embodiments, the substrate is a plurality of aligned fibers. In some embodiments, the substrate comprises a material with insulative properties. In some embodiments, the substrate comprises a material with conductive properties. In some embodiments, the assembly comprises at least one spacer. In some embodiments, the device comprises two or more assemblies. In some embodiments, the device further comprises at least one component selected from the group consisting of a fluid inlet, a fluid outlet, a substrate housing, a gasket, an insert, a viewing area, a delivery device, a collection device, a spacer, and a valve. In some embodiments, the electrode is a pin electrode or a wire mesh. In some embodiments, the electrode is a remote electrode. In some embodiments, the substrate is transverse to the flow of the fluid sample. In some embodiments, the substrate is substantially normal to substantially aligned with the flow of the fluid sample. In some embodiments, the substrate is about 80 degree incidence to about 10 degree incidence to the flow of the fluid sample.
Both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the invention as claimed. The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute part of this specification, illustrate several embodiments of the invention, and together with the description serve to explain the principles of the invention.
DESCRIPTION OF THE DRAWINGSThis invention is further understood by reference to the drawings wherein:
The present invention generally relates to methods and devices for use in assays and separation applications utilizing dielectrophoresis for screening, isolating, or concentrating selected particles from a fluid flow system.
In some embodiments, the present invention employs a porous substrate comprising a plurality of pores and an applied electric field such that the pores produce a nonuniform electric field having local extrema near the pores. In various embodiments, the substrate is a membrane, sheet, film, woven structure of a plurality of fibers, non-woven structure of a plurality of fibers, irregularly spaced array of substantially aligned fibers, or substantially regularly spaced array of substantially aligned fibers. In some embodiments, the substrate comprises a material with insulative properties. In some embodiments, the substrate comprises a material with conductive properties.
The substrates can inhibit particle motion selectively based on the physical and dielectrophoretic properties of the particle. Inhibited particles are constrained to the immediate vicinity of the substrate. By forcing particle-laden fluid through the substrate, inhibition selectively concentrates the particles. If the substrate is arranged so that there is a tangential flow component, particles that are inhibited from crossing the substrate may yet be mobile to flow tangentially along the substrate. This arrangement facilitates “continuous” particle concentration: the system can continuously deliver a stream of selected, concentrated particles to a port or local region of the device.
The invention requires at least two electrodes to apply the electric field and at least one porous substrate. In some embodiments, one of the two electrodes may be the porous substrate. In some embodiments, one or more physically remote electrodes are used. If the substrate comprises a conductive material, in some embodiments the substrate is held at a potential, frequency, and/or phase different from that of another electrode or electrodes so the substrate itself is an electrode. In other embodiments, the substrate is in electrical communication only with the liquid. In some embodiments elements of the invention are sandwiched into assemblies. Various embodiments of these assemblies have wide spacers to separate and substantially isolate the elements, narrow spacers to provide partial isolation, or no spacers so that different elements contact each other.
These assemblies are arranged and housed in a variety of ways to effect selective batch (pulsed) concentration, continuous concentration, or hybrid continuous and batch concentration. Some embodiments employ assemblies or substrates held transverse to the flow. Other embodiments employ assemblies or substrates that are tilted. In some embodiments, the range of the tilt angles is substantially normal to the flow direction to substantially aligned with the flow. In some embodiments, the range of the tilt angle is from about 80 degree incidence to about 10 degree incidence, i.e. between about 10 and 180 degrees with respect to the flow to force inhibited particles to flow along the substrate to be continuously spilled at the downstream end of the assembly. This results in a one-dimensional concentration or immobilization, effecting a two-dimensional concentration (one spatial dimension and time). In some embodiments, the preferred highest incidence is one that allows particles to flow tangentially rather than becoming immobilized. In some embodiments, the lowest incidence angles are selected based on a given throughput and compact architecture design of a desired device. One skilled in the art may readily select suitable minimum and maximum incidence angles in a given device architecture, e.g. a desired porous substrate and electrode arrangement. Other embodiments use two-dimensional focusing geometries, e.g. conical assemblies, to effect a two-dimensional continuous concentration or three-dimensional batch concentration, supporting high concentration factors.
The present invention provides a substrate comprising a plurality of pores and a nonuniform electric field having local extrema located near pores which drive dielectrophoretic separation of particles in a fluid.
The present invention also provides methods and devices for DEP-based assays which combine filtration technology and dielectrophoresis for high-throughput assays of analytes in fluid samples. Specifically, the present invention provides methods and devices which employ at least one substrate comprising a plurality of pores and a nonuniform electric field having local extrema located near pores. The methods and devices of the present invention may be used for concentrating, assaying, isolating, or filtering analytes from fluid samples having high flow rates and volumes.
As used herein, an “analyte” refers to a particle that may be natural or synthetic and includes chemicals and biomolecules, such as amino acids, peptides, proteins, nucleotides, nucleic acids, carbohydrates, lipids, cells, viral particles, bacteria, spores, protozoa, yeast, mold, fungi, pollen, diatoms, and the like, and ligands, supermolecular assemblies, catalytic particles, zeolites, and the like.
As used herein, a “fluid” refers to a continuous substance that tends to flow and to conform to the outline of a container such as a liquid or a gas. Fluids include saliva, mucus, blood, plasma, urine, bile, breast milk, semen, water, liquid beverages, cooking oils, cleaning solvents, ionic fluids, air, and the like. Fluids can also exist in a thermodynamic state near the critical point, as in supercritical fluids. If one desires to test a solid sample for a given analyte according to the present invention, the solid sample may be made into a fluid sample using methods known in the art. For example, a solid sample may be dissolved in an aqueous solution, ground up or liquefied, dispersed in a liquid medium, melted, digested, and the like. Alternatively, the surface of the solid sample may be tested by washing the surface with a solution such as water or a buffer and then testing the solution for the presence of the given analyte.
As used herein, “concentrating” refers to the reduction of fluid volume per particle in the fluid. The methods and devices of the present invention allow a fluid to be concentrated or diluted. When the methods and devices are used to concentrate a fluid, it is noted that particles in one portion of the fluid becomes “concentrated” and that particles in the second portion of the fluid becomes “diluted”. Prior art devices employing filters concentrate and separate selectively based on size separation. Thus, the prior art devices cannot deliver a concentrate downstream of the filtration element, i.e. filter. The methods and devices of the present invention allow batch or continuous concentration or dilution wherein the concentrate or diluent may be delivered downstream of the porous substrate.
As used herein, “spatial separation”, “physical separation”, and “size separation” are used interchangeably to refer to the process by which a particle is filtered, concentrated, immobilized, retarded, or advanced according to the physical shape and size of the particle. As used herein, “assaying” is used interchangeably with “detecting”, “measuring”, “monitoring” and “analyzing”.
As used herein, the word “conductivity” is used to describe the ease of flow of both conduction and displacement current. It is often mathematically described as a complex number that varies with the frequency of the applied electric field. Similarly, “conduction” is used to describe both conventional conduction and conduction of displacement currents.
The term “pore” is used to describe an opening, such as a hole, an opening or an interstitial space in an object, such as the substrate of the present invention, through which fluid can flow.
The term “porous” is used to describe a material or assembly having a plurality of pores through which fluid can flow.
T
As used herein, “insulative” and “conductive” refers to the relative conductivity of the described item with respect to the fluid being concentrated or diluted according to the methods of the present invention. Insulative materials having relatively low conductivity include plastics, epoxies, photoresists, polymers, silicon, silica, quartz, glass, controlled pore glass, carbon, and the like, and combinations thereof. Preferred insulative materials include thermoplastic polymers such as nylon, polypropylene, polyester, polycarbonate and the like. Conductive materials, in comparison, have relatively high conductivity. Conductive materials include bulk, sputtered, and plated metals and semiconductors, carbon nanotubes, and the like.
As provided herein, the methods and devices of the present invention utilize substrates having pores that selectively restrain the transport of particles. When an electric field is applied across the substrate, the pores create a nonuniform electric field having local extrema near the pores. These field nonuniformities attract or repel particles from the substrate dielectrophoretically, according to the geometrical and electrical properties of the particles. Thus, the present invention provides methods and devices for physical separations and assays. The substrates may be films, membranes, sheets, meshes, webs, and the like. The substrates may be produced by methods known in the art.
H
I
P
S
D
As used herein, the term “mobilization field” refers to any force field that influences a particle to pass through an object or an area, such as the substrate according to the present invention. Mobilization fields include hydrodynamic flow fields produced by pressure differences, gravity, linear or centripetal acceleration, electrokinetic flow fields, magnetophoretic and thermophoretic flow fields, and others known in the art.
D
E
C
A
Embodiments of the present invention comprise a least one porous component and at least two conductive components. Each porous component has a plurality of pores capable of producing nonuniformities in an applied electric field such that local field extrema exist near the pores. The porous component at least one insulative or conductive porous substrate described herein.
The two or more conductive components apply an electric field to the fluid within the device. These conductive components can be directly or capacitively coupled to the fluid. One or more of these conductive components can have pores to allow passage of the fluid. Any conductive components having pores could embody a spatially non-uniform substrate as described herein. One or more of these conductive components may be a fluid in electrical communication with an externally applied field. A conductive component may be a solid or coated conductor or semiconductor ring, pin, mesh, grid electrode, or a combination thereof.
A
One skilled in the art may readily employ spacers and select the size of spacers in order to obtain a desired result. Wide spacers ranging from about 100 to about1000 microns, e.g., injection-molded plastic honeycomb or ridges, are sufficiently wide that the applied electric field is substantially uniform before reaching another element. These wide spacers are favored for selectivity since close coupling of non-uniform fields tend to create extra variation in the field concentration and dielectrophoretic effects unless the interacting elements are carefully patterned and aligned. Disadvantages of the wide spacers are higher voltage and power requirements, increased complexity in assembly, and complexity in developing continuous-flow designs, since the spacers must allow particles to flow along porous surfaces. The narrow spacers ranging from about 25 to about 100 microns, e.g., an insulating mesh, are wide enough to minimize contact of the electrodes with immobilized samples, but do not completely eliminate coupling. Close coupling (no spacer) is simplest in many embodiments, but can complicate continuous flow device designs since the electrodes and other elements must be arranged to allow particles to flow and local electrochemical products can affect performance and cell viability.
As used herein, the size range of the spacers is generally related to the pore size, e.g. a “wide” spacer is greater than about 3 times the pore size and a “narrow” spacer is less than about 3 times the pore size. One skilled in the art may readily determine the maximum size for a wide spacer which is limited by the given device, efficiency and practical limits.
In all arrangements of
D
S
Thus, the present invention provides methods and devices for batch (pulsed) and/or continuous separation, isolation, or collection of desired analytes by dielectrophoretic depletion or enhancement in a fluid flow.
The devices of the present invention may be arranged and housed in a variety of ways to effect selective batch (pulsed) concentration, continuous concentration, or hybrid continuous and batch concentration. Some embodiments employ assemblies held transverse to the flow. Other embodiments employ assemblies that are tilted with respect to the flow to force inhibited particles to flow along the substrate to be continuously spilled at the downstream end of the assembly, effecting a one-dimensional concentration, or immobilized, effecting a two-dimensional concentration (one spatial dimension and time). Other embodiments use two-dimensional focusing geometries, e.g. conical assemblies, to effect a two-dimensional continuous concentration or three-dimensional batch concentration, supporting high concentration factors. For example, the assemblies may be arranged within a cylindrical container or the assemblies may be cylindrical and arranged concentrically around a central electrode (in this manner, each concentric substrate will experience a different local field and gradient without the need to employ electrodes between each substrate and its neighbor.
E
The devices of the present invention include various electrode geometries and placements that may be readily realized by those skilled in the art. For example,
Various geometries and device configurations may, according to the present invention, be readily designed by one skilled in the art for desired versatility and performance.
As provided in the Examples, it has been determined that a device according to the present invention can remove organic particles smaller than the nominal pore size of the filter with high efficiency from a stream of fluid (water representative fluid sample) flowing at a rate of about 10 ml/hr. This effect is apparent for 100-nm fluorescent polystyrene beads, (representative analyte) passing through a filtration system having 200-nm-diameter pores when 6000 V DC is applied across the substrate housing. When the voltage is applied, little fluorescence is observed in the effluent flow. When voltage is discontinued, the effluent flow is observed to return to close to its original fluorescence. This experiment qualitatively demonstrates that electric fields can be used to enhance the ability of a macroscopic filtration unit to trap certain types of particles.
The following examples are intended to illustrate but not to limit the invention.
EXAMPLE 1The background solution was prepared by titrating deionized water from a reverse osmosis filter with NaOH to an approximate pH of 8. The resulting solution possessed a conductivity of approximately 2 μS/mm. A suspension of 100-nm rhodamine dyed fluorescent polystyrene spheres (analyte), Fluorospheres® (Molecular Probes, Eugene, Oreg.) was diluted 1:1,000 in the background solution from at 2% wt. stock suspension and sonicated for 2 minutes.
An apparatus similar to the one shown in
About 6000 V was applied across the electrodes for intervals of 3 to 10 minutes. The channel was imaged at the reservoir, where the optical path facilitated fluorescence measurements. Series of 100 images at 10 fps were taken of the system before applying voltage, when the fluorescent intensity of the reservoir was observed to decrease because of particle trapping at the porous substrate when the field was applied. When the field was removed, this fluorescence intensity signal was observed to increase as the particles eluted through the porous substrate. This effect was observed to reproducible and efficient in the removal of this analyte from the system of use.
EXAMPLE 2The background solution was prepared by titrating deionized water from a reverse osmosis filter with NaOH to an approximate pH of 8. The resulting solution possessed a conductivity of approximately 2 μS/mm. A suspension of 2-μm carboxylate modified fluorescent polystyrene spheres (analyte), Fluorospheres® (Molecular Probes, Eugene, Oreg.) was diluted 3:100,000 in the background solution from at 2% wt. stock suspension and sonicated for 2 minutes.
A device similar to the one described in
To the extent necessary to understand or complete the disclosure of the present invention, all publications, patents, and patent applications mentioned herein are expressly incorporated by reference therein to the same extent as though each were individually so incorporated.
Having thus described exemplary embodiments of the present invention, it should be noted by those skilled in the art that the within disclosures are exemplary only and that various other alternatives, adaptations, and modifications may be made within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments as illustrated herein, but is only limited by the following claims.
Claims
1. A substrate comprising a plurality of pores and a nonuniform electric field having local extrema located near the pores.
2. The substrate of claim 1, wherein the substrate is a membrane, a film, or a filter.
3. The substrate of claim 1, wherein the substrate is a woven structure of a plurality of fibers.
4. The substrate of claim 1, wherein the substrate is a non-woven structure of a plurality of fibers.
5. The substrate of claim 1, wherein the substrate is a plurality of aligned fibers.
6. The substrate of claim 1, wherein the substrate comprises a material with insulative properties.
7. The substrate of claim 1, wherein the substrate comprises a material with conductive properties.
8. A method for assaying, inhibiting, or concentrating analytes in a fluid sample which comprises passing the fluid sample through or in the vicinity of a substrate having a plurality of pores and a nonuniform electric field having local extrema located near the pores.
9. The method of claim 8, wherein the analytes having a size larger than the pores are physically entrapped on one side of the substrate.
10. The method of claim 8, wherein the analytes having a size smaller than the pores pass though the pores of the substrate.
11. The method of claim 8, wherein the analytes are immobilized on or constrained in a given area from the surface of the substrate due to dielectrophoretic forces.
12. A device for assaying, inhibiting, or concentrating analytes in a fluid sample which comprises at least one assembly comprising at least one substrate having a plurality of pores capable of a nonuniform electric field gradient having local extrema located near the pores in the presence of an applied field and at least one electrode.
13. The device of claim 12, wherein the substrate is a conductive material.
14. The device of claim 12, wherein the assembly comprises at least one spacer.
15. The device of claim 12, which comprises two or more assemblies.
16. The device of claim 12, and further comprising at least one component selected from the group consisting of a fluid inlet, a fluid outlet, a substrate housing, a gasket, an insert, a viewing area, a delivery device, a collection device, a spacer, and a valve.
17. The device of claim 12, wherein the electrode is a pin electrode or a wire mesh.
18. The device of claim 12, wherein the electrode is a remote electrode.
19. The device of claim 12, wherein the substrate is transverse to the flow of the fluid sample.
20. The device of claim 12, wherein the substrate is substantially normal to substantially aligned with the flow of the fluid sample.
21. The device of claim 12, wherein the substrate is about 80 degree incidence to about 10 degree incidence to the flow of the fluid sample.
Type: Application
Filed: Mar 11, 2005
Publication Date: Sep 14, 2006
Inventors: Blake Simmons (San Francisco, CA), Gregory McGraw (Livermore, CA), Allen Salmi (Escalon, CA), Gregory Fiechtner (Bethesda, MD), Eric Cummings (Livermore, CA), Yolanda Fintschenko (Livermore, CA)
Application Number: 11/076,971
International Classification: B01D 63/00 (20060101);