|Publication number||USRE42249 E1|
|Application number||US 12/217,114|
|Publication date||29 Mar 2011|
|Filing date||1 Jul 2008|
|Priority date||14 Feb 2001|
|Also published as||US6913697, US20030102263, USRE41762, USRE42315|
|Publication number||12217114, 217114, US RE42249 E1, US RE42249E1, US-E1-RE42249, USRE42249 E1, USRE42249E1|
|Inventors||Gabriel P. Lopez, Steven R. J. Brueck, Linnea K. Ista|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (235), Non-Patent Citations (80), Referenced by (6), Classifications (22)|
|External Links: USPTO, USPTO Assignment, Espacenet|
This application is a Continuation-in-Part of and claims priority to U.S. patent application No. 10/073,935, entitled “Nanostructured Devices for Separation and Analysis,” filed on Feb. 14, 2002, now U.S. Pat. No. 6,685,841 B2 issued on Feb. 3, 2004, which claims priority to U.S. Provisional Patent Application No. 60/268,365, entitled “Nanostructured Devices for Separation and Analysis,” filed Feb. 14, 2001. This application also claims priority to U.S. Provisional Patent Application No. 60/347,002, entitled “Nanostructured Devices,” filed on Jan. 11, 2002. The entire contents and disclosures of the above applications are hereby incorporated by reference.
Notice: more than one reissue application has been filed for the reissue of U.S. Pat. No. 6,913,697 B2. The reissue applications include U.S. patent application Ser. Nos. 12/215,893, 12/217,113 (now abandoned), and 12/217,114 (the present application) all of which are Divisional Reissue Applications, filed as divisionals of U.S. patent application Ser. No. 11/825,298, entitled “Nanostructured Separation and Analysis Devices for Biological Membranes,” filed on Jul. 5, 2007, as a Reissue of U.S. Pat. No. 6,913,697 B2 issued on Jul. 5, 2005, which was filed as application Ser. No. 10/338,654 entitled “Nanostructured Separation and Analysis Devices for Biological Membranes,” on Jan. 9, 2003, as a Continuation-in-Part of U.S. patent application Ser. No. 10/073,935, entitled “Nanostructured Devices for Separation and Analysis,” filed on Feb. 14, 2002, now U.S. Pat. No. 6,685,841 B2 issued on Feb. 3, 2004, which claims priority to U.S. Provisional Patent Application No. 60/268,365, entitled “Nanostructured Devices for Separation and Analysis,” filed on Feb. 14, 2001. This application also claims priority to U.S. Provisional Patent Application No. 60/347,002, entitled “Nanostructured Devices,” filed on Jan. 11, 2002. The entire contents and disclosures of the above applications are hereby incorporated by reference.
This invention is made with government support under grant number DAAD19-99-1-0196 awarded by the United States Army Research Office. The government has certain rights in this invention.
1. Field of the Invention
The present invention relates to the fabrication of nanostructured matrices for use in supporting lipid bilayers for the separation and analysis of membrane-associated molecules.
2. Description of the Prior Art
The demand for precise separation of molecules using small sample volumes is increasing. Currently, polyacrylamide gel electrophoresis (PAGE) remains the standard for protein separation and identification in biotechnology. However, the set of separation strategies that rely on this technique are hampered by: (1) inconvenience of preparation of the variety of gels needed for the separations, (2) inherent inconsistencies in production conditions; and therefore, irreproducibility between different batches of gels, (3) susceptibility of the polymer to degradation under high electric fields, (4) lack of reusability, (5) difficulty in incorporation of these techniques into strategies for development of multi-dimensional (multi-technique) integrated separation systems, and (6) limited resolution and dynamic range of biomolecular separations.
Gradient PAGE techniques utilize one-dimensional filtration by manipulating pore-size though control of cross-linker, monomer, and solvent concentrations. Such separation matrices are recognized as having the potential to maintain excellent resolution and dynamic range. However, their utility is greatly hampered by the need for cumbersome gel preparation protocols and lack of reproducibility.
In general, the separation of molecules across matrices or membranes has been known in the art. Such separations are typically achieved by employing barriers that allow cut-offs at a precise molecular weight or by size-exclusion. The art describes structures where molecular transport and filtration take place perpendicular to the surface of the separating material. These currently available systems, however, suffer from a number of drawbacks: (1) the matrices formed are generally composed of non-uniform structures, (2) even where a gradation in size of structures is required, they may be random or at best have to be serially and sequentially arrayed through a cumbersome process of lithography, (3) fabrications of separation devices pose problems in terms of batch-to-batch variations; and consequently, poor reproducibility of results therefrom, (4) lack of efficiency of separation, (5) loss of sample volume, and (6) biomolecules may not be amenable to separation by many of the available systems.
Thus far, the most relevant work has been the development of “Brownian ratchets” in which assymetric diffusion leads to separation of molecules based on their size (van Oudenaarden et al., Science, 285: 1046-1052, 1999, the entire contents and disclosure of which is hereby incorporated by reference). Subsequently, Chou et al. (see, Chou et al., Proc. Natl. Acad. Sci., 96, 13762-13765, 1999, the entire contents and disclosure of which is hereby incorporated by reference) attempted separation of DNA molecules using Microsystems formed by conventional photolithography. Although such prior work demonstrated that relatively simple 3-dimensional architectures could lead to effective separation, the developments have not gained ground with the biotechnological community. The primary reasons for this lack of acceptance being the difficulty of preparation of the nanofluidic systems and the associated high-cost of fabrication.
Similarly, “artificial gels” incorporating regular arrays of nanoscale pillars created through electron beam and/or imprint lithography have been described, for instance, in U.S. Pat. No. 6,110,339 to Yager, et al. and by Turner, et al. (J. Vac. Sci. Technol. B., 16 3835-3840, 1998, the entire contents and disclosure of which is hereby incorporated by reference). Such nanolithographically-defined structures utilize regular arrays of uniform-sized nanostructures throughout the separation matrix. Although these nanolithographic structures are useful in separation, the systems suffer from drawbacks: (1) resolution limitations, (2) flexibility limitations, and (3) difficulty in integrating the system with other, more complex, separation devices. Thus, the need for an efficient, highly-resolving, flexible, cost-efficient, and reproducible molecular-separation matrix, is largely unmet.
The analysis and characterization of biomolecules is further limited by the difficulty in separating membrane-associated molecules. Typically, detergents are used to remove transmembrane molecules, but even mild detergents may denature such molecules, rendering them inactive and/or disrupting necessary functional interactions with other membrane components including other proteins or lipid components. Additionally, the study of biomolecules is limited by the difficulty in fabricating a cellular environment that allows for the interaction of molecules. Such interactions may be useful in studying molecular transport and communication across cell membranes.
Thus far, the most relevant work in this area is the use of synthetic lipid bilayer membranes as separation platforms for biomolecules. Because of their planar structure, such membranes are more amenable to laboratory use. The separation technology is achieved by integrating planar lipid bilayers with varied surfaces to allow for separation of molecules. For instance, synthetic membranes supported on a glass or silica surface allow for the electrophoretic separation of labeled phospholipids and membrane proteins. See, Groves, J. T. and Boxer, S. G., Electric-field-induced concentration gradients in planar supported bilayers, Biophysical Journal, 69: 1972-1975 (1995), and Groves, J. T., Wulfing, C., and Boxer, S. G., Electrical manipulation of glycan phosphatidyl inositol tethered proteins in planar supported bilayers, Biophysical Journal, 71: 2716-2723 (1996), the entire contents and disclosures of which are hereby incorporated by reference. Additionally, lipid bilayer membranes have been incorporated into microstructured devices by lithographically-derived features to partition the supported membrane into separate regions to pattern the distribution of the lipid bilayer over the surface or as a coating for microchannels. See, Cremer, P. S., and Yang, T., Creating spatially addressed arrays of planar supported fluid phospholipid membranes, Proceedings of the National Academy of Sciences, U.S.A., 121: 8130-8131; Nissen, J., Jacobs, K., and Radler, J. O., Interface dynamics of lipid membrane spreading on solid surfaces, Physical Review Letters, 86: 1904-1907 (2001); and Yang, T. L., Jung, S. Y., Mao, H. B., and Cremer, P. S., Fabrication of phospholipid bilayer-coated microchannels for on-chip immunoassays, Analytical Chemistry, 73: 165-169 (2001), the entire contents and disclosures of which are hereby incorporated by reference. Furthermore, lipid bilayers have been supported on nanostructured arrays to produce Brownian ratchets utilized in the electrophoresis of fluorescent phospholipids. See, van Oudenaarden, A., and Boxer, S. G., Brownian ratchets: Molecular separations in lipid bilayers supported on patterned arrays, Science, 285: 1046-1048 (1999), the entire contents and disclosures of which are hereby incorporated by reference. Finally, hybrid lipid bilayers, in which one leaflet (define leaflet) of the supported membrane is formed by an alkane-thiol monolayer on gold, have shown promise for use in bioseparations. See, Plant, A., Supported hybrid bilayer membranes as rugged cell membrane mimics, Langmuir, 15: 5128-5135 (1999), and Hui, et al., U.S. Pat. No. 5,919,576, the entire contents and disclosures of which are hereby incorporated by reference. However, in these techniques, the close proximity or constraint of the lower leaflet to the supporting surface reduces their usefulness in analyzing transmembrane proteins or interactions between cytoplasmic and extracellular components of the membrane.
Also relevant to the technology of the present invention are previous methods for creating suspended lipid bilayers in which regions of the lipid bilayers are freely suspended between two aqueous reservoirs. Such hybrid bilayers are formed so one leaflet of the suspended region of the bilayer is replaced with a methyl terminated self-assembled monolayer, allowing for suspension of free bilayers over gaps as large as 100 um. See, Ogier, S. D., Bushby, R. J., Cheng, Y., Evans, S. D., Evand, S. W., Jenkins, T. A., Knowles, P. F., and Miles, R. E., Langmuir, 16: 5696-5701 (2000), the entire contents and disclosures of which are hereby incorporated by reference. Although these types of suspended bilayers have been used for studying membrane permeability and transmembrane protein function, the use of such suspended lipid bilayers in the separation of transmembrane proteins has not been examined. Thus, the need for technology that utilizes supported and suspended lipid bilayer membranes that allow for (1) separation of membrane-spanning complexes, and (2) cellular interaction is largely unmet.
It is therefore an object of the present invention to provide an efficient nanostructured matrix for separation and analysis of molecules.
It is a further object of the present invention to provide a matrix that enables gradient or non-uniform transport of molecules across a plane parallel to the surface of the matrix.
A further object of the present invention is to enable integration of multi-dimensional multi-technique molecular separation systems into a single platform.
Yet another object of the present invention is to provide for customized fabrication of a nanostructured separation matrix including an array having a gradient property.
It is yet another object of the present invention is to provide a nanostructured matrix that may cater to different ranges of molecular separations, in terms of resolution and dynamics.
Another object of the present invention is to enable consistency in the composition of the nanostructures forming the separation matrix.
Yet another object of the present invention is to enable separation and/or identification of a molecular species.
A further object of the present invention is to enable calibration-free use of the separation/analysis process.
Yet another object of the present invention is to enable multiple use of a single separation matrix.
A further object of the present invention is to enable parallel production of separation matrices at relatively low cost.
In all of the above embodiments, it is an object to provide enhanced reproducibility and resolution in the separation of molecules.
According to a first broad aspect of the present invention, there is provided a nanostructured device comprising a substrate including at least one nanotrough therein; and a lipid bilayer suspended on the substrate.
According to second broad aspect of the invention, there is provided a nanostructured device comprising a substrate including at least one nanotrough therein; and at least one lipid bilayer supported in at least one of the at least one nanotroughs.
According to a third broad aspect of the invention, there is provided a separation method comprising the steps of supporting or suspending a lipid bilayer on a substrate; wherein the substrate comprises at least one nanostructure and wherein the lipid bilayer comprises at least one membrane associated biomolecule; and applying a driving force to the lipid bilayer to separate the at least one membrane associated biomolecule from the lipid bilayer and to drive the at least one membrane associated biomolecule into the at least one nanostructure.
Other objects and features of the present invention will be apparent from the following detailed description of the preferred embodiment.
The invention will be described in conjunction with the accompanying drawings, in which:
It is advantageous to define several terms before describing the invention. It should be appreciated that the following definitions are used throughout this application.
Where the definition of terms departs from the commonly used meaning of the term, applicant intends to utilize the definitions provided below, unless specifically indicated.
For the purposes of the present invention, the term “nanostructure” refers to a protrusion or void having a diameter in al least one direction of 1 to 500 nm.
For the purposes of the present invention, the term “diameter” refers to the distance across a nanostructure through the middle and perpendicular to the axis of the nanostructure, parallel to the plane of the substrate (upon which the nanostructure is located).
For the purposes of the present invention, the tern “axis” refers to a line running along the middle of a nanostructure in the direction the nanostructure's longest dimension parallel to the surface of the substrate on which the nanostructure is located.
For the purposes of the present invention, the term “protrusion” refers to a structure that protrudes from the surface of a substrate or that protrudes from a portion of a substrate that has been etched. The protrusions of the present invention may be any convenient size or shape. The cross-section of a protrusion may be circular, square, rectangular, oval, elliptical, etc.
For the purposes of the present invention, the term “channel” refers to a gap between any two protrusions. The channels of the present invention may be any convenient size or shape.
For the purposes of the present invention, the term “array” refers to an arrangement of nanostructures.
For the purposes of the present invention, the term “gradient” refers to an array where channels, protrusions or other features at one end of the array are larger than those at an opposite end of the array.
For the purposes of the present invention, the term “continuous gradient” refers to a gradient where successive rows of channels, protrusions or other features decrease in size substantially continuously from one end of the gradient to the other end of the gradient.
For the purposes of the present invention, the term “non-continuous gradient” refers to a gradient that includes regions of the gradient having successive rows of channels, protrusions or other features that are substantially the same size.
For the purposes of the present invention, the term “matrix” refers to a substrate having an array of nanostructures present on or in at least a portion of the substrate. A matrix of the present invention preferably has at least one gradient on or in the substrate formed by the nanostructures. Examples of a matrix of the present invention include one or more arrays located on a chip, such as a semiconductor chip, biochip, etc. Methods for making biochips which may be readily adapted for use in making biochips of the present invention are described in U.S. Pat. No. 6,174,683, the entire contents and disclosure of which is hereby incorporated by reference.
For the purposes of the present invention, the term “interferometric lithography” (IL) refers to a process of lithography that involves interference patterns of two (or more) mutually coherent light waves. The angles between the light propagation vectors of the waves are sufficiently large to produce an interference pattern that has a high spatial frequency. The resulting interference pattern may have nanoscale dimensions. Examples of interferometric lithography techniques that may be used in the present invention are described in Chen X L, Brueck S R J, “Imaging interferometric lithography: approaching the limits of optics” in Optics Letters, 24, pp. 124-126 (1999), in “Imaging interferometric lithography: A wavelength division multiplex approach to extending optical lithography, Chen X L, Brueck S R J, Journal of Vacuum Science and Technology B, vol. 16, pp. 3392-3397 (1998), in U.S. Pat. No. 5,759,744 to Brueck et al., in U.S. Pat. No. 6,233,044 to Brueck et al., and U.S. Pat. No. 6,042,998 to Brueck et al, the entire contents and disclosures of which are hereby incorporated by reference.
For the purposes of the present invention, the term “biomolecules” refers to biologically derived molecules such as peptides, small polypeptides, long polypeptides, proteins, antigens, antibodies, tagged proteins, oligonucleotides, nucleotides, polynucleotides, aptamers, DNA, RNA, carbohydrates, etc, and complexes thereof.
For the purposes of the present invention, the term “size exclusion separation process” refers to separating particles, such as biomolecules, by size based on the ability of smaller particles to pass through smaller openings or channels than larger particles.
For the purposes of the present invention, the term “gel electrophoretic mobility separation process” refers to any conventional electrophoresis separation technique such as two-dimensional polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis (PAGE) is used to separate biomolecules, usually proteins or DNA fragments, by the ratio of each biomolecule's mass to charge. Proteins may be separated in either their native state, or denatured by the addition of a detergent such as SDS (Sodium Dodecyl Sulfate). Further resolution may be obtained in some cases by making a gel with a gradient either in the concentration of the acrylamide or in the degree of crosslinking within the gel matrix. An array of the present invention may be used in performing equivalent molecular weight separations, with either electrical currents or flow as the driving force.
For the purposes of the present invention, the term “isoelectric focusing separation process” refers to the separation of charged biomolecules, such as proteins and peptides, by each biomolecule's isoelectric point. A pH gradient is generally generated using a mixture of ampholytes within the separation matrix, usually polycrylamide polyacrylamide. The biomolecules in the mixture then migrate to the region where the pH is equal to a particular biomolecule's isoelectric point, at which time the charged biomolecule becomes electrically neutral. This technique, combined with subsequent separation by SDS-PAGE, is used in traditional two-dimensional gel electrophoresis. Similar pH gradients may be generated using an array of the present invention including a two-dimensional gradient, using traditional isolectric isoelectric focusing with soluble ampholytes or by using chemical patterning techniques, or immobilization of ampholytes after electrical focusing. Examples of capillary-based isoelectric focusing separation processes suitable for use with the present invention are described in Thorman, Tsai, Michaud, Mosher and Bier, Capillary Isoelectric-Focusing: Effects of Capillary, Geometry, Voltage Gradient and Addition of Linear Polymer, J. Chromatography, 398:75-86 (1987), the entire contents and disclosure of which are hereby incorporated by reference.
For the purposes of the present invention, the term “asymmetric diffusion separation process” refers to a separation process in which steric constraints drive diffusion preferentially in one direction. Examples of asymmetric diffusion separation processes suitable for use with the present invention are described in Van Oudenaarden et al., Science, 285: 1046-1052 (1999), the entire contents and disclosure of which are hereby incorporated by reference.
For the purposes of the present invention, the term “entropic trapping separation process” refers to separations using nanostructured devices of alternating thin and thick regions, with the thin regions being smaller than the radius of gyration of the biomolecule being separated. Under an electrical field, the molecules repeatedly change conformation, costing entropic free energy, thus limiting mobility. An example of an entropic trapping separation process suitable for use with the present invention is described in Han J, Craighead H D, Separation of long DNA molecules in a microfabricated entropic trap array, Science, 288:1026-1029 (2000), the entire contents and disclosure of which is hereby incorporated by reference.
For the purposes of the present invention, the term “hydrophobic interaction chromatography separation process” refers to a technique whereby molecules are partitioned between a hydrophobic matrix and a hydrophilic solvent. The degree of hydrophobicity of the target molecule determines the target molecule's retention time. The array of the present invention may be modified to incorporate a gradient of hydrophobicities or to create a milieu in which the hydrophobicity may be rapidly and reversibly changed, thus providing a driving force for molecular movement.
For the purposes of the present invention, the term “affinity chromatography separation process” refers to a chromatography process that takes advantage of specific chemical interactions between a target molecule and a chromatographic matrix. One of the most widely used forms of affinity chromatography employs immunoaffinity in which an antibody or series of antibodies are immobilized on a support. Other affinity agents include enzymes that interact with specific targets or receptors. Another example of affinity chromatography is a molecular recognition separation process such as the separation of long DNA molecules in a microfabricated entropic trap array. An array of the present invention may be used for both the generation of affinity matrices and for the subsequent use of affinity matrices.
For the purposes of the present invention, the term “enantiomeric resolution separation process” refers to a process to separate organic particles, such as biomolecules by chirality. Enantiomeric resolution is especially important in carbohydrate separations where differences between different glycosides are exclusively enantiomeric. Indeed, common chiral selectors are cyclodextrins used in capillary electrophoresis. Macrocyclic antibiotics and crown ethers are commonly used selectors. Selectors may be used either globally or in zones of an array of the present invention to confer yet another means of separation.
For the purposes of the present invention, the term “capillary electrophoresis separation process” refers to a separation process in which separation takes place in a liquid rather than in a gel matrix. Capillary electrophoresis allows for separations to be done on smaller quantities of material and with improved resolution in comparison to conventional gel electrophoresis processes. The channels in an array of the present invention may be arranged to generate a capillary type arrangement in a second direction following separations based on chemical properties (e.g., IEF, affinity, hydrophobic interaction chromatography or enantiomeric separation) or capillaries may be applied as a third dimension.
For the purposes of the present invention, the phrase “comprises Si” refers to silicon and any silicon complex, compound, etc. that includes silicon, such as SiO2, glass, etc.
For the purposes of the present invention, the term “lipid” refers to conventional lipids, phospholipids, etc.
For the purposes of the present invention, the term “lipid bilayer” refers to any double layer of oriented amphipathic lipid molecules in which the hydrocarbon tails face inward to form a continous continuous nonpolar phase.
For the purposes of the present invention, the term “simple bilayer” refers to a conventional lipid bilayer in which the bilayer is formed from micelles of phospholipids with or without membrane proteins.
For the purposes of the present invention, the term “hybrid bilayer” refers to a bilayer that is derived from more than one source, either through mixing of micelles before formation, or post bilayer fusion. These also refer to bilayers in which one component is synthetically derived, or in which one leaflet is supported on the nanotextured surface prior to bilayer formation.
For the purposes of the present invention, the term “self-assembled monolayer hybrid bilayer” refers to a hybrid bilayer in which the synthetic portion is composed of a self-assembled monolayer of silanes or ω-substituted alkanethilates on gold.
For the purposes of the present invention, the term “suspended” refers to bilayers present on a nanostructure and located above nanotroughs in a nanostructure. An example of a suspended bilayer is shown in
For the purposes of the present invention, the term “supported” refers to bilayers located in nanotroughs of a nanostructure. An example of a supported bilayer is shown in FIG. 12.
For the purposes of the present invention, the term “nanotrough” refers to a trough with a void dimension of 1-500 nm, whether uniform or not.
For the purposes of the present invention, the term “leaflet” refers to one half of a fluid bilayer membrane composed of a single layer of phospholipids and any included proteins.
For the purposes of the present invention, the term “filled with at least one fluid” refers to a nanostructure, preferably a nanotrough or channel, containing a fluid that is at least partially contained within said nanostructure. The nanostructure does not need to be completely filled with a fluid according to this definition.
For the purposes of the present invention, the term “membrane associated biomolecule” refers to any membrane associated biomolecule, such as transmembrane proteins, membrane phospholipids, lipophilic biomolecules, complexes thereof, etc.
The present invention provides, in part, for robust, inexpensive and reproducible methods for forming separation matrices for gradient separations based on, for example, electrophoresis and size exclusion that includes all the positive traits of gradient PAGE. These matrices may be adapted for a host of variant separation strategies, including electrophoresis, detergent solubilization, native electrophoresis, isoelectric focusing, 2D-electrophoresis, hydrophobic interaction, and affinity chromatography. More specifically, the present invention provides for the use of such separation matrices as support for lipid bilayers that serve as separation platforms for membrane-associated biomolecules. The methods of fabrication discussed herein may also be adapted to existing microfabrication and integration facilities.
The present invention provides for separation of molecular species across a nanostructured matrix, a method of fabricating nanostructures comprising the matrix and the use of such a matrix for separation and/or analysis of molecules by defining the physical size and/or chemical features of the nanostructures as a means of screening. The nanostructured matrix may be used to separate biological materials, such as proteins, carbohydrates, and nucleic acids as well as non-biological materials, such as synthetic polymers. These nanostructures may be made out of a variety of materials, including silicon, thus providing systems that may be easily chemically modified for additional flexibility. The use of lithography to generate nanostructured separation matrices has advantages over other techniques (such as traditional acrylamide gel polymerization) since it (1) creates highly ordered structures, (2) gives the possibility of creating macroscopic arrays of continually varying size or chemistry across one dimension, (3) is highly reproducible, and (4) may be easily implemented in the creation of complex, integrated separation systems that are disposable or reusable. Furthermore, the use of lithographically defined separation matrices lends itself to the facile implementation of these matrices into multi-level, 3-dimensional separation devices in which different screening mechanisms allow enhanced separations. Particularly, the lithographic nanostructured surfaces may be used to support lipid bilayers or hybrid lipid bilayers for separating membrane-associated molecules and studying cellular interactions. The present invention aims to (1) eliminate some of the current limitations by the fabrication of highly uniform and reproducible nanostructured separation systems prepared by nano- and microlithography, and (2) eliminate some of the current limitations by utilizing the lithographic nanostructured surfaces in conjunction with lipid bilayers to produce separation platforms for membrane-associated molecules.
Using an advanced lithographic technique such as interferometric lithography (IL) capable of producing nanostructures, patterns of nanostructures may be rapidly created over wide, macroscopic areas at low cost (compared to other techniques such as electron beam lithography). In addition, it may be used to easily generate arrays of nanostructures (protrusions or channels) whose dimensions vary semi-continuously in the plane of surface of the material being patterned. IL has advantages over other methods that might be used to construct nanopatterned fluidic structures (e.g., electron beam lithography, X-ray lithography, or local probe lithography) due to the low cost of implementation and the parallel nature of the lithographic technique. Combining IL with conventional lithography allows for the formation of device structures in individual areas and the addition of aperiodic features such as electronic and fluidic connections. Imaging interferometric lithography extends optics to fundamental, deep-subwavelength scales.
It is worthwhile at this point to consider the fundamental limits of optical lithography. For the interference of two plane waves in air, the period is given by λ/(2 sin θ) where λ is the optical wavelength and θ is the angle of incidence. For a 213-nm laser source (fifth harmonic of YAG) this gives a period of ˜150 nm (for θ=80°).
Water and higher-index liquids, including liquid Ar (n˜1.6), may be used to further extend these results into the sub-100-nm period regime that will be important for biological separations.
Nonlinear processes may be used to further reduce the period.
Importantly, all of these results are macroscopic in scale, e.g., covering areas of ˜1 cm2 or larger. A strength of optics is the parallel nature of the exposure, which may be cm's or larger in extent. For a square lattice with a 100-nm pitch and a 1 cm field, there are 1010 features, well beyond the realistic capabilities of serial techniques such as e-beam and scanning probes. In particular embodiments of the present invention, IL may be extended deep into the nanometer regime (either to feature sizes of ˜10 nm or nearest-neighbor distances (aperture sizes) of <10 nm, but not both simultaneously).
A continuously varying channel spacing between nanostructures is desired for many of the bio-separation applications such as various nanofluidic configurations discussed herein.
One approach to a graded structure is to macroscopically vary the intensity across the plane of exposure while keeping the other interference conditions, such as the angles between the light propagation vectors and the polarization, unchanged. One such variation of intensity would be a smooth gradient in intensity of one of the two interfering light waves. This results in interference fringes with uniform spacing but different intensities. The difference in intensity of the fringes leads to differences in exposure of the photoresist used. Because the fringe spacing is not changed, the pitch is uniform. The interference pattern would have even better contrast if both light waves had the same gradient in intensities.
When a positive photoresist is used, the areas corresponding to fringes with stronger intensities leave wider cavities in the photoresist after exposure and developing. The areas corresponding to fringes with weaker intensities leave narrower cavities in the photoresist. When the substrate is etched, these differing widths translate into features in the substrate that have differing widths. The features have the same pitch, however, because the fringe spacing is not altered. This leads to a constant pitch, but a varying line:space ratio. This procedure provides a continuously decreasing channel width that may be accurately controlled over very long distances. Such gradient separation matrices exhibit the favorable traits of gradient gels (high resolution in separation), without the difficulty and irreproducibility associated with their preparation.
Similarly, this technique, when used with a negative photoresist, leaves wider features in the areas corresponding to fringes with weaker intensity and narrower features in the areas corresponding to fringes with stronger intensity.
An alternative approach may produce features with a gradient in width and pitch. This may be easily achieved with IL by using a cylindrical lens in one of the beams, while keeping the other beam as a plane wave. In this case the plane of exposure becomes a chord for a number of circular wavefronts. Because the wavefronts have different radii of curvature (spacing of an optical wavelength), the spacing between the interference fringes, as well as the width of the interference fringes, vary along the length of the plane containing the interference fringes on the surface of the photoresist coating the substrate. Similarly, curved surfaces (sections of Newton's rings) may be formed by interfering a plane wave and a spherical wave or two spherical waves of differing radii of curvature.
Other types of separation systems may involve discontinuous gradients. One such system may have differing aperture sizes that may be produced by separate exposures with different intensities, at different pitches through shadow masks, or by using multiple exposure techniques to eliminate rows and/or columns of pillars in certain areas of a previously exposed uniform nano-structured surface.
Variations in size may also be produced chemically. For example, increasing the oxidation of silicon in certain areas of a chip may result in a swelling of the features, reducing the width of some channels while conserving the pitch of the features. Similarly, macroscopic areas may be selectively functionalized with monolayers, reducing the width of channels in that area.
One may also electrochemically produce silicon carbide on a silicon substrate. Silicon carbide is suitable for sublimation growth, allowing one to control the width of the modified channels in a certain area. Of course, silicon carbide is only one example of surface modifications that may be performed.
One may also selectively heat a substrate, bringing it close to its annealing temperature. At this time the substrate may be placed under a highly controlled stress. The subsequent strain alters the size of channels. A gradient in temperature across the substrate results in a gradient of strain, and therefore a gradient in channel widths. This technique would only be suitable for substrates without a crystalline structure (such as glass or amorphous silicon, for example).
The very high aspect ratios of
Nanostructures that exhibit a gradient in their capacity to transport biomolecular species (through size exclusion or otherwise) may be created by the IL processes discussed herein. Such gradients make separation matrices feasible for highly efficient separation of molecular species. Molecular species may be driven in the direction of the gradient, and thus separated based on their tendency to traverse the gradient, by a variety of driving forces, including, but not limited to, electrophoresis, externally-applied pressure, capillarity, diffusion, and osmosis.
IL represents a convenient method for generating nanostructured separation matrices that contain physical gradients that allow selective transport of chemical species and, thus, may be used to achieve a separation of different chemicals. When compared to other nanolithographic methods of pattern generation (e.g., electron beam lithography, scanning probe lithography), IL is more convenient, efficient and inexpensive because it may be used to generate the entire pattern in one, parallel step and is not a serial “writing” technique. Other parallel techniques (e.g., imprint lithography) rely on a primary patterning technique to generate a master that may then be used to produce replicas of nanostructured features in a parallel fashion. While IL is a preferred method to generate nanostructured gradients for molecular separation, a variety of methods could be employed to generate the nanostructured matrix gradient “artificial gels” of the present invention. Gradients in the chemistry of the separation matrix may be prepared by a variety of methods as well, including those based on IL.
The use of IL allows such nanostructured separation matrices to be produced easily and very inexpensively. Nanostructures in which channels are on the order of the excluded size of dissolved biomolecules allow an enhanced flexibility in separation. Higher resolution may be obtained in combination with any of the following mechanisms namely, size exclusion, electrophoretic mobility, isoelectric point, asymmetric diffusion, entropic trapping, hydrophobic interaction and affinity interaction (molecular recognition), as well as others. The gradient matrices produced allow efficient separation and identification of biomolecules such as native proteins and protein complexes in addition to denatured proteins and nucleic acids.
Nanolithography-generated systems have advantages over conventional systems in terms of (1) the virtually perfect uniformity of pore size and pore size distribution from device to device, and (2) the flexibility to precisely define the required distribution (gradient) of pore sizes and pore chemistries. This high degree of reproducibility and versatility in nanofabrication will result in the ability to construct separation devices that exhibit unprecedented degrees of flexibility (resolution, dynamic range) and reproducibility in their separation characteristics.
The separation gradient may be formed by a variety of means including, for example, nanolithography (e.g., IL, electron beam, local probe, nanoimprint) and pattern transfer (etching, deposition, lift-off) means.
Multiple-exposure IL moiré patterns provide for cyclic gradients that may be used for simultaneous manufacture of multiple structures. Gradients may also be fabricated across uniform patterns by non-uniform deposition or etching using properly designed deposition and/or etching tools and techniques such as oblique incidence of etch/deposition atomic/molecular species (shadowing). Analogous techniques may be used in generation of gradients in surface modification chemistry incorporated into the array.
As an example of channel formation according to the present invention, IL and anisotropic wet etching of Si allow the creation of open, parallel nanostructured channels (e.g., uncapped in the direction perpendicular to the surface) with lateral features on the order of biomolecular length scales (˜1-10 nm) but with overall dimensions reaching the microscopic (˜100 μm) or even macroscopic (˜1 cm or greater) scales. Depending upon the dimensions, molecular transport mechanisms may include diffusion, electrophoresis or bulk-flow. The relatively large vertical scale is sufficient to allow high throughput of molecules and external pumping using either electrokinetic or electro-osmotic forces. Examples of high aspect ratio IL nanostructured samples are shown in
Arrays of nanostructures (either of uniform size or with a gradient of sizes) may be surface-modified with chemical species that enhance the separation characteristics of the matrix. These chemical species may be distributed uniformly formly over the nanostructured separation matrix or may be distributed in a gradient (continuous or discrete) in the direction of separation over the matrix. These chemical species may include small organic molecules, polymers, receptors or other biomolecules.
IL may be used to expose patterns on photoresist on silicon or other materials (which may later be etched). Silicon and some other materials may have an oxide surface that is easily modified with silanization reagents. Synthetic strategies for modification are also available for other materials (besides oxides), including native silicon and noble metals (e.g., gold). Monomolecular layers may be created from a wide range of commercially- or synthetically-available chemical species that may enhance separation characteristics based on the type and degree of interaction of chemical species being separated with the walls of the surface-modified nanostructured separation matrix. Examples of types of surface modifications (either as gradients or uniform) include the use of hydrophilic oligomeric and polymeric species (e.g., poly-ethylene glycol (PEG)) to minimize interactions of chemical species, especially proteins, with nanostructured surfaces; use of hydrophobic molecular or oligomeric species to elicit hydrophobic interaction of chemical species (especially proteins) with nanostructured surfaces; use of mixtures of hydrophobic and hydrophilic species (polar, apolar, H-bonding, ionic) to tune interaction of different chemical species with surfaces; use of ionic molecular species and mixtures of ionic species to tune interaction of different chemical species with surfaces; use of biomolecular or organic receptors to elicit molecular recognition of small molecules, polymers, proteins, DNA, RNA, or oligonucleotides with the surface; use of oligonucleotide probes to tune interactions of DNA, RNA or nucleic-acid binding proteins with the surface; use of cyclodextrins, macrocyclic antibiotics, crown ethers and other chiral selectors to tune enantiomeric interactions of chemical species with the surface; and use of stimuli-responsive (smart) molecules or polymers to allow external control of interaction of chemical species with the nanostructured surface.
Other types of separation systems of the present invention may be thought of as having discontinuous gradients. These separation systems contain areas with different aperture sizes, and may be made either by separate exposures at different intensity, at different pitches through shadow masks, or by using multiple exposure techniques to eliminate rows and/or columns of pillars. Such systems are especially useful in that they will allow recovery of separated compounds (purification).
Microfabricated Integrated Multi-Dimensional, Multi-Technique Separation Systems
The present invention allows a variety of different separation strategies (electrophoresis, iso-electric focusing, affinity chromatography, hydrophobic interaction chromatography, enantiomeric resolution) to be used on a single monolithic device, thus allowing for ease of use and compactness of instrumentation.
The closest existing commonly used multi-technique separation is two-dimensional gel electrophoresis (2DGE). In traditional 2DGE, proteins are first separated according to isoelectric point, followed by resolution by mass-to-charge-ratio using standard polyacrylamide electrophoresis. This process requires that two separate electrophoretic procedures be performed, each requiring manipulation of the sample. A nanostructured matrix of the present invention allows for sequential analysis on a single chip, thus reducing sample loss and diffusion. The wide range of chemical modifications and array architecture allowed by IL devices will also permit separation of proteins by means in addition to size and isoelectric point, either by appropriate chemical patterning and valving of the device, or by addition of a third separation and/or dilution dimension.
Fabrication of separation matrix systems from materials (e.g., Si and quartz) commonly used in the fabrication of integrated circuits is advantageous. They have unique etching and surface modification characteristics that are well established, and may be easily implemented in existing microfabrication facilities for the development of complex separation and detection systems. Other materials with advantageous characteristics may also be used.
The nanostructured matrix of the present invention may be used for two-dimensional gel electrophoresis, and a number of other separation techniques may be combined with size exclusion and/or isoelectric focusing. In addition, the matrix has the capability of expansion beyond two dimensions.
Combining two or more standard types of analysis on a single platform may enhance the analytical potential of a nanostructured matrix of the present invention. Among the possible combinations of separation technologies applicable to this platform are those analogous to PAGE, isoelectric focusing, hydrophobic interaction chromatography, affinity chromatography, enantiomeric resolution and capillary electrophoresis. The matrix lends itself well to carrying out equivalent molecular weight separations, with either electrical currents or flow as the driving force.
The present invention may be useful in proteomics by enabling combinations of different types of analysis, e.g., size exclusion in one dimension with chemical differentiation in the second. A third dimension, oriented perpendicular to the two dimensional array, may then be used for further separation, or for recovery and further characterization of isolated spots.
The present invention may also find use in protein separations for forensic and medical diagnostic tools and in the separation of bioengineered proteins. Forensic analysis and diagnostics, for example, depend heavily upon differentiation between carbohydrate moieties on blood proteins and bacterial cells. Discovery of clinically useful drugs often depends on identifying interactions with specific cellular receptors, which are usually glycoproteins. Capillary electrophoresis has been extremely useful in separations of acid carbohydrates, with derivatization of the column. The present invention allows for the separation of two properties, for example glycoprotein size and carbohydrate content on a single platform, thus eliminating the need for cumbersome recovery between steps and increasing the yield of useful analyte.
Recently, techniques utilizing antibody-based affinity separations have transitioned from clinical laboratories to those for environmental monitoring. The present invention allows sequential analysis of at least two different properties, thus increasing sensitivity of analysis, with particular interest for environmental monitoring.
The present invention allows for separation of a variety of sizes of nucleic acid species, and thus, may be used for separations that are currently done by standard and pulsed-field gel electrophoresis, as well as nucleic acid sequencing. In addition, modification of the device by nucleic acid-binding molecules (e.g., proteins, drugs) allows for isolation of relevant target sequences from previously uncharacterized genomes, or for isolation of a biocomplex formed with a nucleic acid. Because separation may be multidimensional, these devices may be attached in series with a reaction chamber (for example, a PCR thermocycler) and the resultant product directly fed into the separation platform for purification and analysis in a single device.
IL may be used to create nanostructures on a variety of substrates. IL, in combination with other standard lithographic and microfabrication methodologies, may be used to create a variety of nanostructures that may be modified in many ways to produce tools for separation of relevant biomolecules. These have advantages over contemporary molecular separation systems because they exhibit superior performance (resolution, sensitivity, dynamic range, applicability, reproducibility), may be parallel-produced at relatively low cost, and are extremely flexible in terms of chemical modifications. They have defined features that may be reproducibly made, enable flexible and complex separation, and may be used with existing bioseparation and detection strategies.
Supported and Suspended Lipid Bilayers on Nanotextured Surfaces
An additional aspect of the present invention is the use of defined lithographic nanostructures to suspend or support lipid bilayers and hybrid lipid bilayers as a separation platform for membrane-associated biomolecules. This invention expands upon previous methods for (1) incorporating lipid bilayers and nanostructured surfaces for separation techniques, and (2) creating lipid bilayers in which regions of the lipid bilayers are freely suspended or supported between two aqueous reservoirs. Specifically, the present invention utilizes suspended or supported lipid bilayer architecture in the separation of transmembrane molecules.
Of particular interest are nanofabricated arrays of structures that exhibit a gradient in their capacity to transport molecules. The reason being that such gradients allow for separation matrices that eliminate the requirement for detergent solubilization, and thus denaturation, of transmembrane biomolecules prior to separation, which is the current state of the art. Such gradient structures allow molecular species to be driven in the direction of the gradient, thereby separating the molecules based on their tendency to traverse the gradient. Molecular species may be driven across the gradient by forces such as electrophoresis, externally-applied pressure, capillarity, diffusion, and osmosis. Depending on the desired means of separation, several modifications of the nanostructured support that will enhance separation within the supported or suspended bilayer are envisioned. These include, but are not limited to chemical modifications, such as changes in hydrophobicity, charge, or dipole moment which will allow interactions with protein domains exterior to the bilayer, modification with ligands or other biomolecules that have the potential for interacting with a target class of membrane proteins, and other modifications that end users will deem necessary to base separations on membrane protein function.
Two relevant methods for fabricating suspended lipid bilayers have been reported: (1) suspending small unilamellar vesicles that are made and applied directly to an unmodified Si surface, and (2) generating large unilamellar vesicles with direct pipetting of these structures onto a surface. See, Groves, J. T., Wulfing, C., and Boxer, S. G., Electrical manipulation of glycan phosphatidyl inositol tethered proteins in planar supported bilayers, Biophysical Journal, 71: 2716-2723 (1996), and Menger, F. M., and Angelova, M. I., Accounts of Chemical Research, 31: 789-797 (1999), the entire contents and disclosures of which are hereby incorporated by reference. Preliminary studies included forming suspended lipid bilayers to examine their applicability in the present invention.
Since the electrophoretic mobility of transmembrane molecules across suspended lipid bilayers depends on (1) the molecule's mass to charge ratio, and (2) the size of the extramembrane domains relative to the corrugated apertures in the nanostructured support, it is necessary to fabricate a device that allows for preferential movement of molecules. An embodiment of the present invention suspends lipid bilayer membranes over a series of small gaps, approximately 100 nm in size, and utilizes the entire supported membrane as a separation and analysis platform. The small sizes of the gaps between features allows the lipid bilayer membrane to be suspended over the gaps, which allows for preferential movement of membrane phospholipids, transmembrane proteins, and other lipophilic biomolecules, and their complexes. More specifically, the relative fluidity of the lower leaflet of the lipid bilayer in the suspended regions, and resultant lack of steric hindrance of extramembrane protein domains, results in greater mobility of transmembrane molecules. Furthermore, by making the aperture size on the order of the molecular size of the transmembrane molecules, separations may be based on molecular filtering mechanisms in addition to electrophoretic mobility. Because the areas scanning the gaps may be supported on the underside by aqueous media, more room may also be available for intercellular domains. In addition, biophysical studies both of interactions between extra and intercellular domains of a single protein, and/or of interactions between intercellular domains of proteins within the same membrane are provided by the present invention. Thus, suspended lipid bilayer membranes offer several advantages over the current state of the art, particularly in regard to the separation and concentration of transmembrane proteins.
In a modification of the suspended lipid bilayer model, alkane-chain terminated self-assembled monolayers may be formed on the top surfaces of the nanostructured surfaces, either by silane modification of Si substrates or ω-substitued ω-substituted alkanethiols on, for example, gold. It is anticipated that these structures may provide even greater mobility of lipophilic biomolecules in supported and non-supported regions of the lipid bilayer membrane due to the immobility of the chemically fixed lower leaflet in the hybrid region.
Several nanotextured surfaces have also been explored. IL may produce a variety of features, including posts and grooves, in nearly infinite combinations of types and arrangements. Such features may be arranged in a regular array, thus mimicking standard gels, with the features either shaped or arranged in an asymmetric manner, or as semidiscontinuous, or chirped, arrays that vary in their size and/or separation distance along the direction of separation. Furthermore, a combination of grooves and/or posts may be arranged to achieve configurations that allow for two-dimensional separations. The present invention may be used for separation of membranes from osmotically disrupted cells (cell ghosts). This is particularly significant because no previous isolation of membrane-associated biomolecules is necessary, thus preserving the biomolecules' native conformations and complexes.
Although the present invention is primarily concerned with the structures described above, the nanostructured surfaces and lipid bilayer membranes may be combined in such a way to modify only the tops of the features, the lower surface of the nanostructured surface, the sides of the features, or any combination thereof. In addition, lipid bilayer membranes containing different molecules, or derived from different organelles within a cell, may be patterned on the nanostructured surface, thereby conferring a certain level of selectivity within the membrane itself, either due to innate properties of the molecules or the presence of interactive biomolecules within a region of the pattern. Thus, the present invention may be utilized in several manners to facilitate the study of biomolecules.
For instance, the nanostructured surfaces supporting lipid bilayer membranes may be utilized in biophysics to study membrane components. Because, within the suspended regions, neither leaflet of the membrane may be immobilized on the surface, total membrane fluidity may be increased, thus allowing for greater mobility of embedded biomolecules and creating an experimental milieu more closely replicating that found in the cell. Furthermore, interactions between cytoplasmic and extracellular domains may be more easily studied.
In addition, the present device shows promise as a biosensor device. Because the structure allows for proper orientation of the intercellular domains of transmembrane proteins, natural or engineered receptor proteins may take advantage of naturally occurring transduction mechanisms to facilitate signal transduction.
The present invention may further be useful in purification. The nanostructured lipid bilayer device may provide a unique platform on which to purify lipophilic membrane biomolecules. Because the components may be applied from membrane cell ghosts or in lipid micelles, the need for harsh and possibly denaturing detergent extraction may be unnecessary. In addition, complexes of associated proteins may be purified intact, thereby improving the study of pharmaceutical agents.
The present device may also be useful in the crystallization of membrane proteins to provide more pertinent information as to the structure and function of the proteins. The nanostructured lipid bilayer device may be manufactured to produce a gradient of features so that the protein in question aggregates at a single band in the device, thereby accumulating at the critical concentration.
Finally, the present invention may allow for greater flexibility in screening potential pharmaceutical agents. The nanostructured lipid bilayer device may facilitate observation of interactions between target transmembrane molecules and potential therapeutic agents within a defined membrane milieu, as well as allow for in vitro study of the resultant interactions between the drug-bound receptor and other components within the target complexes.
The present invention allows for unprecedented advances in the study of biomembranes and their associated molecules. The fact that membrane associated biomolecules may be applied to the present device, either via cell ghosts or vesicles, without first isolating them in aqueous media using detergent solubilization means that native configurations, associations and, thus, functionality may be preserved.
Design and construction of microscale electrophoresis cells incorporated much of the characteristics of the present invention into a compact system. The cell preferably has the following characteristics: (1) electrochemical current and fluid flow is restricted to occur only through the separation matrix; (2) loading and stacking functions are included; (3) monitoring of mobility and biomolecular detection is possible (e.g., through fluorescence imaging); and (4) for certain applications, separated compounds are recoverable. Simple methods have been used for incorporating nanostructured silicon/silica chips into electrophoresis cells that satisfy criteria (1-3) above. For example, simple methods of rapid prototyping of elastomeric gasket materials have been used.
Supported phospholipid bilayers (SPBs) of egg phosphatidyl choline (Egg PC) were formed by vesicle fusion on nanostructured silicon wafers containing troughs −180 nm in width on a 360 nm pitch. An intercalating dye was introduced, and the membranes were imaged by scanning laser microscopy. The resultant fluorescence micrographs indicated that the SPBs formed uniformly over the surface and simple FRAP measurements indicated that the bilayers were fluid and that recovery of fluorescence was preferentially in the direction parallel to the nanotroughs.
Transmembrane or membrane associated proteins may be incorporated into an SPB, from incorporation in the vesicle stage, insertion on the membrane or through incorporation of cell ghosts (i.e., intact membranes isolated from cells or organelles). The architecture and/or chemistry of the underlying nanotextured support would be then used to guide the movement of membrane proteins through the supported or suspended bilayer, either by size exclusion of the trough over which the membrane is supported, or by chemical interactions with modifications on the nanostructured support.
Although the present invention has been fully described in conjunction with the preferred embodiment thereof with reference to the accompanying drawings, it is to be understood that various changes and modifications may be apparent to those skilled in the art. Such changes and modifications are to be understood as included within the scope of the present invention as defined by the appended claims, unless they depart therefrom.
|Cited Patent||Filing date||Publication date||Applicant||Title|
|US3855133||20 Dec 1972||17 Dec 1974||Sartorius Membranfilter Gmbh||Multi-layer filter membrane|
|US3906929||12 Dec 1974||23 Sep 1975||Lynn Lawrence Augspurger||Processes for reproduction of cellular bodies|
|US4190535||10 Jul 1978||26 Feb 1980||Corning Glass Works||Means for separating lymphocytes and monocytes from anticoagulated blood|
|US4737268||18 Mar 1986||12 Apr 1988||University Of Utah||Thin channel split flow continuous equilibrium process and apparatus for particle fractionation|
|US4801380||23 Dec 1987||31 Jan 1989||The Texas A&M University System||Method of producing a silicon film with micropores|
|US4814082||20 Oct 1986||21 Mar 1989||Memtec North America Corporation||Ultrafiltration thin film membranes|
|US4814088||20 Jul 1988||21 Mar 1989||National Research Council Of Canada||Method for the molecular filtration of predominantly aliphatic hydrocarbon liquids|
|US4902424||9 Aug 1988||20 Feb 1990||Memetc North America Corp.||Ultrafiltration thin film membranes|
|US4906439||21 Dec 1987||6 Mar 1990||Pb Diagnostic Systems, Inc.||Biological diagnostic device and method of use|
|US4915839||30 Dec 1988||10 Apr 1990||Cuno, Incorporated||Process for surface modifying a microporous membrane|
|US4916110||1 Nov 1988||10 Apr 1990||W. L. Gore & Associates, Inc.||Microporous catalytic material and support structure|
|US4935141||23 Aug 1988||19 Jun 1990||Gambro, Dialysatoren Kg||Selectively permeable asymmetric membranes suitable for use in hemodialysis and processes for manufacturing such membranes|
|US4969998||23 Apr 1984||13 Nov 1990||W. L. Gore & Associates, Inc.||Composite semipermeable membrane|
|US4971904||29 Jan 1988||20 Nov 1990||E. I. Du Pont De Nemours And Company||Heterogeneous immunoassay|
|US4977078||22 Dec 1987||11 Dec 1990||Olympus Optical Co., Ltd.||Plate substrate immunoassay device and method for performing a multi-test immunoassay on a specimen|
|US5013337||12 Apr 1990||7 May 1991||Uop||Process for separating a mixture of molecular species using crystalline microporous metal sulfide compositions|
|US5019263||5 Jun 1990||28 May 1991||Mobil Oil Corp.||Membrane composed of a pure molecular sieve|
|US5130025||28 Jun 1989||14 Jul 1992||Unisearch Limited||Membrane separation and purification of compounds|
|US5131998 *||13 Nov 1990||21 Jul 1992||The University Of North Carolina At Chapel Hill||Two-dimensional high-performance liquid chromatography/capillary electrophoresis|
|US5145584||31 Oct 1991||8 Sep 1992||Allied-Signal Inc.||Processes for using a thin film composite ultrafiltration membrane|
|US5147606||6 Aug 1990||15 Sep 1992||Miles Inc.||Self-metering fluid analysis device|
|US5193688||5 Jul 1991||16 Mar 1993||University Of Utah||Method and apparatus for hydrodynamic relaxation and sample concentration NIN field-flow fraction using permeable wall elements|
|US5204239 *||9 Jan 1991||20 Apr 1993||Yeda Research And Development Co., Ltd.||Biosensors including lipid bilayer doped with ion channels anchored to a recording electrode by bridging molecules|
|US5266207||30 Apr 1992||30 Nov 1993||Techsep||Composite nanofiltration membrane|
|US5296375||1 May 1992||22 Mar 1994||Trustees Of The University Of Pennsylvania||Mesoscale sperm handling devices|
|US5304487||1 May 1992||19 Apr 1994||Trustees Of The University Of Pennsylvania||Fluid handling in mesoscale analytical devices|
|US5427663||8 Jun 1993||27 Jun 1995||British Technology Group Usa Inc.||Microlithographic array for macromolecule and cell fractionation|
|US5427946||21 Jan 1994||27 Jun 1995||Trustees Of The University Of Pennsylvania||Mesoscale sperm handling devices|
|US5474675||17 Nov 1989||12 Dec 1995||Herco-Cff Chiralflow Filtertechnik Gmbh||Filter separator for separating a composite fluid|
|US5500071 *||19 Oct 1994||19 Mar 1996||Hewlett-Packard Company||Miniaturized planar columns in novel support media for liquid phase analysis|
|US5541072||18 Apr 1994||30 Jul 1996||Immunivest Corporation||Method for magnetic separation featuring magnetic particles in a multi-phase system|
|US5587070||4 Jun 1993||24 Dec 1996||Pall Corporation||System for processing biological fluid|
|US5622831||7 Jun 1995||22 Apr 1997||Immunivest Corporation||Methods and devices for manipulation of magnetically collected material|
|US5637458||20 Jul 1994||10 Jun 1997||Sios, Inc.||Apparatus and method for the detection and assay of organic molecules|
|US5639669||7 Jun 1995||17 Jun 1997||Ledley; Robert||Separation of fetal cells from maternal blood|
|US5646048 *||24 Jul 1995||8 Jul 1997||Hewlett-Packard Company||Microcolumnar analytical apparatus with microcolumnar flow gating interface and method of using the apparatus|
|US5674592||4 May 1995||7 Oct 1997||Minnesota Mining And Manufacturing Company||Functionalized nanostructured films|
|US5707799||30 Sep 1994||13 Jan 1998||Abbott Laboratories||Devices and methods utilizing arrays of structures for analyte capture|
|US5709943||4 May 1995||20 Jan 1998||Minnesota Mining And Manufacturing Company||Biological adsorption supports|
|US5715946||7 Jun 1995||10 Feb 1998||Reichenbach; Steven H.||Method and apparatus for sorting particles suspended in a fluid|
|US5716527||8 Jul 1994||10 Feb 1998||Exxon Research & Engineering Company||Zeolite membrane with a selectivity enhancing coating|
|US5728457 *||11 Jun 1996||17 Mar 1998||Cornell Research Foundation, Inc.||Porous polymeric material with gradients|
|US5736342||8 Nov 1995||7 Apr 1998||Washington State University Research Foundation||Biosensor for detecting the presence of chosen analytes|
|US5750339||30 Nov 1994||12 May 1998||Thomas Jefferson University||Methods for identifying fetal cells|
|US5753014||14 Nov 1994||19 May 1998||Van Rijn; Cornelis Johannes Maria||Membrane filter and a method of manufacturing the same as well as a membrane|
|US5770029||30 Jul 1996||23 Jun 1998||Soane Biosciences||Integrated electrophoretic microdevices|
|US5798042||14 Jun 1996||25 Aug 1998||Regents Of The University Of California||Microfabricated filter with specially constructed channel walls, and containment well and capsule constructed with such filters|
|US5837115||7 Jun 1995||17 Nov 1998||British Technology Group Usa Inc.||Microlithographic array for macromolecule and cell fractionation|
|US5843767||10 Apr 1996||1 Dec 1998||Houston Advanced Research Center||Microfabricated, flowthrough porous apparatus for discrete detection of binding reactions|
|US5858188||4 Apr 1996||12 Jan 1999||Aclara Biosciences, Inc.||Acrylic microchannels and their use in electrophoretic applications|
|US5858195||1 Aug 1995||12 Jan 1999||Lockheed Martin Energy Research Corporation||Apparatus and method for performing microfluidic manipulations for chemical analysis and synthesis|
|US5866345||5 Mar 1997||2 Feb 1999||The Trustees Of The University Of Pennsylvania||Apparatus for the detection of an analyte utilizing mesoscale flow systems|
|US5871650||8 Jul 1994||16 Feb 1999||Exxon Research And Engineering Company||Supported zeolite membranes with controlled crystal width and preferred orientation grown on a growth enhancing layer|
|US5876830||6 Sep 1996||2 Mar 1999||Board Of Regents Of The University Of Colorado||Method of assembly of molecular-sized nets and scaffolding|
|US5891651||29 Mar 1996||6 Apr 1999||Mayo Foundation For Medical Education And Research||Methods of recovering colorectal epithelial cells or fragments thereof from stool|
|US5922591||27 Jun 1996||13 Jul 1999||Affymetrix, Inc.||Integrated nucleic acid diagnostic device|
|US5928880 *||11 Jun 1997||27 Jul 1999||Trustees Of The University Of Pennsylvania||Mesoscale sample preparation device and systems for determination and processing of analytes|
|US5935822 *||21 Mar 1995||10 Aug 1999||The Regents Of The University Of Colorado||Product and process for membrane and soluble polypeptide segregation|
|US5938923||15 Apr 1997||17 Aug 1999||The Regents Of The University Of California||Microfabricated filter and capsule using a substrate sandwich|
|US5993661||14 Apr 1997||30 Nov 1999||The Research Foundation Of State University Of New York||Macroporous or microporous filtration membrane, method of preparation and use|
|US6007690||30 Jul 1997||28 Dec 1999||Aclara Biosciences, Inc.||Integrated microfluidic devices|
|US6022590||5 Jan 1998||8 Feb 2000||Competitive Technologies, Inc.||Stepwise formation of multilayered nanostructures from macromolecular precursors|
|US6033546||15 Sep 1998||7 Mar 2000||Lockheed Martin Energy Research Corporation||Apparatus and method for performing microfluidic manipulations for chemical analysis and synthesis|
|US6043177||21 Jan 1997||28 Mar 2000||University Technology Corporation||Modification of zeolite or molecular sieve membranes using atomic layer controlled chemical vapor deposition|
|US6044981||25 Aug 1998||4 Apr 2000||The Regents Of The University Of California||Microfabricated filter with specially constructed channel walls, and containment well and capsule constructed with such filters|
|US6051372||9 Sep 1997||18 Apr 2000||Nimbus Biotechnologie Gmbh||Template induced patterning of surfaces and their reversible stabilization using phase transitions of the patterned material|
|US6051517||26 Jan 1999||18 Apr 2000||University Technology Corp.||Modified zeolite membrane|
|US6054034||9 May 1997||25 Apr 2000||Aclara Biosciences, Inc.||Acrylic microchannels and their use in electrophoretic applications|
|US6060415||10 Feb 1997||9 May 2000||National Science Council||Aligned molecular sieve crystals grown on anodic alumina membrane|
|US6074827||5 Feb 1998||13 Jun 2000||Aclara Biosciences, Inc.||Microfluidic method for nucleic acid purification and processing|
|US6090289||10 Jul 1995||18 Jul 2000||Exxon Research & Engineering Co.||Molecular sieves and processes for their manufacture|
|US6100393||14 Mar 1997||8 Aug 2000||Antibioticos, S.A.||Process for purifying 7-substituted aminodeacetoxy-cephalosporins through the use of filtration membranes|
|US6113794||25 Jan 1999||5 Sep 2000||Kumar; Ashwani||Composite solvent resistant nanofiltration membranes|
|US6113795||17 Nov 1998||5 Sep 2000||The University Of Kansas||Process and apparatus for size selective separation of micro- and nano-particles|
|US6132607||15 Dec 1999||17 Oct 2000||The Florida State University||System for continuous magnetic separation of components from a mixture|
|US6143576||3 Mar 1997||7 Nov 2000||Biosite Diagnostics, Inc.||Non-porous diagnostic devices for the controlled movement of reagents|
|US6156270||27 Mar 1997||5 Dec 2000||Biosite Diagnostics, Inc.||Diagnostic devices and apparatus for the controlled movement of reagents without membranes|
|US6177373||13 Mar 1997||23 Jan 2001||Exxon Chemicals Patents Inc||Procedure for preparing molecular sieve films|
|US6187446||28 Mar 1996||13 Feb 2001||Pharmacia Biotech Ab||Carrier matrix for integrated microanalysis systems, method for the production thereof and use of the same|
|US6190638||20 Dec 1996||20 Feb 2001||Exxon Chemical Patents Inc.||Molecular sieves and process for their manufacture|
|US6197523||24 Nov 1997||6 Mar 2001||Robert A. Levine||Method for the detection, identification, enumeration and confirmation of circulating cancer and/or hematologic progenitor cells in whole blood|
|US6200765||4 May 1998||13 Mar 2001||Pacific Northwest Cancer Foundation||Non-invasive methods to detect prostate cancer|
|US6210570 *||21 Aug 1998||3 Apr 2001||Agilent Technologies, Inc.||Monolithic silica column|
|US6241894||10 Oct 1997||5 Jun 2001||Systemix||High gradient magnetic device and method for cell separation or purification|
|US6251691||24 Apr 1997||26 Jun 2001||Bioarray Solutions, Llc||Light-controlled electrokinetic assembly of particles near surfaces|
|US6261928||20 Jul 1998||17 Jul 2001||Commissariat A L 'energie Atomique||Producing microstructures or nanostructures on a support|
|US6264044||18 Mar 1998||24 Jul 2001||Cuno, Inc.||Reinforced, three zone microporous membrane|
|US6265229||10 Mar 1995||24 Jul 2001||Oystein Fodstad||Method and device for detection of specific target cells in specialized or mixed cell populations and solutions containing mixed cell populations|
|US6277489||4 Dec 1998||21 Aug 2001||The Regents Of The University Of California||Support for high performance affinity chromatography and other uses|
|US6309798||3 May 1997||30 Oct 2001||Studiengesellschaft Kohle Mbh||Lithographical process for production of nanostructures on surfaces|
|US6315940||13 May 1999||13 Nov 2001||Nmi Naturwissenschaftliches Und Medizinisches Institut An Der Universitat Tubingen In Reutlingen||Microelement device|
|US6329209||14 Jul 1999||11 Dec 2001||Zyomyx, Incorporated||Arrays of protein-capture agents and methods of use thereof|
|US6344326||10 Feb 2000||5 Feb 2002||Aclara Bio Sciences, Inc.||Microfluidic method for nucleic acid purification and processing|
|US6361958||12 Nov 1999||26 Mar 2002||Motorola, Inc.||Biochannel assay for hybridization with biomaterial|
|US6368871||13 Aug 1997||9 Apr 2002||Cepheid||Non-planar microstructures for manipulation of fluid samples|
|US6383759||15 Jan 1999||7 May 2002||Gerald P. Murphy Cancer Foundation||Non-invasive method to detect prostate cancer|
|US6387290||2 Jul 1999||14 May 2002||University Of Washington||Tangential flow planar microfabricated fluid filter|
|US6432630||4 Sep 1997||13 Aug 2002||Scandinanian Micro Biodevices A/S||Micro-flow system for particle separation and analysis|
|US6444461||20 Sep 2000||3 Sep 2002||Caliper Technologies Corp.||Microfluidic devices and methods for separation|
|US6450047 *||29 Mar 2001||17 Sep 2002||Agilent Technologies, Inc.||Device for high throughput sample processing, analysis and collection, and methods of use thereof|
|US6454938||21 Dec 2000||24 Sep 2002||Kionix, Inc.||Integrated monolithic microfabricated electrospray and liquid chromatography system and method|
|US6465225||28 Jun 1999||15 Oct 2002||Evotec Oai Ag||Method and device for manipulating particles in microsystems|
|US6491823 *||28 Nov 2000||10 Dec 2002||Symyx Technologies, Inc.||Targeted separation protocols for rapid characterizations of polymers|
|US6503452||4 Aug 2000||7 Jan 2003||The Board Of Trustees Of The Leland Stanford Junior University||Biosensor arrays and methods|
|US6540895||21 May 1999||1 Apr 2003||California Institute Of Technology||Microfabricated cell sorter for chemical and biological materials|
|US6570196 *||22 Mar 2000||27 May 2003||Max-Plank-Gesellschaft Zur Forderung Der Wissenschaften||Lipid vesicles or lipid bilayers on chips|
|US6576478||14 Jul 1998||10 Jun 2003||Zyomyx, Inc.||Microdevices for high-throughput screening of biomolecules|
|US6582969||12 May 2000||24 Jun 2003||Zyomyx, Inc.||Microdevices for high-throughput screening of biomolecules|
|US6596144 *||30 Oct 2000||22 Jul 2003||Purdue Research Foundation||Separation columns and methods for manufacturing the improved separation columns|
|US6596545 *||14 Jul 1999||22 Jul 2003||Zyomyx, Inc.||Microdevices for screening biomolecules|
|US6613525||17 Jan 2002||2 Sep 2003||Aclara Biosciences, Inc.||Microfluidic apparatus and method for purification and processing|
|US6632619||16 May 1997||14 Oct 2003||The Governors Of The University Of Alberta||Microfluidic system and methods of use|
|US6635163||25 May 2000||21 Oct 2003||Cornell Research Foundation, Inc.||Entropic trapping and sieving of molecules|
|US6641997 *||24 Mar 1999||4 Nov 2003||The Rockefeller University||Assays for screening compounds which interact with cation channel proteins, mutant prokaryotic cation channel proteins, and uses thereof|
|US6664104||7 Nov 2001||16 Dec 2003||Cepheid||Device incorporating a microfluidic chip for separating analyte from a sample|
|US6685841||14 Feb 2002||3 Feb 2004||Gabriel P. Lopez||Nanostructured devices for separation and analysis|
|US6746503||30 Jan 2003||8 Jun 2004||The Regents Of The University Of California||Precision gap particle separator|
|US6762059||14 Feb 2001||13 Jul 2004||U.S. Genomics, Inc.||Methods and apparatuses for characterization of single polymers|
|US6783647||19 Oct 2001||31 Aug 2004||Ut-Battelle, Llc||Microfluidic systems and methods of transport and lysis of cells and analysis of cell lysate|
|US6794148 *||5 Dec 2001||21 Sep 2004||Perseptive Biosystems, Inc.||High speed, automated, continuous flow, multi-dimensional molecular selection and analysis|
|US6815664||14 Nov 2001||9 Nov 2004||Genoptix, Inc.||Method for separation of particles|
|US6818184||17 Apr 2003||16 Nov 2004||The Regents Of The University Of California||Capillary array and related methods|
|US6872522 *||24 Dec 1998||29 Mar 2005||Michael Mecklenburg||Broad specificity affinity arrays: a qualitative approach to complex sample discrimination|
|US6878271||23 Dec 2002||12 Apr 2005||Cytonome, Inc.||Implementation of microfluidic components in a microfluidic system|
|US6881315||31 Jul 2002||19 Apr 2005||Nec Corporation||Fractionating apparatus having colonies of pillars arranged in migration passage at interval and process for fabricating pillars|
|US6893881||10 Sep 1993||17 May 2005||Abbott Laboratories, Inc.||Method for detection of specific target cells in specialized or mixed cell population and solutions containing mixed cell populations|
|US6911345||18 Jul 2001||28 Jun 2005||California Institute Of Technology||Methods and apparatus for analyzing polynucleotide sequences|
|US6913697||9 Jan 2003||5 Jul 2005||Science & Technology Corporation @ Unm||Nanostructured separation and analysis devices for biological membranes|
|US6958245||28 Nov 2001||25 Oct 2005||Bioarray Solutions Ltd.||Array cytometry|
|US6960449||10 Feb 2000||1 Nov 2005||Cell Works Diagnostics, Inc.||Class characterization of circulating cancer cells isolated from body fluids and methods of use|
|US7150812||23 Oct 2003||19 Dec 2006||The Trustees Of Princeton University||Method for continuous particle separation using obstacle arrays asymmetrically aligned to fields|
|US20010036672||31 Dec 2000||1 Nov 2001||Anderson Rolfe C.||Integrated nucleic acid diagnostic device|
|US20020005354||13 Aug 2001||17 Jan 2002||California Institute Of Technology||Microfabricated cell sorter|
|US20020058332||14 Sep 2001||16 May 2002||California Institute Of Technology||Microfabricated crossflow devices and methods|
|US20020090741||8 Jan 2001||11 Jul 2002||Jurgensen Stewart Russell||Method of separating cells from a sample|
|US20020108859||14 Nov 2001||15 Aug 2002||Genoptix||Methods for modifying interaction between dielectric particles and surfaces|
|US20020115163||14 Nov 2001||22 Aug 2002||Genoptix||Methods for sorting particles by size and elasticity|
|US20020115164||14 Nov 2001||22 Aug 2002||Genoptix||Methods and apparatus for generating and utilizing a moving optical gradient|
|US20020123078||28 Nov 2001||5 Sep 2002||Michael Seul||Array cytometry|
|US20020123112||14 Nov 2001||5 Sep 2002||Genoptix||Methods for increasing detection sensitivity in optical dielectric sorting systems|
|US20020132315||14 Nov 2001||19 Sep 2002||Genoptix||Methods and apparatus for measurement of dielectric constants of particles|
|US20020132316||14 Nov 2001||19 Sep 2002||Genoptix||Methods and apparatus for sorting of bioparticles based upon optical spectral signature|
|US20020172987||19 Feb 2002||21 Nov 2002||Terstappen Leon W.M.M.||Methods and reagents for the rapid and efficient isolation of circulating cancer cells|
|US20030072682||18 Jun 2002||17 Apr 2003||Dan Kikinis||Method and apparatus for performing biochemical testing in a microenvironment|
|US20030077292||16 Sep 2002||24 Apr 2003||The Regents Of The University Of Michigan||Detection and treatment of cancers of the lung|
|US20030159999||4 Feb 2003||28 Aug 2003||John Oakey||Laminar Flow-Based Separations of Colloidal and Cellular Particles|
|US20040018116||3 Jan 2003||29 Jan 2004||Desmond Sean M.||Microfluidic size-exclusion devices, systems, and methods|
|US20040072278||1 Apr 2003||15 Apr 2004||Fluidigm Corporation||Microfluidic particle-analysis systems|
|US20040121343||27 Dec 2002||24 Jun 2004||Biosite Incorporated||Markers for differential diagnosis and methods of use thereof|
|US20040144651||23 Oct 2003||29 Jul 2004||Huang Lotien Richard||Method for continuous particle separation using obstacle arrays asymmetrically aligned to fields|
|US20040232074||19 Mar 2004||25 Nov 2004||Ralf-Peter Peters||Microstructured separating device and microfluidic process for separating liquid components from a particle-containing liquid|
|US20040245102||31 Mar 2004||9 Dec 2004||Gilbert John R.||Implementation of microfluidic components, including molecular fractionation devices, in a microfluidic system|
|US20050049793||30 Oct 2003||3 Mar 2005||Patrizia Paterlini-Brechot||Prenatal diagnosis method on isolated foetal cell of maternal blood|
|US20050092662||27 Sep 2004||5 May 2005||Cytonome, Inc.||Implementation of microfluidic components in a microfluidic system|
|US20050123454||8 Dec 2003||9 Jun 2005||David Cox||Microfluidic device and material manipulating method using same|
|US20050142663||24 May 2004||30 Jun 2005||3M Innovative Properties Company||Methods for nucleic acid isolation and kits using a microfluidic device and concentration step|
|US20050145497||8 Feb 2005||7 Jul 2005||Cytonome, Inc.||Implementation of microfluidic components in a microfluidic system|
|US20050164158||18 May 2004||28 Jul 2005||The Regents Of The University Of California, A California Corporation||Microfluidic sorting device|
|US20050170373||10 Sep 2004||4 Aug 2005||Althea Technologies, Inc.||Expression profiling using microarrays|
|US20050170418||18 Feb 2005||4 Aug 2005||John Moreland||Microfluidic platform of arrayed switchable spin-valve elements for high-throughput sorting and manipulation of magnetic particles and biomolecules|
|US20050175996||9 May 2002||11 Aug 2005||Xiangning Chen||Multiple sequencible and ligatible structures for genomic analysis|
|US20050191636||1 Mar 2004||1 Sep 2005||Biocept, Inc.||Detection of STRP, such as fragile X syndrome|
|US20050211556||25 Mar 2004||29 Sep 2005||Childers Winthrop D||Method of sorting cells on a biodevice|
|US20050236314||29 Jun 2005||27 Oct 2005||Neyer David W||Variable flow rate injector|
|US20050239101||28 Oct 2004||27 Oct 2005||The Johns Hopkins University School Of Medicine||Quantitative multiplex methylation-specific PCR|
|US20050244843||30 Oct 2004||3 Nov 2005||Wen-Tien Chen||Blood test prototypes and methods for the detection of circulating tumor and endothelial cells|
|US20050250111||5 May 2004||10 Nov 2005||Biocept, Inc.||Detection of chromosomal disorders|
|US20050250199||13 Dec 2004||10 Nov 2005||Anderson Rolfe C||Integrated nucleic acid diagnostic device|
|US20050252840||13 May 2004||17 Nov 2005||Eksigent Technologies, Llc||Micromixer|
|US20050262577||23 Sep 2003||24 Nov 2005||Christian Guelly||Polypeptides and nucleic acids encoding these and their use for the prevention, diagnosis or treatment of liver disorders and epithelial cancer|
|US20050266433||3 Mar 2005||1 Dec 2005||Ravi Kapur||Magnetic device for isolation of cells and biomolecules in a microfluidic environment|
|US20050282196||2 May 2005||22 Dec 2005||Jose Costa||Methods and compositions for cancer diagnosis|
|US20060051265||8 Sep 2005||9 Mar 2006||Health Research, Inc.||Apparatus and method for sorting microstructures in a fluid medium|
|US20060060767||11 Nov 2005||23 Mar 2006||Wang Mark M||Methods and apparatus for use of optical forces for identification, characterization and/or sorting of particles|
|US20060134599||29 Sep 2003||22 Jun 2006||Mehmet Toner||Microfluidic device for cell separation and uses thereof|
|US20060160243||18 Jan 2005||20 Jul 2006||Biocept, Inc.||Recovery of rare cells using a microchannel apparatus with patterned posts|
|US20060223178||29 Dec 2005||5 Oct 2006||Tom Barber||Devices and methods for magnetic enrichment of cells and other particles|
|US20060252054||31 Oct 2005||9 Nov 2006||Ping Lin||Methods and compositions for detecting non-hematopoietic cells from a blood sample|
|US20060252087||19 Jul 2006||9 Nov 2006||Biocept, Inc.||Recovery of rare cells using a microchannel apparatus with patterned posts|
|US20070026381||8 Jun 2006||1 Feb 2007||Huang Lotien R||Devices and methods for enrichment and alteration of cells and other particles|
|US20070026413||29 Dec 2005||1 Feb 2007||Mehmet Toner||Devices and methods for enrichment and alteration of circulating tumor cells and other particles|
|US20070026414||29 Dec 2005||1 Feb 2007||Martin Fuchs||Devices and methods for enrichment and alteration of circulating tumor cells and other particles|
|US20070026415||29 Dec 2005||1 Feb 2007||Martin Fuchs||Devices and methods for enrichment and alteration of circulating tumor cells and other particles|
|US20070026416||29 Dec 2005||1 Feb 2007||Martin Fuchs||Devices and methods for enrichment and alteration of circulating tumor cells and other particles|
|US20070026417||29 Dec 2005||1 Feb 2007||Martin Fuchs||Devices and methods for enrichment and alteration of circulating tumor cells and other particles|
|US20070026418||29 Dec 2005||1 Feb 2007||Martin Fuchs||Devices and methods for enrichment and alteration of circulating tumor cells and other particles|
|US20070026419||29 Dec 2005||1 Feb 2007||Martin Fuchs||Devices and methods for enrichment and alteration of circulating tumor cells and other particles|
|US20070026469||29 Dec 2005||1 Feb 2007||Martin Fuchs||Devices and methods for enrichment and alteration of circulating tumor cells and other particles|
|US20070059680||15 Sep 2005||15 Mar 2007||Ravi Kapur||System for cell enrichment|
|US20070059683||15 Sep 2005||15 Mar 2007||Tom Barber||Veterinary diagnostic system|
|US20070059716||15 Sep 2005||15 Mar 2007||Ulysses Balis||Methods for detecting fetal abnormality|
|US20070059718||15 Sep 2005||15 Mar 2007||Mehmet Toner||Systems and methods for enrichment of analytes|
|US20070059719||15 Sep 2005||15 Mar 2007||Michael Grisham||Business methods for prenatal Diagnosis|
|US20070059774||15 Sep 2005||15 Mar 2007||Michael Grisham||Kits for Prenatal Testing|
|US20070059781||15 Sep 2005||15 Mar 2007||Ravi Kapur||System for size based separation and analysis|
|US20070099207||8 Jun 2006||3 May 2007||Martin Fuchs||Devices and methods for enrichment and alteration of circulating tumor cells and other particles|
|DE19712309A1||24 Mar 1997||20 May 1998||Nmi Univ Tuebingen||Mikroelementenanordnung, Verfahren zum Kontaktieren von in einer flüssigen Umgebung befindlichen Zellen und Verfahren zum Herstellen einer Mikroelementenanordnung|
|EP0057907B1||3 Feb 1982||30 Dec 1986||Asahi Kasei Kogyo Kabushiki Kaisha||Apparatus for separating blood components|
|EP0094193B1||4 May 1983||19 Aug 1987||Bar-Ilan University||System and methods for cell selection|
|EP0919812B1||23 Nov 1998||6 Oct 2004||Levine, Robert Aaron||Method for the detection, identification, enumeration and confirmation of circulating cancer cells and/or hemotologic progenitor cells in whole blood|
|EP1221342A2||10 Dec 2001||10 Jul 2002||Becton, Dickinson and Company||Method for seperating cells from a sample|
|EP1338894A2||25 Feb 2003||27 Aug 2003||Agilent Technologies, Inc.||Mobile phase gradient generation microfluidic device|
|EP1418003A1||28 Oct 2003||12 May 2004||Hewlett-Packard Development Company, L.P.||Microfluidic pumping system|
|EP1462800A1||9 Dec 2002||29 Sep 2004||Netech Inc.||Blood cell separation system|
|WO1993022053A1||29 Apr 1993||11 Nov 1993||Trustees Of The University Of Pennsylvania||Microfabricated detection structures|
|WO1994029707A1||3 Jun 1994||22 Dec 1994||British Technology Group Usa Inc.||Microlithographic array for macromolecule and cell fractionation|
|WO1998010267A1||4 Sep 1997||12 Mar 1998||Technical University Of Denmark||A micro flow system for particle separation and analysis|
|WO1999044064A1||24 Feb 1999||2 Sep 1999||Cli Oncology, Inc.||Method and compositions for differential detection of primary tumor cells and metastatic cells|
|WO2000062931A1||21 Apr 2000||26 Oct 2000||Clinical Micro Sensors, Inc.||The use of microfluidic systems in the electrochemical detection of target analytes|
|WO2002012896A1||15 Sep 2000||14 Feb 2002||Aviva Biosciences Corporation||Methods for manipulating moieties in microfluidic systems|
|WO2002028523A2||20 Sep 2001||11 Apr 2002||Aviva Biosciences Corporation||Apparatuses containing multiple force generating elements and uses thereof|
|WO2002030562A1||9 Oct 2001||18 Apr 2002||Aviva Biosciences Corporation||An integrated biochip system for sample preparation and analysis|
|WO2003000418A2||20 Jun 2002||3 Jan 2003||Cytonome, Inc.||Microfluidic system including a virtual wall fluid interface port for interfacing fluids with the microfluidic system|
|WO2003031938A2||10 Oct 2002||17 Apr 2003||Aviva Biosciences Corporation||Methods, compositions, and automated systems for separating rare cells from fluid samples|
|WO2003035894A2||28 Oct 2002||1 May 2003||Immunivest Corporation||Multiparameter analysis of comprehensive nucleic acids and morphological features on the same sample|
|WO2003079006A1||20 Mar 2003||25 Sep 2003||Monica Almqvist||Microfluidic cell and method for sample handling|
|WO2003085379A2||1 Apr 2003||16 Oct 2003||Fluidigm Corporation||Microfluidic particle-analysis systems|
|WO2004004906A1||3 Jul 2003||15 Jan 2004||Nanostream, Inc.||Microfluidic closed-end metering systems and methods|
|WO2004015411A1||8 Aug 2003||19 Feb 2004||Nanostream, Inc.||Systems and methods for high-throughput microfluidic sample analysis|
|WO2004024327A1||5 Sep 2003||25 Mar 2004||Intel Corporation||Microfluidic apparatus with integrated porous-substrates/sensor for real-time(bio)chemical molecule detection|
|WO2004029221A2||29 Sep 2003||8 Apr 2004||The General Hospital Corporation||Microfluidic device for cell separation and uses thereof|
|WO2004037374A2||23 Oct 2003||6 May 2004||The Trustees Of Princeton University||Method for continuous particle separation using obstacle arrays asymmetrically aligned to fields|
|WO2004056978A1||15 Dec 2003||8 Jul 2004||Ivonex Gmbh||Method for the separation of cell fractions|
|WO2004113877A1||9 Jun 2004||29 Dec 2004||The General Hospital Corporation||Microfluidic systems for size based removal of red blood cells and platelets from blood|
|WO2005043121A2||30 Oct 2004||12 May 2005||Vitatex, Inc.||Blood test prototypes and methods for the detection of circulating tumor and endothelial cells|
|WO2005047529A1||15 Sep 2004||26 May 2005||Aviva Biosciences Corporation||Methods, compositions, and automated systems for separating rare cells from fluid samples|
|WO2005049168A2||17 Nov 2004||2 Jun 2005||Immunivest Corporation||Method and apparatus for pre-enrichment and recovery of cells from densified whole blood|
|WO2005058937A2||10 Dec 2004||30 Jun 2005||Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services||A human cytotoxic t-lymphocyte epitope and its agonist epitope from the non-variable number of tandem repeat sequence of muc-1|
|WO2005068503A2||6 Jan 2005||28 Jul 2005||Chiron Corporation||M-csf-specific monoclonal antibody and uses thereof|
|WO2005084374A2||3 Mar 2005||15 Sep 2005||The General Hospital Corporation||Magnetic device for isolation of cells and biomolecules in a microfluidic environment|
|WO2005085861A2||1 Mar 2005||15 Sep 2005||Oridis Biomed Forschungs- Und Entwicklungs Gmbh||Nucleic acids and encoded polypeptides for use in liver disorders and epithelial cancer|
|WO2005108621A1||2 May 2005||17 Nov 2005||Yale University||Methods and compositions for cancer diagnosis|
|WO2005109238A2||3 May 2005||17 Nov 2005||Cygene Laboratories, Inc.||Method and system for a comprehensive knowledge-based anonymous testing and reporting, and providing selective access to test results and report|
|WO2005121362A2||14 Jun 2005||22 Dec 2005||Inserm (Institut National De La Sante Et De La Recherche Medicale||Method for selectively quantifying vegf isoforms in a biological sample and uses thereof.|
|WO2006078470A2||5 Jan 2006||27 Jul 2006||Biocept, Inc.||Cell separation using microchannel having patterned posts|
|1||"Micromechanics Imitate Blood Vessels" Design News 15 (Mar. 22, 1993).|
|2||Archer, et al. Cell Reactions to Dielectrophoretic Manipulation. Biochemical and Biophysical Research Communications. 1999;257:687-98.|
|3||Ashcroft, et al. Solid State Physics. Saunders College Publishing. Orlando, Fl. 1976. (Table of Contents only.).|
|4||Babochkina, T. I. Ph. D. Dissertation-Fetal cells in maternal circulation: Fetal cell separation and FISH analysis. University of Basel, Switzerland. Dec. 8, 2005. (123 pages).|
|5||Babochkina, T. I. Ph. D. Dissertation—Fetal cells in maternal circulation: Fetal cell separation and FISH analysis. University of Basel, Switzerland. Dec. 8, 2005. (123 pages).|
|6||Bauer, J. Advances in cell separation: recent developments in counterflow centrifugal elutriation and continuous flow cell separation. Journal of Chromatography. 1999;722:55-69.|
|7||Becker, et al. Fabrication of Microstructures With High Aspect Rations and Great Structural Heights by Synchrotron Radiation Lithography, Galvanoforming, and Plastic Moulding (LIGA Process). Microelectronic Engineering. 1986;4:35-56.|
|8||Becker, et al. Planar quartz chips with submicron channels for two-dimensional capillary electrophoresis applications. J. Micromech Microeng.1998;9:24-28.|
|9||Beebe et al. Functional Hydrogel Structures for Autonomous Flow Control Inside Microfluidic Channels. Nature. 2000; 404:588-590.|
|10||Benincasa, et al. Cell Sorting by One Gravity SPLITT Fractionation. Analytical Chemistry. 2005; 77(16):5294-5301.|
|11||Berg, H. C. Random Walks in Biology. Ch. 4. Princeton University Press. Princeton, NJ. 1993.pp. 48-64.|
|12||Cao, et al. Fabrication of 10 nm enclosed nanofluidic channels. Applied Physics Letters. 2002; 81(1): 174-6.|
|13||Cao, et al. Gradient nanostructures for interfacing microfluidics and nanofluidics. Applied Physics Letters. 2002; 81(16): 3058-60.|
|14||Carlson, et al. Self-Sorting of White Blood Cells in a Lattice. Phys. Rev. Lett. 79:2149-2152 (1997).|
|15||Chiu, et al. Patterned Deposition of Cells and Proteins Onto Surfaces by Using Three-Dimensional Microfluidic Systems. Proceedings of the National Academy of Sciences of the United States of America. 2000; pp. 2408-2413.|
|16||Chou, et al. A Microfabricated Device for Sizing and Sorting DNA Molecules. Proceedings of the National Academy of Sciences of the United States of America. 1999; pp. 11-13.|
|17||Chou, et al. Imprint of sub-25 nm vias and trenches in polymers. Applied Physics Letters. 1995; 67(21): 3114-6.|
|18||Chou, et al. Sorting by diffusion: An asymmetric obstacle course for continuous molecular separation. PNAS. 1999; 96(24):13762-13765.|
|19||Colburn, et al. Patterning nonflat substrates with a low pressure, room temperature, imprint lithography process. Journal of Vacuum Science & Technology B (Microelectronics and Nanometer Structures). 2001; 19(6): 2162-72.|
|20||Craighead, et al. Nanoelectromechanical systems. Science. 2000; 290(5496): 1532-5.|
|21||Das, et al. Dielectrophoretic segregation of different human cell types on microscope slides. Anal. Chem. 2005; 77:2708-2719.|
|22||De Kretser, et al. The Separation of Cell Populations using Monoclonal Antibodies attached to Sepharose. Tissue Antigens. 1980;16:317-325.|
|23||Delamarche, et al. Microfluidic Networks for Chemical Patterning of Substrates: Design and Application to Bioassays. Journal of the American Chemical Society. 1998; 120:500-508.|
|24||Delamarche, et al. Patterned Delivery of Immunoglobulins to Surfaces Using Microfluidic Networks. Science. 1997; 276:779-781.|
|25||Deshmukh, et al. Continuous Micromixer With Pulsatile Micropumps. Solid-State Sensor and Actuator Workshop. Hilton Head Island, South Carolina; Jun. 4-8, 2000;73-76.|
|26||Doyle, et al. Self-Assembled Magnetic Matrices for DNA Separation Chips. Science 295:2237 (2002).|
|27||Eigen, et al. Sorting Single Molecules: Application to Diagnostics and Evolutionary Biotechnology. Proceedings of the National Academy of Sciences of the United States of America. 1994; 91:5740-5747.|
|28||Evans, et al. The Bubble Spring and Channel (BSAC) Valve: An Actuated, Bi-Stable Mechanical Valve For In-Plane Fluid Control. Transducers '99. Sendai, Japan; Jun. 7-10, 1999.|
|29||Fan, et al. Molecular dynamics simulation of a liquid in a complex nano channel flow. Physics of Fluids. 2002; 14(3): 1146-53.|
|30||Farooqui, et al. Microfabrication of Submicron Nozzles in Silicon Nitride. Journal of Microelectromechanical Systems. 1992; 1(2):86-88.|
|31||Fiedler, et al. Dielectrophoretic Sorting of Particles and Cells in a Microsystem. Analytical Chemistry. 1998; pp. 1909-1915.|
|32||Freemantle, M. Downsizing Chemistry. Chemical analysis and synthesis on microchips promise a variety of potential benefits. Chemical & Engineering News. 1999; pp. 27-36.|
|33||Fu, et al. A Microfabricated Fluorescence-Activated Cell Sorter. Nature Biotechnology.1999; 17:1109-1111.|
|34||Fu, et al. An integrated miscrofabricated cell sorter. Anal Chem. 2002;74:2451-2457.|
|35||Fuhr, et al. Biological Application of Microstructures. Topics in Current Chemistry. 1997; 194:83-116.|
|36||Giddings, J. C. Chemistry ‘Eddy’ Diffusion in Chromatography. Nature. 1959;184:357-358.|
|37||Giddings, J. C. Chemistry 'Eddy' Diffusion in Chromatography. Nature. 1959;184:357-358.|
|38||Giddings, J. C. Field-Flow Fractionation: Analysis of Macromolecular, Colloidal, and Particulate Materials. Science. 1993;260:1456-1465.|
|39||Giddings, J.C. Unified Separation Science. John Wiley & Sons, Inc. 1991; Cover Page & Table of Contents only.|
|40||Han, et al. Separation of Long DNA Molecules in a Microfabricated Entropic Trap Array. Science. 2000;288:1026-1029.|
|41||Harnett, et al. Heat-depolymerizable polycarbonates as electron beam patternable sacrifical layers for nanofluidics. Journal of Vacuum Science & Technology B (Microelectronics and Nanometer Structures). 2001; 19(6): 2842-5.|
|42||Hibara, et al. Nanochannels on a fused-silica microchip and liquid properties invesigation by time-resolved fluorescence measurements. Analytical Chemistry. 2002; 74(24): 6170-6176.|
|43||Huang, et al. A DNA prism for high-speed continuous fractionation of large DNA molecules. Nature Biotechnology. 2002;20:1048-1051.|
|44||Huang, et al. Continuous Particle Separation Through Deterministic Lateral Displacement. Science 304:987-90 (2004).|
|45||Huang, et al. Electric Manipulation of Bioparticles and Macromoledules on Microfabricated Electrodes. Analytical Chemistry. 2001; pp. 1549-1559.|
|46||Huang, et al. Role of Molecular Size in Ratchet Fractionation. 2002; 89(17):178301-1-178301-4.|
|47||Huh, et al. Gravity-driven microhydrodynamics-based cell sorter (microHYCS) for rapid, inexpensive, and efficient cell separation and size-profiling. 2nd Annual International IEEE-EMBS Special Topic Conference on Microtechnology in Medicine and Biology. Madison, Wisconsin USA; May 2-4, 2002:466-469.|
|48||Ivker, M. Direct Observation of Reptation in Artificial Gel Environments. Bachelor of Arts thesis, Princeton University. Spring 1991.|
|49||Jeon, et al. Generation of Solution and surface Gradients Using Microfluidic Systems. Langmuir. 2000, pp. 8311-8316.|
|50||Kamholz, et al. Quantitative Analysis of Molecular Interaction in a Microfluidic Channel: the T-Sensor. Analytical Chemistry. 1999; pp. 5340-5347.|
|51||Kapur, et al., U.S. Appl. No. 11/227,904, entitled "Devices And Methods For Enrichment And Alteration Of Cells And Other Particles," filed Sep. 15, 2005.|
|52||Kenis, et al. Microfabrication Inside Capillaries Using Multiphase Laminar Flow Patterning. Science. 1999; 285:83-85.|
|53||Kim, et al. Polymer microstructures formed by moulding in capillaries. Nature. 1995;376:581-584.|
|54||Kumar, et al. Cell Separation: A Review. Pathology. 1984;16:53-62.|
|55||Li, et al. Transport, Manipulation, and Reaction of Biological Cells On-Chip Using Electrokinetic Effects. Analytical Chemistry., 1997; pp. 1564-1568.|
|56||Mehrishi, et al. Electrophoresis of cells and the biological relevance of surface charge. Electrophoresis.2002;23:1984-1994.|
|57||Mohamed, et al. Development of a rare cell fractionation device; application for cancer detection. IEEE Trans Nanobioscience. 2004; 3(4): 251-6.|
|58||Moore, et al. Lymphocyte fractionation using immunomagnetic colloid and a dipole magnet flow cell sorter. J Biochem Biophys Methods. 1998;37:11-33.|
|59||Oakey et al. Laminar Flow-Based Separations at the Microscale. Biotechnology Progress. 2002; pp. 1439-1442.|
|60||Olson, et al. An In Situ Flow Cytometer for the Optical Analysis of Individual Particles in Seawater. Available at http://www.whoi.edu/science/B/Olsonlab/insitu2001.htm. Accessed Apr. 24, 2006.|
|61||Petersen, et al. The Promise of Miniaturized Clinical Diagnostic Systems. IVD Technol. 4:43-49 (1998).|
|62||Product literature for GEM, a system for blood testing: GEM Premier 3000. Available at http://www.ilus.com/premier_gem300_iqm.asp. Accessed Apr. 24, 2006.|
|63||Raymond, et al. Continuous Separation of High Molecular Weight Compounds Using a Microliter Volume Free-Flow Electrophoresis Microstructure. 1996;68:2515-2522.|
|64||Rice, et al. Electrokinetic flow in a cylindrical capillary. Journal of Physical Chemistry. 1965; 69(11): 4017-4024.|
|65||Sethu, et al. Continuous Flow Microfluidic Device for Rapid Erythrocyte Lysis. Anal. Chem. 76:6247-6253 (2004).|
|66||Studer, et al. Nanoembossing of thermoplastic polymers for microfluidic applications. Applied Physics Letters. 2002; 80(19): 3614-16.|
|67||Takayama, et al. Patterning Cells and Their Environments Using Multiple Laminar Fluid Flows in Capillary Networks. Proceedings of the National Academy of Sciences of the United States of America. 1999:5545-5548.|
|68||Takayama, et al. Subcellular Position of Small Molecues. Nature. 2001; 411:1016.|
|69||Toner, et al. Blood-on-a-Chip. Annu. Rev. Biomed. Eng. 7:77-103, C1-C3 (2005).|
|70||Tong, et al. Low Temperature Wafer Direct Bonding. Journal of Microelectromechanical Systems. 1994;3:29-35.|
|71||Turner, et al. Confinement-Induced Entropic Recoil of Single DNA Molecules in a Nanofluidic Structure. Physical Review Letters.2002;88:128103.1-128103.4.|
|72||Voldman, et al. Holding Forces of Single-Particle Dielectrophoretic Traps. Biophysical Journal.2001;80:531-541.|
|73||Volkmuth, et al. DNA electrophoresis in microlithographic arrays. Nature. 1992; 358:600-602.|
|74||Volkmuth, et al. Observation of Electrophoresis of Single DNA Molecules in Nanofabricated Arrays. Presentation at joint annual meeting of Biophysical Society and the American Society for Biochemistry and Molecular Biology. Feb. 9-13, 1992.|
|75||Weigl, et al. Microfluidic Diffusion-Based Separation and Detection. Science. 1999; pp. 346-347.|
|76||Xu, et al. Dielectrophoresis of human red cells in microchips. Electrophoresis.1999;20:1829-1831.|
|77||Zaidi, et al. Optical properties of nanoscale, one-dimensional silicon grating structures. Journal of Applied Physics. 1996; 80(12): 6997-7008.|
|78||Zankovych, et al. Nanoimprint lithography: challenges and prospects. Nanotechnology. 2001; 12(2): 91-5.|
|79||Zhang, et al. High-speed free-flow electrophoresis on chip. Anal Chem. 2003;75:5759-5766.|
|80||Zuska, P. Microtechnology Opens Doors to the Universe of Small Space, MD&DI Jan. 1997, p. 131.|
|Citing Patent||Filing date||Publication date||Applicant||Title|
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|US8961764||14 Oct 2011||24 Feb 2015||Lockheed Martin Corporation||Micro fluidic optic design|
|US9067207||4 Mar 2011||30 Jun 2015||University Of Virginia Patent Foundation||Optical approach for microfluidic DNA electrophoresis detection|
|US9322054||21 Feb 2013||26 Apr 2016||Lockheed Martin Corporation||Microfluidic cartridge|
|US9541480||28 Jun 2012||10 Jan 2017||Academia Sinica||Capture, purification, and release of biological substances using a surface coating|
|US20070264675 *||8 May 2007||15 Nov 2007||The General Hospital Corporation||Microfluidic device for cell separation and uses thereof|
|U.S. Classification||210/638, 210/656, 209/208, 435/4, 436/161, 209/12.1, 210/806, 209/155, 436/178|
|International Classification||B01D21/00, B01D61/14, B01D11/00|
|Cooperative Classification||Y10T436/255, Y10T436/25375, Y10S977/717, Y10S977/845, B82Y30/00, G01N30/00, G01N2030/8813, G01N30/88, G01N27/44773|