EP1971691A2 - Bead based assays using a liquid crystal reporter - Google Patents
Bead based assays using a liquid crystal reporterInfo
- Publication number
- EP1971691A2 EP1971691A2 EP06733654A EP06733654A EP1971691A2 EP 1971691 A2 EP1971691 A2 EP 1971691A2 EP 06733654 A EP06733654 A EP 06733654A EP 06733654 A EP06733654 A EP 06733654A EP 1971691 A2 EP1971691 A2 EP 1971691A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- substrate
- beads
- analyte
- tube
- magnet
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y25/00—Nanomagnetism, e.g. magnetoimpedance, anisotropic magnetoresistance, giant magnetoresistance or tunneling magnetoresistance
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01F—MAGNETS; INDUCTANCES; TRANSFORMERS; SELECTION OF MATERIALS FOR THEIR MAGNETIC PROPERTIES
- H01F1/00—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties
- H01F1/0036—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties showing low dimensional magnetism, i.e. spin rearrangements due to a restriction of dimensions, e.g. showing giant magnetoresistivity
- H01F1/0045—Zero dimensional, e.g. nanoparticles, soft nanoparticles for medical/biological use
- H01F1/0054—Coated nanoparticles, e.g. nanoparticles coated with organic surfactant
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2446/00—Magnetic particle immunoreagent carriers
-
- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01F—MAGNETS; INDUCTANCES; TRANSFORMERS; SELECTION OF MATERIALS FOR THEIR MAGNETIC PROPERTIES
- H01F1/00—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties
- H01F1/01—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties of inorganic materials
- H01F1/03—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties of inorganic materials characterised by their coercivity
- H01F1/12—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties of inorganic materials characterised by their coercivity of soft-magnetic materials
- H01F1/34—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties of inorganic materials characterised by their coercivity of soft-magnetic materials non-metallic substances, e.g. ferrites
- H01F1/36—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties of inorganic materials characterised by their coercivity of soft-magnetic materials non-metallic substances, e.g. ferrites in the form of particles
Definitions
- the present invention relates to the field of detection of analytes, and in particular to detection of viruses, cells, bacteria, lipid-membrane containing organisms, proteins, nucleic acids, carbohydrates and other biomolecules, organic molecules and inorganic molecules using a liquid crystal- assay format.
- the detection of pathogen, protein, and nucleic acid targets in biological samples forms the basis of the multi-billion dollar in vitro diagnostic industry. Detection of protein and nucleic acid targets can be divided into diagnostic and research based markets.
- the diagnostic market includes the detection and identification of pathogens such as viruses and bacteria, the identification of various genetic markers, and the identification of markers associated with the presence of tumors.
- the research market includes the genomics and proteomics industries, which require analytical, drug discovery, and high-throughput screening technologies.
- the present invention relates to the field of detection of analytes, and in particular to detection of viruses, cells, bacteria, lipid-membrane containing organisms, proteins, nucleic acids, carbohydrates and other biomolecules, organic molecules and inorganic molecules using a liquid crystal assay format. Accordingly, in some embodiments, the present invention provides a system comprising: magnetic beads functionalized with a recognition moiety; and a substrate that , ,, , , , ,
- the system further comprises a magnet (e.g., a permanent magnet or an electric magnet).
- the system further comprises a tube and a pump, wherein the pump is fluidically connected to the tube and the tube is positioned to be exposed to the magnetic field of the magnet.
- the tube has an inner surface with a diameter so that the surface tension energy of a fluid within the tube is larger than the surface energy of the tube so that a pressure gradient between the two ends of the tube induces movement of the fluid plugs inside the tube without wetting the inner surface.
- the tube is movable with respect to the magnet. In other embodiments, the tube is rotatable.
- the magnetic beads are located in the tube so as to be attractable by the magnet.
- the present invention is not limited to a particular substrate.
- a variety of substrates are contemplated for use in the present invention including, but not limited to, a polyimide coated substrate, an anisotropic gold substrate, or a rubbed substrate.
- the substrate comprises microfluidic channels that orient liquid
- the substrate comprises PDMS.
- the tube is made from glass or plastic.
- the system further comprises a stamp.
- the system further comprises mesogens (e.g., 4-cyano-4'-pentylbiphenyl, N-(4methoxybenzylidene)-4-butlyaniline or combinations thereof).
- the recognition moiety is a protein, peptide, polypeptide,
- the Analyte is a protein, peptide, polypeptide, nucleic acid, carbohydrate or an organic compound.
- the present invention further provides a method comprising providing magnetic beads functionalized with a recognition moiety that binds an analyte; a substrate that orients liquid crystals; a sample suspected of containing an analyte; a magnet; and
- the present invention also provides a kit comprising magnetic beads functionalized with a recognition moiety; and a substrate that orients liquids crystals.
- the present invention additionally provides a method comprising providing: a tube comprising magnetic beads functionalized with a recognition moiety that binds an analyte; a magnet (e.g., a permanent magnet or an electric magnet), and a pump (e.g., a manual pump or an automated pump) fluidically connected to the tube and at least one reservoir comprising an elution solution and at least one reservoir containing a solution suspected of containing an analyte; via the pump, drawing an aliquot of the solution suspected of containing an analyte into the tube; via the pump, drawing an aliquot of the treatment solution into the tube so that the aliquot of the solution suspected of containing an analyte is separated from the aliquot of the elution solution by a volume of air; via the pump, moving the aliquot of the solution suspected of containing an analyt
- the pump is fluidically connected to at least one other reservoir containing a wash solution.
- at least one aliquot of the wash solution is loaded into the tube prior to the aliquot of solution suspected of containing an analyte.
- at least one aliquot of the wash solution is loaded into the tube between the aliquot of solution suspected of containing an analyte and the aliquot of the elution solution.
- at least one aliquot of the wash solution is loaded into the tube after the aliquot of the elution solution.
- a plurality of reservoirs containing different wash solutions are fluidically connected to the pump so that aliquots of the different wash solutions may be loaded into the tube before, in between and/or after the aliquots of the solution suspected of containing an analyte and aliquot of elution solution.
- the method further comprises recovering the elution solution.
- the method further comprises detecting the analyte. The present invention is not limited to a particular detection method.
- Exemplary detection methods include, but are not limited to, Northern blotting, Southern blotting, Western blotting, ELISA, fluorescent detection and liquid crystal detection (e.g., by applying the elution solution to a substrate that orients liquid crystals and applying a liquid crystal to the X U S fa / i substrate, wherein the presence of the analyte is indicated by a difference in the ordering of the liquid crystal).
- the liquid crystal assumes a random planar orientation over areas of the substrate having bound analyte.
- the substrate is a polyimide coated substrate, an anisotropic gold substrate, or a rubbed substrate.
- the substrate comprises microfluidic channels that orient liquid crystals.
- the substrate comprises PDMS.
- the analyte is a protein, nucleic acid, virus, bacterium, or carbohydrate.
- the recognition moiety is a protein, polypeptide, peptide, antibody, nucleic acid, or carbohydrate.
- the tube following contacting the beads with an aliquot of solution, the tube is moved to mix the beads with the solution, hi some embodiments, the tube is rotated.
- the tube is glass or plastic.
- the liquid crystal comprises 5CB.
- the present invention provides a kit comprising: magnetic beads; a tube (e.g., a glass tube or a plastic tube); a magnet (e.g., a permanent magnet or a electric magnet); and mesogens.
- the magnetic beads are functionalizable.
- the magnetic beads are functionalized with a ligand.
- the kit further comprises at least one container of wash solution.
- the kit further comprises at least one aliquot of elution solution.
- the kit further comprises instructions for using the kit to detect an analyte.
- the kit further comprises a substrate that orients liquid crystals (e.g., a polyimide coated substrate, an anisotropic gold substrate, or a rubbed substrate).
- the substrate comprises microfluidic channels that orient liquid crystals.
- the substrate comprises PDMS.
- the present invention provides a system for parallel processing of samples suspected of containing an analyte comprising: a plate comprising a plurality of wells (e.g., 16, 24, 96, or 384 wells); a plurality of magnetic beads comprising at least one recognition moiety that binds an analyte, the plurality of magnetic beads positioned in sat least one of the wells; a magnet positionable to attract the magnetic beads; and a substrate that orients a liquid crystal.
- different subsets of wells comprise magnetic beads functionalized with different recognition moieties.
- the subset of wells comprises one or more .
- the wells comprises control magnetic beads.
- the magnet is insertable between the wells so that the magnetic beads are drawn to the sides of the wells.
- the system further comprises a magnet (e.g., a permanent magnet or an electric magnet).
- the substrate that orients liquid crystals is a substrate that anisotropically orients liquid crystals or a substrate that homeotropically orients liquid crystals.
- the substrate is nanostructured gold, polyimide, or rubbed substrates.
- the substrate comprises microchannels.
- the magnetic beads are about 0.8 microns in diameter. In some embodiments, the
- analyte is a protein, polypeptide, peptide, or a nucleic acid.
- the analyte is a cytokine (e.g., interleukin-1 alpha, interleukin-1 beta, interleukin-2, interleukin-4, interleuldn-6, interleukin-7, interleukin-8, interleukin-10, interleukin-12, GM-CSF, interferon-gamma, or TNF-alpha).
- the recognition moiety is a peptide, polypeptide, protein (e.g., antigen binding protein), or nucleic acid.
- the antigen binding protein binds a cytokine.
- the system further comprises a stamp substrate comprising at least one projection insertable into at least one well.
- the at least one projection comprises a distal end having a stamp surface.
- the stamp surface comprises PDMS.
- the stamp surface comprises a 0 recognition moiety.
- the recognition moiety on the magnetic bead binds a class of immunoglobulins and the recognition moiety on the stamp surface is specific for an analyte.
- the magnetic beads comprise a plurality of recognition moieties specific for more than one class of imunoglobulins and the recognition moiety on the stamp surface binds an analyte.
- the 5 immunoglobulin is IgA, IgG, IgM, IgE, or combinations thereof.
- the present invention provides a method of detecting an analyte comprising: providing a solution suspected of containing an analyte, a wash solution, and an elution solution; a multiwell plate comprising a plurality of wells (e.g., 16, 24, 96, or 384 well plates); a first plurality of magnetic beads functionalized with a
- the first plurality of magnetic beads positioned in at least one of the wells; a substrate that orients a liquid crystal; and a magnet (e.g., a permanent magnet or an electric magnet); placing the solution into the at least one well containing the first plurality of magnetic beads so that the magnetic beads bind the analyte; attracting the magnetic beads with the magnet; washing the magnetic beads to provide washed magnetic beads; attracting the washed magnetic beads with the magnet; contacting the washed magnetic beads with aid elution solution so that the analyte is eluted from the beads into the elution solution; contacting the substrate with the elution solution; and applying a liquid crystal to the substrate, wherein the presence of the analyte is indicated by a difference in orientation in a region of the liquid crystal.
- a magnet e.g., a permanent magnet or an electric magnet
- different subsets of wells comprise magnetic beads functionalized with different recognition moieties.
- the subset of wells comprises one or more wells.
- at least one of the wells comprises control magnetic beads.
- the magnet is insertable between the wells so that the magnetic beads are drawn to the sides of the wells.
- the substrate homerotropically orients liquid crystals.
- the substrate is nanostructured gold, polyimide, or a rubbed substrate.
- the substrate comprises microchannels.
- the magnetic beads are about 0.8 microns in diameter.
- the analyte is a protein, polypeptide, peptide, or nucleic acid.
- the analyte is a cytokine (e.g., interleukin-1 alpha, interleukin-1 beta, interleukin-2, interleukin-4, interleukin-6, interleukin-7, interleukin-8, interleukin-10, interleukin-12, GM-CSF, interferon-gamma, or TNF-alpha).
- the recognition moiety is a peptide (e.g., antigen binding protein) polypeptide, protein, or nucleic acid.
- the antigen binding protein binds a cytokine.
- the method further comprises providing a stamp substrate comprising at least one projection insertable into at least one well and transferring the analyte to the the substrate via the stamp.
- the at least one projection comprises a distal end having a stamp surface.
- the stamp surface comprises PDMS. In other embodiments, the stamp surface comprises a recognition moiety.
- the present invention provides a kit comprising: a multiwell plate comprising a plurality of wells; a first plurality of magnetic beads functionalized with a recognition moiety; a magnet (e.g., a permanent magnet or an electric magnet); a container containing wash, solution; a container containing elution solution; and a substrate that orients liquid crystals.
- the magnet is positionable between the wells of the multiwell plate so that the magnetic beads are attracted to the sides of the wells.
- the kit further comprises at least a second plurality of magnetic beads, the second plurality of magnetic beads comprising a different recognition moiety than the first plurality of magnetic beads.
- the kit further comprises a container of mesogens. In certain embodiments, the kit further comprises instructions for the detection of an analyte.
- the substrate homeotropically orients liquid crystals.
- the substrate is nanostructured gold, polyimide, or a rubbed substrate. In other embodiments, the substrate comprises microchannels. In some embodiments, the magnetic beads are approximately 0.8 microns in diameter.
- the recognition moiety is a peptide, polypeptide, protein, or nucleic acid.
- the protein is an antigen binding protein.
- the antigen binding protein binds a cytokine (e.g., interleukin-1 alpha, interleukin-1 beta, interleukin-2, interleukin-4, interleukin-6, interleukin-7, interleukin-8, interleukin-10, interleukin-12, GM-CSF, interferon-gamma, or TNF-alpha).
- the kit further comprises a stamp substrate comprising at least one projection insertable into at least one well.
- the at least one projection comprises a distal end having a stamp surface.
- thee stamp surface comprises PDMS.
- the stamp surface comprises a recognition moiety.
- the present invention provides a method of identifying immunoglobulins of a specific subclass comprising: providing magnetic beads functionalized with antibodies specific for one or more subclasses of immunoglobulins; a solution suspected of containing an immunoglobulin specific for an antigen; a stamp functionalized with the antigen; a substrate that orients liquid crystals; contacting the magnetic beads with the solution so that the magnetic beads bind at least a first subclass of immunoglobulins but do not bind a second subclass of immunoglobulins; contacting the solution with the stamp so that the second subclass of immunoglobulins binds to the stamp; transferring the second class of immunoglobulins to the substrate; and forming a liquid crystal on the substrate, wherein the presence of the second subclass of immunoglobulins is indicated by a region of difference in the orientation of the liquid crystal.
- the at least first subclass of immunoglobulins comprises IgG and the second subclass of immunoglobulins comprises Ig
- the present invention provides a kit for detecting immunoglobulins of a specific subclass comprising: magnetic beads functionalized with antibodies specific for one or more subclasses of immunoglobulins; a stamp functionalized with an antigen; and a substrate that orients liquid crystals.
- the magnetic beads are functionalized with antibodies that bind IgG.
- the magnetic beads are functionalized with antibodies that bind IgG and IgM.
- the substrate homeotropically orients liquid crystals.
- the kit further comprises a multiwell plate.
- the kit further comprises a container of mesogens.
- Figure 1 is an image of an assay using a polyimide coated substrate to non-specifically detect an analyte.
- Figure 2 is a graphic representation of luminosity index for the experiment depicted in Figure 1.
- Figure 3 is an image of an assay using a polyimide coated substrate to non- specifically detect an analyte.
- Figure 4 is an image of the results of the detection of Fl in chicken serum.
- Figure 5 is an image of the results of the same experiment as Figure 4 taken with a polarized microscope.
- Figure 6 is a schematic depiction of a readout device of the present invention.
- Figure 7 presents a schematic of a magneto-fluidic assay.
- Figure 8 is optical photographs of a liquid crystal cell between crossed polarizing films. Spots represented by dashed circles are treated with (A) elution buffer alone, (B) eluant from 50 ⁇ l of 20 ng/mL Fl from human serum, (C) eluant from 50 ⁇ L of 100 ng/mL Fl from human serum, and (D) eluant from a 50 ⁇ L of human serum without Fl . ; ⁇ " u Ii ./ ::...u LiI IB / iu m i
- the volume of the fluids spotted on each case is 10 ⁇ L.
- the top and the bottom rows represent the replica of the same experiment.
- Figure 9 is a depiction of a magnetic base place for use with a 96 well plate.
- Figure 10 is a picture of a modified 96 well plate on a magnetic base.
- Figure 11 is a schematic depiction of attraction of beads to the side of a well.
- Figure 12 is a photograph of a liquid crystal cell. The presence of the analyte (Fl antigen) is indicated by bright field (i.e., random orientation of the liquid crystal).
- Figure 13 shows a schematic diagram illustrating the homeotropic alignment of liquid crystals on the polyimide coated surface.
- the term “recognition moiety” refers to a composition of matter that interacts with an analyte of interest in either a covalent or noncovalent manner.
- the term “virus recognition moiety” refers to any composition of matter that binds specifically to a virus. Examples of “virus recognition moieties” include, but are not limited to antigen binding proteins and nucleic acid aptamers.
- substrate refers to a composition that serves as a base for another composition such as recognition moiety.
- substrates include, but are not 20 limited to, silicon surfaces, glass surfaces, glass beads, magnetic beads, agarose beads, etc.
- analyte refers to a substance or chemical constituent that is undergoing analysis.
- ligand refers to any molecule that binds to or can be 25 bound by another molecule.
- a ligand is any ion, molecule, molecular group, or other substance that binds to another entity to form a larger complex.
- Examples of ligands include, but are not limited to, peptides, carbohydrates, nucleic acids, antibodies, or any molecules that bind to receptors.
- the term "homeotropic director” refers to a topographical feature 30 (e.g., a nanostructure or homeotropic orienting polyimide) of a substrate that homeotropically orients a liquid crystal. ./ . ⁇ i i l
- pathogen refers to disease causing organisms, microorganisms, or agents including, but not limited to, viruses, bacteria, parasites (including, but not limited to, organisms within the phyla Protozoa, Platyhelminthes, Aschelminithes, Acanthocephala, and Arthropoda), fungi, and prions.
- bacteria and “bacterium” refer to all prokaryotic organisms, including those within all of the phyla in the Kingdom Procaryotae.
- Gram negative and Gram positive refer to staining patterns obtained with the Gram-staining process which is well known in the art (See e.g., Finegold and Martin, Diagnostic Microbiology, 6th Ed. (1982), CV Mosby St. Louis, pp 13-15).
- lipid membrane refers to, in its broadest sense, a thin sheet or layer comprising lipid molecules. It is intended that the term encompass all “biomembranes” (i.e., any organic membrane including, but not limited to, plasma membranes, nuclear membranes, organelle membranes, and synthetic membranes). Typically, membranes are composed of lipids, proteins, glycolipids, steroids, sterol and/or other components. As used herein, the term “membrane fragment” refers to any portion or piece of a membrane.
- lipid refers to a variety of compounds that are characterized by their solubility in organic solvents. Such compounds include, but are not limited to, fats, waxes, steroids, sterols, glycolipids, glycosphingolipids (including gangliosides), phospholipids, terpenes, fat-soluble vitamins, prostaglandins, carotenes, and chlorophylls.
- lipid-based materials refers to any material that contains lipids.
- liposome refers to artificially produced spherical lipid complexes that can be induced to segregate out of aqueous media.
- secondary binding agent refers to a molecule or collection of molecules that binds to one of an analyte-recognition moiety complex. It is contemplated that secondary binding agents are useful for amplifying the signal resulting from analyte-recognition moiety binding.
- the term “column media” refers to media used to fill a chromatography column, such as cationic exchange media, anionic exchange media, and immunoaffinity column media.
- the term “detection region” refers to a discreet area on substrate that is designated for detection of an analyte (e.g., a virus of interest) in a sample.
- immobilization refers to the attachment or entrapment, either chemically or otherwise, of a material to another entity (e.g., a solid support) in a manner that restricts the movement of the material.
- material and “materials” refer to, in their broadest sense, any composition of matter.
- the term "antigen binding protein” refers to a glycoprotein evoked in an animal by an immunogen (antigen) and to proteins derived from such glycoprotein (e.g., single chain antibodies and F(ab')2, Fab' and Fab fragments).
- An antibody demonstrates specificity to the immunogen, or, more specifically, to one or more epitopes contained in the immunogen.
- Native antibody comprises at least two light polypeptide chains and at least two heavy polypeptide chains. Each of the heavy and light polypeptide chains contains at the amino terminal portion of the polypeptide chain a variable region (i.e. , VH and VL respectively), which contains a binding domain that interacts with antigen.
- Each of the heavy and light polypeptide chains also comprises a constant region of the polypeptide chains (generally the carboxy terminal portion) which may mediate the binding of the immunoglobulin to host tissues or factors influencing various cells of the immune system, some phagocytic cells and the first component (CIq) of the classical complement system.
- the constant region of the light chains is referred to as the "CL region,” and the constant region of the heavy chain is referred to as the "CH region.”
- the constant region of the heavy chain comprises a CHl region, a CH2 region, and a CH3 region. A portion of the heavy chain between the CHl and CH2 regions is referred to as the hinge region (Le., the "H region").
- the constant region of the heavy chain of the cell surface form of an antibody further comprises a spacer-transmembranal region (Ml) and a cytoplasmic region (M2) of the membrane carboxy terminus.
- Ml spacer-transmembranal region
- M2 cytoplasmic region
- selective binding refers to the binding of one material to another in a manner dependent upon the presence of a particular molecular structure (i.e., specific binding).
- an immunoglobulin will selectively bind an antigen that contains the chemical structures complementary to the ligand binding site(s) of the 5 immunoglobulin. This is in contrast to "non-selective binding,” whereby interactions are arbitrary and not based on structural compatibilities of the molecules.
- polymerization encompasses any process that results in the conversion of small molecular monomers into larger molecules consisting of repeated units. Typically, polymerization involves chemical crosslinking of monomers to one 0 another.
- an antigen refers to any molecule or molecular group that is recognized by at least one antibody.
- an antigen must contain at least one epitope (i.e., the specific biochemical unit capable of being recognized by the antibody).
- immunogen refers to any molecule, compound, or aggregate that induces the 5 production of antibodies.
- an immunogen must contain at least one epitope (i.e., the specific biochemical unit capable of causing an immune response).
- the terms "home testing” and "point of care testing” refer to testing that occurs outside of a laboratory environment. Such testing can occur indoors or outdoors at, for example, a private residence, a place of business, public or private land, 0 in a vehicle, as well as at the patient's bedside.
- virus refers to minute infectious agents, which with certain exceptions, are not observable by light microscopy, lack independent metabolism, and are able to replicate only within a living host cell.
- the individual particles i.e., virions
- the term "virus” encompasses all types of viruses, including animal, plant, phage, and other viruses.
- nanostructures refers to microscopic structures, typically measured on a nanometer scale. Such structures include various three-dimensional assemblies, including, but not limited to, liposomes, films, multilayers, braided, lamellar, 0 helical, tubular, and fiber-like shapes, and combinations thereof. Such structures can, in some embodiments, exist as solvated polymers in aggregate forms such as rods and coils. - 1 , . ..• • ⁇ .. ••
- Such structures can also be formed from inorganic materials, such as prepared by the physical deposition of a gold film onto the surface of a solid, proteins immobilized on surfaces that have been mechanically rubbed, and polymeric materials that have been molded or imprinted with topography by using a silicon template prepared by electron 5 beam lithography.
- inorganic materials such as prepared by the physical deposition of a gold film onto the surface of a solid, proteins immobilized on surfaces that have been mechanically rubbed, and polymeric materials that have been molded or imprinted with topography by using a silicon template prepared by electron 5 beam lithography.
- self-assembling monomers and “lipid monomers” refer to molecules that spontaneously associate to form molecular assemblies. In one sense, this can refer to surfactant molecules that associate to form surfactant molecular assemblies.
- self-assembling monomers includes single molecules (e.g., a
- small molecular assemblies e.g., polymerized lipids
- the individual small molecular assemblies can be further aggregated (e.g., assembled and polymerized) into larger molecular assemblies.
- linker or "spacer molecule” refers to material that links one entity to another.
- a molecule or molecular group can be a linker that is
- bond refers to the linkage between atoms in molecules and between ions and molecules in crystals.
- single bond refers to a bond with two electrons occupying the bonding orbital.
- notations are represented by a single line drawn between two atoms (e.g., C-C).
- double bond refers to a bond that shares two electron pairs. Double bonds are stronger than single bonds and are more reactive.
- triple bond refers to the sharing of three electron pairs.
- ene-yne refers to alternating double and triple bonds.
- amine bond thiol bond
- aldehyde bond
- amine group 25 refers to any bond formed between an amine group (i. e. , a chemical group derived from ammonia by replacement of one or more of its hydrogen atoms by hydrocarbon groups), a thiol group (i.e., sulfur analogs of alcohols), and an aldehyde group (i.e., the chemical group -CHO joined directly onto another carbon atom), respectively, and another atom or molecule.
- amine group i. e. , a chemical group derived from ammonia by replacement of one or more of its hydrogen atoms by hydrocarbon groups
- thiol group i.e., sulfur analogs of alcohols
- aldehyde group i.e., the chemical group -CHO joined directly onto another carbon atom
- the term “covalent bond” refers to the linkage of two atoms by the sharing of two electrons, one contributed by each of the atoms.
- the term “spectrum” refers to the distribution of light energies arranged in order of wavelength.
- visible spectrum refers to light radiation that contains wavelengths from approximately 360 nm to approximately 800 nm.
- substrate refers to a solid object or surface upon which another material is layered or attached. Solid supports include, but are not limited to, glass, metals, gels, and filter paper, among others.
- array and patterned array refer to an arrangement of elements (i.e., entities) into a material or device.
- elements i.e., entities
- ligand binding molecules e.g., antibodies or nucleic acids
- situ refers to processes, events, objects, or information that are present or take place within the context of their natural environment.
- sample is used in its broadest sense. In one sense it can refer to a biopolymeric material. In another sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products, such as plasma, serum and the like. Environmental samples include environmental material such as surface matter, soil, water, crystals and industrial samples. These examples are not to be construed as limiting the sample types applicable to the present invention.
- liquid crystal refers to a thermodynamic stable phase characterized by anisotropy of properties without the existence of a three-dimensional crystal lattice, generally lying in the temperature range between the solid and isotropic liquid phase.
- the term “mesogen” refers to compound(s) that form liquid crystals, and in particular rigid rodlike or disclike molecules that are components of liquid crystalline materials.
- thermotropic liquid crystal refers to liquid crystals that result from the melting of mesogenic solids due to an increase in temperature. Both pure substances and mixtures form thermotropic liquid crystals. . i ... .. • . l '
- Lyotropic refers to molecules that form phases with orientational and/or positional order in a solvent. Lyotropic liquid crystals can be formed using amphiphilic molecules (e.g., sodium laurate, phosphatidylethanolamine, lecithin).
- the solvent can be water.
- heterophilic molecules e.g., sodium laurate, phosphatidylethanolamine, lecithin.
- the solvent can be water.
- heterogenous surface refers to a surface that orients liquid crystals in at least two separate planes or directions, such as across a gradient.
- Nematic refers to liquid crystals in which the long axes of the molecules remain substantially parallel, but the positions of the centers of mass are randomly distributed. Nematic liquid crystals can be substantially oriented by a nearby surface.
- Chiral nematic refers to liquid crystals in which the mesogens are optically active. Instead of the director being held locally constant as is the case for nematics, the director rotates in a helical fashion throughout the sample. Chiral nematic crystals show a strong optical activity that is much higher than can be explained on the bases of the rotatory power of the individual mesogens.
- the director acts like a diffraction grating, reflecting most and sometimes all of the light incident on it. If white light is incident on such a material, only one color of light is reflected and it is circularly polarized. This phenomenon is known as selective reflection and is responsible for the iridescent colors produced by chiral nematic crystals.
- “Smectic,” as used herein refers to liquid crystals which are distinguished from “nematics” by the presence of a greater degree of positional order in addition to orientational order; the molecules spend more time in planes and layers than they do between these planes and layers.
- “Polar smectic” layers occur when the mesogens have permanent dipole moments. In the smectic A2 phase, for example, successive layers show anti ferroelectric order, with the direction of the permanent dipole alternating from layer to layer. If the molecule contains a permanent dipole moment transverse to the long molecular axis, then the chiral smectic phase is ferroelectric. A device utilizing this phase can be intrinsically bistable.
- “Frustrated phases,” as used herein, refers to another class of phases formed by chiral molecules. These phases are not chiral, however, twist is introduced into the phase until
- a cubic lattice of defects (where the director is not defined) exist in a complicated, orientationally ordered twisted structure. The distance between these defects is hundreds of nanometers, so these phases reflect light just as crystals reflect x-rays.
- Discotic phases are formed from molecules that are disc shaped rather than elongated. Usually these molecules have aromatic cores and six lateral substituents. If the molecules are chiral or a chiral dopant is added to a discotic liquid crystal, a chiral nematic discotic phase can form.
- "Viewed between crossed polarizers” means polarizers whose transmission axes are aligned at some angle.
- “Polarizer” means a device, which in the transmission of electro-magnetic radiation, confines the vibration of the electric and magnetic field vectors of light to one plane.
- a chamber means any enclosed space.
- a chamber may be, but not limited to, a tube made of glass or plastic.
- 5 "Pneumatic" means gaseous elements.
- Pressure gap means a space of gaseous elements that separate two or more liquid compositions.
- Mesogen-aligning substrate means a substrate that causes certain mesogens to align in a substantially similarly ordered direction in a liquid crystal when in contact with 0 the substrate.
- the present invention relates to the field of detection of analytes, and in particular 5 to detection of viruses, cells, bacteria, lipid-membrane containing organisms, proteins, nucleic acids, carbohydrates and other biomolecules, organic molecules and inorganic molecules using a liquid crystal assay format.
- Liquid crystal-based assay systems are described in U.S. Pat. No. 6,284,197; WO 01/61357; WO 01/61325; WO 99/63329; Gupta et al, Science 279:2077-2080 (1998); Seung-Ryeol Kim, Rahul R. 0 Shah, and Nicholas L.
- the present invention provides systems, devices, and methods for both direct and indirect detection of analytes.
- the indirect detection systems utilize a first substrate comprising a recognition moiety that interacts with an analyte of interest, preferably
- the first substrate is exposed to a sample suspected of containing an analyte
- analyte interacting with the recognition moieties displayed on the first substrate are transferred to the second substrate.
- the analyte interacts with the second substrate in a non-specific manner.
- the second substrate comprises a detection region that orients mesogens in liquid crystal.
- the second substrate is then contacted with a liquid crystal.
- a disordered liquid crystal is indicative of the presence of an analyte in the detection region.
- the birefringence is not constant over the entire sample surface. At times some of the mesogens manifest a perpendicular alignment while others manifest a planar alignment; therefore, some of the surface areas appear light and others appear dark when viewed between crossed polarizers. The light and dark areas denote regions of differing birefringence.
- the anisotropic nature of liquid crystals can be used to detect the
- polyimide coated surfaces and polydimethylsiloxane (PDMS) [Dow Chemicals] micro fluidic channels align or can be made to align mesogens homeotropically (perpendicular to the surface). If a liquid crystal is fabricated on a sample polyimide surface (or PDMS micro fluidic channel) containing molecules on the sample surface, then the presence of the molecules between
- the mesogens and polyimide coating manifests itself by causing some planar alignment of the mesogens in the areas where the molecules interrupt homeotropic alignment form the polyimide surface.
- the presence of molecules on a spot (or in a micro fluidic channel) of the sample surface will result in areas of light appearance when the liquid crystal substrate is viewed between crossed polarizers.
- WO 01/61357 describes the detection of viruses using liquid crystal based assays. These assays utilize a patterned detection region on a substrate that organizes mesogens in a homeotropic orientation. The assays are designed so that binding of a virus to the detection regions disrupts the homeotropic orientation.
- the devices, systems and methods of the present invention are useful for detecting a variety of analytes, including, but not limited to, the following analytes: biomolecules including polypeptides (e.g., proteins), toxins, polynucleotides (e.g., RNA and DNA), carbohydrates, viruses, mycoplasmas, fungi, bacteria, and protozoa, especially Class A agents such as Variola major (smallpox), Bacillus anthracis (anthrax), Yersinia pestis (plague), Clostridium botulinum (botulism), Francisella tularensis (tularemia), Arenaviruses (Arenaviridae), Ebola hemorrhagic fever virus, Marburg hemorrhagic fever, Lassa fever virus, Junin and related viruses (Argentinian hemorrhagic fever virus, Venezuelan hemorrhagic fever virus, Venezuelan hemorrhagic fever virus), Dengue hemorrhagic fever virus, Brazil
- Alphaviruses such as Venezuelan equine encephalitis virus, Eastern equine encephalitis virus, Western equine encephalitis virus, and toxins such as ricin toxin, epsilin toxin from Clostridium perf ⁇ gens, and Staphylococcus enterotoxin B; and Class C agents such as mutlidrug resistant tuberculosis, Nipah virus, Hantaviruses, Tick- borne hemorrhagic fever viruses, Tick-borne encephalitis viruses, and Yellow fever virus.
- analytes include, but are not limited to, acids, bases, organic ions, inorganic ions, pharmaceuticals, herbicides, pesticides, chemical warfare agents, and noxious gases. These agents can be present as components in mixtures of structurally unrelated compounds, racemic mixtures of stereoisomers, non-racemic mixtures of stereoisomers, mixtures of diastereomers, mixtures of positional isomers or as pure compounds.
- the detection of these analytes, and specific substrates and recognition moieties for such ih ,... f . i i .. • ... l detection is described in more detail in co-pending applications 10/227,974, 10/443,419, and 60/585,275; all of which are incorporated herein by reference in their entirety.
- the present invention provides improved substrates and devices for the detection of analytes.
- the description of the present invention is 5 divided into the following sections: I. Recognition Moieties; II. Substrates; III.
- recognition moieties find use in the present invention.
- the recognition moieties are immobilized on a bead, such as a magnetic bead.
- a "recognition moiety" attached to or associated with a bead is utilized to bind to or otherwise interact with another molecule
- recognition moieties are attached to either ⁇ -functionalized spacer arms or ⁇ -functionalized SAM components which are in turn attached to or associated with a bead.
- the recognition moiety comprises an organic functional group.
- the organic functional group is a
- the recognition moiety is a biomolecule.
- the biomolecule is a protein, antigen binding protein, peptide, nucleic acid (e.g., single nucleotides or nucleosides, oligonucleotides, polynucleotides and single- and
- the recognition moiety is biotin.
- the recognition moieties are antigen binding proteins. Examples of antigen binding proteins finding use in the present invention include, but are not limited to, immunoglobulins, single chain antibodies, chimeric antibodies, polyclonal antibodies, monoclonal antibodies, and
- polyclonal antibodies Various procedures known in the art may be used for the production of polyclonal antibodies.
- various host animals including but not limited to rabbits, mice, rats, sheep, goats, etc., can be immunized by injection with the peptide corresponding to an epitope.
- the peptide is conjugated to an immunogenic carrier (e.g., diphtheria toxoid, bovine serum albumin (BSA), or keyhole limpet hemocyanin (KLH)).
- an immunogenic carrier e.g., diphtheria toxoid, bovine serum albumin (BSA), or keyhole limpet hemocyanin (KLH).
- BSA bovine serum albumin
- KLH keyhole limpet hemocyanin
- adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacte ⁇ um parvum).
- BCG Bacille Calmette-Guerin
- any technique that provides for the production of antibody molecules by continuous cell lines in culture will find use with the present invention (See e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). These include but are not limited to the hybridoma technique originally developed by K ⁇ hler and Milstein (K ⁇ hler and Milstein, Nature 256:495-497 [1975]), as well as the trioma technique, the human B-cell hybridoma technique (See e.g., Kozbor et al, Immunol.
- any technique suitable for producing antibody fragments will find use in generating antibody fragments that are useful recognition moieties.
- fragments include but are not limited to: F(ab')2 fragment that can be produced by pepsin digestion of the antibody molecule; Fab' fragments that can be generated by reducing the disulfide bridges of the F(ab')2 fragment, and Fab fragments that can be generated by treating the antibody molecule with papain and a reducing agent.
- the recognition moiety comprises a phage displaying an antigen binding protein.
- the recognition moiety is a polynucleotide or polypeptide
- a plurality of recognition moieties are arrayed on the substrates using photo activated chemistry, microcontact printing, and ink-jet printing.
- photolithography is utilized (See e.g., U.S. Patent Nos. 6,045,996; 5,925,525; and 5,858,659; each of which is herein incorporated by reference).
- nucleic acid recognition moieties are electronically captured on a suitable substrate (See e.g., U.S. Patent Nos. 6,017,696; 6,068,818; and 6,051,380; each of which are herein incorporated by reference).
- DNA capture probes unique to a given target are electronically placed at, or "addressed" to, specific sites on the microchip. Since DNA has a strong negative charge, it can be electronically moved to an area of positive charge.
- recognition moieties are arrayed on a suitable substrate by utilizing differences in surface tension (See e.g., U.S. Patent Nos. 6,001,311; 5,985,551; and 5,474,796; each of which is herein incorporated by reference).
- oligonucleotide probes are synthesized directly on the chip by ink-jet printing of reagents.
- the array with its reaction sites defined by surface tension is mounted on a X/Y translation stage under a set of four piezoelectric nozzles, one for each of the four standard DNA bases.
- the translation stage moves along each of the rows of the array and the appropriate reagent is delivered to each of the reaction site.
- the A amidite is delivered only to the sites where amidite A is to be coupled during that synthesis step and so on.
- Common reagents and washes are delivered by flooding the ..'" ' ' ' ... ' - • - 1 '»' « entire surface and then removing them by spinning.
- recognition moieties are spotted onto a suitable substrate.
- spotting can be done by hand with a capillary tube or micropipette, or by an automated spotting apparatus such as those available from Affymetrix and Gilson ⁇ See e.g., U.S. Pat. Nos. 5,601,980; 6,242,266; 6,040,193; and 5,700,637; each of which is incorporated herein by reference).
- the recognition moiety is an amine
- the recognition moiety will interact with a structure on the analyte which reacts by binding to the amine (e.g., carbonyl groups, alkylhalo groups).
- the amine is protonated by an acidic moiety on the analyte of interest (e.g., carboxylic acid, sulfonic acid).
- the recognition moiety when the recognition moiety is a carboxylic acid, the recognition moiety will interact with the analyte by complexation (e.g., metal ions). In still other preferred embodiments, the carboxylic acid will protonate a basic group on the analyte (e.g. amine).
- complexation e.g., metal ions
- carboxylic acid will protonate a basic group on the analyte (e.g. amine).
- the recognition moiety is a drug moiety.
- the drug moieties can be agents already accepted for clinical use or they can be drugs whose use is experimental, or whose activity or mechanism of action is under investigation.
- the drug moieties can have a proven action in a given disease state or can be only hypothesized to show desirable action in a given disease state.
- the drug moieties are compounds that are being screened for their ability to interact with an analyte of choice.
- drug moieties that are useful in practicing the instant invention include drugs from a broad range of drug classes having a variety of pharmacological activities. Classes of useful agents include, for example, non-steroidal anti-inflammatory drugs (NSAIDS).
- NSAIDS non-steroidal anti-inflammatory drugs
- the MAIDS can, for example, be selected from the following categories: (e.g., propionic acid derivatives, acetic acid derivatives, fenamic acid derivatives, biphenylcarboxylic acid derivatives and oxicams); steroidal anti-inflammatory drugs including hydrocortisone and the like; antihistaminic drugs (e.g., chlorpheniramine, triprolidine); antitussive drugs (e.g., dextromethorphan, codeine, carmiphen and carbetapentane); antipruritic drugs (e.g., methidilizine and trimeprizine); anticholinergic drugs (e.g., scopolamine, atropine, homatropine, levodopa); anti-emetic and antinauseant drugs (e.g., cyclizine, meclizine, chlorpromazine, buclizine); anorexic drugs (e.g., benzphetamine, phentermine, chlorphentermin
- vasoconstrictor drugs e.g., dihydroergotamine, ergotamine and methylsergide
- antiulcer drugs e.g., ranitidine and cimetidine
- anesthetic drugs e.g., lidocaine, bupivacaine, chlorprocaine, dibucaine
- antidepressant drugs e.g., imipramine, desipramine, amitryptiline, nortryptiline
- tranquilizer and sedative drugs e.g., chlordiazepoxide, benacytyzine, benzquinamide, flurazapam, hydroxyzine, loxapine and promazine
- antipsychotic drugs e.g., chlorprothixene, fluphenazine, haloperidol, molindone, thiori
- Antimicrobial drugs which are preferred for incorporation into the present composition include, for example, pharmaceutically acceptable salts of ⁇ -lactam drugs, quinolone drugs, ciprofloxacin, norfloxacin, tetracycline, erythromycin, amikacin, triclosan, doxycycline, capreomycin, chlorhexidine, chlortetracycline, oxytetracycline, clindamycin, ethambutol, hexamidine isothionate, metronidazole; pentamidine, gentamycin, kanamycin, lineomycin, methacycline, methenamine, minocycline, neomycin, netilmycin, paromomycin, streptomycin, tobramycin, miconazole, and amanfadine.
- drugs e.g., antiandrogens (e.g., leuprolide or flutamide), cytocidal agents .(e.g., adriamycin, doxorubicin, taxol, cyclophosphamide, busulfan, cisplatin, a-2-interferon) anti-estrogens (e.g., tamoxifen), antimetabolites (e.g., fluorouracil, methotrexate, mercaptopurine, thioguanine).
- antiandrogens e.g., leuprolide or flutamide
- cytocidal agents e.g., adriamycin, doxorubicin, taxol, cyclophosphamide, busulfan, cisplatin, a-2-interferon
- anti-estrogens e.g., tamoxifen
- antimetabolites e.g., fluorouraci
- the recognition moiety can also comprise hormones (e.g., medroxyprogesterone, estradiol, leuprolide, megestrol, octreotide or somatostatin); muscle relaxant drugs (e.g., cinnamedrine, cyclobenzaprine, flavoxate, orphenadrine, papaverine, mebeverine, idaverine, ritodrine, dephenoxylate, dantrolene and azumolen); antispasmodic drugs; 5 bone-active drugs (e.g., diphosphonate and phosphonoalkylphosphinate drug compounds); endocrine modulating drugs (e.g., contraceptives (e.g., ethinodiol, ethinyl estradiol, norethindrone, mestranol, desogestrel, medroxyprogesterone), modulators of diabetes (e.g., glyburide or
- estrogens e.g., diethylstilbesterol
- glucocorticoids e.g., triamcinolone, betamethasone, etc.
- progenstogens such as norethindrone, ethynodiol, norethindrone, levonorgestrel
- thyroid agents e.g., liothyronine or levothyroxine
- anti-thyroid agents e.g., methimazole
- 15 antihyperprolactinemic drugs e.g., cabergoline
- hormone suppressors e.g., danazol or goserelin
- oxytocics e.g., methylergonovine or oxytocin
- prostaglandins such as mioprostol, alprostadil or dinoprostone
- immunomodulating drugs e.g., ' antihistamines, mast cell stabilizers, such as lodoxamide and/or cromolyn
- steroids e.g., 20 triamcinolone, beclomethazone, cortisone, dexamethasone, prednisolone, methylprednisolone, beclomethasone, or clobetasol
- histamine H 2 antagonists e.g., famotidine, cimetidine, ranitidine
- immunosuppressants e.g., azathioprine, cyclosporin
- Groups with anti-inflammatory activity such as sulindac, etodolac, ketoprofen and ketorolac, are also of use.
- Other drugs of use in conjunction with the present invention 25 will be apparent to those of skill in the art.
- the recognition moiety is a chelating agent, crown ether or cyclodextrin
- host-guest chemistry will dominate the interaction between the recognition moiety and the analyte.
- the use of host-guest chemistry allows a great degree of recognition-moiety-analyte specificity to be engineered into a device of the invention. 30
- the use of these compounds to bind to specific compounds is well known to those of skill in the art. See, for example, Pitt et al. "The Design of Chelating Agents for the Treatment of Iron Overload," In 3 INORGANIC CHEMISTRY IN BIOLOGY AND MEDICINE;
- the recognition moiety is a polyaminocarboxylate chelating agent such as ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA).
- EDTA ethylenediaminetetraacetic acid
- DTPA diethylenetriaminepentaacetic acid
- the recognition moiety is a biomolecule such as a protein, nucleic acid, peptide or an antibody.
- Biomolecules useful in practicing the present invention can be derived from any source. The biomolecules can be isolated from natural sources or can be produced by synthetic methods. Proteins can be natural proteins or mutated proteins. Mutations can be effected by chemical mutagenesis, site-directed mutagenesis or other means of inducing mutations known to those of skill in the art. Proteins useful in practicing the instant invention include, for example, enzymes, antigens, antibodies and receptors. Antibodies can be either polyclonal or monoclonal.
- Peptides and nucleic acids can be isolated from natural sources or can be wholly or partially synthetic in origin.
- the protein can be tethered to a SAM component or a spacer arm by any reactive peptide residue available on the surface of the protein.
- the reactive / " ; " t *' - ! '' ; ' '"' groups are amines or carboxylates.
- the reactive groups are the e-amine groups of lysine residues.
- these molecules can be adsorbed onto the surface of the substrate or SAM by non-specific interactions (e.g., chemisorption, physisorption).
- Recognition moieties that are antibodies can be used to recognize analytes which are proteins, peptides, nucleic acids, saccharides or small molecules such as drugs, herbicides, pesticides, industrial chemicals and agents of war. Methods of raising antibodies for specific molecules are well-known to those of skill in the art. See, U.S. Pat. Nos. 5,147,786; 5,334,528; 5,686,237; 5,573,922; each of which is incorporated herein by reference. Methods for attaching antibodies to surfaces are also art-known (See, Delamarche et al Langmuir 12: 1944-1946 (1996)).
- Peptides and nucleic acids can be attached to a SAM component or spacer arm. Both naturally-derived and synthetic peptides and nucleic acids are of use in conjunction with the present invention. These molecules can be attached to a SAM component or spacer arm by any available reactive group. For example, peptides can be attached through an amine, carboxyl, sulfhydryl, or hydroxyl group. Such a group can reside at a peptide terminus or at a site internal to the peptide chain. Nucleic acids can be attached through a reactive group on a base (e.g., exocyclic amine) or an available hydroxyl group on a sugar moiety (e.g., 3'- or 5 '-hydroxyl).
- a base e.g., exocyclic amine
- an available hydroxyl group on a sugar moiety e.g., 3'- or 5 '-hydroxyl
- the peptide and nucleic acid chains can be further derivatized at one or more sites to allow for the attachment of appropriate reactive groups onto the chain (See, Chrisey et al. Nucleic Acids Res. 24:3031-3039 (1996)).
- a reactive group or masked reactive group can be incorporated during the process of the synthesis.
- Many derivatized monomers appropriate for reactive group incorporation in both peptides and nucleic acids are know to those of skill in the art (See, for example, THE PEPTIDES: ANALYSIS, SYNTHESIS, BIOLOGY, Vol. 2: "Special Methods in Peptide Synthesis," Gross, E.
- the peptide is attached directly to the substrate (See, Frey et al Anal. Chem. 65:3187-3193 (1996)). In a particularly preferred embodiment, the peptide is attached to a gold substrate through a sulfhydryl group on a cysteine residue.
- the peptide is attached through a thiol to a spacer arm which terminates in, for example, an iodoacetamide, chloroacetamide, benzyl iodide, benzyl bromide, alkyl iodide or alkyl bromide.
- a spacer arm which terminates in, for example, an iodoacetamide, chloroacetamide, benzyl iodide, benzyl bromide, alkyl iodide or alkyl bromide.
- the recognition moiety forms an inclusion complex with the analyte of interest.
- the recognition moiety is a cyclodextrin or modified cyclodextrin.
- Cyclodextrins are a group of cyclic oligosaccharides produced by numerous microorganisms. Cyclodextrins have a ring structure which has a basket-like shape.
- Cyclodextrins are able to form inclusion complexes with an array of organic molecules including, for example, drugs, pesticides, herbicides and agents of war (See, Tenjarla et al, J. Pharm. Sci. 87:425-429 (1998); Switzerlandhul et al, Phctrm. Dev. Techno!.
- cyclodextrins are able to discriminate between enantiomers of compounds in their inclusion complexes.
- the invention provides for the detection of a particular enantiomer in a mixture of enantiomers (See, Koppenhoefer et al J. Chromatogr. A 793:153-164 (1998)).
- the cyclodextrin recognition moiety can be attached to a SAM component, through a spacer arm or directly to the substrate (See, Yamamoto et al, J. Phys. Chem.
- the recognition moieties can be nucleic acids (e.g., RNA or
- nucleic acids are aptatners.
- the isolation of aptamers is described in U.S. Pat. Nos. 5,475,096; 5,270,163; and 5,475,096; and in PCT publications WO 97/38134, WO 98/33941, and WO 99/07724, all of which are herein incorporated by reference.
- recognition moieties are incorporated to detect a variety of bacteria and pathogens.
- recognition moieties include, but not limited to, sialic acid to detect HIV (Wies et al., Nature 333: 426 [1988]), influenza (White et al., Cell 56: 725 [1989]), chlamydia (Infect. Imm.
- ICAM-I Marlin et al., Nature 344: 70 [1990]
- N-CAM N-CAM
- myelin- 0 associated glycoprotein MAb Shephey et al., Proc. Natl. Acad. Sci.
- oligomannose to detect Escherichia coli
- ganglioside GMI to detect Neisseria meningitidis
- antibodies to detect a broad variety of pathogens e.g., 5 Neisseria gonorrhoeae, V. vulnificus, V. parahaemolyticus, V. cholerae, V. alginolyticus , etc.
- the recognition moiety is a ligand that interacts with a binding partner.
- ligands include, but are not limited to, small organic molecules such as steroid molecules and small drug molecules, proteins, polypeptides 0 and peptides, metal ions, and nucleic acids.
- the ligand is recognized by a binding molecule in a sample.
- binding molecules include, but are not limited to, steroids, hormones, proteins, polypeptides, and peptides such immunoglobulin molecules and fragments thereof, nucleic acids, and other organic or non-organic molecules.
- the ligand is recognized by a binding molecule in a body fluid of a test subject.
- the ligand can be a virus- envelope protein or some other antigenic molecule from a pathogenic organism (such as those listed above).
- the antigenic molecule e.g., a protein
- the ligand is protein E from the envelope of West Nile Virus.
- Substrates that are viseful in practicing the present invention can be made of practically any physicochemically stable material.
- the recognitions moieties are attached to a first substrate (such as a bead), but detection with the liquid crystal occurs after transfer to a second substrate (e.g., a detection substrate).
- the substrate material is non-reactive towards the constituents of the mesogenic layer.
- the substrates can be either rigid or flexible and can be either optically transparent or optically opaque.
- the substrates can be electrical insulators, conductors or semiconductors. Further, the substrates can be substantially impermeable to liquids, vapors and/or gases or, alternatively, the substrates can be permeable to one or more of these classes of materials.
- Exemplary substrate materials include, but are not limited to, inorganic crystals, inorganic glasses, inorganic oxides, metals, organic polymers and combinations thereof.
- the substrates have microchannels therein for the delivery of sample and/or other reagents to the substrate surface or detection regions thereon. The design and use of microchannels are described, for example, in U.S. Pat. Nos.
- inorganic crystals and inorganic glasses are utilized as substrate materials (e.g., LiF, NaF, NaCl, KBr, KI, CaF 2 , MgF 2 , HgF 2 , BN, AsS 3 , ZnS, Si 3 N 4 and the like).
- substrate materials e.g., LiF, NaF, NaCl, KBr, KI, CaF 2 , MgF 2 , HgF 2 , BN, AsS 3 , ZnS, Si 3 N 4 and the like.
- the crystals and glasses can be prepared by art standard techniques (See, e.g., Goodman, C.H.L., Crystal Growth Theory and
- the crystals can be purchased commercially (e.g., Fischer Scientific).
- the crystals can be the sole component of the substrate or they can be coated with one or more additional substrate components.
- a crystal can constitute a portion of a substrate which contacts another portion of the substrate made of a different material, or a different physical form (e.g., a glass) of the same material.
- Other useful substrate configurations utilizing inorganic crystals and/or glasses will be apparent to those of skill in the art.
- inorganic oxides are utilized as the . substrate.
- Inorganic oxides of use in the present invention include, for example, Cs 2 O, Mg(OH) 2 , TiO 2 , ZrO 2 , CeO 2 , Y 2 O 3 , Cr 2 O 3 , Fe 2 O 3 , NiO, ZnO, Al 2 O 3 , SiO 2 (glass), quartz, In 2 O 3 , SnO 2 , PbO 2 and the like.
- the inorganic oxides can be utilized in a variety of physical forms such as films, supported powders, glasses, crystals and the like.
- a substrate can consist of a single inorganic oxide or a composite of more than one inorganic oxide.
- a composite of inorganic oxides can have a layered structure (i.e., a second oxide deposited on a first oxide) or two or more oxides can be arranged in a contiguous non-layered structure.
- one or more oxides can be admixed as particles of various sizes and deposited on a support such as a glass or metal sheet.
- a layer of one or more inorganic oxides can be intercalated between two other substrate layers (e.g., metal-oxide-metal, metal-oxide-crystal).
- the substrate is a rigid structure that is impermeable to liquids and gases.
- the substrate consists of a glass plate onto which a metal, such as gold is layered by evaporative deposition.
- the substrate is a glass plate (SiO 2 ) onto which a first metal layer such as titanium has been layered. A layer of a second metal such as gold is then layered on top of the first metal layer.
- metals are utilized as substrates.
- the metal can be used as a crystal, a sheet or a powder.
- the metal can be deposited onto a backing by any method known to those of skill in the art including, but not limited to, evaporative deposition, sputtering, electroless deposition, electrolytic deposition and adsorption or deposition of preform particles of the metal including metallic nanoparticles.
- metals that are chemically inert towards the mesogenic layer will be useful as a substrate in the present invention.
- Metals that are reactive or interactive towards the mesogenic layer will also be useful in the present invention.
- Metals that are presently preferred as substrates include, but are not limited to, gold, silver, platinum, palladium, nickel and copper.
- more than one metal is used. The more than one metal can be present as an alloy or they can be formed into a layered "sandwich" structure, or they can be laterally adjacent to one another.
- the metal used for the substrate is gold. In a particularly preferred embodiment the metal used is gold layered on titanium.
- the metal layers can be either permeable or impermeable to materials such as liquids, solutions, vapors and gases.
- Organic polymers are utilized as substrate materials.
- Organic polymers useful as substrates in the present invention include polymers that are permeable to gases, liquids and molecules in solution. Other useful polymers are those that are impermeable to one or more of these same classes of compounds.
- Organic polymers that form useful substrates include, for example, polyalkenes
- polyacrylics e.g., polyacrylate, n ./ L polymethyl methacrylate, polycyanoacrylate
- polyvinyls e.g., polyvinyl alcohol, polyvinyl acetate, polyvinyl butyral, polyvinyl chloride
- polystyrenes polycarbonates, polyesters, polyurethanes, polyaraides, polyimides, polysulfone, polysiloxanes, polyheterocycles, cellulose derivative (e.g., methyl cellulose, cellulose acetate, nitrocellulose), polysilanes, fluorinated polymers, epoxies, polyethers and phenolic resins ⁇ See, Cognard, J.
- organic polymers include polydimethylsiloxane, polyethylene, polyacrylonitrile, cellulosic materials, polycarbonates and polyvinyl pyridinium.
- the substrate is permeable and it consists of a layer of gold, or gold over titanium, which is deposited on a polymeric membrane, or other material, that is permeable to liquids, vapors and/or gases.
- the liquids and gases can be pure compounds (e.g., chloroform, carbon monoxide) or they can be compounds which are dispersed in other molecules (e.g., aqueous protein solutions, herbicides in air, alcoholic solutions of small organic molecules).
- Useful permeable membranes include, but are not limited to, flexible cellulosic materials (e.g., regenerated cellulose dialysis membranes), rigid cellulosic materials (e.g., cellulose ester dialysis membranes), rigid polyvinylidene fluoride membranes, polydimethylsiloxane and track etched polycarbonate membranes.
- the layer of gold on the permeable membrane is itself permeable.
- the permeable gold layer has a thickness of about 70 Angstroms or less.
- the film can be as thick as is necessary for a particular application.
- the film can be thicker than in an embodiment in which it is necessary for the film to be transparent or semi-transparent to light.
- the film is of a thickness of from about 0.01 nanometer to about 1 micrometer. In a further preferred embodiment, the film is of a thickness of from about 5 nanometers to about 100 nanometers. In yet a further preferred embodiment, the film is of a thickness of from about 10 nanometers to about 50 nanometers.
- a first substrate e.g., a capture substrate such as a bead
- a second substrate e.g., a detection substrate
- the two or more substrates may comprise any of the materials described above.
- the first substrates of the present invention are provided in a variety of formats.
- the first substrate is a stamp.
- the stamp comprises a plurality of analyte binding regions that display recognition moieties.
- the analyte binding regions are extensions from the body of the stamp.
- stamps of the present invention are not limited to any particular material composition.
- the stamps are formed from a pliable material, such as PDMS.
- the first substrates may present planar or curved surfaces or be beads.
- the bead format is especially useful for the indirect detection methods described below.
- the bead substrates of the present invention may comprise any of the substrate materials described above.
- the beads are commercially available beads such as agarose beads, acrylic beads, or latex beads.
- the beads are magnetic.
- the beads are coated with a metal such as silver or gold.
- substrates such column chromatography media may be used to capture analytes.
- protein-A affinity columns such as S-SEPHAROSE, SP-SEPHAROSE, and carboxymethyl cellulose
- anion exchange columns such as DEAE Cellulose, QAE SEPHADEX, and FAST Q SEPHAROSE
- sizing columns such as ULTRAGEL columns, phsospho
- the surface of the substrate (e.g., a first or second substrate as described above) is functionalized so that a recognition moiety is immobilized on the surface of the substrate.
- the immobilized recognition moiety forms a detection region.
- a plurality of detection regions are formed on the surface of the substrate.
- the same recognition moiety is provided on two or more of the plurality of detection regions, while in other embodiments, at least two different recognition moieties are immobilized on one or more of the plurality of detection regions.
- the recognition moieties are arrayed in discreet detection regions on the substrate surfaces by the methods described in more detail below.
- the surface of the substrate is first functionalized by forming a self-assembled monolayer (SAM) on the substrate surface.
- SAM self-assembled monolayer
- Self-assembled monolayers are generally depicted as an assembly of organized, closely packed linear molecules.
- Additional methods include techniques such as depositing a vapor of the monolayer precursor onto a substrate surface and the layer-by-layer deposition of polymers and polyelectrolytes from solution (Ladam et al., Protein Adsorption onto Auto-Assembled Polyelectrolyte Films, Langmuir; 2001; 17(3); 878-882).
- composition of a layer of a SAM useful in the present invention can be varied over a wide range of compound structures and molar ratios.
- the SAM is formed from only one compound.
- the SAM is formed from only one compound.
- SAM is formed from two or more components.
- one component is a long-chain hydrocarbon having a chain length of between 10 and 25 carbons and a second component is a short-chain hydrocarbon having a chain length of between 1 and 9 carbon atoms.
- the SAM is formed from CH 3 (CH 2 )i 5 SH and CH 3 (CH 2 ) 4 SH or CH 3 (CH 2 )i 5 SH and CH 3 (CH 2 ⁇ SH.
- the carbon chains can be functionalized at the ⁇ -terminus (e.g., NH 2 , COOH, OH, CN) 3 at internal positions of the chain (e.g., aza, oxa, thia) or at both the ⁇ -terminus and internal positions of the chain.
- ⁇ -terminus e.g., NH 2 , COOH, OH, CN
- internal positions of the chain e.g., aza, oxa, thia
- a recognition moiety can be attached to the surface of a SAM by any of a large number of art-known attachment methods.
- a reactive SAM component is attached to the substrate and the recognition moiety is subsequently bound to the SAM component via the reactive group on the component and a group of complementary reactivity on the recognition moiety (See, e.g., Hegner et at. Biophys. J. 70:2052-2066 (1996)).
- the recognition moiety is attached to the SAM component prior to immobilizing the SAM component on the substrate surface: the recognition moiety-SAM component cassette is then attached to the substrate.
- the recognition moiety is attached to the substrate via a displacement reaction.
- the SAM is preformed and then a fraction of the SAM components are displaced by a recognition moiety or a SAM component bearing a virus recognition moiety.
- the polypeptide recognition moieties are adsorbed directly onto hydrophobic monolayers such as CH 3 (CH 2 )i 5 SH.
- the recognition moiety is an antibody or other molecule that binds to protein A
- protein A is first attached to the monolayer followed by the antibody, which is bound by protein A.
- reactive SAM components refers to components that have a functional group available for reaction with a recognition moiety or other species following the attachment of the component to the substrate.
- a substrate's surface is functionalized with
- Reactive groups which can be used in practicing the present invention include, for example, amines, hydroxyl groups, carboxylic acids, carboxylic acid derivatives, alkenes, sulfhydryls, siloxanes, etc.
- reaction types are available for the functionalization of a substrate surface.
- substrates constructed of a plastic such as polypropylene can be surface derivatized by chromic acid oxidation, and subsequently converted to hydroxylated or aminomethylated surfaces.
- Substrates made from highly crosslinked divinylbenzene can be surface derivatized by chloromethylation and subsequent functional group manipulation.
- functionalized substrates can be made from etched, reduced polytetrafluoroethylene.
- the surface can be derivatized by reacting the surface Si-OH, SiO-H, and/or Si-Si groups with a functionalizing reagent.
- the substrate is made of a metal film, the surface can be derivatized with a material displaying avidity for that metal.
- the covalent bonding of the reactive group to the glass surface is achieved by conversion of groups on the substrate's surface by a silicon modifying reagent such as:
- R is an alkyl group, such as methyl or ethyl
- R 1 is a linking group between silicon and X and X is a reactive group or a protected reactive group.
- the reactive group can also be a recognition moiety as discussed below.
- Silane derivatives having halogens or other leaving groups beside the displayed alkoxy groups are also useful in the present invention.
- siloxane functionalizing reagents can be used, for example: 1. Hydroxyalkyl siloxanes (Silylate surface, functionalize with diborane, and
- Dimeric secondary aminoalkyl siloxanes a. bis (3-trimethoxysilylpropyl) amine - ⁇ bis(silyloxylpropyl)amine.
- the substrate is at least partially a metal film, such as a gold film, and the reactive group is tethered to the metal surface by an agent displaying avidity for that surface.
- the substrate is at least partially a gold film and the group which reacts with the metal surface comprises a thiol, sulfide or disulfide such as:
- R is a linking group between sulfur and X 2 and X 2 is a reactive group or a protected reactive group.
- X can also be a recognition moiety as discussed below.
- Y is a member selected from the group consisting of H, R 3 and R 3 -S-, wherein R 2 and R 3 are independently selected.
- R 2 and R 3 are the same, symmetrical sulfides and disulfides result, and when they are different, asymmetrical sulfides and disulfides result.
- a large number of functionalized thiols, sulfides and disulfides are commercially available (Aldrich Chemical Co., St. Louis). Additionally, those of skill in the art have available to them a manifold of synthetic routes with which to produce additional such molecules.
- amine-functionalized thiols can be produced from the corresponding halo-amines, halo-carboxylic acids, etc. by reaction of these halo precursors with sodium sulfhydride. See, e.g., Reid, ORGANIC CHEMISTRY of BIVALENT SULFUR, VOL 1, pp. 21-29, 32-35, vol. 5, pp. 27-34, Chemical Publishing Co., New York, 1 ,958, 1963. Additionally, functionalized sulfides can be prepared via alkylthio-de-halogenation with a mercaptan salt (See, Reid, ORGANIC CHEMISTRY OF BIVALENT SULFUR, vol. 2, pp. 16-21, 24-29, vol.
- the functionalizing reagent provides for more than one reactive group per each reagent molecule.
- each reactive site on the substrate surface is, in essence, "amplified" to two or more functional groups:
- R is an alkyl group, such as methyl
- R 2 is a linking group between silicon and X 2
- X is a reactive group or a protected reactive group
- n is an integer between 2 and 50, and more preferably between 2 and 20.
- Similar amplifying molecules are also of use in those embodiments wherein the substrate is at least partially a metal film.
- the group which reacts with the metal surface comprises a thiol, sulfide or disulfide such as in Formula (4):
- R 2 is a linking group between sulfur and X 2 and X 2 is a reactive group or a protected reactive group.
- X 2 can also be a recognition moiety.
- Y is a member selected from the group consisting of H, R 3 and R 3 -S-, wherein R 2 and R 3 are independently selected.
- R groups of use for R 1 , R 2 and R 3 in the above described embodiments of the present invention include, but are not limited to, alkyl, substituted alkyl, aryl, arylalkyl, substituted aryl, substituted arylalkyl, acyl, halogen, hydroxy, amino, alkylamino, acylamino, alkoxy, acyloxy, aryloxy, aryloxyalkyl, mercapto, saturated cyclic hydrocarbon, unsaturated cyclic hydrocarbon, heteroaryl, heteroarylalkyl, substituted heteroaryl, substituted heteroarylalkyl, heterocyclic, substituted heterocyclic and heterocyclicalkyl groups.
- each of R 1 , R 2 and R 3 are either stable or they can be cleaved by chemical or photochemical reactions.
- R groups comprising ester or disulfide bonds can be cleaved by hydrolysis and reduction, respectively.
- R groups which are cleaved by light such as, for example, nitrobenzyl derivatives, phenacyl groups, benzoin esters, etc.
- Other such cleaveable groups are well-known to those of skill in the art.
- the organosulfur compound is partially or entirely halogenated.
- An example of compounds useful in this embodiment include:
- X 1 is a member selected from the group consisting of H, halogen reactive groups and protected reactive groups. Reactive groups can also be recognition moieties as discussed below.
- Q, Q 1 and Q are independently members selected from the group consisting of H and halogen.
- Z 1 is a member selected from the group consisting of -CQ 2 -, -CQ 1 Z -, -CQ 2 2 -, -O-, -S-, NR 4 -, -C(O) NR 4 and R 4 NC(OO-, in which R 4 is a member selected from the group consisting of H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl and heterocyclic groups and m and n are independently a number between 0 and 40. -• • ' -" b . ⁇ • " i
- the organic layer comprises a compound according to Formula 5 above, in which Q, Q 1 and Q 2 are independently members selected from the group consisting of H and fluorine.
- the organic layer comprises compounds having a structure according to Formulae (6) and (7):
- Z 1 and Z 2 are members independently selected from the group consisting of -CH 2 -, -O-, -S-, NR 4 , -C(O)NR 4 and R 4 NC(O)- in which R 4 is a member selected from the group consisting of H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl and heterocyclic groups.
- R 4 is a member selected from the group consisting of H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl and heterocyclic groups.
- the Z groups of adjacent molecules participate in either an attractive (e.g., hydrogen bonding) or repulsive (e.g., van der Waals) interaction.
- m is a number between O and 40
- n is a number between 0 and 40
- o is a number between 0 and 40
- p is a number between 0 and 40.
- the compounds of Formulae 6 and 7 are used in conjunction with an organosulfur compound, either halogentated or unhalogenated, that bears a recognition moiety.
- an organosulfur compound either halogentated or unhalogenated
- the organic layer can comprise a single halogenated compound or more than one halogenated compound having different structures. Additionally, these layers can comprise a non-halogenated organosulfur compound.
- the reactive functional groups (X 1 and X ) are, for example: (a) carboxyl groups and various derivatives thereof including, but not limited to,
- N-hydroxysuccinimide esters N-hydroxybenztriazole esters, acid halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromatic esters;
- haloalkyl groups wherein the halide can be later displaced with a nucleophilic group such as, for example, an amine, a carboxylate anion, thiol anion, carbanion, or an alkoxide ion, thereby resulting in the covalent attachment of a new group at the site of the halogen atom;
- a nucleophilic group such as, for example, an amine, a carboxylate anion, thiol anion, carbanion, or an alkoxide ion
- dienophile groups which are capable of participating in Diels-Alder reactions such as, for example, maleimido groups
- aldehyde or ketone groups such that subsequent derivatization is possible via formation of carbonyl derivatives such as, for example, imines, hydrazones, semicarbazones or oximes, or via such mechanisms as Grignard addition or alkyllithium addition;
- thiol groups which can be converted to disulfides or reacted with acyl halides
- amine or sulfhydryl groups which can be, for example, acylated or alkylated
- alkenes which can undergo, for example, cycloadditions, acylation, Michael addition, etc
- J epoxides which can react with, for example, amines and hydroxyl compounds.
- the reactive moieties can also be recognition moieties.
- the nature of these groups is discussed in greater detail below.
- the reactive functional groups can be chosen such that they do not participate in, or interfere with, the reaction controlling the attachment of the functionalized SAM component onto the substrate's surface.
- the reactive functional group can be protected from participating in the reaction by the presence of a protecting group.
- a protecting group Those of skill in the art will understand how to protect a particular functional group from interfering with a chosen set of reaction conditions. For examples of useful protecting groups, see Greene et al, PROTECTIVE GROUPS IN ORGANIG SYNTHESIS, John Wiley & Sons, New York, 1991.
- the SAM component bearing the recognition moiety is attached directly and essentially irreversibly via a "stable bond" to the surface of the substrate.
- a “stable bond”, as used herein, is a bond which maintains its chemical integrity over a wide range of conditions (e.g., amide, carbamate, carbon-carbon, ether, etc.).
- the SAM component bearing the recognition moiety is attached to the substrate surface by a "cleaveable bond”.
- a “cleaveable bond”, Z as used herein, is a bond that is designed to undergo scission under conditions which do not degrade other bonds in the recognition moiety-analyte complex. Cleaveable bonds include, but are not limited to, disulfide, imine, carbonate and ester bonds.
- the recognition moiety attached to a SAM component having a structure that is different than that of the constituents of the bulk SAM.
- the group to which the recognition moiety is bound is referred to as a "spacer arm” or “spacer.”
- spacer arms the properties of the SAM adjacent to the recognition moiety can be controlled. Properties that are usefully controlled include, for example, hydrophobicity, hydrophilicity, surface-activity and the distance of the recognition moiety from the plane of the substrate and/or the SAM.
- the recognition moiety can be attached to the substrate or the surface of the SAM via an amine terminated poly(ethyleneglycol).
- hydrophilicity of the substrate surface can be enhanced by reaction with polar molecules such as amine-, hydroxyl- and polyhydroxylcontaining molecules.
- polar molecules such as amine-, hydroxyl- and polyhydroxylcontaining molecules.
- Representative examples include, but are not limited to, polylysine, polyethyleneimine, poly(ethyleneglycol) and poly(propyleneglycol).
- Suitable functionalization chemistries and strategies for these compounds are known in the art (See, for example, Dunn, R.L., et al, Eds. POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACS Symposium Series Vol. 469, American Chemical Society, Washington, D.C. 1991).
- the hydrophobicity of the substrate surface can be modulated by using a hydrophobic spacer arm such as, for example, long chain diamines, long chain thiols, ⁇ , ⁇ -amino acids, etc.
- Representative hydrophobic spacers include, but are not limited to, 1,6-hexanediamine, 1,8-octanediamine, 6-aminohexanoic acid and 8-aminooctanoic acid.
- the substrate surface can also be made surface-active by attaching to the substrate surface a spacer which has surfactant properties.
- Compounds useful for this purpose include, for example, aminated or hydroxylated detergent molecules such as, for example, 1-aminododecanoic acid.
- the spacer serves to distance the virus recognition moiety from the substrate or SAM.
- Spacers with this characteristic have several uses. For ,• ⁇ ' ' [ ⁇ • • a example, a recognition moiety held too closely to the substrate or SAM surface may not react with incoming analyte, or it may react unacceptably slowly. When an analyte is itself sterically demanding, the reaction leading to recognition moiety-analyte complex formation can be undesirably slowed, or not occur at all, due to the monolithic substrate hindering the approach of the two components.
- the physicochemical characteristics (e.g., hydrophobicity, hydrophilicity, surface activity, conformation) of the substrate surface and/or SAM are altered by attaching a monovalent moiety which is different in composition than the constituents of the bulk SAM and which does not bear a recognition moiety.
- monovalent moiety refers to organic molecules with only one reactive functional group. This functional group attaches the molecule to the substrate.
- “Monovalent moieties” are to be contrasted with the bifunctional "spacer” groups described above. Such monovalent groups are used to modify the hydrophilicity, hydrophobicity, binding characteristics, etc. of the substrate surface. Examples of groups useful for this purpose include long chain alcohols, amines, fatty acids, fatty acid derivatives, poly(ethyleneglycol) monomethyl ethers, etc.
- the components can be contacted with the substrate as a mixture of SAM components or, alternatively, the components can be added individually.
- the mole ratio of a mixture of the components in solution results in the same ratio in the mixed SAM.
- the two components do not phase segregate into islands ⁇ See, Bain and Whitesides, J. Am. Chem. Soc. 111 :7164 (1989)).
- This feature of SAMs can be used to immobilize recognition moieties or bulky modifying groups in such a manner that certain interactions, such as steric hindrance, between these molecules is minimized.
- a first SAM component is attached to the substrate's surface by "underlabeling" the surface functional groups with less than a stoichiometric equivalent of the first component.
- the first component can be a SAM component liked to a terminal reactive group or recognition group, a spacer arm or a / - -"" ; ⁇ ;il * ⁇ " « 'TM monovalent moiety.
- the second component is contacted with the substrate. This second component can either be added in stoichiometric equivalence, stoichiometric excess or can again be used to underlabel to leave sites open for a third component.
- the substrates are coated with polyimide layer. It is contemplated that polyimide coated substrates are especially useful because in some instances, the surfaces homeotropically orient a liquid crystal, while in other instances the surfaces can be rubbed to provide an anisotropic surface for orient a liquid crystal.
- a substrate such as a silicon wafer is coated with a polyimide.
- the substrate is spin coated with the polyimide.
- a variety of polyimides find use with the present invention, including, but not limited to Nissan 7210, Nissan 3510, Nissan 410, Nissan 3140, Nissan 5291, and Japan Synthetic Rubber JALS 146-Rl 9 for planar alignment of liquid crystals and Nissan 7511 L and SE 1211 for homeotropic orientation of liquid crystals. Surprising, it has been found that the ability of rubbed polyimide surfaces to orient liquid crystals is maintained when a recognition moiety is displayed on the rubbed surface, and then masked when an analyte binds the recognition moiety.
- areas where an analyte is bound have a non-ordered liquid crystal and appear white or bright when viewed through cross polars and areas where analyte is not bound remain ordered and appear dark when viewed through cross polars.
- polyimide surfaces that homeotropically orient liquid crystals can be used to report non-specific binding to the surface.
- areas where an analyte is bound have a disordered liquid crystal appear white or bright when viewed through cross polars and areas where no analyte is bound maintain the homeotropic orientation and appear dark.
- These different polyimides provide different anchoring properties and different binding affinity to different proteins that can be used to probe and report the binding events between the proteins.
- different liquid crystals show different response to the specific binding event. Therefore, it is possible to tune the assays by using different liquid crystalline materials such as, / - ⁇ •• .”
- thermotropic or lyotropic liquid crystals can exist in a number of forms including nematic, chiral nematic, smectic, polar smectic, chiral smectic, frustrated phases and discotic phases.
- the mesogen is a member selected from the group consisting of 4-cyano-4'-pentylbiphenyl, N-(4methoxybenzylidene)-4-butlyaniline and combinations thereof.
- the mesogenic layer can be a substantially pure compound, or it can contain other compounds which enhance or alter characteristics of the mesogen.
- the mesogenic layer further comprises a second compound, for example and alkane, which expands the temperature range over which the nematic and isotropic phases exist. Use of devices having mesogenic layers of this composition allows for detection of the analyte recognition moiety interaction over a greater temperature range.
- the mesogenic layer further comprises a dichroic dye or fluorescent compound. Examples of dichroic dyes and fluorescent compounds useful in the present invention include, but are not limited to, azobenzene, BTBP, polyazo compounds, anthraquinone, perylene dyes, and the like.
- a dichroic dye of fluorescent compound is selected that complements the orientation dependence of the liquid crystal so that polarized light is not required to read the assay.
- the absorbance of the liquid crystal is in the visible range, then changes in orientation can be observed using ambient light without crossed polars.
- the dichroic dye or fluorescent compound is used in combination with a fluorimeter and the changes in fluorescence are used to detect changes in orientation of the liquid crystal.
- the assays of the present find use for the non-specific detection of an analyte following specific capture.
- the analyte is captured by a capture substrate (e.g., a PDMS stamp or bead) displaying a recognition moiety that interacts with the analyte.
- the analyte is then transferred to a detection substrate to which the analyte non-specifically binds.
- the presence of the analyte on the second (e.g., detection) substrate is detected by contacting the second substrate with a liquid crystal. Areas of disorder or order within the liquid crystal are indicative of the presence of analyte.
- the assay devices are configured with electrodes as described above so that ' .,, i the analyte can be transferred to a surface of the assay device by use of an electric current (e.g., by dielectrophoresis).
- the electrodes are also used to measured changes in electrical properties of the device (e.g., dielectric capacitance) as a result of changes in liquid crystal orientation.
- the assays of the present invention are used for the detection of multiple species or genera of animals to a pathogenic organism.
- antibodies specific West Nile Vims have been detected in samples collected from horses, mallard ducks, pigeons, rabbits, and mice. It will be recognized that these assays find use for testing samples from avian species such as crow, blue jay, eagles, sparrows and the more than 150 species of birds present in the US that are known to be infected with West Nile Viral, horses, humans, small mammals such as dogs and cats and other companion animals, rodents such as mice and rats, etc., and other wildlife such as raccoons, skunks, felines, canids, etc.
- avian species such as crow, blue jay, eagles, sparrows and the more than 150 species of birds present in the US that are known to be infected with West Nile Viral, horses, humans, small mammals such as dogs and cats and other companion animals, rodents such as mice and rats, etc., and other wildlife such as raccoons, skunks, felines,
- surfaces of the detection substrate as described above are
- the detection substrates are substrates onto which a metal (e.g., gold) has been obliquely deposited and functionalized with 4-Aminothiophenol (ATP).
- a metal e.g., gold
- ATP 4-Aminothiophenol
- a stamp substrate surface is prepared that displays at least one recognition moiety.
- a stamp substrate is any substrate that can be used to transfer an entity that is covalently or non-covalently bound to the surface of the stamp substrate to another surface. Examples of suitable stamp substrates include, but are not limited to,
- PDMS and other elastomeric materials are arrayed in different areas of the stamp substrate.
- a variety of different recognition moieties e.g., envelope proteins from different enveloped viruses
- multiple recognition moieties in multiple concentrations are arrayed on the stamp substrate surface.
- a control area is included on the stamp ..• t .. . substrate surface. The recognition moiety (or recognition moieties) is then introduced to the stamp substrate surface, preferably in an array.
- the stamp substrate surface is a functionalized surface so that a covalent chemical bond is formed with the recognition moiety.
- a PDMS substrate is functionalized with disuccinimidyl suberate (DSS). Examples of other suitable functionalizing agents include those that are listed above.
- the recognition moiety is attached via the functionalization agent.
- the stamp substrate is then exposed to a test sample under conditions such that an analyte (e.g., binding partner of the recognition moiety) suspected of being contained in the test sample is captured by the recognition moiety on the stamp substrate.
- the test sample comprises a body fluid from a test subject. After a period incubation (e.g., 10 minutes to about 10 hours), the stamp substrate is washed.
- the detection substrate is then contacted with the stamp substrate under conditions such that the analyte (e.g., an antibody) is transferred from the stamp substrate surface to the detection substrate.
- the compound used to functionalize the surface of the detection substrate displays a stronger affinity for the analyte than does the recognition moiety so that the analyte is detached from the recognition moiety and transferred to the detection substrate.
- the analyte is captured on a bead that displays a recognition moiety.
- the beads may be formed from latex, polymers, agarose, or other materials and in some preferred embodiments are magnetic.
- the analyte is then transferred to the detection substrate. The transfer may be accomplished in a variety of ways.
- the analyte is eluted from the beads either directly onto the detection substrate or eluted and the transferred to the detection substrate by a method such as spotting.
- the beads exposed to analyte are contacted with the detection substrate so that the analyte is transferred to the detection substrate.
- the detection substrate surface is functionalized with a moiety with a stronger affinity for the analyte than the recognition moiety on the bead so that the analyte is transferred to the detection substrate.
- the signal from the analyte is amplified by binding one or more additional molecules to the analyte prior to elution.
- analyte used is an antibody
- a secondary anti-species antibody e.g., and anti-Fc antibody for a particular species or rabbit-anti-human antibody, mouse-anti-human antibody, mouse-anti-rabbit antibody, etc.
- Enzyme-antibody conjugates, analyte specific second antibodies, gold sol particles and other molecules and molecule systems may also be utilized.
- the analyte detection assays outlined herein may follow an amplification method such as PCR.
- the detection substrate comprises a rubbed polyimide or a polyimide that homeotropically orients a liquid crystal.
- the detection substrates comprises a nanostructured gold surface (e.g., an anisotropically deposited gold surface).
- the gold surface is coated with an organic layer as described above in detail, preferably an amine-terminated organic layer such as ATP.
- the angle of the deposition of the gold may vary from about 5 degrees to about 80 degrees, and is preferably from about 35 degrees to about 60 degrees, and most preferably about 60 degrees.
- the gold surface is chemically modified with a metal carboxylate, for example, Cr(C10 4 ) 2 .
- a liquid crystal is applied to the detection substrate so that the presence of the binding partner on the detection substrate can be detected.
- a variety of liquid crystal-forming substances can be used, including those listed above.
- 5CB is used.
- the detection substrate is used to form an optical cell with another substrate and the liquid crystal is applied to a chamber formed by the two substrates.
- the foregoing methods can be adapted to detect of variety of analyte-recognition moiety combinations, including protein-protein, protein-nucleic acid, nucleic acid-nucleic acid, and other molecular interactions.
- the detection is label free.
- this system is especially useful for multiplexed assays.
- the capture substrate can be functionalized with a variety of recognition moieties in an array that corresponds to a series of discreet detection regions on the detection substrate. Positive signals on the detection substrate can thus be correlated with the particular recognition moiety on the stamp substrate.
- a first B- :, ⁇ / o detection area on the detection substrate can be specific for a first analyte (e.g., an antibody specific for a particular pathogen), a second detection area on the detection substrate can be specific for a second analyte (e.g., an antibody specific for a second pathogen or a different antibody specific for the first pathogen to provide confirmatory 5 results), and so on.
- a first analyte e.g., an antibody specific for a particular pathogen
- a second detection area on the detection substrate can be specific for a second analyte (e.g., an antibody specific for a second pathogen or a different antibody specific for the first pathogen to provide confirmatory 5 results)
- multiplexing is accomplished by the use of at least first and second pluralities of beads functionalized with different recognition moieties. These beads are used in conjunction with multiwell plates.
- the multiwell plate is a 8, 16, 26, 96 or 384 well plate. In some embodiments of this system,
- 10 different subsets of wells in the multiwells plates contain beads functionalized with different recognition moieties.
- solutions suspected of containing an analyte e.g., proteins, peptides, nucleic acids, carbohydrates
- the beads are magnetic beads and a magnet
- the magnet is positionable in relation to the wells so that the beads are attracted to the sides of the wells (see, e.g., figures 18, 19 and 20).
- the beads are treated with an elution solution so that the analyte is eluted into the solution.
- the solution containing the analyte is then transferred to a substrate that orients liquid crystals as described above.
- a variety of methods of transfer may be used.
- the solution containing the analyte can be spotted onto the substrate or transferred to a microchannel in the substrate.
- a stamp is utilized to transfer the analyte to the substrate.
- the stamp comprising a
- the projections comprise a distal end that preferably comprises a material suitable for transferring the solution and thus the analyte to the substrate.
- the material is PDMS. It will be recognized that other materials described above can also be used for the stamp.
- the substrate is prepared so
- the substrate homeotropically orients the liquid crystal.
- the substrate is used to form a cell with a second substrate and the cell is filled with a liquid crystal.
- the second substrate is also prepared so that is orients a liquid crystal, preferably homeotropically.
- the presence of an analyte on the substrate is indicated by a difference in the orientation of the liquid crystal.
- the presence of the analyte is indicated by a bright field area, or randon orientation of the liquid crystal, when the liquid crystal cell is viewed through cross polar lenses.
- the beads are functionalized with antibodies or other proteins that bind cytokines.
- cytokines are known in the art and many are commercially available.
- the present invention is not limited the detection of any particular cytokine. Indeed, the detection of a variety of different cytokines is possible, including the following: interleukin-1 alpha, interleukin-1 beta, interleukin-2, interleukin-4, interleukin-6, interleukin-7, interleukin-8, interleukin- 10, interleukin-12, GM-CSF, interferon-gamma, and TNF-alpha.
- the cytokine assay is multiplexed so that multiple cytokines can be detected in the same assay.
- one subset of wells will contain beads functionalized with antibodies to a first cytokine
- a second subset of wells will contain beads functionalized with antibodies to a second cytokine, and so on.
- Some of the wells may also be utilized as control wells.
- the present invention provides method for the selective detection of specific classes of antibodies.
- beads are functionalized with antibodies specific for particular subclasses (e.g., IgG (e.g., IgGl, IgG2, IgG2a, IgG2b, IgG2c, IgG3, IgG4); IgM; IgAl; IgA2; IgA sec ; IgD; and IgE and combination thereof).
- a solution suspected of containing an antibody of interest is incubated with beads functionalized with antibodies to immunoglobulins of a particular subclasses to remove those antibodies from the solution.
- the solution is then analyzed, preferably by a liquid crystal assay, for antibodies of the desired subclass.
- the presence of IgM, which produced during active infection is assayed by capturing antibodies to a desired antigen with a stamp or some other means.
- the antibodies are transferred to a container (e.g., a well in a multiwell plate) that contains beads functionalized with anti-IgG antibodies.
- the solution is mixed with the beads under conditions such that the IgG is bound by the beads (i.e., removed from the solution).
- the remaining solution is then analyzed by contacting a substrate that orients liquid crystals with the solution, forming a cell with the substrate, and filling the cell with a liquid crystal.
- the substrate homeotropically orients the liquid crystal and presence of the analyte (in this case IgM immunoglobulins) is indicated by areas of disordered liquid crystal, which appear bright when viewed through cross polar lenses.
- the transfer is performed with a stamp as described above. It will be appreciated that this system can be adapted to detect any desired subclass of immunoglobulins. For example, to detect IgA in a sample, the beads can be functionalized with anti-IgG and anti-IgM to remove IgG and IgM from the sample and so forth.
- the label free detection possible with the present system provides advantages over currently used processes such as ELISA.
- the present system does not require a secondary antibody to detect ligand or antigen specific antibodies from a test subject. This is important because the present system can be utilized to detect antigen/ligand specific antibodies from different species in a single assay because separate secondary antibodies specific for each species are not required. This aspect greatly increases the flexibility of the assays and time needed to respond outbreaks of a disease in a wide or previously unstudied population of subjects.
- the present system does not require a labeling systems such as radioactive, fluorescent, or enzymatic system. These systems are often relatively unstable or have short shelf lives and require specialized equipment (scintillation counters, film) that is not readily adaptable to field use.
- kits for the detection of analytes comprise one or more substrates as described in detail above.
- the kits comprise capture and detection substrates.
- the capture substrates are beads or stamps.
- the kits comprise a substrate that can be used in conjunction with the detection substrate to assemble a liquid crystal cell.
- the kits comprise a vial containing mesogens.
- the kits comprise at least one vial containing a control analyte or analytes.
- the kit comprises instructions for using the reagents contained in the kit for the detection of at least one type of analyte.
- the instructions further comprise the statement of intended use required by the U.S. Food and Drug Administration (FDA) in labeling in vitro diagnostic products.
- FDA U.S. Food and Drug Administration
- the FDA classifies in vitro diagnostics as medical devices and requires that they be approved through the 510(k) procedure.
- Information required in an application under 510(k) includes: 1) The in vitro diagnostic product name, including the trade or proprietary name, the common or usual name, and the classification name of the device; 2) The intended use of the product; 3) The establishment registration number, if applicable, of the owner or operator submitting the 510(k) submission; the class in which the in vitro diagnostic product was placed under section 513 of the FD&C Act, if known, its appropriate panel, or, if the owner or operator determines that the device has not been classified under such section, a statement of that determination and the basis for the determination that the in vitro diagnostic product is not so classified; 4) Proposed labels, labeling and advertisements sufficient to describe the in vitro diagnostic product, its intended use, and directions for use.
- photographs or engineering drawings should be supplied; 5) A statement indicating that the device is similar to and/or different from other in vitro diagnostic products of comparable type in commercial distribution in the U.S., accompanied by data to support the statement; 6) A 510(k) summary of the safety and effectiveness data upon which the substantial equivalence determination is based; or a statement that the 510(k) safety and effectiveness information supporting the FDA finding of substantial equivalence will be made available to any person within 30 days of a written request; 7) A statement that the submitter believes, to the best of their knowledge, that all data and information submitted in the premarket notification are truthful and accurate and that no material fact has been omitted; 8) Any additional information regarding the in vitro diagnostic product requested that is necessary for the FDA to make a substantial equivalency determination. Additional information is available at the Internet web page of the U.S. FDA. .
- a pressure gradient between the two ends of the tube induces movement of the fluidic plugs inside the tube without wetting the surface.
- Magnetic beads suspended in the fluid can be pulled down and secured at a location within the tube by applying of a magnetic field. Mixing the beads inside the tube can be achieved by generating a relative motion between the magnet and the tube.
- the application of a magnetic field, movement of the fluid plugs, and rotation of the tube in the magnetic field allows the magnetic beads to be mixed in the fluid. Moving the fluidic plugs through tube in or out of the magnetic field by applying a pressure or vacuum between in the tube allows for successive treatment of the magnetic beads with different reagents and capture of target from a sample.
- One fluid plug in the tube can be an elution buffer.
- the eluted buffer may be dropped on to a reporting surface that is used in conjunction with liquid crystals for detection.
- the principle of the operation of magneto-fluidic assay is schematically depicted in figure 7.
- the current invention relates to the capture specific biological interactions such as antigen-antibody interactions, protein-protein interactions, etc.
- the presence of sample can be reported using polyimide coated glass substrates.
- the presence of sample can be reported using the other reporting surfaces including but not limited to rubbed polyimide, nanostructured gold surfaces, PDMS channels etc.
- the fluidic tubes can also be integrated directly with PDMS channels so that the additional steps associated with the dropping the eluant from the tube to a reporting surface can be avoided.
- the PDMS channels can be attached to the end of the tube and a two-way valve can direct the excess fluid to a reservoir and the eluant to the PDMS channels for reporting.
- the tube is not limited to a particular kind or shape.
- interior of the tube could be in the shape of a triangle, oval, or square instead of a circle.
- the fluidic tube may have multiple bents or it could be made of a flexible material (e.g. rubber tubing).
- the inner surfaces of these tubes are chemically functionalized to achieve the desired surface characteristics for forming fluidic plugs.
- Sera-Mag beads (0.8 ⁇ M in diameter) were functionalized with either 0.4mg/mL EDC (Aldrich) or 1.1 mg/mL Sulfo-NHS (Pierce).
- EDC Aldrich
- Sulfo-NHS Purifier
- 27 ⁇ l of 5% Sera-Mag beads were diluted in ImL of the functionalizing agent. Reactions were carried out 15 minutes and then quenched with 2-mercaptoethanol. The beads were washed 3 times with 25mM MES and 37.5mM NaCl. The washed beads were centrifuged at 11,300 rpm for 5 minutes. Removal of buffer was followed by the addition of fresh buffer.
- aFlpAb (lOO ⁇ g/mL) was added for 1.5-2 hours during which the beads were rotated and mixed.
- the beads were quenched for amine by adding a final concentration of 1OmM D-glucosamine.
- the beads were then washed in PBS+ D- glucusoamine (1OmM) for 20 minutes.
- the beads were transferred to regular microfuge tubes blocked with BSA to prevent non-specific binding of non-target molecules to the beads.
- Magnetic beads were functionalized with anti-Fl polyclonal antibody as described above.
- a binding solution was prepared from PBS, Tween 0.05%, Heparin 50 ⁇ g/mL, and 10OmM additional NaCl.
- Wash solutions were prepared from PBS/Tween 0.05% and PBS/Tween 0.05%+200mM additional NaCl.
- the beads were functionalized and stored in PBS at 0.135% w/v, the same concentration at which the beads were utilized.
- ImL of bead solution was utilized and bound up to 5 ⁇ g/mL of Fl at over 99.0%.
- An ELISA on Fl samples applied before and after capture on the on the beads was carried out.
- the Fl samples were placed on ELISA strip wells measured for levels of Fl protein using anti-Fl polyclonal antibody.
- the two conditions, start material and depleted material, were then graphed against absorbance 450nm as result of ELISA colorimetric assay. This allowed to estimation of % of Fl captured by the functionalized beads. The data showed that almost all the Fl was absorbed to the beads.
- the Fl antigen was then eluted to a detection substrate surface comprising polyimide.
- the polyimide surface is prepared as follows: Undiluted polyimide 7511 liquid is applied to a slide, spin coated, pre-cured at 8O 0 C for 15 minutes, cured at 180 0 C for one hour and stored at RT.
- lOuL 0.1M pH 2.4 glycine is mixed with the particle beads, and the tubes are then vortexed in a microfuge and placed on a magnetic stand to obtain separation of beads from supernatant.
- the low pH glycine serves to elute bound Fl and its antibody complex from the bead surface.
- lOuL of the eluent is applied to the polyimide surface for the LC assay.
- an optical cell was formed by adding a second substrate separated from the detection substrate by a thin mylar film. Mesogens (5CB) were then introduced into the optical cell.
- aFlpAb beads In the top row, from left to right, are elution samples from aFlpAb beads initially blocked with RNase A treated with BSA 400ng/mL, Fl 500pg/mL and 2.5ng/mL. The bottom row is 25, 100, and 400ng/mL concentrations of Fl. After initial protein binding in binding solution PBS/T/H/N, aFlpAb beads were washed in PBS/Tween 0.05%, 20OmM additional NaCl (PBS/T/N) wash solution.
- Figure 1 demonstrates that with increasing Fl concentration, the disruption of homeotropic alignment increases as indicated by increased white signal against aligned LC black background on homeotropic aligning polyimide surfaces. This type of assay can be used to quantify levels of the Fl antigen.
- Figure 2 is a graphic representation of luminosity index for the experiment depicted in Figure 1. These experiments were carried in triplicate and figure chosen above have intermediate levels of signal with respect to two other replicates. Elution buffer was also applied to one area and used as reference for Luminosity index shown in the graph below. In further experiments, a rubbed polyimide surface was utilized. Figure 3 shows a digitized image taken with a polarized microscope with cross polar filters (0 degrees). Polyimide SE 7210 1.5% slides were rubbed at 2.55ml/m setting at 4.0cm/s table speed with a wheel speed of 343rpm.
- Figure 3 shows the results of elutions to the polyimide surface from functionalized aFlpAb beads that were treated with various concentrations of Fl or BSA as control. After washing, the beads were treated by the addition of rabbit anti-mouse IgG (5 ⁇ g/mL) and anti- mouse FC IgG (6 ⁇ g/mL). The complex was eluted off of the beads with lO ⁇ l acid elution of 0.1M glycine pH 2.3. Elution samples were neutralized by addition of l ⁇ l 1 M Tris pH 7.5. The samples were contacted with the polyimide substrate and an optical cell was constructed by placing a mylar spacer on the substrate clamping another substrate onto the first with bulldog clamp. 5CB was applied in liquid phase and after cooling the digitized image was acquired.
- FIG. 4 provides the results of further experiments demonstrating the detection of an analyte (Fl) from a complex medium (chicken serum).
- chicken serum CS
- BSA blocked beads functionalized as above were used.
- Six 0.5mL suspensions of aFlpAb beads (0.135% concentration) were contacted with the diluted CS. After binding, all tubes were washed with PBS/Tween buffer. The beads were then washed a second time with lOO ⁇ L 0.5X PBS+2mM SDS or lOO ⁇ L O.lMglycine pH 4.0. After mixing, the wash buffers were removed by pelleting the beads in PBS/Tween 0.05%.
- FIG. 4 is a digitized image taken with cross polar filters (0 degrees). In top row are elutions from beads treated contacted with 20% CS with no additional wash, SDS wash, and a 0. IM glycine pH 4.0 wash.
- Figure 5 presents the results of the same experiments as analyzed with a polarized microscope. The digitized images were taken by polarized microscope where the polaroid filters are cross polar at 0 °. The order of the treatments is the same as for Figure 4.
- Magnetic beads typically with diameter of 0.8 micron (Seradyne, Indianapolis, IN), are functionalized with a carboxylic acid group.
- Target molecules specific to a receptor i.e., primary antibody
- BSA bovine serum albumin
- These receptor functionalized and BSA blocked magnetic beads are exposed to sample fluid containing target molecules (i.e., antigen).
- the magnetic beads are washed with PBS/Tween20 solution followed by SDS buffer removing nonspecifically bound proteins including the BSA.
- the magnetic beads are exposed to a sample containing a secondary antibody that also binds to the target molecules.
- a rinse with PBS/Tween20 removes the nonspecifically bound secondary antibody from the beads.
- the magnetic beads are subjected to an elution buffer, a low pH glycine buffer or a urea solution, that breaks the antibody-antigen binding. If the magnetic beads had any target molecules captured, the , eluted sample would have both target molecules and the secondary antibody, The beads are pulled down using a magnet and the eluant is extracted, The eluted buffer is dried onto a polyimide coated surface (SE 1211 Nissan Chemicals) or passed through polydimethylsiloxane (PDMS) [Dow Chemicals] micro fluidic channels both of which align liquid crystals homeotropically (perpendicular to the surface).
- PDMS polydimethylsiloxane
- a liquid crystal cell is fabricated with these surfaces or liquid crystal is passed through the channels. Presence of the target molecules on the sample manifests as a random planar alignment of liquid crystals on the spots or on the micro fluidic channels which is detected by two polarizers set up in series so that their optical axes are parallel.
- Antibody functionalized BSA blocked magnetic beads as described in Example 25 suspended in solvent are placed in a low retention tube.
- a magnet is positioned near the beads on the outside of the tube causing the beads to accumulate near the magnet on the inside of the tube.
- the solvent is removed.
- a binding buffer is added to the tube containing the beads. These beads are ready for capture of an antigen specific to the receptor (i.e., antibody).
- a 1.0 ml serological pipette (Corning Inc.) is attached to a variable flow mini pump (VWR Scientific).
- a plug of elution buffer (0.1 M glycine with pH 2.4) is placed within a pipette using a pressure gradient between two ends of the tube.
- the plug of elution buffer is moved through the tube allowing an air pocket to form at the end of the tube where the elusion buffer entered the tube.
- fluid plugs containing 50-100 ⁇ l of PBS/Tween 20 (0.05%), 50- 100 ⁇ l of antibody specific to the target in binding buffer (PBS/Tween 20 0.05 %/NaCl 100 mM), 50-100 ⁇ l of SDS 2mM/0.4x PBS, 50-100 ⁇ l of PBS/Tween 20 0.05 % with additional 200 mM NaCl, and finally 100 ⁇ l of -0,01 % of the antibody functionalized BSA blocked magnetic beads in the binding buffer.
- All these fluid plugs are separated by ⁇ 100 ⁇ l air pockets.
- a 50 ⁇ l sample containing target molecules (i.e., antigen) in human serum is then injected to the first plug containing the magnetic beads using a syringe. .. .,. ... reckon ,. ⁇ .
- the pipette is mounted near a permanent magnet of strength ⁇ 2kGauss to create a magnetized area within the pipette.
- the tube is slowly rotated. This introduces a rigorous mixing of the beads in the binding buffer to allow the capture of target molecules from the sample. After a few rotations of the pipette, the rotation is ceased, and the beads are pulled down at the bottom of the tube by the magnet.
- the binding buffer plug is then pushed across the pipette away from the magnetic beads using the pump leaving the beads bound to the bottom of the pipette in the magnetized area. The pressure gradient pushing the fluid plug through the pipette is maintained until the next fluid plug comes over to the magnetized area.
- the pump is turned OFF and the tube is rotated again a few times to allow mixing of the beads in the fluid of the plug. Again, the beads are pulled down to the bottom of the pipette and the next fluid plug is brought over the beads in the magnetized area. This process is repeated with all the reagents in each plug until the elution buffer plug comes over to the magnetic beads.
- the PBS/ Tween20 and the SDS buffer remove the non-specific binding whereas the secondary antibody amplifies the signal.
- the elution buffer comes in to contact with the beads and the pipette is rotated. This rotation causes the antibodies to come off of the beads.
- the beads are pulled down and the eluted buffer is dropped onto a polyimide (PI) coated glass slide.
- PI polyimide
- the droplet is allowed to evaporate on the surface, rinsed with water, and a liquid crystal cell is fabricated by pairing this surface with another PI coated surface separated by a 25 micron gap.
- the cavity is then filled with liquid crystal 5CB and the cell is observed between two crossed polarizing films (see figure 8).
- the eluant In presence of the target in the sample, the eluant will have antibodies and target molecules in it which bind to the polyimide surface.
- the invention is not limited to any particular mechanism, we believe the presence of target molecules on the polyimide surface causes the liquid crystals to orient randomly parallel to the surface. When viewed in between crossed polarizers, the homogeneous (planar) texture oriented film of liquid crystal causes the appearance of a bright spot.
- the sample does not contain the specific target molecules nothing comes off the beads to bind to the polyimide surface and the liquid crystals align perpendicular to the surface as dictated by the polyimide surface causing the liquid crystal to appear dark between crossed polarizers.
- the lateral flow system was used to analyze the presence of Fl antigen in a sample.
- a sample as low as 1 ng/mL was readily detected, corresponding to an absolute detection limit of 50pg of Fl antigen.
- the buffers, beads and conditions are as described in Example 22.
- Binding buffer 100 ⁇ L+ 50 ⁇ L of sample Human Sera containing 0, 1, 5, and IOng/mL of Fl was applied to the functionalized magnetic beads.
- lOO ⁇ L washes of PBS+Tween20 0.05%+ 20OmM NaCl and SDS 2mM in 0.4XPBS were performed.
- 100 ⁇ L aFlpAb lO ⁇ g/mL used and finally beads were washed with lOO ⁇ L PBS+Tween20 0.05%.
- the magnetic beads typically with diameter of 0.8 micron (Seradyne, Indianapolis, IN), are functionalized with appropriate chemistries, such as with carboxyl groups.
- Target receptor molecules such as antibodies are then immobilized onto the beads using carbodiimide chemistry. The remaining unoccupied sites are then blocked with some other protein such as bovine serum albumin (BSA) to prevent non specific binding.
- BSA bovine serum albumin
- these beads are washed with PBS/Tween20 solution followed by SDS buffer to remove nonspecifically bound proteins.
- the beads are exposed to a reagent solution containing the secondary antibody capable of binding to the target molecules, and a final rinse with PBS/Tween20 removes the nonspecifically bound secondary antibody from the beads.
- a reagent solution containing the secondary antibody capable of binding to the target molecules
- PBS/Tween20 removes the nonspecifically bound secondary antibody from the beads.
- these beads are subjected to an elution buffer (for example, a low pH glycine buffer or a urea solution) that breaks the antibody-antigen bonds. If the sample has any target molecules captured, the eluate will contain the secondary antibody and some portion of the captured target molecules.
- the captured target molecules may stay bound to the primary antibodies on the bead surfaces and will not be eluted. Since the liquid crystal assay is designed to measure the mass of protein in a sample, the absence of a portion of a relatively low molecular weight target molecule is not considered detrimental to the overall signal generated in the assay. The bulk of the signal will result from the relatively high molecular weight secondary antibody.
- the beads are pulled down using a magnet and the eluate is extracted.
- the eluted sample is applied and dried down onto a polyimide coated surface (for example, SE 1211 Nissan Chemicals) or passed through polydimethylsiloxane (PDMS) [Dow Chemicals] micro fluidic channels both of which align liquid crystals homeotropically, i.e., perpendicular to the surface.
- PDMS polydimethylsiloxane
- a liquid crystal cell is fabricated with these surfaces or liquid crystal is passed through the channels. Presence of the target molecules on the sample manifests as a random planar alignment of liquid crystals on the spots or on the micro fluidic channels.
- the polyimide or PDMS surfaces display a homeotropic liquid crystal alignment yielding a dark appearance when viewed through crossed polarizing filters. In the presence of eluted protein, the surfaces display a random alignment yielding a bright appearance.
- Example 22 The procedure described in Example 22 was conducted in individual 1.5 ml microcentrofuge tubes.
- This present assay has been adapted to be performed in a 96 well plate using strips of 8 wells (for example, Nunc's PlySorp® 96 well plate, Cat# 475086).
- the plate format allows for numerous samples to be simultaneously manipulated using a muiti channel pipette.
- a base plate has been constructed consisting of a plastic base with magnets mounted at set intervals. See Figures 18 and 19.
- the 96 well plate is set onto this magnetic base plate and the beads in each well are of with magnets placed between columns of wells. The beads are attracted by the magnets to the side of the well and held in place as the media is removed for the next step. See Figure 11.
- the 96 well plate is removed from the magnetic base, allowing the beads to resuspend by the addition of media from the next step of the process.
- the plate format also allows for easy multiplexing, allowing for the detection of multiple agents within the same sample.
- the functionalized beads capture and remove only the specified antigen from the sample, leaving all non specific antigens in the sample. Once the beads are separated by the magnet, the sample is transferred to a second well. The second well contains beads functionalized against a different antigen, capturing only those specific antigens.
- This procedure could be carried out several times with each well containing beads functionalized to different antigens. This would allow several tests to be run simultaneously on the same initial (50 - 200 ⁇ l) sample. This multiplexing ability would be extremely useful in settings that are limited by initial sample size, such as small animal veterinary medicine or pediatrics.
- Example 5 Bead-based capture of Ras on nano or micrometer sized beads for detection
- purified Ras and purified non-specific proteins e.g., BSA
- Ras-free cell lysates with purified Ras spiked at known concentrations.
- 3T3 cells are stimulated with EGF (5OnM) and then lysed (ImI of cold lysis buffer) and centrifuged.
- Ras present in the extract is fully activated by addition of 5ml of 1OmM GTPgS, and then incubated with Rafl- Ras binding domain coupled to agarose beads. After gentle mixing of the reaction mixture for 45 minutes at 4 ° C, the beads are separated by centrifugation.
- the supernatant is depleted of Ras, aliquoted, snap frozen and stored at -80 ° C.
- the absence of Ras from the extract is confirmed by using ELISA, polyacrylamide gel and Western blot analysis.
- Ras depleted extract is spiked with known amounts of pure Ras for quantification of Ras binding.
- ELISA is used to quantify the fraction of Ras captured onto beads using the following procedures. First, an ELISA standard curve is determined in order to establish the level of Ras binding to Reacti-BindTM maleic anhydride wells (Pierce, Rockford, IL) using a given anti-Ras antibody concentration. Following the primary antibody, a second anti-species antibody against primary antibody is added which has a HRP conjugate.
- the TMB color developing reagent is added and upon color formation, the reaction is stopped by addition of H2SO4 which also turns the color of the solution in the wells to yellow.
- the absorbance is read at 450nm.
- Antibody to Immobilize on the Magnetic Beads A number of commercial sources of anti-Ras antibodies are available. These antibodies have been validated for use in immunoprecipitation and Western blots. These antibodies are screened for their binding activity when immobilized on the surfaces of magnetic beads.
- the commercial antibodies to be evaluated are:Anti-Ras, Clone RASlO; Catalog # 05-516, Upstate
- Immobilization Chemistry Optimal procedures to immobilize anti-Ras antibodies to the surfaces of magnetic beads presenting carboxylic acids groups and primary amine groups are identified. A number of cross-linking chemistries are evaluated. These cross-linking chemistries lead to the immobilization of antibodies in random orientations.
- the oriented immobilization of antibodies on surfaces e.g., via use of protein A or G, or via reaction with carbohydrates localized on the Fc region of the antibody
- the areal densities of antibodies immobilized in defined orientations on surfaces are lower than the areal densities of antibodies immobilized with random orientations.
- the present methods utilize immobilization of antibodies with random orientations - via primary amines on the surface of the antibodies. Chemistries and procedures leading to immobilization of antibodies without preferred orientations are substantially simpler than procedures and chemistries for oriented immobilization.
- a second criterion used to select the optimal immobilization chemistry is the minimization of non-specific binding of proteins to the beads. Using the ELISA-based procedures described above to determine capture of Ras, the depletion of non-specific proteins from solutions is measured to determine the extent of non-specific binding of proteins to the beads.
- a fundamental and general advantage of magnetic bead-based assays is the 5 rapid mass transport of the target to a surface-immobilized binding group, by virtue of the mobility of the binding group located on a bead as opposed to a macroscopic (immobile) surface.
- the present invention is not limited to a particular mechanism. Nor is an understanding of the mechanism necessary to practice the present invention. Nonetheless, it is contemplated mat because small beads diffuse more rapidly than large
- Nano-magnetic beads 15 sources for nano- and micro-sized magnetic beads that present a variety of surface chemistries, including COOH- and NH2 chemistries for suitable for covalent attachment of anti-Ras antibodies.
- Nano-magnetic beads are purchased from Miltenyi Biotec (50nm average diameter), BD Biosciences (200nm average diameter), Stem-Cell (50nm average diameter) and Immunicon Corporation (200nm average diameter).
- Miltenyi Biotec 50nm average diameter
- BD Biosciences 200nm average diameter
- Stem-Cell 50nm average diameter
- Immunicon Corporation 200nm average diameter
- Additional parameters are assayed to optimize the capture of Ras on magnetic beads.
- different chemistries are employed for immobilization (e.g., maleimide-based chemistries) of the antibodies, including the use of molecular spacers (e.g., ethylene glycol spacers) to increase the distance between the antibodies and surfaces of the beads.
- molecular spacers e.g., ethylene glycol spacers
- alternative blocking strategies including grafted PEG layers on the beads and use of non-ionic surfactants (e.g., Triton surfactants) are assed.
- the second step in conducting a Ras assay is the transfer of the Ras captured by anti-Ras-decorated magnetic beads onto nanostructured surfaces for the detection, signal amplification and reporting of Ras by using liquid crystals.
- the approach to the optimization of the transfer of Ras from the beads to the capture surface is based first on the use of aqueous solutions of purified Ras, and subsequently validated using Ras-free cell lysates doped with known concentrations of Ras.
- a number of procedures for the transfer of Ras from beads to the reporting surfaces are assayed (see below). In some of these procedures, Ras is eluted from the beads into a solution that is subsequently spotted on the reporting surface.
- ELISA assays are used to determine the fraction of Ras captured on beads that is released into the eluant, and the fraction of Ras in the eluant that is not transferred from the eluant to the reporting surface.
- Ras is transferred from the beads directly to the reporting surface by physical contact of the beads with the reporting surface. This procedure has precedent in that contact printing has been shown to permit the transfer of proteins from stamps directly to reporting surfaces.
- fluorescence microscopy in combination with grey scale analysis is used to compare the relative amounts of Ras transferred to the reporting surface by the different procedures.
- the Alexa Fluor 488 label is covalently attached to Ras.
- Ras protein is diluted into 2 mg/ml with 0.1 M sodium bicarbonate, and then incubated with one vial of reactive Alexa Fluor 488 dye (Abs/Em: 494/519 nm) (Molecular Probes; Catalog # A 10235) for 1 hour at room temperature with stirring.
- the reaction mixture is applied onto a Bio-Rad BioGel P-6 size exclusion resin column pre-equilibrated with PBS and eluted with same buffer. Labeled proteins are eluted first then the unincorporated dye. The degree of labeling is determined by measuring the absorbance of the conjugated protein at 494 nm.
- Quantification of labeled Ras is carried using fluorescence microscopy (Axiovert 200 Zeiss fluorescence microscope) in combination with grey scale analysis of captured images. The optimal transfer process is determined by the evaluation of 4 candidate procedures:
- Parameters to be optimized in this procedure are (I) elution conditions (buffer components, pH, temperature, duration), (II) volume of eluant phase relative to bead number for each size of bead evaluated, (III) spotting conditions (volume of eluant containing Ras, adsorption time), (IV) conditions for rinsing of the reporting surface (solvent, volume, procedure).
- elution conditions buffer components, pH, temperature, duration
- volume of eluant phase relative to bead number for each size of bead evaluated are evaluated.
- III spotting conditions (volume of eluant containing Ras, adsorption time)
- IV conditions for rinsing of the reporting surface (solvent, volume, procedure).
- elution was carried out in 0. IM aqueous glycine at pH 2.4. The beads were mixed with 2OuL of solution, for 2 minutes and the elution buffer was separated from the beads using a magnet.
- methyl or amine-terminated monolayers are very effective as adsorbing a wide variety of proteins from solution.
- Nanostructured surfaces supporting methyl- or amine- terminated monolayers are used to capture Ras in the channels.
- the use of the microfiuidic channels enables the quantitation of the amount of Ras in a sample. The greater the amount of Ras, the longer the reaction zone within the channel. PDMS
- beads loaded with labeled Ras are contacted with surfaces presenting amine, carboxylic acid groups and metal salts (prepared by using self-assembled monolayers on gold films), and fluorescence microscopy is used to compare the relative amounts of Ras transferred to the reporting surfaces.
- Addition optimization experiments include altering the chemistry of the nanostructured surface to increase the strength of adsorption of Ras, reducing the flow rate of eluant through the channels by constricting the channel exit (this will increase the time available for adsorption of the Ras in the channel), and blocking of the channel entrance to minimize loss of Ras to the walls of the PDMS.
- the nanostructured surface can also be preloaded with a protein such as BSA.
- This example describes the optimization of the design of nanostructured surfaces for the label-free, direct reporting of Ras using liquid crystals.
- Past studies have established that the orientational ordering of liquid crystals on nanostructured surfaces is influenced by the nanometer-scale topography of the surfaces as well as the chemical functionality of the surfaces. Similarly, the orientational response of liquid crystals to the presence of Ras captured on a surface is influenced by these same variables.
- a range of materials are screened for their ability to report Ras via the orientational ordering of liquid crystals.
- Initial experiments are performed by spotting aqueous solutions of Ras of known concentrations on the surface of the nanostructured materials, rinsing the surfaces free of salts, and then placing liquid crystal into contact with the surface.
- Polarized light microscopy is used to image the orientational order of the liquid crystals on the nanostructured surfaces. Images are captured digitally, and analyzed using image analysis software (Image J, NIH Image). It is also possible to quantify the optical response of the liquid crystal to proteins by use of standard multiwell plate reader technology. Evaluation of the nanostructured reporting surfaces characterizes three parameters of the assay: sensitivity, dynamic range; and reproducibility.
- the following materials containing nanostructured surfaces are screened as candidate interfaces for the reporting of Ras:
- Gold surfaces with nanometer-scale topography Past studies have demonstrated that the physical vapor deposition of gold at oblique angles to the surface can lead to the preparation of gold films that possess a nanometer-scale topography that defines the azimuthal orientation of liquid crystals contacted with these surfaces. Proteins when bound to these surfaces mask the nanometer-scale topography, leading to an orientational ordering of the liquid crystal in the presence of the protein that is distinct from the orientational ordering of the liquid crystal in the absence of bound protein. Parameters to be optimized include (I) angle of deposition of the gold, (II) rate of deposition of the gold, (III) thickness of gold films.
- Titanium surfaces with nanometer-scale topography Titanium surfaces with nanometer-scale topography. Whereas gold films deposited at oblique angles have been widely used in past studies of the orientational behavior of liquid crystals at interfaces, it is contemplated that titanium surfaces may yield nanostructured surfaces that have robust and long-lived topographies that can orient liquid crystals. Because the melting temperature of Ti is substantially higher than gold, these films may be more robust and possess longer shelf-lives than gold films.
- the orientational behavior of liquid crystals on titanium surfaces prepared by oblique deposition is investigated, and the response of the liquid crystals to Ras deposited onto the surface of the Ti. Preliminary results have established the feasibility of orienting liquid crystals on Ti surfaces (which form a native oxide under standard laboratory conditions). ,. ,,, , . .,, ..,.,
- polyimide SEl 211 (Nissan Chemicals) was used to achieve the homeotropic alignment of liquid crystals on the surface.
- these polyimides possess a number of aliphatic side chains (with n >12) attached to the main chain.
- a thin ( ⁇ 20 nm) film of polyamic acid is cured to form polyimide film, a significant number of these aliphatic chains orient normal to the surface.
- These oriented aliphatic chains provide a template for the homeotropic alignment of the liquid crystals supported on them.
- Figure 13 A schematic diagram illustrating the homeotropic alignment of liquid crystals on the polyimide coated surface is shown in Figure 13.
Abstract
Description
Claims
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PCT/US2006/000688 WO2007081326A2 (en) | 2006-01-09 | 2006-01-09 | Bead based assays using a liquid crystal reporter |
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EP1971691A2 true EP1971691A2 (en) | 2008-09-24 |
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EP06733654A Withdrawn EP1971691A2 (en) | 2006-01-09 | 2006-01-09 | Bead based assays using a liquid crystal reporter |
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EP (1) | EP1971691A2 (en) |
AU (1) | AU2006335329A1 (en) |
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JP5844156B2 (en) | 2008-10-27 | 2016-01-13 | アドヴァンテイジアス システムズ エルエルシーAdvantageous Systems,Llc | Water purification using magnetic nanoparticles |
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US20030127396A1 (en) * | 1995-02-21 | 2003-07-10 | Siddiqi Iqbal Waheed | Apparatus and method for processing magnetic particles |
WO2002071929A2 (en) * | 2001-03-14 | 2002-09-19 | Burnstein Technologies, Inc. | Methods of decreasing non-specific binding in dual bead assays and system apparatus for detecting medical targets |
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2006
- 2006-01-09 WO PCT/US2006/000688 patent/WO2007081326A2/en active Application Filing
- 2006-01-09 AU AU2006335329A patent/AU2006335329A1/en not_active Abandoned
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WO2007081326A3 (en) | 2008-10-30 |
AU2006335329A1 (en) | 2007-07-19 |
WO2007081326A2 (en) | 2007-07-19 |
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