Classifications

• • 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
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
• • 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

Definitions

• compositions for Making the Same filed August 16, 2002, in the name of David Quincy, which is incorporated herein by reference.
• FIELD This disclosure concerns a class of reagents and a method for surface functionalization for creating arrays of bioactive compounds on a substrate.
• proteomics This study of proteins and their interactions is termed "proteomics.” Due to advances in molecular biology, proteins of interest can be produced more rapidly than current techniques can characterize the proteins. For example, conventional methods for protein identification in proteomics applications rely upon two-dimensional, polyacrylamide gel electrophoresis (2D-PAGE) technology to isolate proteins, followed by subsequent identification by mass spectrometry. Typically, 2D-PAGE can separate as many as 5000 different proteins. However, identification of each protein is a tedious, labor-intensive process that requires cutting each of the individual separated proteins out of the polyacrylamide gel. Moreover, the method has a relatively low sensitivity. For example, when using silver staining, the detection limit is about 1 nanogram of protein.
• 2D-PAGE Additional drawbacks include a lack of reproducibility, low throughput, low resolution and protein-dependent sensitivity.
• 20- PAGE generally does not resolve all proteins present in a mixture and systems are limited to processing a handful of gels over a two-day period.
• high- molecular-weight, low-molecular- weight and membrane-bound proteins are underrepresented.
• U.S. Patent No. 6,406,921 to Wagner et al. discloses a method of making protein-coated substrates
• U.S. Patent No. 5,620,850 to Bamdad et al. discloses a method for making a surface including a plurality of chelating agents, which can be used to bind metal ions. The bound metal ions are then reportedly used to capture a biological molecule that also includes a chelating agent.
• a number of hurdles must be overcome to provide protein arrays of high quality which produce accurate and reproducible screening results.
• proteins must remain hydrated, be kept at ambient temperatures, and are very sensitive to the physical and chemical properties of the support materials. Thus, maintaining protein activity at the liquid-solid interface requires new strategies for assembling arrays that address the sensitivity of the proteins to the environment.
• reagents and a method for using such reagents to prepare substrates derivatized with affinity agents, such as small molecules, peptides, proteins, nucleic acids and other bioactive molecules.
• affinity agents such as small molecules, peptides, proteins, nucleic acids and other bioactive molecules.
• densely packed affinity agent arrays exhibiting minimal, non-specific binding can be prepared.
• a substrate is functionalized with a reagent having at least two reactive functional groups.
• the first reactive functional group serves to couple the reagent to the substrate and the second reactive functional group is an affinity agent or provides a site for operatively associating an affinity agent.
• non-specific protein adhesion to the arrays is diminished by the incorporation of a protein resistant component, which diminishes such non-specific protein adhesion.
• the disclosure provides reagents and techniques for assembling arrays of affinity agents that exhibit reduced non-specific binding.
• FIG. 1 illustrates one embodiment of the disclosed method for functionalizing a surface.
• FIG. 2 A is a schematic representing one embodiment of the disclosed array.
• FIG 2B provides examples of monolayer components used to prepare such arrays.
• FIG. 3 A illustrates a method for using an asymmetric disulfide reagent to prepare a monolayer.
• FIG. 3B depicts examples of monolayer components prepared via Staudinger ligation.
• FIG. 4 illustrates one example of a process for preparing a novel array.
• FIG. 5 is a schematic of a fluorescence detection unit which may be used to monitor interaction of the proteins of the array with an analyte.
• FIG. 6 is a schematic of an ellipsometric detection unit which may be used to monitor interactions between analytes and the affinity tags of the array.
• FIG. 7 illustrates the synthesis of an asymmetric disulfide having N- hydroxysuccinimide and polyethylene glycol groups.
• substrate refers to a bulk, underlying material used in the arrays and devices disclosed herein.
• an “array” refers to a two-dimensional distribution or pattern.
• polypeptide and “protein” are used interchangeably to refer to an amino acid polymer.
• antibody means an immunoglobulin, whether natural or wholly or partially synthetically produced. All derivatives thereof which maintain specific binding ability are also included in the term. The term also covers any protein having a binding domain which is homologous or largely homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources, or partly or wholly synthetically produced.
• An antibody may be monoclonal or polyclonal. In an exemplary embodiment, the antibody is a glycosylated antibody.
• the antibody may be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE.
• the antibody is of the IgG class.
• the term "antibody fragment" refers to any derivative of an antibody which is less than full-length. In an exemplary embodiment, the antibody fragment retains at least a significant portion of the full-length antibody's specific binding ability. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 , scFv, Fv, dsFv diabody, and Fd fragments.
• the antibody fragment may be produced by any means.
• the antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody or it may be recombinantly produced from a gene encoding the partial antibody sequence.
• the antibody fragment may be wholly or partially synthetically produced.
• the antibody fragment may optionally be a single chain antibody fragment.
• the fragment may comprise multiple chains which are linked together, for instance, by disulfide linkages.
• the fragment may also optionally be a multimolecular complex.
• a functional antibody fragment will typically comprise at least about 50 amino acids and more typically will comprise at least about 200 amino acids.
• Single-chain Fvs are recombinant antibody fragments consisting of only the variable light chain (V L ) and variable heavy chain (N ⁇ ) covalently connected to one another by a polypeptide linker. Either N L or N ⁇ may be the ⁇ H 2 -terminal domain.
• the polypeptide linker may be of variable length and composition so long as the two variable domains are bridged without serious steric interference.
• the linkers are comprised primarily of stretches of glycine and serine residues with some glutamic acid or lysine residues interspersed for solubility.
• a Fv fragment is an antibody fragment which consists of one V H and one V domain held together by noncovalent interactions.
• dsFv is used herein to refer to an Fv with an engineered intermolecular disulfide bond to stabilize the
• a F(ab') 2 fragment is an antibody fragment essentially equivalent to that obtained from immunoglobulins (typically IgG) by digestion with an enzyme pepsin at pH 4.0-4.5. The fragment may be recombinantly produced.
• a Fab' fragment is an antibody fragment essentially equivalent to that obtained by reduction of the disulfide bridge or bridges joining the two heavy chain pieces in the F(ab') 2 fragment. The Fab' fragment may be recombinantly produced.
• a Fab fragment is an antibody fragment essentially equivalent to that obtained by digestion of immunoglobulins (typically IgG) with the enzyme papain. The Fab fragment may be recombinantly produced.
• amino acid refers to both naturally occurring and “unnatural” amino acids.
• Residues of amino acids also are encompassed by the term amino acid.
• Organic thinfilm refers to a thin layer of organic molecules formed directly on a substrate or on a coating on the substrate.
• the organic thinfilms disclosed herein are from about 0.5 nm to about 50 nm thick, and more typically from about 0.5 nm to about 10 nm thick.
• the organic thinfilm can be assembled prior to deposition on the substrate; however, typically the organic thinfilm is assembled as the component molecules are attached to the substrate.
• the organic thinfilm optionally includes associated inorganic ions and chelated metals bound to the thinfilm.
• the organic thinfilm can be homogeneous or heterogeneous and can be composed of one or more monolayers. An example of a thinfilm composed of plural mono layers is a lipid bilayer.
• the organic thinfilm is a monolayer.
• the organic thinfilm can include more than one type of organic thinfilm.
• the organic thinfilms disclosed herein include an affinity agent or a functional group suitable for covalently or noncovalently associating an affinity agent to the thinfilm.
• the organic thinfilm also optionally can bear functional groups that reduce the association of molecules with the thinfilm.
• such functional groups are hydrophilic groups, such as, for example, polyalkylene oxides, including polyethylene glycol (PEG) and polypropylene glycol (PPG), which generally are not bound tightly by proteins.
• PEG and PPG include oligomers of ethylene glycol and propylene glycol, respectively.
• PEG and PPG as used herein refer to polymers having as few as two glycol subunits.
• PEG was used as a protein-resistant component of organic thinfilms to reduce the nonspecific binding of proteins to the organic thinfilm.
• PEG components that were used were not monodisperse, and therefore the specific numbers of ethylene glycol units referred to can also refer to an average number where a polydisperse mixture of PEG units are used.
• Other functional groups of the organic thinfilm serve to tether the thinfilm to the surface of the substrate or a coating on the substrate.
• the term "monolayer” refers to a layer having a single-molecule thickness, which can be an organic thinfilm or portion thereof.
• a monolayer can be ordered or disordered. Typically the monolayer is ordered and densely packed.
• the monolayer can be homogeneous or heterogeneous, however one face of the monolayer will include functional groups that can be chemisorbed or physisorbed onto the surface of the substrate or a coating on the substrate.
• a first monolayer also can be converted into a second monolayer.
• the component molecules in a first monolayer having a first end chemisorbed or physisorbed to a substrate can be functionalized by covalently bonding one or more second molecules to plural second ends of the first monolayer component molecules, thereby converting the first monolayer into a new, second monolayer.
• Arrays of Affinity Agents The present disclosure is directed to arrays of affinity agents and methods for making and using such arrays. Typically the arrays form a two-dimensional display of an affinity agent, which can be used to characterize the interaction of the affinity agent with a soluble molecule of interest.
• the disclosed functionalized substrates are further functionalized with an affinity agent.
• the affinity agent can be any agent that can be bound to the functionalized substrate and that interacts covalently or noncovalently with a molecule of interest.
• the affinity agent is a small molecule, oligonucleotide, peptide or protein that binds to or interacts with a soluble molecule of interest.
• the soluble molecule of interest or analyte can be a small molecule, oligonucleotide, peptide or protein.
• Interactions between the affinity agent and the analyte can be detected by any suitable method, and working embodiments used methods such as optical detection methods including ultraviolet and visible absorption, chemoluminescence, and fluorescence (including lifetime, polarization, fluorescence correlation spectroscopy (FCS), and fluorescence- resonance energy transfer (FRET)).
• optical detection methods including ultraviolet and visible absorption, chemoluminescence, and fluorescence (including lifetime, polarization, fluorescence correlation spectroscopy (FCS), and fluorescence- resonance energy transfer (FRET)).
• an array of affinity agents includes a substrate, an organic thinfilm formed on at least a portion of the surface of the substrate, and including plural copies of at least one affinity agent operatively associated with, e.g., covalently or noncovalently associated with, the underlying organic thinfilm.
• affinity agents operatively associated with, e.g., covalently or noncovalently associated with, the underlying organic thinfilm.
• the different types of affinity agents are grouped in "patches,” so that affinity agents localized in one patch of the array differ from affinity agents localized to another patch.
• the present invention provides an array of glycoproteins containing a substrate, at least one organic thinfilm on some or all of the substrate surface, and a plurality of patches arranged in discrete, known regions on portions of the substrate surface covered by organic thinfilm, wherein each of said patches comprises a protein immobilized on the underlying organic thinfilm.
• the array optionally contains an interlayer between the substrate and coating.
• the array will comprise at least about ten patches. In an exemplary embodiment, the array comprises at least about 50 patches. In another exemplary embodiment the array comprises at least about 100 patches. In alternative exemplary embodiments, the array of affinity agents can comprise more than 10 3 , 10 4 or 10 5 patches.
• the surface area of the substrate covered by each of the patches is no more than about 0.25 mm 2 . In another exemplary embodiment, the area of the substrate surface covered by each of the patches is between about 1 ⁇ m 2 and about 10,000 ⁇ m 2 . In another exemplary embodiment, each patch covers an area of the substrate surface from about 100 ⁇ m 2 to about 2,500 ⁇ m 2 . In an alternative embodiment, a patch on the array may cover an area of the substrate surface as small as about 2,500 nm , although patches of such small size are generally not necessary for the array to be useful.
• the patches of the array may be of any geometric shape.
• the patches may be rectangular or circular.
• the patches of the array may also be irregularly shaped.
• the distance separating the patches of the array can vary.
• the patches of the array are separated from neighboring patches by about 1 ⁇ m to about 500 ⁇ m.
• the distance separating the patches is roughly proportional to the diameter or side length of the patches on the array if the patches have dimensions greater than about 10 ⁇ m. If the patch size is smaller, then the distance separating the patches typically will be larger than the dimensions of the patch.
• the patches of the array are all contained within an area of about 1 cm 2 or less on the surface of the substrate.
• the array comprises 100 or more patches within a total area of about 1 cm 2 or less on the surface of the substrate.
• an exemplary array comprises 10 3 or more patches within a total area of about 1 cm 2 or less.
• An exemplary array may even optionally comprise 10 4 or 10 5 or more patches within an area of about 1 cm 2 or less on the surface of the substrate.
• all of the patches of the array are contained within an area of about 1 cm 2 or less on the surface of the substrate.
• only one type of affinity agent is immobilized on each patch of the array.
• the affinity agent immobilized on one patch differs from the affinity agent immobilized on a second patch of the same array.
• a plurality of different affinity agents can be present on separate patches of the array.
• a single patch comprises two or more affinity agents that bind to the same analyte. Such affinity agents typically bind to different epitopes of the analyte.
• One class of affinity agents that can be used in this aspect is polyclonal antibodies.
• the array comprises at least about ten different affinity agents. In an exemplary embodiment, the array comprises at least about 50 different affinity agents. In another exemplary embodiment, the array comprises at least about 100 different affinity agents. Alternative exemplary arrays comprise more than about 10 3 different affinity agents or more than about 10 4 different affinity agents. The array may even optionally comprise more than about 10 5 different affinity agents.
• each of the patches of the array contains a different affinity agent.
• an array comprising about 100 patches could comprise about 100 different affinity agents.
• an array of about 10,000 patches could comprise about 10,000 different affinity agents.
• each different affinity agent is immobilized on more than one separate patch on the array.
• each different affinity agent can optionally be present on two to six different patches. Therefore an array can comprise about three-thousand affinity agent patches, but only comprise about one thousand different affinity agents, since each different agent is present on three different patches.
• the affinity agent of one patch is different from that of another, the affinity agents are related.
• the two different affinity agents are members of the same protein family.
• the different proteins on the array can be either functionally related or thought to be functionally related.
• the function of the immobilized proteins may be unknown.
• the different glycoproteins on the different patches of the array typically share a similarity in structure or sequence or are thought to sharing a similarity in structure or sequence.
• the immobilized proteins can be fragments of different members of a protein family.
• affinity agent that can be operatively associated with an organic thinfilm on the substrate can be employed in the disclosed arrays.
• Classes of different affinity agents include, without limitation, small molecules, peptides, proteins and nucleic acids, including DNA and RNA.
• an array can include different affinity agents from different classes.
• the proteins can be members of a protein family, such as a receptor family, examples of which include growth factor receptors, catecholamine receptors, amino acid derivative receptors, cytokine receptors, and lectins; a ligand family, examples of which include cytokines and serpins; an enzyme family, examples of which include proteases, kinases, phosphatases, ras-like GTPases, and hydrolases; and transcription factors, examples of which include steroid hormone receptors, heat-shock transcription factors, zinc- finger proteins, leucine-zipper proteins and homeodomain proteins.
• the different immobilized proteins are all HIV proteases or hepatitis C virus (HCV) proteases.
• the associated proteins on the array are all hormone receptors, neurotransmitter receptors, extracellular matrix receptors, antibodies, DNA-binding proteins, intracellular signal transduction modulators and effectors, apoptosis-related factors, DNA synthesis factors, DNA repair factors, DNA recombination factors, or cell-surface antigens.
• Antibodies and antibody fragments are particularly useful affinity agents for use with the disclosed arrays.
• the antibodies optionally can be polyclonal or monoclonal antibodies.
• the production and isolation of antibodies that bind to specific targets, including protein targets, using standard hybridoma technology is known to those of ordinary skill in the art.
• numerous antibodies are available commercially.
• antibodies or antibody fragments can be expressed in bacteriophage.
• Such antibody phage display technologies, including methods for bacteriophage selection, are well known to those of ordinary skill in the art.
• the disclosure provides reagents and techniques for assembling arrays of affinity agents.
• a first heterobifunctional reagent is covalently or non-covalently associated with a substrate, thereby forming a monolayer.
• a second heterobifunctional reagent is coupled to the monolayer to provide an array of reactive functional groups and a third reagent including a protein resistant component is coupled to the monolayer, such that the three reagents form an organic thinfilm including reactive functional groups.
• the reactive functional groups are selected such that an affinity agent can be coupled to the organic thinfilm, thereby forming an affinity agent array having affinity agents operatively coupled to the organic thinfilm.
• Coupling involves covalently or non-covalently associating the affinity agent.
• Non-covalent associate may exploit, without limitation, one or more of Coulombic interactions, hydrogen bonds, Van der Waals interactions and hydrophobic interactions.
• an affinity agent is coupled to an organic thinfilm by a chemoselective ligation reaction.
• Chemoselective ligation reactions generally refer to reactions between functional groups that have orthogonal reactivity to other functional groups present, particularly those functional groups found in many biomolecules. Thus, chemoselective ligation reactions are particularly useful when the affinity agent is a biomolecule.
• chemoselective ligation reactions used with biomolecules employ one or more non-native functional groups to ensure that the reaction is orthogonal to native functional groups.
• a chemoselective ligation reaction is a Staudinger ligation.
• ketones and aldehydes such as the condensation of a hydrazide or aminooxy compound with a ketone or aldehyde to yield the corresponding hydrazone or oxime.
• Another example is the reaction of a thiocarboxylate with an ⁇ -halo carbonyl compound to give a thioester. Versions of these reactions also can be used to attach compounds other than biomolecules with the organic thinfilm.
• Staudinger ligation functions as an amide bond forming reaction and typically involves two reactive components, the first typically having the formula Y-Z-PR 2 R 3 where Z is an aryl group substituted with R 1 , wherein R 1 is preferably in the ortho position on the aryl ring relative to the PR 2 R 3 ; and wherein R 1 is an electrophilic group to trap (e.g., stabilize) an aza-ylide group, including, but not necessarily limited to, a carboxylic acid, an ester (e.g., alkyl ester (e.g., lower alkyl ester, benzyl ester), aryl ester, substituted aryl ester), aldehyde, amide, e.g., alkyl amide (e.g., lower alkyl amide), aryl amide, alkyl halide (e.g.,
• Exemplary affinity agents further include dyes (e.g., fluorescein or modified fluorescein, and the like), antibodies, toxins (including cytotoxins), linkers, peptides, and the like.
• An exemplary and preferred engineered phosphine reactant is 2-diphenylphosphanyl- benzoic acid methyl ester.
• the second reagent comprises an azide. Molecules comprising an azide and suitable for use in the present invention, as well as methods for producing azide- comprising molecules suitable for use in the present invention, are well known to those having ordinary skill in the art. In a working embodiment a monolayer comprising a 2-diphenylphosphanyl- benzoic acid methyl ester derivative was prepared on a gold substrate.
• a first reagent, 11-thio-undecanionic-N-hydroxysuccinimide ester was linked to the substrate via the sulfhydryl group.
• the resulting monolayer was derivatized with compound 25, thereby forming a second monolayer from the first, where the second monolayer is suitable for coupling of an affinity agent containing an azide moiety.
• a fluorescently labeled, azidoalanine-containing peptide was then coupled to the second monolayer via Staudinger ligation.
• a native functional group of a biomolecule is used to attach it to an organic thinfilm.
• proteins that have one or more cysteine residues can be selectively alkylated with reagents, such as ⁇ -halo carbonyl compounds.
• reagents such as ⁇ -halo carbonyl compounds.
• ⁇ -halo carbonyl compounds When an ⁇ -halo carbonyl compound is associated with a thinfilm, this can be a useful reaction for associating certain proteins to the thinfilm.
• a natural or non-natural amino acid can be incorporated into a peptide or protein to provide a functional group for associating the protein with an organic thinfilm.
• an azide-containing amino acid was incorporated into a peptide, which is then attached to an organic thinfilm via a Staudinger ligation protocol.
• the substrate is functionalized with a chelator, such as an N-nitrilotriacetic acid ( ⁇ TA) derivative or an imidodiacetic acid (IDA) derivative, which chelate metals, such as nickel, cobalt, iron and copper.
• a histidine-tagged protein can be noncovalently bound to the substrate via, for example, mutual nickel chelation by both the substrate-associated chelator, such as an ⁇ TA or IDA derivative, and the histidine tag.
• Proteins having histidine tags are known to those of ordinary skill in the art. See, Hochuli, et al., Biotechnology, 1988, 6, 1321.
• the initial, non-covalent association can be followed by covalent bond formation between the affinity agent and the substrate associated organic thinfilm.
• a photoactivatable group such as an aryl azide, particularly haloaryl azides, for example a pentafluorophenyl aryl azide, benzophenones, diazocompounds, particularly diazopyruvates. Additional suitable photoactivatable groups are taught by Hermanson, G.T. Bioconjugate Techniques, Academic Press: San Diego, 1996, which is incorporated herein by reference.
• the first affinity agent is a multivalent protein, and is associated with a substrate-associated organic thinfilm.
• the first affinity agent can then serve as the site of attachment for a second affinity agent having a portion for binding to the first affinity agent and a portion for interacting with a molecule of interest.
• the first affinity agent is streptavidin, which is bound to the substrate via a biotin derivative displayed on the substrate-associated organic thinfilm, and the second affinity agent is a biotin-co ⁇ jugated molecule. Because streptavidin has plural biotin binding sites, the biotin conjugated molecule is then associated with the organic thinfilm via the streptavidin.
• Classes of materials useful as substrate 2 for forming arrays as disclosed herein include inorganic materials, metals, and organic polymers.
• inorganic materials include silicon, silica, quartz, glass, controlled pore glass, carbon, alumina, titanium oxide, tantalum oxide, indium tin oxide, germanium, silicon nitride, gallium arsenide, zeolites, mica and combinations thereof.
• Useful metals include aluminum, copper, gold, platinum, titanium, alloys thereof, and combinations thereof.
• useful polymers include, without limitation, polyethylene, polyethyleneimine, polyvinylethylene, polystyrene, poly(tetra)fluoroethylene, polycarbonate, polymethylmethacrylate, polydimethylsiloxane, polyvinylphenol, polyoxymethylene, polymethacrylamide, polyimide and copolymers of such materials.
• the substrate coated with one or more different materials which typically are selected from those listed above.
• the coating optionally may be a metal film. Possible metal films include aluminum, chromium, titanium, tantalum, nickel, stainless steel, zinc, lead, iron, copper, magnesium, manganese, cadmium, tungsten, cobalt, and alloys or oxides thereof.
• the coating can include silicon, silicon oxide, silicon nitride, titanium oxide, tantalum oxide, silicon nitride, silicon hydride, indium tin oxide, magnesium oxide, alumina, glass, hydroxylated surfaces, and polymers.
• X can be any group that yields chemisorption or physisorption of reagent 4 on substrate 2, thereby forming a monolayer.
• X typically is a group that reacts chemically with substrate 2, and thus X is selected to be compatible with the substrate material.
• the substrate is an oxide, such as silicon oxide, indium tin oxide, magnesium oxide, alumina, quartz, glass, or is a material such as silicon or aluminum having an oxidized surface layer
• silanes and siloxanes are useful groups for functionalizing the substrate.
• halosilanes, including mono-, di- and tri-halosilanes can be used to attach a reagent to such substrates.
• suitable X groups for derivatizing metal substrates or a metal coating on a substrate include those sulfur-bearing compounds, such as thiols, thioethers (sulfides), isothiocyanates, xanthanates, thioacids, thiocarbamate, disulfides (including symmetric and asymmetric disulfides), dithioacids (including symmetric and asymmetric dithioacids) and sulfur-containing heterocycles; selenium bearing molecules, such as selenols, selenides and diselenides (including symmetric and asymmetric diselenides; nitrogen-bearing compounds, such as 1°, 2° and perhaps 3° amines, aminooxides, pyridines, isocyanates, isonitriles, nitriles, and hydroxamic acids; phosphorus-bearing compounds, such as phosphines; and oxygen-bearing compounds, such as carboxylates, hydroxyl-bea
• Sulfur-containing compounds are particularly useful for functionalizing silver, gold and platinum surfaces.
• the substrate is a metal, such as silver, gold or platinum
• thiols and disulfides are preferred reagents for functionalizing the substrate.
• thiols and disulfides were used to attach various reagents to gold surfaces
• the surface of the substrate (or coating thereon) is composed of a metal oxide such as titanium oxide, tantalum oxide, indium tin oxide, magnesium oxide, or alumina and X is a carboxylic acid.
• a metal oxide such as titanium oxide, tantalum oxide, indium tin oxide, magnesium oxide, or alumina
• X is a carboxylic acid.
• the surface of the substrate (or coating thereon) of the device is copper, then X typically is a hydroxamic acid.
• the substrate used in the invention is a polymer
• a coating on the substrate such as a copper coating will be included in the device.
• An appropriate functional group X for the coating would then be chosen for use in the device.
• the surface of the polymer may be plasma-modified to expose desirable surface functionalities for monolayer formation.
• European Patent Publication No. 780423 describes using a monolayer molecule that has an alkene X functionality on a plasma exposed surface.
• Still another possibility for the invention device comprised of a polymer is that the surface of the polymer on which the monolayer is formed is functionalized due to copolymerization of appropriately functionalized precursor molecules.
• X can be a free-radical-producing or free-radical-activated moiety.
• a functional group is especially appropriate when the surface on which the monolayer is formed is a hydrogenated silicon surface.
• free radical producing moieties include, but are not limited to, diacylperoxides, peroxides, and azo compounds.
• unsaturated moieties such as unsubstituted alkenes (particularly ⁇ - ⁇ unsaturated ketones), alkynes, cyano compounds and isonitrile compounds can be used for X as free radical activated moieties, particularly if the reaction with X is accompanied by ultraviolet, infrared, visible, or microwave radiation.
• X can be a vinyl, sulfonyl, phosphoryl or silicon hydride group.
• the linker group in reagent 4 between groups X and Y can be any suitable, chemically compatible spacer group.
• the linker group is selected to promote the self assembly of reagent 4 into monolayer 6, and/or to ensure that the monolayer is ordered and densely packed.
• the factors that contribute to self assembly are known to those of ordinary skill in the art, as described by Laibinis, et al. Science 1989, 245, 845 and Ulman, An Introduction to Ultrathin Organic Films: From Langmuir-Blodgett to Self- Assembly, Academic Press (1991).
• the linker group in reagent can include the functional groups to facilitate crosslinking.
• crosslinking confers additional stability to the monolayer.
• Such methods are familiar to those of ordinary skill in the art (for instance, see Ulman, An Introduction to Ultrathin Organic Films: From Langmuir-Blodgett to Self- Assembly, Academic Press (1991)).
• functionalization of substrate 12 with reagent 4 yields monolayer 6 formed on substrate 2, which is further derivatized with reagents 8 and 10 to yield monolayer 14.
• Examples of functional groups suitable for use as Y include electrophiles, such as activated carboxylic acids, including, for example, acyl imidazolides, acyl azides, and activated esters, such as pentafluorophenol, ?-nitrophenol, N- hydroxysuccinimide and sulfo-N-hydroxysuccinimide esters.
• Additional useful electrophiles include Michael acceptors, such as ⁇ , ⁇ -unsaturated carbonyl groups (including acids, amides, esters, ketones and aldehydes), ⁇ , ⁇ -unsaturated sulfoxides, ⁇ , ⁇ r unsaturated sulfones, and the like.
• Y can be a dienophile used in a Diels- Alder reaction.
• suitable dienophiles include electron poor alkenes or, alternatively, in an inverse-demand Diels-Alder reaction, electron-rich alkenes.
• electron-rich dienes or electron-poor dienes are used in Diels-Alder and inverse-demand Diels-Alder cycloaddition reactions, respectively.
• Azide-containing compounds can be used in several different types of cycloaddition reactions.
• 1,3-dipolar cycloaddition reactions can be used to link a compound to monolayer 6 when Y includes an azide, which can be reacted with a suitable alkene compound to yield, following thermal elimination of nitrogen, the corresponding aziridine compound.
• Y can include an alkene group that can react with an azide-containing compound.
• organometallic reactions include, without limitation, metathesis reactions, for example, in one embodiment an aldehyde- or alkene-functionalized surface can be reacted with reagents 8 and 10 wherein N and V include an aldehyde or alkene functional group.
• organometaliic reactions include, without limitation, metathesis reactions, for example, in one embodiment an aldehyde- or alkene-functionalized surface can be reacted with reagents 8 and 10 wherein N and V include an aldehyde or alkene functional group.
• catalysts and conditions for such metathesis reactions are known to those of ordinary skill in the art.
• Certain examples of such catalysts include the metals titanium, tungsten, molybdenum or ruthenium. See, for example, Ivin, K. J., Mol, J.C. Olefin Metathesis and Metathesis Polymerization; Academic Press: London, 1997.
• Suitable protecting groups that can be used can be selected, installed and removed as is known to those of ordinary skill in the art, and include protecting groups disclosed in Greene, T.W.; Wuts P.G. M. Protective Groups in Organic Synthesis, 3rd ed.; Wiley-Interscience, 2002. In a working embodiment a protected aldehyde was used to produce a masked aldehyde- functionalized substrate.
• Reagent 10 includes functional group V for attachment to monolayer 6 and also includes a protein resistant component W.
• Functional groups N and V can be the same or different functional groups.
• reagents 8 and 10 can be delivered in two different steps.
• reagent 8 could be deposited either before or after reagent 10.
• N and V are optionally covalently linked in, for example, a disulfide bond, such that reagents 8 and 10 having functional groups Z and W, respectively, are delivered in the same step.
• Functional groups N and N' are selected with reference to functional group Y displayed on monolayer 6. As noted above, with respect to functional group Y, N and/or V can form a covalent linkage to monolayer 6, or can associate noncovalently with monolayer 6.
• Functional group Z on monolayer 14 is used to attach an affinity agent to yield monolayer 16 having affinity agent 18 bound thereto.
• Structure 30 includes an aldehyde, which can be used as an electrophile for reaction with a various nucleophilic reagents.
• nucleophilic groups capable of reacting with an aldehyde to produce a covalent bond are useful in the current invention.
• Useful reactive groups include, for example, alkoxides, enolates, carbanion equivalents, such as Grignard-type reactive groups, alcohol groups (yielding the corresponding acetal or hemiacetal), cyanide reactive groups, amines, including primary or secondary amines, hydrazines, phosphorus ylides, phosphine oxide anions, aminooxy reagents, hydrazides thiosemicarbazides, and the like.
• an aldehyde can be used in an organometallic reaction, such as an aldehyde-alkene metathesis reaction.
• FIG. 3 illustrates a method for preparing organic thinfilm arrays by first derivatizing a substrate 34 using an asymmetric disulfide 33.
• the disulfide 33 is asymmetric in the sense that it is composed of two different substituents. Deposition according to this method yields a monolayer 35, which includes two different monolayer components.
• one or more additional symmetric or asymmetric disulfides can be deposited to provide a monolayer including three or more different monolayer components, or simply a different ratio of the two monolayer components provided by the asymmetric disulfide.
• deposition of asymmetric disulfide 33 can be accompanied by deposition of an equal amount of a symmetric disulfide having the formula X-R-S-S-R— X.
• X and Y in monolayer 35 function as an affinity agent and a protein resistant component or as reactive functional groups for incorporating an affinity agent and protein resistant reagent.
• X and Y are such reactive functional groups, they can be the same or different functional groups.
• X is a group that renders the monolayer component an active acylating agent.
• acylating agents can be prepared from the corresponding carboxy group as is known to those of ordinary skill in the art.
• 39 is an activated ester group, such that X is a pentafluorophenol, j»-nitrophenol, N-hydroxysuccinimide, sulfo-N- hydroxysuccinimide, or the like.
• Additional suitable X groups include halides, imidazolides, anhydrides, and the like.
• Structure 41 like structure 39, illustrates a monolayer component formed on a glass or silicon substrate. However, 41 functions as a protein resistant component.
• FIG. 4 illustrates another embodiment for functionalizing a substrate.
• substrate 45 is glass or has a silicon oxide coating, which is derivatized with a 3-maleimidopropyl-l-silane derivative to give the monolayer coated substrate shown having a maleimide (MAL) functional group on one face.
• MAL maleimide
• the maleimide group can be used to attach a sulfhydryl-containing reagent with high efficiency.
• a heterobifunctional reagent having a sulfhydryl group at a first end and a biotin group at a second end is attached to the substrate via the maleimide group.
• a reagent containing a protein-resistant component is then attached via remaining maleimide functional groups.
• the disclosed arrays are useful for characterizing the interaction of soluble analytes with arrayed affinity agents.
• the arrays can be used to identify affinity agents that bind to a molecule of interest, particularly a biomolecule of interest, such as a protein or nucleic acid.
• the arrays are especially useful for high throughput drug screening, using both small molecule and biomolecule arrays.
• a wide range of detection methods is applicable to characterizing the interactions of soluble analytes with the disclosed arrayed affinity agents.
• the arrays can be incorporated into a device that is interfaced with optical detection methods such as absorption in the visible range, chemoluminescence, and fluorescence (including lifetime, polarization, fluorescence correlation spectroscopy (FCS), and fluorescence-resonance energy transfer (FRET)).
• optical detection methods such as absorption in the visible range, chemoluminescence, and fluorescence (including lifetime, polarization, fluorescence correlation spectroscopy (FCS), and fluorescence-resonance energy transfer (FRET)
• built-in detectors such as optical waveguides as disclosed in PCT Publication WO 96/26432 and U.S. Pat. No. 5,677,196, incorporated herein by reference, surface plasmon resonance, and surface charge sensors are compatible with embodiments of the arrays disclosed herein.
• An exemplary type of fluorescence detection unit that may be used to monitor interaction of immobilized affinity agents of an array with an analyte is described in U.S. Patent No.
• FIG. 5 is a schematic diagram of this type of fluorescence detection unit, which may be used to monitor interaction of immobilized affinity agents of an array with an analyte.
• the array 54 is positioned on a base plate 52.
• Light from a 100W mercury arc lamp 62 is directed through an excitation filter 60 and onto a beam splitter 58.
• the light is then directed through a lens 56, such as a Micro Nikkor 55 mm 1:2:8 lens, and onto the array 54. Fluorescence emission from the array returns through the lens 56 and the beam splitter 58.
• the emission is received by a cooled CCD camera 66, such as the Slowscan TE/CCD- 1024SF&SB (Princeton Instruments).
• the camera is operably connected to a CPU 68, which is in turn operably connected to a VCR/monitor 70.
• polarizer 86 plane polarized (polarizer 86) and directed onto the surface of the sample on substrate 82 and detected by a detector 92.
• a compensator 88 changes the elliptically polarized reflected beam to plane-polarized. The corresponding angle is determined by an analyzer 90 and then translated into the ellipsometric parameters Psi and Delta which change upon binding of analyte with the immobilized proteins. Additional information can be found in Azzam, et al., Ellipsometry and Polarized Light, North- Holland Publishing Company: Amsterdam, 1977.
• a Teflon rack holding glass microscope slides (1" by 3", Fisher Scientific) or 1 cm x 2 cm silicon wafers was immersed in the cleaning bath. After about 15 minutes, the slide rack was removed from the bath and placed into a 300 mL bath of nanopure water for 10 minutes with gentle agitation, followed by immersion in a second 300 mL bath of nanopure water for 10 minutes with gentle agitation.
• the slides were stored under nanopure water and dried immediately before use via spin rinse drying, using a cycle of 3 minutes washing at 600 rpm, 10 seconds nitrogen purging at 1500 rpm, followed by two drying cycles of 1.5 and 20 minutes at 2500 and 600 rpm, respectively.
• Example 1 This example describes the formation of an aminoreactive organic thin film on a gold substrate using disulfide thin film precursors.
• a freshly prepared Au(l 11) chip was immersed in a 1 mM solution of asymmetric disulfide compound 120 (the synthetic route to compound 120 is illustrated in FIG. 7 and described in detail below) at room temperature for 1 hour.
• the chip was then rinsed with ethanol (3 x 10 mL). After rinsing, the coated chip was dried under a nitrogen stream and used immediately for reaction with an amine-containing reagent.
• Example 2 This example describes functionalizing an aminoreactive organic thin film to form a biotin-functionalized chip.
• a chip having an aminoreactive coating prepared according to example 1 was incubated with a 5 mM solution of either (+)-biotinyl- 3 ,6-dioxaoctanediamine or (+)-biotinyl-3 ,6,9, 11 -tetraoxatridecanediamine
• Example 3 This example describes the general protocol used for silanization of silicon oxide substrates. Reagents deposited by this method included 11-azidoundecyl-l- triethoxysilane and 3 -maleimidopropyl-1-triethoxy silane, which was prepared according to the protocol of Shaltout et al. Mat Res Soc. Symp. Proc. 1999, 576, 15- 20, which is incorporated herein by reference. The Shaltout procedure also was applied to the synthesis of other maleimide derivatives.
• 3-Maleimidopropyl-l- triethoxysilane reagent was deposited as follows: To a clean, dry 120 mL PTFE vial (Savillex, Minnetonka, Minnesota) was added 50 mL dry toluene (99.8% anhydrous, Aldrich catalog number, 24,451-1, Milwaukee, Wisconsin) and 1 mL hexanoic acid (Aldrich catalog number 15374-5), followed by 0.5 mL of neat silane. The vial was swirled and dry, clean silicon oxide wafers were added to the silane solution. The silanization was allowed to proceed for 24 hours at room temperature on a shaker (ca. 100-150 rpm).
• Example 4 This example describes the general Staudinger ligation protocol used to couple aminoreactive and protein resistant components to the 11-azidoundecyl-l- triethoxysilane functionalized substrate prepared according to the protocol of example 3.
• the protein resistant Staudinger reagent 20 and aminoreactive Staudinger reagent 30 are depicted below. Staudinger reagents 20 and 30 were prepared according to the method disclosed by Saxon and Bertozzi in U.S. Patent No. 6,570,040, which is incorporated herein by reference in its entirety.
• Staudinger reagent was dissolved in 4 mL of oxygen purged (via nitrogen sparging) 3:1 tetrahydrofuran:nanopure (deionized, 18 M ⁇ ) water to give a concentration of 20 mM.
• the 11-azidoundecyl-l-triethoxysilane chip was placed in a teflon container and was immersed in the Staudinger reagent solution. The container was shaken on a shaker for 24 hours at room temperature, and then the chip was removed and rinsed with 30 mL each of 3:1 tetrahydrofuranmanopure water, nanopure water, and absolute ethanol. Each chip was dried under nitrogen and stored until further use. The progress of the Staudinger ligation can be monitored by IR spectroscopy, and the yield can be determined by quantitative IR spectroscopy.
• Example 5 This example describes the functionalization of a 3-maleimidopropyl-l- triethoxysilane substrate with a biotin derivative 40 and a protein resistant component 50.
• a maleimide functionalized chip prepared according the protocol of example 3 was coated with 250 ⁇ L of an 80 ⁇ M solution of compound 40 in buffer (20 mM citrate, 150 mM sodium, 5 mM EDTA, 0.05% Tween 20 buffer at pH 6.5). The chip was incubated at room temperature for 30 minutes with slight agitation on a shaker.
• This example describes the preparation of an aldehyde functionalized organic thinfilm.
• Ethylene glycol 28 mL, 31.4 g, 505 mmol, 1.05 equiv.
• the solution was refluxed under a Dean- Stark trap for 3 hours.
• the wafer Upon removal from the acid bath, the wafer was immersed in 1 M pH 7 NaHCO for 1 minute, removed, immersed in water and agitated for 5 minutes and then dried using a stream of nitrogen gas.
• the deprotection reaction can be followed by X-ray photoelectron spectroscopy (XPS, 45 °C take off angle). The disappearance of the peak corresponding to the C-O bond at 287 eV can be monitored, and the deprotection reaction is nearly complete at 1 hour.
• the resulting aldehyde functionalized surface can be derivatized with 3'-aminofunctionalized oligonucleotides via reductive amination.
• Oligonucleotide 5 ⁇ M is spotted on the aldehyde functionalized surface, and the surface is allowed to dry.
• the spotted surface is rinsed in a solution of 0.2% SDS in deionized water for 2 minutes at room temperature with agitation.
• the surface is then immersed in a fresh reducing solution (1.5 g NaBH 4 , 133 mL absolute ethanol, and 450 mL PBS at pH 7.2-7.4) and soaked for 5 minutes at room temperature.
• IL-8 human Interleukin-8
• AVLPRSAKELRCQCrKT'YSKPFHPKFLKELRVIES GPHCANTEIIVKLSDGRELCLDPKENWNQRNNEKFLKRAENS SEQ ID 1 with a C-terminal Factor Xa cleavage site and Protein Kinase A site (GIEGRRRASV) SEQ ID 2 was created by gene assembly of oligonucleotides.
• the cells were collected by centrifugation, resuspended in 5ml buffer/g with 300mM NaCl, 50mM sodium phosphate, 5mM beta-mercaptoethanol, 5 mM imidazole, pH 8.0 including one Complete Protease Inhibitor Cocktail tablet (Roche Applied Science; Cat No. 1697498) per 50ml and lysed using a micro fluidizer.
• the soluble fraction of IL-8 was purified by metal affinity chromatography using TALON Superflow beads (Clontech, Palo Alto, CA; Cat No. 8908-2) followed by gel filtration in PBS using a Superdex 75 prep grade column (Amersham Biosciences).
• the IL-8 containing fractions were concentrated to 0.4 - 0.5 mg/ml using a model 8400 stirred ultrafiltration cell (Millipore, Bedford, MA; CAT No 5124) with a 3K MWCO membrane and then dialyzed into PBS with 10% glycerol for storage. Identity and Correct secondary structure was confirmed by mass spectrometry, ELISA and circular dichroism (Spectrapolarimeter J-810, Jasco, Easton MD, www.jascoinc.com).
• pepsinolysis Conditions for pepsinolysis were as follows: 30% (by volume) pepsin agarose (settled bed volume, beads washed in 20 mM NaOAc, pH 4.5), 0.5 - 2 mg/ml IgG, 20 mM NaOAc, 260 mM KC1, 0.1% Triton X-100, pH 4.5. Reactions were incubated at 37°C with agitation for an amount of time that had previously been optimized (MAB9647, 12 h; MAB208, 4.5 h;MAB602, 3.5 h). After pepsin- treatment, the fragments were recovered from the pepsin agarose by washing the resin with 0.1M NaOAc, pH 4.5.
• the reduced F(ab)' was treated with 20 mM N-ethylmaleimide or maleimide-activated biotin (Pierce product number 21901) for 2 h at room temperature, and the unincorprated ⁇ EM or biotin-maleimide was then removed by dialysis.
• the samples were concentrated and the Fab' fragments were purified from other fragments by FPLC using a Superdex-75 gel filtration column (Amersham-Pharmacia). In the case of the NEM-treated Fab' fragments, random biotinylation was as described below.
• biotinylation of all capture agents used in this study was verified by Western blot analysis using an HRP-conjugated streptavidin (SA) probe (data not shown).
• HRP-conjugated streptavidin (SA) probe was used in this study.
• the extent of biotinylation was estimated by using a SA resin pull-down assay (data not shown). Biotinylation was 60% or greater in each of these reactions. No attempt was made to remove non biotinylated protein prior to surface immobilization.
• Example 9 This example describes the carbobiotinylation protocol. IgG (3-5 mg/ml) were dialyzed into coupling buffer (0.1 M NaOAc pH 5.5) and then incubated with 20 mM sodium meta periodate, NaI0 4 in the dark for 1 h at 0 °C to oxidize the vicinal diols in the carbohydrate to aldehydes. The reaction was then quenched by the addition of 30 mM glycerol for 10 minutes, filtered to remove insoluble salts, and then dialyzed against coupling buffer for 6 hours.
• coupling buffer 0.1 M NaOAc pH 5.5
• aldehyde reactive probe (ARP, N-(aminooxyacetyl)-N-(D biotinyl) hydrazine, trifluoroacetic acid salt, Molecular Probes, Eugene, OR) is added at 1 mg/ml and incubated at room temperature for 2 h, after which the sample is extensively dialyzed against PBS.
• ARP aldehyde reactive probe
• Example 10 This example describes the random biotinylation of IgG and Fab 'fragment molecules.
• IgG and NEM-treated Fab' fragments were modified with the amine reactive probe, EZ LinkTM Sulfo-NHS-Biotin (Pierce). Reactions were performed with a 20-fold molar excess of biotinylation reagent over protein in IX PBS at room temperature for 2 hours. The biotinylation reagent was then quenched by adding Tris, pH 7.4, to a final concentration of lOmM. The samples were then dialyzed against a 1000-fold excess of PBS 5 times in order to remove free biotin probe.
• biotinylated proteins were analyzed on a gel and tested for extent of biotinylation on UltraLinkTM Plus Immobilized Streptavidin Gel (Pierce). Typically 60% - 100% of the protein would be biotinylated (data not shown).
• Example 11 This example describes using surface plasmon resonance (SPR) to measure surface coverage and activity of affinity agents linked to a thinfilm formed on a gold substrate. All SPR assays were performed in a BIAcore 3000 sing a biotinylated self-assembled monolayer formed on a gold-coated glass surface. The surface was prepared using unsymmetrical alkanedisulfi.de compound 10 according to the protocol of example 1. The prepared surface comprised N-hydroxysuccinimide and methoxy groups on its exposed face. This monolayer was then reacted with (+)- biotinyl-3,6-dioxaoctanediamine (tri-ethyleneglycol amino biotin) reagent to give a biotinylated surface.
• SPR surface plasmon resonance
• biotin groups on the surface allow for the binding of streptavidin (SA). All assays were performed at 25° C in PBS with 0.05% Tween- 20. SA was loaded onto the surface at a flow rate of 20 ⁇ l/min at 0.1 mg/ml. Typically 320 ⁇ l was loaded to achieve a saturated surface of S A whereby a surface coverage of 3.7-4.0 pmol/cm 2 was obtained, as calculated according to Jung et al., Langmuir 14:5636-5648 (1998). After SA deposition, the various capture agents were loaded at 20-100 nM at a flow rate of 20 ⁇ l/min until saturation was observed.
• alkenyl bromide compound 100 is used as the starting material for both "arms" of the asymmetric disulfide.
• 100 is reacted with alkoxide triethylene glycol monomethyl ether (produced by deprotonation with NaH) to afford compound 102 in 60% yield.
• compound 102 is subjected to radical addition to give the terminal thioacetyl compound 104 in 88% yield.
• acetyl group is removed under acidic conditions (4N HCl/dioxane at 75 °C for 4 hours) to afford thiol 106 (95%), which is treated with 2',2'-dipyridyl disulfide to furnish the corresponding 2'-pyridyl mixed disulfide 108 in 98% yield.
• the NHS "arm" of the mixed disulfide 120 is prepared from 100 via reflux with tetraethylene glycol in aqueous NaOH for 24 hours to yield compound 110 in 42% yield.
• Terminal alcohol 110 is treated with NaH and alkylated with t- butylbromoacetate to afford terminal ester compound 112 in 42% yield.
• Compound 112 is converted to thioacetyl compound 114 in 87% yield via free radical addition of thioacetic acid. Removal of the acetyl and t-butyl blocking groups by treatment with 2N aqueous HC1 for 48 to 60 hours at 75 °C produced thiol 116 in 80% yield.
• Compounds 108 and 116 were then combined in the presence of Amberlite
• CG-50 (pH ca. 3-4) for about 12 to about 16 hours at room temperature to yield asymmetric disulfide 118, which was converted to desired compound 120 via DCC- catalyzed condensation with N-hydroxysuccinimide.

Landscapes

• Engineering & Computer Science (AREA)
• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Immunology (AREA)
• Life Sciences & Earth Sciences (AREA)
• Physics & Mathematics (AREA)
• Biomedical Technology (AREA)
• Nanotechnology (AREA)
• Hematology (AREA)
• General Physics & Mathematics (AREA)
• Molecular Biology (AREA)
• Urology & Nephrology (AREA)
• Cell Biology (AREA)
• Pathology (AREA)
• Food Science & Technology (AREA)
• Analytical Chemistry (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• Microbiology (AREA)
• Medicinal Chemistry (AREA)
• Composite Materials (AREA)
• Condensed Matter Physics & Semiconductors (AREA)
• Materials Engineering (AREA)
• Crystallography & Structural Chemistry (AREA)
• Biotechnology (AREA)
• Peptides Or Proteins (AREA)
• Investigating Or Analysing Biological Materials (AREA)

Applications Claiming Priority (3)

Publications (2)

Family

ID=31888306

Family Applications (1)

Country Status (5)

Families Citing this family (12)

Family Cites Families (6)

• 2003
  • 2003-08-18 EP EP03788638A patent/EP1537202A4/de not_active Withdrawn
  • 2003-08-18 CA CA002496141A patent/CA2496141A1/en not_active Abandoned
  • 2003-08-18 WO PCT/US2003/025927 patent/WO2004017042A2/en active Application Filing
  • 2003-08-18 AU AU2003263922A patent/AU2003263922A1/en not_active Abandoned
  • 2003-08-18 JP JP2004529123A patent/JP2005535898A/ja active Pending

Non-Patent Citations (2)

Also Published As

Similar Documents

Legal Events