CA2337654A1 - Arrays of proteins and methods of use thereof - Google Patents
Arrays of proteins and methods of use thereof Download PDFInfo
- Publication number
- CA2337654A1 CA2337654A1 CA002337654A CA2337654A CA2337654A1 CA 2337654 A1 CA2337654 A1 CA 2337654A1 CA 002337654 A CA002337654 A CA 002337654A CA 2337654 A CA2337654 A CA 2337654A CA 2337654 A1 CA2337654 A1 CA 2337654A1
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- Prior art keywords
- array
- protein
- proteins
- substrate
- patches
- 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.)
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Classifications
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- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
Protein arrays for the parallel, in vitro screening of biomolecular activity are provided. Methods of using the protein arrays are also disclosed. On the arrays, a plurality of different proteins, such as different members of a single protein family, are immobilized on one or more organic thin films on the substrate surface. The protein arrays are particularly useful in drug development, proteomics, and clinical diagnostics.
Description
ARRAYS OF PROTEINS AND METHODS OF USE THEREOF
BACKGROUND OF THE INVENTION
a) Field of the Invention The present invention relates generally to arrays of proteins and methods for the parallel, in vitro screening of a plurality of protein-analyte interactions.
More specifically, the present invention relates to uses of the arrays for drug development, proteomics, and clinical diagnostics.
b) Description of Related Art A vast number of new drug targets are now being identified using a combination of genomics, bioinformatics, genetics, and high-throughput biochemistry. Genomics provides information on the genetic composition and the activity of an organism's genes. Bioinformatics uses computer algorithms to recognize and predict structural patterns in DNA and proteins, defining families of related genes and proteins. The information gained from the combination of these approaches is expected to greatly boost the number of drug targets (usually, proteins).
The number of chemical compounds available for screening as potential drugs is also growing dramatically due to recent advances in combinatorial chemistry, the production of large numbers of organic compounds through rapid parallel and automated synthesis. The compounds produced in the combinatorial libraries being generated will far outnumber those compounds being prepared by traditional, manual means, natural product extracts, or those in the historical compound files of large pharmaceutical companies.
Both the rapid increase of new drug targets and the availability of vast libraries of chemical compounds creates an enormous demand for new technologies which improve the screening process. Current technological approaches which attempt to address this need include multiwell-plate based screening systems, cell-based screening systems, microfluidics-based screening systems, and screening of soluble targets against solid-phase synthesized drug components.
Automated multiwell formats are the best developed high-throughput screening systems. Automated 96-well plate-based screening systems are the most widely used. The current trend in plate based screening systems is to reduce the volume of the reaction wells further, thereby increasing the density of the wells per plate (96-well to 384- and 1536-well per plate). The reduction in reaction volumes results in increased throughput, dramatically decreased bioreagent costs, and a decrease in the number of plates which need to be managed by automation.
However, although increases in well numbers per plate are desirable for high throughput efficiency, the use of volumes smaller than 1 microliter in the well format generates significant problems with evaporation, dispensing times, protein inactivation, and assay adaptation. Proteins are very sensitive to the physical and chemical properties of the reaction chamber surfaces. Proteins are prone to denaturation at the liquid/solid and liquid/air interfaces.
Miniaturization of assays to volumes smaller than 1 microliter increases the surface to volume ratio substantially. (Changing volumes from 1 microliter to 10 nanoliter increases the surface ratio by 460%, leading to increased protein inactivation.) Furthermore, solutions of submicroliter volumes evaporate rapidly, within seconds to a few minutes, when in contact with air. Maintaining microscopic volumes in open systems is therefore very difficult.
Other types of high-throughput assays, such as miniaturized cell-based assays are also being developed. Miniaturized cell-based assays have the potential to generate screening data of superior quality and accuracy, due to their in vivo nature. However, the interaction of drug compounds with proteins other than the desired targets is a serious problem related to this approach which leads to a high rate of false positive results.
BACKGROUND OF THE INVENTION
a) Field of the Invention The present invention relates generally to arrays of proteins and methods for the parallel, in vitro screening of a plurality of protein-analyte interactions.
More specifically, the present invention relates to uses of the arrays for drug development, proteomics, and clinical diagnostics.
b) Description of Related Art A vast number of new drug targets are now being identified using a combination of genomics, bioinformatics, genetics, and high-throughput biochemistry. Genomics provides information on the genetic composition and the activity of an organism's genes. Bioinformatics uses computer algorithms to recognize and predict structural patterns in DNA and proteins, defining families of related genes and proteins. The information gained from the combination of these approaches is expected to greatly boost the number of drug targets (usually, proteins).
The number of chemical compounds available for screening as potential drugs is also growing dramatically due to recent advances in combinatorial chemistry, the production of large numbers of organic compounds through rapid parallel and automated synthesis. The compounds produced in the combinatorial libraries being generated will far outnumber those compounds being prepared by traditional, manual means, natural product extracts, or those in the historical compound files of large pharmaceutical companies.
Both the rapid increase of new drug targets and the availability of vast libraries of chemical compounds creates an enormous demand for new technologies which improve the screening process. Current technological approaches which attempt to address this need include multiwell-plate based screening systems, cell-based screening systems, microfluidics-based screening systems, and screening of soluble targets against solid-phase synthesized drug components.
Automated multiwell formats are the best developed high-throughput screening systems. Automated 96-well plate-based screening systems are the most widely used. The current trend in plate based screening systems is to reduce the volume of the reaction wells further, thereby increasing the density of the wells per plate (96-well to 384- and 1536-well per plate). The reduction in reaction volumes results in increased throughput, dramatically decreased bioreagent costs, and a decrease in the number of plates which need to be managed by automation.
However, although increases in well numbers per plate are desirable for high throughput efficiency, the use of volumes smaller than 1 microliter in the well format generates significant problems with evaporation, dispensing times, protein inactivation, and assay adaptation. Proteins are very sensitive to the physical and chemical properties of the reaction chamber surfaces. Proteins are prone to denaturation at the liquid/solid and liquid/air interfaces.
Miniaturization of assays to volumes smaller than 1 microliter increases the surface to volume ratio substantially. (Changing volumes from 1 microliter to 10 nanoliter increases the surface ratio by 460%, leading to increased protein inactivation.) Furthermore, solutions of submicroliter volumes evaporate rapidly, within seconds to a few minutes, when in contact with air. Maintaining microscopic volumes in open systems is therefore very difficult.
Other types of high-throughput assays, such as miniaturized cell-based assays are also being developed. Miniaturized cell-based assays have the potential to generate screening data of superior quality and accuracy, due to their in vivo nature. However, the interaction of drug compounds with proteins other than the desired targets is a serious problem related to this approach which leads to a high rate of false positive results.
Microfluidics-based screening systems that measure in vitro reactions in solution make use of ten to several-hundred micrometer wide channels.
Micropumps, electroosmotic flow, integrated valves and mixing devices control liquid movement through the channel network. Microfluidic networks prevent evaporation but, due to the large surface to volume ratio, result in significant protein inactivation. The successful use of microfluidic networks in biomolecule screening remains to be shown.
Drug screening of soluble targets against solid-phase synthesized drug components is intrinsically limited: The surfaces required for solid state organic synthesis are chemically diverse and often cause the inactivation or non-specific binding of proteins, leading to a high rate of false-positive results.
Furthermore, the chemical diversity of drug compounds is limited by the combinatorial synthesis approach that is used to generate the compounds at the interface.
Another major disadvantage of this approach stems from the limited accessibility of the binding site of the soluble target protein to the immobilized drug candidates.
Miniaturized DNA chip technologies have been developed (for example, see U.S. Patent Nos. 5,412,087, 5,445, 934 and 5,744,305) and are currently being exploited for nucleic acid hybridization assays. However, DNA biochip technology is not transferable to protein arrays because the chemistries and materials used for DNA biochips are not readily transferable to use with proteins.
Nucleic acids withstand temperatures up to 100°C, can be dried and re-hydrated without loss of activity, and can be bound directly to organic adhesion layers supported by materials such as glass while maintaining their activity. In contrast, proteins must remain hydrated, kept at ambient temperatures, and are very sensitive to the physical and chemical properties of the support materials.
Therefore, maintaining protein activity at the liquid-solid interface requires entirely different immobilization strategies than those used for nucleic acids.
Additionally, the proper orientation of the protein at the interface is desirable to ensure accessibility of their active sites with interacting molecules. With miniaturization of the chip and decreased feature sizes the ratio of accessible to non-accessible antibodies becomes increasingly relevant and important.
In addition to the goal of achieving high-throughput screening of compounds against targets, to identify potential drug leads, researchers also need to be able to identify highly specific lead compounds early in the drug discovery process. Analyzing a multitude of members of a protein family or forms of a polymorphic protein in parallel (multitarget screening) enables quick identification of highly specific lead compounds. Proteins within a structural family share similar binding sites and catalytic mechanisms. Often, a compound that effectively interferes with the activity of one family member also interferes with other members of the same family. Using standard technology to discover such additional interactions requires a tremendous effort in time and costs and as a consequence is simply not done.
However, cross-reactivity of a drug with related proteins can be the cause of low efficacy or even side effects in patients. For instance, AZT, a major treatment for AIDS, blocks not only viral polymerises, but also human polymerises, causing deleterious side effects. Cross-reactivity with closely related proteins is also a problem with nonsteroidal anti-inflammatory drugs (NSAIDs} and aspirin. These drugs inhibit cyclooxygenase-2, an enzyme which promotes pain and inflammation. However, the same drugs also strongly inhibit a related enzyme, cyclooxygenase-l, that is responsible for keeping the stomach lining and kidneys healthy, leading to common side-effects including stomach irritation.
For the foregoing reasons, there is a need for miniaturized protein arrays and for methods for the parallel, in vitro, screening of the interactions between a plurality of proteins and one or more analytes in a manner that minimizes reagent volumes and protein inactivation problems.
WO 00/04382 . PCT/US99/15971 SUMMARY OF THE INVENTION
The present invention is directed to miniaturized protein arrays and methods of use thereof that satisfy the need for parallel, in vitro, screening of the interactions between a plurality of proteins and one or more analytes in a manner that minimizes reagent volumes and protein inactivation problems.
In one embodiment, the present invention provides an array of proteins which comprises a substrate, at least one organic thinfilin 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 thinfllin, wherein each of said patches comprises a protein immobilized on the underlying organic thinfilm.
Preferably, a plurality of different proteins are present on separate patches of the array.
In a second embodiment, the invention provides a method for screening a plurality of proteins for their ability to interact with a component of a sample.
The method of this embodiment comprises delivering the sample to the array of proteins of the invention, and detecting, either directly or indirectly, for the interaction of the component with the immobilized protein of each patch.
In a third embodiment, the invention provides a method for screening a plurality of proteins for their ability to bind a particular component of a sample.
The method of this embodiment comprises first delivering the sample to the array of proteins of the invention. In a final step, the method comprises detecting, either directly or indirectly, for the presence or amount of the particular component which is retained at each patch. Optionally, the method comprises the additional step of further characterizing the particular component retained at the site of at least one patch.
In an alternative embodiment, the invention provides a method of assaying for protein-protein binding interactions. The first step of the method of this embodiment comprises delivering a sample comprising at least one protein to be assayed for binding to the protein array of the invention. The last step comprises detecting, either directly or indirectly, for the presence or amount of the protein from the sample which is retained at each patch In another embodiment of the invention, a method for assaying for a plurality of analytes in a sample is provided which comprises delivering the sample to a protein array of the invention and detecting for the interaction of the analytes with the immobilized protein at each patch.
In still another embodiment of the invention, an alternative method for assaying for a plurality of analytes in a sample is provided which cbmprises delivering the fluid sample to a protein array of the invention and detecting either directly or indirectly, for the presence or amount of analyte retained at each patch.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows the top view of an array of protein-reactive patches.
Figure 2 shows the cross section of an individual patch of the array of Figure 1.
Figure 3 shows the cross section of a row of monolayer-covered patches of the array of Figure 1.
Figure 4 shows a thiolreactive monolayer on a substrate.
Figure 5 shows an aminoreactive monolayer on a coated substrate.
Figure 6 shows the immobilization of a protein on a monolayer-coated substrate via an affinity tag.
Figure 7 shows the immobilization of a protein on a monolayer-coated substrate via an affinity tag and an adaptor.
Figure 8 shows a schematic of a fluorescence detection unit which may be used to monitor interaction of the proteins of the array with an analyte.
Figure 9 shows a schematic of an ellipsometric detection unit which may be used to monitor interactions between analytes and the proteins of the array.
DETAILED DESCRIPTION OF THE INVENTION
A variety of protein arrays, methods, and protein-coated substrates useful for drug development, proteomics, clinical diagnostics, and related applications are provided by the present invention.
(a) Definitions A "protein" means a polymer of amino acid residues linked together by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. Typically, however, a protein will be at least six amino acids long. Preferably, if the protein is a short peptide, it will be at least about 10 amino acid residues long. A protein may be naturally occurring, recombinant, or synthetic, or any combination of these. A protein may also be just a fragment of a naturally occurring protein or peptide. A protein may be a single molecule or may be a multi-molecular complex. The term protein may also apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid.
An amino acid polymer in which one or more amino acid residues is an "unnatural" amino acid, not corresponding to any naturally occurring amino acid, is also encompassed by the use of the term "protein" herein.
A "fragment of a protein" means a protein which is a portion of another protein. For instance, fragments of a proteins may be polypeptides obtained by digesting full-length protein isolated from cultured cells. A fragment of a protein will typically comprise at least six amino acids. More typically, the fragment will comprise at least ten amino acids. Preferably, the fragment comprises at least about 16 amino acids.
The term "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. The antibody may be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. Derivatives of the IgG class, however, are preferred in the present invention.
The term "antibody fragment" refers to any derivative of an antibody which is less than full-length. Preferably, 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')Z, scFv, Fv, dsFv diabody, and Fd fragments. The antibody fragment may be produced by any means. For instance, 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. Alternatively, the antibody fragment may be wholly or partially synthetically produced. The antibody fragment may optionally be a single chain antibody fragment.
Alternatively, 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 (scFvs) are recombinant antibody fragments consisting of only the variable light chain (VL) and variable heavy chain {VH) covalently connected to one another by a polypeptide linker. Either VL or VH may be the NH2-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. Typically, the linkers are comprised primarily of stretches of glycine and serine residues with some glutamic acid or lysine residues interspersed for solubility.
"Diabodies" are dimeric scFvs. The components of diabodies typically have shorter peptide linkers than most scFvs and they show a preference for associating as dimers.
An "Fv" fragment is an antibody fragment which consists of one VH and one VL domain held together by noncovalent interactions. The term "dsFv" is used herein to refer to an Fv with an engineered intermolecular disulfide bond to stabilize the VH-VL pair.
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. The heavy chain segment of the Fab fragment is the Fd piece.
The term "protein-capture agent" means a molecule or a mufti-molecular complex which can bind a protein to itself. Protein-capture agents preferably bind their binding partners in a substantially specific manner. Protein-capture agents with a dissociation constant (KD) of less than about 10~ are preferred.
Antibodies or antibody fragments are highly suitable as protein-capture agents. Antigens may also serve as protein-capture agents, since they are capable of binding antibodies.
A receptor which binds a protein ligand is another example of a possible protein-capture agent. Protein-capture agents are understood not to be limited to agents which only interact with their binding partners through noncovalent interactions.
Protein-capture agents may also optionally become covalently attached to the . 10 proteins which they bind. For instance, the protein-capture agent may be photocrosslinked to its binding partner following binding.
The term "binding partner" means a protein which is bound by a particular protein-capture agent, preferably in a substantially specific manner. In some cases, the binding partner may be the protein normally bound in vivo by a protein which is a protein-capture agent. In other embodiments, however, the binding partner may be the protein or peptide on which the protein-capture agent was selected (through in vitro or in vivo selection) or raised (as in the case of antibodies). A binding partner may be shared by more than one protein-capture agent. For instance, a binding partner which is bound by a variety of polyclonal antibodies may bear a number of different epitopes. One protein-capture agent may also bind to a multitude of binding partners (for instance, if the binding partners share the same epitope), "Conditions suitable for protein binding" means those conditions (in terms of salt concentration, pH, detergent, protein concentration, temperature, etc.) which allow for binding to occur between a protein and its binding partner in solution. Preferably, the conditions are not so lenient that a significant amount of nonspecific protein binding occurs.
A "body fluid" may be any liquid substance extracted, excreted, or secreted from an organism or tissue of an organism. The body fluid need not necessarily contain cells. Body fluids of relevance to the present invention include, but are not limited to, whole blood, serum, urine, plasma, cerebral spinal fluid, tears, sinovial fluid, and amniotic fluid.
An "array" is an arrangement of entities in a pattern on a substrate.
Although the pattern is typically a two-dimensional pattern, the pattern may also be a three-dimensional pattern.
The term "substrate" refers to the bulk, underlying, and core material of the arrays of the invention.
ll The terms "micromachining" and "microfabrication" both refer to any number of techniques which are useful in the generation of microstructures (structures with feature sizes of sub-millimeter scale). Such technologies include, but are not limited to, laser ablation, electrodeposition, physical and chemical vapor deposition, photolithography, and wet chemical and dry etching. Related technologies such as injection molding and LIGA (x-ray lithography, electrodeposition, and molding) are also included. Most of these techniques were originally developed for use in semiconductors, microelectronics, and Micro-ElectroMechanical Systems (MEMS) but are applicable to the present invention as well.
The term "coating" means a layer that is either naturally or synthetically formed on or applied to the surface of the substrate. For instance, exposure of a substrate, such as silicon, to air results in oxidation of the exposed surface. In the case of a substrate made of silicon, a silicon oxide coating is formed on the surface upon exposure to air. In other instances, the coating is not derived from the substrate and may be placed upon the surface via mechanical, physical, electrical, or chemical means. An example of this type of coating would be a metal coating that is applied to a silicon or polymer substrate or a silicon nitride coating that is applied to a silicon substrate. Although a coating may be of any thickness, typically the coating has a thickness smaller than that of the substrate.
An "interlayer" is an additional coating or layer that is positioned between the first coating and the substrate. Multiple interlayers may optionally be used together. The primary purpose of a typical interlayer is to aid adhesion between the first coating and the substrate. One such example is the use of a titanium or chromium interlayer to help adhere a gold coating to a silicon or glass surface.
However, other possible functions of an interlayer are also anticipated. For instance, some interlayers may perform a role in the detection system of the array (such as a semiconductor or metal layer between a nonconductive substrate and a nonconductive coating).
An "organic thinfihn" is a thin layer of organic molecules which has been applied to a substrate or to a coating on a substrate if present. Typically, an organic thinfilm is less than about 20 nm thick. Optionally, an organic thinfihn may be less than about 10 nm thick. An organic thinfihn may be disordered or ordered. For instance, an organic thinfilin can be amorphous (such as a chemisorbed or spin-coated polymer) or highly organized (such as a Langmuir-Blodgett film or self assembled monolayer). An organic thinfilm may be heterogeneous or homogeneous. Organic thinfilms which are monolayers are preferred. A lipid bilayer or monolayer is a preferred organic thinfihn.
Optionally, the organic thinfihn may comprise a combination of more than one form of organic thinfilin. For instance, an organic thinfihn may comprise a lipid bilayer on top of a self assembled monolayer. A hydrogel may also compose an organic thinfilm. The organic thinfilm will typically have functionalities exposed on its surface which serve to enhance the surface conditions of a substrate or the coating on a substrate in any of a number of ways. For instance, exposed functionalities of the organic thinfihn are typically useful in the binding or covalent immobilization of the proteins to the patches of the array.
Alternatively, the organic thinfihn may bear functional groups (such as polyethylene glycol (PEG)) which reduce the non-specific binding of molecules to the surface.
Other exposed functionalities serve to tether the thinfilin to the surface of the substrate or the coating: Particular functionalities of the organic thinfilm may also be designed to enable certain detection techniques to be used with the surface.
Alternatively, the organic thinfilm may serve the purpose of preventing inactivation of a protein immobilized on a patch of the array or analytes which are proteins from occurring upon contact with the surface of a substrate or a coating on the surface of a substrate.
A "monolayer" is a single-molecule thick organic thinfilm. A monolayer may be disordered or ordered. A monolayer may optionally be a polymeric compound, such as a polynonionic polymer, a polyionic polymer, or a block-copolymer. For instance, the monolayer may be composed of a poly(amino acid) such as polylysine. A monolayer which is a self assembled monolayer, however, is most preferred. One face of the self assembled monolayer is typically composed of chemical functionalities on the termini of the organic molecules that are chemisorbed or physisorbed onto the surface of the substrate or, if present, the coating on the substrate. Examples of suitable functionalities of monolayers include the positively charged amino groups of poly-L-lysine for use on negatively charged surfaces and thiols for use on gold surfaces. Typically, the other face of the self assembled monolayer is exposed and may bear any number of chemical functionalities (end groups). Preferably, the molecules of the self assembled monolayer are highly ordered.
A "self assembled monolayer" is a monolayer which is created by the spontaneous assembly of molecules. The self assembled monolayer may be ordered, disordered, or exhibit short- to long-range order.
An "affinity tag" is a functional moiety capable of directly or indirectly immobilizing a protein onto an exposed functionality of the organic thinfilm.
Preferably, the affinity tag enables the site-specific immobilization and thus enhances orientation of the protein onto the organic thinfilm. In some cases, the affinity tag may be a simple chemical functional group. Other possibilities include amino acids, poly(amino acid) tags, or full-length proteins. Still other possibilities include carbohydrates and nucleic acids. For instance, the affinity tag may be a polynucleotide which hybridizes to another polynucleotide serving as a functional group on the organic thinfilin ar another polynucleotide serving as an adaptor. The affinity tag may also be a synthetic chemical moiety. If the organic thinfilrn of each of the patches comprises a lipid bilayer or monolayer, then a membrane anchor is a suitable affinity tag. The affinity tag may be covalently or noncovalently attached to the protein. For instance, if the affinity tag is covalently attached to the protein it may be attached via chemical conjugation or as a fusion protein. The affinity tag may also be attached to the protein via a cleavable linkage. Alternatively, the affinity tag may not be directly in contact with the protein. The affinity tag may instead be separated from the protein by an adaptor. The affinity tag may immobilize the protein to the organic thinfilm either through noncovalent interactions or through a covalent linkage.
An "adaptor", for purposes of this invention, is any entity that links an affinity tag to the immobilized protein of a patch of the array. The adaptor may be, but need not necessarily be, a discrete molecule that is noncovalently attached to both the affinity tag and the protein. The adaptor can instead be covalently attached to the affinity tag or the protein or both (via chemical conjugation or as a fusion protein, for instance). Proteins such as full-length proteins, polypeptides, or peptides are typical adaptors. Other possible adaptors include carbohydrates and nucleic acids.
The term "fusion protein" refers to a protein composed of two or more polypeptides that, although typically enjoined in their native state, are joined by their respective amino and carboxyl termini through a peptide linkage to form a single continuous polypeptide. It is understood that the two or more polypeptide components can either be directly joined or indirectly joined through a peptide linker/spacer.
The term "normal physiological condition" means conditions that are typical inside a living organism or a cell. While it is recognized that some organs or organisms provide extreme conditions, the infra-organismal and infra-cellular environment normally varies around pH 7 (i. e., from pH 6.5 to pH 7.5), contains water as the predominant solvent, and exists at a temperature above 0°C
and below 50°C. It will be recognized that the concentration of various salts depends on the organ, organism, cell, or cellular compartment used as a reference:
"Proteomics" means the study of or the characterization of either the proteome or some fraction of the proteome. The "proteome" is the total collection of the intracellular proteins of a cell or population of cells and the proteins secreted by the cell or population of cells. This characterization most typically . 15 includes measurements of the presence, and usually quantity, of the proteins which have been expressed by a cell. The function, structural characteristics (such as post translational modification), and location within the cell of the proteins may also be studied. "Functional proteomics" refers to the study of the functional characteristics, activity level, and structural characteristics of the protein expression products of a cell or population of cells.
(b) Arrays of proteins.
The present invention is directed to arrays of proteins. Typically, the protein arrays comprise micrometer-scale, two-dimensional patterns of patches of proteins immobilized on an organic thi~lm coating on the surface of the substrate.
In one embodiment, the present invention provides an array of proteins which comprises a substrate, at least one organic thinfihn 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.
In most cases, the array will comprise at least about ten patches. In a preferred embodiment, the array comprises at least about SO patches. In a particularly preferred embodiment the array comprises at least about 100 patches.
In alternative preferred embodiments, the array of proteins may comprise more than 103, 104 or 105 patches.
The area of surface of the substrate covered by each of the patches is preferably no more than about 0.25 mm2. Preferably, the area of the substrate surface covered by each of the patches is between about 1 pxn2 and about 10,000 ptn2. In a particularly preferred embodiment, each patch covers an area of the substrate surface from about 100 pmt to about 2,500 Nxnz. In an alternative embodiment, a patch on the array may cover an area of the substrate surface as small as about 2,500 nm2, although patches of such small size are generally not necessary for the use of the array .
The patches of the array may be of any geometric shape. For instance, 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. Preferably, the patches of the array are separated from neighboring patches by about 1 pxn to about 500 ~ln. Typically, 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 p,cn. If the patch size is smaller, then the distance separating the patches will typically be larger than the dimensions of the patch.
In a preferred embodiment of the array, the patches of the array are all contained within an area of about 1 cm2 or less on the surface of the substrate. In one preferred embodiment of the array, therefore, the array comprises 100 or more patches within a total area of about 1 cm2 or less on the surface of the substrate.
Alternatively, a particularly preferred array comprises 103 or more patches within a total area of about 1 cm2 or less. A preferred array may even optionally comprise 104 or 105 or more patches within an area of about 1 cm2 or less on the surface of the substrate. In other embodiments of the invention, all of the patches of the array are contained within an area of about 1 mm2 or less on the surface of the substrate.
Typically, only one type of protein is immobilized on each patch of the array. In a preferred embodiment of the array, the protein immobilized on one patch differs from the protein immobilized on a second patch of the same array.
In such an embodiment, a plurality of different proteins are present on separate patches of the array. Typically the array comprises at least about ten different proteins. Preferably, the array comprises at least about SU different proteins.
More preferably, the array comprises at least about 100 different proteins.
Alternative preferred arrays comprise more than about 103 different proteins or more than about 104 different proteins. The array may even optionally comprise more than about 105 different proteins.
In one embodiment of the array, each of the patches of the array comprises a different protein. For instance, an array comprising about 100 patches could comprise about 100 different proteins. Likewise, an array of about 10,000 patches could comprise about 10,000 different proteins. In an alternative embodiment, however, each different protein is immobilized on more than one separate patch on the array. For instance, each different protein may optionally be present on two to six different patches. An array of the invention, therefore, may comprise about three-thousand protein patches, but only comprise about one thousand different proteins since each different protein is present on three different patches.
In another embodiment of the present invention, although the protein of one patch is different from that of another, the proteins are related. In a preferred embodiment, the two different proteins are members of the same protein family.
The different proteins on the invention array may be either functionally related or just suspected of being functionally related. In another embodiment of the invention array, however, the function of the immobilized proteins may be unknown. In this case, the different proteins on the different patches of the array share a similarity in structure or sequence or are simply suspected of sharing a similarity in structure or sequence. Alternatively, the immobilized proteins may be just fragments of different members of a protein family.
The proteins immobilized on the array of the invention may be members of a protein family such as a receptor family (examples: growth factor receptors, catecholamine receptors, amino acid derivative receptors, cytokine receptors, lectins), ligand family (examples: cytokines, serpins), enzyme family (examples:
proteases, kinases, phosphatases, ras-like GTPases, hydrolases), and transcription factors (examples: steroid hormone receptors, heat-shock transcription factors, zinc-finger proteins, leucine-zipper proteins, homeodomain proteins). In one embodiment, the different immobilized proteins are all HIV proteases or hepatitis C virus (HCV) proteases. In other embodiments of the invention, the immobilized proteins on the patches of 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.
In a preferred embodiment, the protein immobilized on each patch is an antibody or antibody fragment. The antibodies or antibody fragments of the array may optionally be single-chain Fvs, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, dsFvs diabodies, Fd fragments, full-length antigen-specific polyclonal antibodies, or full-length monoclonal antibodies. In a preferred embodiment, the immobilized proteins on the patches of the array are monoclonal antibodies, Fab fragments or single-chain Fvs.
In another preferred embodiment of the invention, the proteins immobilized to each patch of the array are protein-capture agents.
In an alternative embodiment of the invention array, the proteins on different patches are identical.
Biosensors, micromachined devices, and diagnostic devices that comprise the protein arrays of the invention are also contemplated by the present invention.
(c) Substrates, coating, and organic thinfilms.
The substrate of the array may be either organic or inorganic, biological or non-biological, or any combination of these materials. In one embodiment, the substrate is transparent or translucent. The portion of the surface of the substrate on which the patches reside is preferably flat and firm or semi-firm. However, the array of the present invention need not necessarily be flat or entirely two-dimensional. Significant topological features may be present on the surface of the substrate surrounding the patches, between the patches or beneath the patches.
For instance, walls or other barriers may separate the patches of the array.
Numerous materials are suitable for use as a substrate in the array embodiment of the invention. For instance, the substrate of the invention array can comprise a material selected from a group consisting of silicon, silica, quartz, glass, controlled pore glass, carbon, alumina, titania, tantalum oxide, germanium, silicon nitride, zeolites, and gallium arsenide. Many metals such as gold, platinum, aluminum, copper, titanium, and their alloys are also options for substrates of the array. In addition, many ceramics and polymers may also be used as substrates. Polymers which may be used as substrates include, but are not limited to, the following: polystyrene; poly(tetra)fluoroethylene (PTFE);
polyvinylidenedifluoride; polycarbonate; polymethylmethacrylate;
polyvinylethylene; polyethyleneimine; poly(etherether)ketone; polyoxymethylene (POM); polyvinylphenol; polylactides; polymethacrylimide (PMI);
polyalkenesulfone (PAS); polypropylene; polyethylene;
polyhydroxyethylmethacrylate (HEMA); polydimethylsiloxane; polyacrylamide;
polyimide; and block-copolymers. Preferred substrates for the array include silicon, silica, glass, and polymers. The substrate on which the patches reside may also be a combination of any of the aforementioned substrate materials.
An array of the present invention may optionally further comprise a coating between the substrate and organic thinfilm on the array. This coating may either be formed on the substrate or applied to the substrate. The substrate can be modified with a coating by using thin-film technology based, for example, on physical vapor deposition (PVD), thermal processing, or plasma-enhanced chemical vapor deposition (PECVD). Alternatively, plasma exposure can be used to directly activate or alter the substrate and create a coating. For instance, plasma etch procedures can be used to oxidize a polymeric surface (i. e., polystyrene or polyethylene to expose polar functionalities such as hydroxyls, carboxylic acids, aldehydes and the like).
WO 00/04382 PCT/US99/159~1 The coating is optionally 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. In a preferred embodiment, the metal film is a noble metal film.
Noble metals that may be used for a coating include, but are not limited to, gold, platinum, silver, and copper. In an especially preferred embodiment, the coating comprises gold or a gold alloy. Electron-beam evaporation may be used to provide a thin coating of gold on the surface of the substrate. In a preferred embodiment, the metal film is from about 50 nm to about 500 nm in thickness.
In an alternative embodiment, the metal film is from about 1 nm to about 1 pm in thickness.
In alternative embodiments, the coating comprises a composition selected from the group consisting of silicon, silicon oxide, titania, tantalum oxide, silicon nitride, silicon hydride, indium tin oxide, magnesium oxide, alumina, glass, hydroxylated surfaces, and polymers.
In one embodiment of the invention array, the surface of the coating is atomically flat. In this embodiment, the mean roughness of the surface of the coating is less than about 5 angstroms for areas of at least 25 prn2. In a preferred embodiment, the mean roughness of the surface of the coating is less than about 3 angstroms for areas of at least 25 pln2. The ultraflat coating can optionally be a template-stripped surface as described in Hegner et al., Surface Science, 1993, 291:39-46 and Wagner et al., Langmuir, 1995, 11:3867-3875, both of which are incorporated herein by reference.
It is contemplated that the coatings of many arrays will require the addition of at least one adhesion layer between said coating and the substrate.
Typically, the adhesion layer will be at least 6 angstroms thick and may be much thicker.
For instance, a layer of titanium or chromium may be desirable between a silicon wafer and a gold coating. In an alternative embodiment, an epoxy glue such as Epo-tek 377~, Epo-tek 301-2~, (Epoxy Technology Inc., Billerica, Massachusetts) may be preferred to aid adherence of the coating to the substrate.
Determinations as to what material should be used for the adhesion layer would be obvious to one skilled in the art once materials are chosen for both the substrate and coating. In other embodiments, additional adhesion mediators or interlayers may be necessary to improve the optical properties of the array, for instance, in waveguides for detection purposes.
Deposition or formation of the coating (if present) on the substrate is performed prior to the formation of the organic thinfilm thereon. Several different types of coating may be combined on the surface. The coating may cover the wholes surface of the substrate or only parts of it. The pattern of the coating may or may not be identical to the pattern of organic thinfilms used to immobilize the proteins. In one embodiment of the invention, the coating covers the substrate surface only at the site of the patches of the immobilized. Techniques useful for the formation of coated patches on the surface of the substrate which are organic thinfilin compatible are well known to those of ordinary skill in the art. For instance, the patches of coatings on the substrate may optionally be fabricated by photolithography, micromolding (PCT Publication WO 96/29629), wet chemical or dry etching, or any combination of these.
The organic thinfilm on which each of the patches of proteins is immobilized forms a layer either on the substrate itself or on a coating covering the substrate. The organic thinfilin on which the proteins of the patches are immobilized is preferably less than about 20 nm thick. In some embodiments of the invention, the organic thinfilin of each of the patches may be less than about nm thick.
A variety of different organic thinfilms are suitable for use in the present invention. Methods for the formation of organic thinfihns include in situ growth from the surface, deposition by physisorption, spin-coating, chemisorption, self assembly, or plasma-initiated polymerization from gas phase. For instance, a hydrogel composed of a material such as dextran can serve as a suitable organic thinfihn on the patches of the array. In one preferred embodiment of the invention, the organic thinfilm is a lipid bilayer. In another preferred embodiment, the organic thinfilm of each of the patches of the array is a monolayer. A monolayer of polyarginine or polylysine adsorbed on a negatively charged substrate or coating is one option for the organic thinfilm. Another option is a disordered monolayer of tethered polymer chains. In a particularly preferred embodiment, the organic thinfilm is a self assembled monolayer. A monolayer of polylysine is one option for the organic thinfilm. The organic thinfilm is most preferably a self assembled monolayer which comprises molecules of the formula X-R-Y, wherein R is a spacer, X is a functional group that binds R to the surface, and Y is a functional group for binding proteins onto the monolayer. In an alternative preferred embodiment, the self assembled monolayer is comprised of molecules of the formula (X)aR(Y)b where a and b are, independently, integers greater than or equal to 1 and X, R, and Y are as previously defined. In an alternative preferred embodiment, the organic thinfilm comprises a combination of organic thinfilms such as a combination of a lipid bilayer immobilized on top of a self assembled monolayer of molecules of the formula X-R-Y. As another example, a monolayer of polylysine can also optionally be combined with a self assembled monolayer of molecules of the formula X-R-Y (see US Patent No.
Micropumps, electroosmotic flow, integrated valves and mixing devices control liquid movement through the channel network. Microfluidic networks prevent evaporation but, due to the large surface to volume ratio, result in significant protein inactivation. The successful use of microfluidic networks in biomolecule screening remains to be shown.
Drug screening of soluble targets against solid-phase synthesized drug components is intrinsically limited: The surfaces required for solid state organic synthesis are chemically diverse and often cause the inactivation or non-specific binding of proteins, leading to a high rate of false-positive results.
Furthermore, the chemical diversity of drug compounds is limited by the combinatorial synthesis approach that is used to generate the compounds at the interface.
Another major disadvantage of this approach stems from the limited accessibility of the binding site of the soluble target protein to the immobilized drug candidates.
Miniaturized DNA chip technologies have been developed (for example, see U.S. Patent Nos. 5,412,087, 5,445, 934 and 5,744,305) and are currently being exploited for nucleic acid hybridization assays. However, DNA biochip technology is not transferable to protein arrays because the chemistries and materials used for DNA biochips are not readily transferable to use with proteins.
Nucleic acids withstand temperatures up to 100°C, can be dried and re-hydrated without loss of activity, and can be bound directly to organic adhesion layers supported by materials such as glass while maintaining their activity. In contrast, proteins must remain hydrated, kept at ambient temperatures, and are very sensitive to the physical and chemical properties of the support materials.
Therefore, maintaining protein activity at the liquid-solid interface requires entirely different immobilization strategies than those used for nucleic acids.
Additionally, the proper orientation of the protein at the interface is desirable to ensure accessibility of their active sites with interacting molecules. With miniaturization of the chip and decreased feature sizes the ratio of accessible to non-accessible antibodies becomes increasingly relevant and important.
In addition to the goal of achieving high-throughput screening of compounds against targets, to identify potential drug leads, researchers also need to be able to identify highly specific lead compounds early in the drug discovery process. Analyzing a multitude of members of a protein family or forms of a polymorphic protein in parallel (multitarget screening) enables quick identification of highly specific lead compounds. Proteins within a structural family share similar binding sites and catalytic mechanisms. Often, a compound that effectively interferes with the activity of one family member also interferes with other members of the same family. Using standard technology to discover such additional interactions requires a tremendous effort in time and costs and as a consequence is simply not done.
However, cross-reactivity of a drug with related proteins can be the cause of low efficacy or even side effects in patients. For instance, AZT, a major treatment for AIDS, blocks not only viral polymerises, but also human polymerises, causing deleterious side effects. Cross-reactivity with closely related proteins is also a problem with nonsteroidal anti-inflammatory drugs (NSAIDs} and aspirin. These drugs inhibit cyclooxygenase-2, an enzyme which promotes pain and inflammation. However, the same drugs also strongly inhibit a related enzyme, cyclooxygenase-l, that is responsible for keeping the stomach lining and kidneys healthy, leading to common side-effects including stomach irritation.
For the foregoing reasons, there is a need for miniaturized protein arrays and for methods for the parallel, in vitro, screening of the interactions between a plurality of proteins and one or more analytes in a manner that minimizes reagent volumes and protein inactivation problems.
WO 00/04382 . PCT/US99/15971 SUMMARY OF THE INVENTION
The present invention is directed to miniaturized protein arrays and methods of use thereof that satisfy the need for parallel, in vitro, screening of the interactions between a plurality of proteins and one or more analytes in a manner that minimizes reagent volumes and protein inactivation problems.
In one embodiment, the present invention provides an array of proteins which comprises a substrate, at least one organic thinfilin 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 thinfllin, wherein each of said patches comprises a protein immobilized on the underlying organic thinfilm.
Preferably, a plurality of different proteins are present on separate patches of the array.
In a second embodiment, the invention provides a method for screening a plurality of proteins for their ability to interact with a component of a sample.
The method of this embodiment comprises delivering the sample to the array of proteins of the invention, and detecting, either directly or indirectly, for the interaction of the component with the immobilized protein of each patch.
In a third embodiment, the invention provides a method for screening a plurality of proteins for their ability to bind a particular component of a sample.
The method of this embodiment comprises first delivering the sample to the array of proteins of the invention. In a final step, the method comprises detecting, either directly or indirectly, for the presence or amount of the particular component which is retained at each patch. Optionally, the method comprises the additional step of further characterizing the particular component retained at the site of at least one patch.
In an alternative embodiment, the invention provides a method of assaying for protein-protein binding interactions. The first step of the method of this embodiment comprises delivering a sample comprising at least one protein to be assayed for binding to the protein array of the invention. The last step comprises detecting, either directly or indirectly, for the presence or amount of the protein from the sample which is retained at each patch In another embodiment of the invention, a method for assaying for a plurality of analytes in a sample is provided which comprises delivering the sample to a protein array of the invention and detecting for the interaction of the analytes with the immobilized protein at each patch.
In still another embodiment of the invention, an alternative method for assaying for a plurality of analytes in a sample is provided which cbmprises delivering the fluid sample to a protein array of the invention and detecting either directly or indirectly, for the presence or amount of analyte retained at each patch.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows the top view of an array of protein-reactive patches.
Figure 2 shows the cross section of an individual patch of the array of Figure 1.
Figure 3 shows the cross section of a row of monolayer-covered patches of the array of Figure 1.
Figure 4 shows a thiolreactive monolayer on a substrate.
Figure 5 shows an aminoreactive monolayer on a coated substrate.
Figure 6 shows the immobilization of a protein on a monolayer-coated substrate via an affinity tag.
Figure 7 shows the immobilization of a protein on a monolayer-coated substrate via an affinity tag and an adaptor.
Figure 8 shows a schematic of a fluorescence detection unit which may be used to monitor interaction of the proteins of the array with an analyte.
Figure 9 shows a schematic of an ellipsometric detection unit which may be used to monitor interactions between analytes and the proteins of the array.
DETAILED DESCRIPTION OF THE INVENTION
A variety of protein arrays, methods, and protein-coated substrates useful for drug development, proteomics, clinical diagnostics, and related applications are provided by the present invention.
(a) Definitions A "protein" means a polymer of amino acid residues linked together by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. Typically, however, a protein will be at least six amino acids long. Preferably, if the protein is a short peptide, it will be at least about 10 amino acid residues long. A protein may be naturally occurring, recombinant, or synthetic, or any combination of these. A protein may also be just a fragment of a naturally occurring protein or peptide. A protein may be a single molecule or may be a multi-molecular complex. The term protein may also apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid.
An amino acid polymer in which one or more amino acid residues is an "unnatural" amino acid, not corresponding to any naturally occurring amino acid, is also encompassed by the use of the term "protein" herein.
A "fragment of a protein" means a protein which is a portion of another protein. For instance, fragments of a proteins may be polypeptides obtained by digesting full-length protein isolated from cultured cells. A fragment of a protein will typically comprise at least six amino acids. More typically, the fragment will comprise at least ten amino acids. Preferably, the fragment comprises at least about 16 amino acids.
The term "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. The antibody may be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. Derivatives of the IgG class, however, are preferred in the present invention.
The term "antibody fragment" refers to any derivative of an antibody which is less than full-length. Preferably, 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')Z, scFv, Fv, dsFv diabody, and Fd fragments. The antibody fragment may be produced by any means. For instance, 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. Alternatively, the antibody fragment may be wholly or partially synthetically produced. The antibody fragment may optionally be a single chain antibody fragment.
Alternatively, 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 (scFvs) are recombinant antibody fragments consisting of only the variable light chain (VL) and variable heavy chain {VH) covalently connected to one another by a polypeptide linker. Either VL or VH may be the NH2-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. Typically, the linkers are comprised primarily of stretches of glycine and serine residues with some glutamic acid or lysine residues interspersed for solubility.
"Diabodies" are dimeric scFvs. The components of diabodies typically have shorter peptide linkers than most scFvs and they show a preference for associating as dimers.
An "Fv" fragment is an antibody fragment which consists of one VH and one VL domain held together by noncovalent interactions. The term "dsFv" is used herein to refer to an Fv with an engineered intermolecular disulfide bond to stabilize the VH-VL pair.
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. The heavy chain segment of the Fab fragment is the Fd piece.
The term "protein-capture agent" means a molecule or a mufti-molecular complex which can bind a protein to itself. Protein-capture agents preferably bind their binding partners in a substantially specific manner. Protein-capture agents with a dissociation constant (KD) of less than about 10~ are preferred.
Antibodies or antibody fragments are highly suitable as protein-capture agents. Antigens may also serve as protein-capture agents, since they are capable of binding antibodies.
A receptor which binds a protein ligand is another example of a possible protein-capture agent. Protein-capture agents are understood not to be limited to agents which only interact with their binding partners through noncovalent interactions.
Protein-capture agents may also optionally become covalently attached to the . 10 proteins which they bind. For instance, the protein-capture agent may be photocrosslinked to its binding partner following binding.
The term "binding partner" means a protein which is bound by a particular protein-capture agent, preferably in a substantially specific manner. In some cases, the binding partner may be the protein normally bound in vivo by a protein which is a protein-capture agent. In other embodiments, however, the binding partner may be the protein or peptide on which the protein-capture agent was selected (through in vitro or in vivo selection) or raised (as in the case of antibodies). A binding partner may be shared by more than one protein-capture agent. For instance, a binding partner which is bound by a variety of polyclonal antibodies may bear a number of different epitopes. One protein-capture agent may also bind to a multitude of binding partners (for instance, if the binding partners share the same epitope), "Conditions suitable for protein binding" means those conditions (in terms of salt concentration, pH, detergent, protein concentration, temperature, etc.) which allow for binding to occur between a protein and its binding partner in solution. Preferably, the conditions are not so lenient that a significant amount of nonspecific protein binding occurs.
A "body fluid" may be any liquid substance extracted, excreted, or secreted from an organism or tissue of an organism. The body fluid need not necessarily contain cells. Body fluids of relevance to the present invention include, but are not limited to, whole blood, serum, urine, plasma, cerebral spinal fluid, tears, sinovial fluid, and amniotic fluid.
An "array" is an arrangement of entities in a pattern on a substrate.
Although the pattern is typically a two-dimensional pattern, the pattern may also be a three-dimensional pattern.
The term "substrate" refers to the bulk, underlying, and core material of the arrays of the invention.
ll The terms "micromachining" and "microfabrication" both refer to any number of techniques which are useful in the generation of microstructures (structures with feature sizes of sub-millimeter scale). Such technologies include, but are not limited to, laser ablation, electrodeposition, physical and chemical vapor deposition, photolithography, and wet chemical and dry etching. Related technologies such as injection molding and LIGA (x-ray lithography, electrodeposition, and molding) are also included. Most of these techniques were originally developed for use in semiconductors, microelectronics, and Micro-ElectroMechanical Systems (MEMS) but are applicable to the present invention as well.
The term "coating" means a layer that is either naturally or synthetically formed on or applied to the surface of the substrate. For instance, exposure of a substrate, such as silicon, to air results in oxidation of the exposed surface. In the case of a substrate made of silicon, a silicon oxide coating is formed on the surface upon exposure to air. In other instances, the coating is not derived from the substrate and may be placed upon the surface via mechanical, physical, electrical, or chemical means. An example of this type of coating would be a metal coating that is applied to a silicon or polymer substrate or a silicon nitride coating that is applied to a silicon substrate. Although a coating may be of any thickness, typically the coating has a thickness smaller than that of the substrate.
An "interlayer" is an additional coating or layer that is positioned between the first coating and the substrate. Multiple interlayers may optionally be used together. The primary purpose of a typical interlayer is to aid adhesion between the first coating and the substrate. One such example is the use of a titanium or chromium interlayer to help adhere a gold coating to a silicon or glass surface.
However, other possible functions of an interlayer are also anticipated. For instance, some interlayers may perform a role in the detection system of the array (such as a semiconductor or metal layer between a nonconductive substrate and a nonconductive coating).
An "organic thinfihn" is a thin layer of organic molecules which has been applied to a substrate or to a coating on a substrate if present. Typically, an organic thinfilm is less than about 20 nm thick. Optionally, an organic thinfihn may be less than about 10 nm thick. An organic thinfihn may be disordered or ordered. For instance, an organic thinfilin can be amorphous (such as a chemisorbed or spin-coated polymer) or highly organized (such as a Langmuir-Blodgett film or self assembled monolayer). An organic thinfilm may be heterogeneous or homogeneous. Organic thinfilms which are monolayers are preferred. A lipid bilayer or monolayer is a preferred organic thinfihn.
Optionally, the organic thinfihn may comprise a combination of more than one form of organic thinfilin. For instance, an organic thinfihn may comprise a lipid bilayer on top of a self assembled monolayer. A hydrogel may also compose an organic thinfilm. The organic thinfilm will typically have functionalities exposed on its surface which serve to enhance the surface conditions of a substrate or the coating on a substrate in any of a number of ways. For instance, exposed functionalities of the organic thinfihn are typically useful in the binding or covalent immobilization of the proteins to the patches of the array.
Alternatively, the organic thinfihn may bear functional groups (such as polyethylene glycol (PEG)) which reduce the non-specific binding of molecules to the surface.
Other exposed functionalities serve to tether the thinfilin to the surface of the substrate or the coating: Particular functionalities of the organic thinfilm may also be designed to enable certain detection techniques to be used with the surface.
Alternatively, the organic thinfilm may serve the purpose of preventing inactivation of a protein immobilized on a patch of the array or analytes which are proteins from occurring upon contact with the surface of a substrate or a coating on the surface of a substrate.
A "monolayer" is a single-molecule thick organic thinfilm. A monolayer may be disordered or ordered. A monolayer may optionally be a polymeric compound, such as a polynonionic polymer, a polyionic polymer, or a block-copolymer. For instance, the monolayer may be composed of a poly(amino acid) such as polylysine. A monolayer which is a self assembled monolayer, however, is most preferred. One face of the self assembled monolayer is typically composed of chemical functionalities on the termini of the organic molecules that are chemisorbed or physisorbed onto the surface of the substrate or, if present, the coating on the substrate. Examples of suitable functionalities of monolayers include the positively charged amino groups of poly-L-lysine for use on negatively charged surfaces and thiols for use on gold surfaces. Typically, the other face of the self assembled monolayer is exposed and may bear any number of chemical functionalities (end groups). Preferably, the molecules of the self assembled monolayer are highly ordered.
A "self assembled monolayer" is a monolayer which is created by the spontaneous assembly of molecules. The self assembled monolayer may be ordered, disordered, or exhibit short- to long-range order.
An "affinity tag" is a functional moiety capable of directly or indirectly immobilizing a protein onto an exposed functionality of the organic thinfilm.
Preferably, the affinity tag enables the site-specific immobilization and thus enhances orientation of the protein onto the organic thinfilm. In some cases, the affinity tag may be a simple chemical functional group. Other possibilities include amino acids, poly(amino acid) tags, or full-length proteins. Still other possibilities include carbohydrates and nucleic acids. For instance, the affinity tag may be a polynucleotide which hybridizes to another polynucleotide serving as a functional group on the organic thinfilin ar another polynucleotide serving as an adaptor. The affinity tag may also be a synthetic chemical moiety. If the organic thinfilrn of each of the patches comprises a lipid bilayer or monolayer, then a membrane anchor is a suitable affinity tag. The affinity tag may be covalently or noncovalently attached to the protein. For instance, if the affinity tag is covalently attached to the protein it may be attached via chemical conjugation or as a fusion protein. The affinity tag may also be attached to the protein via a cleavable linkage. Alternatively, the affinity tag may not be directly in contact with the protein. The affinity tag may instead be separated from the protein by an adaptor. The affinity tag may immobilize the protein to the organic thinfilm either through noncovalent interactions or through a covalent linkage.
An "adaptor", for purposes of this invention, is any entity that links an affinity tag to the immobilized protein of a patch of the array. The adaptor may be, but need not necessarily be, a discrete molecule that is noncovalently attached to both the affinity tag and the protein. The adaptor can instead be covalently attached to the affinity tag or the protein or both (via chemical conjugation or as a fusion protein, for instance). Proteins such as full-length proteins, polypeptides, or peptides are typical adaptors. Other possible adaptors include carbohydrates and nucleic acids.
The term "fusion protein" refers to a protein composed of two or more polypeptides that, although typically enjoined in their native state, are joined by their respective amino and carboxyl termini through a peptide linkage to form a single continuous polypeptide. It is understood that the two or more polypeptide components can either be directly joined or indirectly joined through a peptide linker/spacer.
The term "normal physiological condition" means conditions that are typical inside a living organism or a cell. While it is recognized that some organs or organisms provide extreme conditions, the infra-organismal and infra-cellular environment normally varies around pH 7 (i. e., from pH 6.5 to pH 7.5), contains water as the predominant solvent, and exists at a temperature above 0°C
and below 50°C. It will be recognized that the concentration of various salts depends on the organ, organism, cell, or cellular compartment used as a reference:
"Proteomics" means the study of or the characterization of either the proteome or some fraction of the proteome. The "proteome" is the total collection of the intracellular proteins of a cell or population of cells and the proteins secreted by the cell or population of cells. This characterization most typically . 15 includes measurements of the presence, and usually quantity, of the proteins which have been expressed by a cell. The function, structural characteristics (such as post translational modification), and location within the cell of the proteins may also be studied. "Functional proteomics" refers to the study of the functional characteristics, activity level, and structural characteristics of the protein expression products of a cell or population of cells.
(b) Arrays of proteins.
The present invention is directed to arrays of proteins. Typically, the protein arrays comprise micrometer-scale, two-dimensional patterns of patches of proteins immobilized on an organic thi~lm coating on the surface of the substrate.
In one embodiment, the present invention provides an array of proteins which comprises a substrate, at least one organic thinfihn 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.
In most cases, the array will comprise at least about ten patches. In a preferred embodiment, the array comprises at least about SO patches. In a particularly preferred embodiment the array comprises at least about 100 patches.
In alternative preferred embodiments, the array of proteins may comprise more than 103, 104 or 105 patches.
The area of surface of the substrate covered by each of the patches is preferably no more than about 0.25 mm2. Preferably, the area of the substrate surface covered by each of the patches is between about 1 pxn2 and about 10,000 ptn2. In a particularly preferred embodiment, each patch covers an area of the substrate surface from about 100 pmt to about 2,500 Nxnz. In an alternative embodiment, a patch on the array may cover an area of the substrate surface as small as about 2,500 nm2, although patches of such small size are generally not necessary for the use of the array .
The patches of the array may be of any geometric shape. For instance, 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. Preferably, the patches of the array are separated from neighboring patches by about 1 pxn to about 500 ~ln. Typically, 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 p,cn. If the patch size is smaller, then the distance separating the patches will typically be larger than the dimensions of the patch.
In a preferred embodiment of the array, the patches of the array are all contained within an area of about 1 cm2 or less on the surface of the substrate. In one preferred embodiment of the array, therefore, the array comprises 100 or more patches within a total area of about 1 cm2 or less on the surface of the substrate.
Alternatively, a particularly preferred array comprises 103 or more patches within a total area of about 1 cm2 or less. A preferred array may even optionally comprise 104 or 105 or more patches within an area of about 1 cm2 or less on the surface of the substrate. In other embodiments of the invention, all of the patches of the array are contained within an area of about 1 mm2 or less on the surface of the substrate.
Typically, only one type of protein is immobilized on each patch of the array. In a preferred embodiment of the array, the protein immobilized on one patch differs from the protein immobilized on a second patch of the same array.
In such an embodiment, a plurality of different proteins are present on separate patches of the array. Typically the array comprises at least about ten different proteins. Preferably, the array comprises at least about SU different proteins.
More preferably, the array comprises at least about 100 different proteins.
Alternative preferred arrays comprise more than about 103 different proteins or more than about 104 different proteins. The array may even optionally comprise more than about 105 different proteins.
In one embodiment of the array, each of the patches of the array comprises a different protein. For instance, an array comprising about 100 patches could comprise about 100 different proteins. Likewise, an array of about 10,000 patches could comprise about 10,000 different proteins. In an alternative embodiment, however, each different protein is immobilized on more than one separate patch on the array. For instance, each different protein may optionally be present on two to six different patches. An array of the invention, therefore, may comprise about three-thousand protein patches, but only comprise about one thousand different proteins since each different protein is present on three different patches.
In another embodiment of the present invention, although the protein of one patch is different from that of another, the proteins are related. In a preferred embodiment, the two different proteins are members of the same protein family.
The different proteins on the invention array may be either functionally related or just suspected of being functionally related. In another embodiment of the invention array, however, the function of the immobilized proteins may be unknown. In this case, the different proteins on the different patches of the array share a similarity in structure or sequence or are simply suspected of sharing a similarity in structure or sequence. Alternatively, the immobilized proteins may be just fragments of different members of a protein family.
The proteins immobilized on the array of the invention may be members of a protein family such as a receptor family (examples: growth factor receptors, catecholamine receptors, amino acid derivative receptors, cytokine receptors, lectins), ligand family (examples: cytokines, serpins), enzyme family (examples:
proteases, kinases, phosphatases, ras-like GTPases, hydrolases), and transcription factors (examples: steroid hormone receptors, heat-shock transcription factors, zinc-finger proteins, leucine-zipper proteins, homeodomain proteins). In one embodiment, the different immobilized proteins are all HIV proteases or hepatitis C virus (HCV) proteases. In other embodiments of the invention, the immobilized proteins on the patches of 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.
In a preferred embodiment, the protein immobilized on each patch is an antibody or antibody fragment. The antibodies or antibody fragments of the array may optionally be single-chain Fvs, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, dsFvs diabodies, Fd fragments, full-length antigen-specific polyclonal antibodies, or full-length monoclonal antibodies. In a preferred embodiment, the immobilized proteins on the patches of the array are monoclonal antibodies, Fab fragments or single-chain Fvs.
In another preferred embodiment of the invention, the proteins immobilized to each patch of the array are protein-capture agents.
In an alternative embodiment of the invention array, the proteins on different patches are identical.
Biosensors, micromachined devices, and diagnostic devices that comprise the protein arrays of the invention are also contemplated by the present invention.
(c) Substrates, coating, and organic thinfilms.
The substrate of the array may be either organic or inorganic, biological or non-biological, or any combination of these materials. In one embodiment, the substrate is transparent or translucent. The portion of the surface of the substrate on which the patches reside is preferably flat and firm or semi-firm. However, the array of the present invention need not necessarily be flat or entirely two-dimensional. Significant topological features may be present on the surface of the substrate surrounding the patches, between the patches or beneath the patches.
For instance, walls or other barriers may separate the patches of the array.
Numerous materials are suitable for use as a substrate in the array embodiment of the invention. For instance, the substrate of the invention array can comprise a material selected from a group consisting of silicon, silica, quartz, glass, controlled pore glass, carbon, alumina, titania, tantalum oxide, germanium, silicon nitride, zeolites, and gallium arsenide. Many metals such as gold, platinum, aluminum, copper, titanium, and their alloys are also options for substrates of the array. In addition, many ceramics and polymers may also be used as substrates. Polymers which may be used as substrates include, but are not limited to, the following: polystyrene; poly(tetra)fluoroethylene (PTFE);
polyvinylidenedifluoride; polycarbonate; polymethylmethacrylate;
polyvinylethylene; polyethyleneimine; poly(etherether)ketone; polyoxymethylene (POM); polyvinylphenol; polylactides; polymethacrylimide (PMI);
polyalkenesulfone (PAS); polypropylene; polyethylene;
polyhydroxyethylmethacrylate (HEMA); polydimethylsiloxane; polyacrylamide;
polyimide; and block-copolymers. Preferred substrates for the array include silicon, silica, glass, and polymers. The substrate on which the patches reside may also be a combination of any of the aforementioned substrate materials.
An array of the present invention may optionally further comprise a coating between the substrate and organic thinfilm on the array. This coating may either be formed on the substrate or applied to the substrate. The substrate can be modified with a coating by using thin-film technology based, for example, on physical vapor deposition (PVD), thermal processing, or plasma-enhanced chemical vapor deposition (PECVD). Alternatively, plasma exposure can be used to directly activate or alter the substrate and create a coating. For instance, plasma etch procedures can be used to oxidize a polymeric surface (i. e., polystyrene or polyethylene to expose polar functionalities such as hydroxyls, carboxylic acids, aldehydes and the like).
WO 00/04382 PCT/US99/159~1 The coating is optionally 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. In a preferred embodiment, the metal film is a noble metal film.
Noble metals that may be used for a coating include, but are not limited to, gold, platinum, silver, and copper. In an especially preferred embodiment, the coating comprises gold or a gold alloy. Electron-beam evaporation may be used to provide a thin coating of gold on the surface of the substrate. In a preferred embodiment, the metal film is from about 50 nm to about 500 nm in thickness.
In an alternative embodiment, the metal film is from about 1 nm to about 1 pm in thickness.
In alternative embodiments, the coating comprises a composition selected from the group consisting of silicon, silicon oxide, titania, tantalum oxide, silicon nitride, silicon hydride, indium tin oxide, magnesium oxide, alumina, glass, hydroxylated surfaces, and polymers.
In one embodiment of the invention array, the surface of the coating is atomically flat. In this embodiment, the mean roughness of the surface of the coating is less than about 5 angstroms for areas of at least 25 prn2. In a preferred embodiment, the mean roughness of the surface of the coating is less than about 3 angstroms for areas of at least 25 pln2. The ultraflat coating can optionally be a template-stripped surface as described in Hegner et al., Surface Science, 1993, 291:39-46 and Wagner et al., Langmuir, 1995, 11:3867-3875, both of which are incorporated herein by reference.
It is contemplated that the coatings of many arrays will require the addition of at least one adhesion layer between said coating and the substrate.
Typically, the adhesion layer will be at least 6 angstroms thick and may be much thicker.
For instance, a layer of titanium or chromium may be desirable between a silicon wafer and a gold coating. In an alternative embodiment, an epoxy glue such as Epo-tek 377~, Epo-tek 301-2~, (Epoxy Technology Inc., Billerica, Massachusetts) may be preferred to aid adherence of the coating to the substrate.
Determinations as to what material should be used for the adhesion layer would be obvious to one skilled in the art once materials are chosen for both the substrate and coating. In other embodiments, additional adhesion mediators or interlayers may be necessary to improve the optical properties of the array, for instance, in waveguides for detection purposes.
Deposition or formation of the coating (if present) on the substrate is performed prior to the formation of the organic thinfilm thereon. Several different types of coating may be combined on the surface. The coating may cover the wholes surface of the substrate or only parts of it. The pattern of the coating may or may not be identical to the pattern of organic thinfilms used to immobilize the proteins. In one embodiment of the invention, the coating covers the substrate surface only at the site of the patches of the immobilized. Techniques useful for the formation of coated patches on the surface of the substrate which are organic thinfilin compatible are well known to those of ordinary skill in the art. For instance, the patches of coatings on the substrate may optionally be fabricated by photolithography, micromolding (PCT Publication WO 96/29629), wet chemical or dry etching, or any combination of these.
The organic thinfilm on which each of the patches of proteins is immobilized forms a layer either on the substrate itself or on a coating covering the substrate. The organic thinfilin on which the proteins of the patches are immobilized is preferably less than about 20 nm thick. In some embodiments of the invention, the organic thinfilin of each of the patches may be less than about nm thick.
A variety of different organic thinfilms are suitable for use in the present invention. Methods for the formation of organic thinfihns include in situ growth from the surface, deposition by physisorption, spin-coating, chemisorption, self assembly, or plasma-initiated polymerization from gas phase. For instance, a hydrogel composed of a material such as dextran can serve as a suitable organic thinfihn on the patches of the array. In one preferred embodiment of the invention, the organic thinfilm is a lipid bilayer. In another preferred embodiment, the organic thinfilm of each of the patches of the array is a monolayer. A monolayer of polyarginine or polylysine adsorbed on a negatively charged substrate or coating is one option for the organic thinfilm. Another option is a disordered monolayer of tethered polymer chains. In a particularly preferred embodiment, the organic thinfilm is a self assembled monolayer. A monolayer of polylysine is one option for the organic thinfilm. The organic thinfilm is most preferably a self assembled monolayer which comprises molecules of the formula X-R-Y, wherein R is a spacer, X is a functional group that binds R to the surface, and Y is a functional group for binding proteins onto the monolayer. In an alternative preferred embodiment, the self assembled monolayer is comprised of molecules of the formula (X)aR(Y)b where a and b are, independently, integers greater than or equal to 1 and X, R, and Y are as previously defined. In an alternative preferred embodiment, the organic thinfilm comprises a combination of organic thinfilms such as a combination of a lipid bilayer immobilized on top of a self assembled monolayer of molecules of the formula X-R-Y. As another example, a monolayer of polylysine can also optionally be combined with a self assembled monolayer of molecules of the formula X-R-Y (see US Patent No.
5,629,213).
In all cases, the coating, or the substrate itself if no coating is present, must be compatible with the chemical or physical adsorption of the organic thinfilm on its surface. For instance, if the patches comprise a coating between the substrate and a monolayer of molecules of the formula X-R-Y, then it is understood that the coating must be composed of a material for which a suitable functional group X
is available. If no such coating is present, then it is understood that the substrate must be composed of a material for which a suitable functional group X is available.
In a preferred embodiment of the invention, the regions of the substrate surface, or coating surface, which separate the patches of proteins are free of organic thinfilin. In an alternative embodiment, the organic thinfilm extends beyond the area of the substrate surface, or coating surface if present, covered by the protein patches. For instance, optionally, the entire surface of the array may be covered by an organic thinfilm on which the plurality of spatially distinct patches of proteins reside. An organic thinfilm which covers the entire surface of the array may be homogenous or may optionally comprise patches of differing exposed functionalities useful in the immobilization of patches of different proteins. In still another alternative embodiment, the regions of the substrate surface, or coating surface if a coating is present, between the patches of proteins are covered by an organic thinfilin, but an organic thinfilm of a different type than that of the patches of proteins. For instance, the surfaces between the patches of proteins may be coated with an organic thinfilm characterized by low non-specific binding properties for proteins and other analytes.
A variety of techniques may be used to generate patches of organic thinfilin on the surface of the substrate or on the surface of a coating on the substrate.
These techniques are well known to those skilled in the art and will vary depending upon the nature of the organic thinfilin, the substrate, and the coating if present. The techniques will also vary depending on the structure of the underlying substrate and the pattern of any coating present on the substrate.
For instance, patches of a coating which is highly reactive with an organic thinflhn may have akeady been produced on the substrate surface. Arrays of patches of organic thinfilin can optionally be created by microfluidics printing, microstamping (US Patent Nos. 5,512,131 and 5,731,152), or microcontact printing (MCP) (PCT Publication WO 96/29629). Subsequent immobilization of proteins to the reactive monolayer patches results in two-dimensional arrays of the agents. Inkjet printer heads provide another option for patterning monolayer X-R-Y molecules, or components thereof, or other organic thinfilm components to manometer or micrometer scale sites on the surface of the substrate or coating (Lemmo et al., Anal Chem., 1997, 69:543-551; US Patent Nos. 5,843,767 and 5,837,860). In some cases, commercially available arrayers based on capillary dispensing (for instance, OmniGridTM from Genemachines, inc, San Carlos, CA, and High-Throughput Microarrayer from Intelligent Bio-Instruments, Cambridge, MA) may also be of use in directing components of organic thinfilms to spatially distinct regions of the array.
Diffusion boundaries between the patches of proteins immobilized on organic thinfilms such as self assembled monolayers may be integrated as topographic patterns (physical barriers) or surface functionalities with orthogonal wetting behavior (chemical barriers). For instance, walls of substrate material or photoresist may be used to separate some of the patches from some of the others or all of the patches from each other. Alternatively, non-bioreactive organic thinfilms, such as monolayers, with different wettability may be used to separate patches from one another.
In a preferred embodiment of the invention, each of the patches of proteins comprises a self assembled monolayer of molecules of the formula X-R-Y, as previously defined, and the patches are separated from each other by surfaces free of the monolayer.
Figure 1 shows the top view of one example of an array of 25 patches reactive with proteins. On the array, a number of patches 15 cover the surface of the substrate 3.
Figure 2 shows a detailed cross section of a patch 15 of the array of Figure 1. This view illustrates the use of a coating 5 on the substrate 3. An adhesion interlayer 6 is also included in the patch. On top of the patch resides a self assembled monolayer 7.
Figure 3 shows a cross section of one row of the patches 15 of the array of Figure 1. This figure also shows the use of a cover 2 over the array. Use of the cover 2 creates an inlet port 16 and an outlet port 17 for solutions to be passed over the array.
A variety of chemical moieties may function as monolayer molecules of the formula X-R-Y in the array of the present invention. However, three major classes of monolayer formation are preferably used to expose high densities of reactive omega-functionalities on the patches of the array: (i) alkylsiloxane monolayers ("silanes") on hydroxylated and non-hydroxylated surfaces (as taught in, for example, US Patent No. 5,405,766, PCT Publication WO 96/38726, US
Patent No. 5,412,087, and US Patent No. 5,688,642); (ii) alkyl-thiol/dialkyldisulfide monolayers on noble metals (preferably Au(111)) (as, for example, described in Allara et al., US 4,690,715; Bamdad et al., US
5,620,850;
Wagner et al., Biophysical Journal, 1996, 70:2052-2066); and (iii) alkyl monolayer formation on oxide-free passivated silicon (as taught in, for example, Linford et al., J. Am. Chem. Soc., 1995, I 17:3145-3155, Wagner et al., Journal of Structural Biology, 1997, 119:189-201, US Patent No. 5,429,708). One of ordinary skill in the art, however, will recognize that many possible moieties may be substituted for X, R, and/or Y, dependent primarily upon the choice of substrate, coating, and affinity tag. Many examples of monolayers are described in Ulman, An Introduction to Ultrathin Organic Films: From Langmuir-Blodgett to Self Assembly, Academic press ( 1991 ).
In one embodiment, the monolayer comprises molecules of the formula (X)aR(Y)b wherein a and b are, independently, equal to an integer between l and about 200. In a preferred embodiment, a and b are, independently, equal to an integer between 1 and about 80. In a more preferred embodiment, a and b are, independently, equal to 1 or 2. In a most preferred embodiment, a and b are both equal to 1 (molecules of the formula X-R-Y).
If the patches of the invention array comprise a self assembled monolayer of molecules of the formula (X)aR(Y)b, then R may optionally comprise a linear or branched hydrocarbon chain from about 1 to about 400 carbons long. The hydrocarbon chain may comprise an alkyl, aryl, alkenyl, alkynyl, cycloalkyl, alkaryl, aralkyl group, or any combination thereof. If a and b are both equal to one, then R is typically an alkyl chain from about 3 to about 30 carbons long.
In a preferred embodiment, if a and b are both equal to one, then R is an alkyl chain from about 8 to about 22 carbons long and is, optionally, a straight alkane.
However, it is also contemplated that in an alternative embodiment, R may readily comprise a linear or branched hydrocarbon chain from about 2 to about 400 carbons long and be interrupted by at least one hetero atom. The interrupting hetero groups can include -O-, -CONH-, -CONHCO-, -NH-, -CSNH-, -CO-, -CS-, -S-, -SO-, -(OCH2CH2)" (where n=1-20), -(CF2)n (where n=1-22), and the like.
Alternatively, one or more of the hydrogen moieties of R can be substituted with deuterium. In alternative, less preferred, embodiments, R may be more than about 400 carbons long.
X may be chosen as any group which affords chemisorption or physisorption of the monolayer onto the surface of the substrate (or the coating, if present). When the substrate or coating is a metal or metal alloy, X, at least prior to incorporation into the monolayer, can in one embodiment be chosen to be an asymmetrical or symmetrical disulfide, sulfide, diselenide, selenide, thiol, isonitrile, selenol, a trivalent phosphorus compound, isothiocyanate, isocyanate, xanthanate, thiocarbamate, a phosphine, an amine, thio acid or a dithio acid.
This embodiment is especially preferred when a coating or substrate is used that is a noble metal such as gold, silver, or platinum.
If the substrate of the array is a material such as silicon, silicon oxide, indium tin oxide, magnesium oxide, alumina, quartz, glass, or silica, then the array of one embodiment of the invention comprises an X that, prior to incorporation into said monolayer, is a monohalosilane, dihalosilane, trihalosilane, trialkoxysilane, dialkoxysilane, or a monoalkoxysilane. Among these silanes, trichlorosilane and trialkoxysilane are particularly preferred.
In a preferred embodiment of the invention, the substrate is selected from the group consisting of silicon, silicon dioxide, indium tin oxide, alumina, glass, and titania; and X, prior to incorporation into said monolayer, is selected from the group consisting of a monohalosilane, dihalosilane, trihalosilane, trichlorosilane, trialkoxysilane, dialkoxysilane, monoalkoxysilane, carboxylic acids, and phosphates.
In another preferred embodiment of the invention, the substrate of the array is silicon and X is an olefin.
In still another preferred embodiment of the invention, the coating (or the substrate if no coating is present) is titania or tantalum oxide and X is a phosphate.
In other embodiments, the surface of the substrate (or coating thereon) is composed of a material such as titanium oxide, tantalum oxide, indium tin oxide, magnesium oxide, or alumina where X is a carboxylic acid or phosphoric acid.
Alternatively, if the surface of the substrate (or coating thereon) of the array is copper, then X may optionally be a hydroxamic acid.
If the substrate used in the invention is a polymer, then in many cases a coating on the substrate such as a copper coating will be included in the array. An appropriate functional group X for the coating would then be chosen for use in the array. In an alternative embodiment comprising a polymer substrate, the surface of the polymer may be plasma-modified to expose desirable surface functionalities for monolayer formation. For instance, EP 780423 describes the use of a monolayer molecule that has an alkene X functionality on a plasma exposed surface. Still another possibility for the invention array comprised of a polymer is that the surface of the polymer on which the monolayer is formed is functionalized by copolymerization of appropriately functionalized precursor molecules.
Another possibility is that prior to incorporation into the monolayer, X can be a free-radical-producing moiety. This functional group is especially appropriate when the surface on which the monolayer is formed is a hydrogenated silicon surface. Possible free-radical producing moieties include, but are not limited to, diacylperoxides, peroxides, and azo compounds. Alternatively, unsaturated moieties such as unsubstituted alkenes, alkynes, cyano compounds and isonitrile compounds can be used for X, if the reaction with X is accompanied by ultraviolet, infrared, visible, or microwave radiation.
In alternative embodiments, X, prior to incorporation into the monolayer, may be a hydroxyl, carboxyl, vinyl, sulfonyl, phosphoryl, silicon hydride, or an ammo group.
The component, Y, of the monolayer is a functional group responsible for binding a protein onto the monolayer. In a preferred embodiment of the invention, the Y group is either highly reactive (activated) towards the protein or is easily converted into such an activated form. In a preferred embodiment, the coupling of Y with the protein occurs readily under normal physiological conditions not detrimental to the activity of the protein. The functional group Y may either form a covalent linkage or a noncovalent linkage with the protein (or its affinity tag, if present). In a preferred embodiment, the functional group Y forms a covalent linkage with the protein or its affinity tag. It is understood that following the attachment of the protein (with or without an affinity tag) to Y, the chemical nature of Y may have changed. Upon attachment of the protein, Y may even have been removed from the organic thinfilm.
In one embodiment of the array of the present invention, Y is a' functional group that is activated in situ. Possibilities for this type of functional group include, but are not limited to, such simple moieties such as a hydroxyl, carboxyl, amino, aldehyde, carbonyl, methyl, methylene, alkene, alkyne, carbonate, aryliodide, or a vinyl group. Appropriate modes of activation would be obvious to one skilled in the art. Alternatively, Y can comprise a functional group that requires photoactivation prior to becoming activated enough to trap the protein.
In an especially preferred embodiment of the array of the present invention, Y is a complex and highly reactive functional moiety that is compatible with monolayer formation and needs no in situ activation prior to reaction with the protein and/or affinity tag. Such possibilities for Y include, but are not limited to, maleimide, N-hydroxysuccinimide (Wagner et al., Biophysical Journal, 1996, 70:2052-2066), nitrilotriacetic acid (LJS Patent No. 5,620,850), activated hydroxyl, haloacetyl, bromoacetyl, iodoacetyl, activated carboxyl, hydrazide, epoxy, aziridine, sulfonylchloride, trifluoromethyldiaziridine, pyridyldisulfide, N-acyl-imidazole, imidazolecarbamate, vinylsulfone, succinimidylcarbonate, arylazide, anhydride, diazoacetate, beiizophenone, isothiocyanate, isocyanate, imidoester, fluorobenzene, and biotin.
Figure 4 shows one example of a monolayer on a substrate 3. In this example, substrate 3 comprises glass. The monolayer is thiokeactive because it bears a maleimidyl functional group Y.
Figure 5 shows another example of a monolayer on a substrate 3 which is silicon. In this case, however, a thinfihn gold coating S covers the surface of the substrate 3. Also, in this embodiment, a titanium adhesion interlayer 6 is used to adhere the coating 5 to the substrate 3. This monolayer is aminoreactive because it bears an N-hydroxysuccinimidyl functional group Y.
In an alternative embodiment, the functional group Y of the array is selected from the group of simple functional moieties. Possible Y functional groups include, but are not limited to, -OH, -NH2, -COOH, -COOR, -RSR, -Pp4 3, -OS03-2, -S03-, -COO-, -SOO-, -CONR2, -CN, -NR2, and the like.
The monolayer molecules of the present invention can optionally be assembled on the surface in parts. In other words, the monolayer need not necessarily be constructed by chemisorption or physisorption of molecules of the formula X-R-Y to the surface of the substrate (or coating). Instead, in one embodiment, X may be chemisorbed or physisorbed to the surface of the substrate (or coating) alone first. Then, R or even just individual components of R can be attached to X through a suitable chemical reaction. Upon completion of addition of the spacer R to the X moiety akeady immobilized on the surface, Y can be attached to the ends of the monolayer molecule through a suitable covalent linkage.
Not all self assembled monolayer molecules on a given patch need be identical to one another. Some patches may comprise mixed monolayers. For instance, the monolayer of an individual patch may optionally comprise at least two different molecules of the formula X-R-Y, as previously described. This second X-R-Y molecule may optionally immobilize the same protein as the first.
In addition, some of the monolayer molecules X-R-Y of a patch may have failed to attach any protein.
As another alternative embodiment of the invention, a mixed, self assembled monolayer of an individual patch on the array may comprise both molecules of the formula X-R-Y, as previously described, and molecules of the formula, X-R-V where R is a spacer, X is a functional group that binds R to the surface, and V is a moiety which is biocompatible with proteins and resistant to the non-specific binding of proteins. For example, V may consist of a hydroxyl, saccharide, or oligo/polyethylene glycol moiety (EP Publication 780423).
In still another embodiment of the invention, the array comprises at least one unreactive patch of organic thinfilm on the substrate or coating surface which is devoid of any protein. For instance, the unreactive patch may optionally comprise a monolayer of molecules of the formula X-R-V, where R is a spacer, X
is a functional group that binds R to the surface, and V is a moiety resistant to the non-specific binding of proteins. The unreactive patch may serve as a control patch or be useful in background binding measurements.
Regardless of the nature of the monolayer molecules, in some arrays it may be desirable to provide crosslinking between molecules of an individual patch's monolayer. In general, crosslinking confers additional stability to the monolayer.
Such methods are familiar to those skilled in the art (for instance, see Ulman, An Introduction to Ultrathin Organic Films: From Langmuir Blodgett to Self Assembly, Academic Press (1991)).
After completion of formation of the monolayer on the patches, the protein may be attached to the monolayer via interaction with the Y-functional group.
Y-functional groups which fail to react with any proteins are preferably quenched prior to use of the array.
(d) Affinity tags and immobilization of the proteins.
In a preferred embodiment, the protein-immobilizing patches of the array further comprise an affinity tag that enhances immobilization of the protein onto the organic thinfilm. The use of an affinity tag on the protein of the array typically provides several advantages. An affinity tag can confer enhanced binding or reaction of the protein with the functionalities on the organic thinfilm, such as Y if the organic thinfilin is a an X-R-Y monolayer as previously described. This enhancement effect may be either kinetic or thermodynamic. The affinity tag/thinfilm combination used in the patches of the array preferably allows for immobilization of the proteins in a manner which does not require harsh reaction conditions that are adverse to protein stability or function. In most embodiments, immobilization to the organic thinfilm in aqueous, biological buffers is ideal.
An affinity tag also preferably offers immobilization on the organic thinfilm that is specific to a designated site or location on the protein (site-specific immobilization). For this to occur, attachment of the affinity tag to the protein must be site-specific. Site-specific immobilization helps ensure that the active site or binding site of the immobilized protein, such as the antigen-binding site of the antibody moiety, remains accessible to ligands in solution. Another advantage of immobilization through affinity tags is that it allows for a common immobilization strategy to be used with multiple, different proteins.
The affinity tag is optionally attached directly, either covalently or noncovalently, to the protein. In an alternative embodiment, however, the affinity tag is either covalently or noncovalently attached to an adaptor which is either covalently or noncovalently attached to the protein.
In a preferred embodiment, the affinity tag comprises at least one amino acid. The affinity tag may be a polypeptide comprising at least two amino acids which is reactive with the functionalities of the organic thinfllm.
Alternatively, the affinity tag may be a single amino acid which is reactive with the organic thinfilm. Examples of possible amino acids which could be reactive with an organic thinfilin include cysteine, lysine, histidine, arginine, tyrosine, aspartic acid, glutamic acid, tryptophan, serine, threonine, and glutamine. A
polypeptide or amino acid affinity tag is preferably expressed as a fusion protein with the immobilized protein of each patch. Amino acid affinity tags provide either a single amino acid or a series of amino acids that can interact with the functionality of the organic thinfllm, such as the Y-functional group of the self assembled monolayer molecules. Amino acid affinity tags can be readily introduced into recombinant proteins to facilitate oriented immobilization by covalent binding to the Y-functional group of a monolayer or to a functional group on an alternative organic thinfilm.
The affinity tag may optionally comprise a poly(amino acid) tag. A
poly(amino acid) tag is a polypeptide that comprises from about 2 to about 100 residues of a single amino acid, optionally interrupted by residues of other amino acids. For instance, the affinity tag may comprise a poly-cysteine, polylysine, poly-arginine, or poly-histidine. Amino acid tags are preferably composed of two to twenty residues of a single amino acid, such as, for example, histidines, lysines, arginines, cysteines, glutamines, tyrosines, or any combination of these.
According to a preferred embodiment, an amino acid tag of one to twenty amino acids includes at least one to ten cysteines for thioether linkage; or one to ten lysines for amide linkage; or one to ten arginines for coupling to vicinal dicarbonyl groups. One of ordinary skill in the art can readily pair suitable affinity tags with a given functionality on an organic thinfilm.
The position of the amino acid tag can be at an amino-, or carboxy-terminus of the protein of a patch of the array, or anywhere in-between, as long as the active site or binding site of the protein remains in a position accessible for ligand interaction. Where compatible with the protein chosen, affinity tags introduced for protein purification are preferentially located at the C-terminus of the recombinant protein to ensure that only full-length proteins are isolated during protein purification. For instance, if intact antibodies are used on the arrays, then the attachment point of the affinity tag on the antibody is preferably located at a C-terminus of the effector (Fc) region of the antibody. If scFvs are used on the arrays, then the attachment point of the affinity tag is also preferably located at the C-terminus of the molecules.
Affinity tags may also contain one or more unnatural amino acids.
Unnatural amino acids can be introduced using suppressor tRNAs that recognize stop codons (i.e., amber) (Noren et al., Science, 1989, 244:182-188; Elhnan et al., Methods Enzym., 1991, 202:301-336; Cload et al., Chem. Biol., 1996, 3:1033-1038). The tRNAs are chemically amino-acylated to contain chemically altered ("unnatural") amino acids for use with specific coupling chemistries (i. e., ketone modifications, photoreactive groups).
In an alternative embodiment the affinity tag can comprise an intact protein, such as, but not limited to, glutathione S-transferase, an antibody, avidin, or streptavidin.
Other protein conjugation and immobilization techniques known in the art may be adapted for the purpose of attaching affinity tags to the protein. For instance, in an alternative embodiment of the array, the affinity tag may be an organic bioconjugate which is chemically coupled to the protein of interest.
Biotin or antigens may be chemically cross linked to the protein.
Alternatively, a chemical crosslinker may be used that attaches a simple functional moiety such as a thiol or an amine to the surface of a protein to be immobilized on a patch on the array. Alternatively, protein synthesis or protein ligation techniques known to those skilled in the art may be used to attach an affinity tag to a protein.
For instance, intein-mediated protein ligation may optionally be used to attach the affinity tag to the protein (Mathys, et al., Gene 231:1-13, 1999; Evans, et al., Protein Science 7:2256-2264, 1998).
In an alternative embodiment of the invention, the organic thinfilm of each of the patches comprises, at least in part, a lipid monolayer or bilayer, and the affinity tag comprises a membrane anchor. Optionally, the Iipid monolayer or bilayer is immobilized on a self assembled monolayer.
Figure 6 shows a detailed cross section of a patch on one embodiment of the invention array. In this embodiment, a protein 10 is immobilized on a monolayer 7 on a substrate 3. An affinity tag 8 connects the protein 10 to the monolayer 7. The monolayer 7 is formed on a coating 5 which is separated from the substrate 3 by an interlayer 6.
In an alternative embodiment of the invention, no affinity tag is used to immobilize the proteins onto the organic thinfilin. An amino acid or other moiety (such as a carbohydrate moiety) inherent to the protein itself may instead be used to tether the protein to the reactive group of the organic thief lm. In preferred embodiments, the immobilization is site-specific with respect to the location of the site of immobilization on the protein. For instance, the sulfhydryl group on the C-terminal region of the heavy chain portion of a Fab' fragment generated by pepsin digestion of an antibody, followed by selective reduction of the disulfide between monovalent Fab' fragments, may be used as the affinity tag. Alternatively, a carbohydrate moiety on the Fc portion of an intact antibody can be oxidized under mild conditions to an aldehyde group suitable for immobilizing the antibody on a monolayer via reaction with a hydrazide-activated Y group on the monolayer.
Examples of immobilization of proteins without any affinity tag can be found in Wagner et al., Biophys. .L, 70:2437-2441, 1996 and the specific examples, Examples 8-10, below.
When the proteins of at least some of the different patches on the array are different from each other, different solutions, each containing a different, preferably, affinity-tagged protein, must be delivered to their individual patches.
Solutions of proteins may be transferred to the appropriate patches via arrayers which are well-known in the art and even commercially available. For instance, microcapillary-based dispensing systems may be used. These dispensing systems are preferably automated and computer-aided. A description of and building instructions for an example of a microarrayer comprising an automated capillary system can be found on the Internet at http://cmgm.stanford.edu/pbrown/axray.html and http://cmgm.stanford.edu/pbrown/mguide/index.html. The use of other microprinting techniques for transferring solutions containing the proteins to the protein-reactive patches is also possible. Ink jet printer heads may also optionally be used for precise delivery of the proteins to the protein-reactive patches.
Representative, non-limiting disclosures of techniques useful for depositing the proteins on the patches may be found, for example, in U.S. Patent Nos. 5,731, (stamping apparatus), 5,807,522 (capillary dispensing device), 5,837,860 (ink jet printing technique, Hamilton 2200 robotic pipetting delivery system), and 5,843,767 (ink jet printing technique, Hamilton 2200 robotic pipetting delivery system), all incorporated by reference herein.
(e) Adaptors.
Another embodiment of the arrays of the present invention comprises an adaptor that links the affinity tag to the immobilized protein. The additional spacing of the protein from the surface of the substrate (or coating) that is afforded by the use of an adaptor is particularly advantageous since proteins are known to be prone to surface inactivation. The adaptor may optionally afford some additional advantages as well. For instance, the adaptor may help facilitate the attachment of the protein to the affinity tag. In another embodiment, the adaptor may help facilitate the use of a particular detection technique with the array. One of ordinary skill in the art will be able to choose an adaptor which is appropriate for a given affinity tag. For instance, if the affinity tag is streptavidin, then the adaptor could be a biotin molecule that is chemically conjugated to the protein which is to be immobilized.
In a preferred embodiment, the adaptor is a protein. In a preferred embodiment, the affinity tag, adaptor, and protein to be immobilized together compose a fusion protein. Such a fusion protein may be readily expressed using standard recombinant DNA technology. Adaptors which are proteins are especially useful to increase the solubility of the protein of interest and to increase the distance between the surface of the substrate or coating and the protein of interest. Use of an adaptor which is a protein can also be very useful in facilitating the preparative steps of protein purification by affinity binding prior to immobilization on the array. Examples of possible adaptors which are proteins include glutathione-S-transferase (GST), maltose-binding protein, chitin-binding protein, thioredoxin, green-fluorescent protein (GFP). GFP can also be used for quantification of surface binding. If the protein immobilized on the patches of the array is an antibody or antibody fragment comprising an Fc region, then the adaptor may optionally be protein G, protein A, or recombinant protein A/G (a gene fusion product secreted from a non-pathogenic form of Bacillus which contains four Fc binding domains from protein A and two from protein G).
Figure 7 shows a crass section of a patch on one particular embodiment of the invention array. The patch comprises a protein 10 immobilized on a monolayer 7 via both an afFlnity tag 8 and an adaptor molecule 9. The monolayer 7 rests on a coating 5. An interlayer 6 is used between the coating 5 and the substrate 3.
(f) Preparation of the proteins of the array.
The proteins immobilized on the array may be produced by any of the variety of means known to those of ordinary skill in the art.
In preparation for immobilization to the arrays of the present invention, the protein can optionally be expressed from recombinant DNA either in vivo or in vitro. The cDNA of the protein to be immobilized on the array is cloned into an expression vector (many examples of which are commercially available) and introduced into cells of the appropriate organism for expression. A broad range of host cells and expression systems may be used to produce the proteins to be immobilized on the array. For in vivo expression of the proteins, cDNAs can be cloned into commercial expression vectors (Qiagen, Novagen, Clontech, for example) and introduced into an appropriate organism for expression.
Expression in vivo may be done in bacteria (for example, Escherichia coli), plants (for example, Nicotiana tabacum), lower eukaryotes (for example, Saccharomyces cerevisiae, Saccharomyces pombe, Pichia pastoris), or higher eukaryotes (for example, bacculovirus-infected insect cells, insect cells, mammalian cells).
For in vitro expression PCR-amplified DNA sequences are directly used in coupled in vitro transcription/translation systems (for instance: Escherichia coli S30 lysates from T7 RNA polymerase expressing, preferably protease-deficient strains; wheat germ lysates; reticulocyte lysates (Promega, Pharmacia, Panvera)).
The choice of organism for optimal expression depends an the extent of post-translational modifications (i. e., glycosylation, lipid-modifications) desired. One of ordinary skill in the art will be able to readily choose which host cell type is most suitable for the protein to be immobilized and application desired.
DNA sequences encoding amino acid affinity tags and adaptor protein sequences are engineered into the expression vectors such that the genes of interest can be cloned in frame either 5' or 3' of the DNA sequence encoding the affinity tag and adaptor.
The expressed proteins are purified by affinity chromatography using commercially available resins.
Preferably, production of families of related proteins involves parallel processing from cloning to protein expression and protein purification. cDNAs for the protein of interest will be amplified by PCR using cDNA libraries or EST
(expressed sequence tag) clones as templates. Any of the in vitro or in vivo expression systems described above can then be used for expression of the proteins to be immobilized on the array.
Escherichia coli-based protein expression is generally the method of choice for soluble proteins that do not require extensive post-translational modifications for activity. Extracellular or intracellular domains of membrane proteins will be fused to protein adaptors for expression and purification.
The entire approach can be performed using 96-well assay plates. PCR
reactions are carried out under standard conditions. Oligonucleotide primers contain unique restriction sites for facile cloning into the expression vectors.
Alternatively, the TA cloning system (Clontech) can be used. Expression vectors contain the sequences for affinity tags and the protein adaptors. PCR products are ligated into the expression vectors (under inducible promoters) and introduced into the appropriate competent Escherichia coli strain by calcium-dependent transformation (strains include: XL-1 blue, BL21, SG13009(lon-)). Transformed Escherichia coli cells are plated and individual colonies transferred into 96-array blocks. Cultures are grown to mid-log phase, induced for expression, and cells collected by centrifugation. Cells are resuspended containing lysozyme and the membranes broken by rapid freeze/thaw cycles, or by sonication. Cell debris is removed by centrifugation and the supernatants transferred to 96-tube arrays.
The appropriate affinity matrix is added, protein of interest bound and nonspecifically bound proteins removed by repeated washing steps using 12 - 96 pin suction devices and centrifugation. Alternatively, magnetic affinity beads and filtration devices can be used (Qiagen). The proteins are eluted and transferred to a new 96-well array. Protein concentrations are determined and an aliquot of each protein is spotted onto a nitrocellulose filter and verified by Western analysis using an antibody directed against the affinity tag. The purity of each sample is assessed by SDS-PAGE and silver staining or mass spectrometry. Proteins are snap-frozen and stored at -80°C.
Saccharomyces cerevisiae allows for core glycosylation and lipid modifications of proteins. The approach described above for Escherichia coli can be used with slight modifications for transformation and cell lysis.
Transformation of Saccharomyces cerevisiae is by lithium-acetate and cell lysis is either by lyticase digestion of the cell walls followed by freeze-thaw, sonication or glass-bead extraction. Variations of post-translational modifications can be obtained by different yeast strains (i.e. Saccharomyces pombe, Pichia pastoris).
The advantage of the bacculovirus system or mammalian cells are the wealth of post-translational modifications that can be obtained. The bacculo-system requires cloning of viruses, obtaining high titer stocks and infection of liquid insect cell suspensions (cells are SF9, SF21). Mammalian cell-based expression requires transfection and cloning of cell lines. Soluble proteins are collected from the medium while intracellular or membrane bound proteins require cell lysis (either detergent solubilization, freeze-thaw). Proteins can then be purified analogous to the procedure described for Escherichia coli.
For in vitro translation the system of choice is Escherichia coli lysates obtained from protease-deficient and T7 RNA polymerase overexpressing strains.
Escherichia coli lysates provide efficient protein expression (30-50 pg/ml lysate).
The entire process is carried out in 96-well arrays. Genes of interest are amplified by PCR using oligonucleotides that contain the gene-specific sequences containing a T7 RNA polymerase promoter and binding site and a sequence encoding the affinity tag. Alternatively, an adaptor protein can be fused to the gene of interest by PCR. Amplified DNAs can be directly transcribed and translated in the Escherichia coli lysates without prior cloning for fast analysis.
The proteins are then isolated by binding to an affinity matrix and processed as described above.
Alternative systems which may be used include wheat germ extracts and reticulocyte extracts. In vitro synthesis of membrane proteins and or post-translationally modified proteins will require reticulocyte lysates in combination with microsomes.
In one preferred embodiment of the invention, the proteins immobilized on the patches of the array are antibodies. Optionally, the immobilized proteins may be monoclonal antibodies. The production of monoclonal antibodies against specific protein targets is routine using standard hybridoma technology. In fact, numerous monoclonal antibodies are available commercially.
As an alternative to obtaining antibodies or antibody fragments which have been produced by cell fusion or from continuous cell lines, the antibody moieties may be expressed in bacteriophage. Such antibody phage display technologies are well known to those skilled in the art. The bacteriophage expression systems allow for the random recombination of heavy- and light-chain sequences, thereby creating a library of antibody sequences which can be selected against the desired antigen. The expression system can be based on bacteriophage ~, or , more preferably, on filamentous phage. The bacteriophage expression system can be used to express Fab fragments, Fv's with an engineered intermolecular disulfide bond to stabilize the VH-VL pair (dsFv's), scFvs, or diabody fragments.
The antibody genes of the phage display libraries may be from pre-immunized donors. For instance, the phage display library could be a display library prepared from the spleens of mice previously immunized with a mixture of proteins (such as a Iysate of human T-cells). Immunization can optionally be used to bias the library to contain a greater number of recombinant antibodies reactive towards a specific set of proteins (such as proteins found in human T-cells).
Alternatively, the library antibodies may be derived from naive or synthetic libraries. The naive libraries have been constructed from spleens of mice which have not been contacted by external antigen. In a synthetic library, portions of the antibody sequence, typically those regions corresponding to the complementarity determining regions (CDR) loops, have been mutagenized or randomized.
The phage display method involves batch-cloning the antibody gene library into a phage genome as a fusion to the gene encoding one of the phage coat proteins (pIII, pVI, or pVIII). The pIII phage protein gene is preferred. When the fusion product is expressed it is incorporated into the mature phage coat. As a result, the antibody is displayed as a fusion on the surface of the phage and is available for binding and hence, selection, on a target protein. Once a phage particle is selected as bearing an antibody-coat protein fusion with the desired affinity towards the target protein, the genetic material within the phage particle which corresponds to the displayed antibody can be amplified and sequenced or otherwise analyzed.
In a preferred embodiment, a phagemid is used as the expression vector in the phage display procedures. A phagemid is a small plasmid vector that carries gene III with appropriate cloning sites and a phage packaging signal and contains both host and phage origins of replication. The phagemid is unable to produce a complete phage as the gene III fusion is the only phage gene encoded on the phagemid. A viable phage can be produced by infecting cells containing the phagemid with a helper phage containing a defective replication origin. A
hybrid phage emerges which contains all of the helper phage proteins as well as the gene III-rAb fusion. The emergent phage contains the phagemid DNA only.
In a preferred embodiment of the invention, the recombinant antibodies used in phage display methods of preparing antibody fragments for the arrays of the invention are expressed as genetic fusions to the bacteriophage gene III
protein on a phagemid vector. For instance, the antibody variable regions encoding a single-chain Fv fragment can be fused to the amino terminus of the gene III
protein on a phagemid. Alternatively, the antibody fragment sequence could be fused to the amino terminus of a truncated pIII sequence lacking the first two N-terminal domains. The phagemid DNA encoding the antibody-pIII fusion is preferably packaged into phage particles using a helper phage such as M13K07 or VCS-M 13, which supplies all structural phage proteins.
To display Fab fragments on phage, either the light or heavy (Fd) chain is fused via its C-terminus to pIII. The partner chain is expressed without any fusion to pIII so that both chains can associate to form an intact Fab fragment.
Any method of selection may be used which separates those phage particles which do bind the target protein from those which do not. The selection method must also allow for the recovery of the selected phages. Most typically, the phage particles are selected on an immobilized target protein. Some phage selection strategies known to those skilled in the art include the following: panning on an immobilized antigen; panning on an immobilized antigen using specific elution;
using biotinylated antigen and then selecting on a streptavidin resin or streptavidin-coated magnetic beads; affinity purification; selection on Western blots (especially useful for unknown antigens or antigens di~cult to purify);
in vivo selection; and pathfinder selection. If the selected phage particles are amplified between selection rounds, multiple iterative rounds of selection may optionally be performed.
Elution techniques will vary depending upon the selection process chosen, but typical elution techniques include washing with one of the following solutions: HCl or glycine buffers; basic solutions such as triethylamine;
chaotropic agents; solutions of increased ionic strength; or DTT when biotin is linked to the antigen by a disulfide bridge. Other typical methods of elution include enzymatically cleaving a protease site engineered between the antibody and gene III, or by competing for binding with excess antigen or excess antibodies to the antigen.
A method for producing an array of antibody fragments therefore comprises first selecting recombinant bacteriophage which express antibody fragments from a phage display library. The recombinant bacteriophage are selected by affinity binding to the desired antigen. (Iterative rounds of selection are possible, but optional.) Next, at least one purified sample of an antibody fragment from a bacteriophage which was selected in the first step is produced.
This antibody production step typically entails infecting E. coli cells with the selected bacteriophage. In the absence of helper phage, the selected bacteriophage then replicate as expressive plasmids without producing phage progeny.
Alternatively, the antibody fragment gene of the selected recombinant bacteriophage is isolated, amplified, and then expressed in a suitable expression system. In either case, following amplification, the expressed antibody fragment of the selected and amplified recombinant bacteriophage is isolated and purified.
In a third step of the method, the earlier steps of phage display selection and purified antibody fragment production are repeated using affinity binding to antigens from before until the desired plurality of purified samples of different antibody fragments with different binding partners are produced. In a final step of the method, the antibody fragment of each different purified sample is immobilized onto organic thinfilm on a separate patch on the surface of a substrate to form a plurality of patches of antibody fragments on discrete, known regions of the substrate surface covered by organic thiiifilm.
For instance, to generate an antibody array with antibody fragments against known antigens, open reading frames of the known protein targets identified in DNA databases are amplified by polymerise chain reaction and transcribed and translated in vitro to produce proteins on which a recombinant bacteriophage expressing single-chain antibody fragments are selected. Once selected, the antibody fragment sequence of the selected bacteriophage is amplified (typically using the polymerise chain method) and recloned into a desirable expression system. The expressed antibody fragments are purified and then printed onto organic thinfilms on substrates to form the high density arrays.
In the preparation of the arrays of the invention, phage display methods analogous to those used for antibody fragments may be used for other proteins which are to be immobilized on an array of the invention as long as the protein is of suitable size to be incorporated into the phagemid or alternative vector and expressed as a fusion with a bacteriophage coat protein. Phage display techniques using non-antibody libraries typically make use of some type of protein host scaffold structure which supports the variable regions. For instance, (3-sheet proteins, oc-helical handle proteins, and other highly constrained protein structures have been used as host scaffolds.
Alternative display vectors may also be used to produce the proteins which are printed on the arrays of the invention. Polysomes, stable protein-ribosome-mRNA complexes, can be used to replace live bacteriophage as the display vehicle for recombinant antibody fragments or other proteins (Hares and Pluckthun, Proc. Natl. Acad. Sci USA, 94:4937-4942, 1997). The polysomes are formed by preventing release of newly synthesized and correctly folded protein from the ribosome. Selection of the polysome library is based on binding of the antibody fragments or other proteins which are displayed on the polysomes to the target protein. mRNA which encodes the displayed protein or antibody having the desired affinity for the target is then isolated. Larger libraries may be used with polysome display than with phage display.
(g) Uses of the arrays.
The present invention also provides for methods of using the invention array. The arrays of the present invention are particularly suited for the use in drug development. Other uses include medical diagnostics, proteomics and biosensors.
Use of one of the protein arrays of the present invention may optionally involve placing the two-dimensional protein array in a flowchamber with approximately 1-10 microliters of fluid volume per 25 mm2 overall surface area.
The cover over the array in the flowchamber is preferably transparent or translucent. In one embodiment, the cover may comprise Pyrex or quartz glass.
In other embodiments, the cover may be part of a detection system that monitors WO 00!04382 PCT/US99/15971 interaction between biological moieties immobilized on the array and an analyte.
The flowchambers should remain filled with appropriate aqueous solutions to preserve protein activity. Salt, temperature, and other conditions are preferably kept similar to those of normal physiological conditions. Analytes and potential drug compounds may be .flushed into the flow chamber as desired and their.
interaction with the immobilized proteins determined. Sufficient time must be given to allow for binding between the immobilized proteins and an analyte to occur. No specialized microfluidic pumps, valves, or mixing techniques are required for fluid delivery to the array.
Alternatively, fluid can be delivered to each of the patches of the array individually. For instance, in one embodiment, the regions of the substrate surface may be microfabricated in such a way as to allow integration of the array with a number of fluid delivery channels oriented perpendicular to the array surface, each one of the delivery channels terminating at the site of an individual protein-coated patch.
The sample which is delivered to the array is typically a fluid.
In general, delivery of solutions containing proteins to be bound by the proteins of the array may optionally be preceded, followed, or accompanied by delivery of a blocking solution. A blocking solution contains protein or another moiety which will adhere to sites of non-specific binding on the array. For instance, solutions of bovine serum albumin or milk may be used as blocking solutions.
A wide range of detection methods is applicable to the methods of the invention. As desired, detection may be either quantitative or qualitative.
The invention array can be interfaced with optical detection methods such as absorption in the visible or infrared range, chemoluminescence, and fluorescence (including lifetime, polarization, fluorescence correlation spectroscopy (FCS), and fluorescence-resonance energy transfer (FRET)). Furthermore, other modes of detection such as those based on optical waveguides (PCT Publication WO
96/26432 and U.S. Patent No. 5,677,196), surface plasmon resonance, surface charge sensors, and surface force sensors are compatible with many embodiments of the invention. Alternatively, technologies such as those based on Brewster angle microscopy (Schaaf et al., Langmuir, 3:1131-1135 (I987)) and ellipsometry (U.S. Patent Nos. 5,141,311 and 5,116,121; Kim, Macromolecules, 22:2682-2685 ( 1984)) can be used in conjunction with the arrays of the invention. Quartz crystal microbalances and desorption processes (see for example, U.S. Patent No.
5,719,060) provide still other alternative detection means suitable for at least some embodiments of the invention array. An example of an optical biosensor system compatible both with some arrays of the present invention and a variety of non-label detection principles including surface plasmon resonance, total internal reflection fluorescence (TIRE), Brewster Angle microscopy, optical waveguide lightmode spectroscopy (OWLS), surface charge measurements, and ellipsometry can be found in U.S. Patent No. 5,313,264.
Although non-label detection methods are generally preferred, some of the types of detection methods commonly used for traditional immunoassays which require the use of labels may be applied to use with at least some of the arrays of the present invention, especially those arrays which are arrays of protein-capture agents. These techniques include noncomperitive immunoassays, competitive immunoassays, and dual label, ratiometric immunoassays. These particular techniques are primarily suitable for use with the arrays of proteins when the number of different proteins with different specificity is small (less than about 100). In the competitive method, binding-site occupancy is determined indirectly.
In this method, the proteins of the array are exposed to a labeled developing agent, which is typically a labeled version of the analyte or an analyte analog. The developing agent competes for the binding sites on the protein with the analyte.
The fractional occupancy of the proteins on different patches can be determined by the binding of the developing agent to the proteins of the individual patches. In the noncompetitive method, binding site occupancy is determined directly. In this method, the patches of the array are exposed to a labeled developing agent capable of binding to either the bound analyte or the occupied binding sites on the protein.
For instance, the developing agent may be a labeled antibody directed against occupied sites (i.e., a "sandwich assay"). Alternatively, a dual label, ratiometric, approach may be taken where the immobilized protein is labeled with one label and the second, developing agent is labeled with a second label (Ekins, et al., Clinica Chimica Acta., 194:91-114, 1990). Many different labeling methods may be used in the aforementioned techniques, including radioisotopic, enzymatic, chemiluminescent, and fluorescent methods. Fluorescent methods are preferred.
Figure 8 shows a schematic diagram of one type of fluorescence detection unit which may be used to monitor interaction of immobilized proteins of an array with an analyte. In the illustrated detection unit, the protein array 21 is positioned on a base plate 20. Light from a 100W mercury arc lamp 25 is directed through an excitation filter 24 and onto a beam splitter 23. The light is then directed through a lens 22, such as a Micro Nikkor 55 mm 1:2:8 lens, and onto the array 21. Fluorescence emission from the array returns through the lens 22 and the beam splitter 23. After next passing through an emission filter 26, the emission is received by a cooled CCD camera 27, such as the Slowscan TE/CCD-1024SF&SB (Princeton Instruments). The camera is operably connected to a CPU
28 which is in turn operably connected to a VCR 29 and a monitor 30.
Figure 9 shows a schematic diagram of an alternative detection method based on ellipsometry. Ellipsometry allows for information about the sample to be determined from the observed change in the polarization state of a reflected light wave. Interaction of an analyte with a layer of immobilized proteins on a patch results in a thickness change and alters the polarization status of a plane-polarized light beam reflected off the surface. This process can be monitored in situ from aqueous phase and, if desired, in imaging mode. In a typical setup, monochromatic light (e.g. from a He-Ne laser, 30) is plane polarized (polarizer 31) and directed onto the surface of the sample and detected by a detector 35.
A
compensator 32 changes the elliptically polarized reflected beam to plane-polarized. The corresponding angle is determined by an analyzer 33 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.
In one embodiment, the invention provides a method for screening a plurality of proteins for their ability to interact with a component of a sample comprising the steps of delivering the sample to a protein array of the invention comprising the proteins to be scteened and detecting for the interaction of the component with the immobilized protein of each patch. Optionally, the component may be a protein.
Possible interactions towards which the present invention may be directed include, but are not limited to, antibody/antigen, antibody/hapten, enzyme/substrate, carrier protein/substrate, lectin/carbohydrate, receptor/hormone, receptor/effector, protein/DNA, protein/RNA, repressor/inducer, or the like.
The interaction may involve binding and/or catalysis. The array of he invention is even suitable for assaying translocation by a membrane through a lipid bilayer. In preferred embodiments of use of the array, the assayed interaction is a binding interaction. The assayed interaction may be between a potenrial drug candidate and a plurality of potential drug targets. For instance, a synthesized organic compound may be tested for its ability to act as an inhibitor to a family of immobilized receptors.
Another aspect of the invention provides for a method for screening a plurality of proteins for their ability to bind a particular component of a sample.
This method comprises delivering the sample to a protein array of the invention comprising the proteins to be screened and detecting, either directly or indirectly, for the presence or amount of the particular component retained at each patch.
In a preferred embodiment, the method further comprises the intermediate step of washing the array to remove any unbound or nonspecifically bound components of the sample from the array before the detection step. In another embodiment, the method further comprises the additional step of further characterizing the particular component retained on at least one patch. The particular component may optionally be a protein.
The optional step of further characterizing the particular component retained on a patch of the array is typically designed to identify the nature of the component bound to the protein of a particular patch. In some cases, the entire identity of the component may not be known and the purpose of the further characterization may be the initial identification of the mass, sequence, structure and/or activity (if any) of the bound component. In other cases, the basic identity of the component may be known, but some information about the component may not be known. For instance it may be known that the component is a particular protein, but the post-translational modification, activation state, or some other feature of the protein may not be known. In one embodiment, the step of further characterizing components which are proteins involves measuring the activity of the proteins. Although in some cases it may be preferable to remove the component from the patch before the step of further characterizing the protein is carried out, in other cases the component can be further characterized while still bound to the patch. In still further embodiments, the proteins of the patch which binds a component can be used to isolate and/or purify the component on a larger scale, such as by purifying a component which is a protein from cells. The purified sample of the component can then be characterized through traditional means such as microsequencing, mass spectrometry, and the like.
In another embodiment of the invention, a method of assaying for protein-protein binding interactions is provided which comprises the following steps:
first, delivering a sample comprising at least one protein to be assayed for binding to the protein array of the invention; and then detecting, either directly, or indirectly, for the presence or amount of the protein from the sample which is retained at each patch. In a preferred embodiment, the method further comprises an additional step prior to the detection step which comprises washing the array to remove unbound or nonspecifically bound components of the sample from the array. Typically, the protein being assayed for binding will be from the same organism as the proteins immobilized on the array.
Another embodiment of the invention provides a method of assaying in parallel for the presence of a plurality of analytes in a sample which can react with one or more of the immobilized proteins on the protein array. This method comprises delivering the sample to the invention array and detecting for the interaction of the analyte with the immobilized protein at each patch.
In still another embodiment of the invention, a method of assaying in parallel for the presence of a plurality of analytes in a sample which can bind one or more of the immobilized proteins on the protein array comprises delivering the fluid sample to the invention array and detecting, either directly or indirectly, for the presence or amount of analyte retained at each patch. In a preferred embodiment, the method further comprises the step of washing the array tot remove any unbound or non-specifically bound components of the sample from the array.
The array may be used in a diagnostic manner when the plurality of analytes being assayed are indicative of a disease condition or the presence of a pathogen in an organism. In such embodiments, the sample which is delivered to the array will then typically be derived from a body fluid or a cellular extract from the organism.
The array may be used for drug screening when a potential drug candidate is screened directly for its ability to bind or otherwise interact with a plurality of proteins on the invention array. Alternatively, a plurality of potential drug candidates may be screened in parallel for their ability to bind or otherwise interact with one or more immobilized proteins on the array. The drug screening process may optionally involve assaying for the interaction, such as binding, of at least one analyte or component of a sample with one or more immobilized proteins on an invention array, both in the presence and absence of the potential drug candidate. This allows for the potential drug candidate to be tested for its ability to act as an inhibitor of the interaction or interactions originally being assayed.
(h) Examples The following specific examples are intended to illustrate the invention and should not be construed as limiting the scope of the claims:
Example 1. Fabrication of a two-dimensional array by photolithography.
In a preferred embodiment of the invention, two-dimensional arrays are fabricated onto the substrate material via standard photolithography and/or thin film deposition. Alternative techniques include microcontact printing.
Usually, a computer-aided design pattern is transferred to a photomask using standard techniques, which is then used to transfer the pattern onto a silicon wafer coated with photoresist.
In a typical example, the array ("chip") with lateral dimensions of 10 x 10 mm comprises squared patches of a bioreactive layer (here: gold as the coating on a silicon substrate) each 0.1 x 0.1 mm in size and separated by hydrophobic surface areas with a 0.2 mm spacing. 4" diameter Si( 100) wafers (Virginia Semiconductor) are used as bulk materials. Si( 100) wafers are first cleaned in a 3:1 mixture of HZS04, cone: 30% HZO2 (90°C, 10 min), rinsed with deionized water (18 MS2cm), finally passivated in 1% aqueous HF, and singed at 150°C for 30 min to become hydrophobic. The wafer is then spincoated with photoresist (Shipley 1813), prebaked for 25 minutes at 90°C, exposed using a Karl Suss contact printer and developed according to standard protocols. The wafer is then dried and postbaked at I IO°C for 25 min. In the next step, the wafer is primed with a titanium layer of 20 nm thickness, followed by a 200 nm thick gold layer.
Both layers were deposited using electron-beam evaporation (5 ~/s). After resist stripping and a short plasma treatment, the gold patches can be further chemically modified to achieve the desired bioreactive and biocompatible properties (see Example 3, below).
Example 2. Fabrication of a two-dimensional array by deposition through a hole mask.
In another preferred embodiment the array of gold patches is fabricated by thin film deposition through a hole mask which is in direct contact with the substrate. In a typical example, Si(100) wafers are first cleaned in a 3:1 mixture of HZS04, cone: 30% H202 (90°C, 10 min), rinsed with deionized water (18 MS2cm), finally passivated in 1% aqueous HF and singed at 150°C for 30 min to become hydrophobic. The wafer is then brought into contact with a hole mask exhibiting the positive pattern of the desired patch array. In the next step, the wafer is primed with a titanium layer of 20 nm thickness, followed by a 200 nm thick gold layer. Both layers were deposited using electron-beam evaporation (5 ~r/s). After removal of the mask, the gold patches can be further chemically modified to achieve the desired bioreactive and biocompatible properties (see Example 3, below).
Example 3. Synthesis of an aminoreactive monolayer molecule (following the procedure outlined in Wagner et al., Biophys. J., 1996, 70:2052-2066).
General. 1H- and 13C-NMR spectra are recorded on Broker instruments (100 to 400 MHz). Chemical shifts (S) are reported in ppm relative to internal standard ((CH3)4Si, 8 = 0.00 (1H- and 13C-NMR)). FAB-mass spectra are recorded on a VG-SABSEQ instrument (Cs+, 20 keV). Transmission infrared spectra are obtained as dispersions in KBr on an FTIR Perkin-Elmer 1600 Series instrument. Thin-layer chromatography (TLC) is performed on precoated silica ge160 F254 plates (MERCK, Darmstadt, FRG), and detection was done using C12/toluidine, PdCl2 and UV-detection under NH3-vapor. Medium pressure liquid chromatography (MPLC) is performed on a Labomatic MD-80 (LABOMATIC
INSTR. AG, Allschwil, Switzerland) using a Buechi column (460x36 mm;
BUECHI, Flawil, Switzerland), filled with silica gel 60 (particle size 15-40 pm) from Merck.
Synthesis of Il, ll'-dithiobis(succinimidylundecanoate) (DSU). Sodium thiosulfate (55.3 g, 350 mmol) is added to a suspension of 11-bromo-undecanoic acid (92.8 g, 350 mmol) in 50 % aqueous 1,4-dioxane (1000 ml). The mixture is heated at reflux (90 °C) for 2 h until the reaction to the intermediate Bunte salt was complete (clear solution). The oxidation to the corresponding disulfide is carried out in situ by adding iodine in portions until the solution retained with a yellow to brown colour. The surplus of iodine is retitrated with 15 % sodium pyrosulfite in water. After removal of 1,4-dioxane by rotary evaporation the creamy suspension is filtered to yield product 11, ll'-dithiobis(undecanoic acid).
RecrystallizaNon from ethyl acetate/THF provides a white solid (73.4 g, 96.5 %):
mp 94 °C; 1H NMR (400 MHz, CDC13 / CD30D 95 : 5): b 2.69 (t, 2H, J =
7.3 Hz), 2.29 (t, 2H, J = 7.5 Hz), 1.76-1.57 (m, 4H), and 1.40-1.29 (m, 12H); FAB-MS (Cs+, 20 keV): m/z (relative intensity) 434 ( 100, M+). Anal. Calcd. for C22H4204S2: C, 60.79; H, 9.74; S, 14.75. Found: C, 60.95; H, 9.82; S, 14.74.
To a solution of 11,11'-dithiobis(undecanoic acid). (1.0 g, 2.3 mmol) in THF (50 ml) is added N-hydroxysuccinimide (0.575 g, 5 mmol) followed by DCC (1.03 g, 5 mmol) at 0 °C. After the reaction mixture is allowed to warm to 23 °C and is stirred for 36 h at room temperature, the dicyclohexylurea (DCU) is filtered.
Removal of the solvent under reduced pressure and recrystallization from acetone/hexane provides ll, ll'-dithiobis(succinimidylundecanoate) as a white solid. Final purification is achieved by medium pressure liquid chromatography (9 bar) using silica gel and a 2:1 mixture of ethyl acetate and hexane. The organic phase is concentrated and dried in vacuum to afford I l, l I '-dithiobis(succinimidylundecanoate) (1.12 g, 78 %): mp 95 °C; 1H NMR
(400 MHz, CDC13): 8 2.83 (s, 4H), 2.68 (t, 2H, J = 7.3 Hz), 2.60 (t, 2H, J = 7.5 Hz), 1.78-1.63 (m, 4H), and 1.43-1.29 (m, 12H); FAB-MS (Cs+, 20 keV): m/z (relative intensity) 514 (100), 628 (86, M+). Anal. Calcd. for C3oH4gN2OgS2: C, 57.30;
H, 7.69; N, 4.45; S, 10.20. Found: C, 57.32; H, 7.60; N, 4.39; S, 10.25.
Example 4. Formation of an aminoreactive monolayer on gold (following the procedure of Wagner et al., Biophys. J., 1996, 70:2052-2066).
Monolayers based on ll, ll'-dithiobis(succinimidylundecanoate) (DSU) can be deposited on Au(111) surfaces of microarrays described under Examples 1 and 2 by immersing them into a 1 mM solution of DSU in chloroform at room temperature for 1 hour. After rinsing with 10 volumes of solvent, the N-hydroxysuccinimidyl-terminated monolayer is dried under a stream of nitrogen and immediately used for protein immobilization.
Example 5. Expression and purification of human caspase fusion proteins.
Caspases are cysteine proteases of the papain superfamily, with a different active site and catalytic mechanism than observed for papain, Wilson, K.P. et al., Nature, 1994 370:270-275. Caspases are important enzymes in the promotion of the cell death pathways and inflammation, Villa, et al., TIBS, 1997, 22:288-392.
Identification of selective caspase inhibitors is essential to prevent cross-inhibition of other caspase-dependent pathways. Caspases 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, Villa, et al., TIBS, 1997, 22:288-392 and new caspase homologs identified by the human genome project are PCR amplified and cloned into an E. coli expression vector containing an N-terminal histidine tag, Hochuli, et al., Biotechnology, 1988 6:1321, a factor Xa cleavage site, a lysine tag and a tri-glycine linker.
Fusion proteins are expressed, purified by nickel-nitrilotriacetic acid (NTA) agarose chromatography, the histidine tag removed by factor Xa cleavage, followed by gel filtration. Caspases are snap-frozen and stored in 20 mM PIPES, pH 7.2, 150 mM
NaCI, 0.1% CHAPS, 10 % sucrose at -80°C.
Example 6. Immobilization of fusion proteins on a 2D-protein array.
Caspase-fusion proteins can be immobilized to the aminoreactive monolayer surface of the bioreactive patches of the two-dimensional array (see Examples I, 2, and 4 above). Caspase fusion proteins can be diluted to concentrations of 1 pg/ml in 20 mM PIPES, pH 7.2, 15U mM NaCI, 0.1%
CHAPS, 10% sucrose and applied onto the bioreactive patches using a computer-aided, capillary-based dispensing system. After an immobilization period of 30 min, the 2D array was rinsed and subjected to analysis. Ultrapure water with a resistance of 18 MS2cm is generally useable for all aqueous buffers (purified by passage through a Barnstead Nanopure~ system).
Example 7. Assay of caspase activity on a two-dimensional array.
Caspase activity can be determined by a binding assay using three fluorescently labeled peptide aldehyde inhibitors that form a reversible thiohemiacetal moiety with the active site cysteine, Thornberry, Methods in Enzymology, 1994, 244:615-631. The peptides are adapted to caspase 1, 3, 4, 7:
Dns (dansyl)-SS- DEVD-CHO, caspase 1: Dns-SS-VDVAD-CHO, caspase 6:
Dns-SS-VQID-CHO, Talanian, J. Biol. Chem., 1997, 272:9677-9682. The affinity for Ac-DEVD-CHO to caspase 1 is determined to be in the low nanomolar range, Thornberry, Methods in Enzymolo~, 1994, 244:615-63 I. The assay buffer is 20 mM PIPES, pH 7.2, 150 mM NaCI, 0.1% CHAPS, 10 % sucrose, Stennicke, and Salvesen, J. Biol. Chem., 1997, 272:25719-25723. Fluorescently labeled peptides are mixed to a final concentration of 1 to 5 nM each, the potential drug compound added and flushed onto the 2D array. Peptides are allowed to bind for 10 - 60 min., unbound peptide removed by washing with buffer and the fluorescence intensity measured (excitation at 360 nm, emission at 470 nm).
Example 8. Formation and use of an array of immobilized Fab' antibody fragments to detect concentrations of soluble proteins prepared from cultured mammalian cells.
Collections of IgG antibodies are purchased from commercial sources (e.g.
Pierce, Rockford, IL). The antibodies are first purified by affinity chromatography based on binding to immobilized protein A. The antibodies are diluted 1:1 in binding buffer( 0.1 M Tris-HCI, 0.15 M NaCI, pH 7.5). A 2 ml minicolumn containing a gel with immobilized protein A is prepared. (Hermanson, et. al., Immobilized Affinity Ligand Techniques, Academic Press, San Diego, 1992.) The column is equilibrated with 10 ml of binding buffer. Less than 10 mg of immunoglobulin is applied to each 2 ml minicolumn and the column is washed with binding buffer until the absorbance at 280 nm is less than 0.02. The bound immunoglobulins are eluted with 0.1 M glycine, 0.15 M NaCI, pH 2.8, and immediately neutralized with 1.0 M Tris-HCI, pH 8.0 to 50 mM final concentration and then dialyzed against 10 mM sodium phosphate, 0.1 S M NaCI, pH 7.2 and stored at 4°C.
The purified immunoglobulin are digested with immobilized pepsin. Pepsin is an acidic endopeptidase and hydrolyzes proteins favorably adjacent to aromatic and dicarboxylic L-amino acid residues. Digestion of IgG with pepsin generates intact F(ab')2 fragments. Immobilized pepsin gel is washed with digestion buffer;
20 mM sodium acetate, pH 4.5. A solution of purified IgG at 10 mg/ml is added to the immobilized pepsin gel and incubated at 37°C for 2 hours. The reaction is neutralized by the addition of 10 mM Tris-HCI, pH 7.5 and centrifuged to pellet the gel. The supernatant liquid is collected and applied to an immobilized protein A column, as described above, to separate the F(ab') 2 fragments from the Fc and undigested IgG. The pooled F(ab')~ is dialyzed against 10 mM sodium phosphate, 0.15 M NaCl, pH 7.2 and stored at 4°C. The quantity of pooled, eluted F(ab') 2 is measured by peak area absorbance at 280 nm.
The purified F(ab') 2 fragments at a concentration of 10 mg/ml are reduced at 37 °C for 1 hour in a buffer of 10 mM sodium phosphate, 0.15 M NaCI, mM 2-mercaptoethylamine, S mM EDTA, pH 6Ø The Fab' fragments are separated from unsplit F(ab')2 fragments and concentrated by application to a Sephadex G-25 column (Mr = 46,000 - 58,000). The pooled Fab' fragments are dialyzed against 10 mM sodium phosphate, 0.15 M NaCI, pH 7.2. The reduced Fab' fragments are diluted to 100 pg/ml and applied onto the bioreactive patches containing exposed aminoreactive functional groups using a computer-aided, capillary-based microdispensing system (for antibody immobilization procedures, see Dammer et al., Biophys. J., 70:2437-2441, 1996). After an immobilization period of 30 minutes at 30°C, the array is rinsed extensively with 10 mM sodium phosphate, 0.15 M NaCI, 5 mM EDTA, pH 7Ø
Transformed human cells grown in culture are collected by low speed centrifugation, briefly washed with ice-cold phosphate-buffered solution (PBS), and then resuspended in ice-cold hypotonic buffer containing DNase/RNase (10 pg/mI each, final concentration) and a mixture of protease inhibitors. Cells are transferred to a microcentrifuge tube, allowed to swell for 5 minutes, and lysed by rapid freezing in liquid nitrogen and thawing in ice-cold water. Cell debris and precipitates are removed by high-speed centrifugation and the supernatant is cleared by passage through a 0.45 pm filter. The cleared lysate is applied to the Fab' fragment array described above and allowed to incubate for 2 hours at 30°C.
After binding the array is washed extensively with 10 mM sodium phosphate, 0.15 M NaCI, 5 mM EDTA, pH 7Ø The location and amount of bound proteins are determined by optical detection.
Example 9. Formation and use of an array of immobilized antibody fragments to detect concentrations of soluble proteins prepared from cultured mammalian cells.
A combinatorial library of filamentous phage expressing scFv antibody fragments is generated based on the technique of McCafferty and coworkers;
McCafferty, et al., Nature, 1990, 348:552-554; Winter and Milstein, Nature, 1991, 349:293-299. Briefly, mRNA is purified from mouse spleens and used to construct a cDNA library. PCR fragments encoding sequences of the variable heavy and light chain immunoglobulin genes of the mouse are amplified from the prepared cDNA. The amplified PCR products are joined by a linker region of DNA encoding the 15 amino acid peptide (Gly4SerG1y2CysGlySerGly4Ser) (SEQ
ID NO: 1) and the resulting full-length PCR fragment is cloned into an expression plasmid (pCANTAB 5 E) in which the purification peptide tag (E Tag) has been replaced by a His6 peptide (SEQ ID NO: 2). Electrocompetent TG 1 E. coli cells are transformed with the expression plasmid by electroporation. The pCANTAB-transformed cells are induced to produced functional filamentous phage expressing scFv fragments by superinfection with M13K07 helper phage. Cells are grown on glucose-deficient medium containing the antibiotics ampicillin (to select for cells with the phagemid) and kanamycin (to select for cells infected with M13K07). In the absence of glucose, the lac promoter present on the phagemid is no longer repressed, and synthesis of the scFv-gene 3 fusion begins.
Proteins from a cell lysate are adsorbed to the wells of a 96-well plate.
Transformed human cells grown in culture are collected by low speed centrifugation and the cells are briefly washed with ice-cold PBS. The washed cells are then resuspended in ice-cold hypotonic buffer containing DNase/RNase (10 pg/ml each, final concentration) and a mixture of protease inhibitors, allowed to swell for 5 minutes, and lysed by rapid freezing in liquid nitrogen and thawing in ice-cold water. Cell debris and precipitates are removed by high-speed centrifugation and the supernatant is cleared by passage through a 0.45 N,m filter.
The cleared lysate is diluted to 10 ~,g/ml in dilution buffer; 20 mM PIPES, 0.15 M
NaCI, 0.1 % CHAPS, 10%, 5 mM EDTA, S mM 2-mercaptoethanol, 2 mM DTT, pH 7.2 and applied to the 96-plate wells. After immobilization for 1 hour at 30°C, the well is washed with the dilution buffer and then incubated with dilution buffer containing 10% nonfat dry milk to block unreacted sites. After the blocking step, the well is washed extensively with the dilution buffer.
Phage expressing displayed antibodies are separated from E. coli cells by centrifugation and then precipitated from the supernatant by the addition of 15%
w/v PEG 8000, 2.5 M NaCI followed by centrifugation. The purified phage are resuspended in the dilution buffer containing 3% nonfat dry milk and applied to the well containing the immobilized proteins described above, and allowed to bind for 2 hours at 37°C, followed by extensive washing with the binding buffer. Phage are eluted from the well with an elution buffer; 20 mM PIPES, 1 M NaCI, 0.1 CHAPS, 10%, 5 mM EDTA, 5 mM 2-mercaptoethanol, 2 mM DTT, pH 7.2. The well is then extensively washed with purge buffer; 20 mM PIPES, 2.5 M NaCI, 0.1 % CHAPS, 10%, 5 mM EDTA, 5 mM 2-mercaptoethanol, 2 mM DTT, pH
In all cases, the coating, or the substrate itself if no coating is present, must be compatible with the chemical or physical adsorption of the organic thinfilm on its surface. For instance, if the patches comprise a coating between the substrate and a monolayer of molecules of the formula X-R-Y, then it is understood that the coating must be composed of a material for which a suitable functional group X
is available. If no such coating is present, then it is understood that the substrate must be composed of a material for which a suitable functional group X is available.
In a preferred embodiment of the invention, the regions of the substrate surface, or coating surface, which separate the patches of proteins are free of organic thinfilin. In an alternative embodiment, the organic thinfilm extends beyond the area of the substrate surface, or coating surface if present, covered by the protein patches. For instance, optionally, the entire surface of the array may be covered by an organic thinfilm on which the plurality of spatially distinct patches of proteins reside. An organic thinfilm which covers the entire surface of the array may be homogenous or may optionally comprise patches of differing exposed functionalities useful in the immobilization of patches of different proteins. In still another alternative embodiment, the regions of the substrate surface, or coating surface if a coating is present, between the patches of proteins are covered by an organic thinfilin, but an organic thinfilm of a different type than that of the patches of proteins. For instance, the surfaces between the patches of proteins may be coated with an organic thinfilm characterized by low non-specific binding properties for proteins and other analytes.
A variety of techniques may be used to generate patches of organic thinfilin on the surface of the substrate or on the surface of a coating on the substrate.
These techniques are well known to those skilled in the art and will vary depending upon the nature of the organic thinfilin, the substrate, and the coating if present. The techniques will also vary depending on the structure of the underlying substrate and the pattern of any coating present on the substrate.
For instance, patches of a coating which is highly reactive with an organic thinflhn may have akeady been produced on the substrate surface. Arrays of patches of organic thinfilin can optionally be created by microfluidics printing, microstamping (US Patent Nos. 5,512,131 and 5,731,152), or microcontact printing (MCP) (PCT Publication WO 96/29629). Subsequent immobilization of proteins to the reactive monolayer patches results in two-dimensional arrays of the agents. Inkjet printer heads provide another option for patterning monolayer X-R-Y molecules, or components thereof, or other organic thinfilm components to manometer or micrometer scale sites on the surface of the substrate or coating (Lemmo et al., Anal Chem., 1997, 69:543-551; US Patent Nos. 5,843,767 and 5,837,860). In some cases, commercially available arrayers based on capillary dispensing (for instance, OmniGridTM from Genemachines, inc, San Carlos, CA, and High-Throughput Microarrayer from Intelligent Bio-Instruments, Cambridge, MA) may also be of use in directing components of organic thinfilms to spatially distinct regions of the array.
Diffusion boundaries between the patches of proteins immobilized on organic thinfilms such as self assembled monolayers may be integrated as topographic patterns (physical barriers) or surface functionalities with orthogonal wetting behavior (chemical barriers). For instance, walls of substrate material or photoresist may be used to separate some of the patches from some of the others or all of the patches from each other. Alternatively, non-bioreactive organic thinfilms, such as monolayers, with different wettability may be used to separate patches from one another.
In a preferred embodiment of the invention, each of the patches of proteins comprises a self assembled monolayer of molecules of the formula X-R-Y, as previously defined, and the patches are separated from each other by surfaces free of the monolayer.
Figure 1 shows the top view of one example of an array of 25 patches reactive with proteins. On the array, a number of patches 15 cover the surface of the substrate 3.
Figure 2 shows a detailed cross section of a patch 15 of the array of Figure 1. This view illustrates the use of a coating 5 on the substrate 3. An adhesion interlayer 6 is also included in the patch. On top of the patch resides a self assembled monolayer 7.
Figure 3 shows a cross section of one row of the patches 15 of the array of Figure 1. This figure also shows the use of a cover 2 over the array. Use of the cover 2 creates an inlet port 16 and an outlet port 17 for solutions to be passed over the array.
A variety of chemical moieties may function as monolayer molecules of the formula X-R-Y in the array of the present invention. However, three major classes of monolayer formation are preferably used to expose high densities of reactive omega-functionalities on the patches of the array: (i) alkylsiloxane monolayers ("silanes") on hydroxylated and non-hydroxylated surfaces (as taught in, for example, US Patent No. 5,405,766, PCT Publication WO 96/38726, US
Patent No. 5,412,087, and US Patent No. 5,688,642); (ii) alkyl-thiol/dialkyldisulfide monolayers on noble metals (preferably Au(111)) (as, for example, described in Allara et al., US 4,690,715; Bamdad et al., US
5,620,850;
Wagner et al., Biophysical Journal, 1996, 70:2052-2066); and (iii) alkyl monolayer formation on oxide-free passivated silicon (as taught in, for example, Linford et al., J. Am. Chem. Soc., 1995, I 17:3145-3155, Wagner et al., Journal of Structural Biology, 1997, 119:189-201, US Patent No. 5,429,708). One of ordinary skill in the art, however, will recognize that many possible moieties may be substituted for X, R, and/or Y, dependent primarily upon the choice of substrate, coating, and affinity tag. Many examples of monolayers are described in Ulman, An Introduction to Ultrathin Organic Films: From Langmuir-Blodgett to Self Assembly, Academic press ( 1991 ).
In one embodiment, the monolayer comprises molecules of the formula (X)aR(Y)b wherein a and b are, independently, equal to an integer between l and about 200. In a preferred embodiment, a and b are, independently, equal to an integer between 1 and about 80. In a more preferred embodiment, a and b are, independently, equal to 1 or 2. In a most preferred embodiment, a and b are both equal to 1 (molecules of the formula X-R-Y).
If the patches of the invention array comprise a self assembled monolayer of molecules of the formula (X)aR(Y)b, then R may optionally comprise a linear or branched hydrocarbon chain from about 1 to about 400 carbons long. The hydrocarbon chain may comprise an alkyl, aryl, alkenyl, alkynyl, cycloalkyl, alkaryl, aralkyl group, or any combination thereof. If a and b are both equal to one, then R is typically an alkyl chain from about 3 to about 30 carbons long.
In a preferred embodiment, if a and b are both equal to one, then R is an alkyl chain from about 8 to about 22 carbons long and is, optionally, a straight alkane.
However, it is also contemplated that in an alternative embodiment, R may readily comprise a linear or branched hydrocarbon chain from about 2 to about 400 carbons long and be interrupted by at least one hetero atom. The interrupting hetero groups can include -O-, -CONH-, -CONHCO-, -NH-, -CSNH-, -CO-, -CS-, -S-, -SO-, -(OCH2CH2)" (where n=1-20), -(CF2)n (where n=1-22), and the like.
Alternatively, one or more of the hydrogen moieties of R can be substituted with deuterium. In alternative, less preferred, embodiments, R may be more than about 400 carbons long.
X may be chosen as any group which affords chemisorption or physisorption of the monolayer onto the surface of the substrate (or the coating, if present). When the substrate or coating is a metal or metal alloy, X, at least prior to incorporation into the monolayer, can in one embodiment be chosen to be an asymmetrical or symmetrical disulfide, sulfide, diselenide, selenide, thiol, isonitrile, selenol, a trivalent phosphorus compound, isothiocyanate, isocyanate, xanthanate, thiocarbamate, a phosphine, an amine, thio acid or a dithio acid.
This embodiment is especially preferred when a coating or substrate is used that is a noble metal such as gold, silver, or platinum.
If the substrate of the array is a material such as silicon, silicon oxide, indium tin oxide, magnesium oxide, alumina, quartz, glass, or silica, then the array of one embodiment of the invention comprises an X that, prior to incorporation into said monolayer, is a monohalosilane, dihalosilane, trihalosilane, trialkoxysilane, dialkoxysilane, or a monoalkoxysilane. Among these silanes, trichlorosilane and trialkoxysilane are particularly preferred.
In a preferred embodiment of the invention, the substrate is selected from the group consisting of silicon, silicon dioxide, indium tin oxide, alumina, glass, and titania; and X, prior to incorporation into said monolayer, is selected from the group consisting of a monohalosilane, dihalosilane, trihalosilane, trichlorosilane, trialkoxysilane, dialkoxysilane, monoalkoxysilane, carboxylic acids, and phosphates.
In another preferred embodiment of the invention, the substrate of the array is silicon and X is an olefin.
In still another preferred embodiment of the invention, the coating (or the substrate if no coating is present) is titania or tantalum oxide and X is a phosphate.
In other embodiments, the surface of the substrate (or coating thereon) is composed of a material such as titanium oxide, tantalum oxide, indium tin oxide, magnesium oxide, or alumina where X is a carboxylic acid or phosphoric acid.
Alternatively, if the surface of the substrate (or coating thereon) of the array is copper, then X may optionally be a hydroxamic acid.
If the substrate used in the invention is a polymer, then in many cases a coating on the substrate such as a copper coating will be included in the array. An appropriate functional group X for the coating would then be chosen for use in the array. In an alternative embodiment comprising a polymer substrate, the surface of the polymer may be plasma-modified to expose desirable surface functionalities for monolayer formation. For instance, EP 780423 describes the use of a monolayer molecule that has an alkene X functionality on a plasma exposed surface. Still another possibility for the invention array comprised of a polymer is that the surface of the polymer on which the monolayer is formed is functionalized by copolymerization of appropriately functionalized precursor molecules.
Another possibility is that prior to incorporation into the monolayer, X can be a free-radical-producing moiety. This functional group is especially appropriate when the surface on which the monolayer is formed is a hydrogenated silicon surface. Possible free-radical producing moieties include, but are not limited to, diacylperoxides, peroxides, and azo compounds. Alternatively, unsaturated moieties such as unsubstituted alkenes, alkynes, cyano compounds and isonitrile compounds can be used for X, if the reaction with X is accompanied by ultraviolet, infrared, visible, or microwave radiation.
In alternative embodiments, X, prior to incorporation into the monolayer, may be a hydroxyl, carboxyl, vinyl, sulfonyl, phosphoryl, silicon hydride, or an ammo group.
The component, Y, of the monolayer is a functional group responsible for binding a protein onto the monolayer. In a preferred embodiment of the invention, the Y group is either highly reactive (activated) towards the protein or is easily converted into such an activated form. In a preferred embodiment, the coupling of Y with the protein occurs readily under normal physiological conditions not detrimental to the activity of the protein. The functional group Y may either form a covalent linkage or a noncovalent linkage with the protein (or its affinity tag, if present). In a preferred embodiment, the functional group Y forms a covalent linkage with the protein or its affinity tag. It is understood that following the attachment of the protein (with or without an affinity tag) to Y, the chemical nature of Y may have changed. Upon attachment of the protein, Y may even have been removed from the organic thinfilm.
In one embodiment of the array of the present invention, Y is a' functional group that is activated in situ. Possibilities for this type of functional group include, but are not limited to, such simple moieties such as a hydroxyl, carboxyl, amino, aldehyde, carbonyl, methyl, methylene, alkene, alkyne, carbonate, aryliodide, or a vinyl group. Appropriate modes of activation would be obvious to one skilled in the art. Alternatively, Y can comprise a functional group that requires photoactivation prior to becoming activated enough to trap the protein.
In an especially preferred embodiment of the array of the present invention, Y is a complex and highly reactive functional moiety that is compatible with monolayer formation and needs no in situ activation prior to reaction with the protein and/or affinity tag. Such possibilities for Y include, but are not limited to, maleimide, N-hydroxysuccinimide (Wagner et al., Biophysical Journal, 1996, 70:2052-2066), nitrilotriacetic acid (LJS Patent No. 5,620,850), activated hydroxyl, haloacetyl, bromoacetyl, iodoacetyl, activated carboxyl, hydrazide, epoxy, aziridine, sulfonylchloride, trifluoromethyldiaziridine, pyridyldisulfide, N-acyl-imidazole, imidazolecarbamate, vinylsulfone, succinimidylcarbonate, arylazide, anhydride, diazoacetate, beiizophenone, isothiocyanate, isocyanate, imidoester, fluorobenzene, and biotin.
Figure 4 shows one example of a monolayer on a substrate 3. In this example, substrate 3 comprises glass. The monolayer is thiokeactive because it bears a maleimidyl functional group Y.
Figure 5 shows another example of a monolayer on a substrate 3 which is silicon. In this case, however, a thinfihn gold coating S covers the surface of the substrate 3. Also, in this embodiment, a titanium adhesion interlayer 6 is used to adhere the coating 5 to the substrate 3. This monolayer is aminoreactive because it bears an N-hydroxysuccinimidyl functional group Y.
In an alternative embodiment, the functional group Y of the array is selected from the group of simple functional moieties. Possible Y functional groups include, but are not limited to, -OH, -NH2, -COOH, -COOR, -RSR, -Pp4 3, -OS03-2, -S03-, -COO-, -SOO-, -CONR2, -CN, -NR2, and the like.
The monolayer molecules of the present invention can optionally be assembled on the surface in parts. In other words, the monolayer need not necessarily be constructed by chemisorption or physisorption of molecules of the formula X-R-Y to the surface of the substrate (or coating). Instead, in one embodiment, X may be chemisorbed or physisorbed to the surface of the substrate (or coating) alone first. Then, R or even just individual components of R can be attached to X through a suitable chemical reaction. Upon completion of addition of the spacer R to the X moiety akeady immobilized on the surface, Y can be attached to the ends of the monolayer molecule through a suitable covalent linkage.
Not all self assembled monolayer molecules on a given patch need be identical to one another. Some patches may comprise mixed monolayers. For instance, the monolayer of an individual patch may optionally comprise at least two different molecules of the formula X-R-Y, as previously described. This second X-R-Y molecule may optionally immobilize the same protein as the first.
In addition, some of the monolayer molecules X-R-Y of a patch may have failed to attach any protein.
As another alternative embodiment of the invention, a mixed, self assembled monolayer of an individual patch on the array may comprise both molecules of the formula X-R-Y, as previously described, and molecules of the formula, X-R-V where R is a spacer, X is a functional group that binds R to the surface, and V is a moiety which is biocompatible with proteins and resistant to the non-specific binding of proteins. For example, V may consist of a hydroxyl, saccharide, or oligo/polyethylene glycol moiety (EP Publication 780423).
In still another embodiment of the invention, the array comprises at least one unreactive patch of organic thinfilm on the substrate or coating surface which is devoid of any protein. For instance, the unreactive patch may optionally comprise a monolayer of molecules of the formula X-R-V, where R is a spacer, X
is a functional group that binds R to the surface, and V is a moiety resistant to the non-specific binding of proteins. The unreactive patch may serve as a control patch or be useful in background binding measurements.
Regardless of the nature of the monolayer molecules, in some arrays it may be desirable to provide crosslinking between molecules of an individual patch's monolayer. In general, crosslinking confers additional stability to the monolayer.
Such methods are familiar to those skilled in the art (for instance, see Ulman, An Introduction to Ultrathin Organic Films: From Langmuir Blodgett to Self Assembly, Academic Press (1991)).
After completion of formation of the monolayer on the patches, the protein may be attached to the monolayer via interaction with the Y-functional group.
Y-functional groups which fail to react with any proteins are preferably quenched prior to use of the array.
(d) Affinity tags and immobilization of the proteins.
In a preferred embodiment, the protein-immobilizing patches of the array further comprise an affinity tag that enhances immobilization of the protein onto the organic thinfilm. The use of an affinity tag on the protein of the array typically provides several advantages. An affinity tag can confer enhanced binding or reaction of the protein with the functionalities on the organic thinfilm, such as Y if the organic thinfilin is a an X-R-Y monolayer as previously described. This enhancement effect may be either kinetic or thermodynamic. The affinity tag/thinfilm combination used in the patches of the array preferably allows for immobilization of the proteins in a manner which does not require harsh reaction conditions that are adverse to protein stability or function. In most embodiments, immobilization to the organic thinfilm in aqueous, biological buffers is ideal.
An affinity tag also preferably offers immobilization on the organic thinfilm that is specific to a designated site or location on the protein (site-specific immobilization). For this to occur, attachment of the affinity tag to the protein must be site-specific. Site-specific immobilization helps ensure that the active site or binding site of the immobilized protein, such as the antigen-binding site of the antibody moiety, remains accessible to ligands in solution. Another advantage of immobilization through affinity tags is that it allows for a common immobilization strategy to be used with multiple, different proteins.
The affinity tag is optionally attached directly, either covalently or noncovalently, to the protein. In an alternative embodiment, however, the affinity tag is either covalently or noncovalently attached to an adaptor which is either covalently or noncovalently attached to the protein.
In a preferred embodiment, the affinity tag comprises at least one amino acid. The affinity tag may be a polypeptide comprising at least two amino acids which is reactive with the functionalities of the organic thinfllm.
Alternatively, the affinity tag may be a single amino acid which is reactive with the organic thinfilm. Examples of possible amino acids which could be reactive with an organic thinfilin include cysteine, lysine, histidine, arginine, tyrosine, aspartic acid, glutamic acid, tryptophan, serine, threonine, and glutamine. A
polypeptide or amino acid affinity tag is preferably expressed as a fusion protein with the immobilized protein of each patch. Amino acid affinity tags provide either a single amino acid or a series of amino acids that can interact with the functionality of the organic thinfllm, such as the Y-functional group of the self assembled monolayer molecules. Amino acid affinity tags can be readily introduced into recombinant proteins to facilitate oriented immobilization by covalent binding to the Y-functional group of a monolayer or to a functional group on an alternative organic thinfilm.
The affinity tag may optionally comprise a poly(amino acid) tag. A
poly(amino acid) tag is a polypeptide that comprises from about 2 to about 100 residues of a single amino acid, optionally interrupted by residues of other amino acids. For instance, the affinity tag may comprise a poly-cysteine, polylysine, poly-arginine, or poly-histidine. Amino acid tags are preferably composed of two to twenty residues of a single amino acid, such as, for example, histidines, lysines, arginines, cysteines, glutamines, tyrosines, or any combination of these.
According to a preferred embodiment, an amino acid tag of one to twenty amino acids includes at least one to ten cysteines for thioether linkage; or one to ten lysines for amide linkage; or one to ten arginines for coupling to vicinal dicarbonyl groups. One of ordinary skill in the art can readily pair suitable affinity tags with a given functionality on an organic thinfilm.
The position of the amino acid tag can be at an amino-, or carboxy-terminus of the protein of a patch of the array, or anywhere in-between, as long as the active site or binding site of the protein remains in a position accessible for ligand interaction. Where compatible with the protein chosen, affinity tags introduced for protein purification are preferentially located at the C-terminus of the recombinant protein to ensure that only full-length proteins are isolated during protein purification. For instance, if intact antibodies are used on the arrays, then the attachment point of the affinity tag on the antibody is preferably located at a C-terminus of the effector (Fc) region of the antibody. If scFvs are used on the arrays, then the attachment point of the affinity tag is also preferably located at the C-terminus of the molecules.
Affinity tags may also contain one or more unnatural amino acids.
Unnatural amino acids can be introduced using suppressor tRNAs that recognize stop codons (i.e., amber) (Noren et al., Science, 1989, 244:182-188; Elhnan et al., Methods Enzym., 1991, 202:301-336; Cload et al., Chem. Biol., 1996, 3:1033-1038). The tRNAs are chemically amino-acylated to contain chemically altered ("unnatural") amino acids for use with specific coupling chemistries (i. e., ketone modifications, photoreactive groups).
In an alternative embodiment the affinity tag can comprise an intact protein, such as, but not limited to, glutathione S-transferase, an antibody, avidin, or streptavidin.
Other protein conjugation and immobilization techniques known in the art may be adapted for the purpose of attaching affinity tags to the protein. For instance, in an alternative embodiment of the array, the affinity tag may be an organic bioconjugate which is chemically coupled to the protein of interest.
Biotin or antigens may be chemically cross linked to the protein.
Alternatively, a chemical crosslinker may be used that attaches a simple functional moiety such as a thiol or an amine to the surface of a protein to be immobilized on a patch on the array. Alternatively, protein synthesis or protein ligation techniques known to those skilled in the art may be used to attach an affinity tag to a protein.
For instance, intein-mediated protein ligation may optionally be used to attach the affinity tag to the protein (Mathys, et al., Gene 231:1-13, 1999; Evans, et al., Protein Science 7:2256-2264, 1998).
In an alternative embodiment of the invention, the organic thinfilm of each of the patches comprises, at least in part, a lipid monolayer or bilayer, and the affinity tag comprises a membrane anchor. Optionally, the Iipid monolayer or bilayer is immobilized on a self assembled monolayer.
Figure 6 shows a detailed cross section of a patch on one embodiment of the invention array. In this embodiment, a protein 10 is immobilized on a monolayer 7 on a substrate 3. An affinity tag 8 connects the protein 10 to the monolayer 7. The monolayer 7 is formed on a coating 5 which is separated from the substrate 3 by an interlayer 6.
In an alternative embodiment of the invention, no affinity tag is used to immobilize the proteins onto the organic thinfilin. An amino acid or other moiety (such as a carbohydrate moiety) inherent to the protein itself may instead be used to tether the protein to the reactive group of the organic thief lm. In preferred embodiments, the immobilization is site-specific with respect to the location of the site of immobilization on the protein. For instance, the sulfhydryl group on the C-terminal region of the heavy chain portion of a Fab' fragment generated by pepsin digestion of an antibody, followed by selective reduction of the disulfide between monovalent Fab' fragments, may be used as the affinity tag. Alternatively, a carbohydrate moiety on the Fc portion of an intact antibody can be oxidized under mild conditions to an aldehyde group suitable for immobilizing the antibody on a monolayer via reaction with a hydrazide-activated Y group on the monolayer.
Examples of immobilization of proteins without any affinity tag can be found in Wagner et al., Biophys. .L, 70:2437-2441, 1996 and the specific examples, Examples 8-10, below.
When the proteins of at least some of the different patches on the array are different from each other, different solutions, each containing a different, preferably, affinity-tagged protein, must be delivered to their individual patches.
Solutions of proteins may be transferred to the appropriate patches via arrayers which are well-known in the art and even commercially available. For instance, microcapillary-based dispensing systems may be used. These dispensing systems are preferably automated and computer-aided. A description of and building instructions for an example of a microarrayer comprising an automated capillary system can be found on the Internet at http://cmgm.stanford.edu/pbrown/axray.html and http://cmgm.stanford.edu/pbrown/mguide/index.html. The use of other microprinting techniques for transferring solutions containing the proteins to the protein-reactive patches is also possible. Ink jet printer heads may also optionally be used for precise delivery of the proteins to the protein-reactive patches.
Representative, non-limiting disclosures of techniques useful for depositing the proteins on the patches may be found, for example, in U.S. Patent Nos. 5,731, (stamping apparatus), 5,807,522 (capillary dispensing device), 5,837,860 (ink jet printing technique, Hamilton 2200 robotic pipetting delivery system), and 5,843,767 (ink jet printing technique, Hamilton 2200 robotic pipetting delivery system), all incorporated by reference herein.
(e) Adaptors.
Another embodiment of the arrays of the present invention comprises an adaptor that links the affinity tag to the immobilized protein. The additional spacing of the protein from the surface of the substrate (or coating) that is afforded by the use of an adaptor is particularly advantageous since proteins are known to be prone to surface inactivation. The adaptor may optionally afford some additional advantages as well. For instance, the adaptor may help facilitate the attachment of the protein to the affinity tag. In another embodiment, the adaptor may help facilitate the use of a particular detection technique with the array. One of ordinary skill in the art will be able to choose an adaptor which is appropriate for a given affinity tag. For instance, if the affinity tag is streptavidin, then the adaptor could be a biotin molecule that is chemically conjugated to the protein which is to be immobilized.
In a preferred embodiment, the adaptor is a protein. In a preferred embodiment, the affinity tag, adaptor, and protein to be immobilized together compose a fusion protein. Such a fusion protein may be readily expressed using standard recombinant DNA technology. Adaptors which are proteins are especially useful to increase the solubility of the protein of interest and to increase the distance between the surface of the substrate or coating and the protein of interest. Use of an adaptor which is a protein can also be very useful in facilitating the preparative steps of protein purification by affinity binding prior to immobilization on the array. Examples of possible adaptors which are proteins include glutathione-S-transferase (GST), maltose-binding protein, chitin-binding protein, thioredoxin, green-fluorescent protein (GFP). GFP can also be used for quantification of surface binding. If the protein immobilized on the patches of the array is an antibody or antibody fragment comprising an Fc region, then the adaptor may optionally be protein G, protein A, or recombinant protein A/G (a gene fusion product secreted from a non-pathogenic form of Bacillus which contains four Fc binding domains from protein A and two from protein G).
Figure 7 shows a crass section of a patch on one particular embodiment of the invention array. The patch comprises a protein 10 immobilized on a monolayer 7 via both an afFlnity tag 8 and an adaptor molecule 9. The monolayer 7 rests on a coating 5. An interlayer 6 is used between the coating 5 and the substrate 3.
(f) Preparation of the proteins of the array.
The proteins immobilized on the array may be produced by any of the variety of means known to those of ordinary skill in the art.
In preparation for immobilization to the arrays of the present invention, the protein can optionally be expressed from recombinant DNA either in vivo or in vitro. The cDNA of the protein to be immobilized on the array is cloned into an expression vector (many examples of which are commercially available) and introduced into cells of the appropriate organism for expression. A broad range of host cells and expression systems may be used to produce the proteins to be immobilized on the array. For in vivo expression of the proteins, cDNAs can be cloned into commercial expression vectors (Qiagen, Novagen, Clontech, for example) and introduced into an appropriate organism for expression.
Expression in vivo may be done in bacteria (for example, Escherichia coli), plants (for example, Nicotiana tabacum), lower eukaryotes (for example, Saccharomyces cerevisiae, Saccharomyces pombe, Pichia pastoris), or higher eukaryotes (for example, bacculovirus-infected insect cells, insect cells, mammalian cells).
For in vitro expression PCR-amplified DNA sequences are directly used in coupled in vitro transcription/translation systems (for instance: Escherichia coli S30 lysates from T7 RNA polymerase expressing, preferably protease-deficient strains; wheat germ lysates; reticulocyte lysates (Promega, Pharmacia, Panvera)).
The choice of organism for optimal expression depends an the extent of post-translational modifications (i. e., glycosylation, lipid-modifications) desired. One of ordinary skill in the art will be able to readily choose which host cell type is most suitable for the protein to be immobilized and application desired.
DNA sequences encoding amino acid affinity tags and adaptor protein sequences are engineered into the expression vectors such that the genes of interest can be cloned in frame either 5' or 3' of the DNA sequence encoding the affinity tag and adaptor.
The expressed proteins are purified by affinity chromatography using commercially available resins.
Preferably, production of families of related proteins involves parallel processing from cloning to protein expression and protein purification. cDNAs for the protein of interest will be amplified by PCR using cDNA libraries or EST
(expressed sequence tag) clones as templates. Any of the in vitro or in vivo expression systems described above can then be used for expression of the proteins to be immobilized on the array.
Escherichia coli-based protein expression is generally the method of choice for soluble proteins that do not require extensive post-translational modifications for activity. Extracellular or intracellular domains of membrane proteins will be fused to protein adaptors for expression and purification.
The entire approach can be performed using 96-well assay plates. PCR
reactions are carried out under standard conditions. Oligonucleotide primers contain unique restriction sites for facile cloning into the expression vectors.
Alternatively, the TA cloning system (Clontech) can be used. Expression vectors contain the sequences for affinity tags and the protein adaptors. PCR products are ligated into the expression vectors (under inducible promoters) and introduced into the appropriate competent Escherichia coli strain by calcium-dependent transformation (strains include: XL-1 blue, BL21, SG13009(lon-)). Transformed Escherichia coli cells are plated and individual colonies transferred into 96-array blocks. Cultures are grown to mid-log phase, induced for expression, and cells collected by centrifugation. Cells are resuspended containing lysozyme and the membranes broken by rapid freeze/thaw cycles, or by sonication. Cell debris is removed by centrifugation and the supernatants transferred to 96-tube arrays.
The appropriate affinity matrix is added, protein of interest bound and nonspecifically bound proteins removed by repeated washing steps using 12 - 96 pin suction devices and centrifugation. Alternatively, magnetic affinity beads and filtration devices can be used (Qiagen). The proteins are eluted and transferred to a new 96-well array. Protein concentrations are determined and an aliquot of each protein is spotted onto a nitrocellulose filter and verified by Western analysis using an antibody directed against the affinity tag. The purity of each sample is assessed by SDS-PAGE and silver staining or mass spectrometry. Proteins are snap-frozen and stored at -80°C.
Saccharomyces cerevisiae allows for core glycosylation and lipid modifications of proteins. The approach described above for Escherichia coli can be used with slight modifications for transformation and cell lysis.
Transformation of Saccharomyces cerevisiae is by lithium-acetate and cell lysis is either by lyticase digestion of the cell walls followed by freeze-thaw, sonication or glass-bead extraction. Variations of post-translational modifications can be obtained by different yeast strains (i.e. Saccharomyces pombe, Pichia pastoris).
The advantage of the bacculovirus system or mammalian cells are the wealth of post-translational modifications that can be obtained. The bacculo-system requires cloning of viruses, obtaining high titer stocks and infection of liquid insect cell suspensions (cells are SF9, SF21). Mammalian cell-based expression requires transfection and cloning of cell lines. Soluble proteins are collected from the medium while intracellular or membrane bound proteins require cell lysis (either detergent solubilization, freeze-thaw). Proteins can then be purified analogous to the procedure described for Escherichia coli.
For in vitro translation the system of choice is Escherichia coli lysates obtained from protease-deficient and T7 RNA polymerase overexpressing strains.
Escherichia coli lysates provide efficient protein expression (30-50 pg/ml lysate).
The entire process is carried out in 96-well arrays. Genes of interest are amplified by PCR using oligonucleotides that contain the gene-specific sequences containing a T7 RNA polymerase promoter and binding site and a sequence encoding the affinity tag. Alternatively, an adaptor protein can be fused to the gene of interest by PCR. Amplified DNAs can be directly transcribed and translated in the Escherichia coli lysates without prior cloning for fast analysis.
The proteins are then isolated by binding to an affinity matrix and processed as described above.
Alternative systems which may be used include wheat germ extracts and reticulocyte extracts. In vitro synthesis of membrane proteins and or post-translationally modified proteins will require reticulocyte lysates in combination with microsomes.
In one preferred embodiment of the invention, the proteins immobilized on the patches of the array are antibodies. Optionally, the immobilized proteins may be monoclonal antibodies. The production of monoclonal antibodies against specific protein targets is routine using standard hybridoma technology. In fact, numerous monoclonal antibodies are available commercially.
As an alternative to obtaining antibodies or antibody fragments which have been produced by cell fusion or from continuous cell lines, the antibody moieties may be expressed in bacteriophage. Such antibody phage display technologies are well known to those skilled in the art. The bacteriophage expression systems allow for the random recombination of heavy- and light-chain sequences, thereby creating a library of antibody sequences which can be selected against the desired antigen. The expression system can be based on bacteriophage ~, or , more preferably, on filamentous phage. The bacteriophage expression system can be used to express Fab fragments, Fv's with an engineered intermolecular disulfide bond to stabilize the VH-VL pair (dsFv's), scFvs, or diabody fragments.
The antibody genes of the phage display libraries may be from pre-immunized donors. For instance, the phage display library could be a display library prepared from the spleens of mice previously immunized with a mixture of proteins (such as a Iysate of human T-cells). Immunization can optionally be used to bias the library to contain a greater number of recombinant antibodies reactive towards a specific set of proteins (such as proteins found in human T-cells).
Alternatively, the library antibodies may be derived from naive or synthetic libraries. The naive libraries have been constructed from spleens of mice which have not been contacted by external antigen. In a synthetic library, portions of the antibody sequence, typically those regions corresponding to the complementarity determining regions (CDR) loops, have been mutagenized or randomized.
The phage display method involves batch-cloning the antibody gene library into a phage genome as a fusion to the gene encoding one of the phage coat proteins (pIII, pVI, or pVIII). The pIII phage protein gene is preferred. When the fusion product is expressed it is incorporated into the mature phage coat. As a result, the antibody is displayed as a fusion on the surface of the phage and is available for binding and hence, selection, on a target protein. Once a phage particle is selected as bearing an antibody-coat protein fusion with the desired affinity towards the target protein, the genetic material within the phage particle which corresponds to the displayed antibody can be amplified and sequenced or otherwise analyzed.
In a preferred embodiment, a phagemid is used as the expression vector in the phage display procedures. A phagemid is a small plasmid vector that carries gene III with appropriate cloning sites and a phage packaging signal and contains both host and phage origins of replication. The phagemid is unable to produce a complete phage as the gene III fusion is the only phage gene encoded on the phagemid. A viable phage can be produced by infecting cells containing the phagemid with a helper phage containing a defective replication origin. A
hybrid phage emerges which contains all of the helper phage proteins as well as the gene III-rAb fusion. The emergent phage contains the phagemid DNA only.
In a preferred embodiment of the invention, the recombinant antibodies used in phage display methods of preparing antibody fragments for the arrays of the invention are expressed as genetic fusions to the bacteriophage gene III
protein on a phagemid vector. For instance, the antibody variable regions encoding a single-chain Fv fragment can be fused to the amino terminus of the gene III
protein on a phagemid. Alternatively, the antibody fragment sequence could be fused to the amino terminus of a truncated pIII sequence lacking the first two N-terminal domains. The phagemid DNA encoding the antibody-pIII fusion is preferably packaged into phage particles using a helper phage such as M13K07 or VCS-M 13, which supplies all structural phage proteins.
To display Fab fragments on phage, either the light or heavy (Fd) chain is fused via its C-terminus to pIII. The partner chain is expressed without any fusion to pIII so that both chains can associate to form an intact Fab fragment.
Any method of selection may be used which separates those phage particles which do bind the target protein from those which do not. The selection method must also allow for the recovery of the selected phages. Most typically, the phage particles are selected on an immobilized target protein. Some phage selection strategies known to those skilled in the art include the following: panning on an immobilized antigen; panning on an immobilized antigen using specific elution;
using biotinylated antigen and then selecting on a streptavidin resin or streptavidin-coated magnetic beads; affinity purification; selection on Western blots (especially useful for unknown antigens or antigens di~cult to purify);
in vivo selection; and pathfinder selection. If the selected phage particles are amplified between selection rounds, multiple iterative rounds of selection may optionally be performed.
Elution techniques will vary depending upon the selection process chosen, but typical elution techniques include washing with one of the following solutions: HCl or glycine buffers; basic solutions such as triethylamine;
chaotropic agents; solutions of increased ionic strength; or DTT when biotin is linked to the antigen by a disulfide bridge. Other typical methods of elution include enzymatically cleaving a protease site engineered between the antibody and gene III, or by competing for binding with excess antigen or excess antibodies to the antigen.
A method for producing an array of antibody fragments therefore comprises first selecting recombinant bacteriophage which express antibody fragments from a phage display library. The recombinant bacteriophage are selected by affinity binding to the desired antigen. (Iterative rounds of selection are possible, but optional.) Next, at least one purified sample of an antibody fragment from a bacteriophage which was selected in the first step is produced.
This antibody production step typically entails infecting E. coli cells with the selected bacteriophage. In the absence of helper phage, the selected bacteriophage then replicate as expressive plasmids without producing phage progeny.
Alternatively, the antibody fragment gene of the selected recombinant bacteriophage is isolated, amplified, and then expressed in a suitable expression system. In either case, following amplification, the expressed antibody fragment of the selected and amplified recombinant bacteriophage is isolated and purified.
In a third step of the method, the earlier steps of phage display selection and purified antibody fragment production are repeated using affinity binding to antigens from before until the desired plurality of purified samples of different antibody fragments with different binding partners are produced. In a final step of the method, the antibody fragment of each different purified sample is immobilized onto organic thinfilm on a separate patch on the surface of a substrate to form a plurality of patches of antibody fragments on discrete, known regions of the substrate surface covered by organic thiiifilm.
For instance, to generate an antibody array with antibody fragments against known antigens, open reading frames of the known protein targets identified in DNA databases are amplified by polymerise chain reaction and transcribed and translated in vitro to produce proteins on which a recombinant bacteriophage expressing single-chain antibody fragments are selected. Once selected, the antibody fragment sequence of the selected bacteriophage is amplified (typically using the polymerise chain method) and recloned into a desirable expression system. The expressed antibody fragments are purified and then printed onto organic thinfilms on substrates to form the high density arrays.
In the preparation of the arrays of the invention, phage display methods analogous to those used for antibody fragments may be used for other proteins which are to be immobilized on an array of the invention as long as the protein is of suitable size to be incorporated into the phagemid or alternative vector and expressed as a fusion with a bacteriophage coat protein. Phage display techniques using non-antibody libraries typically make use of some type of protein host scaffold structure which supports the variable regions. For instance, (3-sheet proteins, oc-helical handle proteins, and other highly constrained protein structures have been used as host scaffolds.
Alternative display vectors may also be used to produce the proteins which are printed on the arrays of the invention. Polysomes, stable protein-ribosome-mRNA complexes, can be used to replace live bacteriophage as the display vehicle for recombinant antibody fragments or other proteins (Hares and Pluckthun, Proc. Natl. Acad. Sci USA, 94:4937-4942, 1997). The polysomes are formed by preventing release of newly synthesized and correctly folded protein from the ribosome. Selection of the polysome library is based on binding of the antibody fragments or other proteins which are displayed on the polysomes to the target protein. mRNA which encodes the displayed protein or antibody having the desired affinity for the target is then isolated. Larger libraries may be used with polysome display than with phage display.
(g) Uses of the arrays.
The present invention also provides for methods of using the invention array. The arrays of the present invention are particularly suited for the use in drug development. Other uses include medical diagnostics, proteomics and biosensors.
Use of one of the protein arrays of the present invention may optionally involve placing the two-dimensional protein array in a flowchamber with approximately 1-10 microliters of fluid volume per 25 mm2 overall surface area.
The cover over the array in the flowchamber is preferably transparent or translucent. In one embodiment, the cover may comprise Pyrex or quartz glass.
In other embodiments, the cover may be part of a detection system that monitors WO 00!04382 PCT/US99/15971 interaction between biological moieties immobilized on the array and an analyte.
The flowchambers should remain filled with appropriate aqueous solutions to preserve protein activity. Salt, temperature, and other conditions are preferably kept similar to those of normal physiological conditions. Analytes and potential drug compounds may be .flushed into the flow chamber as desired and their.
interaction with the immobilized proteins determined. Sufficient time must be given to allow for binding between the immobilized proteins and an analyte to occur. No specialized microfluidic pumps, valves, or mixing techniques are required for fluid delivery to the array.
Alternatively, fluid can be delivered to each of the patches of the array individually. For instance, in one embodiment, the regions of the substrate surface may be microfabricated in such a way as to allow integration of the array with a number of fluid delivery channels oriented perpendicular to the array surface, each one of the delivery channels terminating at the site of an individual protein-coated patch.
The sample which is delivered to the array is typically a fluid.
In general, delivery of solutions containing proteins to be bound by the proteins of the array may optionally be preceded, followed, or accompanied by delivery of a blocking solution. A blocking solution contains protein or another moiety which will adhere to sites of non-specific binding on the array. For instance, solutions of bovine serum albumin or milk may be used as blocking solutions.
A wide range of detection methods is applicable to the methods of the invention. As desired, detection may be either quantitative or qualitative.
The invention array can be interfaced with optical detection methods such as absorption in the visible or infrared range, chemoluminescence, and fluorescence (including lifetime, polarization, fluorescence correlation spectroscopy (FCS), and fluorescence-resonance energy transfer (FRET)). Furthermore, other modes of detection such as those based on optical waveguides (PCT Publication WO
96/26432 and U.S. Patent No. 5,677,196), surface plasmon resonance, surface charge sensors, and surface force sensors are compatible with many embodiments of the invention. Alternatively, technologies such as those based on Brewster angle microscopy (Schaaf et al., Langmuir, 3:1131-1135 (I987)) and ellipsometry (U.S. Patent Nos. 5,141,311 and 5,116,121; Kim, Macromolecules, 22:2682-2685 ( 1984)) can be used in conjunction with the arrays of the invention. Quartz crystal microbalances and desorption processes (see for example, U.S. Patent No.
5,719,060) provide still other alternative detection means suitable for at least some embodiments of the invention array. An example of an optical biosensor system compatible both with some arrays of the present invention and a variety of non-label detection principles including surface plasmon resonance, total internal reflection fluorescence (TIRE), Brewster Angle microscopy, optical waveguide lightmode spectroscopy (OWLS), surface charge measurements, and ellipsometry can be found in U.S. Patent No. 5,313,264.
Although non-label detection methods are generally preferred, some of the types of detection methods commonly used for traditional immunoassays which require the use of labels may be applied to use with at least some of the arrays of the present invention, especially those arrays which are arrays of protein-capture agents. These techniques include noncomperitive immunoassays, competitive immunoassays, and dual label, ratiometric immunoassays. These particular techniques are primarily suitable for use with the arrays of proteins when the number of different proteins with different specificity is small (less than about 100). In the competitive method, binding-site occupancy is determined indirectly.
In this method, the proteins of the array are exposed to a labeled developing agent, which is typically a labeled version of the analyte or an analyte analog. The developing agent competes for the binding sites on the protein with the analyte.
The fractional occupancy of the proteins on different patches can be determined by the binding of the developing agent to the proteins of the individual patches. In the noncompetitive method, binding site occupancy is determined directly. In this method, the patches of the array are exposed to a labeled developing agent capable of binding to either the bound analyte or the occupied binding sites on the protein.
For instance, the developing agent may be a labeled antibody directed against occupied sites (i.e., a "sandwich assay"). Alternatively, a dual label, ratiometric, approach may be taken where the immobilized protein is labeled with one label and the second, developing agent is labeled with a second label (Ekins, et al., Clinica Chimica Acta., 194:91-114, 1990). Many different labeling methods may be used in the aforementioned techniques, including radioisotopic, enzymatic, chemiluminescent, and fluorescent methods. Fluorescent methods are preferred.
Figure 8 shows a schematic diagram of one type of fluorescence detection unit which may be used to monitor interaction of immobilized proteins of an array with an analyte. In the illustrated detection unit, the protein array 21 is positioned on a base plate 20. Light from a 100W mercury arc lamp 25 is directed through an excitation filter 24 and onto a beam splitter 23. The light is then directed through a lens 22, such as a Micro Nikkor 55 mm 1:2:8 lens, and onto the array 21. Fluorescence emission from the array returns through the lens 22 and the beam splitter 23. After next passing through an emission filter 26, the emission is received by a cooled CCD camera 27, such as the Slowscan TE/CCD-1024SF&SB (Princeton Instruments). The camera is operably connected to a CPU
28 which is in turn operably connected to a VCR 29 and a monitor 30.
Figure 9 shows a schematic diagram of an alternative detection method based on ellipsometry. Ellipsometry allows for information about the sample to be determined from the observed change in the polarization state of a reflected light wave. Interaction of an analyte with a layer of immobilized proteins on a patch results in a thickness change and alters the polarization status of a plane-polarized light beam reflected off the surface. This process can be monitored in situ from aqueous phase and, if desired, in imaging mode. In a typical setup, monochromatic light (e.g. from a He-Ne laser, 30) is plane polarized (polarizer 31) and directed onto the surface of the sample and detected by a detector 35.
A
compensator 32 changes the elliptically polarized reflected beam to plane-polarized. The corresponding angle is determined by an analyzer 33 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.
In one embodiment, the invention provides a method for screening a plurality of proteins for their ability to interact with a component of a sample comprising the steps of delivering the sample to a protein array of the invention comprising the proteins to be scteened and detecting for the interaction of the component with the immobilized protein of each patch. Optionally, the component may be a protein.
Possible interactions towards which the present invention may be directed include, but are not limited to, antibody/antigen, antibody/hapten, enzyme/substrate, carrier protein/substrate, lectin/carbohydrate, receptor/hormone, receptor/effector, protein/DNA, protein/RNA, repressor/inducer, or the like.
The interaction may involve binding and/or catalysis. The array of he invention is even suitable for assaying translocation by a membrane through a lipid bilayer. In preferred embodiments of use of the array, the assayed interaction is a binding interaction. The assayed interaction may be between a potenrial drug candidate and a plurality of potential drug targets. For instance, a synthesized organic compound may be tested for its ability to act as an inhibitor to a family of immobilized receptors.
Another aspect of the invention provides for a method for screening a plurality of proteins for their ability to bind a particular component of a sample.
This method comprises delivering the sample to a protein array of the invention comprising the proteins to be screened and detecting, either directly or indirectly, for the presence or amount of the particular component retained at each patch.
In a preferred embodiment, the method further comprises the intermediate step of washing the array to remove any unbound or nonspecifically bound components of the sample from the array before the detection step. In another embodiment, the method further comprises the additional step of further characterizing the particular component retained on at least one patch. The particular component may optionally be a protein.
The optional step of further characterizing the particular component retained on a patch of the array is typically designed to identify the nature of the component bound to the protein of a particular patch. In some cases, the entire identity of the component may not be known and the purpose of the further characterization may be the initial identification of the mass, sequence, structure and/or activity (if any) of the bound component. In other cases, the basic identity of the component may be known, but some information about the component may not be known. For instance it may be known that the component is a particular protein, but the post-translational modification, activation state, or some other feature of the protein may not be known. In one embodiment, the step of further characterizing components which are proteins involves measuring the activity of the proteins. Although in some cases it may be preferable to remove the component from the patch before the step of further characterizing the protein is carried out, in other cases the component can be further characterized while still bound to the patch. In still further embodiments, the proteins of the patch which binds a component can be used to isolate and/or purify the component on a larger scale, such as by purifying a component which is a protein from cells. The purified sample of the component can then be characterized through traditional means such as microsequencing, mass spectrometry, and the like.
In another embodiment of the invention, a method of assaying for protein-protein binding interactions is provided which comprises the following steps:
first, delivering a sample comprising at least one protein to be assayed for binding to the protein array of the invention; and then detecting, either directly, or indirectly, for the presence or amount of the protein from the sample which is retained at each patch. In a preferred embodiment, the method further comprises an additional step prior to the detection step which comprises washing the array to remove unbound or nonspecifically bound components of the sample from the array. Typically, the protein being assayed for binding will be from the same organism as the proteins immobilized on the array.
Another embodiment of the invention provides a method of assaying in parallel for the presence of a plurality of analytes in a sample which can react with one or more of the immobilized proteins on the protein array. This method comprises delivering the sample to the invention array and detecting for the interaction of the analyte with the immobilized protein at each patch.
In still another embodiment of the invention, a method of assaying in parallel for the presence of a plurality of analytes in a sample which can bind one or more of the immobilized proteins on the protein array comprises delivering the fluid sample to the invention array and detecting, either directly or indirectly, for the presence or amount of analyte retained at each patch. In a preferred embodiment, the method further comprises the step of washing the array tot remove any unbound or non-specifically bound components of the sample from the array.
The array may be used in a diagnostic manner when the plurality of analytes being assayed are indicative of a disease condition or the presence of a pathogen in an organism. In such embodiments, the sample which is delivered to the array will then typically be derived from a body fluid or a cellular extract from the organism.
The array may be used for drug screening when a potential drug candidate is screened directly for its ability to bind or otherwise interact with a plurality of proteins on the invention array. Alternatively, a plurality of potential drug candidates may be screened in parallel for their ability to bind or otherwise interact with one or more immobilized proteins on the array. The drug screening process may optionally involve assaying for the interaction, such as binding, of at least one analyte or component of a sample with one or more immobilized proteins on an invention array, both in the presence and absence of the potential drug candidate. This allows for the potential drug candidate to be tested for its ability to act as an inhibitor of the interaction or interactions originally being assayed.
(h) Examples The following specific examples are intended to illustrate the invention and should not be construed as limiting the scope of the claims:
Example 1. Fabrication of a two-dimensional array by photolithography.
In a preferred embodiment of the invention, two-dimensional arrays are fabricated onto the substrate material via standard photolithography and/or thin film deposition. Alternative techniques include microcontact printing.
Usually, a computer-aided design pattern is transferred to a photomask using standard techniques, which is then used to transfer the pattern onto a silicon wafer coated with photoresist.
In a typical example, the array ("chip") with lateral dimensions of 10 x 10 mm comprises squared patches of a bioreactive layer (here: gold as the coating on a silicon substrate) each 0.1 x 0.1 mm in size and separated by hydrophobic surface areas with a 0.2 mm spacing. 4" diameter Si( 100) wafers (Virginia Semiconductor) are used as bulk materials. Si( 100) wafers are first cleaned in a 3:1 mixture of HZS04, cone: 30% HZO2 (90°C, 10 min), rinsed with deionized water (18 MS2cm), finally passivated in 1% aqueous HF, and singed at 150°C for 30 min to become hydrophobic. The wafer is then spincoated with photoresist (Shipley 1813), prebaked for 25 minutes at 90°C, exposed using a Karl Suss contact printer and developed according to standard protocols. The wafer is then dried and postbaked at I IO°C for 25 min. In the next step, the wafer is primed with a titanium layer of 20 nm thickness, followed by a 200 nm thick gold layer.
Both layers were deposited using electron-beam evaporation (5 ~/s). After resist stripping and a short plasma treatment, the gold patches can be further chemically modified to achieve the desired bioreactive and biocompatible properties (see Example 3, below).
Example 2. Fabrication of a two-dimensional array by deposition through a hole mask.
In another preferred embodiment the array of gold patches is fabricated by thin film deposition through a hole mask which is in direct contact with the substrate. In a typical example, Si(100) wafers are first cleaned in a 3:1 mixture of HZS04, cone: 30% H202 (90°C, 10 min), rinsed with deionized water (18 MS2cm), finally passivated in 1% aqueous HF and singed at 150°C for 30 min to become hydrophobic. The wafer is then brought into contact with a hole mask exhibiting the positive pattern of the desired patch array. In the next step, the wafer is primed with a titanium layer of 20 nm thickness, followed by a 200 nm thick gold layer. Both layers were deposited using electron-beam evaporation (5 ~r/s). After removal of the mask, the gold patches can be further chemically modified to achieve the desired bioreactive and biocompatible properties (see Example 3, below).
Example 3. Synthesis of an aminoreactive monolayer molecule (following the procedure outlined in Wagner et al., Biophys. J., 1996, 70:2052-2066).
General. 1H- and 13C-NMR spectra are recorded on Broker instruments (100 to 400 MHz). Chemical shifts (S) are reported in ppm relative to internal standard ((CH3)4Si, 8 = 0.00 (1H- and 13C-NMR)). FAB-mass spectra are recorded on a VG-SABSEQ instrument (Cs+, 20 keV). Transmission infrared spectra are obtained as dispersions in KBr on an FTIR Perkin-Elmer 1600 Series instrument. Thin-layer chromatography (TLC) is performed on precoated silica ge160 F254 plates (MERCK, Darmstadt, FRG), and detection was done using C12/toluidine, PdCl2 and UV-detection under NH3-vapor. Medium pressure liquid chromatography (MPLC) is performed on a Labomatic MD-80 (LABOMATIC
INSTR. AG, Allschwil, Switzerland) using a Buechi column (460x36 mm;
BUECHI, Flawil, Switzerland), filled with silica gel 60 (particle size 15-40 pm) from Merck.
Synthesis of Il, ll'-dithiobis(succinimidylundecanoate) (DSU). Sodium thiosulfate (55.3 g, 350 mmol) is added to a suspension of 11-bromo-undecanoic acid (92.8 g, 350 mmol) in 50 % aqueous 1,4-dioxane (1000 ml). The mixture is heated at reflux (90 °C) for 2 h until the reaction to the intermediate Bunte salt was complete (clear solution). The oxidation to the corresponding disulfide is carried out in situ by adding iodine in portions until the solution retained with a yellow to brown colour. The surplus of iodine is retitrated with 15 % sodium pyrosulfite in water. After removal of 1,4-dioxane by rotary evaporation the creamy suspension is filtered to yield product 11, ll'-dithiobis(undecanoic acid).
RecrystallizaNon from ethyl acetate/THF provides a white solid (73.4 g, 96.5 %):
mp 94 °C; 1H NMR (400 MHz, CDC13 / CD30D 95 : 5): b 2.69 (t, 2H, J =
7.3 Hz), 2.29 (t, 2H, J = 7.5 Hz), 1.76-1.57 (m, 4H), and 1.40-1.29 (m, 12H); FAB-MS (Cs+, 20 keV): m/z (relative intensity) 434 ( 100, M+). Anal. Calcd. for C22H4204S2: C, 60.79; H, 9.74; S, 14.75. Found: C, 60.95; H, 9.82; S, 14.74.
To a solution of 11,11'-dithiobis(undecanoic acid). (1.0 g, 2.3 mmol) in THF (50 ml) is added N-hydroxysuccinimide (0.575 g, 5 mmol) followed by DCC (1.03 g, 5 mmol) at 0 °C. After the reaction mixture is allowed to warm to 23 °C and is stirred for 36 h at room temperature, the dicyclohexylurea (DCU) is filtered.
Removal of the solvent under reduced pressure and recrystallization from acetone/hexane provides ll, ll'-dithiobis(succinimidylundecanoate) as a white solid. Final purification is achieved by medium pressure liquid chromatography (9 bar) using silica gel and a 2:1 mixture of ethyl acetate and hexane. The organic phase is concentrated and dried in vacuum to afford I l, l I '-dithiobis(succinimidylundecanoate) (1.12 g, 78 %): mp 95 °C; 1H NMR
(400 MHz, CDC13): 8 2.83 (s, 4H), 2.68 (t, 2H, J = 7.3 Hz), 2.60 (t, 2H, J = 7.5 Hz), 1.78-1.63 (m, 4H), and 1.43-1.29 (m, 12H); FAB-MS (Cs+, 20 keV): m/z (relative intensity) 514 (100), 628 (86, M+). Anal. Calcd. for C3oH4gN2OgS2: C, 57.30;
H, 7.69; N, 4.45; S, 10.20. Found: C, 57.32; H, 7.60; N, 4.39; S, 10.25.
Example 4. Formation of an aminoreactive monolayer on gold (following the procedure of Wagner et al., Biophys. J., 1996, 70:2052-2066).
Monolayers based on ll, ll'-dithiobis(succinimidylundecanoate) (DSU) can be deposited on Au(111) surfaces of microarrays described under Examples 1 and 2 by immersing them into a 1 mM solution of DSU in chloroform at room temperature for 1 hour. After rinsing with 10 volumes of solvent, the N-hydroxysuccinimidyl-terminated monolayer is dried under a stream of nitrogen and immediately used for protein immobilization.
Example 5. Expression and purification of human caspase fusion proteins.
Caspases are cysteine proteases of the papain superfamily, with a different active site and catalytic mechanism than observed for papain, Wilson, K.P. et al., Nature, 1994 370:270-275. Caspases are important enzymes in the promotion of the cell death pathways and inflammation, Villa, et al., TIBS, 1997, 22:288-392.
Identification of selective caspase inhibitors is essential to prevent cross-inhibition of other caspase-dependent pathways. Caspases 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, Villa, et al., TIBS, 1997, 22:288-392 and new caspase homologs identified by the human genome project are PCR amplified and cloned into an E. coli expression vector containing an N-terminal histidine tag, Hochuli, et al., Biotechnology, 1988 6:1321, a factor Xa cleavage site, a lysine tag and a tri-glycine linker.
Fusion proteins are expressed, purified by nickel-nitrilotriacetic acid (NTA) agarose chromatography, the histidine tag removed by factor Xa cleavage, followed by gel filtration. Caspases are snap-frozen and stored in 20 mM PIPES, pH 7.2, 150 mM
NaCI, 0.1% CHAPS, 10 % sucrose at -80°C.
Example 6. Immobilization of fusion proteins on a 2D-protein array.
Caspase-fusion proteins can be immobilized to the aminoreactive monolayer surface of the bioreactive patches of the two-dimensional array (see Examples I, 2, and 4 above). Caspase fusion proteins can be diluted to concentrations of 1 pg/ml in 20 mM PIPES, pH 7.2, 15U mM NaCI, 0.1%
CHAPS, 10% sucrose and applied onto the bioreactive patches using a computer-aided, capillary-based dispensing system. After an immobilization period of 30 min, the 2D array was rinsed and subjected to analysis. Ultrapure water with a resistance of 18 MS2cm is generally useable for all aqueous buffers (purified by passage through a Barnstead Nanopure~ system).
Example 7. Assay of caspase activity on a two-dimensional array.
Caspase activity can be determined by a binding assay using three fluorescently labeled peptide aldehyde inhibitors that form a reversible thiohemiacetal moiety with the active site cysteine, Thornberry, Methods in Enzymology, 1994, 244:615-631. The peptides are adapted to caspase 1, 3, 4, 7:
Dns (dansyl)-SS- DEVD-CHO, caspase 1: Dns-SS-VDVAD-CHO, caspase 6:
Dns-SS-VQID-CHO, Talanian, J. Biol. Chem., 1997, 272:9677-9682. The affinity for Ac-DEVD-CHO to caspase 1 is determined to be in the low nanomolar range, Thornberry, Methods in Enzymolo~, 1994, 244:615-63 I. The assay buffer is 20 mM PIPES, pH 7.2, 150 mM NaCI, 0.1% CHAPS, 10 % sucrose, Stennicke, and Salvesen, J. Biol. Chem., 1997, 272:25719-25723. Fluorescently labeled peptides are mixed to a final concentration of 1 to 5 nM each, the potential drug compound added and flushed onto the 2D array. Peptides are allowed to bind for 10 - 60 min., unbound peptide removed by washing with buffer and the fluorescence intensity measured (excitation at 360 nm, emission at 470 nm).
Example 8. Formation and use of an array of immobilized Fab' antibody fragments to detect concentrations of soluble proteins prepared from cultured mammalian cells.
Collections of IgG antibodies are purchased from commercial sources (e.g.
Pierce, Rockford, IL). The antibodies are first purified by affinity chromatography based on binding to immobilized protein A. The antibodies are diluted 1:1 in binding buffer( 0.1 M Tris-HCI, 0.15 M NaCI, pH 7.5). A 2 ml minicolumn containing a gel with immobilized protein A is prepared. (Hermanson, et. al., Immobilized Affinity Ligand Techniques, Academic Press, San Diego, 1992.) The column is equilibrated with 10 ml of binding buffer. Less than 10 mg of immunoglobulin is applied to each 2 ml minicolumn and the column is washed with binding buffer until the absorbance at 280 nm is less than 0.02. The bound immunoglobulins are eluted with 0.1 M glycine, 0.15 M NaCI, pH 2.8, and immediately neutralized with 1.0 M Tris-HCI, pH 8.0 to 50 mM final concentration and then dialyzed against 10 mM sodium phosphate, 0.1 S M NaCI, pH 7.2 and stored at 4°C.
The purified immunoglobulin are digested with immobilized pepsin. Pepsin is an acidic endopeptidase and hydrolyzes proteins favorably adjacent to aromatic and dicarboxylic L-amino acid residues. Digestion of IgG with pepsin generates intact F(ab')2 fragments. Immobilized pepsin gel is washed with digestion buffer;
20 mM sodium acetate, pH 4.5. A solution of purified IgG at 10 mg/ml is added to the immobilized pepsin gel and incubated at 37°C for 2 hours. The reaction is neutralized by the addition of 10 mM Tris-HCI, pH 7.5 and centrifuged to pellet the gel. The supernatant liquid is collected and applied to an immobilized protein A column, as described above, to separate the F(ab') 2 fragments from the Fc and undigested IgG. The pooled F(ab')~ is dialyzed against 10 mM sodium phosphate, 0.15 M NaCl, pH 7.2 and stored at 4°C. The quantity of pooled, eluted F(ab') 2 is measured by peak area absorbance at 280 nm.
The purified F(ab') 2 fragments at a concentration of 10 mg/ml are reduced at 37 °C for 1 hour in a buffer of 10 mM sodium phosphate, 0.15 M NaCI, mM 2-mercaptoethylamine, S mM EDTA, pH 6Ø The Fab' fragments are separated from unsplit F(ab')2 fragments and concentrated by application to a Sephadex G-25 column (Mr = 46,000 - 58,000). The pooled Fab' fragments are dialyzed against 10 mM sodium phosphate, 0.15 M NaCI, pH 7.2. The reduced Fab' fragments are diluted to 100 pg/ml and applied onto the bioreactive patches containing exposed aminoreactive functional groups using a computer-aided, capillary-based microdispensing system (for antibody immobilization procedures, see Dammer et al., Biophys. J., 70:2437-2441, 1996). After an immobilization period of 30 minutes at 30°C, the array is rinsed extensively with 10 mM sodium phosphate, 0.15 M NaCI, 5 mM EDTA, pH 7Ø
Transformed human cells grown in culture are collected by low speed centrifugation, briefly washed with ice-cold phosphate-buffered solution (PBS), and then resuspended in ice-cold hypotonic buffer containing DNase/RNase (10 pg/mI each, final concentration) and a mixture of protease inhibitors. Cells are transferred to a microcentrifuge tube, allowed to swell for 5 minutes, and lysed by rapid freezing in liquid nitrogen and thawing in ice-cold water. Cell debris and precipitates are removed by high-speed centrifugation and the supernatant is cleared by passage through a 0.45 pm filter. The cleared lysate is applied to the Fab' fragment array described above and allowed to incubate for 2 hours at 30°C.
After binding the array is washed extensively with 10 mM sodium phosphate, 0.15 M NaCI, 5 mM EDTA, pH 7Ø The location and amount of bound proteins are determined by optical detection.
Example 9. Formation and use of an array of immobilized antibody fragments to detect concentrations of soluble proteins prepared from cultured mammalian cells.
A combinatorial library of filamentous phage expressing scFv antibody fragments is generated based on the technique of McCafferty and coworkers;
McCafferty, et al., Nature, 1990, 348:552-554; Winter and Milstein, Nature, 1991, 349:293-299. Briefly, mRNA is purified from mouse spleens and used to construct a cDNA library. PCR fragments encoding sequences of the variable heavy and light chain immunoglobulin genes of the mouse are amplified from the prepared cDNA. The amplified PCR products are joined by a linker region of DNA encoding the 15 amino acid peptide (Gly4SerG1y2CysGlySerGly4Ser) (SEQ
ID NO: 1) and the resulting full-length PCR fragment is cloned into an expression plasmid (pCANTAB 5 E) in which the purification peptide tag (E Tag) has been replaced by a His6 peptide (SEQ ID NO: 2). Electrocompetent TG 1 E. coli cells are transformed with the expression plasmid by electroporation. The pCANTAB-transformed cells are induced to produced functional filamentous phage expressing scFv fragments by superinfection with M13K07 helper phage. Cells are grown on glucose-deficient medium containing the antibiotics ampicillin (to select for cells with the phagemid) and kanamycin (to select for cells infected with M13K07). In the absence of glucose, the lac promoter present on the phagemid is no longer repressed, and synthesis of the scFv-gene 3 fusion begins.
Proteins from a cell lysate are adsorbed to the wells of a 96-well plate.
Transformed human cells grown in culture are collected by low speed centrifugation and the cells are briefly washed with ice-cold PBS. The washed cells are then resuspended in ice-cold hypotonic buffer containing DNase/RNase (10 pg/ml each, final concentration) and a mixture of protease inhibitors, allowed to swell for 5 minutes, and lysed by rapid freezing in liquid nitrogen and thawing in ice-cold water. Cell debris and precipitates are removed by high-speed centrifugation and the supernatant is cleared by passage through a 0.45 N,m filter.
The cleared lysate is diluted to 10 ~,g/ml in dilution buffer; 20 mM PIPES, 0.15 M
NaCI, 0.1 % CHAPS, 10%, 5 mM EDTA, S mM 2-mercaptoethanol, 2 mM DTT, pH 7.2 and applied to the 96-plate wells. After immobilization for 1 hour at 30°C, the well is washed with the dilution buffer and then incubated with dilution buffer containing 10% nonfat dry milk to block unreacted sites. After the blocking step, the well is washed extensively with the dilution buffer.
Phage expressing displayed antibodies are separated from E. coli cells by centrifugation and then precipitated from the supernatant by the addition of 15%
w/v PEG 8000, 2.5 M NaCI followed by centrifugation. The purified phage are resuspended in the dilution buffer containing 3% nonfat dry milk and applied to the well containing the immobilized proteins described above, and allowed to bind for 2 hours at 37°C, followed by extensive washing with the binding buffer. Phage are eluted from the well with an elution buffer; 20 mM PIPES, 1 M NaCI, 0.1 CHAPS, 10%, 5 mM EDTA, 5 mM 2-mercaptoethanol, 2 mM DTT, pH 7.2. The well is then extensively washed with purge buffer; 20 mM PIPES, 2.5 M NaCI, 0.1 % CHAPS, 10%, 5 mM EDTA, 5 mM 2-mercaptoethanol, 2 mM DTT, pH
7.2. The well is then extensively washed with dilution buffer; 20 mM PIPES, 0.15 M NaCI, 0.1 % CHAPS, 10%, S mM EDTA, S mM 2-mercaptoethanol, 2 mM DTT, pH 7.2. The eluted phage solution is then re-applied to a new well containing adsorbed antigen and the panning enrichment is repeated 4 times.
Finally, the phage are eluted from the well with 2M of NaCI in 20 mM PIPES, 0.1 CHAPS, 10%, 5 mM EDTA, 5 mM 2-mercaptoethanol, 2 mM DTT, pH 7.2.
Eluates are collected and mixed with log-phase TG1 cells, and grown at 37°C for 1 hour and then plated onto SOB medium containing ampicillin and glucose and allowed to grow for 12 - 24 hours.
Individual colonies are picked and arrayed into 96-well 2ml blocks containing SOB medium and M13K07 helper phage and grown for 8 hours with shaking at 37°C. The phage are separated from cells by centrifugation and precipitated with PEG/NaCI as described above. Concentrated phage are used to infect HB2151 E. coli. E. coli TG1 produces a suppressor tRNA which allows readthrough (suppression) of an amber stop codon located between the scFv and phage gene 3 sequences of the pCANTAB 5 E plasmid. Infected HB2151 cells are selected on medium containing ampicillin, glucose, and nalidixic acid. Cells are grown to mid-log and then centrifuged and resuspended in medium lacking glucose and growth continued. Soluble scFv fragments will accumulate in the cell periplasm. A periplasmic extract is prepared from pelleted cells by mild osmotic shock. The soluble scFv released into the supernatant is purified by affinity binding to Ni-NTA activated agarose and eluted with 1 U mM EDTA.
The purified scFv antibody fragments are diluted to 100 pg/ml and applied onto the bioreactive patches with exposed aminoreactive groups using a computer-aided, capillary-based microdispensing system. After an immobilization period of 30 minutes at 30°C, the array is rinsed extensively with 10 mM sodium phosphate, 0.15 M NaCI, 5 mM EDTA, pH 7Ø
Transformed human cells grown in culture are collected by low speed centrifugation, briefly washed with ice-cold PBS, and then resuspended in ice-cold hypotonic buffer containing DNase/RNase ( 10 gg/ml each, final concentration) and mixture of protease inhibitors. Cells are transferred to a microcentrifuge tube, allowed to swell for 5 minutes, and lysed by rapid freezing in liquid nitrogen and thawing in ice-cold water. Cell debris and precipitates are removed by high-speed centrifugation and the supernatant is cleared by passage through a 0.45 ~,un filter. The cleared lysate is applied to the scFv fragment array described above and allowed to incubate for 2 hours at 30°C. After binding, the array is washed extensively with 0.1 M sodium phosphate, 0.15 M NaCI, 5 mM
EDTA pH 7Ø The location and amount of bound proteins are determined by optical detection.
Patterns of binding are established empirically by testing dilutions of a control cell extract. Extracts from experimental cells are diluted to a series of concentrations and then tested against the array. Patterns of protein expression in the experimental cell lysates are compared to protein expression patterns in the control samples to identify proteins with unique expression profiles.
Example 10. Formation and use of an array of immobilized monoclonal antibodies to detect concentrations of soluble proteins prepared from cultured mammalian cells.
Collections of monoclonal antibodies are purchased from commercial suppliers as either raw ascities fluid or purified by chromotography over protein A, protein G, or protein L. If from raw ascites fluid, the antibodies are purified using a HiTrap Protein G or HiTrap Protein A column (Pharmacia) as appropriate for the immunoglobulin subclass and species. Prior to chromotography the ascites are diluted with an equal volume of 10 mM sodium phosphate, 0.9 % NaCI, pH
7.4 (PBS) and clarified by passage through a 0.22 ~cm filter. The filtrate is loaded onto the column in PBS and the column is washed with two column volumes of PBS. The antibody is eluted with 100 mM Glycine-HCI, pH 2.7 (for protein G) or 100 mM citric acid, pH 3.0 (for protein A). The eluate is collected into 1/10 volume 1 M Tris-HCI, pH 8Ø The final pH is 7.5. Fractions containing the antibodies are confirmed by SDS-PAGE and then pooled and dialyzed against PBS.
The different samples of purified antibodies are each diluted to 100 pg/ml.
Each different antibody sample is applied to a separate patch of an array of aminoreactive monolayer patches (see Example 4, above) using a computer-aided, capillary-based microdispensing system. After an immobilization period of 30 minutes at 30°C, the array is rinsed extensively with 10 mM sodium phosphate, 0.15 M NaCI, 5 mM EDTA, pH 7Ø
WO 00/04382 PCfNS99/15971 Transformed human cells grown in culture are collected by low speed centrifugation, briefly washed with ice-cold PBS, and resuspended in ice-cold hypotonic buffer containing Dnase/Rnase ( 10 p,g/ml each, final concentration) and a mixture of protease inhibitors. Cells are transferred to a microcentrifuge tube, allowed to swell for 5 minutes, and lysed by rapid freezing in liquid nitrogen and thawing in ice-cold water. Cell debris and precipitates are removed by high-speed centrifugation and the supernatant is cleared by passage through a 0.45 ~n filter.
The cleared lysate is applied to the monoclonal antibody array described above and allowed to incubate for 2 hours at 30°C. After binding the array is washed extensively as in Example 9, above. The location and amount of bound proteins are determined by optical detection.
All documents cited in the above specification are herein incorporated by reference. In addition, the co-pending U.S. patent application "Arrays of Protein-Capture Agents and Methods of Use Thereof ', filed on July 14, 1999, with the identifier 24406-0006, for the inventors Peter Wagner, Steffen Nock, Dana Ault-Riche, and Christian Itin, is herein incorporated by reference in its entirety.
Various modifications and variations of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the following claims.
Finally, the phage are eluted from the well with 2M of NaCI in 20 mM PIPES, 0.1 CHAPS, 10%, 5 mM EDTA, 5 mM 2-mercaptoethanol, 2 mM DTT, pH 7.2.
Eluates are collected and mixed with log-phase TG1 cells, and grown at 37°C for 1 hour and then plated onto SOB medium containing ampicillin and glucose and allowed to grow for 12 - 24 hours.
Individual colonies are picked and arrayed into 96-well 2ml blocks containing SOB medium and M13K07 helper phage and grown for 8 hours with shaking at 37°C. The phage are separated from cells by centrifugation and precipitated with PEG/NaCI as described above. Concentrated phage are used to infect HB2151 E. coli. E. coli TG1 produces a suppressor tRNA which allows readthrough (suppression) of an amber stop codon located between the scFv and phage gene 3 sequences of the pCANTAB 5 E plasmid. Infected HB2151 cells are selected on medium containing ampicillin, glucose, and nalidixic acid. Cells are grown to mid-log and then centrifuged and resuspended in medium lacking glucose and growth continued. Soluble scFv fragments will accumulate in the cell periplasm. A periplasmic extract is prepared from pelleted cells by mild osmotic shock. The soluble scFv released into the supernatant is purified by affinity binding to Ni-NTA activated agarose and eluted with 1 U mM EDTA.
The purified scFv antibody fragments are diluted to 100 pg/ml and applied onto the bioreactive patches with exposed aminoreactive groups using a computer-aided, capillary-based microdispensing system. After an immobilization period of 30 minutes at 30°C, the array is rinsed extensively with 10 mM sodium phosphate, 0.15 M NaCI, 5 mM EDTA, pH 7Ø
Transformed human cells grown in culture are collected by low speed centrifugation, briefly washed with ice-cold PBS, and then resuspended in ice-cold hypotonic buffer containing DNase/RNase ( 10 gg/ml each, final concentration) and mixture of protease inhibitors. Cells are transferred to a microcentrifuge tube, allowed to swell for 5 minutes, and lysed by rapid freezing in liquid nitrogen and thawing in ice-cold water. Cell debris and precipitates are removed by high-speed centrifugation and the supernatant is cleared by passage through a 0.45 ~,un filter. The cleared lysate is applied to the scFv fragment array described above and allowed to incubate for 2 hours at 30°C. After binding, the array is washed extensively with 0.1 M sodium phosphate, 0.15 M NaCI, 5 mM
EDTA pH 7Ø The location and amount of bound proteins are determined by optical detection.
Patterns of binding are established empirically by testing dilutions of a control cell extract. Extracts from experimental cells are diluted to a series of concentrations and then tested against the array. Patterns of protein expression in the experimental cell lysates are compared to protein expression patterns in the control samples to identify proteins with unique expression profiles.
Example 10. Formation and use of an array of immobilized monoclonal antibodies to detect concentrations of soluble proteins prepared from cultured mammalian cells.
Collections of monoclonal antibodies are purchased from commercial suppliers as either raw ascities fluid or purified by chromotography over protein A, protein G, or protein L. If from raw ascites fluid, the antibodies are purified using a HiTrap Protein G or HiTrap Protein A column (Pharmacia) as appropriate for the immunoglobulin subclass and species. Prior to chromotography the ascites are diluted with an equal volume of 10 mM sodium phosphate, 0.9 % NaCI, pH
7.4 (PBS) and clarified by passage through a 0.22 ~cm filter. The filtrate is loaded onto the column in PBS and the column is washed with two column volumes of PBS. The antibody is eluted with 100 mM Glycine-HCI, pH 2.7 (for protein G) or 100 mM citric acid, pH 3.0 (for protein A). The eluate is collected into 1/10 volume 1 M Tris-HCI, pH 8Ø The final pH is 7.5. Fractions containing the antibodies are confirmed by SDS-PAGE and then pooled and dialyzed against PBS.
The different samples of purified antibodies are each diluted to 100 pg/ml.
Each different antibody sample is applied to a separate patch of an array of aminoreactive monolayer patches (see Example 4, above) using a computer-aided, capillary-based microdispensing system. After an immobilization period of 30 minutes at 30°C, the array is rinsed extensively with 10 mM sodium phosphate, 0.15 M NaCI, 5 mM EDTA, pH 7Ø
WO 00/04382 PCfNS99/15971 Transformed human cells grown in culture are collected by low speed centrifugation, briefly washed with ice-cold PBS, and resuspended in ice-cold hypotonic buffer containing Dnase/Rnase ( 10 p,g/ml each, final concentration) and a mixture of protease inhibitors. Cells are transferred to a microcentrifuge tube, allowed to swell for 5 minutes, and lysed by rapid freezing in liquid nitrogen and thawing in ice-cold water. Cell debris and precipitates are removed by high-speed centrifugation and the supernatant is cleared by passage through a 0.45 ~n filter.
The cleared lysate is applied to the monoclonal antibody array described above and allowed to incubate for 2 hours at 30°C. After binding the array is washed extensively as in Example 9, above. The location and amount of bound proteins are determined by optical detection.
All documents cited in the above specification are herein incorporated by reference. In addition, the co-pending U.S. patent application "Arrays of Protein-Capture Agents and Methods of Use Thereof ', filed on July 14, 1999, with the identifier 24406-0006, for the inventors Peter Wagner, Steffen Nock, Dana Ault-Riche, and Christian Itin, is herein incorporated by reference in its entirety.
Various modifications and variations of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the following claims.
Claims (39)
1. An array of proteins, comprising:
(a) a substrate;
(b) at least one organic thinfilm on some or all of the substrate surface;
and (c) 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.
(a) a substrate;
(b) at least one organic thinfilm on some or all of the substrate surface;
and (c) 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.
2. The array of Claim 1 which comprises at least about 10 of said patches.
3. The array of Claim 2 which comprises at least about 100 of said patches.
4. The array of Claim 3 which comprises at least about 103 of said patches.
5. The array of Claim 1 which comprises at least about 10 different immobilized proteins.
6. The array of Claim 5 which comprises at least about 100 different immobilized proteins.
7. The array of Claim 6 which comprises at least about 1000 different immobilized proteins.
8. The array of Claim 1, wherein the area of the substrate surface covered by each of the patches is no more than about 0.25 mm2.
9. The array of Claim 8, wherein the area of the substrate surface covered by each of the patches is between about 1 µm2 and about 10,000 µm2.
10. The array of Claim 1, wherein the patches are all contained within an area of about 1 cm2 or less on the surface of the substrate.
11. The array of Claim 1, wherein all of the proteins immobilized on the array are functionally related.
12. The array of Claim 1, wherein all of the proteins immobilized on the array are structurally related.
13. The array of Claim 1, wherein all of the proteins immobilized on the array are members of the same family.
14. An array of Claim 13, wherein said family is selected from the group consisting of growth factor receptors, hormone receptors, neurotransmitter receptors, catecholamine receptors, amino acid derivative receptors, cytokine receptors, extracellular matrix receptors, antibodies, lectins, cytokines, serpins, proteases, kinases, phosphatases, ras-like GTPases, hydrolases, steroid hormone receptors, transcription factors, heat-shock transcription factors, DNA-binding proteins, zinc-finger proteins, leucine-zipper proteins, homeodomain proteins, intracellular signal transduction modulators and effectors, apoptosis-related factors, DNA synthesis factors, DNA repair factors, DNA recombination factors, cell-surface antigens, hepatitis C virus (HCV) proteases and HIV proteases.
15. The array of Claim 1, wherein the proteins are antibodies or antibody fragments.
16. The array of Claim 1, wherein the proteins are protein-capture agents.
17. The array of Claim 1, wherein the organic thinfilm on the array is less than about 20 nm thick.
18. The array of Claim 1, wherein the organic thinfilm on the array comprises a monolayer.
19. The array of Claim 18, wherein the monolayer comprises a self-assembled monolayer comprising molecules of the formula (X)a R(Y)b wherein R is a spacer, X is a functional group that binds R to the surface, Y
is a functional group for binding the protein onto the monolayer, and a and b are, independently, integers.
is a functional group for binding the protein onto the monolayer, and a and b are, independently, integers.
20. The array of Claim 19, wherein both a and b are 1.
21. The array of Claim 19, wherein:
said substrate is selected from the group consisting of silicon, silicon dioxide, indium tin oxide, alumina, glass, and titania; and X, prior to incorporation into said monolayer, is selected from the group consisting of a monohalosilane, dihalosilane, trihalosilane, trichlorosilane, trialkoxysilane, dialkoxysilane, monoalkoxysilane, carboxylic acids, and phosphates.
said substrate is selected from the group consisting of silicon, silicon dioxide, indium tin oxide, alumina, glass, and titania; and X, prior to incorporation into said monolayer, is selected from the group consisting of a monohalosilane, dihalosilane, trihalosilane, trichlorosilane, trialkoxysilane, dialkoxysilane, monoalkoxysilane, carboxylic acids, and phosphates.
22. The array of Claim 19, wherein the substrate comprises silicon and X is an olefin.
23. The array of Claim 1, wherein the substrate comprises a polymer.
24. The array of Claim 19, further comprising at least one coating between said substrate and said monolayer, wherein said coating is formed on the substrate or applied to the substrate.
25. The array of Claim 24, wherein:
the coating comprises a noble metal film; and X, prior to incorporation into said monolayer, is a functional group selected from the group consisting of an asymmetrical or symmetrical disulfide, sulfide, diselenide, selenide, thiol, isonitrile, selenol, trivalent phosphorus compounds, isothiocyanate, isocyanate, xanthanate, thiocarbamate, phosphines, amines, thio acid and dithio acid.
the coating comprises a noble metal film; and X, prior to incorporation into said monolayer, is a functional group selected from the group consisting of an asymmetrical or symmetrical disulfide, sulfide, diselenide, selenide, thiol, isonitrile, selenol, trivalent phosphorus compounds, isothiocyanate, isocyanate, xanthanate, thiocarbamate, phosphines, amines, thio acid and dithio acid.
26. The array of Claim 24, wherein the coating comprises titania or tantalum oxide and X is a phosphate group.
27. The array of Claim 1, wherein each protein is immobilized on the organic thinfilm by an affinity tag.
28. A biosensor comprising an array of proteins of Claim 1.
29. A micromachined device comprising an array of proteins of Claim 1.
30. A diagnostic device comprising an array of proteins of Claim 1.
31. A method for screening a plurality of proteins for their ability to interact with a component of a sample, comprising:
(a) delivering the sample to the array of Claim 1 comprising the proteins to be screened; and (b) detecting, either directly or indirectly, for the interaction of said component with the immobilized protein of each patch.
(a) delivering the sample to the array of Claim 1 comprising the proteins to be screened; and (b) detecting, either directly or indirectly, for the interaction of said component with the immobilized protein of each patch.
32. The method of Claim 31, wherein the component is a protein.
33. A method for screening a plurality of proteins for their ability to bind a particular component of a sample, comprising:
(a) delivering said sample to the array of Claim 1 comprising the proteins to be screened; and (b) detecting, either directly or indirectly, for the presence or amount of said particular component retained at each patch.
(a) delivering said sample to the array of Claim 1 comprising the proteins to be screened; and (b) detecting, either directly or indirectly, for the presence or amount of said particular component retained at each patch.
34. The method of Claim 33, wherein said particular component is a protein.
35. The method of Claim 33, further comprising the step:
(d) further characterizing said particular component retained on at least one patch.
(d) further characterizing said particular component retained on at least one patch.
36. A method of assaying for protein-protein binding interactions, comprising:
(a) delivering a sample comprising at least one protein to be assayed for binding to the array of Claim 1; and (b) detecting, either directly or indirectly, for the presence or amount of the protein from the sample which is retained at each patch.
(a) delivering a sample comprising at least one protein to be assayed for binding to the array of Claim 1; and (b) detecting, either directly or indirectly, for the presence or amount of the protein from the sample which is retained at each patch.
37. A method of assaying in parallel for a plurality of analytes in a sample, comprising:
(a) delivering the sample to the array of Claim 1, wherein at least one of the immobilized proteins of said array can react with each of said analytes;
and (b) detecting for the interaction of the analytes with the immobilized protein at each patch.
(a) delivering the sample to the array of Claim 1, wherein at least one of the immobilized proteins of said array can react with each of said analytes;
and (b) detecting for the interaction of the analytes with the immobilized protein at each patch.
38. A method of assaying in parallel for a plurality of analytes in a sample, comprising:
(a) delivering the fluid sample to the array of Claim 1, wherein at least one of the immobilized proteins of said array can bind each of said analytes;
and (b) detecting, either directly or indirectly, for the presence or amount of analyte retained at each patch.
(a) delivering the fluid sample to the array of Claim 1, wherein at least one of the immobilized proteins of said array can bind each of said analytes;
and (b) detecting, either directly or indirectly, for the presence or amount of analyte retained at each patch.
39. The method of Claim 38, further comprising the step:
(d) further characterizing the analyte retained on at least one patch.
(d) further characterizing the analyte retained on at least one patch.
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Families Citing this family (768)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5776748A (en) * | 1993-10-04 | 1998-07-07 | President And Fellows Of Harvard College | Method of formation of microstamped patterns on plates for adhesion of cells and other biological materials, devices and uses therefor |
ZA971261B (en) * | 1996-02-16 | 1998-12-03 | Smith & Nephew Inc | Graft anchor |
ATE366418T1 (en) | 1996-04-25 | 2007-07-15 | Bioarray Solutions Ltd | LIGHT-REGULATED, ELECTROKINETIC COMPOSITION OF PARTICLES ON SURFACES |
US7041510B2 (en) * | 1996-04-25 | 2006-05-09 | Bioarray Solutions Ltd. | System and method for programmable illumination pattern generation |
US7098320B1 (en) | 1996-07-29 | 2006-08-29 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
ES2287956T3 (en) | 1996-07-29 | 2007-12-16 | Nanosphere Inc. | NANOPARTICLES THAT HAVE OLIGONUCLEOTIDES UNITED TO THE SAME AND USES OF THE SAME. |
US6506564B1 (en) * | 1996-07-29 | 2003-01-14 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US6984491B2 (en) | 1996-07-29 | 2006-01-10 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US6582921B2 (en) | 1996-07-29 | 2003-06-24 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses thereof |
US6750016B2 (en) | 1996-07-29 | 2004-06-15 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US7169556B2 (en) | 1996-07-29 | 2007-01-30 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US6368877B1 (en) * | 1997-06-25 | 2002-04-09 | Massachusetts Institute Of Technology | Self-assembling peptide surfaces for cell patterning and interactions |
US6974669B2 (en) * | 2000-03-28 | 2005-12-13 | Nanosphere, Inc. | Bio-barcodes based on oligonucleotide-modified nanoparticles |
US20050037397A1 (en) * | 2001-03-28 | 2005-02-17 | Nanosphere, Inc. | Bio-barcode based detection of target analytes |
US20020144905A1 (en) | 1997-12-17 | 2002-10-10 | Christian Schmidt | Sample positioning and analysis system |
JP3486171B2 (en) | 1997-12-17 | 2004-01-13 | エコル・ポリテクニック・フェデラル・ドゥ・ロザンヌ(エ・ペー・エフ・エル) | Positioning and electrophysiological characterization of single cell and reconstituted membrane systems on microstructured carriers |
US7244349B2 (en) | 1997-12-17 | 2007-07-17 | Molecular Devices Corporation | Multiaperture sample positioning and analysis system |
WO1999039210A1 (en) * | 1998-01-29 | 1999-08-05 | Miller, Samuel | High density arrays for proteome analysis and methods and compositions therefor |
EP1965213A3 (en) | 1998-02-04 | 2009-07-15 | Invitrogen Corporation | Microarrays and uses therefor |
GB9812783D0 (en) * | 1998-06-12 | 1998-08-12 | Cenes Ltd | High throuoghput screen |
US6897073B2 (en) * | 1998-07-14 | 2005-05-24 | Zyomyx, Inc. | Non-specific binding resistant protein arrays and methods for making the same |
US20020119579A1 (en) * | 1998-07-14 | 2002-08-29 | Peter Wagner | Arrays devices and methods of use thereof |
US20030138973A1 (en) * | 1998-07-14 | 2003-07-24 | Peter Wagner | Microdevices for screening biomolecules |
US6576478B1 (en) * | 1998-07-14 | 2003-06-10 | Zyomyx, Inc. | Microdevices for high-throughput screening of biomolecules |
US6780582B1 (en) | 1998-07-14 | 2004-08-24 | Zyomyx, Inc. | Arrays of protein-capture agents and methods of use thereof |
US6406921B1 (en) * | 1998-07-14 | 2002-06-18 | Zyomyx, Incorporated | Protein arrays for high-throughput screening |
US7132247B1 (en) * | 1998-09-17 | 2006-11-07 | Regents Of The University Of Minnesota | Composite devices incorporating biological material and methods |
EP1121425B1 (en) * | 1998-10-13 | 2005-06-29 | The University Of Georgia Research Foundation, Inc. | Stabilized bioactive peptides and methods of identification, synthesis and use |
US20030190740A1 (en) | 1998-10-13 | 2003-10-09 | The University Of Georgia Research Foundation, Inc | Stabilized bioactive peptides and methods of identification, synthesis, and use |
JP2002532066A (en) * | 1998-11-16 | 2002-10-02 | ジェンウェイ バイオテック, インコーポレイテッド | Antibody production using polynucleotide vaccines in birds |
US6635311B1 (en) | 1999-01-07 | 2003-10-21 | Northwestern University | Methods utilizing scanning probe microscope tips and products therefor or products thereby |
US6827979B2 (en) | 1999-01-07 | 2004-12-07 | Northwestern University | Methods utilizing scanning probe microscope tips and products therefor or produced thereby |
US7166475B2 (en) * | 1999-02-26 | 2007-01-23 | Cyclacel Ltd. | Compositions and methods for monitoring the modification state of a pair of polypeptides |
CA2365431A1 (en) * | 1999-03-10 | 2000-09-14 | Hui Ge | Universal protein array system |
WO2000056774A1 (en) * | 1999-03-19 | 2000-09-28 | Duke University | Methods of using bioelastomers |
DE19916867A1 (en) * | 1999-04-14 | 2000-10-19 | Fraunhofer Ges Forschung | Making a molecular array on which molecules are immobilized, using micro-compartments or microcapillary reactors on planar substrates with sensor elements employs microelectronic production techniques |
US20040053290A1 (en) * | 2000-01-11 | 2004-03-18 | Terbrueggen Robert Henry | Devices and methods for biochip multiplexing |
US7638464B2 (en) * | 1999-04-26 | 2009-12-29 | Biocept, Inc. | Three dimensional format biochips |
CN1169188C (en) | 1999-04-29 | 2004-09-29 | 赛弗根生物系统股份有限公司 | Sample holder with hydrophobic coating for gas phase mass spectrometers |
JP4493125B2 (en) * | 1999-05-07 | 2010-06-30 | 独立行政法人理化学研究所 | Method for detecting interacting proteins |
US6690399B1 (en) * | 1999-05-07 | 2004-02-10 | Tropix, Inc. | Data display software for displaying assay results |
US6824987B1 (en) * | 1999-05-11 | 2004-11-30 | President And Fellows Of Harvard College | Small molecule printing |
US7932213B2 (en) * | 1999-05-11 | 2011-04-26 | President And Fellows Of Harvard College | Small molecule printing |
US20020042081A1 (en) * | 2000-10-10 | 2002-04-11 | Eric Henderson | Evaluating binding affinities by force stratification and force panning |
US20030073250A1 (en) * | 1999-05-21 | 2003-04-17 | Eric Henderson | Method and apparatus for solid state molecular analysis |
US6573369B2 (en) | 1999-05-21 | 2003-06-03 | Bioforce Nanosciences, Inc. | Method and apparatus for solid state molecular analysis |
US20030186311A1 (en) * | 1999-05-21 | 2003-10-02 | Bioforce Nanosciences, Inc. | Parallel analysis of molecular interactions |
DE60044923D1 (en) * | 1999-05-28 | 2010-10-21 | Yokogawa Electric Corp | Biochip reader |
US7179638B2 (en) * | 1999-07-30 | 2007-02-20 | Large Scale Biology Corporation | Microarrays and their manufacture by slicing |
ATE516492T1 (en) * | 1999-08-13 | 2011-07-15 | Bayer Technology Services Gmbh | DEVICE AND METHOD FOR MULTIANALYTE DETERMINATION |
US8111401B2 (en) * | 1999-11-05 | 2012-02-07 | Robert Magnusson | Guided-mode resonance sensors employing angular, spectral, modal, and polarization diversity for high-precision sensing in compact formats |
US7167615B1 (en) | 1999-11-05 | 2007-01-23 | Board Of Regents, The University Of Texas System | Resonant waveguide-grating filters and sensors and methods for making and using same |
ES2275563T3 (en) * | 1999-11-29 | 2007-06-16 | Unilever N.V. | IMMOBILIZATION OF PROTEINS THROUGH THE USE OF A POLIPEPTIDIC SEGMENT. |
CA2396320A1 (en) * | 2000-01-11 | 2001-07-19 | Maxygen, Inc. | Integrated systems and methods for diversity generation and screening |
WO2001051665A2 (en) | 2000-01-13 | 2001-07-19 | Nanosphere Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US7816098B2 (en) | 2000-01-31 | 2010-10-19 | Sense Proteomic Limited | Methods of making and using a protein array |
GB2361698B (en) * | 2000-01-31 | 2003-06-25 | Sense Proteomic Ltd | Modification of DNA molecules |
WO2001057198A2 (en) * | 2000-01-31 | 2001-08-09 | Sense Proteomic Limited | Methods of generating protein expression arrays and the use thereof in rapid screening |
US20080280771A1 (en) * | 2000-02-08 | 2008-11-13 | Regents Of The University Of Michigan | Protein MicroarraySystem |
US20050230315A1 (en) * | 2003-01-13 | 2005-10-20 | Regents Of The University Of Michigan | Protein microarray system |
CA2401118A1 (en) | 2000-02-23 | 2001-08-30 | Zyomyx, Inc. | Microfluidic devices and methods |
US6897015B2 (en) * | 2000-03-07 | 2005-05-24 | Bioforce Nanosciences, Inc. | Device and method of use for detection and characterization of pathogens and biological materials |
US7102024B1 (en) | 2000-08-01 | 2006-09-05 | Schwartz David A | Functional biopolymer modification reagents and uses thereof |
US6686461B1 (en) | 2000-03-22 | 2004-02-03 | Solulink Bioscience, Inc. | Triphosphate oligonucleotide modification reagents and uses thereof |
US6541071B1 (en) * | 2000-03-23 | 2003-04-01 | Corning Incorporated | Method for fabricating supported bilayer-lipid membranes |
EP1294513A4 (en) | 2000-03-27 | 2005-11-23 | Zyomyx Inc | Site-specific, covalent bioconjugation of proteins |
WO2001075150A2 (en) * | 2000-03-30 | 2001-10-11 | Infineon Technologies Ag | Biosensor, biosensor array, method for producing an electrode of a biosensor, method for producing a biosensor |
US20020045194A1 (en) * | 2000-04-10 | 2002-04-18 | Cravatt Benjamin F. | Proteomic analysis |
US20010041349A1 (en) * | 2000-04-17 | 2001-11-15 | Andrew Patron | Protein expression system arrays and use in biological screening |
DE10020704B4 (en) * | 2000-04-27 | 2006-09-28 | Bioref Gmbh | Biochip for archiving and laboratory medical analysis of biological sample material, process for its production and its use in diagnostic procedures |
CN1654956A (en) * | 2000-05-04 | 2005-08-17 | 耶鲁大学 | High density protein arrays for screening of protein activity |
DE10027397A1 (en) * | 2000-06-02 | 2001-12-13 | Graffinity Pharm Design Gmbh | Surface for the immobilization of ligands |
US7153682B2 (en) * | 2000-06-05 | 2006-12-26 | Chiron Corporation | Microarrays on mirrored substrates for performing proteomic analyses |
ES2281424T3 (en) * | 2000-06-05 | 2007-10-01 | Novartis Vaccines And Diagnostics, Inc. | MICROMATRICES TO CARRY OUT PROTEOMIC ANALYSIS. |
US7148058B2 (en) * | 2000-06-05 | 2006-12-12 | Chiron Corporation | Protein microarrays on mirrored surfaces for performing proteomic analyses |
CA2412361C (en) * | 2000-06-14 | 2011-08-23 | Vistagen, Inc. | Toxicity typing using liver stem cells |
AU2001269906A1 (en) * | 2000-06-19 | 2002-01-02 | Zyomyx, Inc. | Methods for immobilizing polypeptides |
US7892854B2 (en) | 2000-06-21 | 2011-02-22 | Bioarray Solutions, Ltd. | Multianalyte molecular analysis using application-specific random particle arrays |
US9709559B2 (en) | 2000-06-21 | 2017-07-18 | Bioarray Solutions, Ltd. | Multianalyte molecular analysis using application-specific random particle arrays |
US20020115068A1 (en) * | 2000-06-23 | 2002-08-22 | Ian Tomlinson | Matrix screening method |
DE10031587A1 (en) * | 2000-06-29 | 2002-01-10 | Basf Ag | Dosing of microscale suspensions for the production of material samples in combinatorial materials research and their testing |
US7023547B2 (en) * | 2000-07-11 | 2006-04-04 | Maven Technologies, Llc | Apparatus including a biochip for imaging of biological samples and method |
US6602669B2 (en) | 2000-07-11 | 2003-08-05 | Northwestern University | Method of detection by enhancement of silver staining |
US7518724B2 (en) * | 2000-07-11 | 2009-04-14 | Maven Technologies | Image acquisition, processing, and display |
EP1301632A2 (en) * | 2000-07-19 | 2003-04-16 | Pointilliste, Inc. | Nested sorting and high throughput screening |
WO2002010358A2 (en) * | 2000-07-31 | 2002-02-07 | Maxygen, Inc. | Nucleotide incorporating enzymes |
JP2004506201A (en) * | 2000-08-03 | 2004-02-26 | マサチューセッツ・インスティチュート・オブ・テクノロジー | Microarray of functional biomolecules and uses thereof |
US7270730B2 (en) | 2000-08-04 | 2007-09-18 | Essen Instruments, Inc. | High-throughput electrophysiological measurement system |
US7067046B2 (en) | 2000-08-04 | 2006-06-27 | Essen Instruments, Inc. | System for rapid chemical activation in high-throughput electrophysiological measurements |
US6977155B2 (en) | 2000-08-10 | 2005-12-20 | Corning Incorporated | Arrays of biological membranes and methods and use thereof |
US7678539B2 (en) | 2000-08-10 | 2010-03-16 | Corning Incorporated | Arrays of biological membranes and methods and use thereof |
AU2001284899A1 (en) * | 2000-08-11 | 2002-02-25 | Qianjin Hu | Methods and universal monoclonal antibody array |
AU2001284867A1 (en) * | 2000-08-14 | 2002-02-25 | Surface Logix, Inc. | Biomolecule arrays |
AU2002245009B2 (en) * | 2000-08-15 | 2007-05-17 | Bioforce Nanoscience, Inc. | Nanoscale molecular arrayer |
PT1309861E (en) | 2000-08-15 | 2006-10-31 | Discerna Ltd | FUNCTIONAL ARRAYS OF PROTEINS |
JP2004509645A (en) * | 2000-08-17 | 2004-04-02 | センス・プロテオミック・リミテッド | Method |
US7094568B2 (en) * | 2000-08-17 | 2006-08-22 | Sense Proteomic Ltd. | Method for producing proteins tagged at the N- or C-terminus |
EP1184349A1 (en) * | 2000-09-01 | 2002-03-06 | A.S.B.L. Facultes Universitaires Notre-Dame De La Paix | Method for obtaining a surface activation of a solid support for building biochips microarrays |
EP2071481A3 (en) * | 2000-09-14 | 2009-09-02 | Reverse Proteomics Research Institute Co., Ltd | Method system, apparatus and device for discovering and preparing chemical compounds for medical and other uses |
US20040115726A1 (en) * | 2001-09-14 | 2004-06-17 | Renpei Nagashima | Method, system, apparatus and device for discovering and preparing chemical compounds for medical and other uses. |
WO2002023163A1 (en) | 2000-09-15 | 2002-03-21 | California Institute Of Technology | Microfabricated crossflow devices and methods |
GB0022978D0 (en) * | 2000-09-19 | 2000-11-01 | Oxford Glycosciences Uk Ltd | Detection of peptides |
US7354721B2 (en) | 2000-09-22 | 2008-04-08 | Clontech Laboratories, Inc. | Highly sensitive proteomic analysis methods, and kits and systems for practicing the same |
EP1195606A1 (en) * | 2000-10-03 | 2002-04-10 | VBC-Genomics Forschungsges.m.b.H. | Allergen-microarray assay |
US7348182B2 (en) * | 2000-10-03 | 2008-03-25 | Mirari Biosciences, Inc. | Directed microwave chemistry |
US20040209303A1 (en) * | 2000-10-03 | 2004-10-21 | Martin Mark T. | Methods and compositions for directed microwave chemistry |
AU2002211317A1 (en) * | 2000-10-03 | 2002-04-15 | Mirari Biosciences, Inc. | Methods and compositions for directed microwave chemistry |
AU2002239740A1 (en) * | 2000-10-20 | 2002-06-11 | Chad A. Mirkin | Nanolithography methods and products therefor and produced thereby |
EP1409127A2 (en) | 2000-10-24 | 2004-04-21 | Fatemeh Mojtabai | Ordered two- and three-dimensional structures of amphiphilic molecules |
US20050100951A1 (en) * | 2000-10-26 | 2005-05-12 | Biocept, Inc. | 3D format biochips and method of use |
US7306827B2 (en) * | 2000-10-30 | 2007-12-11 | Sru Biosystems, Inc. | Method and machine for replicating holographic gratings on a substrate |
US7023544B2 (en) * | 2000-10-30 | 2006-04-04 | Sru Biosystems, Inc. | Method and instrument for detecting biomolecular interactions |
US7217574B2 (en) * | 2000-10-30 | 2007-05-15 | Sru Biosystems, Inc. | Method and apparatus for biosensor spectral shift detection |
US7101660B2 (en) * | 2000-10-30 | 2006-09-05 | Sru Biosystems, Inc. | Method for producing a colorimetric resonant reflection biosensor on rigid surfaces |
US7371562B2 (en) * | 2000-10-30 | 2008-05-13 | Sru Biosystems, Inc. | Guided mode resonant filter biosensor using a linear grating surface structure |
US20030092075A1 (en) * | 2000-10-30 | 2003-05-15 | Sru Biosystems, Llc | Aldehyde chemical surface activation processes and test methods for colorimetric resonant sensors |
US7615339B2 (en) * | 2000-10-30 | 2009-11-10 | Sru Biosystems, Inc. | Method for producing a colorimetric resonant reflection biosensor on rigid surfaces |
US7153702B2 (en) * | 2000-10-30 | 2006-12-26 | Sru Biosystems, Inc. | Label-free methods for performing assays using a colorimetric resonant reflectance optical biosensor |
US7300803B2 (en) * | 2000-10-30 | 2007-11-27 | Sru Biosystems, Inc. | Label-free methods for performing assays using a colorimetric resonant reflectance optical biosensor |
US7202076B2 (en) * | 2000-10-30 | 2007-04-10 | Sru Biosystems, Inc. | Label-free high-throughput optical technique for detecting biomolecular interactions |
US7118710B2 (en) * | 2000-10-30 | 2006-10-10 | Sru Biosystems, Inc. | Label-free high-throughput optical technique for detecting biomolecular interactions |
US7264973B2 (en) * | 2000-10-30 | 2007-09-04 | Sru Biosystems, Inc. | Label-free methods for performing assays using a colorimetric resonant optical biosensor |
US7875434B2 (en) * | 2000-10-30 | 2011-01-25 | Sru Biosystems, Inc. | Label-free methods for performing assays using a colorimetric resonant reflectance optical biosensor |
US7142296B2 (en) * | 2000-10-30 | 2006-11-28 | Sru Biosystems, Inc. | Method and apparatus for detecting biomolecular interactions |
US7575939B2 (en) | 2000-10-30 | 2009-08-18 | Sru Biosystems, Inc. | Optical detection of label-free biomolecular interactions using microreplicated plastic sensor elements |
US7175980B2 (en) * | 2000-10-30 | 2007-02-13 | Sru Biosystems, Inc. | Method of making a plastic colorimetric resonant biosensor device with liquid handling capabilities |
DE10054055A1 (en) * | 2000-10-31 | 2002-05-23 | Nmi Univ Tuebingen | Methods for analyzing proteins |
US7232109B2 (en) * | 2000-11-06 | 2007-06-19 | California Institute Of Technology | Electrostatic valves for microfluidic devices |
US20050084908A1 (en) * | 2000-11-06 | 2005-04-21 | Chugai Seiyaku Kabushiki Kaisha | Methods for detecting binding of low-molecular-weight compound and its binding partner molecule |
US7374906B2 (en) | 2000-11-08 | 2008-05-20 | Surface Logix, Inc. | Biological assays using gradients formed in microfluidic systems |
US7123764B2 (en) | 2000-11-08 | 2006-10-17 | Surface Logix Inc. | Image processing method for use in analyzing data of a chemotaxis or haptotaxis assay |
US6699665B1 (en) | 2000-11-08 | 2004-03-02 | Surface Logix, Inc. | Multiple array system for integrating bioarrays |
WO2002040998A2 (en) * | 2000-11-17 | 2002-05-23 | Zeptosens Ag | Kit and method for determining multiple analytes |
JP2002153272A (en) * | 2000-11-24 | 2002-05-28 | Inst Of Physical & Chemical Res | Biomolecule microarray |
CA2764288A1 (en) | 2000-11-27 | 2002-05-30 | Intelligent Medical Devices, Inc. | Clinically intelligent diagnostic devices and methods |
US6905816B2 (en) | 2000-11-27 | 2005-06-14 | Intelligent Medical Devices, Inc. | Clinically intelligent diagnostic devices and methods |
US20040053340A1 (en) * | 2000-12-13 | 2004-03-18 | De Haard Johannes Joseph | Protein arrays |
TWI314212B (en) | 2000-12-14 | 2009-09-01 | Stroobant Paul | Differential phage capture proteomics |
FR2818287B1 (en) * | 2000-12-14 | 2003-01-17 | Commissariat Energie Atomique | SOLID SUPPORT FOR THE IMMOBILIZATION OF OLIGONUCLEOTIDES |
US6798521B2 (en) * | 2000-12-29 | 2004-09-28 | Texas Instruments Incorporated | Robust integrated surface plasmon resonance sensor |
AU2002246978A1 (en) * | 2001-01-10 | 2002-07-24 | Symyx Technologies, Inc. | Polymer brushes for immobilizing molecules to a surface |
EP1362126A4 (en) * | 2001-01-18 | 2004-07-21 | Kemmons A Tubbs | An integrated high throughput system for the analysis of biomolecules |
JP4880188B2 (en) * | 2001-01-23 | 2012-02-22 | プレジデント アンド フェロウズ オブ ハーバード カレッジ | Nucleic acid programmed protein array |
US20020169562A1 (en) * | 2001-01-29 | 2002-11-14 | Gregory Stephanopoulos | Defining biological states and related genes, proteins and patterns |
US7332286B2 (en) | 2001-02-02 | 2008-02-19 | University Of Pennsylvania | Peptide or protein microassay method and apparatus |
US7138238B2 (en) * | 2001-02-06 | 2006-11-21 | Auburn University | Ligand sensor devices and uses thereof |
JP4797196B2 (en) | 2001-02-14 | 2011-10-19 | 株式会社 フューエンス | Microchip |
US6913697B2 (en) * | 2001-02-14 | 2005-07-05 | Science & Technology Corporation @ Unm | Nanostructured separation and analysis devices for biological membranes |
US20020165675A1 (en) * | 2001-03-03 | 2002-11-07 | Golovlev Valeri V. | Method and microelectronic device for multi-site molecule detection |
US20040191246A1 (en) | 2003-02-26 | 2004-09-30 | Connelly Patrick R. | Process for in vivo treatment of specific biological targets in bodily fluid |
WO2002071067A1 (en) * | 2001-03-07 | 2002-09-12 | Bio-Rad Laboratories, Inc. | Assay system for simultaneous detection and measurement of multiple modified cellular proteins |
US20020137106A1 (en) * | 2001-03-09 | 2002-09-26 | Ciphergen Biosystems, Inc. | Detection of biological pathway components |
CA2441086A1 (en) * | 2001-03-19 | 2002-09-26 | Wisconsin Alumni Research Foundation | Identification of gene expression alterations underlying the aging process in mammals |
WO2002074979A2 (en) * | 2001-03-20 | 2002-09-26 | Ortho-Clinical Diagnostics, Inc. | Expression profiles and methods of use |
US20060040286A1 (en) * | 2001-03-28 | 2006-02-23 | Nanosphere, Inc. | Bio-barcode based detection of target analytes |
AU2002253995A1 (en) * | 2001-03-29 | 2002-10-15 | Hybrigen, Inc. | Improved hybrid gene libraries and uses thereof |
EP1384022A4 (en) | 2001-04-06 | 2004-08-04 | California Inst Of Techn | Nucleic acid amplification utilizing microfluidic devices |
US20040253634A1 (en) * | 2001-04-10 | 2004-12-16 | Denong Wang | Novel microarrays and methods of use thereof |
AU2002303384A1 (en) * | 2001-04-17 | 2002-10-28 | William J. Dower | Epitope-captured antibody display |
EP1385998A1 (en) * | 2001-04-19 | 2004-02-04 | Ciphergen Biosystems, Inc. | Biomolecule characterization using mass spectrometry and affinity tags |
EP1379545A2 (en) * | 2001-04-19 | 2004-01-14 | Gesellschaft für biotechnologische Forschung mbH (GBF) | Method for producing stable, regeneratable antibody arrays |
US20030054408A1 (en) * | 2001-04-20 | 2003-03-20 | Ramamoorthi Ravi | Methods and systems for identifying proteins |
WO2002088388A1 (en) * | 2001-04-26 | 2002-11-07 | Ruebben Albert | A method and a device for quantification of mutation loads |
JP2004530879A (en) * | 2001-05-03 | 2004-10-07 | シグマ−ジェノシス リミテッド | How to build a protein microarray |
IL158822A0 (en) * | 2001-05-11 | 2004-05-12 | Univ Yale | Global analysis of protein activities using proteome chips |
US20080220441A1 (en) | 2001-05-16 | 2008-09-11 | Birnbaum Eva R | Advanced drug development and manufacturing |
US9157875B2 (en) * | 2001-05-16 | 2015-10-13 | Benjamin P. Warner | Drug development and manufacturing |
US7147687B2 (en) * | 2001-05-25 | 2006-12-12 | Nanosphere, Inc. | Non-alloying core shell nanoparticles |
US7238472B2 (en) * | 2001-05-25 | 2007-07-03 | Nanosphere, Inc. | Non-alloying core shell nanoparticles |
US7262063B2 (en) | 2001-06-21 | 2007-08-28 | Bio Array Solutions, Ltd. | Directed assembly of functional heterostructures |
EP1409733A4 (en) * | 2001-06-26 | 2005-05-25 | Wisconsin Alumni Res Found | Gene expression alterations underlying the retardation of aging by caloric restriction in mammals |
US6844028B2 (en) * | 2001-06-26 | 2005-01-18 | Accelr8 Technology Corporation | Functional surface coating |
AU2002318434B2 (en) * | 2001-06-29 | 2008-05-15 | Proteome Sciences Plc | Methods and compositions for determining the purity of and purifying chemically synthesized nucleic acids |
WO2003058193A2 (en) * | 2001-07-02 | 2003-07-17 | The Board Of Trustees Of The Leland Stanford Junior University | Microarrays for cell phenotyping and manipulation |
US20060019235A1 (en) * | 2001-07-02 | 2006-01-26 | The Board Of Trustees Of The Leland Stanford Junior University | Molecular and functional profiling using a cellular microarray |
CN100386627C (en) * | 2001-07-03 | 2008-05-07 | 包刚 | Filtration-based microarray chip |
US20060073610A1 (en) * | 2001-07-06 | 2006-04-06 | Millpore Corporation | Patterned composite membrane and stenciling method for the manufacture thereof |
WO2003006676A2 (en) * | 2001-07-13 | 2003-01-23 | Nanosphere, Inc. | Method for immobilizing molecules onto surfaces |
US20030013208A1 (en) * | 2001-07-13 | 2003-01-16 | Milagen, Inc. | Information enhanced antibody arrays |
WO2005108625A2 (en) * | 2001-07-13 | 2005-11-17 | Nanosphere, Inc. | Method for preparing substrates having immobilized molecules and substrates |
US7297553B2 (en) * | 2002-05-28 | 2007-11-20 | Nanosphere, Inc. | Method for attachment of silylated molecules to glass surfaces |
DK1410029T3 (en) * | 2001-07-16 | 2008-01-07 | Protein Forest Inc | Arrays of buffers for analysis of biomolecules at their isoelectric point |
ATE421091T1 (en) * | 2001-07-16 | 2009-01-15 | Caprotec Bioanalytics Gmbh | CAUGHT COMPOUNDS, THEIR COLLECTION AND METHODS FOR ANALYZING THE PROTEOME AND COMPLEX COMPOSITIONS |
US7517496B2 (en) * | 2001-07-17 | 2009-04-14 | Bio-Rad Laboratories, Inc. | Latex based adsorbent chip |
WO2003079402A2 (en) * | 2001-07-17 | 2003-09-25 | Ciphergen Biosystems, Inc. | Latex based adsorbent chip |
US7172804B2 (en) * | 2001-07-17 | 2007-02-06 | Northwestern University | Film-immobilized capture particles |
AU2002329606A1 (en) * | 2001-07-17 | 2003-03-03 | Bioforce Nanosciences, Inc. | Combined molecular blinding detection through force microscopy and mass spectrometry |
US6444318B1 (en) * | 2001-07-17 | 2002-09-03 | Surmodics, Inc. | Self assembling monolayer compositions |
US20030143612A1 (en) * | 2001-07-18 | 2003-07-31 | Pointilliste, Inc. | Collections of binding proteins and tags and uses thereof for nested sorting and high throughput screening |
US6977138B2 (en) | 2001-07-24 | 2005-12-20 | Massachusetts Institute Of Technology | Reactive polymer coatings |
EP1279963A1 (en) * | 2001-07-27 | 2003-01-29 | Université de Nantes | Protein-target screening method using near-infrared fluorescent dyes |
EP1281966A3 (en) * | 2001-07-30 | 2003-06-18 | Fuji Photo Film Co., Ltd. | Method and apparatus for conducting a receptor-ligand reaction |
EP1414443B1 (en) * | 2001-08-01 | 2006-11-15 | Merck & Co., Inc. | BENZIMIDAZO 4,5-f|ISOQUINOLINONE DERIVATIVES |
US7172905B2 (en) * | 2001-08-07 | 2007-02-06 | The University Of Chicago | Polypeptide immobilization |
US20100104631A1 (en) * | 2001-08-13 | 2010-04-29 | Lipella Pharmaceuticals Inc. | Method of treatment for bladder dysfunction |
US20030049626A1 (en) * | 2001-08-14 | 2003-03-13 | Milagen, Inc. | Antibody-based analysis of matrix protein arrays |
US20030040129A1 (en) * | 2001-08-20 | 2003-02-27 | Shah Haresh P. | Binding assays using magnetically immobilized arrays |
US6767731B2 (en) * | 2001-08-27 | 2004-07-27 | Intel Corporation | Electron induced fluorescent method for nucleic acid sequencing |
JP2005509737A (en) * | 2001-08-27 | 2005-04-14 | サーフェイス ロジックス,インコーポレイティド | Immobilization of biological molecules on monolayer-coated surfaces |
US7075162B2 (en) * | 2001-08-30 | 2006-07-11 | Fluidigm Corporation | Electrostatic/electrostrictive actuation of elastomer structures using compliant electrodes |
CA2460212C (en) | 2001-09-06 | 2013-01-22 | Genomic Profiling Systems, Inc. | Rapid and sensitive detection of cells and viruses |
US20030068655A1 (en) * | 2001-09-12 | 2003-04-10 | Protiveris, Inc. | Microcantilever apparatus and methods for detection of enzymes |
DE10145226A1 (en) * | 2001-09-13 | 2003-04-10 | Lifebits Ag | Manufacture of carrier-bound molecules |
DE10145700A1 (en) * | 2001-09-17 | 2003-04-10 | Infineon Technologies Ag | Biochip arrangement, sensor arrangement and method for operating a biochip arrangement |
JP2003099614A (en) * | 2001-09-21 | 2003-04-04 | Daiwa Securities Group Inc | Holding number inside sale processor, holding number inside sale processing system and program |
US7042488B2 (en) | 2001-09-27 | 2006-05-09 | Fujinon Corporation | Electronic endoscope for highlighting blood vessel |
WO2003038033A2 (en) * | 2001-10-02 | 2003-05-08 | Northwestern University | Protein and peptide nanoarrays |
US20030073104A1 (en) * | 2001-10-02 | 2003-04-17 | Belcher Angela M. | Nanoscaling ordering of hybrid materials using genetically engineered mesoscale virus |
US7192629B2 (en) * | 2001-10-11 | 2007-03-20 | California Institute Of Technology | Devices utilizing self-assembled gel and method of manufacture |
US20050069962A1 (en) | 2001-10-12 | 2005-03-31 | Archer Robert M | Antibody complexes and methods for immunolabeling |
US8323903B2 (en) * | 2001-10-12 | 2012-12-04 | Life Technologies Corporation | Antibody complexes and methods for immunolabeling |
CN100494395C (en) | 2001-10-15 | 2009-06-03 | 生物芯片技术有限公司 | polymorphic locus multiplexed analysis by concurrent probing and enzyme-mediated detection |
US20050153298A1 (en) * | 2001-10-23 | 2005-07-14 | Gembitsky Dmitry S. | Protein micro-arrays and multi-layered affinity interaction detection |
US8440093B1 (en) | 2001-10-26 | 2013-05-14 | Fuidigm Corporation | Methods and devices for electronic and magnetic sensing of the contents of microfluidic flow channels |
WO2003081202A2 (en) | 2001-11-09 | 2003-10-02 | Nanosphere, Inc. | Bioconjugate-nanoparticle probes |
EP1451579A4 (en) * | 2001-11-19 | 2005-12-28 | Protometrix Inc | Method of using a non-antibody protein to detect and measure an analyte |
DE60232688D1 (en) * | 2001-11-20 | 2009-07-30 | Univ Duke | BOUNDARY-BIOMATERIALS |
US7125669B2 (en) * | 2001-11-27 | 2006-10-24 | Compound Therapeutics, Inc. | Solid-phase immobilization of proteins and peptides |
WO2003048295A1 (en) | 2001-11-30 | 2003-06-12 | Fluidigm Corporation | Microfluidic device and methods of using same |
US7361310B1 (en) | 2001-11-30 | 2008-04-22 | Northwestern University | Direct write nanolithographic deposition of nucleic acids from nanoscopic tips |
US6797393B2 (en) | 2001-11-30 | 2004-09-28 | Eastman Kodak Company | Method for making biochip substrate |
US7691333B2 (en) | 2001-11-30 | 2010-04-06 | Fluidigm Corporation | Microfluidic device and methods of using same |
DK1456668T3 (en) * | 2001-12-05 | 2007-09-10 | Sense Proteomic Ltd | Protein arrays for allelic variants and their applications |
US20050112616A1 (en) * | 2001-12-10 | 2005-05-26 | William Lee | Functionalized materials and libraries thereof |
EP1319954A1 (en) * | 2001-12-12 | 2003-06-18 | Centre National de Genotypage | Methods for protein analysis using protein capture arrays |
JP4382339B2 (en) * | 2001-12-14 | 2009-12-09 | 富士フイルム株式会社 | Measuring chip |
GB0130318D0 (en) * | 2001-12-19 | 2002-02-06 | Univ Leeds | Membrane |
EP1456659B1 (en) * | 2001-12-21 | 2008-03-12 | GE Healthcare Bio-Sciences AB | Immobilization of binding agents |
KR20030057219A (en) * | 2001-12-28 | 2003-07-04 | 삼성에스디아이 주식회사 | Compound forming intermediated layer on matrix, composition for intermediated layer, and biochip using the same |
KR100450202B1 (en) * | 2002-01-07 | 2004-09-24 | 삼성에스디아이 주식회사 | A method for forming a pattern of functional group for immobilizing physiological material |
US20030134433A1 (en) * | 2002-01-16 | 2003-07-17 | Nanomix, Inc. | Electronic sensing of chemical and biological agents using functionalized nanostructures |
US20030157540A1 (en) * | 2002-01-16 | 2003-08-21 | Xianqiang Li | Methods for isolating and characterizing short-lived proteins and arrays derived therefrom |
US20060228723A1 (en) * | 2002-01-16 | 2006-10-12 | Keith Bradley | System and method for electronic sensing of biomolecules |
US20070178477A1 (en) * | 2002-01-16 | 2007-08-02 | Nanomix, Inc. | Nanotube sensor devices for DNA detection |
US7056665B2 (en) | 2002-01-16 | 2006-06-06 | Panomics, Inc. | Screening methods involving the detection of short-lived proteins |
CA2472030A1 (en) * | 2002-01-24 | 2003-07-31 | Pointilliste, Inc. | Use of collections of binding sites for sample profiling and other applications |
JP2003222589A (en) * | 2002-01-31 | 2003-08-08 | Communication Research Laboratory | Dual-wavelength surface plasmon resonance spectroscopic device |
SE0200269D0 (en) * | 2002-01-31 | 2002-01-31 | Ellem Bioteknik Ab | Material for implantation |
CA2474530A1 (en) * | 2002-02-07 | 2003-08-14 | Eastern Virginia Medical School Of The Medical College Of Hampton Roads | Diagnostic microarray and method of use thereof |
WO2003074691A1 (en) * | 2002-03-01 | 2003-09-12 | National Institute Of Advanced Industrial Science And Technology | Immobilized cells and liposomes and method of immobilizing the same |
US20050266455A1 (en) * | 2002-03-02 | 2005-12-01 | Sci Tec, Inc. | Method and microelectronic device for multi-site molecule detection |
US6815078B2 (en) | 2002-03-06 | 2004-11-09 | Eastman Kodak Company | Substrate for protein microarray containing functionalized polymer |
IL163880A0 (en) * | 2002-03-07 | 2005-12-18 | Zephyr Proteomix Ltd | Microarrays of cellulose binding chimeric proteinsand methods of use thereof |
ATE421088T1 (en) * | 2002-03-11 | 2009-01-15 | Caprotec Bioanalytics Gmbh | COMPOUNDS AND METHODS FOR ANALYZING THE PROTEOME |
US6703216B2 (en) | 2002-03-14 | 2004-03-09 | The Regents Of The University Of California | Methods, compositions and apparatuses for detection of gamma-hydroxybutyric acid (GHB) |
US20030228709A1 (en) * | 2002-03-25 | 2003-12-11 | Kozlowski Roland Zbignieiw | Arrays |
CA2480728A1 (en) | 2002-04-01 | 2003-10-16 | Fluidigm Corporation | Microfluidic particle-analysis systems |
US20030215858A1 (en) * | 2002-04-08 | 2003-11-20 | Baylor College Of Medicine | Enhanced gene expression system |
US20030194709A1 (en) * | 2002-04-10 | 2003-10-16 | Xing Yang | Hydrophobic zone device |
US20040033546A1 (en) * | 2002-04-10 | 2004-02-19 | The Trustees Of Columbia University In The City Of New York | Novel microarrays and methods of use thereof |
US7687256B2 (en) * | 2002-04-11 | 2010-03-30 | Spire Corporation | Surface activated biochip |
WO2003102547A2 (en) * | 2002-04-26 | 2003-12-11 | Genencor International, Inc. | Methods and articles for high throughput analysis of biomolecular interactions |
US20040171034A1 (en) | 2002-05-03 | 2004-09-02 | Brian Agnew | Compositions and methods for detection and isolation of phosphorylated molecules |
WO2004042347A2 (en) * | 2002-05-03 | 2004-05-21 | Molecular Probes, Inc. | Compositions and methods for detection and isolation of phosphorylated molecules |
US7445894B2 (en) * | 2002-05-03 | 2008-11-04 | Molecular Probes, Inc. | Compositions and methods for detection and isolation of phosphorylated molecules |
US20030211488A1 (en) * | 2002-05-07 | 2003-11-13 | Northwestern University | Nanoparticle probs with Raman spectrocopic fingerprints for analyte detection |
US20030211478A1 (en) * | 2002-05-08 | 2003-11-13 | Gentel Corporation | Transcription factor profiling on a solid surface |
US20030208936A1 (en) * | 2002-05-09 | 2003-11-13 | Lee Charles Hee | Method for manufacturing embroidery decorated cards and envelopes |
AU2003222676B2 (en) * | 2002-05-10 | 2009-04-23 | Anteo Technologies Pty Ltd | Generation of surface coating diversity |
US7460960B2 (en) * | 2002-05-10 | 2008-12-02 | Epitome Biosystems, Inc. | Proteome epitope tags and methods of use thereof in protein modification analysis |
US20060014212A1 (en) * | 2002-05-10 | 2006-01-19 | Epitome Biosystems, Inc. | Proteome epitope tags and methods of use thereof in protein modification analysis |
EP1532439A4 (en) * | 2002-05-10 | 2006-10-18 | Epitome Biosystems Inc | Unique recognition sequences and methods of use thereof in protein analysis |
US7618788B2 (en) * | 2002-05-10 | 2009-11-17 | Millipore Corporation | Proteome epitope tags and methods of use thereof in protein modification analysis |
DE60329548D1 (en) * | 2002-05-13 | 2009-11-19 | Becton Dickinson Co | Sample plate with distributor attachment |
US8785179B2 (en) * | 2002-05-22 | 2014-07-22 | Texas Instruments Incorporated | Biosensor and method |
US20050239193A1 (en) * | 2002-05-30 | 2005-10-27 | Bioforce Nanosciences, Inc. | Device and method of use for detection and characterization of microorganisms and microparticles |
US20040091498A1 (en) * | 2002-05-31 | 2004-05-13 | Aaron Diamond Aids Research Center | Defensins: use as antiviral agents |
EP1369692A3 (en) * | 2002-06-04 | 2003-12-17 | Interuniversitaire Microelectronica Centrum vzw ( IMEC) | Sensor surface |
EP1413886A1 (en) * | 2002-10-25 | 2004-04-28 | Interuniversitair Microelektronica Centrum ( Imec) | Sensor surface |
US7135343B2 (en) * | 2002-06-17 | 2006-11-14 | Agilent Technologies, Inc. | Biomolecule resistant and their methods of use in assays |
CA2490522A1 (en) * | 2002-06-20 | 2003-12-31 | Paul Stroobant | Improved methods for performing differential capture proteomics |
US7948041B2 (en) | 2005-05-19 | 2011-05-24 | Nanomix, Inc. | Sensor having a thin-film inhibition layer |
JP2005532456A (en) * | 2002-07-02 | 2005-10-27 | ナノスフェアー インコーポレイテッド | Nanoparticle polyanion complex and its use in the detection of analytes |
USH2223H1 (en) * | 2002-07-11 | 2008-09-02 | The United States Of America As Represented By The Secretary Of The Navy | Patterned, micrometer-sized antibody features |
US20040009528A1 (en) * | 2002-07-11 | 2004-01-15 | Shyh-Yu Shaw | Protein chips |
US20050008828A1 (en) * | 2002-07-25 | 2005-01-13 | Trustees Of Stevens Institute Of Technology | Patterned polymer microgel and method of forming same |
JP4099822B2 (en) * | 2002-07-26 | 2008-06-11 | セイコーエプソン株式会社 | Dispensing device, dispensing method, and biological sample-containing solution ejection failure detection method |
CA2833718C (en) * | 2002-07-29 | 2017-01-03 | Applied Biomimetic A/S | Biomimetic membrane suitable for use in a solar cell |
US20040067597A1 (en) * | 2002-07-31 | 2004-04-08 | Caliper Technologies Corp. | High density reagent array preparation methods |
US20050074898A1 (en) * | 2002-07-31 | 2005-04-07 | Caliper Technologies Corp. | High density reagent array preparation methods |
EP1540344A4 (en) * | 2002-08-02 | 2007-02-21 | Applera Corp | Fluorescence polarization assay |
AU2003257109A1 (en) * | 2002-08-05 | 2004-02-23 | Invitrogen Corporation | Compositions and methods for molecular biology |
EP1535042A2 (en) * | 2002-08-13 | 2005-06-01 | Discovery Partners International | Spotting pattern for placement of compounds in an array |
GB2391867A (en) * | 2002-08-13 | 2004-02-18 | Smartbead Technologies Ltd | Analysis system using coded supports |
WO2004016802A2 (en) * | 2002-08-15 | 2004-02-26 | Proteoplex, Inc. | Methods and apparatus for preparing and assaying biological samples to determine protein concentration |
WO2004017374A2 (en) * | 2002-08-16 | 2004-02-26 | Clinical Microarrays, Inc. | Reading of fluorescent arrays |
US20050233473A1 (en) * | 2002-08-16 | 2005-10-20 | Zyomyx, Inc. | Methods and reagents for surface functionalization |
EP1537202A4 (en) * | 2002-08-16 | 2007-09-19 | Zyomyx Inc | Methods and reagents for surface functionalization |
US7384742B2 (en) * | 2002-08-16 | 2008-06-10 | Decision Biomarkers, Inc. | Substrates for isolating reacting and microscopically analyzing materials |
WO2004020065A2 (en) * | 2002-08-28 | 2004-03-11 | Mt Technologies, Inc. | Microfluidic affinity system using polydimethylsiloxane and a surface modification process |
WO2004038354A2 (en) * | 2002-08-29 | 2004-05-06 | Bioscale, Inc. | Resonant sensor and sensing system |
US20040043508A1 (en) * | 2002-09-03 | 2004-03-04 | Frutos Anthony G. | Polymer-coated substrates for binding biological molecules |
EP1556695B1 (en) * | 2002-09-03 | 2011-08-10 | Bayer Technology Services GmbH | Analytical platform and identification method with analytes, which are to be identified in a sample optionally after fractionation and which serve as immobilized specific binding partners |
JP2005537486A (en) * | 2002-09-03 | 2005-12-08 | ツェプトゼンス アクチエンゲゼルシャフト | Analytical platform and detection method in which analyte is measured as a specific binding partner immobilized in a sample |
US7429492B2 (en) * | 2002-09-09 | 2008-09-30 | Sru Biosystems, Inc. | Multiwell plates with integrated biosensors and membranes |
US7927822B2 (en) | 2002-09-09 | 2011-04-19 | Sru Biosystems, Inc. | Methods for screening cells and antibodies |
JP4426968B2 (en) * | 2002-09-17 | 2010-03-03 | オリンパス株式会社 | Method and apparatus for disposing a liquid reaction component on the surface of a substrate to detect a target material by reaction between a plurality of components on the substrate, and an article for use in this method |
JP2004105070A (en) * | 2002-09-18 | 2004-04-08 | Inst Of Physical & Chemical Res | Method for producing ligand-bonded protein using cell-free protein synthetic system and use thereof |
US20050064508A1 (en) | 2003-09-22 | 2005-03-24 | Semzyme | Peptide mediated synthesis of metallic and magnetic materials |
WO2004027379A2 (en) * | 2002-09-20 | 2004-04-01 | Novus Molecular, Inc. | Methods and devices for active bioassay |
EP1551753A2 (en) | 2002-09-25 | 2005-07-13 | California Institute Of Technology | Microfluidic large scale integration |
JP5695287B2 (en) | 2002-10-02 | 2015-04-01 | カリフォルニア インスティテュート オブ テクノロジー | Nucleic acid analysis of microfluids |
US7252954B2 (en) * | 2002-10-15 | 2007-08-07 | Abmetrix, Inc. | Sets of digital antibodies directed against short epitopes, and methods using same |
US20040137526A1 (en) * | 2002-10-15 | 2004-07-15 | The Regents Of The University Of Michigan | Multidimensional protein separation system |
DE10249608A1 (en) * | 2002-10-18 | 2004-05-06 | Gkss-Forschungszentrum Geesthacht Gmbh | Device and method for structural analysis and detection of complex glycostructures |
DE60322742D1 (en) * | 2002-10-25 | 2008-09-18 | Sense Proteomic Ltd | ENZYMARRAY AND ENZYMTEST |
US20040081969A1 (en) * | 2002-10-29 | 2004-04-29 | Ilsley Diane D. | Devices and methods for evaulating the quality of a sample for use in an array assay |
WO2004042019A2 (en) * | 2002-10-30 | 2004-05-21 | Pointilliste, Inc. | Systems for capture and analysis of biological particles and methods using the systems |
US20030153013A1 (en) * | 2002-11-07 | 2003-08-14 | Ruo-Pan Huang | Antibody-based protein array system |
CA2545304A1 (en) * | 2002-11-08 | 2004-05-21 | University Of Copenhagen | Method of immobilising a protein to a zeolite |
WO2004047007A1 (en) | 2002-11-15 | 2004-06-03 | Bioarray Solutions, Ltd. | Analysis, secure access to, and transmission of array images |
US20040096914A1 (en) * | 2002-11-20 | 2004-05-20 | Ye Fang | Substrates with stable surface chemistry for biological membrane arrays and methods for fabricating thereof |
WO2004053460A2 (en) * | 2002-12-11 | 2004-06-24 | New England Biolabs, Inc. | Carrier-ligand fusions and uses thereof |
US20050059030A1 (en) * | 2002-12-12 | 2005-03-17 | Nanosphere, Inc. | Direct SNP detection with unamplified DNA |
EP1570043B1 (en) * | 2002-12-12 | 2013-07-24 | Novartis Vaccines and Diagnostics, Inc. | Device and method for in-line blood testing using biochips |
EP1573044A4 (en) | 2002-12-18 | 2006-07-05 | Ciphergen Biosystems Inc | Serum biomarkers in lung cancer |
US20040121339A1 (en) * | 2002-12-19 | 2004-06-24 | Jizhong Zhou | Special film-coated substrate for bio-microarray fabrication and use thereof |
US7642085B2 (en) * | 2002-12-22 | 2010-01-05 | The Scripps Research Institute | Protein arrays |
US7785601B2 (en) | 2002-12-31 | 2010-08-31 | Sygnis Bioscience Gmbh & Co. Kg | Methods of treating neurological conditions with hematopoietic growth factors |
US7695723B2 (en) | 2002-12-31 | 2010-04-13 | Sygnis Bioscience Gmbh & Co. Kg | Methods of treating neurological conditions with hematopoietic growth factors |
WO2004060044A2 (en) * | 2003-01-02 | 2004-07-22 | Bioforce Nanosciences, Inc. | Method and apparatus for molecular analysis in small sample volumes |
KR100523212B1 (en) * | 2003-01-04 | 2005-10-24 | 한국과학기술원 | A Protein Chip for Analyzing Interaction Between Protein and Substrate Peptide Therefor |
US7736909B2 (en) * | 2003-01-09 | 2010-06-15 | Board Of Regents, The University Of Texas System | Methods and compositions comprising capture agents |
DE602004026157D1 (en) * | 2003-01-10 | 2010-05-06 | Protein Crystal Co Ltd | PROTEIN COMPLEX, METHOD FOR THE PRODUCTION AND USE THEREOF |
US7422865B2 (en) * | 2003-01-13 | 2008-09-09 | Agilent Technologies, Inc. | Method of identifying peptides in a proteomic sample |
US20040137158A1 (en) * | 2003-01-15 | 2004-07-15 | Kools Jacques Constant Stefan | Method for preparing a noble metal surface |
US20060051879A9 (en) * | 2003-01-16 | 2006-03-09 | Hubert Koster | Capture compounds, collections thereof and methods for analyzing the proteome and complex compositions |
KR100994566B1 (en) * | 2003-01-20 | 2010-11-15 | 삼성전자주식회사 | An array device comprising a photoresist film having immobilization regions and a method using the same |
AU2003290561A1 (en) * | 2003-02-10 | 2004-09-06 | Dana Ault-Riche | Self-assembling arrays and uses thereof |
US20050008851A1 (en) * | 2003-02-18 | 2005-01-13 | Fuji Photo Film Co., Ltd. | Biosensor |
ATE418146T1 (en) * | 2003-02-25 | 2009-01-15 | Yeda Res & Dev | NANOSCOPIC STRUCTURE AND DEVICE USING THE SAME |
JP2007524347A (en) * | 2003-02-27 | 2007-08-30 | ナノスフェアー インコーポレイテッド | Label-free gene expression profiling using universal nanoparticle probes in microarray format assays |
CN1514243A (en) * | 2003-04-30 | 2004-07-21 | 成都夸常科技有限公司 | Method of preceeding qualitative and lor quantitative analysis against target substance its device and marker and detecting reagent box |
EP1606419A1 (en) | 2003-03-18 | 2005-12-21 | Quantum Genetics Ireland Limited | Systems and methods for improving protein and milk production of dairy herds |
US20040185445A1 (en) * | 2003-03-19 | 2004-09-23 | Ye Fang | Universal readout for target identification using biological microarrays |
US7191068B2 (en) | 2003-03-25 | 2007-03-13 | Proteogenix, Inc. | Proteomic analysis of biological fluids |
US8068990B2 (en) * | 2003-03-25 | 2011-11-29 | Hologic, Inc. | Diagnosis of intra-uterine infection by proteomic analysis of cervical-vaginal fluids |
WO2004087323A1 (en) * | 2003-03-28 | 2004-10-14 | Mergen Ltd. | Multi-array systems and methods of use thereof |
US20040197841A1 (en) * | 2003-04-02 | 2004-10-07 | Apffel James Alexander | Methods and reagents for multiplexed analyses |
JP5419248B2 (en) | 2003-04-03 | 2014-02-19 | フルイディグム コーポレイション | Microfluidic device and method of use thereof |
US8828663B2 (en) | 2005-03-18 | 2014-09-09 | Fluidigm Corporation | Thermal reaction device and method for using the same |
US20050145496A1 (en) | 2003-04-03 | 2005-07-07 | Federico Goodsaid | Thermal reaction device and method for using the same |
US7476363B2 (en) | 2003-04-03 | 2009-01-13 | Fluidigm Corporation | Microfluidic devices and methods of using same |
US7604965B2 (en) | 2003-04-03 | 2009-10-20 | Fluidigm Corporation | Thermal reaction device and method for using the same |
US20040248205A1 (en) * | 2003-04-16 | 2004-12-09 | Stern Lawrence J. | Major histocompatibility complex (MHC)-peptide arrays |
EP1616181B1 (en) | 2003-04-17 | 2009-08-12 | Vermillion, Inc. | Polypeptides related to natriuretic peptides and methods of their identification and use |
WO2004094989A2 (en) * | 2003-04-22 | 2004-11-04 | Ciphergen Biosystems, Inc. | Methods of host cell protein analysis |
WO2004097368A2 (en) * | 2003-04-28 | 2004-11-11 | Ciphergen Biosystems, Inc. | Improved immunoassays |
US7425700B2 (en) | 2003-05-22 | 2008-09-16 | Stults John T | Systems and methods for discovery and analysis of markers |
KR100547015B1 (en) * | 2003-05-23 | 2006-01-26 | 주식회사 올메디쿠스 | Biosensor for analyzing quantitatively analyte with a predetermined size and larger than, and manufacturing method thereof |
US20050250094A1 (en) * | 2003-05-30 | 2005-11-10 | Nanosphere, Inc. | Method for detecting analytes based on evanescent illumination and scatter-based detection of nanoparticle probe complexes |
US20040248323A1 (en) * | 2003-06-09 | 2004-12-09 | Protometrix, Inc. | Methods for conducting assays for enzyme activity on protein microarrays |
KR20060019589A (en) * | 2003-06-10 | 2006-03-03 | 가부시키가이샤 시마즈세이사쿠쇼 | Extract from cultured mammalian cell, process for preparation thereof and method of cell-free protein synthesis using the extract |
WO2004113872A2 (en) * | 2003-06-24 | 2004-12-29 | The Trustees Of Columbia University In The City Of New York | Covalent methods for immobilization of thiolated biomolecules on siliceous and metallic surfaces |
US20040265921A1 (en) * | 2003-06-30 | 2004-12-30 | National University Of Singapore | Intein-mediated attachment of ligands to proteins for immobilization onto a support |
US9243275B1 (en) * | 2003-07-10 | 2016-01-26 | Polytechnic Institute Of New York University | Biosensor and method of making same |
WO2005007677A2 (en) * | 2003-07-10 | 2005-01-27 | The Institute For Systems Biology | Affinity capture of peptides by microarray and related methods |
EP1660858A4 (en) * | 2003-07-21 | 2007-10-24 | Amplified Proteomics Inc | Multiplexed analyte detection |
US20060014003A1 (en) * | 2003-07-24 | 2006-01-19 | Libera Matthew R | Functional nano-scale gels |
US20050059024A1 (en) | 2003-07-25 | 2005-03-17 | Ambion, Inc. | Methods and compositions for isolating small RNA molecules |
ATE554166T1 (en) | 2003-07-25 | 2012-05-15 | Life Technologies Corp | METHOD AND COMPOSITIONS FOR PRODUCING RNA FROM A FIXED SAMPLE |
JP2007511738A (en) | 2003-08-08 | 2007-05-10 | ジーンニュース インコーポレーテッド | Biomarkers for osteoarthritis and uses thereof |
WO2005014805A1 (en) * | 2003-08-08 | 2005-02-17 | Regents Of The University Of Minnesota | A structured material for the production of hydrogen |
US7413712B2 (en) | 2003-08-11 | 2008-08-19 | California Institute Of Technology | Microfluidic rotary flow reactor matrix |
US7223609B2 (en) * | 2003-08-14 | 2007-05-29 | Agilent Technologies, Inc. | Arrays for multiplexed surface plasmon resonance detection of biological molecules |
CN1864068B (en) * | 2003-08-18 | 2010-10-13 | 特提斯生物科学公司 | Methods for reducing complexity of a sample using small epitope antibodies |
US7754497B2 (en) * | 2003-08-29 | 2010-07-13 | Reverse Proteomics Research Institute Co., Ltd. | Method for immobilizing proteins |
EP1668364A4 (en) | 2003-09-03 | 2012-06-06 | Zyomix Inc | Ion detection using a pillar chip |
US20050176026A1 (en) * | 2003-09-05 | 2005-08-11 | Franck Carl P. | Liquid mixing reactor for biochemical assays |
US20050053949A1 (en) * | 2003-09-08 | 2005-03-10 | Silin Vitalii Ivanovich | Biochip for proteomics applications |
US20050059083A1 (en) * | 2003-09-15 | 2005-03-17 | Becton Dickinson And Company | High throughput method to identify ligands for cell attachment |
US7927796B2 (en) | 2003-09-18 | 2011-04-19 | Bioarray Solutions, Ltd. | Number coding for identification of subtypes of coded types of solid phase carriers |
US9492820B2 (en) | 2003-09-19 | 2016-11-15 | Applied Biosystems, Llc | High density plate filler |
US7998435B2 (en) | 2003-09-19 | 2011-08-16 | Life Technologies Corporation | High density plate filler |
US7695688B2 (en) * | 2003-09-19 | 2010-04-13 | Applied Biosystems, Llc | High density plate filler |
US7407630B2 (en) | 2003-09-19 | 2008-08-05 | Applera Corporation | High density plate filler |
KR100518953B1 (en) * | 2003-09-19 | 2005-10-12 | 주식회사 제노포커스 | Method for Whole Surrounding Surface Display of Target Proteins Using Exosporium of Bacillus cereus Group |
US8277760B2 (en) | 2003-09-19 | 2012-10-02 | Applied Biosystems, Llc | High density plate filler |
US8298780B2 (en) * | 2003-09-22 | 2012-10-30 | X-Body, Inc. | Methods of detection of changes in cells |
EP1664722B1 (en) | 2003-09-22 | 2011-11-02 | Bioarray Solutions Ltd | Surface immobilized polyelectrolyte with multiple functional groups capable of covalently bonding to biomolecules |
US20070017870A1 (en) | 2003-09-30 | 2007-01-25 | Belov Yuri P | Multicapillary device for sample preparation |
EP1677886A1 (en) * | 2003-09-30 | 2006-07-12 | Chromba, Inc. | Multicapillary column for chromatography and sample preparation |
US20050069462A1 (en) * | 2003-09-30 | 2005-03-31 | International Business Machines Corporation | Microfluidics Packaging |
US20050069949A1 (en) * | 2003-09-30 | 2005-03-31 | International Business Machines Corporation | Microfabricated Fluidic Structures |
US20050079507A1 (en) * | 2003-10-09 | 2005-04-14 | Ye Fang | Target evaluation using biological membrane arrays |
WO2005047851A2 (en) * | 2003-10-15 | 2005-05-26 | The Trustees Of Columbia University In The City Of New York | Device for measuring nanometer level pattern-dependent binding reactions |
US20050084981A1 (en) * | 2003-10-16 | 2005-04-21 | Magdalena Ostrowski | Method of depositing a bioactive material on a substrate |
US20050112650A1 (en) * | 2003-10-20 | 2005-05-26 | Ciphergen Biosystems, Inc. | Reactive polyurethane-based polymers |
WO2005040800A1 (en) * | 2003-10-23 | 2005-05-06 | Consejo Superior De Investigaciones Científicas | Method for producing and using a new protein array, said protein array and the applications thereof |
JP2006514299A (en) * | 2003-10-27 | 2006-04-27 | センス プロテオミック リミテッド | Enzyme arrays and assays |
EP1692298A4 (en) | 2003-10-28 | 2008-08-13 | Bioarray Solutions Ltd | Optimization of gene expression analysis using immobilized capture probes |
PT1694859E (en) | 2003-10-29 | 2015-04-13 | Bioarray Solutions Ltd | Multiplexed nucleic acid analysis by fragmentation of double-stranded dna |
US20050095648A1 (en) * | 2003-10-30 | 2005-05-05 | Mario Geysen | Method for designing linear epitopes and algorithm therefor and polypeptide epitopes |
JP2007510928A (en) * | 2003-11-06 | 2007-04-26 | エス アール ユー バイオシステムズ,インコーポレイテッド | High density amine functionalized surface |
EP1694816B1 (en) | 2003-11-07 | 2013-08-28 | Ciphergen Biosystems, Inc. | Biomarkers for alzheimer's disease |
US20060007515A1 (en) * | 2003-11-13 | 2006-01-12 | Dmitri Simonian | Surface lubrication in microstructures |
US7153896B2 (en) | 2003-11-14 | 2006-12-26 | Eastman Kodak Company | Element for protein microarrays |
US7332355B2 (en) | 2003-11-18 | 2008-02-19 | California Institute Of Technology | Method and compositions for the detection of protein glycosylation |
US7754500B2 (en) | 2003-11-21 | 2010-07-13 | Anp Technologies, Inc. | Asymmetrically branched polymer conjugates and microarray assays |
US20090246889A1 (en) * | 2003-11-22 | 2009-10-01 | Samsung Electronics Co., Ltd. | Substrate having oxide layer, method for detecting target material using the substrate, and optical sensor including the substrate |
KR100580631B1 (en) * | 2003-11-22 | 2006-05-16 | 삼성전자주식회사 | A substrate having an oxide layer, method for detecting a target substance using the same and optical sensor containing the same |
US20050109622A1 (en) * | 2003-11-26 | 2005-05-26 | Peter Peumans | Method for controlling electrodeposition of an entity and devices incorporating the immobilized entity |
WO2005055812A2 (en) | 2003-12-05 | 2005-06-23 | Ciphergen Biosystems, Inc. | Serum biomarkers for chagas disease |
WO2005057462A1 (en) * | 2003-12-12 | 2005-06-23 | Bio-Layer Pty Limited | A method for designing surfaces |
WO2005059552A1 (en) | 2003-12-15 | 2005-06-30 | University Of Pennsylvania | Method and devices for running reactions on a target plate for maldi mass spectrometry |
US20050170445A1 (en) * | 2004-01-07 | 2005-08-04 | Duke University | Methods of establishing profiles for use in evaluating wound healing and biocompatibility of implant materials and microarrays useful therefor |
WO2005067980A2 (en) * | 2004-01-12 | 2005-07-28 | Pointilliste, Inc. | Design of therapeutics and therapeutics |
JP2005204609A (en) * | 2004-01-26 | 2005-08-04 | Canon Inc | Kit for immobilizing organic material, structure in which organic material is immobilized and method for producing the same structure |
US20050208597A1 (en) * | 2004-01-26 | 2005-09-22 | The Board Of Trustees Of The Leland Stanford Junior University | Microarray analysis of post-translational modifications |
EP1562046A1 (en) * | 2004-02-03 | 2005-08-10 | B.R.A.H.M.S Aktiengesellschaft | Method of diagnosing sepsis by detecting selectively the concentration of superoxide dismutase 1 (SOD-1) in samples |
MXPA06009452A (en) | 2004-02-19 | 2007-03-15 | Univ Alberta | Leptin promoter polymorphisms and uses thereof. |
JP2005269902A (en) * | 2004-03-22 | 2005-10-06 | Seiko Epson Corp | Method for immobilizing cell on solid-phase surface |
US7276283B2 (en) * | 2004-03-24 | 2007-10-02 | Wisconsin Alumni Research Foundation | Plasma-enhanced functionalization of carbon-containing substrates |
US7723126B2 (en) * | 2004-03-24 | 2010-05-25 | Wisconsin Alumni Research Foundation | Plasma-enhanced functionalization of inorganic oxide surfaces |
CN100357738C (en) * | 2004-03-26 | 2007-12-26 | 博奥生物有限公司 | Method of detecting small molecule compound and its special biochip |
JP2005312425A (en) * | 2004-03-31 | 2005-11-10 | Toyo Kohan Co Ltd | Method for immobilizing polypeptide and solid support having polypeptide immobilized thereon, method for detection and purification of polypeptide using the same and solid support for immobilizing polypeptide |
US7371331B2 (en) * | 2004-04-01 | 2008-05-13 | Valerie J Marty | Method of creating a patterned monolayer on a surface |
JP4451193B2 (en) * | 2004-04-12 | 2010-04-14 | 大日本印刷株式会社 | Method for producing pattern forming body |
EP1737982A4 (en) * | 2004-04-14 | 2009-09-23 | Harvard College | Nucleic-acid programmable protein arrays |
EP1745149A4 (en) | 2004-04-15 | 2008-08-06 | Univ Florida | Neural proteins as biomarkers for nervous system injury and other neural disorders |
BRPI0510266A (en) | 2004-04-26 | 2007-10-30 | Childrens Medical Center | methods for detecting an angiogenic disease or disorder in an individual, for detecting cancer in an individual, for treating an individual affected with an angiogenic disease or disorder, for determining the likelihood of efficacy of an anti-angiogenic therapy , to determine the efficacy of a test therapy in modulating the levels of angiogenic platelet regulators, to create a platelet record or profile for an angiogenic disease or disorder, and to monitor the efficacy of a therapy in an individual with an angiogenic disease or disorder |
EP1742054A4 (en) * | 2004-04-28 | 2008-01-16 | Japan Science & Tech Agency | Biochip producing method, biochip, biochip analyzing device, biochip analyzing method |
NZ551782A (en) | 2004-06-03 | 2010-03-26 | Athlomics Pty Ltd | Agents and methods for diagnosing stress |
US20060251795A1 (en) * | 2005-05-05 | 2006-11-09 | Boris Kobrin | Controlled vapor deposition of biocompatible coatings for medical devices |
DE102004031258A1 (en) * | 2004-06-29 | 2006-02-09 | Jennissen, Herbert P., Prof. Dr. | Protein hybrids with polyhydroxyaromatic amino acid epitopes |
JP4897676B2 (en) | 2004-07-02 | 2012-03-14 | バイオ‐レイヤー ピーティーワイ リミティッド | How to use metal complexes |
US20060009623A1 (en) * | 2004-07-06 | 2006-01-12 | National University Of Singapore | C-terminal attachment of ligands to proteins for immobilization onto a support |
US20060014155A1 (en) * | 2004-07-16 | 2006-01-19 | Wisconsin Alumni Research Foundation | Methods for the production of sensor arrays using electrically addressable electrodes |
WO2006007664A1 (en) * | 2004-07-22 | 2006-01-26 | Genomics Research Partners Pty Ltd | Agents and methods for diagnosing osteoarthritis |
WO2006015365A1 (en) * | 2004-07-30 | 2006-02-09 | Mount Sinai School Of Medicine Of New York University | Npc1l1 and npc1l1 inhibitors and methods of use thereof |
US7848889B2 (en) | 2004-08-02 | 2010-12-07 | Bioarray Solutions, Ltd. | Automated analysis of multiplexed probe-target interaction patterns: pattern matching and allele identification |
AU2005269227A1 (en) * | 2004-08-04 | 2006-02-09 | Axela Inc. | Patterned surfaces with chemical crosslinkers for use in diffraction-based sensing |
US20060040377A1 (en) * | 2004-08-17 | 2006-02-23 | Biocept, Inc. | Protein microarrays |
US20130040840A1 (en) * | 2004-09-02 | 2013-02-14 | Bioarray Solutions, Ltd. | Nucleic acid amplification with integrated multiplex detection |
US20060052948A1 (en) * | 2004-09-09 | 2006-03-09 | Jorn Gorlach | Method of identifying drugs, targeting moieties or diagnostics |
US20060051348A1 (en) * | 2004-09-09 | 2006-03-09 | Jorn Gorlach | Method of producing a plurality of isolated antibodies to a plurality of cognate antigens |
US20060094039A1 (en) * | 2004-09-20 | 2006-05-04 | Ron Rosenfeld | Diagnosis of fetal aneuploidy |
ITVR20040149A1 (en) * | 2004-09-22 | 2004-12-22 | Sanitaria Scaligera Spa | RAPID MONITORING SYSTEM OF THE BLOOD GROUP AND FOR THE DETECTION OF IMMUNOHEMATOLOGICAL REACTIONS |
US7592139B2 (en) * | 2004-09-24 | 2009-09-22 | Sandia National Laboratories | High temperature flow-through device for rapid solubilization and analysis |
JP2006095452A (en) * | 2004-09-30 | 2006-04-13 | Fuji Photo Film Co Ltd | Spin coat film forming method |
SE0402476D0 (en) * | 2004-10-13 | 2004-10-13 | Biacore Ab | Preparation and use of a reactive solid support surface |
WO2006041392A1 (en) * | 2004-10-13 | 2006-04-20 | Biacore Ab | Preparation and use of a reactive solid support surface |
WO2006047417A2 (en) | 2004-10-21 | 2006-05-04 | University Of Florida Research Foundation, Inc. | Detection of cannabinoid receptor biomarkers and uses thereof |
US20110077164A1 (en) * | 2004-10-23 | 2011-03-31 | Andras Guttman | Expression profiling platform technology |
WO2006043179A1 (en) * | 2004-10-23 | 2006-04-27 | Biosystems International Sas | Expression profiling platform technology |
WO2006049498A1 (en) * | 2004-11-05 | 2006-05-11 | Modiquest B.V. | Means and methods for isolating and/or identifying a target molecule |
US20060246467A1 (en) * | 2004-11-15 | 2006-11-02 | California Institute Of Technology | Biomarker sensors and method for multi-color imaging and processing of single-molecule life signatures |
US20060223195A1 (en) * | 2004-11-16 | 2006-10-05 | Meyer Grant D | Stress based removal of nonspecific binding from surfaces |
ATE390562T1 (en) * | 2004-11-19 | 2008-04-15 | Ebm Papst St Georgen Gmbh & Co | ARRANGEMENT WITH A FAN AND A PUMP |
US7745143B2 (en) * | 2004-11-19 | 2010-06-29 | Plexera, Llc | Plasmon resonance biosensor and method |
WO2006062427A1 (en) * | 2004-11-24 | 2006-06-15 | Institut Molekulyarnoi Biologii Im. V.A.Engelgardta Rossiiskoi Akademii Nauk | Method for quantitatively detecting biological toxins |
WO2006056490A1 (en) * | 2004-11-29 | 2006-06-01 | Centre National De La Recherche Scientifique | Trichloro silyl alkyl isocyanate molecule and surface modified mineral substrate |
WO2006063174A2 (en) * | 2004-12-08 | 2006-06-15 | Lyotropic Therapeutics, Inc. | Compositions for binding to assay substrata and methods of using |
US20080003599A1 (en) * | 2004-12-28 | 2008-01-03 | Dary Ekaterina L | Biological Microchip for Multiple Parallel Immunoassay of Compounds and Immunoassay Metods Using Said Microchip |
EP1838867B1 (en) * | 2005-01-06 | 2011-03-09 | Eastern Virginia Medical School | Apolipoprotein a-ii isoform as a biomarker for prostate cancer |
JP4736439B2 (en) * | 2005-01-25 | 2011-07-27 | 東レ株式会社 | Nucleic acid immobilization carrier |
JP2008529008A (en) | 2005-01-28 | 2008-07-31 | チルドレンズ メディカル センター コーポレイション | Methods for diagnosis and prognosis of epithelial cancer |
US7396689B2 (en) * | 2005-02-04 | 2008-07-08 | Decision Biomarkers Incorporated | Method of adjusting the working range of a multi-analyte assay |
EP2520669A3 (en) | 2005-02-07 | 2013-02-27 | GeneNews Inc. | Mild osteoathritis biomarkers and uses thereof |
JP2006335912A (en) * | 2005-06-03 | 2006-12-14 | Fujifilm Holdings Corp | Immobilizing agent for physiologically active substance |
EP1696235B1 (en) * | 2005-02-23 | 2009-10-28 | FUJIFILM Corporation | Biosensor |
US20060234265A1 (en) * | 2005-03-21 | 2006-10-19 | Jim Richey | Microarrays having multi-functional, compartmentalized analysis areas and methods of use |
JP4435709B2 (en) * | 2005-03-22 | 2010-03-24 | 富士フイルム株式会社 | Biosensor |
WO2006110314A2 (en) | 2005-03-25 | 2006-10-19 | Ambion, Inc. | Methods and compositions for depleting abundant rna transcripts |
US8048638B2 (en) | 2005-04-01 | 2011-11-01 | University Of Florida Research Foundation, Inc. | Biomarkers of liver injury |
AU2006232370B2 (en) | 2005-04-01 | 2011-10-06 | Banyan Biomarkers | Biomakers of liver injury |
WO2006113785A2 (en) * | 2005-04-18 | 2006-10-26 | Brigham Young University | Laser modification and functionalization of substrates |
DK1877773T3 (en) * | 2005-04-26 | 2015-01-19 | Bayer Ip Gmbh | NEW APPARATUS AND PROCEDURE FOR THE COATING OF CARRIER SUBSTRATES FOR ANALYTICAL DETECTION BY THE AFFINITY DETECTION PROCEDURE |
DE602006019709D1 (en) | 2005-05-02 | 2011-03-03 | Anp Technologies Inc | |
US7648844B2 (en) * | 2005-05-02 | 2010-01-19 | Bioscale, Inc. | Method and apparatus for detection of analyte using an acoustic device |
US7611908B2 (en) * | 2005-05-02 | 2009-11-03 | Bioscale, Inc. | Method and apparatus for therapeutic drug monitoring using an acoustic device |
US7300631B2 (en) * | 2005-05-02 | 2007-11-27 | Bioscale, Inc. | Method and apparatus for detection of analyte using a flexural plate wave device and magnetic particles |
US7749445B2 (en) * | 2005-05-02 | 2010-07-06 | Bioscale, Inc. | Method and apparatus for analyzing bioprocess fluids |
US20070003954A1 (en) * | 2005-05-12 | 2007-01-04 | The Board Of Regents Of The University Of Texas System | Protein and antibody profiling using small molecule microarrays |
US8486629B2 (en) | 2005-06-01 | 2013-07-16 | Bioarray Solutions, Ltd. | Creation of functionalized microparticle libraries by oligonucleotide ligation or elongation |
US20070021433A1 (en) | 2005-06-03 | 2007-01-25 | Jian-Qiang Fan | Pharmacological chaperones for treating obesity |
NZ564243A (en) | 2005-06-08 | 2011-03-31 | Dana Farber Cancer Inst Inc | Methods and compositions for the treatment of persistent infections by inhibiting the programmed cell death 1 (PD-1) pathway |
US20100056392A1 (en) * | 2005-06-15 | 2010-03-04 | Matthew Greving | Microstructure and microdomain microarrays, methods of making same and uses thereof |
US20070154903A1 (en) * | 2005-06-23 | 2007-07-05 | Nanosphere, Inc. | Selective isolation and concentration of nucleic acids from complex samples |
EP2993474B1 (en) | 2005-06-24 | 2019-06-12 | Vermillion, Inc. | Biomarkers for ovarian cancer: beta-2 microglobulin |
US20090264305A1 (en) | 2005-07-07 | 2009-10-22 | Athlomics Pty Ltd. | Polynucleotide Marker Genes and their Expression, for Diagnosis of Endotoxemia |
EP2360473A1 (en) | 2005-08-22 | 2011-08-24 | Cornell Research Foundation | Compositions and methods for analyzing protein interactions |
US7464580B2 (en) * | 2005-09-26 | 2008-12-16 | Oakland University | Ionic liquid high temperature gas sensors |
WO2007038478A2 (en) | 2005-09-26 | 2007-04-05 | Rapid Micro Biosystems, Inc | Cassette containing growth medium |
FR2891279B1 (en) * | 2005-09-27 | 2007-12-14 | Centre Nat Rech Scient | NEW CHIPS FOR SURFACE PLASMON DETECTION (SPR) |
US20070202515A1 (en) * | 2005-10-12 | 2007-08-30 | Pathologica, Llc. | Promac signature application |
US20070141627A1 (en) * | 2005-10-19 | 2007-06-21 | Behrens Timothy W | Systemic Lupus Erythematosus |
CN102260742A (en) | 2005-10-21 | 2011-11-30 | 基因信息股份有限公司 | Method and apparatus for correlating levels of biomarker products with disease |
CA2627360C (en) | 2005-10-29 | 2014-06-10 | Bayer Technology Services Gmbh | Process for determining one or more analytes in samples of biological origin having complex composition, and use thereof |
FR2893130B1 (en) * | 2005-11-08 | 2008-05-02 | Thales Sa | IMAGING DEVICE FOR BIOPUCE, AND BIOPUCE THEREFOR |
EP1951710A4 (en) | 2005-11-09 | 2010-08-25 | Univ Columbia | Photochemical methods and photoactive compounds for modifying surfaces |
US8372596B2 (en) | 2005-11-10 | 2013-02-12 | National University Of Corporation Hiroshima University | Asbestos detection method, asbestos detection agent, asbestos detection kit, method for screening candidate for agent aiming at preventing or treating disease for which asbestos is causative or worsening factor |
US7960312B2 (en) * | 2005-11-10 | 2011-06-14 | National University Of Corporation Hiroshima University | Method and agent for immobilizing protein via protein bound to silicon oxide-containing substance |
DE602006019128D1 (en) * | 2005-11-10 | 2011-02-03 | Bristol Myers Squibb Pharma Co | MOESIN, CAVEOLIN 1 AND YES-ASSOCIATED PROTEIN 1 AS PREDICTIVE MARKERS OF THE RESPONSE TO DASATINIB IN BREAST CANCER |
US7889347B2 (en) * | 2005-11-21 | 2011-02-15 | Plexera Llc | Surface plasmon resonance spectrometer with an actuator driven angle scanning mechanism |
US7463358B2 (en) * | 2005-12-06 | 2008-12-09 | Lumera Corporation | Highly stable surface plasmon resonance plates, microarrays, and methods |
WO2007070809A2 (en) * | 2005-12-12 | 2007-06-21 | Mcgill University | Biomarkers for babesia |
US8841255B2 (en) | 2005-12-20 | 2014-09-23 | Duke University | Therapeutic agents comprising fusions of vasoactive intestinal peptide and elastic peptides |
US20130172274A1 (en) | 2005-12-20 | 2013-07-04 | Duke University | Methods and compositions for delivering active agents with enhanced pharmacological properties |
CN101384272B (en) | 2005-12-20 | 2013-05-01 | 杜克大学 | Methods and compositions for delivering active agents with enhanced pharmacological properties |
JP2007171003A (en) * | 2005-12-22 | 2007-07-05 | Fujifilm Corp | Substrate for mass spectrometry, analytical method, and apparatus |
US7781203B2 (en) * | 2005-12-29 | 2010-08-24 | Corning Incorporated | Supports for assaying analytes and methods of making and using thereof |
WO2007076580A1 (en) * | 2005-12-30 | 2007-07-12 | Bio-Layer Pty Limited | Binding of molecules |
CA2635929A1 (en) * | 2006-01-03 | 2008-01-31 | David W. Barnes | Small molecule printing |
US7709227B2 (en) * | 2006-01-04 | 2010-05-04 | Phasebio Pharmaceuticals, Inc. | Multimeric ELP fusion constructs |
US7648834B2 (en) * | 2006-01-17 | 2010-01-19 | Moore Wayne E | Plasmon fluorescence augmentation for chemical and biological testing apparatus |
US20090011428A1 (en) * | 2006-01-18 | 2009-01-08 | The Regents Of The University Of California | Fluid Membrane-Based Ligand Display System for Live Cell Assays and Disease Diagnosis Applications |
AU2007209980A1 (en) * | 2006-01-27 | 2007-08-09 | Eastern Virginia Medical School | Proteomic fingerprinting of human IVF-derived embryos: identification of biomarkers of developmental potential |
JP4833679B2 (en) * | 2006-01-31 | 2011-12-07 | 富士通株式会社 | Method and apparatus for producing molecular film with adjusted density |
US8008067B2 (en) | 2006-02-13 | 2011-08-30 | University Of Maryland, Baltimore County | Microwave trigger metal-enhanced chemiluminescence (MT MEC) and spatial and temporal control of same |
CN101421622A (en) | 2006-02-17 | 2009-04-29 | 儿童医学中心公司 | Free NGAL as a biomarker for cancer |
US20070207504A1 (en) * | 2006-03-06 | 2007-09-06 | The Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College | Proteomic methods for the identification of differentiated adipose cells and adipose derived adult stem cells |
WO2008076139A1 (en) * | 2006-03-10 | 2008-06-26 | Tethys Bioscience, Inc. | Multiplex protein fractionation |
EP1996949A4 (en) | 2006-03-11 | 2010-01-20 | Univ Leland Stanford Junior | Beta-2 microglobulin as a biomarker for peripheral artery disease |
US7855057B2 (en) * | 2006-03-23 | 2010-12-21 | Millipore Corporation | Protein splice variant/isoform discrimination and quantitative measurements thereof |
US7909928B2 (en) | 2006-03-24 | 2011-03-22 | The Regents Of The University Of Michigan | Reactive coatings for regioselective surface modification |
EP2007514A2 (en) * | 2006-03-28 | 2008-12-31 | Inanovate, Inc. | Nano-particle biochip substrates |
US8375768B2 (en) * | 2006-03-30 | 2013-02-19 | Oakland University | Ionic liquid thin layer sensor for electrochemical and/or piezoelectric measurements |
US7886577B2 (en) | 2006-03-30 | 2011-02-15 | Oakland University | Devices with surface bound ionic liquids and method of use thereof |
WO2007115207A2 (en) * | 2006-03-31 | 2007-10-11 | Regents Of The University Of Minnesota | Irf-5 haplotypes in systemic lupus erythematosus |
US20080003694A1 (en) * | 2006-04-18 | 2008-01-03 | Swanson Basil I | Robust, self-assembled, biocompatible films |
US7923054B2 (en) | 2006-04-19 | 2011-04-12 | Gore Enterprise Holdings, Inc. | Functional porous substrates for attaching biomolecules |
US20100216657A1 (en) * | 2006-05-16 | 2010-08-26 | Arcxis Biotechnologies, Inc. | Pcr-free sample preparation and detection systems for high speed biologic analysis and identification |
WO2007140388A2 (en) * | 2006-05-31 | 2007-12-06 | The Johns Hopkins University | Ablation based laser machining of biomolecule patterns on substrates |
US7947148B2 (en) | 2006-06-01 | 2011-05-24 | The Regents Of The University Of Michigan | Dry adhesion bonding |
KR100931027B1 (en) * | 2006-06-27 | 2009-12-10 | 한국생명공학연구원 | Cysteine-tagged Protein shock Variant at the N-terminus |
US20090297401A1 (en) * | 2006-06-28 | 2009-12-03 | Rgb Technologies Ab | Sensor kit and a system for detecting an analyte in a test environment |
US8178316B2 (en) * | 2006-06-29 | 2012-05-15 | President And Fellows Of Harvard College | Evaluating proteins |
FR2903590B1 (en) * | 2006-07-13 | 2013-05-10 | Commissariat Energie Atomique | CELL SENSING DEVICE BY CONTACT |
EP2442108B1 (en) | 2006-07-14 | 2016-11-16 | The Regents of The University of California | Cancer biomarkers and methods of use thereof |
US8343539B2 (en) * | 2006-07-14 | 2013-01-01 | Regents Of The University Of Minnesota | Compounds that bind α5β1 integrin and methods of use |
KR100813262B1 (en) * | 2006-07-25 | 2008-03-13 | 삼성전자주식회사 | Method for producing a patterned spot microarray using photocatalyst and a microarray produced by the method |
WO2008015645A2 (en) * | 2006-08-02 | 2008-02-07 | Koninklijke Philips Electronics N.V. | A method of determining the concentration of an analyte using analyte sensor molecules coupled to a porous membrane |
GB0617429D0 (en) | 2006-09-05 | 2006-10-18 | Electrophoretics Ltd | Markers of renal transplant rejection and renal damage |
CN101611318B (en) | 2006-09-07 | 2015-03-04 | 奥塔哥创新有限公司 | Biomarkers |
US8658573B2 (en) * | 2006-09-11 | 2014-02-25 | The Trustees Of Columbia University In The City Of New York | Photo-generated carbohydrate arrays and the rapid identification of pathogen-specific antigens and antibodies |
WO2008039774A1 (en) | 2006-09-25 | 2008-04-03 | Mayo Foundation For Medical Education And Research | Extracellular and membrane-associated prostate cancer markers |
US20090087925A1 (en) * | 2007-10-01 | 2009-04-02 | Zyomyx, Inc. | Devices and methods for analysis of samples with depletion of analyte content |
US20080085512A1 (en) * | 2006-10-05 | 2008-04-10 | D Andrade Petula N | Array assay devices and methods for making and using the same |
EP2511844B1 (en) * | 2006-10-10 | 2015-08-12 | XRpro Sciences, Inc. | X-ray microscope |
EP2074230B1 (en) | 2006-10-11 | 2012-11-28 | Janssen Pharmaceutica NV | Compositions and methods for treating and diagnosing irritable bowel syndrome |
US20080167532A1 (en) * | 2006-10-13 | 2008-07-10 | Mayo Foundation For Medical Education And Research | Assessing cancer treatment responsiveness |
JP2008105973A (en) * | 2006-10-24 | 2008-05-08 | Toyo Kohan Co Ltd | Method of preserving polypeptide immobilized support |
AU2007313830A1 (en) * | 2006-10-31 | 2008-05-08 | Sru Biosystems, Inc. | Method for blocking non-specific protein binding on a functionalized surface |
JP2008171800A (en) * | 2006-10-31 | 2008-07-24 | Fei Co | Protective layer for charged particle beam processing |
WO2008058018A2 (en) | 2006-11-02 | 2008-05-15 | Mayo Foundation For Medical Education And Research | Predicting cancer outcome |
WO2008070570A2 (en) * | 2006-12-01 | 2008-06-12 | Cedars-Sinai Medical Center | Positive selection of serum proteins for proteomic analysis |
US8029902B2 (en) * | 2006-12-11 | 2011-10-04 | Wisconsin Alumni Research Foundation | Plasma-enhanced functionalization of substrate surfaces with quaternary ammonium and quaternary phosphonium groups |
JP5623747B2 (en) * | 2006-12-27 | 2014-11-12 | エモリー ユニバーシティ | Compositions and methods for treating infections and tumors |
KR100894419B1 (en) * | 2006-12-29 | 2009-04-24 | 삼성전자주식회사 | Biochip kit and method of testing biological sample |
US8664364B2 (en) | 2007-01-24 | 2014-03-04 | Carnegie Mellon University | Optical biosensors |
US20090053690A1 (en) * | 2007-02-02 | 2009-02-26 | California Institute Of Technology | Surface chemistry and deposition techniques |
US7867783B2 (en) | 2007-02-22 | 2011-01-11 | Maven Technologies, Llc | Apparatus and method for performing ligand binding assays on microarrays in multiwell plates |
TW200837349A (en) * | 2007-03-07 | 2008-09-16 | Nat Univ Tsing Hua | Biochip and manufacturing method thereof |
KR100836206B1 (en) | 2007-03-20 | 2008-06-09 | 연세대학교 산학협력단 | Peg hydrogel for fabrication of micropattern and process thereof |
US8399047B2 (en) | 2007-03-22 | 2013-03-19 | The Regents Of The Univeristy Of Michigan | Multifunctional CVD coatings |
JP4850855B2 (en) * | 2007-03-22 | 2012-01-11 | 信越化学工業株式会社 | Manufacturing method of substrate for producing microarray |
WO2008123948A1 (en) | 2007-03-27 | 2008-10-16 | Vermillion, Inc. | Biomarkers for ovarian cancer |
US20080242559A1 (en) * | 2007-03-28 | 2008-10-02 | Northwestern University | Protein and peptide arrays |
JP5656339B2 (en) * | 2007-03-28 | 2015-01-21 | Jsr株式会社 | Protein-immobilized carrier and method for producing the same |
US7863037B1 (en) | 2007-04-04 | 2011-01-04 | Maven Technologies, Llc | Ligand binding assays on microarrays in closed multiwell plates |
US8093039B2 (en) | 2007-04-10 | 2012-01-10 | The Trustees Of The Stevens Institute Of Technology | Surfaces differentially adhesive to eukaryotic cells and non-eukaryotic cells |
WO2008128333A1 (en) * | 2007-04-19 | 2008-10-30 | The Governors Of The University Of Alberta | A method to distinguish antibody-mediated tissue rejection from t cell-mediated tissue rejection |
CA2683082A1 (en) * | 2007-04-19 | 2008-10-30 | Sru Biosystems, Inc. | Method for employing a biosensor to detect small molecules that bind directly to immobilized targets |
US20080274458A1 (en) * | 2007-05-01 | 2008-11-06 | Latham Gary J | Nucleic acid quantitation methods |
EP2639315A1 (en) | 2007-05-11 | 2013-09-18 | The Johns Hopkins University | Biomarkers for melanoma |
US20090041633A1 (en) * | 2007-05-14 | 2009-02-12 | Dultz Shane C | Apparatus and method for performing ligand binding assays on microarrays in multiwell plates |
WO2008143351A1 (en) * | 2007-05-18 | 2008-11-27 | Fujirebio Inc. | Chemical surface nanopatterns to increase activity of surface-immobilized biomolecules |
US7799558B1 (en) | 2007-05-22 | 2010-09-21 | Dultz Shane C | Ligand binding assays on microarrays in closed multiwell plates |
EP2160478B1 (en) * | 2007-06-06 | 2014-08-27 | Siemens Healthcare Diagnostics Inc. | Predictive diagnostics for kidney disease |
KR100927886B1 (en) * | 2007-06-18 | 2009-11-23 | 한국생명공학연구원 | Protein shock-oligonucleotide conjugates |
CA2692171C (en) | 2007-06-22 | 2019-10-22 | Randolph Watnick | Methods and uses thereof of prosaposin |
WO2009003273A1 (en) * | 2007-06-29 | 2009-01-08 | The Governors Of The University Of Alberta | Assessing tissue rejection |
CA2693700A1 (en) * | 2007-07-11 | 2009-01-15 | Sru Biosystems, Inc. | Methods for identifying modulators of ion channels |
US9134307B2 (en) | 2007-07-11 | 2015-09-15 | X-Body, Inc. | Method for determining ion channel modulating properties of a test reagent |
JP5363481B2 (en) * | 2007-07-20 | 2013-12-11 | ザ ユニバーシティ オブ ユタ リサーチ ファウンデーション | Biomarker identification and quantification to assess the risk of preterm birth |
DE102007034993A1 (en) | 2007-07-26 | 2009-01-29 | Hanna Diehl | Soluble Cadherin 17 for the diagnosis and risk stratification of colon or colon cancer |
US20090060786A1 (en) * | 2007-08-29 | 2009-03-05 | Gibum Kim | Microfluidic apparatus for wide area microarrays |
US8354280B2 (en) | 2007-09-06 | 2013-01-15 | Bioscale, Inc. | Reusable detection surfaces and methods of using same |
WO2009039173A2 (en) | 2007-09-19 | 2009-03-26 | Applied Biosystems Inc. | SiRNA SEQUENCE-INDEPENDENT MODIFICATION FORMATS FOR REDUCING OFF-TARGET PHENOTYPIC EFFECTS IN RNAi, AND STABILIZED FORMS THEREOF |
JP5743135B2 (en) | 2007-09-28 | 2015-07-01 | エックスアールプロ・サイエンシーズ・インコーポレーテッド | Method and apparatus for measuring post-translational modifications of proteins |
WO2009058867A2 (en) * | 2007-10-29 | 2009-05-07 | Primorigen Biosciences, Llc | Affinity measurements using frameless multiplexed microarrays |
EP3536336A1 (en) * | 2007-11-30 | 2019-09-11 | Siemens Healthcare Diagnostics Inc. | Adiponectin receptor fragments and methods of use |
US8004669B1 (en) | 2007-12-18 | 2011-08-23 | Plexera Llc | SPR apparatus with a high performance fluid delivery system |
US20090181857A1 (en) * | 2008-01-15 | 2009-07-16 | Academia Sinica | System and method for producing a label-free micro-array biochip |
KR100959831B1 (en) | 2008-01-18 | 2010-05-28 | 포항공과대학교 산학협력단 | Pattern-Recognition Type Plate Detecting Multi-Biomolecule |
US20090196852A1 (en) * | 2008-02-04 | 2009-08-06 | Watkinson D Tobin | Compositions and methods for diagnosing and treating immune disorders |
DE102008011850A1 (en) * | 2008-02-29 | 2009-09-03 | Michael Grzendowski | Biomarker for the diagnosis of brain tumor |
US20110160070A1 (en) | 2008-03-10 | 2011-06-30 | Lineagen, Inc. | Copd biomarker signatures |
JP2011516038A (en) | 2008-03-12 | 2011-05-26 | オタゴ イノベーション リミテッド | Biomarker |
WO2009113879A1 (en) | 2008-03-12 | 2009-09-17 | Christopher Joseph Pemberton | Biomarkers |
WO2009118343A1 (en) * | 2008-03-27 | 2009-10-01 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and kits for determining the occurrence of a liver disease in a subject |
EP2274098B1 (en) * | 2008-03-28 | 2013-12-25 | Biotix, Inc. | Multicapillary sample preparation devices and methods for processing analytes |
US8257936B2 (en) | 2008-04-09 | 2012-09-04 | X-Body Inc. | High resolution label free analysis of cellular properties |
US20090286692A1 (en) * | 2008-04-15 | 2009-11-19 | Wainwright Norman R | Cartridge and Method for Sample Analysis |
US20090263905A1 (en) * | 2008-04-18 | 2009-10-22 | Kim Scheuringer | Detection test assembly for detecting the presence of a substance in a sample |
US9029508B2 (en) | 2008-04-29 | 2015-05-12 | Abbvie Inc. | Dual variable domain immunoglobulins and uses thereof |
US8039270B2 (en) * | 2008-05-22 | 2011-10-18 | Maven Technologies, Llc | Apparatus and method for performing ligand binding assays on microarrays in multiwell plates |
US7981664B1 (en) | 2008-05-22 | 2011-07-19 | Maven Technologies, Llc | Apparatus and method for performing ligand binding assays on microarrays in multiwell plates |
EP2291553A4 (en) | 2008-05-28 | 2011-12-14 | Genomedx Biosciences Inc | Systems and methods for expression-based discrimination of distinct clinical disease states in prostate cancer |
US10407731B2 (en) | 2008-05-30 | 2019-09-10 | Mayo Foundation For Medical Education And Research | Biomarker panels for predicting prostate cancer outcomes |
US20090298082A1 (en) * | 2008-05-30 | 2009-12-03 | Klee George G | Biomarker panels for predicting prostate cancer outcomes |
EP3002299A1 (en) | 2008-06-03 | 2016-04-06 | AbbVie Inc. | Dual variable domain immunoglobulins and uses thereof |
US20100015721A1 (en) * | 2008-06-04 | 2010-01-21 | Sru Biosystems, Inc. | Detection of Promiscuous Small Submicrometer Aggregates |
US8617863B2 (en) * | 2008-06-04 | 2013-12-31 | Grifols Therapeutics Inc. | Composition, method, and kit for preparing plasmin |
CA2726894A1 (en) | 2008-06-27 | 2009-12-30 | Duke University | Therapeutic agents comprising elastin-like peptides |
US8021850B2 (en) * | 2008-07-14 | 2011-09-20 | Ribo Guo | Universal tandem solid-phases based immunoassay |
WO2010123608A2 (en) | 2009-01-29 | 2010-10-28 | The Regents Of The University Of California | A spatial biomarker of disease and detection of spatial organization of cellular recptors |
WO2010011860A1 (en) * | 2008-07-23 | 2010-01-28 | Diabetomics, Llc | Methods for detecting pre-diabetes and diabetes |
US11865534B2 (en) | 2008-09-24 | 2024-01-09 | First Light Diagnostics, Inc. | Imaging analyzer for testing analytes |
GB2463401B (en) | 2008-11-12 | 2014-01-29 | Caris Life Sciences Luxembourg Holdings S A R L | Characterizing prostate disorders by analysis of microvesicles |
HUE028582T2 (en) * | 2008-11-28 | 2016-12-28 | Univ Emory | Methods for determining the efficacy of pd-1 antagonists |
JP4911639B2 (en) * | 2008-12-02 | 2012-04-04 | 学校法人早稲田大学 | Biosensing method and immobilization method |
US8349325B2 (en) | 2008-12-23 | 2013-01-08 | Abbott Laboratories | Soluble FMS-like tyrosine kinase-1 (sFLT-1) antibody and related composition, kit, methods of using, and materials and method for making |
US20100166739A1 (en) * | 2008-12-30 | 2010-07-01 | Lipella Paharmaceuticals Inc. | Methods and Compositions for Diagnosing Urological Disorders |
EP2384367A4 (en) | 2008-12-30 | 2013-07-10 | Janssen Biotech Inc | Serum markers predicting clinical response to anti-tnf antibodies in patients with ankylosing spondylitis |
WO2010088187A1 (en) | 2009-01-27 | 2010-08-05 | Proteogenix, Inc. | Biomarkers for detection of neonatal sepsis in biological fluid |
WO2010087985A2 (en) | 2009-01-28 | 2010-08-05 | Yale University | Novel markers for detection of complications resulting from in utero encounters |
WO2010096331A1 (en) | 2009-02-11 | 2010-08-26 | Duke University | Sensors incorporating antibodies and methods of making and using the same |
US9206410B2 (en) | 2009-03-03 | 2015-12-08 | Grifols Therapeutics Inc. | Compositions, methods and kits for preparing plasminogen and plasmin prepared therefrom |
RU2015132478A (en) | 2009-03-05 | 2015-12-10 | Эббви Инк. | BINDING IL-17 PROTEINS |
WO2010106535A1 (en) | 2009-03-15 | 2010-09-23 | Technion Research And Development Foundation Ltd. | Soluble hla complexes for use in disease diagnosis |
CN102388308B (en) | 2009-04-23 | 2015-08-19 | 西门子医疗保健诊断公司 | The adiponectin receptor fragments of monomer and dimeric forms and using method |
US20100273185A1 (en) * | 2009-04-27 | 2010-10-28 | Sru Biosystems, Inc. | Detection of Biased Agonist Activation |
US8741581B2 (en) | 2009-04-27 | 2014-06-03 | Technion Research And Development Foundation Ltd. | Markers for cancer detection |
WO2010127247A1 (en) * | 2009-05-01 | 2010-11-04 | University Of Utah Research Foundation | Methods and compositions for measuring high affinity interactions with kinetic imaging of single molecule interaction (kismi) |
CN102460171A (en) * | 2009-05-15 | 2012-05-16 | Sru生物系统公司 | Detection of changes in cell populations and mixed cell populations |
CA2763685A1 (en) * | 2009-05-29 | 2010-12-02 | The Board Of Regents Of The University Of Texas System | Peptoid ligands for isolation and treatment of autoimmune t-cells |
KR20120122869A (en) * | 2009-06-02 | 2012-11-07 | 보드 오브 리전츠, 더 유니버시티 오브 텍사스 시스템 | Identification of small molecules recognized by antibodies in subjects with neurodegenerative diseases |
EP2443459B1 (en) | 2009-06-19 | 2018-12-26 | The Arizona Board of Regents, A Body Corporate Of the State of Arizona acting for and on behalf Of Arizona State University | Compound arrays for sample profiling |
GB0912231D0 (en) * | 2009-07-14 | 2009-08-26 | Imp Innovations Ltd | Method and apparatus for determining an analyte parameter |
WO2011025602A1 (en) * | 2009-07-20 | 2011-03-03 | The Board Of Regents Of The University Of Texas System | Combinational multidomain mesoporous chips and a method for fractionation, stabilization, and storage of biomolecules |
IN2012DN02120A (en) | 2009-08-14 | 2015-08-21 | Phasebio Pharmaceuticals Inc | |
EP2504024A2 (en) | 2009-09-27 | 2012-10-03 | Ruhr-Universität Bochum | Method for the therapy and diagnosis of alzheimer's disease |
TW201124726A (en) * | 2009-10-16 | 2011-07-16 | Univ Texas | Compositions and methods for producing coded peptoid libraries |
KR101105328B1 (en) * | 2009-11-23 | 2012-01-16 | 한국표준과학연구원 | Apparatus and method for quantifying the binding and dissociation kinetics of molecular interactions |
US8663576B2 (en) | 2009-11-25 | 2014-03-04 | Hologic, Inc. | Detection of intraamniotic infection |
EP3925670A1 (en) | 2009-12-17 | 2021-12-22 | Children's Medical Center, Corp. | Saposin-a derived peptides and uses thereof |
US8355133B2 (en) * | 2009-12-30 | 2013-01-15 | Maven Technologies, Llc | Biological testing with sawtooth-shaped prisms |
EP2524059A4 (en) | 2010-01-13 | 2013-11-20 | Caris Life Sciences Luxembourg Holdings | Detection of gastrointestinal disorders |
CN102725637B (en) * | 2010-01-25 | 2015-02-25 | 松下健康医疗控股株式会社 | A method for immobilizing protein A on a self-assembled monolayer |
US9682132B2 (en) | 2010-01-26 | 2017-06-20 | Banyan Biomarkers, Inc | Compositions and methods relating to argininosucccinate synthetase |
EP2542696B1 (en) | 2010-03-01 | 2016-09-28 | Caris Life Sciences Switzerland Holdings GmbH | Biomarkers for theranostics |
JP5828195B2 (en) | 2010-03-11 | 2015-12-02 | ユニバーシティ オブ ルイスビル リサーチ ファンデーション,インコーポレイテッドUniversity Of Louisville Research Foundation,Inc. | Methods for predicting pregnancy loss risk and methods for reducing pregnancy loss risk |
US20110229921A1 (en) | 2010-03-18 | 2011-09-22 | Abbott Laboratories | METHODS OF ASSAYING URINARY NEUTROPHIL GELATINASE-ASSOCIATED LIPOCALIN (uNGAL) IN THE PROGNOSIS OF CADAVERIC KIDNEY TRANSPLANT FUNCTION IN A PATIENT, INCLUDING A PATIENT DIAGNOSED WITH DELAYED GRAFT FUNCTION (DGF), A METHOD OF ASSAYING uNGAL IN THE ASSESSMENT OF RISK OF DGF IN A PATIENT DIAGNOSED WITH EARLY GRAFT FUNCTION (EGF), AND RELATED KITS |
EP2553151A4 (en) * | 2010-03-26 | 2013-07-31 | X Body Inc | Use of induced pluripotent cells and other cells for screening compound libraries |
AU2011237669B2 (en) | 2010-04-06 | 2016-09-08 | Caris Life Sciences Switzerland Holdings Gmbh | Circulating biomarkers for disease |
EP3508854A1 (en) | 2010-04-27 | 2019-07-10 | The Regents of The University of California | Cancer biomarkers and methods of use thereof |
ES2635594T3 (en) | 2010-05-14 | 2017-10-04 | Abbvie Inc. | IL-1 binding proteins |
US9211542B2 (en) * | 2010-05-21 | 2015-12-15 | Eidgenossische Technische Hochschule Zurich | High-density sample support plate for automated sample aliquoting |
WO2011163558A1 (en) | 2010-06-25 | 2011-12-29 | Abbott Laboratories | Materials and methods for assay of anti-hepatitis c virus (hcv) antibodies |
WO2012006500A2 (en) | 2010-07-08 | 2012-01-12 | Abbott Laboratories | Monoclonal antibodies against hepatitis c virus core protein |
UY33492A (en) | 2010-07-09 | 2012-01-31 | Abbott Lab | IMMUNOGLOBULINS WITH DUAL VARIABLE DOMAIN AND USES OF THE SAME |
ES2629850T3 (en) | 2010-07-19 | 2017-08-16 | Otago Innovation Limited | Signal biomarkers |
KR101045209B1 (en) * | 2010-07-26 | 2011-06-30 | 삼성전자주식회사 | An array device comprising a photoresist film having immobilization regions and a method using the same |
NZ607480A (en) | 2010-08-03 | 2014-10-31 | Abbott Lab | Dual variable domain immunoglobulins and uses thereof |
WO2012029202A1 (en) * | 2010-08-30 | 2012-03-08 | Panasonic Corporation | A method for immobilizing streptavidin on a self-assembled monolayer |
JP2013545439A (en) | 2010-09-17 | 2013-12-26 | プレジデント・アンド・フェロウズ・オブ・ハーバード・カレッジ | Functional genomics assay to characterize the usefulness and safety of pluripotent stem cells |
EP2628013B1 (en) | 2010-10-14 | 2019-06-12 | The Johns Hopkins University | Biomarkers of brain injury |
WO2012053138A1 (en) | 2010-10-19 | 2012-04-26 | パナソニック株式会社 | Method for immobilizing glucose oxidase on self-assembled film |
US20120115244A1 (en) | 2010-11-09 | 2012-05-10 | Abbott Laboratories | Materials and methods for immunoassay of pterins |
CN102486474B (en) * | 2010-12-06 | 2014-05-28 | 北京大学人民医院 | Protein chip for chronic hepatitis C outcome prediction in chronic hepatitis C interferon treatment |
WO2012121775A2 (en) | 2010-12-21 | 2012-09-13 | Abbott Laboratories | Dual variable domain immunoglobulins and uses thereof |
BR112013023724A2 (en) | 2011-03-15 | 2019-09-24 | Univ Utah Res Found | methods for treating disease or symptom, screening for an agent or a combination of agents, and for determining the effectiveness of an agent and treatment |
US8735175B2 (en) * | 2011-03-18 | 2014-05-27 | Chris D. Geddes | Multicolor microwave-accelerated metal-enhanced fluorescence (M-MAMEF) |
EP2694968B1 (en) | 2011-04-08 | 2018-11-21 | The General Hospital Corporation | Fungal-derived carbohydrate-conjugated scaffold |
AU2012242684B2 (en) | 2011-04-15 | 2017-04-20 | Children's Medical Center Corporation | Diagnostic markers and therapeutic targets of Kawasaki disease |
KR101158362B1 (en) | 2011-04-20 | 2012-06-22 | 한국과학기술원 | Method for analyzing single protein-protein interactions in whole cell lysates |
US20140141458A1 (en) | 2011-05-12 | 2014-05-22 | The Johns Hopkins University | Assay reagents for a neurogranin diagnostic kit |
WO2012170524A1 (en) | 2011-06-06 | 2012-12-13 | Phasebio Pharmaceuticals, Inc. | Use of modified vasoactive intestinal peptides in the treatment of hypertension |
WO2012168988A1 (en) * | 2011-06-10 | 2012-12-13 | パナソニック株式会社 | Method for affixing antibodies to self-assembled monolayer |
US10261081B2 (en) | 2011-06-13 | 2019-04-16 | Indevr, Inc. | Low density microarrays for vaccine related protein quantification, potency determination and efficacy evaluation |
CN102279261B (en) * | 2011-06-20 | 2013-09-18 | 东南大学 | Inkjet printing preparation method of pattern code microcarrier |
AU2012275841A1 (en) | 2011-06-27 | 2014-01-16 | The Jackson Laboratory | Methods and compositions for treatment of cancer and autoimmune disease |
WO2013005269A1 (en) * | 2011-07-05 | 2013-01-10 | パナソニック株式会社 | Method for immobilizing albumin on self-assembled monolayer |
WO2013008280A1 (en) * | 2011-07-08 | 2013-01-17 | パナソニック株式会社 | Method for immobilizing protein on self-assembled film |
US20140235492A1 (en) * | 2011-09-20 | 2014-08-21 | Institut National De La Sante Et De La Recherche Medicate (Inserm) | Methods for preparing single domain antibody microarrays |
WO2013055829A1 (en) * | 2011-10-11 | 2013-04-18 | Nestec S.A. | Proximity-based assays for the detection of signaling protein expression and activation |
TW201323440A (en) | 2011-10-24 | 2013-06-16 | Abbvie Inc | Immunobinders directed against sclerostin |
EP2771361A1 (en) | 2011-10-24 | 2014-09-03 | AbbVie Inc. | Bispecific immunobinders directed against tnf and il-17 |
PL2776550T3 (en) | 2011-11-07 | 2018-05-30 | Rapid Micro Biosystems, Inc. | Cassette for sterility testing |
KR102035877B1 (en) | 2011-11-14 | 2019-10-23 | 알파시그마 에스.피.에이. | Assays for selecting a treatment regimen for a subject with depression and methods for treatment |
EP2791359B1 (en) | 2011-12-13 | 2020-01-15 | Decipher Biosciences, Inc. | Cancer diagnostics using non-coding transcripts |
WO2013096868A2 (en) | 2011-12-22 | 2013-06-27 | Children's Medical Center Corporation | Saposin-a derived peptides and uses thereof |
US8993248B2 (en) | 2011-12-31 | 2015-03-31 | Abbott Laboratories | Truncated human vitamin D binding protein and mutation and fusion thereof and related materials and methods of use |
NZ629074A (en) | 2012-01-20 | 2016-09-30 | Adelaide Res & Innovation Pty | Biomarkers for gastric cancer and uses thereof |
JP6012767B2 (en) * | 2012-02-07 | 2016-10-25 | ヴィブラント ホールディングス リミテッド ライアビリティ カンパニー | Substrates, peptide arrays, and methods |
CN104470942B (en) | 2012-03-20 | 2018-12-14 | 奥塔哥创新有限公司 | Biomarker |
EP4060016A1 (en) | 2012-04-16 | 2022-09-21 | Rapid Micro Biosystems, Inc. | Cell culturing device |
EP2866928A1 (en) | 2012-06-29 | 2015-05-06 | Danmarks Tekniske Universitet | A method of charging a test carrier and a test carrier |
UY34905A (en) | 2012-07-12 | 2014-01-31 | Abbvie Inc | IL-1 UNION PROTEINS |
CA2884737A1 (en) | 2012-08-16 | 2014-02-20 | The Trustees Of Columbia University In The City Of New York | Diagnostic markers of indolent prostate cancer |
US11035005B2 (en) | 2012-08-16 | 2021-06-15 | Decipher Biosciences, Inc. | Cancer diagnostics using biomarkers |
US10006909B2 (en) | 2012-09-28 | 2018-06-26 | Vibrant Holdings, Llc | Methods, systems, and arrays for biomolecular analysis |
WO2014068408A2 (en) | 2012-10-23 | 2014-05-08 | Caris Life Sciences Switzerland Holdings, S.A.R.L. | Aptamers and uses thereof |
US10942184B2 (en) | 2012-10-23 | 2021-03-09 | Caris Science, Inc. | Aptamers and uses thereof |
EP2935628B1 (en) | 2012-12-19 | 2018-03-21 | Caris Life Sciences Switzerland Holdings GmbH | Compositions and methods for aptamer screening |
AU2013202668B2 (en) | 2012-12-24 | 2014-12-18 | Adelaide Research & Innovation Pty Ltd | Inhibition of cancer growth and metastasis |
US9086412B2 (en) | 2012-12-31 | 2015-07-21 | University Of Louisville Research Foundation, Inc. | Extracellular vesicle-associated protein markers of cancer |
US9790478B2 (en) | 2013-03-14 | 2017-10-17 | Abbott Laboratories | HCV NS3 recombinant antigens and mutants thereof for improved antibody detection |
CA2906421C (en) | 2013-03-14 | 2022-08-16 | George J. Dawson | Hcv antigen-antibody combination assay and methods and compositions for use therein |
CN105378099B (en) | 2013-03-14 | 2021-05-11 | 雅培制药有限公司 | HCV core lipid binding domain monoclonal antibodies |
US9157910B2 (en) | 2013-03-15 | 2015-10-13 | Abbott Laboratories | Assay with increased dynamic range |
US9005901B2 (en) | 2013-03-15 | 2015-04-14 | Abbott Laboratories | Assay with internal calibration |
CA2914918C (en) | 2013-05-10 | 2023-10-10 | Johns Hopkins University | Compositions and methods for ovarian cancer assessment having improved specificity |
DE102013210138A1 (en) | 2013-05-30 | 2014-12-04 | Boehringer Ingelheim Vetmedica Gmbh | Method for generating a plurality of measuring ranges on a chip and chip with measuring ranges |
JP2014235115A (en) | 2013-06-04 | 2014-12-15 | ウシオ電機株式会社 | Microchip and method for forming metal thin film in microchip |
JP6611710B2 (en) | 2013-07-17 | 2019-11-27 | ザ・ジョンズ・ホプキンス・ユニバーシティ | Multiprotein biomarker assay for detection and outcome of brain injury |
US20160319361A1 (en) | 2013-08-28 | 2016-11-03 | Caris Life Sciences Switzerland Holdings Gmbh | Oligonucleotide probes and uses thereof |
JP2017508457A (en) | 2014-02-27 | 2017-03-30 | ザ・ブロード・インスティテュート・インコーポレイテッド | T cell balance gene expression, composition and method of use thereof |
WO2015187227A2 (en) | 2014-03-13 | 2015-12-10 | Duke University | Electronic platform for sensing and control of electrochemical reactions |
EP3139949B1 (en) | 2014-05-08 | 2020-07-29 | Phasebio Pharmaceuticals, Inc. | Compositions comprising a vip-elp fusion protein for use in treating cystic fibrosis |
EP3916390A1 (en) | 2014-06-04 | 2021-12-01 | Indevr, Inc. | Universal capture array for multiplexed subtype-specific quantification and stability determination of influenza proteins |
AU2015276899B2 (en) | 2014-06-19 | 2021-08-12 | Memorial Sloan-Kettering Cancer Center | Biomarkers for response to EZH2 inhibitors |
US9694518B2 (en) | 2014-06-20 | 2017-07-04 | The Regents Of The University Of Michigan | Breath-activated images and anti-counterfeit authentication features formed of nanopillar arrays |
BR112016030008A2 (en) | 2014-06-27 | 2017-10-24 | Abbott Lab | method for detecting human pegivirus 2 infection in an individual, for detecting human pegivirus 2 nucleic acid and for detecting human pegivirus 2 in a sample, and, composition |
WO2016011383A1 (en) | 2014-07-17 | 2016-01-21 | The Trustees Of The University Of Pennsylvania | Methods for using exosomes to monitor transplanted organ status |
US20160032281A1 (en) * | 2014-07-31 | 2016-02-04 | Fei Company | Functionalized grids for locating and imaging biological specimens and methods of using the same |
US10222386B2 (en) | 2014-09-19 | 2019-03-05 | The Johns Hopkins University | Biomarkers of congnitive dysfunction |
ES2828713T3 (en) | 2014-10-29 | 2021-05-27 | Abbott Lab | Target Anti-HCV Antibody Detection Assays Using NS3 Capture Peptides |
KR101687950B1 (en) * | 2014-11-14 | 2016-12-21 | 연세대학교 산학협력단 | Surface modified micropate, menufacturing method for the surface modified micropate and immunoassays using the surface modified micropate |
EP3224381B1 (en) | 2014-11-25 | 2019-09-04 | The Brigham and Women's Hospital, Inc. | Method of identifying a person having a predisposition to or afflicted with a cardiometabolic disease |
US10683552B2 (en) | 2014-11-25 | 2020-06-16 | Presidents And Fellows Of Harvard College | Clonal haematopoiesis |
WO2016090323A1 (en) | 2014-12-05 | 2016-06-09 | Prelude, Inc. | Dcis recurrence and invasive breast cancer |
WO2016109378A1 (en) | 2014-12-29 | 2016-07-07 | North Carolina State University | Multiplexed diagnostic to recognize concentrations of related proteins and peptides |
ES2822598T3 (en) | 2015-02-09 | 2021-05-04 | Phasebio Pharmaceuticals Inc | Methods and Compositions for Treating Muscle Diseases and Disorders |
WO2016134365A1 (en) | 2015-02-20 | 2016-08-25 | The Johns Hopkins University | Biomarkers of myocardial injury |
WO2016138488A2 (en) | 2015-02-26 | 2016-09-01 | The Broad Institute Inc. | T cell balance gene expression, compositions of matters and methods of use thereof |
CN107533069A (en) * | 2015-03-06 | 2018-01-02 | 泰摩拉分析运营有限责任公司 | Array for the chemically functionalization of analyzing proteins modification |
WO2016145128A1 (en) | 2015-03-09 | 2016-09-15 | Caris Science, Inc. | Oligonucleotide probes and uses thereof |
US10294451B2 (en) | 2015-04-22 | 2019-05-21 | University Of Maryland, Baltimore County | Flow and static lysing systems and methods for ultra-rapid isolation and fragmentation of biological materials by microwave irradiation |
WO2017004243A1 (en) | 2015-06-29 | 2017-01-05 | Caris Science, Inc. | Therapeutic oligonucleotides |
CA2992139A1 (en) | 2015-07-10 | 2017-01-19 | West Virginia University | Markers of stroke and stroke severity |
US11561180B2 (en) | 2015-07-22 | 2023-01-24 | University Of Maryland, Baltimore County | Hydrophilic coatings of plasmonic metals to enable low volume metal-enhanced fluorescence |
WO2017019918A1 (en) | 2015-07-28 | 2017-02-02 | Caris Science, Inc. | Targeted oligonucleotides |
DE102015114026A1 (en) | 2015-08-24 | 2017-03-02 | Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V. | Biomarker panel for the diagnosis of recurrent prostate cancer |
US10758886B2 (en) | 2015-09-14 | 2020-09-01 | Arizona Board Of Regents On Behalf Of Arizona State University | Conditioned surfaces for in situ molecular array synthesis |
CN108513586A (en) | 2015-09-30 | 2018-09-07 | 因姆内克斯普雷斯私人有限公司 | Pathogenic organisms marker and application thereof |
CN108367289B (en) | 2015-12-02 | 2022-01-18 | 勃林格殷格翰维特梅迪卡有限公司 | Method for producing a plurality of measurement regions on a chip and chip having a plurality of measurement regions |
EP3386594B1 (en) | 2015-12-11 | 2023-05-03 | The General Hospital Corporation | Combination of a platelet-derived growth factor receptor alpha inhibitor and a histone lysine demethylase inhibitor for treating drug-tolerant glioblastoma |
EP3998345A1 (en) | 2015-12-24 | 2022-05-18 | ImmuneXpress Pty Ltd | Triage biomarkers and uses therefor |
CN109715802A (en) | 2016-03-18 | 2019-05-03 | 卡里斯科学公司 | Oligonucleotide probe and application thereof |
AU2017271579B2 (en) | 2016-05-25 | 2023-10-19 | Caris Science, Inc. | Oligonucleotide probes and uses thereof |
US11747334B2 (en) | 2016-06-20 | 2023-09-05 | Cowper Sciences Inc. | Methods for differential diagnosis of autoimmune diseases |
US11774446B2 (en) | 2016-06-20 | 2023-10-03 | Cowper Sciences Inc. | Methods for diagnosis and treatment of autoimmune diseases |
JP6420281B2 (en) * | 2016-07-04 | 2018-11-07 | 花王株式会社 | Solid phase carrier for protein analysis and method for producing the same |
AU2017315425B2 (en) | 2016-08-24 | 2023-11-09 | The Regents Of The University Of Michigan | Use of genomic signatures to predict responsiveness of patients with prostate cancer to post-operative radiation therapy |
WO2018089858A1 (en) | 2016-11-11 | 2018-05-17 | Healthtell Inc. | Methods for identifying candidate biomarkers |
JP7132233B2 (en) | 2016-11-11 | 2022-09-06 | アセンダント ディーエックス, エルエルシー | Compositions and Methods for Diagnosing and Differentiating Systemic Juvenile Idiopathic Arthritis and Kawasaki Disease |
WO2018132916A1 (en) | 2017-01-20 | 2018-07-26 | Genomedx Biosciences, Inc. | Molecular subtyping, prognosis, and treatment of bladder cancer |
CA3055925A1 (en) | 2017-03-09 | 2018-09-13 | Decipher Biosciences, Inc. | Subtyping prostate cancer to predict response to hormone therapy |
WO2018200489A1 (en) | 2017-04-25 | 2018-11-01 | The Brigham And Women’S Hospital, Inc. | Il-8, il-6, il-1b and tet2 and dnmt3a in atherosclerosis |
US11078542B2 (en) | 2017-05-12 | 2021-08-03 | Decipher Biosciences, Inc. | Genetic signatures to predict prostate cancer metastasis and identify tumor aggressiveness |
US10538808B2 (en) | 2017-05-26 | 2020-01-21 | Vibrant Holdings, Llc | Photoactive compounds and methods for biomolecule detection and sequencing |
EA202090427A1 (en) | 2017-08-16 | 2020-06-08 | МЕДИММЬЮН, ЭлЭлСи | COMPOSITIONS AND METHODS OF TREATING ATOPIC DERMATITIS AND CHOICE OF TREATMENT |
US11709155B2 (en) | 2017-09-18 | 2023-07-25 | Waters Technologies Corporation | Use of vapor deposition coated flow paths for improved chromatography of metal interacting analytes |
US11709156B2 (en) | 2017-09-18 | 2023-07-25 | Waters Technologies Corporation | Use of vapor deposition coated flow paths for improved analytical analysis |
WO2019094798A1 (en) | 2017-11-10 | 2019-05-16 | The Trustees Of Columbia University In The City Of New York | Methods and compositions for promoting or inducing hair growth |
EP3791188A1 (en) | 2018-05-10 | 2021-03-17 | The Methodist Hospital | Methods for prognosis and management of disease |
EP3803415A1 (en) | 2018-06-04 | 2021-04-14 | Avon Products, Inc. | Protein biomarkers for identifying and treating aging skin and skin conditions |
CA3112792A1 (en) | 2018-09-14 | 2020-03-19 | Prelude Corporation | Method of selection for treatment of subjects at risk of invasive breast cancer |
SG11202104619WA (en) | 2018-11-07 | 2021-06-29 | Seer Inc | Compositions, methods and systems for protein corona analysis and uses thereof |
GB201904697D0 (en) | 2019-04-03 | 2019-05-15 | Vib Vzw | Means and methods for single molecule peptide sequencing |
MX2022001519A (en) | 2019-08-05 | 2022-04-06 | Seer Inc | Systems and methods for sample preparation, data generation, and protein corona analysis. |
US11918936B2 (en) | 2020-01-17 | 2024-03-05 | Waters Technologies Corporation | Performance and dynamic range for oligonucleotide bioanalysis through reduction of non specific binding |
EP4074820A1 (en) | 2021-04-16 | 2022-10-19 | The Trustees of The University of Pennsylvania | Micro-engineered models of the human eye and methods of use |
WO2023288108A1 (en) * | 2021-07-16 | 2023-01-19 | The University Of Chicago | Biocompatible surface for quantum sensing and methods thereof |
WO2023122723A1 (en) | 2021-12-23 | 2023-06-29 | The Broad Institute, Inc. | Panels and methods for diagnosing and treating lung cancer |
Family Cites Families (163)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4071409A (en) | 1976-05-20 | 1978-01-31 | Corning Glass Works | Immobilization of proteins on inorganic support materials |
US4722896A (en) | 1981-01-26 | 1988-02-02 | The Beth Israel Hospital Association | Method for affinity purification of hybridoma antibodies |
US4444879A (en) * | 1981-01-29 | 1984-04-24 | Science Research Center, Inc. | Immunoassay with article having support film and immunological counterpart of analyte |
US4690715A (en) | 1982-06-18 | 1987-09-01 | American Telephone And Telegraph Company, At&T Bell Laboratories | Modification of the properties of metals |
US4514508A (en) * | 1982-07-06 | 1985-04-30 | Biond Inc. | Assaying for a multiplicity of antigens or antibodies with a detection compound |
US4973493A (en) | 1982-09-29 | 1990-11-27 | Bio-Metric Systems, Inc. | Method of improving the biocompatibility of solid surfaces |
US4994373A (en) * | 1983-01-27 | 1991-02-19 | Enzo Biochem, Inc. | Method and structures employing chemically-labelled polynucleotide probes |
US4591570A (en) * | 1983-02-02 | 1986-05-27 | Centocor, Inc. | Matrix of antibody-coated spots for determination of antigens |
US4816567A (en) * | 1983-04-08 | 1989-03-28 | Genentech, Inc. | Recombinant immunoglobin preparations |
GB8314523D0 (en) | 1983-05-25 | 1983-06-29 | Lowe C R | Diagnostic device |
US4608112A (en) * | 1984-05-16 | 1986-08-26 | The United States Of America As Represented By The Secretary Of The Air Force | Mask aligner for solar cell fabrication |
AU600885B2 (en) | 1984-05-25 | 1990-08-30 | Zymogenetics Inc. | Stable DNA constructs |
US5096807A (en) | 1985-03-06 | 1992-03-17 | Murex Corporation | Imaging immunoassay detection system with background compensation and its use |
US5523215A (en) * | 1985-03-28 | 1996-06-04 | Chiron Corporation | Enhanced purification and expression of insoluble recombinant proteins |
US5866363A (en) * | 1985-08-28 | 1999-02-02 | Pieczenik; George | Method and means for sorting and identifying biological information |
US4894146A (en) * | 1986-01-27 | 1990-01-16 | University Of Utah | Thin channel split flow process and apparatus for particle fractionation |
US4802951A (en) * | 1986-03-07 | 1989-02-07 | Trustees Of Boston University | Method for parallel fabrication of nanometer scale multi-device structures |
US5643948A (en) * | 1986-06-11 | 1997-07-01 | Procyon Pharmaceuticals, Inc. | Protein kinase C modulators. K. |
US5637489A (en) * | 1986-08-23 | 1997-06-10 | Hoechst Aktiengesellschaft | Phosphinothricin-resistance gene, and its use |
US4859538A (en) | 1986-11-20 | 1989-08-22 | Ribi Hans O | Novel lipid-protein compositions and articles and methods for their preparation |
US5079600A (en) | 1987-03-06 | 1992-01-07 | Schnur Joel M | High resolution patterning on solid substrates |
US4928112A (en) * | 1987-03-23 | 1990-05-22 | Howtek, Inc. | Ink curing apparatus |
US5154808A (en) | 1987-06-26 | 1992-10-13 | Fuji Photo Film Co., Ltd. | Functional organic thin film and process for producing the same |
US4987032A (en) | 1987-06-26 | 1991-01-22 | Fuji Photo Film Co., Ltd. | Functional organic thin film and method of manufacture thereof |
DE3733190A1 (en) * | 1987-10-01 | 1989-04-13 | Kugelfischer G Schaefer & Co | MULTI-ROW BALL OR ROLLER BEARING OR COMBINED BALL ROLLER BEARING |
US6692751B1 (en) * | 1988-05-06 | 2004-02-17 | New York Blood Center | Methods and systems for producing recombinant viral antigens |
US4908112A (en) | 1988-06-16 | 1990-03-13 | E. I. Du Pont De Nemours & Co. | Silicon semiconductor wafer for analyzing micronic biological samples |
US5281540A (en) * | 1988-08-02 | 1994-01-25 | Abbott Laboratories | Test array for performing assays |
US5720928A (en) * | 1988-09-15 | 1998-02-24 | New York University | Image processing and analysis of individual nucleic acid molecules |
SE462454B (en) | 1988-11-10 | 1990-06-25 | Pharmacia Ab | METHOD FOR USE IN BIOSENSORS |
JPH02272081A (en) * | 1989-04-14 | 1990-11-06 | Fuji Photo Film Co Ltd | Functional organic thin film |
IT1229691B (en) | 1989-04-21 | 1991-09-06 | Eniricerche Spa | SENSOR WITH ANTIGEN CHEMICALLY LINKED TO A SEMICONDUCTIVE DEVICE. |
US5424186A (en) * | 1989-06-07 | 1995-06-13 | Affymax Technologies N.V. | Very large scale immobilized polymer synthesis |
US5744101A (en) * | 1989-06-07 | 1998-04-28 | Affymax Technologies N.V. | Photolabile nucleoside protecting groups |
US5143854A (en) | 1989-06-07 | 1992-09-01 | Affymax Technologies N.V. | Large scale photolithographic solid phase synthesis of polypeptides and receptor binding screening thereof |
US5800992A (en) * | 1989-06-07 | 1998-09-01 | Fodor; Stephen P.A. | Method of detecting nucleic acids |
US6346413B1 (en) * | 1989-06-07 | 2002-02-12 | Affymetrix, Inc. | Polymer arrays |
US5252743A (en) * | 1989-11-13 | 1993-10-12 | Affymax Technologies N.V. | Spatially-addressable immobilization of anti-ligands on surfaces |
DE3939973A1 (en) | 1989-12-02 | 1991-06-06 | Basf Ag | TWO-DIMENSIONAL CRYSTALLIZED MACROMOLECULAR LAYERS |
US5283173A (en) * | 1990-01-24 | 1994-02-01 | The Research Foundation Of State University Of New York | System to detect protein-protein interactions |
US5858188A (en) | 1990-02-28 | 1999-01-12 | Aclara Biosciences, Inc. | Acrylic microchannels and their use in electrophoretic applications |
DK0484472T3 (en) | 1990-04-12 | 1997-08-11 | Max Dolder | Method for light-induced immobilization of biomolecules on chemically "inert" surfaces |
US5861254A (en) * | 1997-01-31 | 1999-01-19 | Nexstar Pharmaceuticals, Inc. | Flow cell SELEX |
US5665582A (en) * | 1990-10-29 | 1997-09-09 | Dekalb Genetics Corp. | Isolation of biological materials |
WO1992008788A1 (en) * | 1990-11-19 | 1992-05-29 | The Board Of Trustees Of The University Of Illinois | Mutant orientable proteins and coated substrates |
US5294369A (en) | 1990-12-05 | 1994-03-15 | Akzo N.V. | Ligand gold bonding |
EP0834576B1 (en) * | 1990-12-06 | 2002-01-16 | Affymetrix, Inc. (a Delaware Corporation) | Detection of nucleic acid sequences |
US5384886A (en) * | 1991-04-01 | 1995-01-24 | Xerox Corporation | Process for electronically printing envelopes |
US5763170A (en) * | 1991-04-16 | 1998-06-09 | Amersham International Plc | Method for forming an array of biological particles |
ATE161964T1 (en) * | 1991-10-15 | 1998-01-15 | Multilyte Ltd | BINDING TEST USING A LABELED REAGENT |
US5605662A (en) | 1993-11-01 | 1997-02-25 | Nanogen, Inc. | Active programmable electronic devices for molecular biological analysis and diagnostics |
US6051380A (en) * | 1993-11-01 | 2000-04-18 | Nanogen, Inc. | Methods and procedures for molecular biological analysis and diagnostics |
IL103674A0 (en) * | 1991-11-19 | 1993-04-04 | Houston Advanced Res Center | Method and apparatus for molecule detection |
US5412087A (en) | 1992-04-24 | 1995-05-02 | Affymax Technologies N.V. | Spatially-addressable immobilization of oligonucleotides and other biological polymers on surfaces |
US5384261A (en) | 1991-11-22 | 1995-01-24 | Affymax Technologies N.V. | Very large scale immobilized polymer synthesis using mechanically directed flow paths |
DE69233331T3 (en) * | 1991-11-22 | 2007-08-30 | Affymetrix, Inc., Santa Clara | Combinatorial Polymersynthesis Strategies |
PT1024191E (en) * | 1991-12-02 | 2008-12-22 | Medical Res Council | Production of anti-self antibodies from antibody segment repertoires and displayed on phage |
EP0544969B1 (en) | 1991-12-06 | 1997-03-05 | Ciba-Geigy Ag | Apparatus and method for electrophoretic separation |
CA2064683A1 (en) | 1992-03-26 | 1993-09-27 | Krishna Mohan Rao Kallury | Formation of thermostable enzymes with extra-ordinary heat tolerance by immobilization on phospholipid matrices |
EP0637384B1 (en) | 1992-04-22 | 1996-10-02 | Ecole Polytechnique Federale De Lausanne | Lipid membrane sensors |
US5637469A (en) | 1992-05-01 | 1997-06-10 | Trustees Of The University Of Pennsylvania | Methods and apparatus for the detection of an analyte utilizing mesoscale flow systems |
US5304487A (en) | 1992-05-01 | 1994-04-19 | Trustees Of The University Of Pennsylvania | Fluid handling in mesoscale analytical devices |
US5726026A (en) | 1992-05-01 | 1998-03-10 | Trustees Of The University Of Pennsylvania | Mesoscale sample preparation device and systems for determination and processing of analytes |
JPH0641183A (en) | 1992-07-23 | 1994-02-15 | Mitsubishi Kasei Corp | Monomolecular film of oligonucleotide |
DE4237113B4 (en) * | 1992-11-03 | 2006-10-12 | "Iba Gmbh" | Peptides and their fusion proteins, expression vector and method of producing a fusion protein |
CA2108705A1 (en) * | 1992-11-06 | 1994-05-07 | Richard Barner | Biologically recognizing layers on new ti02 waveguide for biosensors |
US5472881A (en) | 1992-11-12 | 1995-12-05 | University Of Utah Research Foundation | Thiol labeling of DNA for attachment to gold surfaces |
US5532142A (en) * | 1993-02-12 | 1996-07-02 | Board Of Regents, The University Of Texas System | Method of isolation and purification of fusion polypeptides |
US5677196A (en) | 1993-05-18 | 1997-10-14 | University Of Utah Research Foundation | Apparatus and methods for multi-analyte homogeneous fluoro-immunoassays |
US5512492A (en) | 1993-05-18 | 1996-04-30 | University Of Utah Research Foundation | Waveguide immunosensor with coating chemistry providing enhanced sensitivity |
EP1347493A3 (en) | 1993-05-28 | 2005-11-23 | Baylor College Of Medicine | Method and apparatus for desorption and ionization of analytes |
US5861242A (en) | 1993-06-25 | 1999-01-19 | Affymetrix, Inc. | Array of nucleic acid probes on biological chips for diagnosis of HIV and methods of using the same |
US5837832A (en) | 1993-06-25 | 1998-11-17 | Affymetrix, Inc. | Arrays of nucleic acid probes on biological chips |
AU7516694A (en) * | 1993-07-30 | 1995-02-28 | Affymax Technologies N.V. | Biotinylation of proteins |
US5441876A (en) | 1993-07-30 | 1995-08-15 | The United States Of America As Represented By The Secretary Of The Navy | Process for the preparation of headgroup-modified phospholipids using phosphatidylhydroxyalkanols as intermediates |
RU2041262C1 (en) * | 1993-08-11 | 1995-08-09 | Институт молекулярной биологии им.В.А.Энгельгардта РАН | Method for immobilization of water soluble bioorganic compounds on capillary-porous carrier |
JPH0784372A (en) | 1993-09-17 | 1995-03-31 | Res Dev Corp Of Japan | Organic silane modified oxide and modified surface light patterning oxide |
DE4332003C2 (en) | 1993-09-21 | 1996-02-22 | Seeger Stefan | Process for coating surfaces with biomolecules and other receptor molecules |
US5512131A (en) | 1993-10-04 | 1996-04-30 | President And Fellows Of Harvard College | Formation of microstamped patterns on surfaces and derivative articles |
JP3488465B2 (en) * | 1993-10-28 | 2004-01-19 | ヒューストン・アドバンスド・リサーチ・センター | Microfabricated flow-through porosity device for separately detecting binding reactions |
US5429708A (en) | 1993-12-22 | 1995-07-04 | The Board Of Trustees Of The Leland Stanford Junior University | Molecular layers covalently bonded to silicon surfaces |
DE4435728A1 (en) * | 1994-01-19 | 1995-07-20 | Boehringer Mannheim Gmbh | Biotin silane compounds and binding matrix containing these compounds |
ATE221660T1 (en) * | 1994-01-19 | 2002-08-15 | Roche Diagnostics Gmbh | BIOTINE SILANE COMPOUNDS AND BINDING MATRIX CONTAINING THESE COMPOUNDS |
US5623055A (en) * | 1994-01-28 | 1997-04-22 | Prolinx, Inc. | Phenylboronic acid complexes derived from aminosalicylic acid for bioconjugate preparation |
US5594111A (en) * | 1994-01-28 | 1997-01-14 | Prolinx, Inc. | Phenylboronic acid complexes for bioconjugate preparation |
JPH09509485A (en) | 1994-02-09 | 1997-09-22 | アボツト・ラボラトリーズ | Diagnostic flow cell device |
US5514501A (en) | 1994-06-07 | 1996-05-07 | The United States Of America As Represented By The Secretary Of Commerce | Process for UV-photopatterning of thiolate monolayers self-assembled on gold, silver and other substrates |
US6287850B1 (en) * | 1995-06-07 | 2001-09-11 | Affymetrix, Inc. | Bioarray chip reaction apparatus and its manufacture |
US5807522A (en) | 1994-06-17 | 1998-09-15 | The Board Of Trustees Of The Leland Stanford Junior University | Methods for fabricating microarrays of biological samples |
EP0770210A1 (en) | 1994-07-14 | 1997-05-02 | Technobiochip | Biosensor and method and instrument for deposition of alternating monomolecular layers |
US5498545A (en) * | 1994-07-21 | 1996-03-12 | Vestal; Marvin L. | Mass spectrometer system and method for matrix-assisted laser desorption measurements |
SE9403245D0 (en) | 1994-09-26 | 1994-09-26 | Pharmacia Biosensor Ab | Improvements relating to bilayer lipid membranes |
US5620850A (en) | 1994-09-26 | 1997-04-15 | President And Fellows Of Harvard College | Molecular recognition at surfaces derivatized with self-assembled monolayers |
US5571410A (en) | 1994-10-19 | 1996-11-05 | Hewlett Packard Company | Fully integrated miniaturized planar liquid sample handling and analysis device |
US5585069A (en) | 1994-11-10 | 1996-12-17 | David Sarnoff Research Center, Inc. | Partitioned microelectronic and fluidic device array for clinical diagnostics and chemical synthesis |
US5603351A (en) | 1995-06-07 | 1997-02-18 | David Sarnoff Research Center, Inc. | Method and system for inhibiting cross-contamination in fluids of combinatorial chemistry device |
US5688642A (en) | 1994-12-01 | 1997-11-18 | The United States Of America As Represented By The Secretary Of The Navy | Selective attachment of nucleic acid molecules to patterned self-assembled surfaces |
US5622826A (en) | 1994-12-22 | 1997-04-22 | Houston Advanced Research Center | Method for immobilization of molecules on platinum solid support surfaces |
US5814565A (en) | 1995-02-23 | 1998-09-29 | University Of Utah Research Foundation | Integrated optic waveguide immunosensor |
EP0812434B1 (en) | 1995-03-01 | 2013-09-18 | President and Fellows of Harvard College | Microcontact printing on surfaces and derivative articles |
US5629213A (en) | 1995-03-03 | 1997-05-13 | Kornguth; Steven E. | Analytical biosensor |
JPH11503005A (en) | 1995-03-08 | 1999-03-23 | アクゾ ノーベル ナムローゼ フェンノートシャップ | Surface-modified affinity separation membrane |
CA2213854C (en) * | 1995-03-10 | 2010-08-10 | Meso Scale Technologies, Llc | Multi-array, multi-specific electrochemiluminescence testing |
US6140045A (en) * | 1995-03-10 | 2000-10-31 | Meso Scale Technologies | Multi-array, multi-specific electrochemiluminescence testing |
US6309820B1 (en) * | 1995-04-07 | 2001-10-30 | University Of North Carolina At Chapel Hill | Polypeptides having a functional domain of interest and methods of identifying and using same |
US5624711A (en) * | 1995-04-27 | 1997-04-29 | Affymax Technologies, N.V. | Derivatization of solid supports and methods for oligomer synthesis |
US5625184A (en) * | 1995-05-19 | 1997-04-29 | Perseptive Biosystems, Inc. | Time-of-flight mass spectrometry analysis of biomolecules |
US5700642A (en) | 1995-05-22 | 1997-12-23 | Sri International | Oligonucleotide sizing using immobilized cleavable primers |
WO1996038726A1 (en) | 1995-05-30 | 1996-12-05 | Ecole Polytechnique Federale De Lausanne (Epfl) | Covalently immobilized phospholipid bilayers on solid surfaces |
US5776674A (en) | 1995-06-05 | 1998-07-07 | Seq, Ltd | Chemical biochemical and biological processing in thin films |
US6720149B1 (en) * | 1995-06-07 | 2004-04-13 | Affymetrix, Inc. | Methods for concurrently processing multiple biological chip assays |
EP0836418A1 (en) | 1995-06-07 | 1998-04-22 | The Regents Of The University Of California | Microfabricated devices for diagnostic applications |
US5545531A (en) * | 1995-06-07 | 1996-08-13 | Affymax Technologies N.V. | Methods for making a device for concurrently processing multiple biological chip assays |
US6518168B1 (en) | 1995-08-18 | 2003-02-11 | President And Fellows Of Harvard College | Self-assembled monolayer directed patterning of surfaces |
DE69632989T2 (en) * | 1995-10-17 | 2005-08-25 | Combichem, Inc., San Diego | Template for the synthesis of combinatorial libraries in solution |
DE19543232A1 (en) | 1995-11-07 | 1997-05-15 | Knoell Hans Forschung Ev | Production of matrix-bound miniaturised combinatorial polymer and oligomer library |
US5763263A (en) * | 1995-11-27 | 1998-06-09 | Dehlinger; Peter J. | Method and apparatus for producing position addressable combinatorial libraries |
EP0876601B1 (en) | 1995-12-01 | 2004-07-14 | Innogenetics N.V. | Impedimetric detection system and method of production thereof |
DE19548152A1 (en) | 1995-12-22 | 1997-06-26 | Boehringer Mannheim Gmbh | Process for covering a surface with a film of an oligoethylene glycol derivative |
AU723909B2 (en) | 1996-03-15 | 2000-09-07 | President And Fellows Of Harvard College | Method of forming articles and patterning surfaces via capillary micromolding |
CA2250212C (en) | 1996-04-03 | 2010-02-09 | The Perkin-Elmer Corporation | Device and method for multiple analyte detection |
US5942443A (en) | 1996-06-28 | 1999-08-24 | Caliper Technologies Corporation | High throughput screening assay systems in microscale fluidic devices |
US5925552A (en) | 1996-04-25 | 1999-07-20 | Medtronic, Inc. | Method for attachment of biomolecules to medical devices surfaces |
US6165335A (en) | 1996-04-25 | 2000-12-26 | Pence And Mcgill University | Biosensor device and method |
DE69719817T2 (en) | 1996-04-25 | 2003-12-24 | Pence Inc | BIOSENSOR DEVICE AND METHOD |
US5731152A (en) | 1996-05-13 | 1998-03-24 | Motorola, Inc. | Methods and systems for biological reagent placement |
US6075875A (en) * | 1996-09-30 | 2000-06-13 | Microsoft Corporation | Segmentation of image features using hierarchical analysis of multi-valued image data and weighted averaging of segmentation results |
US20030017149A1 (en) * | 1996-10-10 | 2003-01-23 | Hoeffler James P. | Single chain monoclonal antibody fusion reagents that regulate transcription in vivo |
WO1998023948A1 (en) | 1996-11-29 | 1998-06-04 | The Board Of Trustees Of The Leland Stanford Junior University | Arrays of independently-addressable supported fluid bilayer membranes and methods of use thereof |
GB9624927D0 (en) * | 1996-11-29 | 1997-01-15 | Oxford Glycosciences Uk Ltd | Gels and their use |
US5905024A (en) * | 1996-12-17 | 1999-05-18 | University Of Chicago | Method for performing site-specific affinity fractionation for use in DNA sequencing |
US5837860A (en) | 1997-03-05 | 1998-11-17 | Molecular Tool, Inc. | Covalent attachment of nucleic acid molecules onto solid-phases via disulfide bonds |
US6180288B1 (en) * | 1997-03-21 | 2001-01-30 | Kimberly-Clark Worldwide, Inc. | Gel sensors and method of use thereof |
WO1998050773A2 (en) | 1997-05-08 | 1998-11-12 | University Of Minnesota | Microcantilever biosensor |
US6190619B1 (en) * | 1997-06-11 | 2001-02-20 | Argonaut Technologies, Inc. | Systems and methods for parallel synthesis of compounds |
NZ516848A (en) * | 1997-06-20 | 2004-03-26 | Ciphergen Biosystems Inc | Retentate chromatography apparatus with applications in biology and medicine |
US5948621A (en) * | 1997-09-30 | 1999-09-07 | The United States Of America As Represented By The Secretary Of The Navy | Direct molecular patterning using a micro-stamp gel |
US6548021B1 (en) * | 1997-10-10 | 2003-04-15 | President And Fellows Of Harvard College | Surface-bound, double-stranded DNA protein arrays |
US6061476A (en) * | 1997-11-24 | 2000-05-09 | Cognex Corporation | Method and apparatus using image subtraction and dynamic thresholding |
US6232066B1 (en) * | 1997-12-19 | 2001-05-15 | Neogen, Inc. | High throughput assay system |
EP1965213A3 (en) | 1998-02-04 | 2009-07-15 | Invitrogen Corporation | Microarrays and uses therefor |
US6087103A (en) * | 1998-03-04 | 2000-07-11 | Lifespan Biosciences, Inc. | Tagged ligand arrays for identifying target-ligand interactions |
JP4163383B2 (en) * | 1998-04-14 | 2008-10-08 | カリフォルニア・インスティテュート・オブ・テクノロジー | Method and system for determining analyte activity |
US6287765B1 (en) * | 1998-05-20 | 2001-09-11 | Molecular Machines, Inc. | Methods for detecting and identifying single molecules |
US6682942B1 (en) * | 1998-07-14 | 2004-01-27 | Zyomyx, Inc. | Microdevices for screening biomolecules |
US6406921B1 (en) * | 1998-07-14 | 2002-06-18 | Zyomyx, Incorporated | Protein arrays for high-throughput screening |
US6576478B1 (en) * | 1998-07-14 | 2003-06-10 | Zyomyx, Inc. | Microdevices for high-throughput screening of biomolecules |
US20020119579A1 (en) * | 1998-07-14 | 2002-08-29 | Peter Wagner | Arrays devices and methods of use thereof |
US6897073B2 (en) * | 1998-07-14 | 2005-05-24 | Zyomyx, Inc. | Non-specific binding resistant protein arrays and methods for making the same |
US6197599B1 (en) * | 1998-07-30 | 2001-03-06 | Guorong Chin | Method to detect proteins |
US6190908B1 (en) * | 1998-08-12 | 2001-02-20 | The Scripps Research Institute | Modulation of polypeptide display on modified filamentous phage |
AU3387700A (en) | 1999-03-02 | 2000-09-21 | Chiron Corporation | Microarrays for identifying pathway activation or induction |
CA2365431A1 (en) | 1999-03-10 | 2000-09-14 | Hui Ge | Universal protein array system |
AU3883300A (en) | 1999-03-11 | 2000-09-28 | Combimatrix Corporation | Microarrays of peptide affinity probes for analyzing gene products and methods for analyzing gene products |
KR100379411B1 (en) * | 1999-06-28 | 2003-04-10 | 엘지전자 주식회사 | biochip and method for patterning and measuring biomaterial of the same |
US6899137B2 (en) * | 1999-06-28 | 2005-05-31 | California Institute Of Technology | Microfabricated elastomeric valve and pump systems |
US6406840B1 (en) * | 1999-12-17 | 2002-06-18 | Biomosaic Systems, Inc. | Cell arrays and the uses thereof |
CA2401118A1 (en) * | 2000-02-23 | 2001-08-30 | Zyomyx, Inc. | Microfluidic devices and methods |
US6531283B1 (en) * | 2000-06-20 | 2003-03-11 | Molecular Staging, Inc. | Protein expression profiling |
US7094568B2 (en) * | 2000-08-17 | 2006-08-22 | Sense Proteomic Ltd. | Method for producing proteins tagged at the N- or C-terminus |
US6699665B1 (en) * | 2000-11-08 | 2004-03-02 | Surface Logix, Inc. | Multiple array system for integrating bioarrays |
EP1451579A4 (en) * | 2001-11-19 | 2005-12-28 | Protometrix Inc | Method of using a non-antibody protein to detect and measure an analyte |
US20050026215A1 (en) * | 2003-07-17 | 2005-02-03 | Predki Paul F. | Method for the prediction of an epitope |
-
1998
- 1998-07-14 US US09/115,455 patent/US6406921B1/en not_active Expired - Lifetime
-
1999
- 1999-07-14 JP JP2000560456A patent/JP2002520620A/en active Pending
- 1999-07-14 JP JP2000560449A patent/JP2002520618A/en active Pending
- 1999-07-14 DE DE69938867T patent/DE69938867D1/en not_active Expired - Lifetime
- 1999-07-14 WO PCT/US1999/015971 patent/WO2000004382A1/en active IP Right Grant
- 1999-07-14 AT AT99935571T patent/ATE397752T1/en not_active IP Right Cessation
- 1999-07-14 EP EP99935573A patent/EP1097380A1/en not_active Ceased
- 1999-07-14 CA CA002337654A patent/CA2337654A1/en not_active Abandoned
- 1999-07-14 CA CA002337075A patent/CA2337075A1/en not_active Abandoned
- 1999-07-14 US US09/353,555 patent/US6329209B1/en not_active Expired - Lifetime
- 1999-07-14 AU AU51025/99A patent/AU765508B2/en not_active Ceased
- 1999-07-14 AU AU51023/99A patent/AU773068B2/en not_active Ceased
- 1999-07-14 WO PCT/US1999/015968 patent/WO2000004389A2/en active IP Right Grant
- 1999-07-14 EP EP99935571A patent/EP1097377B1/en not_active Expired - Lifetime
- 1999-07-14 US US09/353,215 patent/US6475808B1/en not_active Expired - Fee Related
-
2000
- 2000-05-12 US US09/570,363 patent/US6630358B1/en not_active Expired - Fee Related
- 2000-05-12 US US09/570,588 patent/US6475809B1/en not_active Expired - Fee Related
- 2000-05-18 US US09/574,748 patent/US6365418B1/en not_active Expired - Lifetime
-
2002
- 2002-03-26 US US10/107,122 patent/US20030003599A1/en not_active Abandoned
- 2002-03-29 US US10/113,964 patent/US20020110933A1/en not_active Abandoned
- 2002-03-29 US US10/112,840 patent/US20020106702A1/en not_active Abandoned
-
2004
- 2004-08-04 US US10/911,945 patent/US20050008674A1/en not_active Abandoned
- 2004-08-04 US US10/911,877 patent/US20050014292A1/en not_active Abandoned
-
2006
- 2006-03-28 US US11/392,262 patent/US20060228701A1/en not_active Abandoned
-
2008
- 2008-08-15 US US12/192,321 patent/US20090131278A1/en not_active Abandoned
-
2010
- 2010-11-19 US US12/950,698 patent/US20110086779A1/en not_active Abandoned
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ATE397752T1 (en) | 2008-06-15 |
US20110086779A1 (en) | 2011-04-14 |
DE69938867D1 (en) | 2008-07-17 |
US20020106702A1 (en) | 2002-08-08 |
US20090131278A1 (en) | 2009-05-21 |
EP1097377B1 (en) | 2008-06-04 |
JP2002520620A (en) | 2002-07-09 |
EP1097380A1 (en) | 2001-05-09 |
CA2337075A1 (en) | 2000-01-27 |
US20020110933A1 (en) | 2002-08-15 |
WO2000004382A8 (en) | 2001-03-15 |
AU773068B2 (en) | 2004-05-13 |
US6630358B1 (en) | 2003-10-07 |
US6406921B1 (en) | 2002-06-18 |
JP2002520618A (en) | 2002-07-09 |
AU765508B2 (en) | 2003-09-18 |
AU5102399A (en) | 2000-02-07 |
WO2000004389A3 (en) | 2000-04-27 |
WO2000004389A2 (en) | 2000-01-27 |
AU5102599A (en) | 2000-02-07 |
US6329209B1 (en) | 2001-12-11 |
US6365418B1 (en) | 2002-04-02 |
US6475809B1 (en) | 2002-11-05 |
US20030003599A1 (en) | 2003-01-02 |
US6475808B1 (en) | 2002-11-05 |
WO2000004382A1 (en) | 2000-01-27 |
EP1097377A2 (en) | 2001-05-09 |
US20050008674A1 (en) | 2005-01-13 |
US20060228701A1 (en) | 2006-10-12 |
US20050014292A1 (en) | 2005-01-20 |
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