JP2005535898A - Methods and reagents for surface functionalization - Google Patents

Abstract

Reagents and methods for creating an array of affinity agents are disclosed. Also disclosed is a method of using an array of affinity agents. Arrays are particularly useful for high throughput drug screening and clinical diagnostic applications.

Description

Cross-reference to related applications This application is a US Provisional Application No. 60 / 403,971 entitled “Biochip Surface Configurations and Methods and Compositions for Making the Same” filed on August 16, 2002 in the name of David Quincy. Claims the benefit of (incorporated herein by reference).

FIELD This disclosure relates to a class of reagents and methods for surface functionalization to create arrays of bioactive compounds on a substrate.

Background A comprehensive understanding of disease states requires the identification and characterization of all proteins involved in a particular disease pathway. This study of proteins and their interactions is termed “proteomics”. Advances in molecular biology can produce the protein of interest faster than the current technology can determine protein properties. For example, conventional protein identification methods in proteomics applications rely on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) techniques for protein isolation, followed by identification by mass spectrometry as follows. Typically, 2D-PAGE can separate as many as 5000 different proteins. However, the identification of each protein is a tedious and tedious process that requires each individual separated protein to be cleaved from the polyacrylamide gel. Furthermore, this method is relatively insensitive. For example, when silver staining is used, the detection limit is about 1 nanogram of protein.

  Further disadvantages of 2D-PAGE include lack of reproducibility, low throughput, low resolution, and sensitivity of the protein. For example, 2D-PAGE generally does not degrade all proteins present in the mixture, and the system is limited to a slight gel treatment in 2 days. In addition, lower molecular weight, lower molecular weight, and membrane bound proteins are shown.

  Attempts have been made to use protein arrays for high-throughput characterization of proteins. For example, Wagner et al., US Pat. No. 6,406,921, discloses a method for making a protein-coated substrate, and Bamdad et al., US Pat. No. 5,620,850, describes multiple chelating agents that can be used to bind metal ions. Is disclosed. It is then reported that bound metal ions are used to capture biomolecules (including chelating agents).

  In order to provide a high quality protein array that produces accurate and reproducible screening results, a number of difficulties must be overcome. Typically, proteins must be maintained in a hydrated state and maintained at ambient temperature, and are very sensitive to the physical and chemical properties of the support material. Thus, maintaining protein activity at the liquid-solid interface requires new strategies for assembly of arrays that address the sensitivity of the protein to the environment.

SUMMARY Disclosed herein are reagents and methods for using such reagents to prepare substrates derivatized with affinity agents such as small molecules, peptides, proteins, nucleic acids, and other bioactive molecules. . As disclosed herein, densely packed affinity agent arrays that exhibit minimal and non-specific binding can be prepared.

  In one embodiment of the method, the substrate is functionalized with a reagent having at least two reactive functional groups. The first reactive functional group acts to couple the reagent to the substrate, and the second reactive functional group is an affinity agent or operatively acts on the affinity agent. Provides a site for binding.

  In a second embodiment of the method, non-specific protein attachment to the array is reduced by the introduction of a protein resistant component that reduces such non-specific protein attachment. Accordingly, in one aspect, the present disclosure provides reagents and techniques for assembling arrays of affinity agents that exhibit reduced non-specific binding.

DETAILED DESCRIPTION In order to better describe the compounds, compositions and methods of the present invention and to guide those skilled in the art in practicing the present disclosure, the following explanations of terms and methods are provided. The terminology used in the present disclosure is intended to describe only specific aspects and specific examples and is not intended to be limiting.

A. Definitions As used herein, the term “substrate” refers to the body or base material used in the arrays and devices disclosed herein.

  “Array” refers to a two-dimensional distribution or pattern.

  The terms “polypeptide” and “protein” are used interchangeably to refer to an amino acid polymer.

  The term “antibody” refers to a naturally occurring or fully or partially synthetically produced immunoglobulin. All of its derivatives that maintain specific binding ability are included in the term. The term also encompasses any protein having a binding domain that is homologous or nearly homologous to an immunoglobulin binding domain. These proteins can be derived from natural sources or can be partially or fully synthetically produced. The antibody can be monoclonal or polyclonal. In an exemplary embodiment, the antibody is a glycosylated antibody. The antibody can be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. In an exemplary embodiment, the antibody is of the IgG class.

The term “antibody fragment” refers to any derivative of an antibody that is shorter than its full length. In an exemplary embodiment, the antibody fragment retains at least a significant portion of the full-length antibody's specific binding ability. Specific examples of antibody fragments include, but are not limited to, Fab, Fab ′, F (ab ′) ₂ , scFv, Fv, dsFv bispecific antibodies (diabody), and Fd fragments. Antibody fragments can be generated by any means. For example, antibody fragments can be generated enzymatically or chemically by fragmenting intact antibodies, or recombinantly generated from a gene encoding a partial sequence of the antibody. Alternatively, antibody fragments can be generated fully or partially synthetically. The antibody fragment is optionally a single chain antibody fragment. Alternatively, a fragment can include multiple chains linked by, for example, disulfide bonds. The fragment can optionally be a multimolecular complex. A functional antibody fragment typically comprises at least about 50 amino acids and more typically comprises at least about 200 amino acids. Single chain Fvs (scFvs) are recombinant antibody fragments consisting only of a variable light chain (V _L ) and a variable heavy chain (V _H ) covalently linked together by a polypeptide linker. Either V _L or V _H may be an NH ₂ terminal domain. Polypeptide linkers can be of various lengths and compositions as long as the two variable domains are cross-linked without severe steric hindrance. Typically, the linker consists mainly of a stretch of glycine and serine residues with several glutamic or lysine residues interspersed for solubility.

An Fv fragment is an antibody fragment consisting of one V _H and one _VL domain held together by non-covalent interactions. As used herein, the term “dsFv” refers to an Fv having an intermolecular disulfide bond that has been engineered to stabilize the V _H -V _L pair.

F (ab ′) ₂ fragments are antibody fragments that are essentially equivalent to fragments obtained from immunoglobulins (typically IgG) by digestion with the enzyme pepsin at pH 4.0-4.5. Fragments can be produced recombinantly.

A Fab ′ fragment is an antibody fragment that is essentially equivalent to a fragment resulting from the reduction of a disulfide bridge connecting two heavy chain pieces in an F (ab ′) ₂ fragment. Fab ′ fragments can be produced recombinantly.

  A Fab fragment is an antibody fragment that is essentially equivalent to a fragment obtained from digestion of an immunoglobulin (typically IgG) with the enzyme papain. Fab fragments can be produced recombinantly.

  “Amino acid” refers to both naturally occurring and “non-natural” amino acids. Amino acid residues are also encompassed in the term amino acid.

  “Organic thin film” refers to a thin layer of organic molecules formed directly on a substrate or formed on a coating on the substrate. Typically, the organic thin films disclosed herein are about 0.5 nm to about 50 nm thick, more typically about 0.5 nm to about 10 nm thick. The organic film can be assembled prior to attachment to the substrate, but is typically assembled when the component molecules are attached to the substrate. The organic thin film optionally includes bound inorganic ions and a chelated metal bound to the thin film. The organic thin film can be uniform or non-uniform and can consist of one or more monolayers. An example of a thin film composed of a plurality of monomolecular layers is a lipid bilayer. However, typically, the organic thin film is a monolayer. In some cases, the organic film can include more than one type of organic film.

  Typically, the organic thin film disclosed herein comprises an affinity agent or functional group suitable for covalently or non-covalently binding an affinity agent to the thin film. The organic thin film may optionally have a functional group that reduces molecular bonds with the thin film. Typically, such functional groups are hydrophilic groups such as polyalkylene oxides including polyethylene glycol (PEG) and polypropylene glycol (PPG), which are generally not tightly bound by proteins. PEG and PPG include oligomers of ethylene glycol and propylene glycol, respectively. Thus, PEG and PPG as used herein refer to a polymer having as few as two glycol subunits. In an embodiment, PEG was used as an anti-protein component of the organic thin film to reduce non-specific binding of proteins to the organic thin film. In some examples, the PEG component used was not monodispersed, so the specific number of ethylene glycol units referred to can also refer to the average number when using a polydisperse mixture of PEG units. . Other functional groups of the organic thin film act to tether the thin film to the surface of the substrate or to the coating on the substrate.

  The term “monomolecular layer” refers to a layer having a monomolecular thickness that can be an organic thin film or a portion thereof. The monolayer may be regular or may not be regular. Typically, monolayers are regularly ordered and densely packed. Monolayers can be uniform or non-uniform, but one side of the monolayer contains functional groups that can chemisorb or physisorb onto the surface of the substrate or a coating on the substrate.

  It is also possible to convert the first monolayer into a second monolayer. For example, a component molecule in a first monolayer having a first end that is chemisorbed or physisorbed to a substrate, one or more second molecules, a plurality of first monolayer component molecules It can be functionalized by covalent bonding to the second end, thereby converting the first monolayer into a new second monolayer.

B. Arrays of Affinity Agents The present disclosure relates to arrays of affinity agents and methods for making and using such arrays. Typically, the array forms a two-dimensional display of affinity agents that can be used to characterize the interaction between the affinity agent and the soluble molecule of interest.

  Thus, in one aspect, the disclosed functionalized substrate is further functionalized with an affinity agent. An affinity agent can be any agent that can bind to a functionalized substrate and interact covalently or non-covalently with a molecule of interest. Typically, an affinity agent is a small molecule, oligonucleotide, peptide, or protein that binds or interacts with a soluble molecule of interest. Similarly, a soluble molecule or analyte of interest can be a small molecule, oligonucleotide, peptide, or protein. The interaction between the affinity agent and the analyte can be detected by any suitable method, and in embodiments, ultraviolet and visible absorption, chemiluminescence, and fluorescence (lifetime, polarization, fluorescence correlation spectroscopy ( FCS) and optical detection methods including fluorescence resonance energy transfer (FRET) were used.

  In one aspect, the array of affinity agents is formed on at least a portion of the substrate, the substrate surface, and is operably linked to the underlying organic film (eg, covalently or non-covalently bound). A) comprising an organic film comprising a plurality of copies of at least one affinity agent.

  In general, when multiple types of affinity agents are used in the same array, different types of affinity agents are grouped together in a “patch” so that the affinity effect is localized in a patch of the array. The substance is different from the affinity agent that is localized in another patch. In one aspect, the present invention provides a plurality of substrates disposed in separate known regions on a substrate, at least one organic film on some or all of the substrate surface, and a portion of the substrate surface covered by the organic film. Of glycoproteins, each containing a protein immobilized on an underlying organic film. The array optionally contains an intermediate layer between the substrate and the coating.

In most cases, the array contains at least about 10 patches. In an exemplary embodiment, the array includes at least about 50 patches. In another exemplary embodiment, the array comprises at least about 100 patches. In exemplary alternative embodiments, the array of affinity agents can comprise a number of more than 10 ³ , 10 ⁴ or 10 ⁵ patches.

In an exemplary embodiment, the surface area of the substrate covered by each patch is about 0.25 mm ² or less. In another exemplary embodiment, the substrate surface area covered by each patch is between about 1 μm ² and about 10,000 μm ² . In another exemplary embodiment, each patch covering a substrate surface area of about 100 [mu] m ² ~ about 2,500 ^2. In an alternative embodiment, the patch on the array may cover the substrate surface area of only about 2,500 nm ^2, patches of such small size, the array is not generally necessary in order to be useful.

  The patches of the array can be any geometric shape. For example, the patch can be rectangular or circular. Array patches can also be irregularly shaped.

  The distance separating the patches of the array can vary. For example, the patches in the array are about 1 μm to about 500 μm away from neighboring patches. If the patch has a size greater than about 10 μm, typically the distance separating the patches is approximately proportional to the diameter of the patch or the length of the side on the array. If the patch size is smaller, the distance separating the patches is typically greater than the patch dimensions.

In an exemplary embodiment of the array, the array patches are all contained within an area of about 1 cm ² or less on the substrate surface. Thus, in one exemplary embodiment of the array, the array comprises 100 or more patches within a total area of about 1 cm ² or less on the surface of the substrate. Alternatively, a typical array includes 10 ³ or more patches within a total area of about 1 cm ² or less. Exemplary arrays may further optionally include 10 ⁴ or 10 ⁵ or more patches in an area of about 1 cm ² or less on the substrate surface. In other embodiments of the invention, all patches of the array are contained within an area of about 1 cm ² or less on the substrate surface.

  In one embodiment, only one type of affinity agent is immobilized on each patch of the array. In an exemplary embodiment of the array, the affinity agent immobilized on one patch is different from the affinity agent immobilized on a second patch of the same array. In such embodiments, a plurality of different affinity agents can be present on separate patches of the array. In another embodiment, a single patch includes two or more affinity agents that bind to the same analyte. Such affinity agents typically bind to different epitopes of the analyte. One class of affinity agents that can be used in this embodiment are polyclonal antibodies.

Typically, the array includes at least about 10 different affinity agents. In an exemplary embodiment, the array includes at least about 50 different affinity agents. In another exemplary embodiment, the array comprises at least about 100 different affinity agents. Alternative exemplary arrays include more than about 10 ³ different affinity agents or more than about 10 ⁴ different affinity agents. The array further optionally includes more than about 10 ⁵ different affinity agents.

  In one aspect of the array, each of the patches of the array contains a different affinity agent. For example, an array comprising about 100 patches could contain about 100 different affinity agents. Similarly, an array of about 10,000 patches could contain about 10,000 different affinity agents. However, in an alternative embodiment, each different affinity agent is immobilized on one or more separate patches on the array. For example, each different affinity agent can optionally be present on 2-6 different patches. Thus, an array can contain approximately 3000 affinity agent patches, but only about 1000 different affinity agents since each different agent is present on 3 different patches. It is.

  In another embodiment, the affinity agent of one patch is different from the affinity agent of another patch, but the affinity agent is related. In an exemplary embodiment, the two different affinity agents are members of the same protein family. Different proteins on the array can be considered functionally related or some functionally related. However, in another embodiment of the array of the present invention, the function of the protein to be immobilized may not be known. In this case, different glycoproteins on different patches of the array typically share similarities in structure or sequence, or appear to share similarities in structure or sequence. Alternatively, the protein to be immobilized can be a fragment of a different member of the protein family.

  Any affinity agent that can be operatively associated with an organic film on a substrate can be used in the disclosed array. Different classes of affinity agents include, but are not limited to, small molecules, peptides, proteins, and nucleic acids including DNA and RNA. In some cases, the array can include different classes of different affinity agents.

  If a protein is selected as an affinity agent, the protein is a receptor family (examples include growth factor receptors, catecholamine receptors, amino acid derivative receptors, cytokine receptors, and lectins); ligand families (Examples include cytokines and serpins); enzyme families (examples include proteases, kinases, phosphatases, ras-like GTPases, and hydrolases); and transcription factors (specific examples include steroids) Hormone members, heat shock transcription factors, zinc finger proteins, leucine zipper proteins, and homeodomain proteins)). In one aspect, the different immobilized proteins are all HIV protease or hepatitis C virus (HCV) protease. In another embodiment, all bound proteins on the array are hormone receptors, neurotransmitter receptors, extracellular matrix receptors, antibodies, DNA-binding proteins, intracellular signaling regulators and effectors, apoptosis-related factors, DNA synthesis factor, DNA repair factor, DNA recombination factor, or cell surface antigen.

  Antibodies and antibody fragments are particularly useful affinity agents for use in the disclosed arrays. The antibody can optionally be a polyclonal or monoclonal antibody. Production and isolation of antibodies that bind to specific targets, including protein targets, using standard hybridoma technology are known to those skilled in the art. In addition, many antibodies are commercially available. Alternatively, the antibody or antibody fragment can be expressed in a bacteriophage. Such antibody phage display techniques, including bacteriophage selection methods, are well known to those skilled in the art.

C. Reagents and Techniques for Preparing Arrays of Affinity Agents In one aspect, the disclosure provides reagents and techniques for assembly of an array of affinity agents. According to one aspect of the method, the first heterobifunctional reagent is covalently or non-covalently bound to the substrate, thereby forming a monolayer. Reactive by coupling a second heterobifunctional reagent to a monolayer to provide an array of reactive functional groups and a third reagent containing an anti-protein component to the monolayer An organic thin film containing a functional group is formed by three reagents. Reactive functional groups are selected such that the affinity agent is coupled to the organic film, thereby forming an affinity agent array in which the affinity agent is operatively coupled to the organic film. Coupling involves covalently or non-covalently binding affinity agents. Non-covalent bonds can use, but are not limited to, one or more of Coulomb interactions, hydrogen bonds, van der Waals interactions, and hydrophobic interactions.

  In one aspect, the affinity agent is coupled to the organic film by a chemoselective ligation reaction. Chemoselective ligation generally involves a reaction between functional groups that have orthogonal reactivity to other functional groups present, particularly those that are found in many biological molecules. Say. Thus, chemoselective ligation is particularly useful when the affinity agent is a biomolecule. Typically, chemoselective ligation reactions used with biomolecules use one or more non-native functional groups to ensure that the reaction is orthogonal to the native functional group. One specific example of a chemoselective ligation reaction is Staudinger ligation. Other examples include the reaction of ketones and aldehydes, such as condensing a hydrazide or aminooxy compound with a ketone or aldehyde to produce the corresponding hydrazone or oxime. Another example is a reaction in which a thioester is obtained by reacting a thiocarboxylate with an α-halocarbonyl compound. Using these reaction types, compounds other than biomolecules can be attached to the organic thin film.

In a representative method disclosed herein, a reagent or affinity agent is bound to an organic film using a Staudinger ligation reaction. The Staudinger linkage functions as an amide bond forming reaction and typically involves two reactive components (the first typically has the formula YZ-PR ² R ³ (wherein , Z is an aryl group substituted with R ¹ , where R ¹ is preferably ortho to the PR ² R ³ on the aryl ring; where R ¹ is an aza-ylide group Electrophilic groups for trapping (eg, stabilizing) (carboxylic acids, esters (eg, alkyl esters (eg, lower alkyl esters, benzyl esters), aryl esters, substituted aryl esters), aldehydes, amides, eg , Alkylamides (eg lower alkylamides), arylamides, halogenated alkyls (eg halogenated lower alkyls), thioesters, sulfonyl esters, alkyl ketones (eg For example, lower alkyl ketones), aryl ketones, substituted aryl ketones, halosulfonyl, nitriles, nitro, etc., but are not necessarily limited); R ² and R ³ are generally aryl groups ( Substituted aryl groups or cycloalkyl groups (including cyclohexyl groups, for example), wherein R ² and R ³ may be the same or different, preferably the same; Y is H, antiprotein A reactive group that facilitates covalent attachment of a sex group, affinity agent or molecule of interest, where Y is at any position on the aryl group (eg, para, meta, ortho) Where representative reactive groups include carboalkyl, amine (eg, alkylamine (eg, lower alkylamine), arylamine), ester (eg, alkylester) For example, lower alkyl ester, benzyl ester), aryl ester, substituted aryl ester), thioester, sulfonyl halide, alcohol, thiol, succinimidyl ester, isothiocyanate, iodoacetamide, maleimide, hydrazine, etc. Exemplary affinity agents further include dyes (eg, fluorescein or modified fluorescein), antibodies, toxins (including cytotoxins), linkers, peptides, etc. Representative and preferred. The engineered phosphine reactant is 2-diphenylformanyl-benzoic acid methyl ester.

  The second reagent includes azide. Molecules containing azide and suitable for use in the present invention and methods for producing azide-containing molecules suitable for use in the present invention are well known to those skilled in the art.

In one embodiment, a monolayer comprising a 2-diphenylformanyl-benzoic acid methyl ester derivative was prepared on a gold substrate. According to this embodiment, the first reagent, 11-thio-undecanionic-N-hydroxysuccinimide ester, was bound to the substrate via a sulfhydryl group. If the second monolayer is suitable for coupling an affinity agent containing an azide moiety, the resulting monolayer is derivatized with compound 25, thereby causing the first monolayer to the second monolayer. A molecular layer was formed. The fluorescently labeled azidoalanine-containing peptide was then coupled to the second monolayer by Staudinger ligation.

  In another embodiment of the method, it is attached to the organic film using the native functional group of the biomolecule. For example, a protein having one or more cysteine residues can be selectively alkylated with a reagent such as an α-halocarbonyl compound. When the α-halocarbonyl compound is bound to the thin film, this can be a useful reaction for binding certain proteins to the thin film.

  In a parallel manner, natural or unnatural amino acids can be introduced into peptides or proteins to provide functional groups for attaching proteins to organic thin films. For example, as discussed above, in an embodiment, an azide-containing amino acid is introduced into a peptide and then the peptide is attached to the organic film by a Staudinger ligation protocol.

  In one aspect, the substrate is functionalized with a chelating agent such as an N-nitrilotriacetic acid (NTA) derivative or an imidodiacetic acid (IDA) derivative that chelates metals such as nickel, cobalt, iron, and copper. With such derivatives, histidine-labeled proteins are non-covalently bound to the substrate, for example, by mutual nickel chelation with both a substrate-bound chelator (such as an NTA or IDA derivative) and a histidine label. Proteins with a histidine tag are known to those skilled in the art. See Hochuli, et al., Biotechnology, 1988, 6, 1321. In another embodiment of this method of attaching an affinity agent, a covalent bond can be formed between the affinity agent and the substrate-bound organic film following the initial non-covalent bond. This can be achieved, for example, by using photoactivatable groups such as aryl azides, in particular haloaryl azides, for example pentafluorophenyl aryl azide, benzophenone, diazo compounds, in particular diazopyruvate. Further suitable photoactivatable groups are taught by Hermanson, G.T. Bioconjugate Techniques, Academic Press: San Diego, 1996 (incorporated herein by reference).

  In the second example, the first affinity agent is a multivalent protein that is bound to the substrate-bound organic thin film. The first affinity agent then serves as an attachment site for a second affinity agent having a portion for binding to the first affinity agent and a portion for interacting with the molecule of interest. Works. In a specific example, the first affinity agent is streptavidin bound to the substrate via a biotin derivative presented on the substrate-bound organic thin film, and the second affinity agent is a biotin conjugated molecule. It is. In that case, since streptavidin has a plurality of biotin binding sites, the biotin-conjugated molecule binds to the organic thin film via streptavidin.

  One aspect of the method includes one or more optionally performed “quenching” steps. Typically, the quench step is performed to inactivate reactive functional groups that are bound to the organic film. In an embodiment, cysteine was used to quench the thiol reactive functional group attached to the film. In other embodiments, glycine was used to quench the amino reactive functional group attached to the film. Suitable quenching agents and procedures can be selected as known to those skilled in the art, but are typically similar to the functional groups used to attach the reagent or affinity agent to the organic film. A quenching reagent having a functional group having a property can be selected.

  In one embodiment of the array of affinity agents, an array of specifically oriented immobilized antibodies is provided. The antibody is immobilized by conjugating a biotin molecule to the antibody. In one example, the antibody is first conjugated to a biotin molecule by oxidizing the vicinal diol functionality on the antibody glycosyl moiety to form the corresponding aldehyde. The aldehyde of the glycosyl moiety is reacted with a biotin molecule functionalized with an aminooxy group. This reaction results in an antibody-biotin compound covalently bonded via an oxime bond. Finally, the antibody-biotin compound is immobilized on an organic thin film containing a streptavidin compound.

  Additional reagents and functional groups useful for monolayer assembly and conjugation of affinity reagents to organic films are disclosed in Hermanson, GT Bioconjugate Techniques, Academic Press: San Diego, 1996 (incorporated herein by reference). You can choose from

  One disclosed embodiment for derivatizing a substrate is shown in FIG. Referring to FIG. 1, the substrate 2 is functionalized with a reagent 4 having at least two functional groups X and Y.

  The classes of materials useful as the substrate 2 to form the arrays disclosed herein include inorganic materials, metals, and organic polymers. Examples of inorganic materials include silicon, silica, quartz, glass, controlled pore glass, carbon, alumina, titanium oxide, tantalum oxide, indium tin oxide, germanium, silicon nitride, gallium arsenide, zeolite, Mica and combinations thereof are included. Useful metals include aluminum, copper, gold, platinum, titanium, alloys thereof, and combinations thereof. Examples of useful polymers include polyethylene, polyethyleneimine, polyvinylethylene, polystyrene, poly (tetra) fluoroethylene, polycarbonate, polymethyl methacrylate, polydimethylsiloxane, polyvinylphenol, polyoxymethylene, polymethacrylamide, polyimide, and Copolymers of such materials are included, but are not limited. In some cases, the substrate is typically coated with one or more different materials selected from those listed above. For example, the coating can optionally be a metal film. Possible metal films include aluminum, chromium, titanium, tantalum, nickel, stainless steel, zinc, lead, iron, copper, magnesium, manganese, cadmium, tungsten, cobalt, and alloys or oxides thereof. In an exemplary embodiment, the metal film is a noble metal film. Noble metals that can be used for coating include, but are not limited to, gold, platinum, silver, and copper. Electron or ion beam evaporation can be used to apply a thin coating of gold on the substrate surface. In another exemplary embodiment, the coating comprises a precious metal alloy. The metal film has a thickness in the range of about 50 nm to about 1,000 nm, and more specifically has a thickness of about 100 nm to about 500 nm. In an alternative aspect, the coating may comprise silicon, silicon oxide, silicon nitride, titanium oxide, tantalum oxide, silicon nitride, silicon hydride, indium tin oxide, magnesium oxide, alumina, glass, hydroxylated surface, and polymer. it can.

  X can be any group that causes chemical or physical adsorption of reagent 4 on substrate 2 and thereby forms a monolayer. X is typically a group that reacts chemically with the substrate 2, and thus X is selected to be compatible with the substrate material. For example, if the substrate is an oxide such as silicon oxide, indium tin oxide, magnesium oxide, alumina, quartz, glass, or a material such as silicon or aluminum having an oxidized surface layer, silane and siloxane are A group useful for functionalizing a substrate. For example, halosilanes (including monohalosilanes, dihalosilanes, and trihalosilanes) can be used to attach reagents to such substrates. Useful siloxanes that can be used to attach functional groups to a substrate include monoalkoxysilanes, dialkoxysilanes, and trialkoxysilanes. In an embodiment, when the substrate 2 was silicon oxide, X was a siloxane such as triethoxysiloxane.

  Examples of suitable X groups for derivatizing metal substrates or metal coatings on substrates include sulfur-containing compounds (thiols, thioethers (sulfides), isothiocyanates, xanthanates, thioacids, thiocarbamates, disulfides ( Including symmetric and asymmetric disulfides), dithioacids (including symmetric and asymmetric dithioacids), and sulfur-containing heterocycles; selenium-containing molecules (including selenols, selenides, and diselenides (including symmetric and asymmetric diselenides)) Nitrogen containing compounds (such as primary, secondary, and possibly tertiary amines, amino oxides, pyridines, isocyanates, isonitriles, nitriles, and hydroxamic acids); phosphorus containing compounds (such as phosphines); and oxygen containing Compound (carbochelate), hydroxyl-containing compound (Al Etc. Lumpur), and mixtures thereof.

  Sulfur-containing compounds are particularly useful for functionalizing silver, gold, and platinum surfaces. When the substrate is a metal such as silver, gold, or platinum, thiols and disulfides are preferred reagents for functionalizing the substrate. In an embodiment, various reagents were attached to the gold surface using thiols and disulfides.

  In other embodiments, the substrate surface (or coating thereon) comprises a metal oxide such as titanium oxide, tantalum oxide, indium tin oxide, magnesium oxide, or alumina, and X is a carboxylic acid. Alternatively, if the device substrate surface (or coating thereon) is copper, X is typically hydroxamic acid.

  When the substrate used in the present invention is a polymer, a coating on the substrate, such as a copper coating, is often included in the device. The appropriate functional group X for coating is then selected for use in the device. In an alternative embodiment involving a polymeric substrate, the surface of the polymer can be plasma modified to expose the desired surface functional groups for monolayer formation. For example, European Patent Publication No. 780423 describes the use of monolayer molecules having an alkene X functionality on the plasma exposed surface. Yet another possibility for a device of the invention consisting of a polymer is to functionalize the polymer surface on which the monolayer has been formed by copolymerization of appropriately functionalized precursor molecules.

  Another possibility is that before introduction into the monolayer, X can be a free radical generating or free radical activating moiety. Such functional groups are particularly suitable when the surface on which the monolayer is formed is a silicon hydride surface. Possible free radical generating moieties include, but are not limited to, diacyl peroxide, peroxide, and azo compounds. Or unsaturated moieties such as unsubstituted alkenes (especially α, β-unsaturated ketones), alkynes, cyano compounds, and isonitrile compounds, especially reactions with X involve ultraviolet, infrared, visible, or microwave irradiation In some cases, X can be used as a free radical activating moiety.

  In alternative embodiments, X can be a vinyl, sulfonyl, phosphoryl, or silicon hydride group.

  The linker group between groups X and Y in reagent 4 can be any suitable chemically compatible spacer group. In one aspect, the linker group is selected to promote self-assembly of reagent 4 into monolayer 6 and / or to ensure that the monolayer is ordered and packed with high density. Laibinis, et al. Science 1989, 245, 845 and Ulman, An Introduction to Ultrathin Organic Films: From Langmuir-Blodgett to Self-Assembly, Academic Press (1991). Known to vendors.

  Typically, the linker group in reagent 4 between functional groups X and Y is a hydrocarbon chain, optionally branched and / or containing heteroatoms such as oxygen. When the linker group is a hydrocarbon chain, the group typically contains 2 to about 22 carbon atoms. Thus, as used herein, the term “hydrocarbon chain” typically refers to a chain of carbon atoms that typically contains from 2 to about 22 carbon atoms. The chain can include aliphatic and aryl groups, and can include linear, branched, and / or cyclic groups. In one embodiment, the linker group between X and Y was a 3 carbon chain, and in another embodiment the linker group was an 11 carbon unbranched hydrocarbon chain.

  In one aspect, it may be desirable to provide cross-linking between monolayer component molecules regardless of the nature of the monolayer molecule. In this case, the linker group in the reagent can include a functional group for facilitating crosslinking. In general, crosslinking provides additional stability to the monolayer. Such methods are well known to those skilled in the art (see, eg, Ulman, An Introduction to Ultrathin Organic Films: From Langmuir-Blodgett to Self-Assembly, Academic Press (1991)).

  With continued reference to FIG. 1, the monolayer 6 formed on the substrate 2 is obtained by functionalizing the substrate 12 with the reagent 4, and the monolayer 14 is further derivatized with the reagents 8 and 10. Have gained. Accordingly, the Y component of the monomolecular layer 6 is a functional group responsible for the attachment of the second set of reagents (eg, 8 and 10). Y can function to form a covalent bond with reagents 8 and 10, or can interact non-covalently with 8 and 10, where 8 and 10 are operatively associated with monolayer 6 It will be. In some cases, a reversible covalent bond can be formed so that reagents 8, 10 or both can dissociate from monolayer 6.

  When 8 and / or 10 can be covalently added to the monolayer 6 and thereby form a new monolayer, Y is typically a functional group on reagents 8 and 10, respectively. Reactive with V and V ′ or easily activated for reaction with V and V ′.

  Examples of functional groups suitable for use as Y include activated carboxylic acids (including, for example, acyl imidazolides, acyl azides) and activated esters (pentafluorophenol, p-nitrophenol, N-hydroxysuccinimide, and sulfo Electrophilic reagents such as -N-hydroxysuccinimide ester). Additional useful electrophiles include Michael, such as α, β-unsaturated carbonyl groups (including acids, amides, esters, ketones, and aldehydes), α, β-unsaturated sulfoxides, α, β-unsaturated sulfones. Receptors are included. Preferred Michael acceptors include maleimides and maleimide derivatives. Further examples of electrophiles useful as Y groups include aldehydes and alkylating agents (epoxides, aziridines, and alkyl halides (such as iodinated, brominated, and alkyl chlorides, particularly α-haloacetyl groups and primary and Tertiary iodide, bromide, and alkyl chloride)) and the like. Similarly, alkylating agents such as alkyl sulfonic acid esters are useful alkylating agents, examples of which include methylene sulfonic acid esters, trifluoromethylene sulfonic acid esters, and p-toluene sulfonic acid esters.

  Further examples of functional groups suitable for use as Y include nucleophilic groups such as hydroxyl, peroxide, peracid, sulfhydryl, thioic acid, selenol, amino, aminooxy, hydrazide, and thiosemicarbazide groups. .

  Another class of reactive Y groups that can be used includes functional groups that participate in cycloaddition reactions. Examples of useful cycloaddition reactions include, but are not limited to, 2 + 2, 3 + 2, especially 1,3-dipolar cycloaddition and 4 + 2 cycloaddition reactions. For example, Y can be a dienophile used in the Diels-Alder reaction. Examples of suitable dienophiles include electron deficient alkenes or electron rich alkenes in the reverse demand Diels-Alder reaction. Conversely, electron-rich or electron-deficient dienes are used in Diels-Alder and reverse-requested Diels-Alder cycloaddition reactions, respectively.

  Azide-containing compounds can be used in several different types of cycloaddition reactions. For example, if Y contains an azide, the compound can be attached to the monolayer 6 using a 1,3-dipolar cycloaddition reaction, which is reacted with a suitable alkene compound to generate nitrogen. After heat removal, the corresponding aziridine compound can be generated. Conversely, Y can include an alkene group that reacts with an azide-containing compound.

  Azides can also be used to react with ketene formed in situ with Staudinger-type cycloaddition, resulting in a β-lactam containing product. Suitable precursors and conditions for functionalizing the monolayer 6 using a cycloaddition reaction can be selected by those skilled in the art.

  Another class of reactions that can be used to covalently functionalize the monolayer 6 includes organometallic reactions. Certain organometallic reactions include, but are not limited to, metathesis reactions, for example, in one aspect, an aldehyde functionalized or alkene functionalized surface, and a reagent 8 where V and V ′ include an aldehyde or alkene functional group. And 10 can be reacted. Examples of catalysts and conditions for such metathesis reactions are known to those skilled in the art. Certain examples of such catalysts include metallic titanium, tungsten, molybdenum, or ruthenium. See, for example, Ivin, K.J., Mol, J.C. Olefin Metathesis and Metathesis Polymerization; Academic Press: London, 1997.

  When Y is a functional group that is activated in situ, the possibilities for Y include azide, hydroxyl, carboalkyl, amino, aldehyde, carbonyl, methyl, methylene, alkene, alkyne, carbonate, aryl iodide, or vinyl. Examples include, but are not limited to, groups such as groups. Suitable activated forms of such functional groups are known to those skilled in the art. For example, when Y is a carboalkyl group, acylation can be achieved by converting Y to an activated ester (such as pentafluorophenol, p-nitrophenol, N-hydroxysuccinimide, or sulfo-N-hydroxysuccinimide ester). So Y can be activated in situ. Moreover, such activated esters readily react with various suitable nucleophiles (amines, thiols, alcohols, etc.). In one aspect, Y can be a protective functional group that can be removed in situ to unmask the reactive functional group. Suitable protecting groups that can be used can be selected, introduced and removed as known to those skilled in the art, Greene, TW; Wuts PGM Protective Groups in Organic Synthesis, 3rd ed .; Wiley-Interscience, 2002 Including the protecting groups disclosed in. In one embodiment, a protected aldehyde was used to create a masked aldehyde functionalized substrate.

  In an alternative embodiment, the functional group Y of the monolayer is selected from the group of charged moieties. Possible charged Y functional groups include, but are not limited to, phosphates, sulfates, carboxylates, and the like. If reagents 8, 10 or both contain an ionic group (such as polylysine) that coats the exposed portion of the monolayer, simple groups such as these can be used for Y.

  With continued reference to FIG. 1, reagents 8 and 10 are attached to the monomolecular layer 6, thereby newly forming the second monomolecular layer 14 including the components of the first monomolecular layer 6. The reagent 8 has a first functional group V for attachment to the monolayer 6 and a second functional group Z that can be used, for example, to attach an affinity reagent. The reagent 10 includes a functional group V ′ for attachment to the monolayer 6 and also includes an anti-protein component W. The functional groups V and V ′ can be the same or different functional groups. In some cases, reagents 8 and 10 can be delivered in two different steps. Thus, reagent 8 can be attached either before or after reagent 10. In one aspect, V and V ′ are optionally covalently bonded, eg, with a disulfide bond, such that reagents 8 and 10 having functional groups Z and W, respectively, are delivered in the same step. The functional groups V and V ′ are selected with reference to the functional group Y presented on the monolayer 6. As described above, for the functional group Y, V and / or V ′ can form a covalent bond to the monomolecular layer 6 or can be non-covalently bonded to the monomolecular layer 6. In general, V and V ′ are selected to be complementary to the functional group Y, so, for example, when Y is an electrophilic group, V and V ′ are typically nucleophilic groups; Conversely, when Y is a nucleophilic group, V and V ′ are typically electrophilic groups.

  The functional group Z on the monolayer 14 is used to attach an affinity agent, resulting in a monolayer 16 (with an affinity agent 18 attached thereto).

  FIG. 2 includes a graphical representation 20 of the array disclosed herein and also shows some representative reactive monolayer functional groups. Referring to array 20, substrate 22 can be any suitable substrate. X represents a functional group for binding a reagent to the substrate 22 noncovalently or covalently (reversibly or irreversibly). In some cases, one or more functional groups X shown in the array 20 are directly or indirectly attached to each other. For example, the group X can be attached prior to attachment to the substrate 22 by a polymer backbone such as a poly-L-lysine backbone. Alternatively, X can be (or derived from) a group such as siloxane that can react with one or more during or after bonding to the substrate 22.

  R represents a linker group, such as a linker group of the type discussed with respect to reagent 4 above. Typically, R is a substantially unbranched chain, such as an unbranched hydrocarbon chain. When R comprises a hydrocarbon chain, the chain typically has 2 to about 22 carbon atoms, and more typically R has 3 to about 18 carbon atoms. In embodiments, the various hydrocarbon chains used included hydrocarbon chains having 3, 4, 10, 11, and 12 carbon atoms.

  When the second reagent is covalently or non-covalently bound to the monolayer bound to the substrate, Y typically represents a bond formed as disclosed herein. When Y represents a covalent bond, Y is typically of two functional groups (the first can be described as electrophilic and the second can be described as nucleophilic) Formed by reaction. Useful examples of electrophilic and nucleophilic groups include those discussed above with respect to FIG. Other functional groups that can react to form Y and typically do not characterize either nucleophilic or electrophilic include functional groups involved in cycloaddition and organometallic reactions Is included.

  With continued reference to FIG. 2, structures 24, 26, 28, 30, and 32 are representatives used in embodiments for introducing at least a second reagent into a monolayer formed on a glass or silicon substrate. Represents a functional group of a typical monolayer. For example, the maleimide functional group in structure 24 can be used as an electrophile in a Michael reaction or in a cycloaddition reaction such as a Diels-Alder reaction.

  The azide moiety in structure 26 is activated by being reduced to the corresponding amine to function as a nucleophile or to achieve activation, reduction, and acylation in a single step in situ. Can be used in a Staudinger ligation reaction. Alternatively, the azide functional group can be used directly in a cycloaddition reaction to react with, for example, a ketene reagent formed in situ.

  Structure 28 contains a bromoacetate moiety that is an excellent electrophile (bromine is easily nucleophilic substituted by various nucleophiles, especially thiolates). Referring to structure 28, X can be nitrogen or oxygen.

  Structure 30 includes an aldehyde that can be used as an electrophile for reaction with various nucleophilic reagents. Various nucleophilic groups that can react with aldehydes to form covalent bonds are useful in the present invention. Useful reactive groups include, for example, alkoxides, enolates, carbanion equivalents (such as Grignard type reactive groups), alcohol groups (those that produce the corresponding acetals or hemiacetals), cyanide reactive groups, amines (primary Hydrazine, phosphorus ylide, phosphine oxide anion, aminooxy reagent, hydrazide thiosemicarbazide and the like. Alternatively, aldehydes can be used in organometallic reactions such as aldehyde-alkene metathesis reactions.

  Structure 32 represents an acylating group such as an activated ester. Thus, X is an easily substituted group such as halide, N-hydroxysuccinimide, sulfo-N-hydroxysuccinimide, pentafluorophenol, p-nitrophenol, etc. as is known to those skilled in the art.

  FIG. 3 illustrates a method for preparing an organic thin film array by first derivatizing the substrate 34 with an asymmetric disulfide 33. Disulfide 33 is asymmetric in the sense that it consists of two different substituents. The deposition by this method results in a monolayer 35 containing two different monolayer components. In some cases, one or more additional symmetric or asymmetric disulfides are attached to provide a monolayer comprising three or more different monolayer components or two monolayer components that simply differ in the ratio given by the asymmetric disulfide can do. For example, if it is desired that the ratio of X and Y groups be 3 to 1, attachment of asymmetric disulfide 33 can be accompanied by attachment of an equivalent amount of asymmetric disulfide having the formula X—R—S—S—R—X. X and Y in the monomolecular layer 35 function as an affinity agent and an antiprotein component, or as a reactive functional group for introducing an affinity agent and an antiprotein reagent. In some cases, when X and Y are such reactive functional groups, they can be the same or different functional groups.

  FIG. 3 also shows representative monolayer components formed by the Staudinger linking method disclosed herein. Referring to structure 37, X is a functional group suitable for bonding a monolayer or organic thin film to a substrate 36. As illustrated by structure 37, X is typically attached to a hydrocarbon linking group, although other linking groups can be used for such structures. Typically, the selected hydrocarbon chain has from 2 to about 22 carbon atoms, and thus “n” is from 2 to about 22. More typically, n is 3 to about 18. G can be any group useful for covalently or non-covalently binding an affinity agent or anti-protein reagent, or can be covalent or non-covalent with an affinity agent or anti-protein reagent. It can also be another reactive functional group that can be used to bind covalently.

  Referring again to FIG. 3, structures 39 and 41 represent monolayer components formed on the substrate 36. Referring to structure 39, substrate 36 is functionalized with siloxane functional groups attached to hydrocarbon chains. The illustrated hydrocarbon chain contains "n" methylene groups (n is typically 2 to about 22, more typically 3 to about 18). In one embodiment, n was 11. The hydrocarbon chain is functionalized with an aminoacyl aryl phosphine oxide moiety introduced by the Staudinger ligation protocol. The acylamino group attached to the polyethylene glycol chain containing “m” ethylene glycol units is in the para position relative to the hydrocarbon aminoacyl group. The ethylene glycol chain can be of any length, but m is typically 2 to about 100, more typically 2 to about 70 ethylene glycol units. Most typically, m is about 2 to about 20. One example included a monolayer component with structure 39 where m is 6.

  With continued reference to structure 39, X is a group that provides an active acylating agent to the monolayer component. Such acylating agents are prepared from the corresponding carboxy group as is known to those skilled in the art. Typically, 39 is an activated ester group such that X is pentafluorophenol, p-nitrophenol, N-hydroxysuccinimide, sulfo-N-hydroxysuccinimide, and the like. Further suitable X groups include halides, imidazolides, anhydrides, and the like. Structure 41, like structure 39, shows a monolayer component formed on a glass or silicon substrate. However, 41 functions as an antiprotein component. Referring to 41, “p” is from 2 to about 100 ethylene glycol units, more typically from 2 to about 70 ethylene glycol units. Most typically, “p” is from 2 to about 20 ethylene glycol units. R represents an alkyl group, hydrogen, or a hydroxyl bearing group. Typically, R is either a methyl group or hydrogen.

  FIG. 4 shows another embodiment for functionalizing the substrate. In this embodiment, the substrate 45 is glass or has a silicon oxide coating and is derivatized with a 3-maleimidopropyl-1-silane derivative to show maleimide (MAL) functionality on one side. A monolayer coated substrate is obtained. The maleimide group can be used to attach sulfhydryl-containing reagents with high efficiency. In the illustrated example, a heterobifunctional reagent having a sulfhydryl group at the first end and a biotin group at the second end is attached to the substrate via a maleimide group. The reagent containing the antiprotein component is then attached via the remaining maleimide functionality.

D. Uses of Disclosed Arrays The disclosed arrays are useful for characterizing the interaction of soluble analytes with sequenced affinity agents. Specifically, the array can be used to identify affinity agents that bind to a molecule of interest, in particular a biomolecule of interest such as a protein or nucleic acid. Arrays are particularly useful for high-throughput drug screening using both small and biomolecular arrays.

  A very wide variety of detection methods are applicable to characterize the interaction of soluble analytes with the disclosed sequenced affinity agents. For example, the array can be a device that works with optical detection methods such as absorption in the visible range, chemiluminescence, and fluorescence (including lifetime, polarization, fluorescence correlation spectroscopy (FCS), and fluorescence resonance energy transfer (FRET)) Can be introduced. In addition, an integrated detector such as an optical waveguide, surface plasmon resonance, and surface charge as disclosed in PCT Publication No. WO96 / 26432 and US Pat. No. 5,677,196 (incorporated herein by reference) The sensor is compatible with the array embodiments disclosed herein.

  An exemplary type of fluorescence detection unit that can be used to monitor the interaction between an immobilized affinity agent on an array and an analyte is described in US Pat. No. 6,406,921 to Wagner et al. (Wagner). (Incorporated herein by reference in its entirety). FIG. 5 is a diagram of this type of fluorescence detection unit that can be used to monitor the interaction between the immobilized affinity agent of the array and the analyte. In the illustrated detection unit, the array 54 is located on the base plate 52. Light from the 100 W mercury arc lamp 62 is sent to the beam splitter 58 through the excitation filter 60. The light is then sent to the array 54 through a lens 56, such as a Micro Nikkor 55mm 1: 2: 8 lens. Fluorescence emission from the array is sent back through lens 56 and beam splitter 58. The emitted light then passes through the emission filter 64 and is then received by a cooled CCD camera 66 such as Slowscan TE / CCD-1024SF & SB (Princeton Instruments). The camera is operably connected to the CPU 68, and conversely, the CPU 68 is operably connected to the VCR / monitor 70.

  FIG. 6 is a diagram of an alternative detection method based on elliptical polarization. Elliptical polarization allows information about the sample to be determined from changes observed in the polarization state of the reflected light wave. The interaction between the analyte and the immobilized affinity agent layer on the substrate changes the thickness and changes the polarization state of the plane polarized beam reflected from the surface. This process can be monitored in situ from the aqueous phase in image mode if desired. Referring to FIG. 6, in a typical facility 80, monochromatic light (eg, from a He--Ne laser, 84) is plane-polarized (polarizer 86) and sent over the sample surface on a substrate 82 to detect a detector 92. To detect. A compensator 88 changes the elliptically polarized reflected beam into a plane polarized beam. The corresponding angle is determined by the analyzer 90 and then converted into elliptical polarization parameters Psi and Delta that change when bound to the analyte and the immobilized protein. Additional information can be found in Azzam, et al., Ellipsometry and Polarized Light, North-Holland Publishing Company: Amsterdam, 1977.

E. Examples The foregoing disclosure is further illustrated by the following non-limiting examples. Efforts have been made to ensure the accuracy of numerical values (eg, amounts, temperature, etc.) but some errors and deviations should be taken as a matter of course. Unless indicated otherwise, parts are parts by weight, temperature is expressed in ° C. or is ambient, and pressure is at or near atmospheric.

General Methods After glass and silicon substrate materials were cleaned, they were derivatized according to the following procedure; 200 mL 30% H ₂ 0 ₂ (Aldrich, Milwaukee, Wis.) And 200 mL concentrated NH ₄ 0H (Aldrich) were added to nanopure water (resistance value). 18M?) Was added to a 65 ° C bath containing 1000 mL. A glass microscope slide (1 "wide 3", Fisher Scientific) or a Teflon rack holding a 1 cm x 2 cm silicon wafer was immersed in a clean bath. After about 15 minutes, the slide rack was removed from the bath and placed in a 300 mL bath of nanopure water for 10 minutes with gentle stirring, and then immersed in a second 300 mL bath of nanopure water for 10 minutes with gentle stirring. Store slides under nanopure water and immediately before use with a 3 minute wash cycle at 600 rpm, a 10 second nitrogen purge at 1500 rpm, followed by two drying cycles of 1.5 and 20 minutes at 2500 and 600 rpm, respectively. Drying by spin rinse drying used.

Example 1
This example describes the formation of amino reactive organic thin film gold on a gold substrate using a disulfide thin film precursor. A newly prepared Au (111) chip was immersed in a 1 mM solution of asymmetric disulfide compound 120 (the synthetic route to compound 120 is illustrated in FIG. 7 and described in detail below) for 1 hour at room temperature. The chip was then rinsed with ethanol (3 × 10 mL). After rinsing, the coated chips were dried under a stream of nitrogen and used immediately for reaction with amine-containing reagents.

Example 2
This example describes functionalizing an amino-reactive organic thin film to form a biotin functionalized chip. A chip with an amino-reactive coating prepared according to Example 1 was prepared using 50 mM phosphorus containing 0.05% Tween-20 (polyoxyethylene (20) sorbitan monolaurate) commercially available from Sigma-Aldrich (Milwaukee, Wis.). (+)-Biotinyl-3,6-dioxaoctanediamine or (+)-biotinyl-3,6,9,11-tetraoxatridecanediamine (Pierce, Illinois respectively) in acid buffer (pH 7.5) Rockford and Molecular BioSciences (commercially available from Colorado, Colorado) for 10 minutes at room temperature. The chip was washed with 500 mM ionic strength PBS buffer (pH 7.5) (total wash volume 30 mL). The chip was then immersed in a 1 M glycine solution at 37 ° C. to inactivate any residual amino reactive NHS groups.

Example 3
This example describes a general protocol used for silanization of silicon oxide substrates. The reagent attached by this method is 11-azidoundecyl-1 prepared according to the protocol of Shaltout et al., Mat Res Soc. Symp. Proc. 1999, 576, 15-20 (incorporated herein by reference). -Triethoxysilane and 3-maleimidopropyl-1-triethoxysilane were included. The Shaltout procedure was also applied to the synthesis of other maleimide derivatives. A 3-maleimidopropyl-1-triethoxysilane reagent was deposited as follows. To a clean, dry 120 mL PTFE vial (Savillex, Minnetonka, MN), add 50 mL dry toluene (99.8% anhydrous, Aldrich catalog number 24,451-1, Milwaukee, Wis.) And 1 mL hexanoic acid (Aldrich catalog number 15374-5), followed by Fresh silane was added. The vial was swirled and a dry clean silicon oxide wafer was added to the silane solution. Silanization was allowed to proceed for 24 hours at room temperature on a shaker (about 100-150 rpm). The wafer was rinsed with dry toluene and absolute ethanol and then sonicated for 10 minutes under absolute ethanol. The sonicated wafer was dried under nitrogen and stored in a fluoroware container in a desiccator under argon. The silanized chip could be used for about 2-3 weeks.

シュタウディンガー試薬を酸素パージした（窒素散布による）3:1テトラヒドロフラン：ナノ純水（脱イオン水、18M?）4mLに溶解し、20mMの濃度を得た。11-アジドウンデシル-1-トリエトキシシランチップをテフロン容器中に置き、シュタウディンガー試薬溶液中に浸漬した。容器を振盪機上で24時間室温で振盪し、次いでチップを取り出し、各々30mLの3:1テトラヒドロフラン：ナノ純水、ナノ純水、及び無水エタノールで洗浄した。各チップを窒素下で乾燥し、さらに使用するまで保存した。シュタウディンガー連結の進行はIR分光法によりモニタリングすることができ、収率は定量的IR分光法により決定することができる。 Example 4
This example illustrates a general Stauw used to couple an amino-reactive and anti-protein component to a substrate functionalized with 11-azidoundecyl-1-triethoxysilane prepared according to the protocol of Example 3. Describes the Dinger ligation protocol. The anti-protein Staudinger reagent 20 and the amino-reactive Staudinger reagent 30 are shown below. Staudinger reagents 20 and 30 were prepared according to the method disclosed by Saxon and Bertozzi in US Pat. No. 6,570,040, which is incorporated herein by reference in its entirety.

The Staudinger reagent was dissolved in 4 mL of 3: 1 tetrahydrofuran: nanopure water (deionized water, 18M?) Purged with oxygen (by nitrogen sparge) to give a concentration of 20 mM. An 11-azidoundecyl-1-triethoxysilane chip was placed in a Teflon container and immersed in the Staudinger reagent solution. The vessel was shaken on a shaker for 24 hours at room temperature, then the chip was removed and washed with 30 mL each of 3: 1 tetrahydrofuran: nanopure water, nanopure water, and absolute ethanol. Each chip was dried under nitrogen and stored until further use. The progress of Staudinger ligation can be monitored by IR spectroscopy and the yield can be determined by quantitative IR spectroscopy.

Example 5
This example describes the functionalization of a 3-maleimidopropyl-1-triethoxysilane substrate with biotin derivative 40 and antiprotein component 50. A maleimide functionalized chip prepared according to the protocol of Example 3 was coated with 250 μL of an 80 μM solution of compound 40 in buffer (20 mM citrate, 150 mM sodium, 5 mM EDTA, 0.05% Tween20 buffer, pH 6.5). . The chip was incubated for 30 minutes at room temperature with slight agitation on a shaker. The chip was washed with buffer (0.5 M sodium in 1X PBS, 0.05% Tween 20, pH 7.5) and then in buffer (20 mM citrate, 150 mM sodium, 5 mM EDTA, 0.05% Tween 20 buffer, pH 6.5) Incubate with 150 μL of an 80 μM solution of compound 50 for 30 minutes at room temperature with slight agitation. The chip was washed with buffer (0.5 M sodium in 1X PBS, 0.05% Tween 20, pH 7.5), then cysteine quench solution (0.1 M cysteine, 20 mM citrate, 150 mM sodium, 5 mM EDTA, 1 mM Tris- (2- (Carboxyethyl) phosphine hydrochloride (TCEP), 0.05% Tween20, pH 6.5) for 30 minutes at 37 ° C. The quenched chip was washed with 30 mL of buffer (0.5 M sodium in 1 × PBS, 0.05% Tween 20, pH 7.5) and 30 mL of nanopure water. The chip was spin dried and used immediately. Biotin functionalized chips prepared according to this protocol can be stored at 4 ° C. under argon for up to about 3 weeks.

  Using a biotin-functionalized surface prepared as described above in Example 5, different biotin derivatization reagents can be immobilized on the surface by reciprocal streptavidin binding. The following examples describe four different antibody immobilization strategies. Four types are random biotinylated IgG; oriented IgG (biotinylated on carbohydrate on Fc domain); oriented Fab ′ fragment (biotinylated in hinge region), and random biotinylated Fab ′ fragment is there. Methods for preparing these biotinylated antibodies are described below.

11-トリエトキシシリルウンデシルエチレングリコールアセタール90をアルケンアセタール70（38mL、34.2g、161mmol）から調製し、100mL丸底フラスコに加えた。Pt(Ph₃P)₄(0.20g、0.161mmol、0.001当量）を加え、続いてPtオクタナール/オクタノール（2.5Pt溶液、1.25mL、0.161mmol、0.001当量）を加えた。トリエトキシシラン（29.75mL、26.5g、161mmol、1.0当量）を周囲温度でアルケン触媒溶液に加えると、わずかに発熱した。溶液を周囲温度で1.5時間攪拌し、次いで反応物を2時間105℃に加熱した。¹HNMRスペクトル中のアルケン共鳴の消滅をモニタリングすることより、反応の完了を決定した。114〜130℃及び13mTorrで蒸留することにより、所望の生成物を得た。無色透明な液体が34mL得られた。

Example 6
This example describes the preparation of organic thin films functionalized with aldehydes. Ethylene glycol (28 mL, 31.4 g, 505 mmol, 1.05 eq) is added to a 1 L round bottom flask containing undecylaldehyde 60, toluene (500 mL), followed by p-toluenesulfonic acid hydrate (13.7 g, 72 mmol, 0.15 Equivalent) was added and the solution was heated to 125 ° C. The solution was refluxed for 3 hours under a Dean-Stark trap. After collecting about 13 mL of water in the trap, the solution was cooled to room temperature, diluted with toluene, and washed with aqueous sodium bicarbonate (1N, 2 × 300 mL) and brine (about 400 mL). The organic solution was dried over Na ₂ SO ₄ and concentrated in vacuo to a brown oil. The crude material was purified by distillation at 85-88 ° C. and 48 mTorr. Compound 70 was isolated as a clear liquid (about 110 mL).

11-Triethoxysilylundecylethylene glycol acetal 90 was prepared from alkene acetal 70 (38 mL, 34.2 g, 161 mmol) and added to a 100 mL round bottom flask. Pt (Ph ₃ P) ₄ (0.20 g, 0.161 mmol, 0.001 equiv) was added, followed by Pt octanal / octanol (2.5 Pt solution, 1.25 mL, 0.161 mmol, 0.001 equiv). When triethoxysilane (29.75 mL, 26.5 g, 161 mmol, 1.0 equiv) was added to the alkene catalyst solution at ambient temperature, a slight exotherm occurred. The solution was stirred at ambient temperature for 1.5 hours and then the reaction was heated to 105 ° C. for 2 hours. The completion of the reaction was determined by monitoring the disappearance of the alkene resonance in the ¹ HNMR spectrum. The desired product was obtained by distillation at 114-130 ° C. and 13 mTorr. 34 mL of a colorless and transparent liquid was obtained.

  Silanization was carried out as follows. To a glass Copeland jar pre-rinsed with a small amount of dry toluene was added 50 mL dry toluene and 0.5 mL aldehyde silane. The jar was placed on a shaker to mix the solution and 5 clear glass microscope slides were placed inside the slot. After gentle stirring for 24 hours, the solution was decanted and replaced with 50 mL dry toluene and the jar was shaken for 5 minutes. This procedure was repeated twice. After decanting the final toluene rinse, 50 mL of ethanol was added and the jar was shaken for 5 minutes. After decanting the initial ethanol rinse, an additional 50 mL of ethanol was added and the jar was sonicated for 10 minutes. Finally, the slide was removed from the Copeland jar and dried under nitrogen. Slides were stored under nitrogen to avoid aldehyde oxidation.

A silicon wafer coated with acetal silane 90 was immersed in 0.1 M HCl solution and stirred for 0.25 hours, 1 hour, 4 hours, 8 hours, and 16 hours. Upon removal from the acid bath, the wafer was immersed in 1M pH7 NaHCO ₃ for 1 minute, removed, immersed in water, stirred for 5 minutes, and then dried using a stream of nitrogen gas.

The deprotection reaction can be followed by X-ray electron spectroscopy (XPS, 45 ° C. take-off angle). The disappearance of the peak corresponding to the CO bond could be monitored at 287 eV and the deprotection reaction was completed in approximately 1 hour. The resulting aldehyde functionalized surface can be derivatized by reductive amination with a 3′-amino functionalized oligonucleotide. 5 μM oligonucleotide was spotted onto the aldehyde functionalized surface and the surface was dried. The spotted surface was rinsed for 2 minutes at room temperature with stirring in a solution of 0.2% SDS in deionized water. The surface was then immersed in a new reducing solution (NaBH ₄ 1.5 g, absolute ethanol 133 mL, and PBS 450 mL, pH 7.2-7.4) and soaked at room temperature for 5 minutes.

Example 7
This example describes the preparation of 11-triethoxysilylundecyl-2-bromoacetic acid. 10-Undecenyl alcohol (100 g, 0.59 mol) and α-bromoacetic acid (90 g, 0.65 mol) were weighed into a 1 L round bottom flask and dissolved in 600 mL of dichloromethane. Dicyclohexylcarbodiimide (DCC, 133.3 g, 0.65 mol) was dissolved in 250 mL of dichloromethane and added in several portions to the acid / alcohol mixture. A white precipitate formed immediately and some heat was generated. The mixture was stirred overnight and then the precipitate (dicyclohexylurea, DCU) was removed by filtration. The filtrate was concentrated to an oil and taken up in 500 mL of hexane to precipitate more DCU, which was removed by filtration. The solution was concentrated to give a colorless oil and the product was distilled (74 ° C., about 4.0 mTorr) to give a colorless liquid (115.77 g, 68% yield).

Undecenyl α-bromoacetate (20 g, 0.07 mol) and 280 mg hydrogen hexachloroplatinate (1 mol%) with a purity greater than 97% were added to the round bottom flask. Triethoxysilane (12.46 g, 76 mmol) was added via syringe under Ar. The mixture was placed in a preheated (90 ° C.) oil bath. During the reaction (12 hours), the mixture turned black. The product was purified by distillation at 150 ° C. at 6.0 mTorr. The product was isolated as a clear colorless liquid (13.6 g, 43.5% yield).

  As with the aldehyde silane (Example 6), except that the deposition time was only 2 hours (instead of 24 hours) and care was taken to store all jars in the dark. Silanization was performed using bromoacetal silane. Slides coated with bromoacetylsilane were stored in a desiccator in the dark.

をコードするDNA配列及び配列番号：2のプロテインキナーゼA部位（GIEGRRRASV）をオリゴヌクレオチドの遺伝子組み立てにより作成した。この構築物をpET24a（Novagen、ウィスコンシン州マディソン）のNdeI及びXhoI部位に挿入して、C最末端にHis(6)標識をさらに付加した（LEHHHHHH（配列番号：3）；ここで、LEコドンはXhoI部位を含む)。次いで、このプラスミドを用いて、1mM IPTGをOD0.6で添加することによりEZMix修飾テリフィックブロス（Terrific Broth）（Sigma、ミズーリ州セントルイス）;カタログ番号T-9179）中で増殖し、かつ、BioFlo 3000発酵槽（New Brunswick Scientific、ニュージャージー州エジソン）中で30℃において4時間増殖したBL21 DE3細胞（Novagen）中でIL-8を直接発現させた。細胞を遠心分離により回収し、50mlあたり完全プロテアーゼ阻害剤カクテル錠(Roche Applied Science; カタログ番号1697498)を1個含む5ml緩衝液/g（300mM NaCl、50mMリン酸ナトリウム、5mMベータ-メルカプトエタノール、5mMイミダゾールを有する（pH8.0））に再懸濁し、マイクロ流動化装置（microfluidizer）を用いて溶解した。IL-8の可溶性画分を、TALON Superflowビーズ（Clontech、カリフォルニア州パロアルト）；カタログ番号8908-2）を用いて金属アフィニティークロマトグラフィーにより精製し、続いてSuperdex 75 分取グレードカラム（Amersham Biosciences)を用いてPBS中のゲル濾過により精製した。3K MWCO膜を有するモデル8400攪拌限外濾過細胞（Millipore、マサチューセッツ州ベッドフォード；カタログ番号5124）を用いてIL-8含有画分を0.4〜0.5mg/mlまで濃縮し、次いで貯蔵のために10%グリセロールを有するPBS中に透析した。同定及び正しい2次構造を質量分析、ELISA、及び円偏光二色性（スペクトル偏光計（Spectrapolarimeter） J-810、Jasco、メリーランド州イーストン、www.jascoinc.com）により確認した。 Materials of Examples 8-10
MAB9647 is a mouse IgG ₁ raised against human IL-8 and is produced by Covance Inc. (Princeton, NJ) from mouse ascites using the hybridoma cell line HB-9647 obtained from ATCC (Manassas, VA). And purified with G protein. MAB208 is a mouse IgG ₁ raised against human IL-8 by R & D Systems (Minneapolis, MN) and purified with G protein from mouse ascites (clone number 6271.111, catalog number MAB208). MAB602 is a mouse IgG _2A produced by R & D Systems against human IL-2 and purified from mouse ascites with the G protein (clone number 5355.111, catalog number MAB602). R-phycoerythrin (PE) conjugated mouse anti-human IL-8 antibody was used as a detection antibody for the IL-8 assay (BD Pharmingen, catalog number 20795A). Two detection antibodies for IL-2 assay: goat anti-human IL-2 antibody (R & D, catalog number AF-202-NA) and R-PE-conjugated donkey anti-goat IgGF (ab ′) ₂ (Jackson Immuno Research, Catalog number 705-116-147) was used. Unless otherwise noted, chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Pepsin-agarose was purchased from Pierce (Rockford, Ill.) (Product number 20343). PNGase F was obtained from New England Biolabs (Beverly, Mass.) (Product Number P0704). Aldehyde reactive probes were obtained from Molecular Probes (Eugene, OR). Human interleukin-2 (IL-2) was purchased from Leinco Technologies (St. Louis, MO) (Product Number 011R455) and phosphate buffered saline (PBS; 11.9 mM sodium phosphate, 137 mM NaCl, 2.7 mM KC1, pH 7) .4) Restructured in. The mature human interleukin-8 of SEQ ID NO: 1 having a factor Xa cleavage site at the C-terminus

The DNA sequence coding for and the protein kinase A site of SEQ ID NO: 2 (GIEGRRRASV) were created by oligonucleotide gene assembly. This construct was inserted into the NdeI and XhoI sites of pET24a (Novagen, Madison, Wis.) To further add a His (6) tag at the C-terminus (LEHHHHHH (SEQ ID NO: 3); where the LE codon is XhoI Site). This plasmid was then used to grow in EZMix modified Terrific Broth (Sigma, St. Louis, Mo .; catalog number T-9179) by adding 1 mM IPTG at OD 0.6, and BioFlo IL-8 was directly expressed in BL21 DE3 cells (Novagen) grown for 4 hours at 30 ° C. in a 3000 fermentor (New Brunswick Scientific, Edison, NJ). Cells were harvested by centrifugation and 5 ml buffer / g (300 mM NaCl, 50 mM sodium phosphate, 5 mM beta-mercaptoethanol, 5 mM) containing 1 complete protease inhibitor cocktail tablet (Roche Applied Science; catalog number 1697498) per 50 ml. It was resuspended in imidazole (pH 8.0) and dissolved using a microfluidizer. The soluble fraction of IL-8 was purified by metal affinity chromatography using TALON Superflow beads (Clontech, Palo Alto, Calif .; catalog number 8908-2), followed by a Superdex 75 preparative grade column (Amersham Biosciences). And purified by gel filtration in PBS. Using a model 8400 stirred ultrafiltration cell with 3K MWCO membrane (Millipore, Bedford, Mass .; catalog number 5124), the IL-8 containing fraction was concentrated to 0.4-0.5 mg / ml and then stored for 10 Dialyzed into PBS with% glycerol. Identification and correct secondary structure were confirmed by mass spectrometry, ELISA, and circular dichroism (Spectrapolarimeter J-810, Jasco, Easton, MD, www.jascoinc.com).

Example 8
This example describes the preparation of a biotinylated Fab ′ fragment. Conserved N-linked glycosylation of IgG 'was removed from the antibody under the following reaction conditions (1-4 mg / ml antibody in 10 mM sodium phosphate, pH 7.5, 10-20 U / μl PNGase F (New (Obtained from England Biolabs, using their unit definitions), 24-48 hours at 37 ° C). After deglycosylation, the antibody was buffer exchanged in 20 mM sodium acetate (pH 4.5) (using ultrafiltration or dialysis). The conditions for pepsin degradation were as follows (30% by volume pepsin agarose (beads washed in fixed bed volume, 20 mM NaOAc, pH 4.5), 0.5-2 mg / ml IgG, 20 mM NaOAc, 260 mM KC1 0.1% Triton X-100, pH 4.5). The reaction was incubated at 37 ° C. with agitation for the previously optimized amount of time (12 hours for MAB9647; 4.5 hours for MAB208; 3.5 hours for MAB602). After pepsin treatment, the fragment was recovered from pepsin agarose by washing the resin with 0.1 M NaOAc (pH 4.5). The product of pepsin cleavage was then concentrated and exchanged into 0.1 M Na ₂ HPO ₄ , 5 mM EDTA, pH 6.0, then treated with 20 mM 2-mercaptoethylamine (MEA) for 90 minutes at 37 ° C. in the same buffer. did. The MEA was then removed by dialysis against 0.1M Na ₂ PO ₄ , 5 mM EDTA for 6 hours at 4 ° C. using a 10 kD cut-off membrane, and the residual MEA was then removed from the desalting column (PD-10, Amersham- The sample was removed by running the sample over Pharmacia, Piscataway, NJ. Immediately following this step, the reduced F (ab) ′ was treated with 20 mM N-ethylmaleimide or maleimide activated biotin (Pierce product number 21901) for 2 hours at room temperature, and then unintroduced NEM or biotinmaleimide was removed by dialysis. . Samples were concentrated and Fab ′ fragments were purified from other fragments by FPLC using a Superdex-75 gel filtration column (Amersham-Pharmacia). For NEM-treated Fab ′ fragments, random biotinylation was as described below. Dilute 1: 1 sample of FPLC purified Fab 'fragment with non-reducing protein loading buffer (62.5 mM Tris HCl, 25% glycerol, 2% SDS, 0.01% bromophenol blue), 4-20% gradient SDS polyacrylamide Loaded on gel (Product No. 161-1123, Biorad, Hercules, CA). SDS-PAGE was performed according to Laemmli (1970). In each case, an approximately 50 kD band corresponding to Fab ′ was observed, with only observable contaminants corresponding to the size of the light chain and the cleaved heavy chain. These contaminants migrated with the Fab 'in the high resolution gel filtration column, so the light chain and the cleaved heavy chain (the disulfide bonds that normally bind them are reduced). Possibly corresponding to a non-covalent or otherwise structurally native complex). These fragments are likely functional because there are no sulfide bonds between the heavy and light chains in the antigen binding site. Those fragments corresponded to approximately 20% of the purified protein and some of their Fab ′ fragments were more than 80% active.

  Biotinylation of all capture agents used in this study was verified by Western blot analysis (data not shown) using a streptavidin (SA) probe conjugated with HRP. In addition, the degree of biotinylation was assessed using SA resin pull-down assay (data not shown). In each of these reactions, biotinylation was greater than 60%. No attempt was made to remove non-biotinylated protein prior to surface immobilization.

Example 9
This example describes a carbobiotinylation protocol. IgG (3-5 mg / ml) is dialyzed into coupling buffer (0.1M NaOAc pH5.5) and then incubated with 20 mM sodium metaperiodate (NaIO ₄ ) in the dark for 1 hour at 0 ° C. The vicinal diol inside was oxidized to an aldehyde. The reaction was then quenched by the addition of 30 mM glycerol for 10 minutes, filtered to remove soluble salts, and then dialyzed against coupling buffer for 6 hours. Add an aldehyde-reactive probe (ARP, N- (aminooxyacetyl) -N ′-(D biotinyl) hydrazine, trifluoroacetate, Molecular Probes, Eugene Oregon) at 1 mg / ml and incubate for 2 hours at room temperature, The sample was then dialyzed vigorously against PBS.

Example 10
This example describes random biotinylation of IgG and Fab ′ fragment molecules. IgG and NEM treated Fab ′ fragments were modified with an amine reactive probe, EZ Link® sulfo-NHS-biotin (Pierce). The reaction was carried out for 2 hours at room temperature with a 20-fold molar excess of biotinylation reagent relative to the protein in 1 × PBS. The biotinylation reagent was then quenched by adding Tris (pH 7.4) to a final concentration of 10 mM. The sample was then dialyzed 5 times against 1000-fold excess of PBS to remove free biotin probe. After dialysis, biotinylated proteins were analyzed on a gel and tested for biotinylation on an UltraLink® plus immobilized streptavidin gel (Pierce). Typically, 60% to 100% of the protein is biotinylated (data not shown).

Example 11
This example describes using surface plasmon resonance (SPR) to measure the surface coverage and activity of an affinity agent bound to a thin film formed on a gold substrate. All SPR assays were performed in a BIAcore 3000 that analyzes biotinylated self-assembled monolayers formed on gold-coated glass surfaces. The surface was prepared with asymmetric alkane disulfide compound 10 according to the protocol of Example 1. The prepared surface contained N-hydroxysuccinimide and methoxy groups on its exposed surface. This monolayer was then reacted with (+)-biotinyl-3,6-dioxaoctanediamine (triethylene glycol aminobiotin) reagent to obtain a biotinylated surface. The biotin group on the surface allows for the binding of streptavidin (SA). All assays were performed at 25 ° C in PBS with 0.05% Tween-20. SA was loaded on the surface at a flow rate of 20 μl / min at 0.1 mg / ml. Typically, 320 μl is loaded to achieve a saturated SA surface, which gives a surface coverage of 3.7-4.0 pmol / cm ² calculated according to Jung et al., Langmuir 14: 5636-5648 (1998). It was. After SA attachment, 20-100 nM of various capture reagents were loaded at a flow rate of 20 μl / min until saturation was observed. Unless otherwise stated, a flow rate of 80 μL / min was used for analyte binding. Various analyte concentrations over a wide range were assayed to determine the upper limit of analyte binding. For comparison, non-specific binding of analytes to SA was tested independently at all analyte concentrations studied.

Example 12
This example describes the synthesis of asymmetric NHS disulfide 120. Referring to FIG. 7, alkenyl bromide compound 100 is used as the starting material for both “arms” of the asymmetric disulfide. To generate the antiprotein arm, 100 was reacted with alkoxide triethylene glycol monomethyl ether (generated by deprotonation with NaH) to give compound 102 in 60% yield. Compound 102 was subjected to radical addition to give terminal thioacetyl compound 104 in 88% yield. Removal of the acetyl group under acidic conditions (4N HCl / dioxane, 4 h at 75 ° C.) gave thiol 106 (95%), which was treated with 2 ′, 2′-dipyridyl disulfide to give the corresponding 2 ′ -Pyridyl mixed disulfide 108 was obtained in 98% yield.

  The NHS “arm” of mixed disulfide 120 was prepared from 100 by refluxing in aqueous NaOH with tetraethylene glycol for 24 hours to give compound 110 in 42% yield. Terminal alcohol 110 was treated with NaH and alkylated with t-butyl bromoacetate to give terminal ester compound 112 in 42% yield. Compound 112 was converted to thioacetyl compound 114 in 87% yield by free radical addition of thioacetic acid. Removal of the acetyl and t-butyl protecting groups by treatment with 2N aqueous HCl at 75 ° C. for 48-60 hours produced thiol 116 in 80% yield.

  Compounds 108 and 116 are then combined in the presence of Amberlite CG-50 (about pH 3-4) at room temperature for about 12 to about 16 hours to give asymmetric disulfide 118, which is condensed with N-hydroxysuccinimide with a DCC catalyst. To the desired compound 120.

1 illustrates one embodiment of the disclosed surface functionalization method. FIG. 3 is a diagram illustrating one embodiment of the disclosed array. Examples of monolayer components used in the preparation of such arrays are provided. 3 shows how to use an asymmetric disulfide reagent to prepare a monolayer. An example of a monolayer component prepared by Staudinger ligation is shown. An example of a process for preparing a novel array is shown. FIG. 2 is a diagram of a fluorescence detection unit that can be used to monitor the interaction between an array of proteins and an analyte. FIG. 3 is an elliptical polarization detection unit that can be used to monitor the interaction between the analyte and the affinity label of the array. 3 shows the synthesis of an asymmetric disulfide with N-hydroxysuccinimide and polyethylene glycol groups.

Claims (37)

Providing a substrate;
Contacting the substrate with a first reagent having at least first and second reactive functional groups such that the first reagent is operatively associated with the substrate by the first functional group, thereby Forming a layer;
Binding a first functional group for binding to the monolayer and a second reagent comprising at least a second functional group to the monolayer; and binding an antiprotein reagent to the monolayer. A method for functionalizing a substrate, comprising:   2. The method of claim 1, wherein the first reagent is covalently bound to the substrate.   The first reagent binds to the substrate with a functional group selected from the group consisting of a sulfur-containing group, siloxane, phosphine, phosphoric acid; phosphonic acid; hydroxamic acid, carboxylic acid, and combinations thereof. Method.   The first reagent is a sulfur-containing group, ester, activated ester, Michael acceptor, siloxane, silyl halide, phosphine, amine, azide, α, β-unsaturated ketone, hydroxyl, sulfhydryl, thiosemicarbazide, hydrazide, amino 2. The method of claim 1, comprising one or more functional groups selected from the group consisting of oxy groups, aldehydes, alkyl halides, hydroxamic acids, and carboxylic acids.   2. The method of claim 1, wherein the second reagent is covalently added to the monolayer by alkylation, chemoselective linking, Michael addition, organometallic reaction, cycloaddition, or acylation reaction.   The second reagent comprises one or more of phosphine, amine, azide, α, β-unsaturated ketone, hydroxyl, sulfhydryl, thiosemicarbazide, hydrazide, aminooxy group, aldehyde, or alkyl halide. the method of.   2. The method of claim 1, wherein the antiproteinaceous component is covalently added to the monolayer by alkylation, Michael, Staudinger ligation, organometallic, cycloaddition, or acylation reactions.   The substrate is selected from the group consisting of gold, platinum, silver, copper, glass, silicon, silicon oxide, silicon nitride, tantalum oxide, titanium oxide, indium tin oxide, magnesium oxide, alumina, quartz, silica, and combinations thereof. The method according to claim 1.   2. The method of claim 1, wherein the substrate comprises a noble metal and the first functional group of the first reagent is a sulfur-containing group.   10. The method of claim 9, wherein the sulfur containing group comprises a sulfhydryl group or a disulfide group.   2. The method of claim 1, wherein the substrate comprises glass or silicon and the first functional group of the first reagent comprises a siloxane or halogenated silyl group.   The second reactive functional group of the first reagent is one or more azide, amine, maleimide, phosphine, amine, azide, α, β-unsaturated ketone, hydroxyl, sulfhydryl, thiosemicarbazide, hydrazide, aminooxy group 2. The method of claim 1, comprising an aldehyde, alkyl halide, carboxy, or activated ester group.   2. The method of claim 1, wherein the antiprotein reagent comprises at least one of a polyalkylene oxide group, a polysulfone, a polysaccharide, or a phosphocholine group.   2. The method of claim 1, wherein the antiprotein reagent comprises a polyethylene glycol group.   2. The method of claim 1, further comprising binding an affinity agent to the second functional group of the second reagent.   16. The method of claim 15, further comprising quenching the remaining second functional group.   16. The method of claim 15, wherein the affinity agent comprises a protein.   16. The method of claim 15, wherein coupling the affinity agent to the second functional group of the second reagent includes forming a covalent bond.   19. The method of claim 18, wherein the covalent bond is formed by a reaction selected from the group consisting of alkylation, chemoselective linking, Michael addition, organometallic reaction, cycloaddition, and acylation reaction.   19. The method of claim 18, wherein the covalent bond is formed by a Staudinger ligation reaction.   16. The method of claim 15, wherein the second functional group of the second reagent comprises a biotin group and the affinity agent is streptavidin.   24. The method of claim 21, further comprising binding a biotinylated affinity agent to streptavidin.   23. The method of claim 22, wherein the biotinylated affinity agent is a biotinylated antibody. Providing a substrate;
A method of forming a monolayer comprising the step of contacting a substrate with an asymmetric disulfide having a reactive functional group and an anti-protein functional group, thereby forming a monolayer on the substrate. 25. The method of claim 24, wherein the asymmetric disulfide has the formula:

25. The method of claim 24, wherein the asymmetric disulfide has the formula:

  25. The method of claim 24, further comprising contacting the substrate with a second disulfide, thereby introducing two corresponding sulfides into the monolayer. Substrate;
A plurality of first reagents having first and second ends (the first ends of the first reagents are operatively associated with the substrate);
A plurality of second reagents (each of the second reagents has first and second ends, and the first end of the second reagent is operatively associated with the second end of the first reagent); And an organic comprising a third reagent having a first end and a second end operatively linked to a second end of the first reagent and comprising an anti-protein functional group Thin film array.   The substrate is selected from the group consisting of gold, platinum, silver, copper, glass, silicon, silicon oxide, silicon nitride, tantalum oxide, titanium oxide, indium tin oxide, magnesium oxide, alumina, quartz, silica, and combinations thereof. 30. The array of claim 28.   30. The array of claim 28, wherein the first reagent is covalently bound to the substrate.   30. The array of claim 28, wherein the first reagent is attached to the substrate by a sulfur-containing group, siloxane, phosphine, hydroxamic acid, carboxylic acid, and combinations thereof.   30. The array of claim 28, wherein the first reagent is bound to the substrate by at least one interaction comprising at least one of a metal-ligand bond, a siloxane bond, or a Coulomb interaction.   30. The array of claim 28, wherein the second reagent is covalently attached to the first reagent.   34. The array of claim 33, wherein the second reagent is covalently added to the first reagent by alkylation, chemoselective ligation, Michael addition, organometallic reaction, cycloaddition, or acylation reaction. .   34. The array of claim 33, wherein the second reagent is covalently attached to the first reagent by a Staudinger linkage.   34. The array of claim 33, wherein the second reagent is covalently attached to the first reagent by an amide, ether, ester, imide, sulfide, or carbon-carbon bond. A substrate having an array of discrete array regions formed on the surface;
A plurality of first reagents having first and second ends (the first ends of the first reagents are operatively associated with the substrate);
A plurality of second reagents (each of the second reagents has first and second ends, and the first end of the second reagent is operatively associated with the second end of the first reagent); Array device including).

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