Polypeptide arrays and methods of attaching polypeptides to array

Abstract

To provide improved peptide arrays and improved peptide array manufacturing methods.SOLUTION: A method of attaching a biomolecule to a surface comprises: obtaining a surface comprising a plurality of attachment groups attached to the surface; attaching a photoactivatable conjugation compound to the attachment group; contacting the surface with a biomolecule: and selectively exposing the surface to electromagnetic radiation, where the electromagnetic radiation activates the attached photoactivatable conjugation compound and where the attached activated photoactivatable conjugation compound binds to the biomolecule, thereby attaching the biomolecule to the surface.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. 119(e) of prior co-pending U.S. Provisional Patent Application No. 62 / 132,405, filed March 12, 2015, the disclosure of which is incorporated herein by reference in its entirety. [Background technology]

[0002] background A typical microarray system generally consists of biomolecular probes (e.g., DNA, proteins, or peptides) formatted on a solid surface such as a glass, plastic, or silicon chip, along with the equipment required to handle the sample (automated robotics), read the reporter molecules (scanners), and analyze the data (bioinformatics tools). Microarray technology facilitates monitoring many probes per square centimeter. Advantages of using multiple probes include, but are not limited to, speed, adaptability, comprehensiveness, and relatively low mass production costs. Applications of such arrays include, but are not limited to, diagnostic microbiology, including pathogen detection and identification, antimicrobial resistance research, epidemiological strain classification, cancer genetic research, analysis of microbial infections using host genome expression, and polymorphism profiling.

[0003] Recent advances in genomics have led to the sequencing of the entire genomes of several organisms, including humans. However, genomics alone cannot fully understand the cellular processes involved in disease, development, and other biological phenomena because such processes are often directly mediated by polypeptides. Given the vast number of polypeptides encoded by an organism's genome, the development of high-throughput techniques for analyzing polypeptides is of paramount importance.

[0004] Peptide arrays with distinct analyte detection regions or probes can be fabricated on a single support by techniques well known to those skilled in the art. Various methods are available for fabricating peptide microarrays. These methods include (a) chemoselective immobilization; and (b) in situ parallel synthesis, which can be further divided into (1) SPOT synthesis and (2) photolithography synthesis.

[0005] These methods are labor intensive and not amenable to large-scale processing. These peptide arrays are expensive to produce, have poor reproducibility, can be unstable, require strict storage conditions, take a long time to produce, and have other limitations. Therefore, what is needed are improved peptide arrays and improved methods for fabricating peptide arrays. Summary of the Invention

[0006] overview Disclosed herein are preparations, supports, and arrays.In certain embodiments, disclosed is a method for attaching biomolecules to arrays using photoactivated conjugation compounds.Also disclosed is a method for site-specifically attaching biomolecules to arrays.Also disclosed are arrays produced by these methods and methods for using these arrays.

[0007] In some versions, the method includes obtaining a surface comprising a plurality of attachment groups attached to the surface, attaching photoactivatable conjugation compounds to the attachment groups, contacting the surface with a biomolecule, and selectively exposing the surface to electromagnetic radiation, wherein the electromagnetic radiation activates the attached photoactivatable conjugation compounds and the attached, activated photoactivatable conjugation compounds bind to the biomolecule, thereby attaching the biomolecule to the surface.

[0008] The method also includes attaching a polypeptide to a surface by obtaining a surface comprising a plurality of free amine groups attached to the surface, attaching a conjugation compound to the surface by contacting the surface with a conjugation solution comprising a conjugation compound, wherein the conjugation compound comprises an activated carboxylic acid group, and the activated carboxylic acid group binds to the free amine groups attached to the surface; contacting the surface with a polypeptide; and selectively exposing the surface to electromagnetic radiation, wherein the electromagnetic radiation activates the attached conjugation compound and the attached, activated conjugation compound binds to the polypeptide, thereby attaching the polypeptide to the surface.

[0009] The methods disclosed herein also include a step of attaching a polypeptide to a surface, comprising the steps of: obtaining a surface comprising a plurality of free carboxylic acid groups attached to the surface; contacting the surface with a carboxylic acid activating solution, thereby activating the carboxylic acid groups for conjugation with amine groups; attaching a conjugation compound to the surface by contacting the surface with a conjugation solution comprising a conjugation compound, wherein the conjugation compound comprises an amine group, and the amine group binds to the activated carboxylic acid groups attached to the surface; contacting the surface with a polypeptide; and selectively exposing the surface to electromagnetic radiation, wherein the electromagnetic radiation activates the attached conjugation compound, and the attached, activated conjugation compound binds to the polypeptide, thereby attaching the polypeptide to the surface. [The present invention 1001] 1. A method for attaching a biomolecule to a surface, comprising the steps of: obtaining a surface having a plurality of attachment groups attached to said surface; attaching a photoactivatable conjugating compound to the attachment group; contacting the surface with a biomolecule; and Selectively exposing the surface to electromagnetic radiation, which activates the attached photoactivatable conjugation compounds, and the attached, activated photoactivatable conjugation compounds bind to the biomolecules, thereby attaching the biomolecules to the surface. [The present invention 1002] 1001. The method of claim 1001, wherein the photoactivatable conjugating compound comprises a functional group selected from the group consisting of NHS ester, sulfo-NHS ester, amine, primary alcohol, secondary alcohol, phenol, thiol, aniline, hydroxamic acid, primary amide, aliphatic amine, and sulfonamide. [The present invention 1003] 1001. The method of claim 1001, wherein the photoactivatable conjugated compound comprises an ester. [The present invention 1004] 1001. The method of claim 1001, wherein the photoactivatable conjugated compound comprises a carboxylic acid group. [The present invention 1005] The method of claim 1004, wherein the carboxyl group is activated. [The present invention 1006] 1001. The method of claim 1001, wherein the photoactivatable conjugated compound comprises an N-hydroxysuccinimide moiety. [The present invention 1007] 1001. The method of claim 1001, wherein the photoactivatable conjugated compound comprises an amine group. [The present invention 1008] 1001. The method of claim 1001, wherein the photoactivatable conjugating compound comprises a photoactivatable group selected from the group consisting of diazirine, aryl azide, and benzophenone. [The present invention 1009] 1001. The method of claim 1001, wherein the photoactivatable conjugation compound comprises a photoactivated conjugation moiety selected from the group consisting of a diazirine moiety, an aryl azide moiety, and a benzophenone moiety. [The present invention 1010] 1001. The method of claim 1001, wherein the photoactivatable conjugated compound comprises an N-hydroxysuccinimide moiety attached to an ester. [The present invention 1011] 1001. The method of claim 1001, wherein the photoactivatable conjugated compound comprises an N-hydroxysuccinimide ester functionally attached to a moiety selected from the group consisting of a diazirine moiety, an aryl azide moiety, and a benzophenone moiety. [The present invention 1012] 1001. The method of claim 1001, wherein the attachment group is an amine group. [The present invention 1013] 1001. The method of claim 1001, wherein the attachment group is a carboxylic acid group. [The present invention 1014] The method of claim 1013, wherein a carboxyl group is activated to couple to an amine group. [The present invention 1015] 1001. The method of claim 1001, wherein the biomolecule is a polypeptide. [The present invention 1016] The method of claim 10, wherein the polypeptide is a protein. [The present invention 1017] 1001. The method of claim 1001, wherein the biomolecule is selected from the group consisting of small molecules, proteins, carbohydrates, oligomers, peptides, biomolecules derived from cell lysates, antibodies, proteases, and enzymes. [The present invention 1018] 1001. The method of claim 1001, wherein the wavelength of the electromagnetic radiation is 248 nm. [The present invention 1019] 1001. The method of the present invention, wherein the wavelength of the electromagnetic radiation is 330 to 370 nm. [The present invention 1020] 1001. The method of claim 1001, wherein the surface is porous and the attachment groups are oriented in multiple directions. [The present invention 1021] 1001. The method of claim 1001, wherein the surface is a surface of pillars operatively coupled to positioned locations of a planar layer of a support. [The present invention 1022] The method of claim 1021, wherein each pillar has a flat upper surface extending from a planar layer of support. [The present invention 1023] 1022. The method of claim 1022, wherein the distance between the top surface of each pillar of the support and the planar layer is 1,000 to 5,000 angstroms. [The present invention 1024] 1021. The method of claim 1021, wherein the pillar is one of a plurality of pillars present on a substrate. [The present invention 1025] Multiple pillars 10,000 / cm 2 The method of the present invention 1024, which is present at a density exceeding 1024. [The present invention 1026] The method of claim 1024, wherein the center of each pillar is at least 2,000 angstroms from the center of any other pillar. [The present invention 1027] The method of claim 1024, wherein a surface of each pillar is parallel to the top surface of the planar layer. [The present invention 1028] The method of claim 1024, wherein a surface of each pillar is substantially parallel to the top surface of the planar layer. [The present invention 1029] Each pillar has a surface area of ​​at least 1 μm 2 The method of the present invention 1021. [The present invention 1030] The total surface area of ​​each pillar is 10,000 μm 2 The method of the present invention 1021, wherein the [The present invention 1031] The method of claim 1021, wherein each pillar comprises silicon dioxide or silicon nitride. [The present invention 1032] 1021. The method of claim 1021, wherein each pillar is at least 98-99% by weight silicon dioxide. [The present invention 1033] 1021. The method of claim 1021, wherein each of the defined locations comprises a plurality of identical biomolecules. [The present invention 1034] The method of claim 1033, wherein each of the defined locations comprises a plurality of identical sequences that are different from the other defined locations. [This invention 1035] The method of invention 1021, wherein each of the positioned locations is a location that is distinguishable. [The present invention 1036] The method of claim 1001, wherein the biomolecule is covalently attached to the surface. [This invention 1037] 1001. The method of claim 1001, wherein the attachment efficiency of the biomolecule attachment on the selectively exposed surface is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. [The present invention 1038] The method of the present invention 1001, wherein the attachment of biomolecules to the array is highly stable. [This invention 1039] The method of invention 1001, optionally repeated to produce a surface comprising a plurality of unique biomolecules attached to selected locations on the surface. [The present invention 1040] 1001. The method of claim 1001, wherein the surface comprises at least two unique biomolecules attached to selected locations on the surface. [This invention 1041] The method of claim 1040, wherein the surface comprises at least 10 unique biomolecules attached to selected locations on the surface. [The present invention 1042] 1041. The method of claim 1041, wherein the surface comprises at least 100 unique biomolecules attached to selected locations on the surface. [This invention 1043] The method of claim 1042, wherein the surface comprises at least 1,000 unique biomolecules attached to selected locations on the surface. [This invention 1044] The method of claim 1043, wherein the surface comprises at least 10,000 unique biomolecules attached to selected locations on the surface. [This invention 1045] 1. A method for attaching a polypeptide to a surface, the method comprising the steps of: obtaining a surface comprising a plurality of free amine groups attached to said surface; attaching a conjugated compound to the surface by contacting the surface with a conjugation solution comprising a conjugated compound, wherein the conjugated compound comprises an activated carboxylic acid group, which binds to a free amine group attached to the surface; contacting the surface with a polypeptide; and Selectively exposing the surface to electromagnetic radiation, which activates the attached conjugation compound, and the attached, activated conjugation compound binds to the polypeptide, thereby attaching the polypeptide to the surface. [The present invention 1046] 1. A method for attaching a polypeptide to a surface, the method comprising the steps of: obtaining a surface comprising a plurality of free carboxylic acid groups attached to said surface; contacting the surface with a carboxylic acid activating solution, thereby activating the carboxylic acid groups for bonding with amine groups; attaching a conjugation compound to the surface by contacting the surface with a conjugation solution containing a conjugation compound, wherein the conjugation compound comprises an amine group, which binds to an activated carboxylic acid group attached to the surface; contacting the surface with a polypeptide; and Selectively exposing the surface to electromagnetic radiation, which activates the attached conjugation compound, and the attached, activated conjugation compound binds to the polypeptide, thereby attaching the polypeptide to the surface. [This invention 1047] The method of any one of claims 1045 to 1046, further comprising the step of washing the surface after attaching the conjugated compound to the surface. [This invention 1048] 1046. The method of claim 1046, further comprising the step of washing the surface after activating the surface-attached carboxylic acid groups. [This invention 1049] The method of any one of claims 1045 to 1046, wherein the activated conjugation compound is attached to a site on the polypeptide selected from the group consisting of a peptide backbone, a side chain, an amine group, and a carboxylic acid group. [The present invention 1050] 1047. The method of claim 1045 or 1046, wherein contacting the surface with the conjugation solution comprises spin-coating the conjugation solution onto the surface. [This invention 1051] The method of any one of claims 1045 to 1046, wherein the wavelength of the electromagnetic radiation is 248 nm. [This invention 1052] The method of claim 1045 or 1046, wherein the wavelength of the electromagnetic radiation is 330 to 370 nm. [This invention 1053] The method of any one of claims 1045 to 1046, wherein the conjugated compound comprises a photoactivated conjugated moiety selected from the group consisting of a diazirine moiety, an aryl azide moiety, and a benzophenone moiety. [This invention 1054] The method of claim 1046, wherein the conjugated compound comprises an N-hydroxysuccinimide moiety attached to an ester. [This invention 1055] The method of claim 1046, wherein the conjugated compound comprises an N-hydroxysuccinimide ester operatively attached to a moiety selected from the group consisting of a diazirine moiety, an aryl azide moiety, and a benzophenone moiety. [This invention 1056] The method of any one of claims 1045 to 1046, wherein the conjugation solution comprises a polymer. [This invention 1057] The method of claim 1056, wherein the polymer is polyvinylpyrrolidone. [This invention 1058] The method of claim 1046, wherein the carboxylic acid activating solution comprises a carbodiimide or N-hydroxysuccinimide. [This invention 1059] 1046. The method of claim 1046, wherein the carboxylic acid activating solution comprises a compound selected from the group consisting of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N,N'-diisopropylcarbodiimide, (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, bromo(tripyrrolidin-1-yl)phosphonium hexafluorophosphate, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, O-benzotriazole-N,N,N',N'-tetramethyluronium-hexafluorophosphate, and O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate. [The present invention 1060] The process of any one of claims 1045 to 1046, wherein the carboxylic acid activating solution comprises a compound selected from the group consisting of N,N-diisopropylethylamine, 1-hydroxy-7-azabenzotriazole, and hydroxybenzotriazole. [This invention 1061] The method of any one of claims 1045 to 1046, wherein the polypeptide is a protein. [This invention 1062] The method of claim 1045 or 1046, wherein the attachment efficiency of the biomolecule attachment on the selectively exposed surface is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. [This invention 1063] The method of claim 1045 or 1046, wherein the attachment of biomolecules to the array is highly stable. [This invention 1064] The method of invention 1045 or 1046, optionally repeated to produce a surface comprising a plurality of unique biomolecules attached to selected locations on the surface. [This invention 1065] The method of any one of claims 1045 to 1046, wherein the surface comprises at least two unique biomolecules attached to selected locations on the surface. [The present invention 1066] The method of claim 1065, wherein the surface comprises at least 10 unique biomolecules attached to selected locations on the surface. [This invention 1067] The method of claim 1066, wherein the surface comprises at least 100 unique biomolecules attached to selected locations on the surface. [The present invention 1068] 1067. The method of claim 1067, wherein the surface comprises at least 1,000 unique biomolecules attached to selected locations on the surface. [This invention 1069] The method of claim 1068, wherein the surface comprises at least 10,000 unique biomolecules attached to selected locations on the surface. [The present invention 1070] An array comprising a plurality of attachment groups on an array surface, at least one of the plurality of attachment groups being covalently linked to a photoactivatable conjugating compound. [This invention 1071] An array of the present invention 1070 comprising biomolecules attached to photoactivatable conjugation compounds. [This invention 1072] 1071. The array of claim 1071, wherein the biomolecule is a polypeptide. [This invention 1073] The array of the present invention 1072, wherein the polypeptide is a protein. [This invention 1074] 1071. The array of claim 1071, wherein the biomolecules are selected from the group consisting of small molecules, proteins, carbohydrates, oligomers, peptides, biomolecules derived from cell lysates, antibodies, proteases, and enzymes. [This invention 1075] 1cm 2 Arrays of the invention 1071 comprising at least 100, 1,000, 10,000, 100,000, 1,000,000, or 10,000,000 biomolecules per array. [This invention 1076] The surface area of ​​the array is 1 μm 2 ~10mm 2Array of the present invention 1070. [This invention 1077] Array of the present invention 1070, wherein the photoactivatable conjugating compound comprises a functional group selected from the group consisting of NHS ester, sulfo-NHS ester, amine, primary alcohol, secondary alcohol, phenol, thiol, aniline, hydroxamic acid, primary amide, aliphatic amine, and sulfonamide. [This invention 1078] The array of the present invention 1070, wherein the photoactivatable conjugated compound comprises an ester. [This invention 1079] The array of the present invention 1070, wherein the photoactivatable conjugated compound comprises a carboxylic acid group. [The present invention 1080] Arrays of the present invention 1079, wherein the carboxylic acid groups are activated. [This invention 1081] 1070. The array of the present invention, wherein the photoactivatable conjugated compound comprises an N-hydroxysuccinimide moiety. [This invention 1082] The array of the present invention 1070, wherein the photoactivatable conjugated compound comprises an amine group. [This invention 1083] 1070. The array of claim 1070, wherein the photoactivatable conjugated compound comprises a photoactivatable group selected from the group consisting of diazirine, aryl azide, and benzophenone. [This invention 1084] Array of the invention 1070, wherein the photoactivatable conjugated compounds are activated by electromagnetic radiation comprising a wavelength of 248 nm. [This invention 1085] Array of the invention 1070, wherein the photoactivatable conjugated compounds are activated by electromagnetic radiation comprising wavelengths of 330-360 nm. [The present invention 1086] 1070. The array of claim 1070, wherein the photoactivatable conjugation compound comprises a photoactivated conjugation moiety selected from the group consisting of a diazirine moiety, an aryl azide moiety, and a benzophenone moiety. [This invention 1087] 1070. An array of the invention, wherein the photoactivatable conjugated compound comprises an N-hydroxysuccinimide moiety attached to an ester. [This invention 1088] 1070. The array of claim 1070, wherein the photoactivatable conjugated compound comprises an N-hydroxysuccinimide ester functionally attached to a moiety selected from the group consisting of a diazirine moiety, an aryl azide moiety, and a benzophenone moiety. [This invention 1089] An array of the present invention 1070, wherein the surface is porous and the attachment groups are oriented in multiple directions. [The present invention 1090] Array of the invention 1070, wherein the surface is a surface of pillars operatively coupled at defined locations to a planar layer of support. [This invention 1091] An array of the present invention 1090, wherein each pillar has a flat upper surface extended from a planar layer of support. [This invention 1092] Array of the present invention 1091, wherein the distance between the top surface of each pillar and the planar layer of the support is 1,000 to 5,000 angstroms. [This invention 1093] The array of the present invention 1090, wherein the pillar is one of a plurality of pillars present on a support. [This invention 1094] Pillars: 10,000 / cm 2 Arrays of the present invention 1093 present at densities exceeding 1093. [This invention 1095] Array of the present invention 1093, wherein the center of each pillar is at least 2,000 angstroms away from the center of any other pillar. [This invention 1096] An array of the present invention 1093, wherein the surface of each pillar is parallel to the top surface of the planar layer. [This invention 1097] An array of the present invention 1093, wherein the surface of each pillar is substantially parallel to the top surface of the planar layer. [This invention 1098] Each pillar has a surface area of ​​at least 1 μm 2 The array of the present invention 1090. [This invention 1099] The total surface area of ​​each pillar is 10,000 μm 2 Arrays of the present invention that are less than 1090. [The present invention 1100] An array of the present invention 1090, wherein each pillar comprises silicon dioxide or silicon nitride. [The present invention 1101] Array of the present invention 1090, wherein each pillar is at least 98-99% silicon dioxide by weight. [The present invention 1102] An array of the invention 1090, wherein each defined location contains a plurality of identical biomolecules. [The present invention 1103] An array of the present invention 1102, wherein each of the defined locations contains a plurality of identical sequences that are different from other defined locations. [The present invention 1104] An array of the present invention 1090, wherein each of the defined locations is a location-distinguishable location. [This invention 1105] 1cm 2 Arrays of the present invention 1090 comprising at least 100, 1,000, 10,000, 100,000, 1,000,000, or 10,000,000 pillars per array. [The present invention 1106] Arrays of the invention 1070, wherein the surface comprises at least two unique biomolecules attached to selected locations on the surface. [This invention 1107] Arrays of the invention 1106, wherein the surface comprises at least 10 unique biomolecules attached to selected locations on the surface. [This invention 1108] Arrays of the invention 1107, wherein the surface comprises at least 100 unique biomolecules attached to selected locations on the surface. [This invention 1109] Arrays of the invention 1108, wherein the surface comprises at least 1,000 unique biomolecules attached to selected locations on the surface. [The present invention 1110] 1109. An array of the invention, wherein the surface comprises at least 10,000 unique biomolecules attached to selected locations on the surface. [The present invention 1111] The array of the present invention 1070, wherein the attachment groups are amine groups. [The present invention 1112] The array of the present invention 1070, wherein the attachment group is a carboxylic acid group. [The present invention 1113] An array of the invention 1112, wherein the carboxyl groups are activated to bind to the amine groups. [This invention 1114] 1. A method for detecting a biomolecule in a sample, comprising the steps of: A step of preparing a support according to any one of the present inventions 1070 to 1113; contacting the support with the sample; and Detecting a binding event between a biomolecule present in the sample and a biomolecule attached to the support. [Brief explanation of the drawings]

[0010] A brief description of some of the figures in the drawing These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description and accompanying drawings.

[0011] [Figure 1] 1 shows the synthesis of a pillar-containing support. [Figure 2] 1 shows the attachment of a first protein to an amine-derivatized array using a conjugation compound. [Figure 3] 1 shows the attachment of a second protein to an amine-derivatized array using a conjugation compound. [Figure 4] 1 shows the attachment of a first protein to a carboxylic acid-derivatized array using a conjugation compound. [Figure 5] 1 shows the attachment of a second protein to a carboxylic acid-derivatized array using a conjugation compound. [Figure 6] 1 shows the synthesis of a support comprising pillars with hydroxylated upper surfaces. [Figure 7] 1 shows fluorescence measurements of binding of anti-IL-6 and anti-TNFα antibodies to IL-6 and TNFα proteins attached to a support via a conjugation group. DETAILED DESCRIPTION OF THE INVENTION

[0012] Detailed Description Terms used in the claims and specification, unless otherwise specified, are defined as set forth below.

[0013] As used herein, the term "wafer" refers to a thin slice of semiconductor material, e.g., silicon or germanium crystal, commonly used in integrated circuit fabrication. Wafers may vary in size, e.g., from 25.4 mm (1 inch) to 300 mm (11.8 inches) along one dimension, and may be, e.g., from 275 μm to 775 μm thick.

[0014] As used herein, the terms "photoresist" or "resist" or "photoactive material" refer to a photosensitive material that changes solubility in solution when exposed to ultraviolet or deep ultraviolet light. Photoresists are typically organic or inorganic compounds that are divided into two types: positive resists and negative resists. Positive resists are a type of photoresist in which the exposed photoresist portions become soluble in a photoresist developer. The unexposed photoresist portions remain insoluble in the photoresist developer. Negative resists are a type of photoresist in which the exposed photoresist portions become insoluble in a photoresist developer. The unexposed photoresist portions are dissolved by the photoresist developer.

[0015] As used herein, the term "photomask" or "reticle" or "mask" refers to a light-opaque plate with a light-transmitting pattern or holes that allow light to pass through. In a typical exposure process, the pattern of the photomask is transferred onto a photoresist. The photomask or reticle or mask is used to create a pattern for exposure to electromagnetic radiation and thus site-specifically activate, for example, a photoactive compound or a photoactivatable conjugation group.

[0016] The term "photoactive compound" as used herein refers to a compound that is modified when exposed to electromagnetic radiation. These compounds include, for example, cationic photoinitiators, such as photoacid generators that generate an acid when exposed to electromagnetic radiation, or photobase generators that generate a base when exposed to electromagnetic radiation. Photoinitiators are compounds that are specifically added to formulations to convert electromagnetic radiation into chemical energy in the form of initiating chemical species, such as free radicals or cations. The acid, base, or other product of the photoactive compound exposed to electromagnetic radiation can then react with another compound in a chain reaction to produce a desired chemical reaction. Thus, the spatial direction in which these chemical reactions occur is determined by the pattern of electromagnetic radiation to which the solution or surface containing the photoactive compound is exposed. This pattern may be determined, for example, by a photomask or reticle.

[0017] The term "conjugation compound," as used herein, refers to a compound that binds to a functional group on a support and, upon activation, can bind to a biomolecule, thereby attaching the biomolecule to the support. A photoactivatable or photoactive conjugation compound refers to a compound that is activated and conjugated to a biomolecule upon exposure to electromagnetic radiation. These compounds include, for example, compounds containing a diazirine moiety, an aryl azide moiety, or a benzophenone moiety.

[0018] As used herein, the term "coupling molecule" or "monomer molecule" includes any natural or synthetic amino acid in which the amino group is protected with a fluorenylmethyloxycarbonyl or t-butoxycarbonyl group. These amino acids may optionally have protected side chains. Examples of coupling molecules include Boc-Gly-Oh and Fmoc-Trp-Oh. Other examples are described below.

[0019] As used herein, the terms "coupling" or "coupling process" or "coupling step" refer to the process of forming a bond between two or more molecules, such as linker molecules or coupling molecules. The bond may be a covalent bond, such as a peptide bond. A peptide bond may be a chemical bond formed between two molecules when a carboxyl group of one coupling molecule reacts with an amino group of another coupling molecule, releasing a water molecule (HO). This is a dehydration synthesis reaction (also known as a condensation reaction) and typically occurs between amino acids. The resulting CO-NH bond is called a peptide bond, and the resulting molecule is an amide.

[0020] As used herein, the term "coupling efficiency" refers to the likelihood that a monomer will successfully add to a reactive site (e.g., at the end of a polymer) that can bind to the monomer. For example, during the growth of a peptide chain in the N→C direction, a polypeptide with a free carboxyl group will, under appropriate conditions, bind to a peptide with a free amine group. Coupling efficiency indicates the likelihood that a free peptide will add to a free carboxyl group under certain conditions. Coupling efficiency may also be determined en masse, for example, by monitoring the simultaneous addition of one type of monomer to several unique reactive sites.

[0021] As used herein, the terms "biomolecule," "polypeptide," "peptide," or "protein" are used interchangeably to describe a chain or polymer of amino acids linked by bonds. Thus, as used herein, the term "peptide" includes dipeptides, tripeptides, oligopeptides, and polypeptides. The term "peptide" is not limited to any particular number of amino acids. In some aspects, a peptide contains about 2 to about 50 amino acids, about 5 to about 40 amino acids, or about 5 to about 20 amino acids. Molecules, such as proteins or polypeptides, including enzymes, may be "native" or "wild-type" molecules, meaning that they occur naturally in nature, or they may be "mutants," "variants," "derivatives," or "modified," meaning that they are made from, altered, obtained from, or are different or changed in some way from a native molecule or another molecule, e.g., a mutant.

[0022] As used herein, the term "linker molecule" or "spacer molecule" includes any molecule that does not add any function to the resulting peptide, but that spaces the peptide, extending it from the support, thus increasing the distance between the support surface and the growing peptide. This generally reduces steric hindrance with the support for reactions involving the peptide (including unimolecular folding and multimolecular binding reactions), thereby improving the performance of assays that measure one or more aspects of peptide function.

[0023] The term "developer" as used herein refers to a solution that can selectively dissolve exposed or unexposed material. Typically, developers are water-based solutions with trace amounts of base added. An example includes tetramethylammonium hydroxide dissolved in a water-based developer. Developers are used for initial pattern definition when commercial photoresists are used. The use of developers is described in Example 1 below.

[0024] As used herein, the term "protecting group" includes groups introduced into molecules by chemical modification of functional groups to achieve chemoselectivity in subsequent chemical reactions. "Chemoselectivity" refers to directing a chemical reaction along a desired pathway to obtain a preselected product over another product. For example, when tboc is used as a protecting group, the protecting group can be selectively removed through the chemoselectivity of peptide synthesis using a photomask and a photoacid generator, allowing a predetermined, direct peptide coupling reaction to occur at a location defined by the photomask.

[0025] As used herein, the term "microarray" refers to a support on which different probe molecules of proteins or specific DNA binding sequences are attached in an ordered manner at discrete locations, thus forming a minute array.

[0026] As used herein, the term "microarray system" refers to a system that typically consists of biomolecular probes formatted on a solid surface such as a glass, plastic, or silicon chip, along with the equipment needed to handle the samples (automated robotics), read the reporter molecules (scanners), and analyze the data (bioinformatics tools).

[0027] As used herein, the terms "patterned area" or "pattern" or "location" refer to areas on a substrate where various features are grown. These patterns can be defined using a photomask.

[0028] As used herein, the term "derivatization" refers to the process of chemically modifying a surface to make it suitable for biomolecular synthesis. Typically, derivatization involves the following steps: rendering the support hydrophilic, adding aminosilane groups, and attaching linker molecules.

[0029] As used herein, the term "capping" or "capping process" or "capping step" refers to the addition of a molecule that prevents further reaction of the attached molecule. For example, amino groups are typically capped with acetic anhydride molecules to prevent further formation of peptide bonds. In other embodiments, ethanolamine is used.

[0030] As used herein, the term "diffusion" refers to the spread of photoacid via random motion from an area of ​​high concentration to an area of ​​low concentration.

[0031] As used herein, the term "dye molecule" refers to a dye, which is typically a colored substance that can be bound to a support. Dye molecules can be useful in detecting binding between features on an array and a molecule of interest.

[0032] As used herein, the terms "immunological binding" and "immunological binding properties" refer to the type of non-covalent interactions that occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific.

[0033] As used herein, the term "biological sample" refers to a sample derived from biological tissue or fluid that can be assayed for an analyte of interest. Such samples include, but are not limited to, sputum, amniotic fluid, blood, blood cells (e.g., white blood cells), tissue or fine needle biopsy samples, urine, ascites, and pleural fluid, or cells derived therefrom. Biological samples may also include tissue sections, such as frozen sections taken for histological purposes. While samples are typically taken from human patients, the assay can be used to detect an analyte of interest in samples from any organism (e.g., mammals, bacteria, viruses, algae, or yeast) or mammals, such as dogs, cats, sheep, cattle, and pigs. The sample may be prepared by diluting with an appropriate buffer, if necessary, and may be concentrated if desired.

[0034] As used herein, the term "assay" refers to a type of biochemical test that measures the presence or concentration of a substance of interest in a solution that may contain a complex mixture of substances.

[0035] As used herein, the term "antigen" refers to a molecule that elicits an immune response by a subject's immune system, e.g., the production of antibodies by the immune system. Antigens may be exogenous, endogenous, or self-antigens. Exogenous antigens are antigens that enter the body from the outside through inhalation, ingestion, or injection. Endogenous antigens are antigens that arise within previously normal cells as a result of normal cellular metabolism or as a result of viral or intracellular bacterial infection. Self-antigens are normal proteins or protein complexes present in the host body, but are capable of stimulating an immune response.

[0036] As used herein, the term "epitope" or "immunoreactive region" refers to a distinct molecular surface feature of an antigen that can be bound by a component of the adaptive immune system, such as an antibody or a T-cell receptor. An antigen molecule can present several surface features that can act as interaction points for specific antibodies. All such distinct molecular features can constitute an epitope. Thus, an antigen has the ability to be bound by several distinct antibodies, each of which is specific for a particular epitope.

[0037] As used herein, the term "antibody" or "immunoglobulin molecule" refers to a molecule that is naturally secreted by a particular type of immune system cell: B cells. There are five different natural isotypes of antibodies: IgA, IgM, IgG, IgD, and IgE.

[0038] As used herein, the term "activated carboxylic acid group" refers to a carboxylic acid group to which a leaving group is attached so that it readily couples to an amine group. In some embodiments, a carbodiimide or N-hydroxysuccinimide activates the carboxylic acid group to increase the probability of coupling to an amine group. In some embodiments, an activated carboxylic acid group refers to an ester or carbonyl attached to a group that interacts with an amine group and is thus removed upon formation of a covalent bond between the ester or carbonyl group and the amine group.

[0039] It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0040] composition formulation Disclosed herein are formulations, such as photoactive conjugate solutions and polypeptide formulations, which may be useful, for example, in the manufacture and / or use of the supports and / or polypeptide arrays disclosed herein.

[0041] Photoactive conjugation solution Disclosed herein are photoactive conjugation solutions. In some aspects, the photoactive conjugation solution may include components such as a solvent, a photoactive conjugation compound, and a polymer.

[0042] In one aspect, the photoactive conjugation solution may include a photoactive conjugation compound (i.e., a conjugation compound). The photoactive conjugation compound includes a chemically inert moiety that becomes reactive when exposed to ultraviolet or visible light. Exposure of the photoactive conjugation compound to electromagnetic radiation is the initial photochemical event that generates the compound that binds to the polypeptide. The photoactive conjugation solution may include a photoactive conjugation compound that includes a radiation-sensitive bond precursor that includes chemical groups that can react by elimination, addition, or rearrangement, and any additives that improve performance or processability.

[0043] In some aspects, the photoactive coupling formulation comprises a photoactive conjugated compound in a polymer matrix dispersed in a solvent. In some aspects, the polymer in the photoresist composition is generally inert and non-crosslinkable.

[0044] In some aspects, the photoactive compound may have an amine group or a carboxylic acid group. In some embodiments, the carboxylic acid group is activated by binding to a strong leaving group to induce covalent bonding with the amine group. In some embodiments, the amine group is used to bind to the carboxylic acid group attached to the surface of the array. The photoactive compound includes a photoactive moiety that converts absorbed light energy, UV or visible light, into chemical energy in the form of an initiating chemical species, such as a free radical or cation.

[0045] In one aspect, the photoactive conjugation compound is used as a heterobifunctional crosslinker for attaching a polypeptide to the array surface. In one aspect, the photoactive conjugation compound further comprises an amine group for coupling to a carboxylic acid group attached to the array surface. In another aspect, the photoactive conjugation compound further comprises a carboxylic acid group that is activated to couple to an amine group attached to the array surface. Once attached to the array surface, the photoactive conjugation group is activated to site-specifically conjugate a desired polypeptide, protein, or other biomolecule.

[0046] In some aspects, photoactivatable compounds contain aryl azide, diazirine, or benzophenone moieties. Aryl azides (also called phenyl azides) form nitrene groups upon exposure to UV light. The nitrene groups can initiate addition reactions with double bonds or insertions into CH and NH sites, or undergo ring expansion to react with nucleophiles (e.g., primary amines). Reactions can be performed in a variety of amine-free buffer solutions to conjugate proteins or even molecules lacking useful functional "handles." The diazirine (azipentanoate) moiety has superior photostability to the phenyl azide group and is activated more easily and efficiently by long-wave UV light (330-370 nm) than the phenyl azide group. Photoactivation of the diazirine generates a reactive carbene intermediate. Such intermediates can form covalent bonds with any amino acid side chain or peptide backbone via addition reactions, at distances corresponding to the spacer arm length of the particular reagent. Diazirine analogs of amino acids can be incorporated into protein structures during translation, thereby activating certain recombinant proteins as cross-linkers.

[0047] In some embodiments, the conjugation solution comprises a conjugation compound, a solvent, and a polymer. In one embodiment, the conjugation compound is an NHS ester of an aryl azide, a diazirine, or a benzophenone. In another embodiment, the conjugation compound is an amine group functionally linked to an aryl azide, a diazirine, or a benzophenone. In some aspects, the carbodiimide precursor is present in the activation solution at a concentration of 2.5% by weight. In some aspects, the conjugated compound is present in the conjugation solution at 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4% by weight of the total formulation concentration. , 2.5 wt%, 2.6 wt%, 2.7 wt%, 2.8 wt%, 2.9 wt%, 3.0 wt%, 3.1 wt%, 3.2 wt%, 3.3 wt%, 3.4 wt%, 3.5 wt%, 3.6 wt%, 3.7 wt%, 3.8 wt%, 3.9 wt%, 4.0 wt%, 4.1 wt%, 4.2 wt%, 4.3 wt%, 4.4 wt%, 4.5 wt%, 4.6 wt%, 4.7 wt%, 4.8 wt%, 4.9 wt%, 5.0 wt%, or 5.0 wt%.

[0048] In some aspects, the polymer is an inert polymer that is not crosslinked. In some aspects, the polymer is polyvinylpyrrolidone. The general structure of polyvinylpyrrolidone is: TIFF2025126208000001.tif28128 where n is any positive integer greater than 1.

[0049] In some aspects, the polymer is a polymer of vinylpyrrolidone. In some aspects, the polymer is polyvinylpyrrolidone. Polyvinylpyrrolidone is soluble in water and other polar solvents. Polyvinylpyrrolidone is a light, flaky powder when dry and typically readily absorbs up to 40% of its weight in atmospheric water. In the dissolved state, polyvinylpyrrolidone has excellent wettability and readily forms thin films. In some aspects, the polymer is vinylpyrrolidone or vinyl alcohol. In some aspects, the polymer is polymethyl methacrylate.

[0050] In some aspects, the polymer is 2.5-5% by weight of the total formulation. In some aspects, the polymer is about 0.5-5% by weight of the total formulation. In some aspects, the polymer is about 0.1%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6.0%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7.0%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%. less than 5% by weight, 2.6% by weight, 2.7% by weight, 2.8% by weight, 2.9% by weight, 3.0% by weight, 3.1% by weight, 3.2% by weight, 3.3% by weight, 3.4% by weight, 3.5% by weight, 3.6% by weight, 3.7% by weight, 3.8% by weight, 3.9% by weight, 4.0% by weight, 4.1% by weight, 4.2% by weight, 4.3% by weight, 4.4% by weight, 4.5% by weight, 4.6% by weight, 4.7% by weight, 4.8% by weight, 4.9% by weight, 5.0% by weight or more than 5.0% by weight.

[0051] In some aspects, the solvent is water, ethyl lactate, n-methylpyrrolidone, or a combination thereof. In some aspects, the ethyl lactate can be dissolved in water up to 50% to form the solvent. In some aspects, the solvent may be about 10% propylene glycol methyl ether acetate (PGMEA) and about 90% DI water. In some aspects, the solvent may include up to about 20% PGMEA. In some aspects, the solvent may include 50% ethyl lactate and 50% n-methylpyrrolidone. In some aspects, the solvent is n-methylpyrrolidone. In some aspects, the solvent is water, an organic solvent, or a combination thereof. In some aspects, the organic solvent is N-methylpyrrolidone, dimethylformamide, or a combination thereof.

[0052] In some aspects, the solvent is about 80-90% by weight of the total formulation, or less than about 70%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% by weight of the total formulation.

[0053] Carboxylic acid activated preparation Disclosed herein is an activation formulation for activating carboxylic acid so that the carboxylic acid reacts with the free amine group of a biomolecule, such as a conjugated compound. The activation formulation may include components such as a carboxylic acid group-activating compound and a solvent. In one embodiment, the carboxylic acid group-activating compound is a carbodiimide or a carbodiimide precursor. In some aspects, the carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. In some embodiments, the carboxylic acid group-activating compound is N-hydroxysuccinimide (NHS). In some embodiments, the solvent is water. In some embodiments, the carboxylic acid group-activating compound converts the carboxylic acid into a carbonyl group (i.e., carboxylic acid group activation). In some embodiments, the carboxylic acid group is activated after 5, 10, 15, 20, 30, 45, or 60 minutes of exposure to the activation formulation.

[0054] In some aspects, the activation formulation comprises 4% by weight 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 2% by weight N-hydroxysuccinimide (NHS) dissolved in deionized water.

[0055] In some embodiments, the carboxylic acid group-activating compound is a carbodiimide precursor. In one aspect, the carbodiimide precursor is converted to a carbodiimide by exposure to radiation, for example, ultraviolet light. In one embodiment, the carbodiimide precursor is a thione. The carbodiimide precursor may be referred to as a photoactivated carbodiimide. In one embodiment, the photoactivated carbodiimide is used to site-specifically activate the carboxylic acid groups on the array by spatially controlling the exposure of the photoactivated carbodiimide solution to electromagnetic radiation of a preferred activation wavelength. In some embodiments, the preferred activation wavelength is 248 nm.

[0056] In one embodiment, the carbodiimide precursor is a thione that is converted to a carbodiimide via photoactivation. In one aspect, the thione is converted to a hydroxymethylphenylcarbodiimide after exposure to electromagnetic radiation. In some embodiments, the thione is 4,5-dihydro-4-(hydroxymethyl)-1-phenyl-1H-tetrazole-5-thione, 1-ethyl-4-dimethylaminopropyltetrazole-5-thione, 1,3-Bis(2,2-dimethyl-1,3-dioxolan-4-ylmethyl)-5-thione, 4-cyclohexyl-1H-tetrazole-5(4H)-thione, or 1-phenyl-4-(piperidinomethyl)tetrazole-5(4H)-thione.

[0057] In some embodiments, the activation solution comprises a carbodiimide precursor, a solvent, and a polymer. In one embodiment, the carbodiimide precursor is 4,5-dihydro-4-(hydroxymethyl)-1-phenyl-1H-tetrazole-5-thione, 1-ethyl-4-dimethylaminopropyltetrazole-5-thione, or 1,3-Bis(2,2-dimethyl-1,3-dioxolan-4-ylmethyl)-5-thione. In some aspects, the carbodiimide precursor is present in the activation solution at a concentration of 2.5% by weight. In some aspects, the carbodiimide precursor is present in an amount of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5% by weight of the total formulation concentration. , 2.6 wt%, 2.7 wt%, 2.8 wt%, 2.9 wt%, 3.0 wt%, 3.1 wt%, 3.2 wt%, 3.3 wt%, 3.4 wt%, 3.5 wt%, 3.6 wt%, 3.7 wt%, 3.8 wt%, 3.9 wt%, 4.0 wt%, 4.1 wt%, 4.2 wt%, 4.3 wt%, 4.4 wt%, 4.5 wt%, 4.6 wt%, 4.7 wt%, 4.8 wt%, 4.9 wt%, 5.0 wt%, or 5.0 wt%.

[0058] In some embodiments, the solvent is water. In some aspects, the solvent is about 80-90% by weight of the total formulation. In some aspects, the solvent is less than about 70%, 70%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more by weight of the total formulation.

[0059] In some aspects, the polymer is polyvinylpyrrolidone and / or polyvinyl alcohol. In some aspects, the polymer is about 0.5 to 5% by weight of the total formulation. In some aspects, the polymer is about 0.1%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5% by weight of the total formulation. %, 2.6 wt%, 2.7 wt%, 2.8 wt%, 2.9 wt%, 3.0 wt%, 3.1 wt%, 3.2 wt%, 3.3 wt%, 3.4 wt%, 3.5 wt%, 3.6 wt%, 3.7 wt%, 3.8 wt%, 3.9 wt%, 4.0 wt%, 4.1 wt%, 4.2 wt%, 4.3 wt%, 4.4 wt%, 4.5 wt%, 4.6 wt%, 4.7 wt%, 4.8 wt%, 4.9 wt%, 5.0 wt%, or less than or more than 5.0 wt%.

[0060] In some aspects, the coupling reagent is a carbodiimide. In some aspects, the coupling reagent is a triazole. In some aspects, the coupling reagent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. In some aspects, the coupling reagent is about 0.5 to 5% by weight of the total formulation. In some aspects, the coupling reagent is about 0.1%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, or 2.5 wt%, 2.6 wt%, 2.7 wt%, 2.8 wt%, 2.9 wt%, 3.0 wt%, 3.1 wt%, 3.2 wt%, 3.3 wt%, 3.4 wt%, 3.5 wt%, 3.6 wt%, 3.7 wt%, 3.8 wt%, 3.9 wt%, 4.0 wt%, 4.1 wt%, 4.2 wt%, 4.3 wt%, 4.4 wt%, 4.5 wt%, 4.6 wt%, 4.7 wt%, 4.8 wt%, 4.9 wt%, 5.0 wt%, or less or more than 5.0 wt%.

[0061] Linker formulation Linker formulations are also disclosed herein. The linker formulation may include components such as a solvent, a polymer, a linker molecule, and a coupling reagent. In some aspects, the polymer is 1% by weight polyvinyl alcohol and 2.5% by weight polyvinylpyrrolidone, the linker molecule is 1.25% by weight polyethylene oxide, the coupling reagent is 1% by weight 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and the solvent includes water. In some aspects, the polymer is 0.5-5% by weight polyvinyl alcohol and 0.5-5% by weight polyvinylpyrrolidone, the linker molecule is 0.5-5% by weight polyethylene oxide, the coupling reagent is 0.5-5% by weight 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and the solvent includes water.

[0062] In some aspects, the solvent is water, an organic solvent, or a combination thereof. In some aspects, the organic solvent is N-methylpyrrolidone, dimethylformamide, dichloromethane, dimethyl sulfoxide, or a combination thereof. In some aspects, the solvent is about 80 to 90% by weight of the total formulation concentration. In some aspects, the solvent is less than about 70%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more by weight of the total formulation concentration.

[0063] In some aspects, the polymer is polyvinylpyrrolidone and / or polyvinyl alcohol. The general structure of polyvinyl alcohol is: TIFF2025126208000002.tif22128 where n is any positive integer greater than 1.

[0064] In some aspects, the polymer is about 0.5-5% by weight of the total formulation. In some aspects, the polymer is about 0.1%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6.0%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7.0%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8.1%. less than 5% by weight, 2.6% by weight, 2.7% by weight, 2.8% by weight, 2.9% by weight, 3.0% by weight, 3.1% by weight, 3.2% by weight, 3.3% by weight, 3.4% by weight, 3.5% by weight, 3.6% by weight, 3.7% by weight, 3.8% by weight, 3.9% by weight, 4.0% by weight, 4.1% by weight, 4.2% by weight, 4.3% by weight, 4.4% by weight, 4.5% by weight, 4.6% by weight, 4.7% by weight, 4.8% by weight, 4.9% by weight, 5.0% by weight or more than 5.0% by weight.

[0065] A linker molecule may be inserted between the surface disclosed herein and the peptide being synthesized via a coupling molecule. The linker molecule does not necessarily impart functionality, such as molecular recognition, to the resulting peptide; instead, it can increase the distance between the surface and the peptide to enhance exposure of the peptide's functional region on the surface. In some aspects, the linker may be about 4 to about 40 atoms long to provide exposure. The linker molecule may be, for example, arylacetylene, ethylene glycol oligomer (PEG) containing 2 to 10 monomer units, diamine, diacid, amino acid, or combinations thereof. Examples of diamines include ethylenediamine and diaminopropane. Alternatively, the linker may be the same molecular type as the molecule being synthesized (e.g., the nascent polymer or various coupling molecules), for example, a polymer of polypeptide and amino acid derivatives, such as aminohexanoic acid. In some aspects, the linker molecule has a carboxyl group at one end and a protecting group at the second end. In some aspects, the protecting group is a t-Boc protecting group or an F-Moc protecting group. In some aspects, the linker molecule is or includes an aryl-acetylene, polyethylene glycol, a nascent polypeptide, a diamine, a diacid, a peptide, or a combination thereof. In some aspects, the linker molecule is about 0.5 to 5% by weight of the total formulation concentration.In some aspects, the linker molecule is present in an amount of about 0.1%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.10%, 3.11%, 3.12%, 3.13%, 3.14%, 3.15%, 3.16%, 3.17%, 3.18%, 3.19%, 3.20%, 3.21%, 3.22%, 3.23%, 3.24%, 3.25%, 3.26%, 3.27%, 3.28%, 3.29%, 3.30%, 3.31%, 3.32%, 3.33%, 3.34%, 3.35%, 3.36%, 3.37%, 3.38%, 3.39%, 3.40%, 3.41%, 3.42%, 3.43%, 3.44%, 3.45%, 3.46%, 3.47%, 3.48%, 3.49%, 3.50%, 3.51%, 3.52%, 3.53%, 3.54%, 3.55%, 3.56%, 3.57%, 3.58%, 3.59%, 3.60%, 3.61%, 3.62%, 3.63%, 3.64%, 3.65%, 3.66 0.5 wt%, 2.6 wt%, 2.7 wt%, 2.8 wt%, 2.9 wt%, 3.0 wt%, 3.1 wt%, 3.2 wt%, 3.3 wt%, 3.4 wt%, 3.5 wt%, 3.6 wt%, 3.7 wt%, 3.8 wt%, 3.9 wt%, 4.0 wt%, 4.1 wt%, 4.2 wt%, 4.3 wt%, 4.4 wt%, 4.5 wt%, 4.6 wt%, 4.7 wt%, 4.8 wt%, 4.9 wt%, 5.0 wt%, or less than or more than 5.0 wt%.

[0066] The unattached portion of the linker molecule, i.e., the free end of the linker molecule, may have a reactive functional group that is blocked, protected, or otherwise rendered unavailable for reaction by a removable protecting group, such as the aforementioned t-Boc or F-Moc. Protecting groups may be attached to the monomer, polymer, or linker molecule to protect the reactive functional group therein. Protecting groups that can be used include all acid-labile and base-labile protecting groups. For example, peptide amine groups may be protected with t-butoxycarbonyl (t-BOC or BOC) or benzyloxycarbonyl (CBZ), both of which are acid-labile, or with 9-fluorenylmethoxycarbonyl (FMOC), which is base-labile.

[0067] Additional protecting groups that can be used include acid labile groups for protecting amino moieties: tert-amyloxycarbonyl, adamantyloxycarbonyl, 1-methylcyclobutyloxycarbonyl, 2-(p-biphenyl)propyl(2)oxycarbonyl, 2-(p-phenylazophenylyl)propyl(2)oxycarbonyl, α,α-dimethyl-3,5-dimethyloxybenzyloxycarbonyl, 2-phenylpropyl(2)oxycarbonyl, 4-methyloxybenzyloxycarbonyl, furfuryloxycarbonyl, triphenylmethyl(trityl), p-toluenesulfenylaminocarbonyl, dimethylphosphinothioyl, diphenylphosphinothioyl, 2-benzoyl-1-methylvinyl, o-nitrophenylsulfenyl, and 1-naphthylidene; and base labile groups for protecting amino moieties. For protecting amino moieties that are unstable when reduced, groups include dithiasuccinoyl, p-toluenesulfonyl, and piperidino-oxycarbonyl; for protecting amino moieties that are unstable when oxidized, groups include (ethylthio)carbonyl; for protecting amino moieties that are unstable to a wide variety of reagents, suitable agents are shown in parentheses after the group: phthaloyl (hydrazine), trifluoroacetyl (piperidine), and chloroacetyl (2-aminothiophenol); for protecting carboxylic acids, groups include tert-butyl ester; and for protecting hydroxyl groups, groups include dimethyltrityl. (See also Greene, TW, Protective Groups in Organic Synthesis, Wiley-Interscience, NY, (1981))

[0068] support Supports are also disclosed herein. In some aspects, the support surface is planar (i.e., two-dimensional). In some aspects, the support may include a porous layer (i.e., a three-dimensional layer) containing functional groups for binding the first monomer building block. In some aspects, the support surface includes pillars for attaching or synthesizing peptides. In some embodiments, the porous layer is added to the top of the pillars.

[0069] porous layer support The porous layer that can be used is a flat, permeable polymer material with a porous structure that has carboxylic acid functional groups (either inherent in the component polymer or introduced into the porous layer) for attaching the first peptide building block. For example, the porous layer can be made of porous silicon, with functional groups attached to the surface of the porous silicon for attaching the polymer building block. In another example, the porous layer can include a cross-linked polymer material. In some embodiments, the porous layer can be made of polystyrene, sucrose, dextran, polyacryloylmorpholine, polyacrylate, polymethylacrylate, polyacrylamide, polyacrylolpyrrolidone, polyvinyl acetate, polyethylene glycol, agarose, Sepharose, and other conventional chromatographic materials, as well as derivatives and mixtures thereof. In some embodiments, the porous layer is made of a material selected from poly(vinyl alcohol), dextran, sodium alginate, poly(aspartic acid), poly(ethylene glycol), poly(ethylene oxide), poly(vinylpyrrolidone), poly(acrylic acid), poly(acrylic acid)-sodium salt, poly(acrylamide), poly(N-isopropylacrylamide), poly(hydroxyethyl acrylate), poly(acrylic acid), poly(sodium styrenesulfonate), poly(2-acrylamido-2-methyl-1-propanesulfonic acid), polysaccharides, and cellulose derivatives. The porosity of the porous layer is preferably 10 to 80%. In one embodiment, the thickness of the porous layer is 0.01 μm to approximately 1,000 μm. The pore size of the porous layer may be 2 nm to approximately 100 μm.

[0070] According to another aspect of the present invention, there is provided a support comprising a porous polymeric material having a porosity of 10-80%, with reactive groups chemically bonded to the pore surfaces, adapted for use in interacting with, for example, reactive species, e.g., deprotected monomer building blocks or polymer chains, by chemical bonding. In one embodiment, the reactive groups are carboxylic acid groups, which are free to bond, for example, to unprotected amine groups on peptides or polypeptides.

[0071] In some embodiments, the porous layer is in contact with a support layer. The support layer may comprise, for example, a metal, plastic, silicon, silicon oxide, or silicon nitride. In other embodiments, the porous layer may be in contact with a patterned surface, such as a patterned surface overlying a pillar support, as described below.

[0072] Pillar support In some embodiments, the support comprises a planar layer comprising a metal and having an upper surface and a lower surface, and a plurality of pillars operatively connected to defined locations on the layer, wherein each pillar has a planar surface extending from the layer, the distance between the surface of each pillar and the upper surface of the layer is about 1,000-5,000 angstroms, and the plurality of pillars has a spacing of about 10,000 / cm. 2 It exists at a density exceeding

[0073] In some aspects, the distance between the surface of each pillar and the top surface of the layer can be less than about 1,000 angstroms, about 2,000 angstroms, about 3,000 angstroms, about 3,500 angstroms, about 4,500 angstroms, about 5,000 angstroms, or greater than about 5,000 angstroms (or any integer greater or less than that in between).

[0074] In some aspects, the surface of each pillar is parallel to the top surface of the layer. In some aspects, the surface of each pillar is substantially parallel to the top surface of the layer.

[0075] In some aspects, the plurality of pillars has a density of 500 / cm 2 , 1,000 / cm 2 , 2,000 / cm 2 , 3,000 / cm 2 , 4,000 / cm 2 , 5,000 / cm 2 , 6,000 / cm 2 , 7,000 / cm 2 , 8,000 / cm 2 , 9,000 / cm 2 , 10,000 / cm 2 , 11,000 / cm 2 , or 12,000 / cm 2 In some aspects, the plurality of pillars are present at a density greater than 10,000 / cm (or any integer in between). 2 In some aspects, the pillars are present at a density of greater than about 10,000 / cm 2 ~About 2.5 million / cm 2 (or any integer in between). In some aspects, the pillars are present at a density of 2.5 million / cm 2 It exists at a density exceeding

[0076] In some aspects, the surface area of ​​each pillar is at least 1 μm 2 In some aspects, the surface area of ​​each pillar is at least 0.1 μm 2 , 0.5 μm 2 , 12 μm 2 , 3 μm 2 , 4 μm 2 , 5 μm 2 , 6 μm 2 , 7 μm 2 , 8 μm 2 , 9 μm 2 , 10 μm 2 , 15 μm 2 , 20 μm 2 , 25 μm 2 , 30 μm 2 , 35 μm 2 , 40 μm 2 , 45 μm 2 , or 50 μm 2(or any integer in between). In some aspects, the total surface area of ​​the surfaces of each pillar is 10,000 μm 2 In some aspects, the total surface area of ​​the surfaces of each pillar is less than 500 μm 2 , 1,000 μm 2 , 2,000 μm 2 , 3,000 μm 2 , 4,000 μm 2 , 5,000 μm 2 , 6,000 μm 2 , 7,000 μm 2 , 8,000 μm 2 , 9,000 μm 2 , 10,000 μm 2 , 11,000 μm 2 , or 12,000 μm 2 (or any integer in between).

[0077] In some aspects, the distance between the surface of each pillar and the lower surface of the layer is 2,000 to 7,000 angstroms. In some aspects, the distance between the surface of each pillar and the lower surface of the layer is less than about 500 angstroms, about 1,000 angstroms, about 2,000 angstroms, about 3,000 angstroms, about 4,000 angstroms, about 5,000 angstroms, about 6,000 angstroms, about 7,000 angstroms, about 8,000 angstroms, about 9,000 angstroms, about 10,000 angstroms, about 11,000 angstroms, about 12,000 angstroms, or more than about 12,000 angstroms (or any integer less than or greater than about 12,000 angstroms). In some aspects, the distance between the surface of each pillar and the bottom surface of the layer is 7,000 angstroms, 3,000 angstroms, 4,000 angstroms, 5,000 angstroms, 6,000 angstroms, or 7,000 angstroms (or any integer therebetween).

[0078] In some aspects, the layer is 1,000 to 2,000 angstroms thick. In some aspects, the layer is less than about 500 angstroms, about 1,000 angstroms, about 2,000 angstroms, about 3,000 angstroms, about 4,000 angstroms, about 5,000 angstroms, about 6,000 angstroms, about 7,000 angstroms, about 8,000 angstroms, about 9,000 angstroms, about 10,000 angstroms, about 11,000 angstroms, about 12,000 angstroms thick, or more than about 12,000 angstroms thick (or any integer less than or greater than about 12,000 angstroms thick).

[0079] In some aspects, the center of each pillar is at least 2,000 angstroms away from the center of any other pillar. In some aspects, the center of each pillar is at least about 500 angstroms, about 1,000 angstroms, about 2,000 angstroms, about 3,000 angstroms, or about 4,000 angstroms (or any integer therebetween) away from the center of any other pillar. In some aspects, the center of each pillar is at least about 2 μm to 200 μm away from the center of any other pillar.

[0080] In some aspects, the metal is chromium. In some aspects, the metal is chromium, titanium, aluminum, tungsten, gold, silver, tin, lead, thallium, indium, or a combination thereof. In some aspects, the layer is at least 98.5-99% metal. In some aspects, the layer is 100% metal. In still other embodiments, the layer is at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 98.5%, or greater than about 99% metal. In some aspects, the layer is a uniform metal layer.

[0081] In some aspects, the or each pillar comprises silicon. In some aspects, the or each pillar comprises silicon dioxide or silicon nitride. In some aspects, the or each pillar is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, or 99% silicon dioxide.

[0082] In some aspects, the support may include a linker molecule having a free amino terminus attached to the surface of each pillar. In some aspects, the support may include a linker molecule having a free amino terminus attached to the surface of at least one pillar. In some aspects, the support may include a linker molecule having a protecting group attached to the surface of each pillar. In some aspects, the support may include a linker molecule having a protecting group attached to the surface of at least one pillar. In some aspects, the support may include a coupling molecule attached to the surface of at least one pillar. In some aspects, the support may include a coupling molecule attached to the surface of each pillar. In some aspects, the support may include a polymer in contact with the surface of at least one pillar. In some aspects, the support may include a polymer in contact with the surface of each pillar. In some aspects, the support may include a gelatinous polymer in contact with the surface of at least one pillar. In some aspects, the support may include a solid polymer in contact with the surface of at least one pillar.

[0083] In some aspects, the surface of at least one pillar of the support is derivatized. In some aspects, the support may include a polymer chain attached to the surface of at least one pillar. In some aspects, the polymer chain includes a peptide chain. In some aspects, the attachment to the surface of at least one pillar is via a covalent bond.

[0084] In some aspects, the surface of each pillar is square or rectangular in shape. In some aspects, the support can be coupled to a silicon dioxide layer. The silicon dioxide layer can be about 0.5 μm to 3 μm thick. In some aspects, the support can be coupled to a wafer, e.g., a silicon wafer. The silicon dioxide layer can be about 700 μm to 750 μm thick.

[0085] array Arrays are also disclosed herein. In some aspects, the array may be a three-dimensional array, for example, a porous array, in which features are attached to the surface of the porous array. The surface of the porous layer includes an outer surface and a surface that defines the pore volume within the porous array. In some aspects, the three-dimensional array may include features attached to the surface at defined locations, each of which includes a collection of peptide chains of determinable sequence and intended length. In one embodiment, a portion of the polypeptides in the array are characterized by an average polypeptide conjugation efficiency of more than 98% for each coupling step.

[0086] In some aspects, the average polypeptide conjugation efficiency is at least 98.5%. In some aspects, the average polypeptide conjugation efficiency is at least 99%. In some aspects, the average polypeptide conjugation efficiency of each coupling step is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%.

[0087] In some aspects, the array may comprise at least 2, 10, 100, or 1,000 different polypeptide chains attached to the surface. In some aspects, the array may comprise at least 10,000 different polypeptide chains attached to the surface. In some aspects, the array may comprise at least 100, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, or more than 10,000 different polypeptide chains (or any integer in between) attached to the surface.

[0088] In some aspects, each of the defined locations is at a different known location that is physically separated from each of the other defined locations. In some aspects, each of the defined locations is a distinct location. In some aspects, each determinable sequence is a known sequence. In some aspects, each determinable sequence is a distinct sequence.

[0089] In some aspects, the features are covalently attached to the surface, hi some aspects, the peptide chains are attached to the surface via linker or coupling molecules.

[0090] In some aspects, the feature comprises a plurality of distinct, nested, overlapping peptide chains comprising subsequences derived from a source protein having a known sequence. In some aspects, each peptide chain in the plurality of overlapping peptide chains is substantially the same length. In some aspects, each peptide chain in the plurality of overlapping peptide chains is the same length. In some aspects, each peptide chain in the plurality of overlapping peptide chains is at least 5 amino acids in length. In some aspects, each peptide chain in the plurality of overlapping peptide chains is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids in length. In some aspects, each peptide chain in the plurality of overlapping peptide chains is less than 5 amino acids in length or at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 amino acids in length. , 31 amino acids, 32 amino acids, 33 amino acids, 34 amino acids, 35 amino acids, 36 amino acids, 37 amino acids, 38 amino acids, 39 amino acids, 40 amino acids, 41 amino acids, 42 amino acids, 43 amino acids, 44 amino acids, 45 amino acids, 46 amino acids, 47 amino acids, 48 ​​amino acids, 49 amino acids, 50 amino acids, 51 amino acids, 52 amino acids, 53 amino acids, 54 amino acids, 55 amino acids, 56 amino acids, 57 amino acids, 58 amino acids, 59 amino acids, 60 amino acids, or longer than 60 amino acids. In some aspects, at least one peptide chain in the plurality of overlapping peptide chains is at least 5 amino acids in length. In some aspects, at least one peptide chain in the plurality of overlapping peptide chains is at least 5 amino acids in length, 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, 50 amino acids, 55 amino acids, or 60 amino acids in length.In some aspects, at least one peptide chain in the plurality of overlapping peptide chains is less than 5 amino acids in length or at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 amino acids in length. 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 amino acids, or longer than 60 amino acids. In some aspects, each polypeptide in a feature is substantially the same length. In some aspects, each polypeptide in a feature is the same length. In some aspects, a feature comprises multiple peptide chains, each peptide chain having a random, determinable sequence of amino acids.

[0091] method Method for manufacturing a support Also disclosed herein are methods for making supports. In some aspects, the method for producing a support may include coupling a porous layer to a support layer. The support layer may comprise any metal, plastic, silicon, silicon oxide, or silicon nitride. In one embodiment, the support comprises a plurality of carboxylic acid supports attached to the support for binding peptides during peptide synthesis and protein coupling. In some aspects, the method for producing a support may include coupling a porous layer to a plurality of pillars, the porous layer comprising functional groups for attaching compounds to the support, the plurality of pillars coupled to defined locations on the planar layer, each pillar having a flat surface extending from the planar layer, the distance between the surface of each pillar and the top surface of the planar layer being about 1,000 to 5,000 angstroms, and the plurality of pillars having a surface area of ​​about 10,000 / cm. 2 It exists at a density exceeding

[0092] In some aspects, a surface of each pillar is parallel to the top surface of the planar layer. In some aspects, a surface of each pillar is substantially parallel to the top surface of the planar layer.

[0093] In some aspects, a method of preparing a substrate surface can include obtaining a surface comprising silicon dioxide and contacting the surface with a photoactive coupling formulation comprising a photoactive compound, a coupling molecule, a coupling reagent, a polymer, and a solvent; and exposing defined locations on the surface and in contact with the photoactive formulation to ultraviolet light, wherein the total surface area of ​​each of the defined locations on the surface is about 10,000 / μm. 2 In some aspects, the method may include removing a photoactive formulation located outside the defined location. In some aspects, the method may include reducing a thickness of an upper portion of the surface located outside the defined location. In some aspects, the method may include depositing a metal layer on top of the reduced-thickness surface. In some aspects, the method may include removing a photoactive formulation located on top of the surface and in contact with the defined location.

[0094] In one embodiment, Figures 1A-1E show a process for producing a support.

[0095] In Figure 1A, the first step in support preparation is priming the starting wafer to promote good adhesion between the photoactive formulation (e.g., photoresist) and the surface. Wafer cleaning can also be performed and may include steps such as oxidation, oxide strip, and ionic clean. A deionized (DI) water rinse is typically used to remove contaminants from the wafer surface. In wafer fabrication, silane attachment is generally required to promote chemical adhesion between the organic compound (photoresist) and the non-organic support (wafer). The silane acts as a kind of "bridge" that has bonding properties to both the photoresist and the wafer surface. Hexamethyldisilizane (HMDS) is typically used. HMDS is an organosilicon compound that is typically applied to a heated support either in the gas phase in a spray module or by puddle and spin in the liquid phase in a developer module, followed by a bake step. In the puddle spin method, HMDS is agitated and spun onto the wafer for a specified time and baked for 1-2 minutes at a typical temperature of 110-130°C. In the spray module, HMDS vapor is applied to a heated wafer support at 200-220°C for 30-50 seconds.

[0096] In Figure 1A, after wafer priming, the wafer can be coated with deep ultraviolet (DUV) photoresist in a photoresist coater module. DUV resists are typically polyhydroxystyrene-based polymers containing a photoacid generator to alter their solubility. DUV resists can also contain an optional photosensitizer. The matrix in the polymer consists of protecting groups, such as tboc, attached to its end groups.

[0097] DUV resist is spin-coated onto wafers in a photoresist coater module, which includes a vacuum chuck held in a cup. The wafer is mechanically placed on the chuck, for example by a robotic arm, and then rotated at the required speed specified by the manufacturer to obtain the optimal thickness.

[0098] In Figure 1A, wafers are preheated in a preheat module, which typically includes a hotplate that can be set to the temperature required for the corresponding DUV resist, as specified by the manufacturer. Heating can also be performed by microwave for a batch of wafers.

[0099] In FIG. 1A, the wafer is then exposed through a patterned photomask in a deep UV exposure tool.

[0100] In FIG. 1A, the wafer is then heated in a post-exposure bake module. This post-exposure causes chemical amplification. Resist manufacturers provide typical post-exposure bake temperatures and times for their respective products. A wafer coated with DUV photoresist is exposed to a 248 nm light source through a reticle, generating an initial photoacid or photobase. The photoresist is baked to promote diffusion of the photoacid or photobase. The exposed portions of the resist become soluble in the developer, thereby enabling patterning down to 0.25 micron dimensions. The post-exposure bake module includes a hotplate set to the required temperature specified by the manufacturer. This may consist of three vacuum pins onto which the wafer is positioned, for example, by a robotic arm. In other embodiments, the resist process does not use chemical amplification.

[0101] In Figure 1B, the wafer is then developed in a developer module. The developer module typically consists of a vacuum chuck that can hold the wafer and a pressure nozzle that can dispense developer onto the wafer. The dispense mode can be either paddle-spin mode or spin-rinse mode. Paddle-spin mode means that the wafer remains stationary on the chuck for approximately 30 seconds to 1 minute while the developer is dispensed. This agitates the developer above the wafer. After 1 minute, the developer is removed by spinning. In spin-rinse mode, the developer is dispensed while the wafer is spinning.

[0102] In FIG. 1C, the oxide is then etched in the developed areas by a wet or dry etch process. Etching is a process in which material is removed from a silicon substrate or from a thin film on the substrate surface. When a mask layer is used to protect certain areas of the wafer surface, the goal of etching is to precisely remove the material not covered by the mask. Etching is typically categorized into two types: dry etching and wet etching. Wet etching uses liquid chemicals, primarily acids, to etch material. In contrast, dry etching uses gases in an excited state to etch material. These methods are well known to those skilled in the art. These processes can be controlled to achieve etch depths of, for example, 1000 Å to 2000 Å.

[0103] In Figure ID, a metal is deposited on the wafer. This metal is typically chromium, titanium, or aluminum. In some embodiments, the metal is deposited by a process called sputter deposition, which is a physical vapor deposition (PVD) method in which a thin film is deposited by sputtering, where material is ejected from a source "target" and then deposited on the wafer. The thickness of the metal deposition is ensured to be at least 500 Å on top of the support, if desired.

[0104] In Figure 1E, the illustrated process can be used to lift off the photoresist between the metal layer and the oxide. In some aspects, this process includes lifting off the resist when a metal layer is present on the wafer without affecting the metal layer previously deposited on the silicon dioxide. This process lifts off the photoresist and metal deposited on top of the support pillars, resulting in silicon dioxide pillars that rise above the metal-coated bases separating adjacent pillars. The wafer is immersed in an oxidizer solution overnight and then soaked in piranha solution, typically for one hour. Piranha solution is a 1:1 mixture of sulfuric acid and hydrogen peroxide. This can be used to remove all organic residue from the support. This mixture is a strong oxidizer, removing most of the organic material and hydroxylating most surfaces (adding OH groups), making them hydrophilic. This process can also include an additional plasma ashing step.

[0105] surface derivatization The support can be surface-derivatized in a semiconductor module as described in U.S. Patent Application No. 20100240555, which is incorporated herein by reference in its entirety for all purposes. Exemplary supports of the present invention have oxide pillars ready for surface derivatization. Surface derivatization is a method of adding aminosilane groups to a support to make free amino groups available for coupling of biomolecules. In some aspects, the first molecule attached to a surface-derivatized support is a tboc-protected glycine. This coupling procedure is similar to the standard Merrifield solid-phase peptide synthesis procedure, which is well known to those skilled in the art.

[0106] Methods for fabricating arrays Also disclosed herein are methods for fabricating arrays. In some aspects, the arrays disclosed herein can be synthesized in situ on a surface, such as the support disclosed herein. In some cases, the arrays are fabricated using photolithography. For example, the support is contacted with a photoactive conjugation solution. The photoactive compounds in the photoactive conjugation solution bind to attachment groups (e.g., carboxylic acid or amine groups) attached to the surface of the array. A mask can be used to control the exposure of specific locations on the surface to radiation or light. The conjugation compound is activated in the exposed locations, resulting in the creation of one or more newly reactive moieties on the conjugation compound. The desired biomolecule or polypeptide is then linked to the conjugation compound. This process may be repeated to synthesize multiple features at specific locations or defined positions on the surface (see, e.g., U.S. Pat. No. 5,143,854 to Pirrung et al., U.S. Patent Application Publication Nos. 2007 / 0154946 (filed December 29, 2005), 2007 / 0122841 (filed November 30, 2005), 2007 / 0122842 (filed March 30, 2006), 2008 / 0108149 (filed October 23, 2006), and 2010 / 0093554 (filed June 2, 2008), each of which is incorporated herein by reference).

[0107] In some aspects, a method for producing a two-dimensional array of features includes the steps of obtaining a support including a planar layer comprising a metal and having a top surface and a bottom surface, and a plurality of pillars operatively coupled to defined locations on the layer, each pillar having a flat surface extending from the layer, the distance between the surface of each pillar and the top surface of the layer being about 1,000-5,000 angstroms, and the plurality of pillars having a density of about 10,000 / cm. 2and coupling features to a plurality of pillars via a series of coupling reactions, wherein each feature comprises a known biomolecule or polypeptide. In some embodiments, the average coupling efficiency of biomolecules conjugated to the conjugation compounds attached to the array is at least about 98%. In some embodiments, the average coupling efficiency of biomolecules conjugated to the conjugation compounds attached to the array is greater than 98%. In some aspects, the features are coupled to the pillars using a conjugation solution comprising a conjugation compound, a polymer, and a solvent. The conjugation solution is added to the array, and the conjugation compound is attached to the array. The conjugation solution is removed from the array, for example, by washing with water. A solution containing the features (e.g., biomolecules) is added to the array. The array is selectively exposed to electromagnetic radiation, for example, through a photomask or reticle. Sites exposed to electromagnetic radiation have attached, activated conjugation compounds that bind to the features in solution. This process may be repeated such that sites that did not bind to the feature are activated to bind a different feature from a new solution of features. In one aspect, arrays containing at least two distinct features are generated. In one aspect, arrays containing at least 10 distinct features are generated. In one aspect, arrays containing at least 100 distinct features are generated. In one aspect, arrays containing at least 1,000 distinct features are generated. In one aspect, arrays containing at least 10,000 distinct features are generated. In one aspect, arrays containing at least 2, 5, 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, or 1,000,000 distinct features are generated.

[0108] In some aspects, a method of preparing a surface for attachment of a feature (e.g., a biomolecule) can include obtaining a surface and attaching a linker molecule to the surface using a linker formulation comprising a solvent, a polymer, a linker molecule, and a coupling reagent. In some aspects, the linker molecule comprises a protecting group.

[0109] In some aspects, a method of attaching a coupling reagent to a support comprises the steps of obtaining a support comprising a planar layer comprising a metal and having an upper surface and a lower surface, and a plurality of pillars operably coupled to the layer at defined locations, each pillar having a planar surface extending from the layer, the distance between the surface of each pillar and the upper surface of the layer being between 1,000 and 5,000 angstroms, a linker molecule attached to the surface of each pillar, and the plurality of pillars having a density of 10,000 / cm 2 and attaching the conjugated compound to one or more linker molecules. In some aspects, the conjugated compound is attached to one or more linker molecules using a conjugation solution comprising a solvent, a polymer, and the conjugated compound. In some aspects, the conjugated compound is attached to one or more linker molecules using a conjugation solution disclosed herein. In some aspects, at least one linker molecule is a deprotected linker molecule. In some aspects, the conjugated compound is an NHS ester of a photoactive compound. In some aspects, the conjugated compound is a carbodiimide ester of a photoactive compound. In some aspects, the conjugated compound is an amine of a photoactive compound. In some aspects, the conjugated compound includes a protecting molecule. In some aspects, the conjugation solution is removed using water. In some aspects, the conjugated compound is activated at a site-specific location (e.g., selected pillar) using ultraviolet light or light. In some aspects, the activated conjugated compound is added with a feature and bound to a support. In some aspects, the surface of each pillar is parallel to the top surface of the layer. In some aspects, the surface of each pillar is substantially parallel to the top surface of the layer.

[0110] In some aspects, a method for generating a three-dimensional (e.g., porous) array of features may include obtaining a porous layer attached to a surface, the surface comprising attachment groups, and attaching conjugation groups to the attachment groups. The conjugation groups are then site-selectively activated via electromagnetic radiation through a photomask or reticle, and the activated conjugation groups bind to desired polypeptides added to the surface of the array. A portion of the polypeptide binding to the conjugated groups is characterized by an average conjugation efficiency of at least about 98%. In some aspects, the features are attached to the surface using a photoactivatable conjugation solution comprising a photoactivatable conjugation compound, a polymer, and a solvent, after which a polypeptide is added and the attached conjugation compound is activated.

[0111] In one embodiment, Figures 2 and 3 illustrate a process for fabricating an array. In Figure 2A, a surface containing attached amine groups is provided. The surface is contacted with a conjugation solution containing a photoactive conjugation compound, a polymer, and a solvent (Figure 2B). The photoactive conjugation compound contains activated carboxylic acid groups for coupling to the amine groups on the surface of the array, allowing the conjugation compound to bind to the amine groups on the surface of the array (Figure 2C). The conjugation solution is then removed from the array. The surface is contacted with a biomolecular coupling solution containing a biomolecule, a polymer, and a solvent (Figure 2D). The surface is exposed to UV light in a deep UV scanner tool according to a pattern defined by a photomask. In this case, the locations exposed to UV light undergo photoactivation of the photoactive conjugation compound (Figure 2E). To activate the photoactive conjugation compound, the exposure energy is 1 mJ / cm. 2 ~100mJ / cm 2 In one aspect, activation of the photoactive conjugation compound generates a carbene group that reacts favorably with any XH bond present on the biomolecule.

[0112] The surface is post-baked in a post-exposure bake module after exposure. The post-baking temperature can range from 75°C to 115°C, depending on the thickness of the surface, for at least 60 seconds, but rarely longer than 120 seconds. The photoactivated conjugation compound couples to the biomolecules, resulting in site-specific coupling of the biomolecules to the array surface (Figure 2F). This surface can be porous.

[0113] This entire cycle can be repeated as desired, each time using a different coupling molecule, to obtain the desired sequence (Figure 3A-D).

[0114] Optionally, a cap film solution coat is applied onto the surface to prevent unreacted amino groups on the support from reacting with biomolecules. The cap film coat solution can be prepared as follows: solvent, polymer, and coupling molecule.

[0115] This process is carried out in a capping spin module. The capping spin module may be equipped with a single nozzle that can be configured to dispense the cap film coat solution onto the substrate. This solution may be dispensed by pressurizing a cylinder that stores the cap film coat solution or through a pump that precisely dispenses the required amount. In some aspects, a pump is used to dispense approximately 5-8 cc of cap coat solution onto the substrate. The substrate is spun in a vacuum chuck for 15-30 seconds, and the coupling formulation is dispensed. The rotation speed can be set to 2000-2500 rpm.

[0116] The support containing the capping solution is baked in a cap bake module. The capping bake module is a hot plate dedicated to receiving the wafer immediately after the capping film coat is applied. In some aspects, the present invention provides a method for baking the spin-coated capping coat solution on a hot plate to significantly accelerate the capping reaction. Generally, hot plate baking shortens the capping time of amino acids to less than 2 minutes.

[0117] Byproducts of the capping reaction are removed in a stripper module. The stripper module may include several nozzles, typically up to 10, configured to dispense organic solvents such as acetone, isopropyl alcohol, N-methylpyrrolidone, dimethylformamide, and DI water. In some aspects, nozzles may be configured to dispense acetone followed by isopropyl alcohol onto the rotating wafer. The rotation speed is set to 2000-2500 rpm for approximately 20 seconds.

[0118] In one embodiment, Figures 4 and 5 describe a process for fabricating an array. In Figure 4A, a surface is provided that includes attached carboxylic acid groups. The carboxylic acid groups are activated by adding a carboxylic acid activating solution (Figure 4B). In one embodiment, the carboxylic acid activating solution includes a carbodiimide or succinimide. In one embodiment, the carboxylic acid group activation solution comprises 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N,N'-diisopropylcarbodiimide, (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, bromo(tripyrrolidin-1-yl)phosphonium hexafluorophosphate, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, or O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate. The surface is contacted with a conjugation solution comprising a photoactive conjugation compound, a polymer, and a solvent (Figure 4C). The photoactive conjugation compound comprises an amine group for binding to the activated carboxylic acid groups on the surface of the array. After coupling to the activated carboxylic acid, excess photoactivatable conjugation solution is washed away (Figure 4D). The surface is then contacted with a biomolecule coupling solution containing biomolecules, polymers, and solvents (Figure 4E). The surface is exposed to UV light in a deep-UV scanner tool according to a pattern defined by a photomask. In this case, the UV-exposed locations undergo photoactivation of the photoactivatable conjugation compound (Figure 4F). To activate the photoactivatable conjugation compound, the exposure energy is 1 mJ / cm. 2 ~100mJ / cm 2 In one aspect, activation of the photoactive conjugation compound generates a carbene group that reacts favorably with any XH bond present on the biomolecule.

[0119] The surface is post-baked in a post-exposure bake module after exposure. The post-baking temperature can range from 75°C to 115°C, depending on the thickness of the surface, for at least 60 seconds, but rarely longer than 120 seconds. The photoactivated conjugation compound couples to the biomolecules, resulting in site-specific coupling of the biomolecules to the array surface (Figure 4G). This surface can be porous.

[0120] This entire cycle can be repeated as desired, each time using a different coupling molecule, to obtain the desired sequence (Figure 5A-C).

[0121] Optionally, a cap film solution coat is applied to the surface to prevent unreacted amino groups on the support from reacting with biomolecules. The cap film coat solution can be prepared as follows: solvent, polymer, and coupling molecule. Solvents that can be used include organic solvents such as N-methylpyrrolidone, dimethylformamide, or combinations thereof. The capping molecule is typically acetic anhydride, and the polymer can be polyvinylpyrrolidone, polyvinyl alcohol, polymethyl methacrylate, poly(methylisopropenyl)ketone, or poly(2-methylpentene-1-sulfone). In some aspects, the capping molecule is ethanolamine.

[0122] This process is carried out in a capping spin module. The capping spin module may be equipped with a single nozzle that can be configured to dispense the cap film coat solution onto the substrate. This solution may be dispensed by pressurizing a cylinder that stores the cap film coat solution or through a pump that precisely dispenses the required amount. In some aspects, a pump is used to dispense approximately 5-8 cc of cap coat solution onto the substrate. The substrate is spun in a vacuum chuck for 15-30 seconds, and the coupling formulation is dispensed. The rotation speed can be set to 2000-2500 rpm.

[0123] The support containing the capping solution is baked in a cap bake module. The capping bake module is a hot plate dedicated to receiving the wafer immediately after the capping film coat is applied. In some aspects, the present invention provides a method for baking the spin-coated capping coat solution on a hot plate to significantly accelerate the capping reaction. Generally, hot plate baking shortens the capping time of amino acids to less than 2 minutes.

[0124] Byproducts of the capping reaction are removed in a stripper module. The stripper module may include several nozzles, typically up to 10, configured to dispense organic solvents such as acetone, isopropyl alcohol, N-methylpyrrolidone, dimethylformamide, and DI water. In some aspects, nozzles may be configured to dispense acetone followed by isopropyl alcohol onto the rotating wafer. The rotation speed is set to 2000-2500 rpm for approximately 20 seconds.

[0125] How to use Also disclosed herein are methods of using the supports, formulations, and / or arrays. Uses of the arrays disclosed herein may include research applications, therapeutic purposes, medical diagnostics, and / or stratification of one or more patients.

[0126] Any of the arrays described herein can be used as a research tool or in research applications. In one aspect, the array can be used for high-throughput screening assays. For example, enzyme substrates (i.e., polypeptides present on the peptide arrays described herein) can be tested by subjecting the array to an enzyme and identifying the presence or absence of the enzyme substrate on the array, for example, by detecting at least one change in the characteristics of the array.

[0127] Arrays can also be used in ligand-binding screening assays to confirm substrate specificity or to identify polypeptides that inhibit or activate proteins. Labeling methods, protease assays, and binding assays useful for carrying out these methods are widely known to those of skill in the art.

[0128] In some aspects, arrays are used for high-throughput screening of one or more genetic factors. Proteins associated with genes can serve as potential antigens, and antibodies against these gene-associated proteins can be used to assess the relationship between genes and diseases.

[0129] In another example, the array can be used to identify one or more biomarkers. Biomarkers can be used in the diagnosis, prognosis, treatment, and management of disease. Biomarkers can be expressed, absent, or at different levels in an individual depending on the disease state, disease stage, and response to disease treatment. Biomarkers can be, for example, DNA, RNA, proteins (e.g., enzymes such as kinases), sugars, salts, fats, lipids, or ions.

[0130] Arrays can also be used for therapeutic purposes, e.g., to identify one or more bioactive agents. A method for identifying a bioactive agent can include applying a plurality of test compounds to the array and identifying at least one test compound as a bioactive agent. The test compound can be a small molecule, an aptamer, an oligonucleotide, a chemical, a natural extract, a peptide, a protein, an antibody fragment, an antibody-like molecule, or an antibody. The bioactive agent can be a therapeutic agent or a modulator of a therapeutic target. Therapeutic targets can include phosphatases, proteases, ligases, signaling molecules, transcription factors, protein transporters, protein sorters, cell surface receptors, secreted factors, and cytoskeletal proteins.

[0131] In another aspect, the array can be used to identify therapeutic drug candidates. For example, when one or more epitopes for a specific antibody are identified by an assay (e.g., a binding assay such as ELISA), the epitopes can be used to develop drugs (e.g., monoclonal neutralizing antibodies) against the target antibody in a disease.

[0132] In one aspect, also provided is an array for use in medical diagnosis.The array can be used to confirm the response to the administration of a drug or a vaccine.For example, the response of an individual to a vaccine can be confirmed by detecting the antibody level of the individual using an array with peptides that present the epitopes recognized by the antibodies produced by the induced immune response.Another diagnostic use is to test an individual for the presence of biomarkers.Here, a sample is taken from a subject and tested for the presence of one or more biomarkers.

[0133] Arrays can also be used to stratify patient populations based on the presence or absence of biomarkers that indicate the likelihood that a subject will respond to a therapeutic treatment. Arrays can be used to identify known biomarkers to ascertain appropriate treatment groups. For example, a sample from a subject with a condition can be applied to the array. Binding to the array may indicate the presence of a biomarker for the condition. Previous studies may show that a biomarker is associated with a positive outcome after treatment, while the absence of a biomarker is associated with a negative or neutral outcome after treatment. Because a patient has a biomarker, the patient may be stratified by a medical professional into groups that will receive treatment.

[0134] In some aspects, a method for detecting the presence or absence of a protein of interest (e.g., an antibody) in a sample is disclosed herein and may include obtaining an array contacted with a sample suspected of containing the protein of interest; and determining whether the protein of interest is present in the sample by detecting the presence or absence of binding to one or more features of the array. In some aspects, the protein of interest may be obtained from a body fluid, such as amniotic fluid, aqueous humor, vitreous humor, bile, serum, breast milk, cerebrospinal fluid, earwax, chyle, endolymph, perilymph, feces, female vaginal fluid, gastric acid, gastric juice, lymph, mucus, ascites, pleural fluid, pus, saliva, sebum, semen, sweat, synovial fluid, tears, vaginal secretions, vomit, or urine.

[0135] In some aspects, a method of identifying a vaccine candidate may include obtaining an array as disclosed herein contacted with a sample obtained from a subject to whom the vaccine candidate has been previously administered, wherein the sample comprises a plurality of antibodies; and ascertaining the binding specificity of the plurality of antibodies to one or more features of the array. [Example]

[0136] The following are examples of specific embodiments for carrying out the present invention. These examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0137] The practice of the present invention will employ, unless otherwise specified, conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology within the skill of the art. Such techniques are fully explained in the literature. See, for example, T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3 rd Ed. (Plenum Press) Vols A and B (1992).

[0138] Example 1: Production of pillar supports This example describes the construction of supports with surfaces on top of pillars. A visual overview of this process is provided in Figure 1. Silicon wafers with 2.4 μm of thermally grown oxide were obtained from University Wafers. These wafers were first primed with a primer in a spray module. Hexamethyldisilazane (HMDS) was obtained from Sigma-Aldrich Inc. The wafers were then placed in a photoresist coat module and spin-coated to a thickness of 6000 Å using commercial deep-UV photoresist P5107 from Rohm and Haas or AZ DX7260p 700 from AZ Electronic Materials. The wafers were then baked on a hotplate at 120° C. for 60 seconds.

[0139] A photomask with patterned areas to create features was used to image the array onto the substrate surface. The wafer was then exposed to light in a 248 nm deep UV radiation scanner tool, Nikon S203. The exposure energy was 18 mJ / cm. The wafer was then post-exposure baked on a hotplate at 110°C for 120 seconds and developed for 60 seconds using a commercially available NMD-3 developer obtained from Tokyo Ohka Kogyo Co., Ltd.

[0140] The oxide was then etched using either a wet etch process or a dry plasma etch process. Standard semiconductor etching techniques were used. The oxide etch depth was 1000 Å to 2000 Å.

[0141] After etching, chromium was deposited using physical deposition techniques to a thickness of 500 Å to 1500 Å. Standard etching and metal deposition techniques were used.

[0142] After the chromium deposition, the resist was lifted off using the following process: The wafer was placed in a Nanostrip obtained from Cyantek Inc. overnight and then immersed in piranha solution for 90 minutes. Piranha solution is a 50:50 mixture of sulfuric acid and hydrogen peroxide. The sulfuric acid and hydrogen peroxide were obtained from Sigma Aldrich Corp. Plasma ashing was performed to oxidize remaining impurities. This process resulted in a substrate with silicon dioxide pillars separated by metal.

[0143] Alternatively, the deposited chromium was polished to a thickness of 500 Å to 1500 Å, depending on the deposition. Polishing was performed to obtain silicon dioxide pillars separated by metal. The center-to-center distance between each pillar was 70,000 Å. The surface area of ​​the top of each pillar was 3,500 Å x 3,500 Å.

[0144] Example 2: Surface derivatization with amine groups The wafers from Example 1 were surface derivatized using the following method: Aminopropyltriethoxysilane (APTES) was obtained from Sigma-Aldrich. Ethanol, 200 proof, was obtained from VWR. The wafers were first cleaned with ethanol for 5 minutes, then with 1 wt% APTES / ethanol for 20-30 minutes to grow a silane layer. The wafers were then placed in a nitrogen bake oven at 110°C for curing to grow a silane monolayer containing -NH groups for attaching linker molecules.

[0145] Example 3: Surface derivatization with carboxylic acid groups Silicon wafers with nickel 1000A deposited on a silicon substrate were obtained from University Wafers. Dextran Bio Xtra (MW 40000) was obtained from Sigma Aldrich. Bis-polyethylene glycol carboxymethyl ether was obtained from Sigma Aldrich. Polyvinylpyrrolidone 1000000 was obtained from Poly Sciences Inc. The above three polymers were dissolved in a 2:2:1 weight ratio in a solvent composition of 50 wt% ethyl lactate / 50 wt% water along with 2 wt% of the photoacid generator dimethyl-2,4-dihydroxyphenylsulfonium triflate obtained from Oakwood Chemicals Inc. This solution was spin-coated onto the silicon wafer with nickel 1000A deposited on a silicon substrate.

[0146] The coated wafer was spun at 3000 rpm to obtain a uniform coat of 100 nm thickness. The wafer was then placed in a Nikon S 203 deep UV scanner at 250 mJ / cm. 2 The wafer was exposed to UV light and then baked on a hot plate at 65°C for 90 seconds. The coating was then removed from the wafer using acetone and isopropyl alcohol, followed by a deionized water rinse. The support now has a matrix of free COOH groups ready for activation and coupling with proteins or amino acids for peptide synthesis.

[0147] The above derivatization was carried out on the surface of pillars derived from the pillar support of Example 1.

[0148] Example 4: Preparation of dextran-based porous supports coated with carboxylic acid groups Compared to planar supports, porous supports allow for increased two-dimensional COOH group concentration along the layer. Dextran was coupled onto the surface-derivatized wafer. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (Pierce Scientific) and N-hydroxysuccinimide (NHS) (Pierce Scientific) were dissolved in deionized water at molar concentrations of 0.2 M and 0.1 M, respectively, along with 10 wt% dextran. The coupling solution was spin-coated onto the wafer at 3000 rpm and baked at 65°C for 90 seconds until complete coupling to the dextran-COOH support was achieved. A crosslinking solution was added, followed by crosslinking, to obtain a multidimensional COOH support.

[0149] Example 5: Preparation of PEG-based porous supports coated with carboxylic acid groups Bis-polyethylene glycol carboxymethyl ether was coupled onto the surface-derivatized wafer. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (Pierce Scientific) and N-hydroxysuccinimide (NHS) (Pierce Scientific) were dissolved in deionized water at molar concentrations of 0.2 M and 0.1 M, respectively, along with 10 wt% polyethylene glycol (PEG). The coupling solution was spin-coated onto the wafer at 3000 rpm and baked at 65 °C for 90 seconds until complete coupling to the PEG-COOH support was achieved. A crosslinking solution was added, followed by crosslinking, to yield a multidimensional COOH support.

[0150] Example 6: Generation of hydroxyl-derivatized pillar surfaces on supports Silicon wafers were obtained from University Wafers. In Figure 34A(6), a metal was deposited on the wafer. The metal was selected from chromium, titanium, or aluminum. The metal was deposited by a process called sputter deposition, a physical vapor deposition (PVD) method in which thin films are deposited by sputtering material from a source "target," which then deposits the material onto the wafer. The thickness of the metal deposition was guaranteed to be at least 500 Å on top of the substrate.

[0151] In Figure 6B, silicon dioxide was deposited on the wafer. This oxide was deposited by a process called sputter deposition, a physical chemical vapor deposition (PECVD) method in which thin films are deposited by sputtering material from a source "target," which then deposits the material onto the wafer. The thickness of the oxide deposition was ensured to be at least 500 Å on top of the substrate.

[0152] In Figure 6C, the first step in support preparation is priming the starting wafer to promote good adhesion between the photoactive formulation (e.g., photoresist) and the surface. Wafer cleaning, including oxidation, oxide strip, and ion clean, was also performed. A DI water rinse was used to remove contaminants from the wafer surface. During wafer fabrication, silane deposition was used to promote chemical adhesion between the organic compound (photoresist) and the non-organic support (wafer). The silane acts as a kind of "bridge" that has the properties to bond to both the photoresist and the wafer surface. Typically, hexamethyldisilizane (HMDS) was used. HMDS is an organosilicon agent applied to the heated support in the spray module in the vapor phase or in the liquid phase by puddle and spin in the developer module. This was followed by a bake step. In the puddle and spin method, HMDS was agitated on the wafer for the specified time, then spun and baked at temperatures between 110°C and 130°C for 1–2 minutes. In the spray module, HMDS vapor was applied to a heated wafer support at 200°C to 220°C for 30 to 50 seconds.

[0153] In Figure 6C, after wafer priming, the wafer was coated with a deep ultraviolet (DUV) photoresist in a photoresist coater module. Our DUV resist included a polyhydroxystyrene-based polymer containing a photoacid generator to alter its solubility. The DUV resist also included a photosensitizer. The matrix in the polymer included a protecting group, e.g., tboc, attached to the end group.

[0154] DUV resist was spin-coated onto the wafer in a photoresist coating module, which consisted of a vacuum chuck held in a cup. The wafer was mechanically placed on the chuck by a robotic arm and then spun at the required speed specified by the manufacturer to obtain the optimum thickness.

[0155] In Figure 6C, wafers were preheated in a preheating module. The preheating module was equipped with a hot plate that could be set to the temperature required for the corresponding DUV resist, as specified by the manufacturer. When heating a batch of wafers, we used microwaves for heating.

[0156] In Figure 6D, the wafer was exposed through a patterned photomask in a deep UV exposure tool.

[0157] In Figure 6E, the wafer was heated in a post-exposure bake module. This post-exposure led to chemical amplification. Resist manufacturers provided representative post-exposure bake temperatures and times for their respective products. When a wafer coated with DUV photoresist was exposed to a 248 nm light source through a reticle, the initial photoacid or photobase was generated. The exposed portions of the resist became soluble in the developer, thereby enabling the creation of patterns with 0.25 micron dimensions. The post-exposure bake module contained a hotplate set to the required temperature specified by the manufacturer. This module also contained three vacuum pins onto which the wafer was positioned by a robotic arm.

[0158] In Figure 6E, the wafer was developed in the developer module. The developer module contained a vacuum chuck that held the wafer and a pressure nozzle that dispensed developer onto the wafer. The dispense mode could be either paddle-spin mode or spin-rinse mode. During paddle-spin mode, the wafer was held stationary on the chuck for approximately 30 seconds to 1 minute while the developer was dispensed. This agitated the developer above the wafer. After 1 minute, the developer was removed by spinning. During spin-rinse mode, the developer was dispensed while the wafer was rotating.

[0159] In Figure 6F, the oxide was etched in the developed areas by either a wet or dry etch process. Etching is a process in which material is removed from a silicon substrate or from a thin film on the substrate surface. When a mask layer is used to protect certain areas of the wafer surface, the goal of etching is to precisely remove the material not covered by the mask. Etching is typically categorized into two types: dry etching and wet etching. Wet etching uses liquid chemicals, primarily acids, to etch material. In contrast, dry etching uses gases in an excited state to etch material. For example, these processes were engineered to achieve an etch depth of 500 Å.

[0160] In Figure 6G, the wafer was immersed in an oxidizer solution overnight and then immersed in a piranha solution, typically for one hour. The piranha solution used was a 1:1 mixture of sulfuric acid and hydrogen peroxide. This solution was used to remove all organic residue from the substrate. Because this mixture is a strong oxidizer, it removed most of the organic material and also hydroxylated most of the surface (adding OH groups to the surface), making the surface hydrophilic. This process also included an additional plasma ashing step.

[0161] Example 7: Conjugation of IL-6 and TNFα proteins to amine-group derivatized surfaces Wafers were surface derivatized to provide amine groups on the support, as described in Example 2 (Figure 2A). Photoconjugation groups, such as diazirine, aryl azide, or carboxylic NHS ester of benzophenone, were obtained from Sigma-Aldrich. To prepare the conjugation solution, 0.1 mM NHS-diazirine was dissolved in 1% PVP / water. PVP (polyvinylpyrrolidone) was obtained from Polysciences. The conjugation solution was spin-coated onto the wafer at 2000 rpm for 30 seconds and left for 30 minutes until complete coupling was achieved (Figure 2B). This coupling process can also be performed in a bake oven or microwave-heated to improve coupling efficiency or shorten the time. To quench unreacted NHS, the wafer containing the conjugation solution was washed with tris-buffered saline (VWR) (Figure 2C). The capping solution was prepared as follows: 50% acetic anhydride (Spectrum Chemicals) and 50% N-methylpyrrolidone (VWR) are mixed. The capping solution is coated onto the wafer and baked at 75°C for 90 seconds to cap unreacted amines. The wafer is then cleaned with N-methylpyrrolidone, followed by rinsing with DI water and drying. Recombinant IL-6 was obtained from Life Tech. IL-6 coupling solution is prepared by dissolving 50µg / ml IL-6 and 1% PVP in deionized water. This protein coupling solution is spin-coated onto the wafer at 2000 rpm for 30 seconds (Figure 2D). The wafer is then placed in a Nikon S203 scanner and scanned with 248nm far-UV light at 100mJ / cm using a reticle. 2The array is exposed to UV light (Figure 2E). This can also be done with a digital micromirror or other maskless lithography-based device. During exposure, UV photolysis of the diazirene forms a carbene that is highly reactive with any XH bond in proteins such as IL-6, forming a stable covalent bond. The protein coupling solution is then washed off the array, leaving the bound IL-6 in its site-specific location (Figure 2F). This process completes one round of protein conjugation.

[0162] To couple TNFα to a site-specific spot different from the spot coupled to IL-6, the above steps are repeated using a different reticle to expose a different spot (Figures 3A-3D). These steps can be repeated several times to couple selected polypeptides to specific spots on the array.

[0163] To test the binding of IL-6 and TNFα to the array, anti-TNFα and anti-IL-6 antibodies were added at a 1:1000 dilution in PBST buffer and mixed together. All antibodies and buffers were obtained from Life Technologies. The assay was performed as follows: The chip was washed three times with PBST buffer for 5 minutes. The antibodies were then added and incubated in the dark at 37°C for 1 hour. The chip was then washed three times with PBST buffer for 5 minutes, followed by three washes with deionized water for 5 minutes. Finally, the chip was scanned using a fluorescent scanner.

[0164] The fluorescent signal intensity of IL-6 was measured at 45,000 and that of TNFα at 43,500, compared to a fluorescent signal intensity of 1,500 in the absence of protein (Figure 7). This result demonstrates that two or more proteins can be coupled in the array.

[0165] Because the intermediate carbene formed is highly reactive with XH bonds, this microarray-based photoconjugation can be extended to cover small molecules and any chemical or biomolecule containing XH bonds. In the case of benzophenone, photolysis in far-UV light causes the benzophenone to react with C—H bonds. Therefore, photoconjugation of proteins one at a time in a microarray format can be used not only to create arrays containing antibodies and other biopolymers, but also to develop arrays containing small molecules.

[0166] Example 8: Conjugation of IL-6 and TNFα proteins to carboxylic acid group derivatized surfaces Surface-derivatized wafers are prepared to have COOH groups on the support (FIG. 4A) as described in Example 6. The wafers are activated with EDC / NHS (Sigma-Aldrich) for 10 minutes at room temperature (FIG. 4B).

[0167] Conjugation groups such as aminodiazirine, aryl azide, or benzophenone were obtained from Life Tech. 0.1 mM aminodiazirine was dissolved in 1% PVP / water. PVP (polyvinylpyrrolidone) was obtained from Polysciences. This conjugation solution was spin-coated onto the wafer at 2000 rpm for 30 seconds and left for 30 minutes for complete coupling (Figure 4C). This coupling process can also be performed in a bake oven or microwave-heated to improve coupling efficiency or shorten the time. The wafer was washed with tris-buffered saline (VWR) (Figure 4D). The capping solution was prepared as follows: 1 M ethanolamine (Sigma-Aldrich) was dissolved in DI water and 1% PVP and spin-coated onto the wafer. The coating was left at room temperature for 10 minutes. The wafer was then rinsed with deionized water and dried. Recombinant IL-6 was obtained from Life Tech. Prepare IL-6 coupling solution by dissolving 50 μg / ml IL-6 and 1% PVP in deionized water. This protein coupling solution was spin-coated onto the wafer at 2000 rpm for 30 seconds (Figure 4E). The wafer was then scanned in a Nikon S203 scanner using a reticle and exposed to 100 mJ / cm of 248 nm far-UV light. 2 The wafer was exposed to UV light (Figure 4F). This can be done with a digital micromirror or other maskless lithography-based device, as well as a 365 nm stepper / scanner. During exposure, UV photolysis of the diazirene forms a carbene that is highly reactive with any XH bond present in proteins such as IL-6, forming a stable covalent bond between IL-6 and the conjugated compound. Excess protein coupling solution was then washed off the wafer. This process completes one round of protein coupling (Figure 4G).

[0168] To couple TNFα to a site-specific spot different from the spot coupled to IL-6, the above steps are repeated using a different reticle to expose a different spot (Figures 5A-5C). These steps can be repeated several times to couple selected polypeptides to specific spots on the array.

[0169] To test the binding of IL-6 and TNFα to the array, anti-TNFα and anti-IL-6 antibodies were added at a 1:1000 dilution in PBST buffer and mixed together. All antibodies and buffers were obtained from Life Technologies. The assay was performed as follows: The chip was washed three times with PBST buffer for 5 minutes. The antibodies were then added and incubated for 1 hour at 37°C in the dark. The chip was then washed three times with PBST buffer for 5 minutes, followed by three washes with deionized water for 5 minutes. Finally, the chip was scanned using a fluorescent scanner.

[0170] The fluorescent signal intensity of IL-6 was measured at 45,000 and that of TNFα at 43,500, compared to a fluorescent signal intensity of 1,500 in the absence of protein (Figure 7). This result demonstrates that two or more proteins can be coupled in the array.

[0171] Because the intermediate carbene formed is highly reactive with XH bonds, this microarray-based photoconjugation can be extended to cover small molecules and any chemical or biomolecule containing XH bonds. In the case of benzophenone, photolysis in far-UV light causes the benzophenone to react with C—H bonds. Therefore, photoconjugation of proteins one at a time in a microarray format can be used not only to create arrays containing antibodies and other biopolymers, but also to develop arrays containing small molecules.

[0172] While the present invention has been shown and described in detail with respect to preferred and various alternative embodiments, it will be understood by those skilled in the relevant art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.

[0173] All references, issued patents, and patent applications cited within the body of this specification are hereby incorporated by reference in their entirety for all purposes.

Claims

1. 1. A method for attaching a biomolecule to a surface, comprising the steps of: obtaining a surface having a plurality of attachment groups attached to said surface; attaching a photoactivatable conjugating compound to the attachment group; contacting the surface with a biomolecule; and Selectively exposing the surface to electromagnetic radiation, which activates the attached photoactivatable conjugation compounds, and the attached, activated photoactivatable conjugation compounds bind to the biomolecules, thereby attaching the biomolecules to the surface.

2. 2. The method of claim 1, wherein the photoactivatable conjugating compound comprises a functional group selected from the group consisting of NHS ester, sulfo-NHS ester, amine, primary alcohol, secondary alcohol, phenol, thiol, aniline, hydroxamic acid, primary amide, aliphatic amine, and sulfonamide.

3. 10. The method of claim 1, wherein the photoactivatable conjugated compound comprises an ester.

4. 10. The method of claim 1, wherein the photoactivatable conjugated compound comprises a carboxylic acid group.

5. 5. The method of claim 4, wherein the carboxyl group is activated.

6. 10. The method of claim 1, wherein the photoactivatable conjugated compound comprises an N-hydroxysuccinimide moiety.

7. 10. The method of claim 1, wherein the photoactivatable conjugated compound comprises an amine group.

8. 10. The method of claim 1, wherein the photoactivatable conjugating compound comprises a photoactivatable group selected from the group consisting of diazirine, aryl azide, and benzophenone.

9. 10. The method of claim 1, wherein the photoactivatable conjugation compound comprises a photoactivated conjugation moiety selected from the group consisting of a diazirine moiety, an aryl azide moiety, and a benzophenone moiety.

10. 10. The method of claim 1, wherein the photoactivatable conjugated compound comprises an N-hydroxysuccinimide moiety attached to an ester.

11. 10. The method of claim 1, wherein the photoactivatable conjugated compound comprises an N-hydroxysuccinimide ester functionally attached to a moiety selected from the group consisting of a diazirine moiety, an aryl azide moiety, and a benzophenone moiety.

12. 10. The method of claim 1, wherein the attachment group is an amine group.

13. 10. The method of claim 1, wherein the attachment group is a carboxylic acid group.

14. 14. The method of claim 13, wherein the carboxyl group is activated to couple to the amine group.

15. The method of claim 1, wherein the biomolecule is a polypeptide.

16. 16. The method of claim 15, wherein the polypeptide is a protein.

17. 2. The method of claim 1, wherein the biomolecule is selected from the group consisting of small molecules, proteins, carbohydrates, oligomers, peptides, biomolecules derived from cell lysates, antibodies, proteases, and enzymes.

18. 10. The method of claim 1, wherein the wavelength of the electromagnetic radiation is 248 nm.

19. 10. The method of claim 1, wherein the wavelength of the electromagnetic radiation is between 330 and 370 nm.

20. 10. The method of claim 1, wherein the surface is porous and the attachment groups are oriented in multiple directions.

21. 10. The method of claim 1, wherein the surface is a surface of pillars operatively coupled to a planar layer of a support at defined locations.

22. 22. The method of claim 21, wherein each pillar has a flat upper surface extending from a planar layer of the support.

23. 23. The method of claim 22, wherein the distance between the top surface of each pillar of the support and the planar layer is 1,000 to 5,000 angstroms.

24. 22. The method of claim 21, wherein the pillar is one of a plurality of pillars present on a substrate.

25. Multiple pillars 10,000 / cm 2 25. The method of claim 24, wherein the composition is present at a density of greater than 1000 mg / kg.

26. 25. The method of claim 24, wherein the center of each pillar is at least 2,000 angstroms away from the center of any other pillar.

27. 25. The method of claim 24, wherein a surface of each pillar is parallel to a top surface of the planar layer.

28. 25. The method of claim 24, wherein a surface of each pillar is substantially parallel to a top surface of the planar layer.

29. Each pillar has a surface area of ​​at least 1 μm 2 22. The method of claim 21, wherein:

30. The total surface area of ​​each pillar is 10,000 μm 2 22. The method of claim 21, wherein the

31. 22. The method of claim 21, wherein each pillar comprises silicon dioxide or silicon nitride.

32. 22. The method of claim 21, wherein each pillar is at least 98-99% by weight silicon dioxide.

33. 22. The method of claim 21, wherein each of the defined locations comprises a plurality of identical biomolecules.

34. 34. The method of claim 33, wherein each of the defined locations comprises a plurality of identical sequences that differ from other defined locations.

35. 22. The method of claim 21, wherein each of the positioned locations is a distinct location.

36. 10. The method of claim 1, wherein the biomolecule is covalently attached to the surface.

37. 10. The method of claim 1, wherein the attachment efficiency of the biomolecule attachment on the selectively exposed surface is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

38. The method of claim 1, wherein the attachment of the biomolecules to the array is highly stable.

39. 10. The method of claim 1, optionally repeated to produce a surface comprising a plurality of unique biomolecules attached to selected locations on the surface.

40. 10. The method of claim 1, wherein the surface comprises at least two unique biomolecules attached to selected locations on the surface.

41. 41. The method of claim 40, wherein the surface comprises at least 10 unique biomolecules attached to selected locations on the surface.

42. 42. The method of claim 41, wherein the surface comprises at least 100 unique biomolecules attached to selected locations on the surface.

43. 43. The method of claim 42, wherein the surface comprises at least 1,000 unique biomolecules attached to selected locations on the surface.

44. 44. The method of claim 43, wherein the surface comprises at least 10,000 unique biomolecules attached to selected locations on the surface.

45. 1. A method for attaching a polypeptide to a surface, the method comprising the steps of: obtaining a surface comprising a plurality of free amine groups attached to said surface; attaching a conjugated compound to the surface by contacting the surface with a conjugation solution containing a conjugated compound, wherein the conjugated compound comprises an activated carboxylic acid group, which binds to a free amine group attached to the surface; contacting the surface with a polypeptide; and Selectively exposing the surface to electromagnetic radiation, which activates the attached conjugation compound, and the attached, activated conjugation compound binds to the polypeptide, thereby attaching the polypeptide to the surface.

46. 1. A method for attaching a polypeptide to a surface, the method comprising the steps of: obtaining a surface having a plurality of free carboxylic acid groups attached thereto; contacting the surface with a carboxylic acid activating solution, thereby activating the carboxylic acid groups for bonding with amine groups; attaching a conjugation compound to the surface by contacting the surface with a conjugation solution containing a conjugation compound, wherein the conjugation compound comprises an amine group, which binds to an activated carboxylic acid group attached to the surface; contacting the surface with a polypeptide; and Selectively exposing the surface to electromagnetic radiation, which activates the attached conjugation compound, and the attached, activated conjugation compound binds to the polypeptide, thereby attaching the polypeptide to the surface.

47. 47. The method of claim 45 or 46, further comprising washing the surface after attaching the conjugated compound to the surface.

48. 47. The method of claim 46, further comprising washing the surface after activating the surface-attached carboxylic acid groups.

49. 47. The method of claim 45 or 46, wherein the activated conjugated compound is attached to a site on the polypeptide selected from the group consisting of a peptide backbone, a side chain, an amine group, or a carboxylic acid group.

50. 47. The method of claim 45 or 46, wherein contacting the surface with the conjugation solution comprises spin-coating the conjugation solution onto the surface.

51. 47. The method of claim 45 or 46, wherein the wavelength of the electromagnetic radiation is 248 nm.

52. 47. The method of claim 45 or 46, wherein the wavelength of the electromagnetic radiation is 330 to 370 nm.

53. 47. The method of claim 45 or 46, wherein the conjugated compound comprises a photoactivated conjugated moiety selected from the group consisting of a diazirine moiety, an aryl azide moiety, and a benzophenone moiety.

54. 47. The method of claim 46, wherein the conjugated compound comprises an N-hydroxysuccinimide moiety attached to an ester.

55. 47. The method of claim 46, wherein the conjugated compound comprises an N-hydroxysuccinimide ester operatively attached to a moiety selected from the group consisting of a diazirine moiety, an aryl azide moiety, and a benzophenone moiety.

56. 47. The method of claim 45 or 46, wherein the conjugation solution comprises a polymer.

57. 57. The method of claim 56, wherein the polymer is polyvinylpyrrolidone.

58. 47. The method of claim 46, wherein the carboxylic acid activating solution comprises a carbodiimide or N-hydroxysuccinimide.

59. 47. The method of claim 46, wherein the carboxylic acid activating solution comprises a compound selected from the group consisting of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N,N'-diisopropylcarbodiimide, (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, bromo(tripyrrolidin-1-yl)phosphonium hexafluorophosphate, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, O-benzotriazole-N,N,N',N'-tetramethyluronium-hexafluorophosphate, and O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate.

60. 47. The method of claim 45 or 46, wherein the carboxylic acid activating solution comprises a compound selected from the group consisting of N,N-diisopropylethylamine, 1-hydroxy-7-azabenzotriazole, and hydroxybenzotriazole.

61. 47. The method of claim 45 or 46, wherein the polypeptide is a protein.

62. 47. The method of claim 45 or 46, wherein the attachment efficiency of the biomolecule attachment on the selectively exposed surface is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

63. 47. The method of claim 45 or 46, wherein the attachment of the biomolecules to the array is highly stable.

64. 47. The method of claim 45 or 46, optionally repeated to produce a surface comprising a plurality of unique biomolecules attached to selected locations on the surface.

65. 47. The method of claim 45 or 46, wherein the surface comprises at least two unique biomolecules attached to selected locations on the surface.

66. 66. The method of claim 65, wherein the surface comprises at least 10 unique biomolecules attached to selected locations on the surface.

67. 67. The method of claim 66, wherein the surface comprises at least 100 unique biomolecules attached to selected locations on the surface.

68. 68. The method of claim 67, wherein the surface comprises at least 1,000 unique biomolecules attached to selected locations on the surface.

69. 69. The method of claim 68, wherein the surface comprises at least 10,000 unique biomolecules attached to selected locations on the surface.

70. An array comprising a plurality of attachment groups on an array surface, at least one of the plurality of attachment groups being covalently linked to a photoactivatable conjugating compound.

71. 71. The array of claim 70, comprising biomolecules attached to photoactivatable conjugation compounds.

72. 72. The array of claim 71, wherein the biomolecule is a polypeptide.

73. 73. The array of claim 72, wherein the polypeptide is a protein.

74. 72. The array of claim 71, wherein the biomolecules are selected from the group consisting of small molecules, proteins, carbohydrates, oligomers, peptides, biomolecules derived from cell lysates, antibodies, proteases, and enzymes.

75. 1cm 2 72. The array of claim 71, comprising at least 100, 1,000, 10,000, 100,000, 1,000,000, or 10,000,000 biomolecules per array.

76. The surface area of ​​the array is 1 μm 2 ~10mm 2 71. The array of claim 70, wherein:

77. 71. The array of claim 70, wherein the photoactivatable conjugating compound comprises a functional group selected from the group consisting of NHS ester, sulfo-NHS ester, amine, primary alcohol, secondary alcohol, phenol, thiol, aniline, hydroxamic acid, primary amide, aliphatic amine, and sulfonamide.

78. 71. The array of claim 70, wherein the photoactivatable conjugated compound comprises an ester.

79. 71. The array of claim 70, wherein the photoactivatable conjugated compound comprises a carboxylic acid group.

80. 80. The array of claim 79, wherein the carboxylic acid groups are activated.

81. 71. The array of claim 70, wherein the photoactivatable conjugated compound comprises an N-hydroxysuccinimide moiety.

82. 71. The array of claim 70, wherein the photoactivatable conjugated compound comprises an amine group.

83. 71. The array of claim 70, wherein the photoactivatable conjugating compound comprises a photoactivatable group selected from the group consisting of diazirine, aryl azide, and benzophenone.

84. 71. The array of claim 70, wherein the photoactivatable conjugated compounds are activated by electromagnetic radiation comprising a wavelength of 248 nm.

85. 71. The array of claim 70, wherein the photoactivatable conjugated compounds are activated by electromagnetic radiation comprising wavelengths between 330 and 360 nm.

86. 71. The array of claim 70, wherein the photoactivatable conjugation compound comprises a photoactivated conjugation moiety selected from the group consisting of a diazirine moiety, an aryl azide moiety, and a benzophenone moiety.

87. 71. The array of claim 70, wherein the photoactivatable conjugated compound comprises an N-hydroxysuccinimide moiety attached to an ester.

88. 71. The array of claim 70, wherein the photoactivatable conjugated compound comprises an N-hydroxysuccinimide ester functionally attached to a moiety selected from the group consisting of a diazirine moiety, an aryl azide moiety, and a benzophenone moiety.

89. 71. The array of claim 70, wherein the surface is porous and the attachment groups are oriented in multiple directions.

90. 71. The array of claim 70, wherein the surface is a surface of pillars operatively coupled to positioned locations on a planar layer of a support.

91. 91. The array of claim 90, wherein each pillar has a flat upper surface extending from the planar layer of the support.

92. 92. The array of claim 91, wherein the distance between the top surface of each pillar and the planar layer of the support is between 1,000 and 5,000 angstroms.

93. 91. The array of claim 90, wherein the pillar is one of a plurality of pillars present on a support.

94. Pillars: 10,000 / cm 2 94. The array of claim 93, wherein the array is present at a density of greater than

95. 94. The array of claim 93, wherein the center of each pillar is at least 2,000 angstroms away from the center of any other pillar.

96. 94. The array of claim 93, wherein a surface of each pillar is parallel to a top surface of the planar layer.

97. 94. The array of claim 93, wherein a surface of each pillar is substantially parallel to a top surface of the planar layer.

98. Each pillar has a surface area of ​​at least 1 μm 2 91. The array of claim 90, wherein:

99. The total surface area of ​​each pillar is 10,000 μm 2 91. The array of claim 90, wherein said array is less than 90%.

100. 91. The array of claim 90, wherein each pillar comprises silicon dioxide or silicon nitride.

101. 91. The array of claim 90, wherein each pillar is at least 98-99% silicon dioxide by weight.

102. 91. The array of claim 90, wherein each of the defined locations comprises a plurality of identical biomolecules.

103. 103. The array of claim 102, wherein each of the defined locations comprises a plurality of identical sequences that differ from other defined locations.

104. 91. The array of claim 90, wherein each of the defined locations is a distinct location.

105. 1cm 2 91. The array of claim 90, comprising at least 100, 1,000, 10,000, 100,000, 1,000,000, or 10,000,000 pillars per array.

106. 71. The array of claim 70, wherein said surface comprises at least two unique biomolecules attached to said surface at selected locations.

107. 107. The array of claim 106, wherein said surface comprises at least 10 unique biomolecules attached to said surface at selected locations.

108. 108. The array of claim 107, wherein said surface comprises at least 100 unique biomolecules attached to said surface at selected locations.

109. 109. The array of claim 108, wherein said surface comprises at least 1,000 unique biomolecules attached to said surface at selected locations.

110. 110. The array of claim 109, wherein said surface comprises at least 10,000 unique biomolecules attached to said surface at selected locations.

111. 71. The array of claim 70, wherein the attachment groups are amine groups.

112. 71. The array of claim 70, wherein the attachment groups are carboxylic acid groups.

113. 113. The array of claim 112, wherein said carboxyl groups are activated to bind to said amine groups.

114. 1. A method for detecting a biomolecule in a sample, comprising the steps of: providing a support according to any one of claims 70 to 113; contacting the support with the sample; and Detecting a binding event between a biomolecule present in the sample and a biomolecule attached to the support.

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