Patent Grant
8658573
All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety.
This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.
Diseases must be diagnosed quickly in order to maximize the chances for their successful medical treatment and containment. One method of diagnosis employs biomarkers linked to specific diseases. The efficient identification of biomarkers for specific diseases will greatly facilitate quick diagnoses, the discovery of new biomarkers, and the development of new vaccines.
The alarming rate of appearance of drug resistant diseases underscores the need to expand our methods to treat diseases, including by vaccines. However, it is often difficult to determine or predict the effectiveness of a vaccine. A quick and efficient means to determine the ability of a vaccine to stimulate an immune response would greatly facilitate the search for novel vaccines.
Another pressing concern is the threat of bioterror attacks such as with anthrax. Anthrax is an often-fatal infectious disease caused by the bacterium Bacillus anthracis (B. anthracis), which begins by the entry of spores into the mammalian host. To combat the use of B. anthracis spores as a biological weapon, a rapid and specific method to detect B. anthracis spores is needed. In addition, the serious side effects accompanying currently used anthrax vaccines emphasize the need to find a safer anthrax vaccine.
Cell-surface carbohydrates show promise as biomarkers to study immune responses. Yet carbohydrates have not been efficiently harnessed as biomarkers for disease detection, biomarker identification, or vaccine development.
In one embodiment, the invention relates to an array that includes:
a surface;
a compound of formula (I):
immobilized on at least a part of the surface; and
one or more carbohydrates attached to the surface through a covalent bond to the compound of formula (I);
wherein
one or more of the carbohydrates are capable of binding to an agent, wherein the agent is capable of indicating a presence of a disease or a pathogen;
n is an integer from 1 to 100;
X is R2, —CO2R3, —C(O)NR3R3, —SR4, —CN, —OR3, a halogen, a β-diketone, a silane, a phosphate, a phosphonate, a polymer, or block copolymer;
ring A is substituted with one or more R1 groups;
R1 is independently a halogen, a hydroxyl, an aryl, an amide, a cyano, a nitro, —R2, —C(O)R3, —CO2R3, —OC(O)R3, or —OR3,
R2 is independently a hydrogen, a substituted or unsubstituted straight- or branched-chain alkyl which contains 1-6 carbons, a substituted or unsubstituted alkene which contains 2-4 carbons, a substituted or unsubstituted alkyne which contains 2-4 carbons, or —OC(O)R5;
R3 is independently a hydrogen, a substituted or unsubstituted C1-C10 straight-chain or branched-chain alkyl, or a substituted or unsubstituted alkene which contains 2-4 carbons;
R4 is independently a hydrogen, —S-pyridyl, —SR3, —SO2R3, or SR8, wherein —SR8 and the rest of formula (I) combine to form a bis-disulfide; and
R5 is independently a hydrogen, an unsubstituted straight- or branched-chain alkyl that contains 1-6 carbons, or a straight- or branched-chain alkyl that contains 1-6 carbons and is substituted by an alkyne.
In one embodiment, the invention relates to a method for determining the presence of a pathogen or for diagnosing a disease in a subject, where the method includes:
exposing an array as described herein to a sample from a subject; and
determining the presence of the agent bound to one or more of the carbohydrates, wherein the presence of a bound agent indicates the presence of a pathogen in the sample.
In another embodiment, the invention relates to an array that includes:
a surface;
a compound of Formula (I) immobilized on the surface:
a compound of Formula (III) immobilized on the surface:
one or more carbohydrates attached to the surface through a covalent bond to the compound of formula (I),
wherein:
one or more of the carbohydrates are capable of binding to an agent, wherein the agent is capable of indicating a presence of a disease or a pathogen;
n is an integer from 1 to 100;
X is R2, —CO2R3, —C(O)NR3R3, —SR4, —CN, —OR3, a halogen, a β-diketone, a silane, a phosphate, a phosphonate, a polymer, or block copolymer;
ring A is substituted with one or more R1 groups;
R1 is independently a halogen, a hydroxyl, an aryl, an amide, a cyano, a nitro, —R2, —C(O)R3, —CO2R3, —OC(O)R3, or —OR3;
R2 is independently a hydrogen, a substituted or unsubstituted straight- or branched-chain alkyl which contains 1-6 carbons, a substituted or unsubstituted alkene which contains 2-4 carbons, a substituted or unsubstituted alkyne which contains 2-4 carbons, or —OC(O)R5;
R3 is independently a hydrogen, a substituted or unsubstituted C1-C10 straight-chain or branched-chain alkyl, or a substituted or unsubstituted alkene which contains 2-4 carbons;
R4 is independently a hydrogen, —S-pyridyl, —SR3, —SO2R3, or SR8, wherein —SR8 and the rest of formula (I) combine to form a bis-disulfide;
R5 is independently a hydrogen, an unsubstituted straight- or branched-chain alkyl that contains 1-6 carbons, or a straight- or branched-chain alkyl that contains 1-6 carbons and is substituted by an alkyne; and
Y is —NR3R3, —OH, —SH, —C(O)NR3R3, —CO2H, an ammonium, or a salt thereof.
In one embodiment, the invention relates to a method for making an array, where the method includes:
forming on at least a part of a surface a self-assembled monolayer comprising a compound of formula (I):
depositing one or more carbohydrates onto at least a part of the monolayer; and
irradiating the carbohydrate, monolayer, and optionally the surface,
wherein:
a covalent bond is formed between the carbohydrate and the compound of formula (I);
the carbohydrate is capable of binding to an agent, wherein the agent is capable of indicating a presence of a disease or a pathogen;
n is an integer from 1 to 100;
X is R2, —CO2R3, —C(O)NR3R3, —SR4, —CN, —OR3, a halogen, a β-diketone, a silane, a phosphate, a phosphonate, a polymer, or block copolymer;
ring A is substituted with one or more R1 groups;
R1 is independently a halogen, a hydroxyl, an aryl, an amide, a cyano, a nitro, —R2, —C(O)R3, —CO2R3, —OC(O)R3, or —OR3;
R2 is independently a hydrogen, a substituted or unsubstituted straight- or branched-chain alkyl which contains 1-6 carbons, a substituted or unsubstituted alkene which contains 2-4 carbons, a substituted or unsubstituted alkyne which contains 2-4 carbons, or —OC(O)R5;
R3 is independently a hydrogen, a substituted or unsubstituted C1-C10 straight-chain or branched-chain alkyl, or a substituted or unsubstituted alkene which contains 2-4 carbons;
R4 is independently a hydrogen, —S-pyridyl, —SR3, —SO2R3, or SR8, wherein —SR8 and the rest of formula (I) combine to form a bis-disulfide; and
R5 is independently a hydrogen, an unsubstituted straight- or branched-chain alkyl that contains 1-6 carbons, or a straight- or branched-chain alkyl that contains 1-6 carbons and is substituted by an alkyne.
In one embodiment, the invention relates to a method for making an array, where the method includes:
forming on at least a part of a surface a self-assembled monolayer, wherein the monolayer comprises a compound of Formula (I)
and a compound of Formula (III)
depositing at least one carbohydrate onto at least a part of the monolayer; and
irradiating the carbohydrate, monolayer, and optionally the surface,
wherein:
a covalent bond is formed between the carbohydrate and the compound of formula (I);
the carbohydrate is capable of binding to an agent, wherein the agent is capable of indicating a presence of a disease or a pathogen;
n is an integer from 1 to 100;
X is R2, —CO2R3, —C(O)NR3R3, —SR4, —CN, —OR3, a halogen, a β-diketone, a silane, a phosphate, a phosphonate, a polymer, or block copolymer;
ring A is substituted with one or more R1 groups;
R1 is independently a halogen, a hydroxyl, an aryl, an amide, a cyano, a nitro, —R2, —C(O)R3, —CO2R3, —OC(O)R3, or —OR3;
R2 is independently a hydrogen, a substituted or unsubstituted straight- or branched-chain alkyl which contains 1-6 carbons, a substituted or unsubstituted alkene which contains 2-4 carbons, a substituted or unsubstituted alkyne which contains 2-4 carbons, or —OC(O)R5;
R3 is independently a hydrogen, a substituted or unsubstituted C1-C10 straight-chain or branched-chain alkyl, or a substituted or unsubstituted alkene which contains 2-4 carbons;
R4 is independently a hydrogen, —S-pyridyl, —SR3, —SO2R3, or SR8, wherein —SR8 and the rest of formula (I) combine to form a bis-disulfide;
R5 is independently a hydrogen, an unsubstituted straight- or branched-chain alkyl that contains 1-6 carbons, or a straight- or branched-chain alkyl that contains 1-6 carbons and is substituted by an alkyne; and
Y is —NR3R3, —OH, —SH, —C(O)NR3R3, —CO2H, an ammonium, or a salt thereof.
In one embodiment, the invention relates to a method for detecting a molecule that inhibits an agent-carbohydrate interaction, where the method includes:
depositing an agent onto a carbohydrate on an array;
washing the structure to substantially remove any unbound agent;
incubating the bound agent with a molecule, wherein the molecule is capable of binding to the agent and displacing the first carbohydrate;
incubating the array with an anti-agent antibody;
washing the structure to substantially remove any unbound anti-agent antibody;
treating the array with a labeled secondary antibody;
reading the array with a label reader to determine an amount of bound labeled secondary antibody, wherein a non-zero amount of bound labeled secondary antibody indicates that the molecule inhibits agent-carbohydrate interactions; and
wherein the array comprises:
a surface;
a compound of formula (I):
immobilized on at least a part of the surface; and
one or more carbohydrates covalently attached to the compound of formula (I); and wherein
one or more of the carbohydrates are capable of binding to an agent;
n is an integer from 1 to 100;
X is R2, —CO2R3, —C(O)NR3R3, —SR4, —CN, —OR3, a halogen, a β-diketone, a silane, a phosphate, a phosphonate, a polymer, or block copolymer;
ring A is substituted with one or more R1 groups;
R1 is independently a halogen, a hydroxyl, an aryl, an amide, a cyano, a nitro, —R2, —C(O)R3, —CO2R3, —OC(O)R3, or —OR3;
R2 is independently a hydrogen, a substituted or unsubstituted straight- or branched-chain alkyl which contains 1-6 carbons, a substituted or unsubstituted alkene which contains 2-4 carbons, a substituted or unsubstituted alkyne which contains 2-4 carbons, or —OC(O)R5;
R3 is independently a hydrogen, a substituted or unsubstituted C1-C10 straight-chain or branched-chain alkyl, or a substituted or unsubstituted alkene which contains 2-4 carbons;
R4 is independently a hydrogen, —S-pyridyl, —SR3, —SO2R3, or SR8, wherein —SR8 and the rest of formula (I) combine to form a bis-disulfide; and
R5 is independently a hydrogen, an unsubstituted straight- or branched-chain alkyl that contains 1-6 carbons, or a straight- or branched-chain alkyl that contains 1-6 carbons and is substituted by an alkyne.
For the purposes of promoting an understanding of the principles of the invention, reference will now be made to specific embodiments. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended. Alteration and further modifications of the invention, and further applications of the principles of the invention as illustrated herein, as would normally occur to one skilled in the art to which the invention relates, are also within the scope of the invention.
As used herein, the term “subject” includes a plant or an animal, such as a mammal, including a human.
As used herein, the term “agent” includes molecules, such as biomarkers and proteins. The term also includes antibodies.
As used herein, the term “monolayer” includes monolayers and multilayers.
The invention provides compounds of formula (I):
immobilized on at least a part of the surface; and
at least one carbohydrate covalently attached to the compound of formula (I);
wherein:
the agent is capable of binding to the carbohydrate;
n is an integer from 1 to 1000, such as 1 to 100, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 5 to 20, 5 to 15, 5 to 10, or 15 to 30, or n is 8, 9, 10, 11, 12, or 13;
X is R2, —CO2R3, —C(O)NR3R3, —SR4, —CN, —OR3, a halogen, a β-diketone, a silane, a phosphate, a phosphonate, a polymer, or block copolymer;
ring A is substituted with one or more R1 groups;
each R1 is independently a hydrogen, a halogen, a hydroxyl, an aryl, an amide, a cyano, —R2, —C(O)R3, —CO2R3, —OC(O)R3, —OR3.
each R2 is independently a hydrogen, a substituted or unsubstituted straight- or branched-chain alkyl which contains 1-6 carbons, a substituted or unsubstituted alkene which contains 2-4 carbons, a substituted or unsubstituted alkyne which contains 2-4 carbons, or —OC(O)R5;
each R3 is independently a hydrogen, a substituted or unsubstituted C1-C10 straight-chain or branched-chain alkyl, or a substituted or unsubstituted alkene;
each R4 is independently a hydrogen, —S-pyridyl, —SR3, —SO2R3, or SR8, where the —SR8 and the rest of formula (I) combine to form a bis-disulfide; and
each R5 is independently a hydrogen, an unsubstituted straight- or branched-chain alkyl that contains 1-6 carbons, or a straight- or branched-chain alkyl that contains 1-6 carbons and is substituted by an alkyne.
Examples of compounds of formula (I) are:
The invention also provides compounds of formula (II):
wherein X, R1, and n are as defined above for formula (I); and A is -, —CH2, —C(O)—, —OC(O)—, —C(O)O—, —C(O)NR3—, or —NR3C(O)—.
Examples of Formula (II) are:
In some embodiments, the invention provides photoactive compounds that can form radicals, which lead to the formation of covalent bonds. For example, exposure to UV light can allow a photoactive compound to undergo a radical-based hydrogen abstraction reaction (e.g., abstracting a hydrogen atom from a nearby molecule, such as in.
In other embodiments, the nature of the compounds of the invention preclude the need for protecting groups on either the molecules to be attached or the photoactive compounds, which facilitates the generation of the array and the syntheses of the compounds. Additional reagents may not be needed as the radicals form upon irradiation, and then readily form covalent bonds with nearby molecules, such as carbohydrates.
The invention also provides compounds of Formula (III):
wherein X can be —R2, —CO2R3, —C(O)NR3R3, —SR4, —CN, —OR3, a halogen, a β-diketone, a silane, a phosphate, a phosphonate, a polymer, or block copolymer; Y can be —NR3R3, —OH, —SH, —C(O)NR3R3, —CO2H, a carboxylate, an ammonium, or a salt thereof; and n can be an integer from 1 to 1000, such as 1 to 100, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 5 to 20, 5 to 15, 5 to 10, or 15 to 30, or n is 8, 9, 10, 11, 12, or 13. Each R2 can independently be hydrogen, a substituted or unsubstituted straight- or branched-chain alkyl which contains 1-6 carbons, a substituted or unsubstituted alkene which contains 2-4 carbons, a substituted or unsubstituted alkyne which contains 2-4 carbons, or —OC(O)R5, wherein R5 can independently be a hydrogen, an unsubstituted straight- or branched-chain alkyl which contains 1-6 carbons, or a straight- or branched-chain alkyl which contains 1-6 carbons and is substituted by an alkyne. Each R3 can independently be a hydrogen, a substituted or unsubstituted C1-C10 straight-chain or branched-chain alkyl, or a substituted or unsubstituted alkene. Each R4 can independently be a hydrogen, —S-pyridyl, —SR3, —SO2R3, or SR8, where the —SR8 and the rest of formula (III) can combine to form a bis-disulfide. In one embodiment, Y is a polar group or a group that has a charge, such as a positive or negative charge. Examples of a compound of formula (III) include those compounds shown below and derivatives thereof:
In some embodiments, one or more compounds of Formula (I), (II), and (III), or any combination thereof, can be immobilized on a surface. The compound of Formula (III) can be mixed together with a compound of Formula (I) or (II), or both, prior to or concurrently with addition to the surface. The compound of Formula (III) improves the biomolecular compatibility and/or binding affinity for molecules to be immobilized, such as a carbohydrate, relative to the surface coated with compounds of Formula (I) or (II).
In some embodiments, the ratio of compound of Formula (III) to compound of Formula (I) and/or (II) may be from about 100:1 to about 1:1. For example, the ratio of compound of Formula (III) to compound of Formula (I) and/or (II) may be 80:1, 60:1, 50:1, 30:1, 20:1, 10:1, 5:1, 2:1, and the like.
It is to be appreciated that other photoactive groups may replace the phthalimide or benzophenone of Formula (I) or (II), respectively. Such photoactive groups may include other aromatic or non-aromatic ketone-containing groups, such as xanthones, acetone-type ketones, or derivatives thereof.
In one embodiment, the invention provides a method of immobilizing molecules, such as carbohydrates, on a surface. The method includes forming a self-assembled monolayer on a surface of a substrate, where the self-assembled monolayer includes a compound capable of forming covalent bonds with nearby molecules after irradiation, such as a molecule of Formula I or II, optionally in further combination with a molecule of Formula III; applying carbohydrates on the self-assembled monolayer; and irradiating the system. A photochemical reaction between compounds in the self-assembled monolayer and the carbohydrate results in covalent links between the carbohydrates and the monolayer, immobilizing the carbohydrates at the surface. In some embodiments, the irradiation of the compound may be through a photomask, resulting in a patterned array of carbohydrates. In other embodiments, the carbohydrates may be spotted by hand or using an automated or robotic spotter, such as disclosed in the examples, below. The amount of compound added will depend on the desired amount for each application, as can be readily determined by one skilled in the art. For example, the photoactive compound or mixture thereof, can be in a 0.01% to 10% molar solution, a 0.5% to 5% solution, or a 1% solution.
Other embodiments of the invention also relate to methods for immobilizing molecules on a surface. The methods may generate an array, such as a microarray. Methods of the invention include immobilizing on a surface a photoactive compound capable of forming covalent bonds with nearby molecules after irradiation; applying a molecule to the photoactive compound, the array, the surface, or any combination thereof; and irradiating the photoactive compound, the array, the surface, or any combination thereof, wherein a photochemical reaction between the photoactive compound and the molecule results in covalent links between the molecule and the photoactive compound, which can immobilize the molecule near or on the surface. In certain embodiments, the photoactive compound is immobilized on the surface as a self-assembled monolayer, or a self-assembled mixed-monolayer, or a multilayer (for example, see
In another embodiment of the invention, the compound capable of forming covalent bonds with nearby molecules after irradiation may be a compound of formula (I), such as a phthalimide or a derivative thereof, a compound of formula (II), such as a mono-benzophenone or a derivative thereof. In some embodiments, the compound may be added to a surface or substrate by spin-coating, or by dissolution in an appropriate organic or aqueous solvent, such as toluene, and addition of the solution to a substrate or surface.
Substrates suitable for the invention include inorganic substrates and organic substrates. In some embodiments, the substrate may be a silicon wafer, glass slide, or polymer slide, or may be made of silica, glass, quartz, silicon, titanium, titania, gold, carbon, alumina, titania, tantalum oxide, germanium, silicon nitride, zeolites, gallium arsenide, gold, platinum, aluminum, copper, titanium, polyacrylamide, nylon, polyethylene, polypropylene, PTFE, PVDF polycarbonate, polystyrene, poly(tert-butyl acrylate), poly(vinyl alcohol), nitrocellulose, polymethylmethacrylate, polyvinylethylene, polyethyleneimine, poly(ethylether)ketone, polyoxymethylene (POM), polyvinylphenol, polylactides, polymethacrylimide (PMI), polyalkenesulfone (PAS), polypropylethylene, polyhydroxyethylmethacrylate (HEMA), polydimethylsiloxane, polyacrylamide, polyimide, or block-copolymers thereof. In an embodiment the substrate may comprise a surface on a sensor. The array of the invention may include or be part of a chip, a plate, a sensor, a slide, or a combination thereof.
In one embodiment, irradiation may be accomplished with light. In more specific embodiments, ultraviolet (UV) light can be used. In other embodiments, light of wavelengths from about 280 to about 400 nm, such as from about 290 to about 305 nm, about 305 to about 315 nm, about 315 to about 350 nm, or about 300 nm, can be used in the invention.
In another embodiment, the carbohydrate may be a monosaccharide, an oligosaccharide, or a polysaccharide. In yet another embodiment, the self-assembled monolayer may comprise a compound of formula (I), such as a mono-phthalimide, a compound of formula (II), such as a mono-benzophenone, or a derivative thereof, optionally in combination with a compound of formula (III).
In some embodiments, methods of the invention include immobilizing on a surface a composition that includes a photoactive compound capable of forming covalent bonds with nearby molecules after irradiation and a second compound that increases the affinity of the nearby molecules for the composition; applying a molecule to the composition; and irradiating the composition, where a photochemical reaction occurs between the photoactive compound and the molecule, resulting in covalent links between the molecule and the photoactive compound and immobilizing the molecule on or near the surface. In certain embodiments, the composition can be immobilized on the surface as a self-assembled monolayer. The second compound may include a polar or charged group, which can be presented at the air-monolayer interface. In a specific embodiment, the group may be an amine or ammonium, or a carboxylic acid or carboxylate. In a more specific embodiment, the second compound is attached to the surface and terminates in a polar group at the end that is not attached to the surface. The photoactive compound and the second compound may be attached to the surface through the same or similar means.
In a specific embodiment, the invention provides a mixed monolayer including a photoactive compound and a second compound capable of increasing the affinity of a nearby molecule for the monolayer comprising the photoactive compound. The second compound may have a polar group, such as an amine. The second compound may be of Formula (III), and the ratio of the second compound to the photoactive compound, such as a compound of Formula (I) or (II), may be from about 1:1 to about 100:1, about 2:1 to about 50:1, about 1:1 to about 25:1, about 2:1 to about 25:1, about 5:1 to about 20:1, about 26:1 to about 50:1, or about 51:1 to about 100:1.
In one embodiment, a self-assembled monolayer containing phthalimide chromophores is capable of photochemically immobilizing carbohydrates on a flat surface. An illustrative method requires no chemical modification of the carbohydrates prior to deposition. Further, because covalent attachment is involved, carbohydrates of all molecular weights can be immobilized. The photochemical nature of the technique allows simple arrays to be created with or without a robot and makes the method adaptable to photolithography. In some embodiments, multiple carbohydrate patterns can be immobilized by repeating the photochemical reaction with a different carbohydrate in a previously masked region, or spotting carbohydrates in alternate, known locations. In conjunction with a microarray spotter, large libraries of carbohydrates may be immobilized on a surface. The versatility and ease of the method provides an opportunity for biologists, chemists and engineers to investigate biological phenomena and create new biological materials.
In one embodiment, the term “array” as used herein includes a microarray.
Compounds of Formula (I) or (II) can also abstract electrons from suitable donors such as amines, sulfides, AIBN, and the like. Electron transfer to the photoactive compound may be followed by proton transfer and covalent bond formation in a manner similar to the radical hydrogen abstraction in
In some embodiments, the photoactive compound can further include a functional group capable of being immobilized on a surface. Some examples of functional group capable of being immobilized on a surface include a carboxylic acid, thiol, β-diketone, silane, phosphate, phosphonate, alkyl, alkene, or alkyne, polymer, block co-polymer, and the like. In some other embodiments, the photoactive compound can be incorporated into polymers and/or hydrogels, for example to modify the molecule-surface interfacial tension or to modify steric constraints and make the photoactive portion of the molecule more accessible to molecules that are intended to be attached to the photoactive compound.
In one embodiment, the invention does not require the chemical modification of each molecule prior to deposition and it is not dependent on the molecular weight of the deposited molecule. Moreover, the invention can utilize bonds, including sp3 bonds such as C—H bonds, S—H bonds, Si—H bonds, and the like, sp2 bonds such as are present in alkenes, and sp bonds, such as are present in alkynes, which are present in many molecules (e.g., C—H bonds are readily found in carbohydrates). Reactive sp3 bonds, such as anomeric C—H bonds that are present in carbohydrates, are useful in the invention. In a specific embodiment, the invention requires no chemical reagents beyond the monolayer or multilayer and generates very few byproducts.
The invention has a wide number of applications. For example, the invention can be utilized in tissue engineering, sensor fabrication, glycome sequencing, and in microarray construction for high-throughput characterization, such as the characterization of carbohydrate-related enzyme activity and carbohydrate interactions with cells, antibodies, proteins and microorganisms. Moreover, the surfaces may be used as biological sensors for identifying biological agents including antibodies and biological weapons.
In specific embodiments, the present invention provides a platform and method for screening antibody activity, such as binding activity towards various pathogens; photochemically linking at least one carbohydrate; and glycomic or proteomic studies, such as those aimed at the identification of biological agents, the discovery of new drugs, and understanding cellular processes.
In one embodiment, the invention provides methods for immobilizing an array of molecules on a surface. For example, a substrate can be coated with a photoactive monolayer. Molecules can be placed on the monolayer, for example at discrete locations. The molecules can be linked to the monolayer through a photochemical reaction, such as with irradiation, which causes covalent bonds to form between the molecules and the photoactive monolayer. The system, array, molecules, and/or monolayer may be irradiated through exposure to light, such as UV light. Unbound molecules can then be removed, such as by washing with appropriate aqueous or organic solvents. The photoactive monolayer can include compounds of Formulas (I), (II), or (III), or combinations thereof.
In another embodiment, the invention provides methods for immobilizing a patterned array of molecules on a surface. A mask containing the desired pattern or image can be placed over the photo-active molecule coated surface and irradiated though the mask. Alternatively, a robotic spotter can be utilized to place a photoactive compound and a molecule in a pattern on a photoactive surface, and the resultant patterned array can be irradiated. Other suitable methods to form a patterned array of immobilized carbohydrates will be readily apparent to one of ordinary skill in the art.
In one embodiment, the attachment of molecules onto a surface includes coating the molecules onto the surface.
The molecules to be attached to the surface can be attached at known, discrete locations on a surface. The compounds of formulas (I), (H), or (III) can be attached to an entire surface or one or more parts of the surface. Also, molecules can be attached to the compounds of Formula (I) or (II) at one or more parts of the surface, such as known, discrete and non-overlapping locations. The same molecule may be located at more than one part of the surface, and different molecules may be located at more than one part of the surface. The molecules can be attached to a photoactive compound of Formula (I) or (II) at one or more discrete parts of a surface at known locations.
The molecules used in the invention can be carbohydrates. The carbohydrates can be associated with, or present in or on on, pathogens, including bacteria, viruses, or parasites. Alternatively, the carbohydrates can bind to agents that recognize or bind to pathogens or toxins.
Illustrative pathogens or toxins can include Bacillus anthracis, Vibrio cholerae, E. coli, yeast, the causative organisms of tetanus and botulism, HIV such as HIV-1, orthomyoxyiridae, influenza viruses, SARS-CoV, Bacillus cereus, Brucella abortus, Brucella melitensis, Brucella suds, Campylobacter jejuni, Clostridium botulirum, Clostridium perfringens, Enterohemorrhagic E. Coli, Enterotoxigenic E. coli, Listeria monocytogenes, Salmonella, Shigella, Staphylococcus aureus, Vibrio parahaemolyticus, Vibrio vulnificus, Yersinia enterocolytica and Yersinia pseudotuberculosis, hepatitis A, norwalk-like viruses, rotavirus, astroviruses, calciviruses, adenoviruses, and parvoviruses, Cryptosporidium parvum, Cyclospora cayetanensis, Entamoeba histolytica, Giardia lamblia, Toxoplasma gondii, Trichinella spiralis, Clostridium botulinum, Yersinia pestis, Francisella tullarensis, Brucella species epsilon toxin from Clostridium perfringens, Salmonella species, Escherichia coli 0157:H7, Shigella, Cryptosporidium parrum, Burkholderia mallet, Burkholderia pseudomallei, Chlamydia psittaci, Coxiella burnetii, Ricin toxin from Ricinus communis, Staphylococcal enterotoxin B., Rickettsia prowazekii, ciguatera toxin, shellfish toxins, floviruses, ebola virus, Marburg virus, arenaviruses, Lassa virus, Machupo virus, hantavirus, variola major, hemorrhagic fever virus, Nipah virus, alphaviruses, Venezuelan equine encephalitis, eastern equine encephalitis, and western equine encephalitis, and toxins therefrom.
A biomarker in the present invention can be an antibody or a carbohydrate. A biomarker can include an immunoglobulin, a monoclonal antibody, polyclonal antibody, Fv fragment, single chain Fv (scFv) fragment, Fab′ fragment, F(ab′)2 fragment, single domain antibody, camelized antibody, humanized antibody, diabodies, tribodies, and tetrabodies. In one embodiment, a biomarker can be an agent.
In a more specific embodiment, the carbohydrates used in the invention may correspond to carbohydrates from natural sources or synthetic carbohydrates. The carbohydrates can correspond to those present on or isolated from pathogens, such as Bacillus anthracis. The carbohydrates listed below and described in Table 1, and glycosides and combinations thereof, can be employed. In one embodiment, carbohydrates may include both anomers. In a more specific embodiment, carbohydrates may include both anomers of each monosaccharide present in an oligo- or polysaccharide, in any ratio, or anomers of a subset of all of the monosaccharides present in an oligo- or polysaccharide. Carbohydrates used in the invention may be derivatized or underivatized, and may be obtained from commercial and non-commercial sources, such as the NIH, or may be synthesized according to synthetic procedures available in the literature.
In another aspect, the invention also provides carbohydrate arrays formed by the methods of the invention. The array of the invention can use any number of carbohydrates, including a plurality of carbohydrates. In more specific embodiments, the array includes from 5 to 1,000,000; 5 to 1,000; 10 to 100; or 35 carbohydrates. In another embodiment, the array includes carbohydrates specific to pathogens or which are recognized by antibodies to pathogens.
In one embodiment, the invention includes an array that includes one or more of the following carbohydrates:
glycosides thereof; or combinations thereof.
In another embodiment, the carbohydrates listed in Table 1 can be used in the invention.
Streptococcus pneumoniae type 23 capsular polysaccharide (D-
The carbohydrates of the invention can include the α- and β-anomers of each monosaccharide, or a combination of both, unless otherwise specified. Other molecules than can be immobilized on a surface and used in the invention will be readily apparent to one of skill in the art. In some embodiments, each molecule to be deposited on the surface may be deposited in more than one spot.
In some embodiments, the deposition of a molecule on a surface or a monolayer, is repeated at least twice in at least two different locations, creating at least two identical spots on the array. An array can thus be generated with three, four, five, or more identical spots of a molecule, such as a carbohydrate. A molecule can be spotted on the array at differing concentrations as measured by weight percentages or concentrations or by molar percentages or concentrations. In one embodiment of the invention, the molecules deposited on the surface or monolayer are carbohydrates.
One aspect of the present invention is a method for determining the presence of an antibody that specifically binds Bacillus anthracis in a sample, which includes contacting the sample with a carbohydrate array of the invention; and determining whether a carbohydrate at a known location on the surface has an antibody that specifically binds Bacillus anthracis bound thereto. The sample is from a subject, such as a human. The determination may be accomplished using secondary antibodies to the B. anthracis-binding antibody, where the secondary antibody is labeled with a detectable probe, such as a fluorescent or radioactive molecule or an enzyme conjugate that is used in a colorimetric assay.
Some embodiments of the invention relate to methods for immobilizing molecules on a surface. Methods of the invention can include immobilizing on a surface of a substrate a one or more photoactive compounds that are capable of forming at least one covalent bond with a nearby molecule after irradiation; applying at least one molecule, such as a carbohydrate, onto the photoactive compounds on the surface; and irradiating the photoactive compounds, enabling a photochemical reaction between the photoactive compounds and the carbohydrate, resulting in at least one covalent bond between the photoactive compound and the carbohydrate and immobilizing the carbohydrate at the surface.
In one embodiment, a plurality of different carbohydrates are covalently attached to a one or more compounds of Formula I that have been immobilized on a surface and that form a photoactive surface on top of the surface to which the compounds are immobilized. Each type of different carbohydrate can be spatially segregated on the photoactive surface.
Other embodiments of the invention also relate to methods for immobilizing molecules on a surface. Methods of the invention can include (1) immobilizing on a surface a photoactive compound capable of forming covalent bonds with nearby molecules after irradiation; (2) applying a molecule to the photoactive compound; and (3) irradiating the photoactive compound, wherein a photochemical reaction between the photoactive compound and the molecule results in covalent links between the molecule and the photoactive compound to immobilize the molecule near the surface. In certain embodiments, the photoactive compound can be immobilized on the surface as a self-assembled monolayer (see
In other embodiments, methods of the invention can include (1) immobilizing on a surface a composition that includes a photoactive compound capable of forming covalent bonds with nearby molecules after irradiation and a second compound that can increase the affinity of desired molecules to the composition; (2) applying a molecule to the composition; and (3) irradiating the composition, wherein a photochemical reaction between the photoactive compound and the molecule results in covalent links between the molecule and the photoactive compound to immobilize the molecule near the surface. In certain embodiments, the composition can be immobilized on the surface as a self-assembled monolayer. In other embodiments, the photoactive compound can be immobilized on the surface as a multilayer.
In some embodiments, the molecule that forms covalent links with the photoactive compound can be a carbohydrate.
In certain embodiments, the photoactive compound can be immobilized on the surface of the substrate as a self-assembled monolayer (SAM). In one embodiment, a photochemical reaction described herein includes a radical. In another embodiment, the photochemical reaction does not include a carbene. In yet another embodiment, the substrate does not include a protein.
One embodiment of the invention is the photo-generation of epitope-specific carbohydrate arrays. In one embodiment, a photoactive surface is utilized for the covalent immobilization and patterning of carbohydrates onto a substrate such as glass. This method can employ a glass slide coated with a self-assembled monolayer that presents photoactive chromophores, such as phthalimides, at the air-monolayer interface. Upon exposure to UV radiation, for example at a wavelength of 280 nm to 400 nm or 300 nm, the phthalimide end-groups graft the carbohydrates by hydrogen abstraction and radical recombination.
A radical-quenching substituent on the photoactive moiety is believed to result in inferior reactivity of the photoactive moiety and consequently poorer immobilization of any molecule to the photoactive surface. For example, it is believed that direct amino substitution on the photoactive phthalimide moiety results in poor immobilization of a molecule to the photoactive surface.
One advantage of the invention is that the efficacy of carbohydrate immobilization to the instant array is independent of the molecular weights of spotted carbohydrates. Another advantage of photochemical immobilization is the ability to produce epitope-specific carbohydrate arrays using unmodified carbohydrates. This technology was applied to display a panel of carbohydrate structures, including synthetic fragments and derivatives of the anthrose-containing tetrasaccharide of the B. anthracis exosporium and a number of control carbohydrate antigens (see Table 1), for immunological characterization.
The invention also provides methods for immobilizing a patterned array of molecules on a surface. In one embodiment, a mask containing the desired pattern or image can be placed over the coated surface and the surface can be irradiated though the mask. Alternatively, a robotic spotter can be utilized. The robotic spotter can be used to create patterned areas containing a photoactive compound and a molecule, either concurrently or step-wise, and the patterned array can be irradiated. Other suitable methods to form a patterned array of immobilized carbohydrates will be readily apparent to one of ordinary skill in the art.
The photoactive compound and/or the immobilized molecule can be formed as a coating on a surface, and at various thicknesses. For example, the photoactive compound and/or the immobilized molecule may form a coating with a thickness of less than 1 nm, 2 nm, 5 nm, 10 nm, 20 nm, 50 nm, 100 nm and the like. For example, if a monolayer of photoactive compound and/or the immobilized molecule is formed on the surface, the thickness can depend on the molecular weight of the photoactive compound and/or the immobilized molecule. In some embodiments, the photoactive compound and/or the immobilized molecule may be applied to the surface by spin coating, spray coating, robotic spotting, or any other conventional techniques known in the art to obtain a desired coating of a compound or molecule.
In other embodiments, the molecules are linked to a compound of Formula (I) or (II) on a surface through a radical reaction. The radical reaction can be initiated using light, such as ultraviolet light, or by single-electron transfer. The radical reaction may be initiated by electron transfer to a molecule of Formula (I) or (II) an electron from a molecule such as AIBN, tributyl tin hydride, or an amine or sulfide, such as a thioether, or the like.
In some embodiments, the photoactive compound can further include a functional group capable of being immobilized on a surface. In some cases, this immobilization is through a covalent bond. Some examples of functional group capable of being immobilized on a surface include a carboxylic acid, thiol, β-diketone, silane, phosphate, phosphonate, alkyl, alkene, alkyne, polymer, block co-polymer, and the like. In other embodiments, the photoactive compound can be incorporated into polymers and/or hydrogels to modify the molecule-surface interfacial tension or to modify steric constraints that may make the photoactive portion of the molecule inaccessible. In specific embodiments, the compound may be immobilized via a chemical reaction as follows, a thiol-containing compound can be covalently attached to a gold surface, a silane, phosphate, or phosphonate (such as an silane ether or phosphate or phosphonate ester) can be covalently attached to a surface comprising terminal hydroxyl groups via ether or ester exchange. In a specific embodiment, the photoactive compound contains a silane such as a silane ether, and the covalent immobilization is accomplished through exchange of an ether group thereon for a surface hydroxyl group, using methods as described herein and methods as will be apparent to one of skill in the art.
Any suitable molecule may be covalently bonded to the surface. In one embodiment, carbohydrates, glycolipids, glycopolymers, proteoglycans, and glycoproteins are useful in the invention. For example, carbohydrates, such as monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, polysaccharides, glycosides thereof, and the like, can be utilized in the invention. In some embodiments, the carbohydrate can be a simple carbohydrate, such as glucose or sucrose. In other embodiments, one or more dextrans ranging from 20-2000 kDA can be utilized. In some embodiments, the carbohydrates can be underivatized carbohydrates, without chemical modification. Similar molecules that can be immobilized on a surface will be readily apparent to one of ordinary skill in the art.
In some embodiments the molecule that can be covalently bonded to the surface elicits an immune response in an organism, such as an animal, including a mammal, and further including a human. Such a molecule can be a carbohydrate. In some cases, the response elicited will be the production of antibodies against the molecule.
In one embodiment, the invention does not require the chemical modification of a molecule prior to deposition. The derivatization also may not be dependent on the molecular weight of the molecule. In some embodiments, the photochemical reaction of the invention can utilize bonds, including sp3 bonds such as C—H bonds, S—H bonds, and Si—H bonds, sp2 bonds such as are present in alkenes, and sp bonds, such as are present in alkynes, and the like, which are present in many molecules (e.g., C—H bonds are readily found in carbohydrates). In other embodiments, the invention requires no chemical reagents beyond the photoactive monolayer and generates very few byproducts.
In one embodiment, the invention uses biomarkers unique to B. anthracis.
Another embodiment of the invention is the development of new, safer anthrax vaccines to block the anthrax infection. An efficient means to determine the immune response elicited by a candidate vaccine would greatly facilitate such a development. If B. anthracis spores express potent immunogenic carbohydrate moieties, immunization with the spores should elicit antibodies specific for these carbohydrate structures. Such antibody reactivities could then be detected by carbohydrate arrays that display the carbohydrate structures recognized by the antibodies. One embodiment of the invention is to identify highly specific immunogenic targets, such as those displayed on B. anthracis spores. Highly specific immunogenic targets such as surface-exposed carbohydrate moieties characteristic for a given microbe may serve as key biomarkers for pathogen identification, diagnosis, and vaccine development.
The invention has a wide number of applications. For example, the invention can be utilized in pathogen detection, the determination of exposure to a pathogen, the screening of vaccine candidates, the determination of the immune response elicited by a vaccine or vaccine candidate, the elucidation of the binding epitope of carbohydrate-binding proteins including antibodies, sensor fabrication, glycome sequencing, high-throughput array construction, and high-throughput characterization of carbohydrate enzyme activity and carbohydrate interactions with cells, antibodies, proteins and microorganisms.
In another embodiment, the surfaces may serve as sensors for identifying biological entities, such as cancer cells, pathogens, or biological weapons. The invention also provides for screening antibody activity towards various organisms, including pathogens; photopatterning a carbohydrate, biological or synthetic; glycomic and proteomic studies aimed at the discovery of new drugs and vaccines, and the understanding of cellular processes.
Arrays of the invention may also be used to screen for agents that recognize carbohydrates. The agents may be proteins, and the agents may be associated with pathogens, including agents that are present on a pathogen or agents that bind to a pathogen. The carbohydrates may be specific to pathogens, and the agents may be biomarkers, proteins, such as antibodies, and/or synthetic molecules. The arrays may be used to screen for or otherwise develop compositions which interfere with, or inhibit, pathogen binding. In one embodiment, the carbohydrate can be a biomarker for a pathogen or a disease.
The invention also includes methods for diagnosing a disease or determining the exposure to one or more pathogens in a subject and/or a sample, such as a sample of biological origin, including from a subject. The subject can be an animal, such as a mammal, including a human. The invention also includes methods for detecting or determining the presence of antibodies to a disease or pathogen in a subject or a sample, such as a biological sample, including a sample from a subject. In one embodiment, a carbohydrate or an antibody can be a biomarker for a disease.
The invention will be further described with reference to the following examples; however, it is to be understood that the invention is not limited to such examples. Rather, in view of the present disclosure that describes the current best mode for practicing the invention, many modifications and variations would present themselves to those of skill in the art without departing from the scope and spirit of the invention. All changes, modifications, and variations coming within the meaning and range of equivalency of the claims are to be considered within their scope.
Further description of some examples disclosed herein can be found in U.S. Provisional Patent Application Nos. 60/776,096, 60/735,402, and 60/843,674, each of which are hereby incorporated by reference in their entireties.
A 3.3 mmol portion of 11-bromoundecanetrimethoxysilane (Gelest) was added to a solution of an equimolar amount of potassium phthalimide (Aldrich) in 60 mL of anhydrous DMF (Aldrich). The solution was stirred overnight at room temperature (RT) under argon. Chloroform (50 mL) was added. The solution was transferred to a separatory flask containing 50 mL of H2O. The aqueous layer was separated and then extracted with two 20 mL portions of chloroform. The combined chloroform extract was washed with several 20 mL portions of H2O. The chloroform was removed by rotoevaporation, and residual DMF was removed on a high vacuum line to give a pale yellow liquid (0.99 g, 72% yield). The compound was used without further purification. For self-assembly experiments, residual DMF was not removed. 1H NMR: (CDCl3) δ 7.82 (m, 2H), 7.69 (m, 2H), 3.66 (t, J=7 Hz, 2H), 3.55 (s, 9H), 1.44-1.15 (m, 18H), 0.71-0.51 (m, 2H). LRMS-FAB+ (m/z): (M-H) 420.2 (experimental), 420.2 (calculated); (M-OCH3) 390.1 (experimental), 390.2 (calculated).
Fabrication of Mixed Monolayers:
A robotic spotter was used to deliver polysaccharides to the surface. However, the thermodynamic parameters of the surface needed to be adjusted in order to transfer a detectable amount of carbohydrates from the pin of the spotter to SAM 1 (below). In order to make the surface more attractive to carbohydrates, mixed monolayers were made from a solution containing a 5:1 ratio of aminopropyltrimethoxy silane to compound I-1. Presumably, the hydrophilic amine group interacts more favorably with the carbohydrates as compared to the more hydrophobic phenyl ring of compound I-1, decreasing the interfacial tension between the carbohydrate and the surface, allowing for a sufficient amount of carbohydrates to be adsorbed to the surface for subsequent photo-immobilization. Alternatively, a 20:1 mixture was used.
Preparation of SAM 1:
Substrates consisted of glass (ArrayIt), quartz (SPI) or silicon (wafer world). Substrates and glassware were cleaned by boiling in a “piranha” solution (7:3 sulfuric acid:H2O2) for one hour followed by an extensive rinse with water and methanol. Substrates were dried with a stream of argon and a 1 mmol solution of compound I-1 in anhydrous toluene (Aldrich) and a solution containing a 5× molar amount of aminopropyltrimethoxy silane (Gelest) relative to compound I-1 were simultaneously added to the substrate. The solution was kept under argon and left undisturbed for twelve hours. The surface was then removed and baked for two hours at 110° C. The resulting self-assembled monolayers were rinsed with toluene and sonicated three times for two minutes each in toluene, toluene:methanol 1:1, and methanol, yielding SAM 1. Coated substrates were kept in argon-purged vials until further use.
Alternatively, SAM 2, containing a 20:1 ratio of aminopropyltrimethoxy silane to phthalimide was constructed. Mixed monolayers were formed by the method for SAM 1, above, substituting a solution containing a 20:1 ratio of aminopropyltrimethoxy silane to compound I-1 in anhydrous toluene for the 5:1 solution used above. The solution was mixed in a vial containing anhydrous toluene. The solution was then transferred to a vial, capped with a rubber septum, containing the microscope slide under argon. The procedure then continued as above.
Microarray Construction
The carbohydrates of Table 1 were individually dissolved in saline (0.9% NaCl) at a given concentration and were spotted in triplicate in parallel. The initial amount of carbohydrate spotted was 0.35 ng per spot and was further diluted by serial dilutions of 1:5 thereafter. A high-precision robot designed to produce cDNA microarrays (PIXSYS 5500C, Cartesian Technologies Irvine, Calif.) was utilized to spot carbohydrate antigens onto chemically modified glass slides as described.
Photo-Coupling of Carbohydrates
After microarray spotting, the SAM 1 slides were air-dried and placed in a quartz tube. The sealed tube was subsequently purged with argon or nitrogen before irradiation. UV irradiation was conducted by placing the quartz tube under a desktop lamp containing a 300 nm Rayonet bulb for one hour. Precaution was made to avoid skin and eye contact with the radiation during the irradiation process.
Microarray Screening
Immediately before use, the printed microarrays were rinsed and washed with PBS (PH 7.4) two times with five minutes of incubation in each washing step. They were then “blocked” by incubating the slides in 1% BSA in PBS containing 0.05% NaN3 at room temperature (RT) for 30 minutes. Antibody staining was conducted at RT for one hour at given dilutions in 1% BSA PBS containing 0.05% NaN3 and 0.05% Tween 20. The stained slides were rinsed five times with PBS containing 0.05% Tween 20 after each staining step. A ScanArray 5000A Standard Biochip Scanning System (PerkinElmer, Torrance, Calif.) equipped with multiple lasers, emission filters and ScanArray Acquisition Software was used to scan the microarray. Fluorescence intensity values for each array spot and its background were calculated using ScanArray Express (PerkinElmer, Torrance, Calif.). The screening data are depicted in
Instrumental Measurements
UV-vis spectra were obtained using a Shimadzu (UV-2401PC) UV-vis recording spectrophotometer. Contact angle measurements were performed with a Rame-Hart 100-00 contact angle goniometer using Millipore Mili-Q water. At least three droplets were measured on each sample and averaged. Thicknesses were measured with a Beaglehole ellipsometer in variable angle mode. A refractive index of 1.5 was used for all samples. Measurements were performed three times in different locations on the surface and averaged. Fluorescence spectra were obtained using a Jobin Yvon Fluorolog 3 spectrofluorimeter in front face mode. The surface was placed at an angle of 20° to a line parallel to the plane of the detector.
The above exemplary method to make a carbohydrate microarray has been repeated using the photoactive benzophenone compound II-1 in place of the photoactive phthalimide compound I-1.
The following documents are hereby incorporated by reference in their entireties.
As various changes can be made in the above methods and compositions without departing from the scope and spirit of the invention as described, it is intended that all subject matter contained in the above description, shown in the accompanying drawings, or defined in the appended claims be interpreted as illustrative and not limiting.
This application claims priority to U.S. Provisional Application Nos. 60/858,069, filed Nov. 9, 2006, and 60/843,674, filed Sep. 11, 2006, each of which are hereby incorporated by reference in their entirety.
The work described herein was supported in whole, or in part, by National Institute of Health Grant No. AI064104; U.S. Army Research Laboratory and the U.S. Army Research Office Grant No. DA W911NF-04-1-0282 and the National Science Foundation Grant Nos. DMR-02-14263, IGERT-02-21589 and CHE-04-15516. Thus, the United States Government has certain rights to the invention.
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| Number | Date | Country | |
|---|---|---|---|
| 20100331198 A1 | Dec 2010 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60843674 | Sep 2006 | US | |
| 60858069 | Nov 2006 | US |
